Datasets:
ManuelAlv
/
Cancer

Dataset card Data Studio Files Community
1
input
stringlengths
600
32.7k
label
stringclasses
3 values
chrysoperla nipponensis okamoto which has the unique diapause phenotype distinguishable from nondiapause adult is an ideal model anism for studying the mechanism of reproductive diapause however there is no reliable and effective reference genes used for the reproductive diapause study of c a0nipponensis therefore in this study we evaluated the expression stability of candidate reference genes tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin αtub in adults under diapause and nondiapause induction conditions using four statistical algorithms including genorm normfinder bestkeeper and ˆ†ct method results showed that arp3 and tub1 were the most stable reference genes in all samples and in the adult tissues group arp3 and rps5 were the most stable reference genes in the development degree group αtub and ef1a were unstable reference genes under the conditions of this study meanwhile to verify the reliability of the reference genes we evaluated the relative expression levels of vg and vgr in different treatments significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was respectively observed when using arp3 as the reference gene but not when using an unstable reference gene the reference genes identified in this work provided not only the basis for future functional genomics research in diapause of c a0nipponensis and will also identify reliable normalization factors for realtime quantitative realtime polymerase chain reaction data for other related insectskey words chrysoperla nipponensis okamoto reference genes qrtpcr reproduction diapausedue to the advantages of high sensitivity rapidity specificity and accuracy bustin et a0al valasek et a0al vanguilder et a0al shakeel et a0al quantitative realtime polymerase chain reaction qrtpcr has been widely used in the study of animals plants and microanisms roy et a0al jia et a0al zhang et a0 al ding et a0 al sun et a0 al qrtpcr is the most commonly used method for the expression analysis of target genes however the reliability of qrtpcr results in different samples is determined by a variety of factors among which the use of stably expressed reference genes is an important link for accurate detection of gene expression changes by qrtpcr bustin et a0 al at present several commonly used reference genes for data normalization include tubulin actin ribosomal protein elongation factor 1α glyceraldehyde3phosphate dehydrogenase 18s ribosomal rna and other genes bustin vanguilder et a0al however more and more studies have found that these reference genes do not show consistent expression patterns under different experimental conditions and even affect the reliability of experimental results therefore in order to obtain stable and reliable normalization factors reference genes with stable expression are usually used for correction and standardization to reduce errors between samples selection and evaluation of reference genes have become a necessary step before quantifying the expression of target genes accuratelynowadays there have been many studies on the selection of reference genes for insects such as sesamia inferens helicoverpa armigera aphis gossypii myzus persicae etc lu et a0 al shakeel et a0al zhang et a0al ma et a0al kang et a0al in these studies four statistical algorithms including genorm normfinder bestkeeper and ˆ†ct method were mainly used to analyze the ct values obtained by qrtpcr of reference genes under various experimental conditions shakeel et a0 al finally the stability of the candidate reference genes was determined according to the geometric mean value of each gene ranked using different the authors published by oxford university press on behalf of entomological society of americathis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly cited for commercial reuse please contact spermissionsoupcom 0c of insect science vol no algorithms and the most suitable reference gene was selected for the target gene expression analysis xiao et a0al kang et a0al chrysoperla nipponensis okamoto as one of the important predatory natural enemies of agricultural and forestry pests prefers to eat aphids thrips and other pests okamoto nie et a0al because of its characteristically wide geographical distribution and broad range of host prey niijima syed et a0al it has good prospects for widespread application in biological control mcewen et a0 al memon et a0 al reproductive diapause is an important way for c a0nipponensis adults to escape from adverse environments xu et a0al at present there have been many reports on the diapause of c a0nipponensis xu et a0al found that the body color of c a0nipponensis was green during the reproductive period but turned brown and yellow during the diapause period chrysoperla nipponensis belongs to the photoperiodic sensitive insect the adult diapause was induced by short photoperiods xu et a0al chen et a0al found that different photoperiods affected the material content eg protein and glycogen of c a0nipponensis diapause induced by the short photoperiod was beneficial to the storage of c a0nipponensis chen et a0al we expect an exponential increase of diapause research on c a0nipponensis at the molecular level in the near future thus stable and reliable reference genes are important for accurately quantifying gene expression of c a0nipponensisribosomal proteins and ribosomal rna have been used as reference genes in previous diapause studies for example williams et a0al used ribosomal protein rp49 as a reference gene to study the natural variation of drosophila melanogaster diapause williams et a0 al and sim and denlinger used ribosomal protein large subunit rpl19 as a reference gene in a study of ovarian development of culex pipiens during overwintering diapause sim and denlinger this indicates that under the same experimental conditions the selected reference genes in different species research are also differentin this research candidate reference genes were selected including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub whose expression profiles were measured by the qrtpcr the stability was analyzed by four statistical algorithms genorm normfinder bestkeeper and ˆ†ct method in different developmental stages reproductive and diapause of adults and among different tissues the optimal reference genes under different conditions were determined which contributed to the accurate expression of target genes for future researchmaterials and a0methodsinsecta stable population of c a0 nipponensis was maintained in an artificial climate chamber rsz intelligent artificial climate chamber changzhou guohua jiangsu province under the following conditions a0± °c temperature a0± relative humidity rh and long photoperiod of l d h in our laboratory the eggs were collected by cutting the stalk and incubated in fingertip tubes a0cm in diameter and a0cm in height the primary hatching larvae were fed megoura japonica matsumura whose host plant was vicia faba l a0the adults were paired immediately after emergence in a bottle a0cm in diameter and a0cm in height fed a dry brewer™s yeast feed mixed with sucrose in a ratio of and then minced in a mortar and sifted through mesh and honey water the diapause adults used in this study were kept under the conditions of short photoperiod of ld h in all processes from eggs larvae pupae to adults whereas the nondiapause adults were kept under the conditions of long photoperiod of ld a0hsample collection development degree samples from individuals from varying developmental stages included female adults in the diapause induction period “ d under the short photoperiod the diapause maintenance period “ d under the short photoperiod the diapause termination period “ d under the short photoperiod and the reproduction period “ d under the long photoperiod each sample which included three to four females was independently replicated three times as three biological replicates adult tissues reproduction seven different tissues were collected from reproductive adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of reproductive adults under long photoperiod were collected on the 10th day after emergence each tissue required about “ a0mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues diapause seven different tissues were collected from diapause adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of diapause adults under short photoperiod were collected on the 20th day after emergence each tissue required about “ a0 mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues samples from reproductive and diapause adult tissues all samples samples from group and all treatments were immediately frozen with liquid nitrogen and stored in an ultralow temperature refrigerator at ˆ’°c prior to rna extractiontotal rna extraction and cdna synthesisin this study total rna was extracted using minibest universal rna extraction kit takara japan and dnase i a0 was used for digestion of the membrane rna integrity was estimated by agarose gel electrophoresis rna concentration and purity were measured with a nanodrop one spectrophotometer thermo scientific then 1μg rna was reversetranscribed into the firststrand complementary dna cdna according to the hiscript ii q rt supermix for qpcr gdna wipers vazyme nanjing china instructions and stored at ˆ’°c all cdna was diluted 10fold with dnasernasefree sterile water before usecandidate reference gene selection and primer a0designaccording to several commonly used reference genes candidate reference gene sequences were obtained by screening from the existing transcriptome of c a0 nipponensis in the laboratory namely tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub in order to ensure the predictive accuracy of the selected sequences we conducted blast alignment all primers were designed using primer premier based on the following criteria gc content “ annealing temperature “°c and primers length “ a0 bp and the specificity of each pair of amplicons was determined by qrtpcr followed by agarose gel electrophoresis and melting curve analysis the amplification efficiency of the pcr was calculated by using the formula e a0 a0 ˆ’slopeˆ’ the slope was obtained by the standard curve which was generated by qrtpcr of a series of continuously diluted cdna samples 0c of insect science vol no realtime qrtpcr analysisthe 20µl total reaction volume were configured according to the protocol of chamq sybr qpcr master mix vazyme contained 10µl a0× chamq sybr qpcr master mix a0µl a0μm of each gene specific primer a0µl of cdna and a0µl of ddh2o the amplification reaction program was set as follows predenaturation at °c for a0s followed by cycles of denaturation at °c for a0s annealing at °c for a0 s the melting curves were analyzed in the “°c temperature range after amplification step the reaction was performed on a roche lightcycler96 instrument to obtain ct values amplification curves melting curves and standard curves all samples were carried out in four technical and three biological replicates and the negative control no template was performed in paralleldata analysisct values for all samples were exported into an excel spreadsheet and were used to analyze the stability of candidate reference gene expression by genorm normfinder bestkeeper and ˆ†ct method the comprehensive ranking were performed following methods adapted from xiao et a0 al the optimal number of genes was determined by the pairwise variation vnn1 between the normalization factors calculated by genorm among the four algorithms genorm and normfinder need to convert the original ct value according to the corresponding requirements before analysis in genorm the stability ranking of genes was determined by the expression stability value m value in bestkeeper the stability ranking of genes was determined by the coefficient of variation cv and sd in normfinder the stability ranking of genes was determined by the gene expression stability value sv in ˆ†ct method the stability of genes was ranked according to the sd of genes ˆ†ct values in four statistical algorithms genes with the lowest value were the most stably expressedvalidation of reference a0genesin most insects vitellogenin vg and vitellogenin receptor vgr play important roles in the reproductive process of female insects vg is taken up by developing oocytes through receptormediated endocytosis rme thereby promoting the development of oocytes and the formation of eggs in this process vgr is the main receptor mediating endocytosis previous studies have shown that reproductive diapause arrests development of oogenesis and vitellogenesis tatar and yin and the expression levels of the vg and the vgr in nondiapause female were significantly higher than those in reproductive diapause female jiang et a0al in order to evaluate the effectiveness of the selected reference genes the expression levels of the target genes vg and vgr were respectively detected by qrtpcr in the different development degree and tissues of adults and the most unstable reference gene was used for comparison in parallel the reaction system and program were the same as for qrtpcr of reference genes and four technical and three biological replicates were performed for each treatment the relative expression levels of vg and vgr were respectively calculated in excel using the ˆ†ˆ†ct method the differences of target genes™ expression levels were analyzed by tukey™s test using spss software spss inc under different experimental conditionsresultsselection and primer performance of candidate reference a0genesthe candidate reference genes including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub were selected to identify the normalization factors for qrtpcr analysis and the sequence information has been submitted to genbank and the accession numbers are shown in supp table online only to determine the amplification specificity of the primers agarose gel electrophoresis and melting curve analysis were performed all primer pairs showed a single band and a single peak fig a0 to obtain correlation coefficient r2 and amplification efficiency e of pcr a standard curve was generated with the 10fold dilution series of cdna the results showed that the amplification efficiency ranged from to and the correlation coefficient varied from to supp table [online a0only]expression profiling of candidate reference a0genesto evaluate expression levels of the candidate reference genes the cycle threshold ct values under different groups were obtained by qrtpcr and represented by boxplot fig a0 the results indicated that ct values of all candidate reference genes were different under different conditions and also varied under the same condition overall ct values ranged from to among them the genes with higher expression abundance were tub1 “ rps26e “ and actin “ followed by 128up “ arp3 “ arpc5 “ rps5 “ and ef1a “ the genes with lower expression abundance were αtub “ and gapdh “ according to the ct value range of each candidate reference gene the genes with relatively stable expression were tub1 and arp3 whereas the most unstable genes were ef1a actin and αtubexpression stability of candidate reference genes under different conditionsin this study four statistical algorithms were used to analyze the expression stability of the candidate reference genes under different conditions including genorm normfinder bestkeeper and ˆ†ct method as different statistical algorithms would generate different ranking patterns the comprehensive ranking of genes was finally determined through the geometric mean of sequencing bestkeeper the original ct values were used for analysis evaluated the stabilities of the candidate reference genes according to the cv and sd of the ct values ˆ†ct method the difference values of original ct values were used for analysis performed stability ordering according to the mean sd of ˆ†ct value genorm and normfinder the original ct values were converted for analysis sequenced the candidate reference genes according to the stability values the lower the stability value the more stable the gene expressiondevelopment degree of a0adultsthe expression stability of the candidate reference genes at different periods of reproductive and diapause female showed that the top four ranked genes identified by the genorm bestkeeper normfinder and δct method were similar but the rank order was slightly different arp3 and rps5 were the first and second stably expressed genes in the four statistical algorithms supp table [online only] and comprehensive ranking analysis fig a0 as for the third and fourth ranked gene tub1 and actin identified by comprehensive ranking analysis were the same as those generated by genorm and normfinder while bestkeeper selected arpc5 and tub1 ˆ†ct method selected actin and rps26e supp table [online only] αtub was ranked by genorm bestkeeper normfinder and δct method as the least stable gene among the candidate reference 0c of insect science vol no fig specificity a and product length b of qrtpcr amplification for ten candidate reference genesgenes during different development degrees of adults supp table [online a0only]adult a0tissuesthe expression stability ranking of candidate reference genes in different tissues of reproductive and diapause females was varied according to the four statistical algorithms in different tissues of reproductive females the top four genes were rps5 arp3 arpc5 and tub1 respectively fig a0 but the rank order of the four genes was significantly different among different statistical algorithms rps5 was ranked first by genorm and ˆ†ct method and was ranked third and fourth by normfinder and bestkeeper respectively arp3 was ranked first by bestkeeper and was ranked second third and fifth by normfinder δct method and genorm respectively arpc5 was ranked first and second by genorm and δct method and was ranked fourth and fifth by normfinder and bestkeeper respectively tub1 was ranked first and second by normfinder and bestkeeper and was ranked fifth and sixth by δct method and genorm respectively supp table [online only] however the four statistical algorithms found that ef1a was ranked as the least stable gene in the tissues from reproductive females supp table [online only]in different tissues of diapause females the top four genes were different from those of different tissues of reproductive females tub1 was ranked first by the comprehensive ranking followed by rps26e arp3 and 128up fig a0 through analysis it was found that ˆ†ct method and normfinder displayed the same rankings for expression stability of candidate reference genes under this condition supp table [online only] tub1 was identified as the most stably expressed gene by genorm ˆ†ct method and normfinder although it was ranked fifth by bestkeeper rps26e ranked steadily among the four statistical algorithms was ranked second by ˆ†ct method and normfinder whereas it was ranked first and third by genorm and bestkeeper respectively arp3 was ranked first by bestkeeper with the smallest coefficient of variation whereas it was ranked third by ˆ†ct method and normfinder in addition to being ranked third by genorm 128up was ranked fourth by bestkeeper ˆ†ct method and normfinder supp table [online only] however in tissue from diapause females actin was consistently identified as the gene with the most unstable expression by the four statistical algorithms supp table [online only]in the reproductive and diapause female tissues the expression stability of the candidate reference genes was different in order to accurately determine the expression of the target genes the expression stability of candidate reference genes under the two conditions was analyzed according to the comprehensive ranking 0c of insect science vol no fig expression profiles of candidate reference genes in c a0nipponensis expression data are displayed as ct values for each reference gene using a box and whisker plot in different experimental conditions the line across the box is the median the box indicates the 25th and 75th percentiles the whiskers represent the 10th and 90th percentilesfig expression stability and comprehensive ranking of reference gene measured by the geomean method a a0lower geomean value indicates more stable expressiontub1 and ef1a were the most stable and unstable genes respectively whereas arp3 128up and arpc5 were ranked second third and fourth respectively fig a0 tub1 was the best candidate reference gene identified by normfinder and ˆ†ct method and was ranked third and sixth by bestkeeper and genorm respectively arp3 was the most suitable candidate reference gene selected by bestkeeper and was ranked second by normfinder and ˆ†ct method and fifth by genorm 128up and arpc5 were the most stable candidate reference genes identified by genorm whereas they were ranked separately fifth and sixth by normfinder and ˆ†ct method and seventh and fifth by bestkeeper respectively supp table [online only] ef1a was identified as the least stable gene by genorm normfinder and δct method although bestkeeper selected actin as the least stable gene supp table [online only]all a0samplesin order to determine the best reference gene suitable for the different conditions of adults the stability of the ten candidate reference genes was ranked for all samples arp3 was identified as the most stable gene by the comprehensive ranking followed by tub1 arpc5 and rps5 whereas ef1a was identified as the least stable gene fig a0 0c of insect science vol no but the most and least stable genes identified by different statistical algorithms were slightly different arp3 was selected as the most stable reference gene by bestkeeper and ˆ†ct method although tub1 and arpc5 were selected as the most stable reference gene by normfinder and genorm respectively ef1a was selected as the least stable reference gene by genorm and ˆ†ct method despite it being ranked ninth by bestkeeper and normfinder supp table [online only]the best combination of candidate reference genes under different conditionsaccording to the pairwise variation vnn1 between the normalization factors and cutoff value calculated by genorm the number of reference genes required for optimum normalization in each experimental condition was determined the cutoff value of vnn1 a0 suggested that n reference genes were enough to make gene expression normalization otherwise n reference genes were needed the analysis results showed that all v23 were indicated that the optimal number of reference genes under each condition was two fig a0 more specifically arp3 and rps5 were the most stable gene combinations under adult developmental stage and reproductive adult tissues conditions tub1 and rps26e were the most stable gene combinations under adult diapause tissues conditions and arp3 and tub1 were the most stable gene combinations under adult tissues and all samples groups table a0relative expression levels of target genes vg and vgr genbank mt308983 mt522179 in the whole adult and from tissues of reproductive and diapause adults respectively when the most stable reference genes arp3 andor rps5 were used as normalization factors at different periods of reproduction and diapause the expression patterns of the target genes vg and vgr were consistent with low expression in the diapause period and rich expression in the late diapause and reproduction period however when the most unstable reference gene αtub was used as a normalization factor neither the target gene vg nor vgr showed a consistent expression pattern fig a0 under different tissue conditions when the most stable reference genes tub1 andor arp3 were used as the normalization factors the expression level of the target gene vg in the fat body of the reproductive female was significantly higher than that in the ovary of the reproductive female the expression level of the target gene vgr in the fat body of the reproductive female was lower than the ovary of the diapause female while when the most unstable reference gene ef1a was used as the normalization factor the expression pattern differed with normalization by tub1 and tub1arp3 fig a0 in general when the most stable reference genes were used as the normalization factors the accurate expression pattern of the target gene could be obtainedvalidation of reference a0genesthe stability of reference gene is very important for the analysis of expression level of target gene vg and vgr which are important for insect reproduction were selected to verify the applicability of the selected reference gene we examined the discussionqrtpcr has become an important means to explore gene expression level due to its high sensitivity rapidity specificity and accuracy and was widely used in physiology studies that investigated insect diapause such as drosophila melanogaster williams fig pairwise variations vnn1 was calculated by genorm to determine the optimal number of reference genes for accurate normalization in different conditions the cut off values under indicate that no additional genes are required for the normalization 0c of insect science vol no et a0al culex pipiens sim and denlinger leptinotarsa decemlineata lehmann et a0 al chrysopa septempunctata liu et a0al and pieris melete wu et a0al however the selection of appropriate reference genes was the key to accurately analyze the gene expression level for example under conditions of injury heatstressed and experimentally varied diets table recommendation for the best combination of reference genes based on the genorm and comprehensive rankings under various experimental conditionsgroupreference genemostdevelopment degreeadult tissues reproductionadult tissues diapauseadult tissuesall samplesarp3rps5tub1tub1 arp3 rps5arp3rps26earp3tub1leastαtubef1aactinef1aef1athe best reference gene was different in drosophila melanogaster ponton et a0al under biotic factors and abiotic stress inappropriate selection of the reference genes in locusta migratoria resulted in significant differences in the expression level of the target gene chitin synthase chs1 yang et a0al diapause of most insects was mainly affected by photoperiod and temperature in past studies the screening of reference genes of drosophila melanogaster ponton et a0al leptinotarsa decemlineata shi et a0al helicoverpa armigera zhang et a0al bombyx mori guo et a0al and harmonia axyridis qu et a0al under main environmental factors was completed by genorm normfinder bestkeeper and ˆ†ct method in this study the expression profiles of candidate reference genes of c a0nipponensis were analyzed under different conditions by the same four statistical algorithms genorm vandesompele et a0 al normfinder andersen et a0al bestkeeper pfaffl and ˆ†ct method silver et a0al different algorithms produced different stability rankings in order to obtain statistically consistent and accurate results we finally ranked the gene based on their stabilities determined by fig validation of selected reference genes under different periods a and tissues b of reproductive and diapause female in c a0 nipponensis relative expression levels of the vg and vgr in different samples using different normalization factors the most and least stable genes asterisks indicate significant differences in the expression levels of the vg and vgr r reproduction period d1 the diapause induction period d2 the diapause maintenance period d3 the diapause termination period 0c of insect science vol no comprehensive analysis method xiao et a0 al and selected the most stable reference genes under each condition as far as we know actin which played an important role in cell contraction and cytoskeletal maintenance was found in virtually all eukaryotic cells and was considered as an ideal reference gene for many anisms sürencastillo et a0al shakeel et a0al for example actin was used as a reference gene for normalization in the determination of genes related to reproductive and nutritional signaling such as vitellogenin of chrysopa septempunctata liu et a0al however in this study three genes related to actin were selected for analysis among which actin was similar to actin of c a0septempunctata which was also a member of the neuroptera in our study actin was the most unstable gene in the diapause female tissues but the actinrelated protein arp3 which was structurally homologous with actin showed better stability arp3 was selected as the most stable reference gene in adults of different developmental levels and all samples while it was the second most stable gene in the reproductive adult tissues and all adult tissues although arp3 was ranked as the third most stable gene in the diapause adult tissues it showed relatively stable expression in the expression profile tubulin which played an essential role in maintaining cell shape movement and intracellular material transport was also often used as a reference gene but different types of tubulin have different stability for example in the study of helicoverpa armigera the expression of βtub was relatively stable compared with that of αtub under almost all conditions zhang et a0 al similarly in this study αtub showed unstable expression and was the least stably expressed gene in adults of different developmental stages whereas tub1 was considered to be the most stably expressed reference gene in diapausing adult tissues and all adult tissues and was the second most stable gene in all samples ribosomal protein rp widely distributed in various tissues played an important role in protein biosynthesis and was widely used as a reference gene in many insects lu et a0al koyama et a0 al sun et a0 al in this study rps5 was considered to be stable in the tissues of reproductive females and ranked second among different developmental stages of adults elongation factors ef was a protein factor which promoted polypeptide chain to extension during the translation of mrna and was recommended as the ideal reference gene under different conditions of a variety of insects chapuis et a0 al ponton et a0 al however some studies showed that ef1α was one of the most unstable genes under certain conditions fu et a0 al in our study ef1a was found to be the most unstable gene in the reproductive adult tissues all tissues and all samples and the second least stable gene in the adults of different developmental stages and diapause adult tissues therefore it was not suitable for the study of c a0nipponensisrecently an increasing number of studies have demonstrated the importance of using multiple stably expressed reference genes for the accuracy of qrtpcr analysis ling and salvaterra yuan et a0 al kang et a0 al however this does not mean that the more reference genes increase the reliability of the results the study has indicated that either too few o
Colon_Cancer
" lymph node staging of ductal adenocarcinoma of the pancreatic head pdac by crosssectionalimaging is limited the aim of this study was to determine the diagnostic accuracy of expanded criteria in nodalstaging in pdac patientsmethods sixtysix patients with histologically confirmed pdac that underwent primary surgery were included inthis retrospective irbapproved study crosssectional imaging studies ct andor mri were evaluated by aradiologist blinded to histopathology number and size of lymph nodes were measured shortaxis diameter andcharacterized in terms of expanded morphological criteria of border contour spiculated lobulated and indistinctand texture homogeneous or inhomogeneous sensitivities and specificities were calculated with histopathologyas a reference standardresults fortyeight of patients had histologically confirmed lymph node metastases pn sensitivityspecificity and youden™s index for the criterion œsize were and for œinhomogeneous signal intensity and and for œborder contour and respectively there was a significant associationbetween the number of visible lymph nodes on preoperative ct and lymph node involvement pn p lymph node staging in pdac is mainly limited due to low sensitivity for detection of metastatic diseaseusing expanded morphological criteria instead of size did not improve regional nodal staging due to sensitivityremaining low combining specific criteria yields improved sensitivity with specificity and ppv remaining highkeywords ductal adenocarcinoma of the pancreatic head staging lymph nodes computed tomography magneticresonance imaging crosssectional imaging neoadjuvant therapy correspondence florianlochcharitede1charit “ universittsmedizin berlin corporate member of freie universittberlin humboldtuniversitt zu berlin and berlin institute of healthdepartment of surgery campus benjamin franklin hindenburgdamm berlin germanyfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cloch world of surgical oncology page of pancreatic cancer remains one of the most lethal malignancies being the fourth leading cause of cancerdeath in the usa and predicted to be the secondleading cause of cancer death by the overall5year survival after diagnosis is and at thetime of diagnosis the main proportion of patients hasadvancedstage disease leaving only “ qualifiedfor resective surgery pancreatic cancer is locatedin the head of the pancreas in of the cases even after successful resective surgery in patients withcancer of the pancreatic head the 5year survival remains as low as these data underline theimportance of establishing multimodaltherapeuticconcepts for patients with pancreatic cancer as perother entities of abdominal cancerapart from the potential to increase the resectabilityrate of pancreatic cancer by neoadjuvant therapy [ ]there is evidence that patients which are successfullydownstaged from nodepositive disease cn1 to nodenegative disease ypn0 prior to surgery benefit in termsof higher 5year survival rate this would qualifynodalinvolvement as a sufficient basis for indicatingneoadjuvant therapy yet even given advanced imagingtechnologies identifying lymph node metastasis remainschallenging consequently the indication of a potentiallyeffective neoadjuvant therapy cn with side effects inlymph nodepositive patients cn is mainly based onunreliable clinical stagingthe established criterion for lymph node involvement in pancreatic cancer is size using the size underlies the assumption that tumor spread to regionallymph nodes leads to an enlargement of the respective lymph node the usual cutoff value is a shortaxis diameter of mm [ “] it has been shownthough that lymph nodes of ‰¥ mm are not seenmore frequently in patients with histopathologicallymph node involvement pn in various othertumor entities expanded morphological criteria suchas texture and border contour oflymph nodes areused for the assessment oflymph node malignancyon both computed tomography ct and magneticresonance mr imaging this is utilized in order toimprove the accuracy of lymph node staging [“]by applying morphological criteria instead of brown size criterion alone the sensitivity was improvedfrom to and the specificity from to inlymph node staging of rectal cancer thus the aim of this study was to determine thelymph node staging in patients withaccuracy ofductal adenocarcinoma ofthe pancreatic head byboth computed tomography and magnetic resonanceimaging using sizeand expanded morphologicalcriteriamaterial and methodspatientsin this retrospective singlecenter study approved by thelocal ethics committee consecutive patients with histologically proven ductal adenocarcinoma of the pancreatichead that underwent primary surgery between february and november at the department of surgerycampus benjamin franklin charit”university medicine berlin germany were included patients were retrieved from the database of our pancreatic cancercenter certified by the german cancer society n inclusion criteria were primary oncologic tumor resection and the presence of preoperative crosssectional imaging of sufficient quality see below exclusion criteriawere neoadjuvant therapy presence of a potential simultaneous cause of lymphadenopathy of the upper abdominal region eg abdominal lymphoma neuroendocrinetumor and main tumor mass located outside the pancreatic head on histopathology the process of patientselection with the respective reasons for inclusion andexclusion is shown in fig crosssectional imagingall images were retrospectively analyzed for the purposeof this study by a single abdominal radiologist with morethan years of experience in staging of tumors of the visceral ans blinded to the results of histopathologyall crosssectional imaging studies were assessed forsufficient image quality by the radiologist prior to commencement for ct imaging the minimum quality wasdefined as either thinsection ct ‰ mm reconstructedslice thickness or contrastenhanced ct with a slicethickness of ‰ mm for mri minimum quality was defined as availability of an axial t2weighted sequencewith fat suppression slice thickness ‰ mm in combination with a venous phase postcontrast 3d gradientecho sequence slice thickness ‰ mmfor lymph node assessment all visible regional lymphnodes in the field of view were recorded on a score chartand the total number of visible lymph nodes per patientwas calculated then for each patient all lymph nodeswere characterized in terms of size long and shortaxisdiameter in millimeters and the expanded morphological criteria border contour lobulated spiculated indistinct or unaltered and texture homogeneous orinhomogeneous fig based on kim regional lymph nodes of the pancreas are defined asthe following lymph node station numbers 8a8p 11p 11d 12a 12b 12p13a 13b14p14d 17a17b and in all cases in which a lymph node wasnot definitively regional correlation with postoperativecrosssectional imaging was performed to assess whetherthe lymph node was resected or not only resectedlymph nodes were analyzed in this study 0cloch world of surgical oncology page of fig flowchart of patient recruitment the process of patient selection with the respective reasons for inclusion and exclusion is showna second radiologist with more than years of experience in staging of tumors of the visceral ans alsoblinded to the results of histopathology evaluated thect examinations of a representative subgroup of patients for evaluation of interobserver agreementsurgeryall patients underwent primary oncologic pyloruspreserving pancreaticoduodenectomy or whipple procedure with complete lymphadenectomy of the regionallymph nodes mentioned aboveformalinembedded surgicalhistopathologythe original histopathological reportsfor the studyspecimens wereusingreviewed cancer of the pancreatic head was defined as amalignant tumor located within the pancreas to the rightof the superior mesenteric vein and portal vein each patient with histologically proven lymph node metastaseswas classified as nodepositive pn regardless of thenumber of metastatic lymph nodes patients without anymetastatic lymph nodes were classified as nodenegativepn the ratio of metastatic lymph nodes vs the totalnumber of retrieved lymph nodes was documented infig morphological characterization of lymph nodes based on kim the morphological criteria for lymph node assessment used inthis study are shown smooth and homogeneous lymph nodes were considered normal 0cloch world of surgical oncology page of the histopathological report eg or tumorswere classified according to their respective tnm stageusing the 8th edition of tnm classification of malignant tumors table demographic data of patients with ductal adenocarcinomaof the pancreatic head undergoing primary tumor resectionpatientsagen comparison of crosssectional imaging andhistopathologysensitivity specificity and positive predictive value ofthe nodal status using ct and mri with histopathologyas a reference standard were calculated for lymph nodeinvolvement using size and morphological criteria specifically nodal involvement criteria were based on eithersize shortaxis diameter altered border contour lobulated spiculated or indistinct and inhomogeneous signal intensity fig ct and mri examinations wereconsidered nodepositive cnif at least one lymphnode met one of the respective criteria used for involvement if no lymph node with the respective criteria wasseen on ct or mri then the examination was considered nodenegative cnstatistical analysissensitivities specificities and positive predictive valueppv for the size criterion and all morphological criteria were calculated for their respective cutoff valuesan index summarizing the sensitivity and specificity foryouden™s index was calculated sensitivity specificityˆ’ the number of lymph nodes visible on ctand mr images in the group with pn and withoutpn nodal metastases was compared using the mannwhitney u test when calculating the association between ct and mri criteria and lymph node positivitythe χ2test was used interobserver agreement was calculated using cohen™s kappa statistic a p value of ‰ was considered to indicate a statistically significantdifferenceresultspatientssixtysix patients were included in the study fig with the characteristics of the patients presented intable sixty of these patients were staged by preoperative ct twelve of which had additional stagingby mri and six patients were staged by only mri intwo patientsthe mri examinations were excludeddue to insufficient imaging quality both patients hadsufficient staging by ct and were therefore includedin the study of the patients patients receivedpreoperative biliary drainage eight ofthem werestaged by ct only and two by mri onlycomputed tomography ctlymph nodes were detected by ct in ofthe patients the median number of visible lymph nodesmedian age yearsage range yearssexfemalemalecrosssectional imagingct onlyct and mrimri onlyhistopathological stagingpnpnpt1pt2pt3“ was range “ the smallest visible lymph node was mm of size whereas the largest measured mmshortaxis diameter the mean time between ct andsurgery was days with a median of days range “the slice thickness in of the ct examinations was mm or less in five ct examinations slice thickness was mmsize criterion for lymph node involvement onpreoperative ctfigure shows the percentage of patients with pnand without pn lymph node metastases in which alymph node of the respective size was visible “ mmin table sensitivity specificity and youden™s indexare presented for the respective cutoff values lymphnodes of small and medium size “ mm were visiblein patients with “ “ pn and withoutlymph node metastases “ “ pnineven frequency large lymph nodes “ mm wereseen more frequently in the lymph nodepositive group“ “ pnthan in the lymph nodenegative group “ “ pn the maximumvalue of youden™s index for the size criterion was j ci when a cutoff value of mmwas applied yielding a sensitivity of and specificityof additionallylymph nodesgreater than mm on preoperative ct and the histopathological confirmation of a lymph node metastasispn showed a trend towards significance p the presence of 0cloch world of surgical oncology page of fig graph showing lymph node size and morphological criteria of lymph nodepositive and negative patients frequency of regional lymphnodes of the pancreatic head in percent xaxis with different shortaxis diameters and morphological features yaxis in patients with pn redbars or without histologically proven lymph node metastases pn blue bars on preoperative ct imagingexpanded morphological criteria for lymph node involvementon preoperative ctfigure shows the percentage of patients with pnand pn without lymph node metastases in which alymph node of the respective morphological criterionwas visible and table shows the sensitivity specificityand youden™s index of the respective criterionlymph nodes of lobulated border contour were visiblewith a similar frequency in patients with pn and without lymph node metastases pn lymph nodes of spiculated or indistinct bordercontour were only occasionally detected in both groups vs in the lymph nodepositivegroup pn and vs in the lymphnodenegative group pnlymph nodes of inhomogeneous signal intensity were detected in only one patient of the lymph nodenegative group pn and more frequently in patients of the lymphnodepositive group pn resulting in the maximum value of youden™s index for the morphological criteriaj ci consisting of a sensitivity of and a specificity of the ppv was comparison of size with expanded morphological criteriathe maximum value of the youden™s index of the œsizecriterion was j ci cutoff mmwhich is not inferior to the maximum value of the morphological criteria j ci inhomogeneoussignal intensity figure displays respective ct images ofpatients with and without lymph node metastasesfig ct images of patients with and without lymph node metastases a patient with enlarged suspicious lymph node adjacent to the portalvein and hepatic artery who had no lymph node metastases on pathology b patient with enlarged suspicious lymph node adjacent to thehepatic artery who had lymph node metastases on pathology c patient with multiple suspicious lymph nodes based on size and inhomogeneitywho had lymph node metastases on pathology 0cloch world of surgical oncology page of table sensitivity specificity ppv and youden™s index for cutoff values and morphological criteria by ct and mrisensitivity specificity ppvyouden™s indexctsize cutoff value mm mm mm mm mm mm mm mmmorphological criterionlobulatedspiculatedindistinctinhomogeneous mrisize cutoff value mm mm mm mm mm mm mm mm mmmorphological criterionlobulatedspiculatedindistinctnot visibleinhomogeneousnot visible ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ number of visible lymph nodesthere was a significant association between number of visible lymph nodes seen on preoperativect and histopathologicallymph node involvementpn p seven or more lymph nodes were seen on preoperative ct in of patients with lymph nodemetastases pn and in of patients without lymph node metastasis pn p this resulted in a specificity of youden™s index of and ppv of combination of number size and expanded morphologiccriteria on preoperative ctcombining the size criterion and the morphological criterion with the respective highest youden™s index cutoff mm and œinhomogeneous signalintensity andthe criterion œvisible lymph nodes n ‰¥  was significantly associated with nodal metastases pn p for this combined criterion specificity was sensitivity ppv and youden™s index ci interobserver agreementinterobserver agreement was calculated for patientspn pn vs pnfor the criteria sizemorphology and number of visible lymph nodes withthe respective highest youden™s indexinterobserveragreement was substantial for size mm cutoff κ p moderate for the presence of seven ormore lymph nodes κ p and fair forthe morphological criterion inhomogeneous signal intensity κ p magnetic resonance imaging mrilymph nodes were detected in of patientson preoperative mri the median number of visiblelymph nodes was range “ there was no significantassociation between number of visible lymph nodes seenon preoperative mri and histopathological lymph nodeinvolvement pn p the smallest visible lymph node was mm of sizewhereas the largest measured mm shortaxis diameter the mean time between mri and surgery was days with a median of days range “the cutoff values of the highest diagnostic value were mm or mm for the œsize criterion sensitivity specificity youden™s index the presence ofa lymph node of these sizes was not associated with lymphnode metastases pn p lobulated and spiculated lymph nodes were only seen in a few patients n and n and indistinct and inhomogeneous lymphnodes were not seen at all table discussionin this retrospective singlecenter study on lymph nodestaging by ct in ductal adenocarcinoma of the pancreatic head we could show that the morphologic criteriaœinhomogeneous signal intensity and œsize are specificfor regional nodal metastatic disease replacing the sizecriterion by morphologic criteria however did not improve diagnostic accuracy due to sensitivity remaininglow combining specific criteria yields improved sensitivity with specificity remaining highby ct lymph nodes of “ mm in shortaxis diameterwere seen just as often in patients with and without 0cloch world of surgical oncology page of lymph node metastases resulting in poor discriminationlarger lymph nodes mm had a higher prevalence inthe lymph nodepositive group leading to high specificity however these lymph nodes mm or mmwere seen infrequently resulting in a rather low sensitivity the maximum value of the youden™s index for thesize criterion of was achieved when a cutoff valueof mm was applied consisting of a specificity of and sensitivity of yielding a ppv of as for morphologic criteria lymph nodes of lobulatedborder contour were seen in about half of the patients ofboth groups pn and pn and therefore isa criterion that is not suitable to differentiate betweenthe groups lymph nodes of spiculated and indistinctborder contour were seen in few cases in both patientgroups only pn and versus pn and making them poor diagnostic criteria however lymphnodes of inhomogeneous signal intensity were visible in of patients with lymph node metastases pn andonly in of the patients without lymph node metastases pn resulting in a youden™s index of whichwas the maximum value for the morphological criteriaand a ppv of ideally a good discriminator for nodal metastases isnegative in patients without nodal involvement and positive for tumors with lymph node metastases in ourstudy each criterion ie size as well as different morphological features only met one of these prerequisitesthe size criterion mm as well as the presence of alymph node of inhomogeneous signal intensity as morphological criterion turned out to be negative in patientswithout nodal involvement pn and therefore highlyspecific yet lymph nodes of the respective characteristicwere not positive in a sufficiently high number of tumorswith lymph node metastases pn to reach high levelsof sensitivity and consequently did not have a significantdiagnostic valuethe maximum value of the youden™s index for the sizecriterion was when a cutoff value of mm wasapplied and for the morphological criteria whenthe criterion œinhomogeneous signal intensity was usedshowing that morphologic criteria do not yield in higherdiagnostic value than lymph node size in adenocarcinoma of the pancreatic head pdac patients this iscontrary to the findings of brown in rectal cancer one reason might be that brown used mri toassess morphologic criteria which has a higher soft tissuecontrast compared to ct which was used in most patients in our studyinterestingly we could show that with preoperativect the presence of seven or more lymph nodes wasseen more often in patients with lymph node metastasispn than in those without metastasis pn p when applying this as a sole criterion cn for lymphnode metastasis pn this led to a sensitivity of aspecificity of ppv of and youden™s indexof in diagnostic test analysis criteria can be combinedin mainly two ways sensitive criteria can be taken together to improve specificity or specific criteria canbe accumulated to improve sensitivity when combining the highly specific criteria size cutoff value mm inhomogeneous signal intensity and number ofvisible lymph nodes n ‰¥ a highly significant association with nodal metastases pn p wasfound consequently the ct examination was considered nodepositive cn when at least one of thesecriteria was met the application of this criterion improved the sensitivity to with a remaining specificity of and ppv resulting in an alsoimproved youden™s index of the results of the mri examinations must be viewedin a rather descriptive manner since the sample size waslimited n lymph nodes were detected in the majority of examinations generally allowing theevaluation of lymph nodes by mri as well upper abdominal mri generally has a lower spatial resolutionbut a higher soft tissue contrast compared to ct forthe size criterion a cutoff value of mm or mm ledto the best diagnostic results sensitivity specificity youden™s index lymph nodes of abnormal morphological criteria were seen in only veryfew patients table the main limitation of this study is the retrospectivestudy design in which a nodebynode comparison ofcrosssectionalimaging with histopathology was notpossible this was of minor importance though sincelow sensitivity was the main factor that led to compromised diagnostic performance in our study we werealso able to correlate with postoperative crosssectionalimaging in all cases in which it was unclear whether alymph node had been resected during surgery or notalso in our cohort only patients who had subsequentsurgery were included presuming lower tumor stage ascompared to the average patient who undergoes imagingfor presurgical workupthe strength of our singlecenter study is reinforcedby a defined number of surgeons a high standardizationof the ct technique and an experienced radiologist whoperformed the analysisthe results of our study are consistent with recent andinitial data demonstrating that clinical staging by lowsensitivity underestimates histopathological lymph nodeinvolvement pn [ “] however by adding thecriterion œinhomogeneous signal intensity and œnumberof visible lymph nodes to the size criterion we wereable to increase the sensitivity to in comparison toprevious findings roche nanashima 0cloch world of surgical oncology page of and cao with specificity remainingsufficientan additional imaging modality that has shown the potential to improve the sensitivity of detecting metastaticdisease is positron emission tomographycomputed tomography petct however a beneficial role ofpetct in locoregional nodal staging could not be established to date the majority of initial as well as recentstudies show very limited sensitivities for nodal status between and [“] the petpanc study evaluated the incremental diagnostic accuracy and impact ofpetct in addition to multidetector ct in patients withsuspected pancreatic cancer in a prospective multicenterstudy that included patients in this study significantlymore patients with stage iib disease pn were correctlystaged by petct than by multidetector ct p but this only led to a moderate sensitivity of for petct versus for multidetector ct endoscopic ultrasonography eus is a wellestablisheddiagnostic procedure in pancreatic cancer with the benefitof a dynamic diagnostic examination that allows fineneedle aspiration for cytologic diagnosis two metaanalyses evaluating diagnostic accuracy of eus for locoregional nodal staging the pooled sensitivities and specificities were and li studies n patients and and nawaz studiesn patients advanced techniques such ascontrastenhanced eus cheus and eus elastographyare currently in evaluation to date ct remains the standard staging imaging modality recommended by nccn guidelines for locoregional staging of pancreatic cancer neither petctnor eus yields reliable diagnostic accuracy for nodalstagingan advantageous effect on resectability and overallsurvival os in unresectable cases including both borderline resectable and unresectable of pdac by multimodality therapy including neoadjuvanttherapy hasalready been described in several studies the benefit of neoadjuvant therapy in cases of primarily resectable disease at diagnosis is yet less revealedseveral phase ii trials showed that patients who completed neoadjuvant chemoradiation without progressivedisease at restaging had a higher chance of achieving r0resection and consequently higher median and oswhen compared to historical data as seen in othertumor entities a potential benefit of neoadjuvant therapyon the basis of positive nodal status cn is stronglyimplied cao found that the of patients thatwere successfully downstaged from nodepositive diseasecn1 to nodenegative disease ypn0 by neoadjuvanttherapy benefit in terms of higher rates of 5year survivalypn0 vs ypn1 p this is consistent with the findings of portuondo 5yearsurvival ypn0 vs ypn1 p the nccn guidelines for pancreatic adenocarcinomaappreciates these results by stating that considerationcan be given to neoadjuvant therapy for selected patientswith resectable tumor but poor prognostic features suchas large regional lymph nodes markedly elevated ca large primary tumors extreme pain and excessiveweight loss further clarification on this matter isexpected to come from the ongoing neonax trialnct02047513 a phase ii study comparing neoadjuvant plus adjuvant with only adjuvant nabpaclitaxelplus gemcitabine therapy forresectable pancreaticcancer theiii neopa trialphasenct01900327 compares neoadjuvant chemoradiotherapy with upfront surgery of resectable pancreatichead cancer a subgroup analysis in terms of nodalstatus would present reliable dataongoinggiven the suggested benefit of neoadjuvant therapybased on lymph node staging there is an urgent need tofind criteria and modalities to further improve the diagnostic value of lymph node staging by pretherapeuticcrosssectional imaging in patients with ductal adenocarcinoma of the pancreatic head to date none of theexisting modalities and criteria accomplishes reliablenodal staging larger prospective studies are ongoingand necessary to get a more precise idea of the prognostic advantage of neoadjuvant therapy in patients with regionalcn of pdac inpretherapeutic staginglymph node metastasisslymph node staging in pdac patients when using ctmorphological criteria such as border contour or homogeneity compared to diameter cutoff values does notlead to reliable diagnostic value diagnostic accuracy islimited due to low sensitivity for detection of metastaticdisease combining specific criteria yields improved sensitivity with specificity and ppv remaining high theseresults suggest an attentive interpretation of the resultsof pretherapeutic lymph node staging particularly incases in which lymph node metastases are absentabbreviationspdac adenocarcinoma of the pancreatic head ct computed tomographymri magnetic resonance imaging pn histopathologically involved lymphnodes pn histopathologically no involved lymph nodes cn clinicallyinvolved lymph nodes cn clinically no involved lymph nodes ppv positivepredictive value petct positron emission tomographycomputed tomography eus endoscopic ultrasonography cheus contrastenhancedendoscopic ultrasonography os overall survivalacknowledgementswe acknowledge support from the german research foundation dfg andthe open access publication funds of charit “ universittsmedizin berlinauthors™ contributionsall authors were involved in data acquisition and manuscript revision theconception of the design of the study and drafting of the manuscript was 0cloch world of surgical oncology page of done by fn loch c kamphues and p asbach image analysis was performedby p asbach and assisted by fn loch image analysis of the subgroup foranalysis of interobserver agreement was performed by m haas all authorshave approved the submitted version of the manuscript and account fortheir own contribution and the accuracy as well as the integrity of the workpresentedfundingthe study was not fundedavailability of data and materialsthe datasets used during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study was approved by the local ethics committee of the charituniversity medicine berlin informed consent of participation was waived bythe irb given the retrospective study design irb no ea411418consent for publicationconsent for publication is not applicable for this studycompeting intereststhe authors declare that they have no competing interestsauthor details1charit “ universittsmedizin berlin corporate member of freie universittberlin humboldtuniversitt zu berlin and berlin institute of healthdepartment of surgery campus benjamin franklin hindenburgdamm berlin germany 2charit “ universittsmedizin berlin corporatemember of freie universitt berlin humboldtuniversitt zu berlin and berlininstitute of health department of radiology campus benjamin franklinhindenburgdamm berlin germany 3the johns hopkins universityschool of medicine department of surgery n wolfe street blalock baltimore md usareceived december accepted july zeman rk cooper c zeiberg as kladakis a silverman pm marshall jl tnm staging of pancreatic carcinoma using helical ct am jroentgenol “ m¼ller mf meyenberger c bertschinger p schaer r marincek b pancreatictumors evaluation with endoscopic us ct and mr imaging radiology“ midwinter mj beveridge cj wilsdon jb bennett mk baudouin cj charnleyrm correlation between spiral computed tomography endoscopicultrasonography and findings at operation in pancreatic and ampullarytumours br j surg “ r¶sch t braig c gain t feuerbach s siewert jr schusdziarra v staging of pancreatic and ampullary carcinoma by endoscopicultrasonography comparison with conventional sonography computedtomography and angiography gastroenterology “ prenzel kl h¶lscher ah vallb¶hmer d drebber u gutschow ca m¶nig sp lymph node size and metastatic infiltration in adenocarcinoma of thepancreatic head eur j surg oncol “ rollvn e blomqvist l ¶istm¶ e hjern f csanaky g abrahamnordling mmorphological predictors for lymph node metastases on computedtomography in colon cancer abdom radiol “ brown g richards cj bourne mw newcombe rg radcliffe ag dallimorens morphologic predictors of lymph node status in rectal cancer withuse of highspatialresolution mr imaging with histopathologic comparisonradiology “kim jh beets gl kim mj kessels agh beetstan rgh highresolution mrimaging for nodal staging in rectal cancer are there any criteria in additionto the size eur j radiol “isaji s murata y kishiwada m new japanese classification of pancreaticcancer in neoptolemos j urrutia r abbruzzese j b¼chler mw editorspancreatic cancer new york springer p “ brierley jd gospo
Colon_Cancer
" preproofsevere acute respiratory syndrome coronavirus sarscov2 is a highly contagious zoonotic pathogen that has exacted heavy public health social and economic tolls in february the world health anization acronymed the disease caused by sarscov2 as covid19 for coronavirus disease the number of confirmed covid19 infections which has been detected in at least countries has reached worldwide as of april with deaths according to the us centers for disease control and prevention cdc1 many cases of covid19 resolve quickly however the disease which like other respiratory pathogens that cause common cold symptoms is believed to be transmitted through respiratory droplets infection with covid19 can also lead to significant morbidity and death this is particularly the case for cancer patients moreover because the signs and symptoms of covid are easily misattributed to the sequelae of cancer itself such as pulmonary embolism or its treatment such as nausea and diarrhea diagnosis may be delayed or missed potential covid rule out criteria based on the wells˜ criteria for pulmonary embolism another protean disease entity are provided as a decisionmaking aid this review summarizes the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis rationale to treat the cancer or not treatment and prevention of covid19 with an emphasis on implications in cancer keywords covid19 sarscov2 cancer introduction sarscov2 the rna virus responsible for the illness which has been named covid19 for coronavirus disease2019 the year it was diagnosed has sent shockwaves and dominated the news cycle due to its pandemic spread from the point of origin in wuhan china to the rest of the world declared a public health emergency of international concern pheic by the world health anization who2 sarscov2 is the third highly pathogenic novel zoonotic bat coronavirus sonamed because of the crownlike spikes on its surface to have emerged the first was sars coronavirus now named sarscov1 in with a fatality rate of and 0c preproofthe second was middle east respiratory syndrome mers in with a fatality rate of where the camel was the intermediate host3 sarscov1 merscov and sarscov2 belong to the betacoronavirus genus which are enveloped positivestranded rna viruses whose approximately nucleotide genome serves as an mrna template for the translation of viral proteins4 the virion contains four proteins spike envelope membrane and nucleocapsid and the host receptor with which the spike surface glycoprotein of sarscov2 engages is angiotensin converting enzyme ace25 the biology of sarscov2 is described in more detail in figure a zinc metallopeptidase enzyme ace2 which is abundantly present in lung and gastrointestinal epithelial cells6 not only mediates viral entry through receptormediated endocytosis7 but also the efficiency of viral replication8 its expression is upregulated with older age smoking the antihypertensives angiotensin converting enzyme ace inhibitors and angiotensin receptor blockers arbs thiazolidinediones tzds a class of oral antidiabetic drugs and ibuprofen risk factors which may increase susceptibility to the covid19 virus infection and which are common in generally elderly multimorbid cancer patients9 preproofthe clinical spectrum of sarscov2 ranges from mild upper respiratory tract infection with fever sore throat headache cough and potentially nausea and diarrhea the majority of cases recover without serious complications to severe pneumonia with sequelae that include acute respiratory distress syndrome ards cytokine storm and death10 because sars is an acronym for severe acute respiratory syndrome digestive manifestations including inappetence nausea abdominal pain and diarrhea of covid19 resulting from binding of the virus to the ace2 receptor in the gi tract may precede respiratory symptoms11 gastrointestinal manifestations are potentially underappreciated and overlooked as sentinel symptoms that herald the onset or persistence of disease especially in cancer patients and may contribute to delayed or missed opportunities for testing diagnosis and containment unlike sarscov1 which seems to have disappeareddied out12 and mers which reappears only sporadically sarscov2 is less lethal with a fatality rate between although this number is highly uncertain and debated13 but much more infective consequently public panic and economic disruption have ensued resulting in wartimelike mobilization efforts to mitigate its spread old age smoking and comorbidities such as diabetes morbid obesity immunosuppression frailty and cardiovascular disease appear to predispose to worse outcomes possibly secondary to impaired t and b cell responses notably covid19 infection is associated with lymphopenia and delayed development of the adaptive immune response which appears to correlate with prolonged virus clearance and more severe disease progression15 the first pillar of defense against infection is hand washing avoidance of face touching and minimization of close contact ie social distancing with selfquarantine and selfisolation16 in case of exposure or evidence of covid19 symptoms respectively the second prophylactic pillar is vaccination with specific viral antigens or mrnas which are not yet publicly available although the company moderna has reportedly started testing an mrna vaccine in healthy volunteers and multiple other vaccination strategiesplatforms appear to be in progressunder development17 0c transmission and prevention according to the world health anization who21 sarscov2 is spread persontoperson mainly via aerosol inhalation from sneezing coughing or exhalation 22and via fomitetoface contact since depending on the surface material the virus may remain viable and infectious for hours to days figure fecaloral transmission has also been hypothesized because diarrhea was a common feature with sars and mers and diarrhea and other digestive issues have also been reported in patients with covid1925 26notably transmission of sarscov2 is not limited to symptomatic individuals ie those with fever cough sore throat myalgias or dyspnea but also to asymptomatic or subclinically infected carriers of the virus which is problematic from the perspective of disease control 27and highlights the importance of containment measures including isolation and quarantine the basic reproduction number r0 of sarscov2 is which means that on average for every patient an additional individuals are infected because coronaviruses may persist on inanimate surfaces like metal glass or plastic for up to days careful disinfection with or greater ethanol for small surfaces or sodium hypochlorite for larger surfaces is recommended preproofdiagnosis and clinical features preproofthe available evidence is limited but clinical courses and outcomes of covid19 are likely to be worse in patients with cancer especially given the clear association between severity of disease and older age and higher levels of comorbidity the overall case fatality rate cfr for the general covid19infected population is around but in cancer it rises to overall and to in italy19 this cfr in cancer patients compares to for no comorbid conditions for cardiovascular disease for diabetes for hypertension and for chronic respiratory disease the aim of this review is to summarize and condense the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis treatment and prevention of covid19 with a special focus on cancer in the united states the test of choice for sarscov2 is a nasopharyngeal swab specimen or sputum if a productive cough is present on which a reversetranscriptase pcr rtpcr assay or an enzymelinked immunoassay eia directed particularly at the envelope e rnadependent rna polymerase rdrp spike protein s and nucleocapsid n genes is performed the fda has also approved an antibody test29 a positive test for sarscov2 in a symptomatic patient generally confirms the diagnosis of covid19 with the caveat that false positive and false negative tests have been documented if initial testing is negative but a high index of suspicion and pretest probability for covid19 remains on the basis of patient signs and symptoms then retesting is indicated in patients with high indexes of clinical suspicion and equivocal or negative test results the who recommends that lower respiratory tract specimens which contain the highest viral loads should be obtained since nasopharyngeal swabs may miss some infections30 according to cdc guidelines disease is excluded on the basis of two 0c preproofconsecutive negative tests respiratory tests separated by ‰¥ hours however in the presence of suggestive symptoms rectal swabs may also be indicated since the ace2 enzyme to which the virus binds is abundantly present in rectal epithelia cells31 the differential diagnosis for sarscov2 in cancer is extremely broad and includes conditions such as foreign body aspiration toxicities from chemotherapy and radiation tumor progression postobstructive pneumonia malignant obstruction atelectasis pulmonary embolism pneumonitis pulmonary edemafluid overload immunotherapyrelated pneumonitis copd exacerbation q fever adenovirus bocavirus coronavirus 229e hcov 229e coronavirus hku1 hcov hku1 coronavirus nl63 hcov nl63 coronavirus oc43 hcov oc43 human metapneumovirus hmpv influenza a influenza a subtype h1n1pdm09 influenza a subtypes h1 and h3 influenza b parainfluenza virus ““ piv “ respiratory syncytial virus ab rsv ab rhinovirusenterovirus hrvev bordetella pertussis legionella pneumophila and mycoplasma pneumoniae32 the diagnosis of sarscov2 is complicated by the possibility of simultaneous coinfection with other respiratory viruses33 which is especially true for immunosuppressed cancer patients whose susceptibility to microanisms is increased the heightened infectious risk for cancer patients underscores the importance of screening them at presentation with extended viral respiratory panel testing given that coinfection may impact management decisions since conceptually at least the morbidity of covid19 and the risk of severe illness should increase in the presence of a second or third virus preproofunlike infection with influenza for example covid19 signs and symptoms may vary considerably depending on the dose of viral inoculum route of inoculation concomitant medications and underlying health status34 to include fever dry cough fatigue sputum production shortness of breath sore throat headache myalgia or arthralgia chills nausea or vomiting nasal congestion diarrhea hemoptysis and conjunctival congestion with an incubation period of to days after exposure36 presymptomatic or minimally symptomatic infection may majorly drive transmission especially since detected viral loads are similar in both symptomatic and asymptomatic patients3738 populations of concern include the elderly smokers vapers and dual users those of any age with preexisting chronic medical conditions those receiving particular medications or therapies which upregulate the ace2 receptor or suppress the immune system and those from lower socioeconomic classes a conglomeration of factors which are often present in cancer patients as depicted in figure while the surveillance focus for covid19 is on the respiratory tract enteric symptoms are a potentially underappreciated overlooked and misattributed manifestation of disease as stated earlier and this is especially the case for cancer patients where gastrointestinal toxicity occurs routinely from chemotherapy ie cisplatincarboplatinoxaliplatin irinotecan 5fluorouracil ifosfamide from targeted agents ie erlotinib imatinib bortezomib temsirolimus sunitinib regorafenibsorafenib and bevacizumab39 and from locally advanced or metastatic disease therefore abdominal complaints in cancer patients which are potentially but not automatically attributable to underlying disease justify further investigation especially if persistent worsening or new particularly because sarscov2 transmission may occur via the fecal“oral route40 0c preproofabnormal laboratory findings in covid19 include lymphopenia percent prolonged prothrombin time percent elevated lactate dehydrogenase percent elevated ast and alt “ percent elevated highly sensitive hs crp and elevated procalcitonin however because these parameters routinely fall well outside of the normal reference range in cancer patients it is difficult to confirm or refute the presence of disease on this basis alone chest radiographs and chest ct abnormalities are similarly nonspecific since the most common features multifocal groundglass opacities and consolidation mimic other pneumonias41 significant antibody production is observed after infection but it is unknown whether this helps or harms since antibodydependent enhancement ade may potentiate viral entry and the induction of a severe inflammatory response42 universal screening of cancer patients for covid19 is desirable but logistically impossible for the foreseeable future since diagnostic tests are in short supply and simply not always readily available43 hence covid19 rule out criteria are proposed in table as a potential decisionmaking aide mémoire which separates patients into low and highrisk groups by analogy to the wells˜ criteria for pulmonary embolism4445 preventive measures focus on selfisolation social distancing with a 6foot 2m separation46 frequent hand washing with soap and water andor use of hand sanitizers patient isolation during clinical care use of masks to help prevent aerosol transmission and flushing with the lid closed to control socalled •toilet plume– in an asco guidance immunocompromised cancer patients are advised to minimize exposure to sick contacts and large crowds48 for healthcare personnel the use of personal protective equipment such as n95 masks ffp3 masks gowns eye protection gloves and gowns is mandated49 preproof vaccination and immunity vaccination efforts and the related topic of whether those who have recovered from covid19 develop protective immunity have drawn great attention the latter has implications on whether people who test positive for sarscov2 antibodies can be safely assumed to be immune and at negligible risk of contracting or transmitting the disease there have been case reports of patients who have recovered from covid19 and had recurrence of rtpcr positivity approximately one month after initial diagnosis with only one patient exhibiting significant clinical symptoms and another having a mild intermittent cough50 but while not zero the risk of transmissibility or recurrence of symptomatic disease in recovered patients has yet to be quantified and the paucity of currently available reports of recurrence in the setting of a pandemic suggests that it is low a separate practical question will be whether antibodybased tests prove to have sufficient sensitivity and specificity to identify people who had asymptomatic infections developed immunity and can return to normal activities without jeopardizing disease containment efforts immunity may be due to antibodies cell mediated immunity or a combination of the two previous experience with using plasma from convalescent patients to treat severe cases of the first sars and mers as well as limited experience with covid19 suggests that antibody mediated immunity alone is clinically beneficial even during acute infection51 safety concerns about antibodies have been raised based on preclinical studies of sarscov vaccination in 0c preproofferrets showing hepatotoxicity52 and of vaccination against feline infectious peritonitis virus another coronavirus leading to more severe disease when kittens were subsequently challenged with the virus53 although animal models may not be representative of human hostpathogen interactions the nature of sarscov and sarscov2 antibodies are likely different as crossneutralization was not observed invitro54 and experience with convalescent plasma has not borne evidence of antibody mediated enhancement of infection in acutely infected patients the potential risk deserves attention if vaccination is proposed for the entire population t cell responses are also readily observable in patients who recover from coronavirus infections55 and memory t cell responses alone were protective in mice56 with the potential advantage of longer persistence of memory t cell responses compared to humoral immunity when clinical data on vaccine candidates becomes available cancer patients may face different considerations surrounding vaccination than the general population particularly patients with hematologic malignancies being treated with agents targeting b cells who would derive greater benefit from vaccines eliciting cell mediated than antibody responses preproofpathogenesis and pathology relating to ace2 and ras signaling the ace2 enzyme a key regulator of the reninangiotensin system ras57 to which the virus binds through its surface spike proteins is particularly abundant in the digestive tract lungs kidney heart and blood vessels where pathology from sarscov2 occurs58 a peptidase that catalyzes the conversion of angiotensin ii angii referred to as •the quintessential perpetrator of inflammation– to angiotensin ang ace2 mediates antiproliferative and vasodilatory functions that oppose the vasoconstrictive and inflammatory functions of angiotensin converting enzyme ace60 the binding of sarscov2 to ace2 leads to downregulation of ace2 expression potentially through increased internalization and shedding from the cell surface with decreased ang1“ generation and increased ang ii levels as a consequence61 this unfavorably shifts the balance of the renin angiotensin system ras from the vasoprotective ace2ang17 axis to the aceang iiangiotensin at1 receptor axis and drives a proinflammatory profibrotic and proliferative response62 as shown in figure fang et al63 contend that because thiazolidinediones ibuprofen and angiotensin converting enzyme ace inhibitors and angiotensin ii typei receptor blockers arbs substantially increase the expression of ace2 they facilitate sarscov2 infection and therefore the risk of severe and fatal covid19 in contrast alghatrif et al64 present a diametrically opposed hypothesis that downregulated ace2 signaling is responsible for sarscov2induced acute lung injury ali acute respiratory distress syndrome ards and cytokine storm and that aceis and arbs are beneficial precisely because they increase ace2 expression and activity furthermore according to alghatrif et al lower ace2 levels and hence higher baseline oxidative stress and inflammation6566 are present in older comorbid individuals such as cancer patients which renders them more susceptible to severe covid19 than younger noncomorbid individuals with increased ace2 levels and lower baseline inflammation as shown in figure furthermore low ace2 may promote tumor progression and conversely ace2 overexpression is associated with antiangiogenesis and tumor regression67 in summary then 0c preproofdespite the concerns and controversy68 surrounding the use and continuation of aceisarbs during the sarscov2 epidemic it is likely that the pros outweigh the cons especially in cancer patients due to their potential antitumor and anticovid19 effects69 in line with ras involvement emerging data suggest that sarscov2 infection may induce serious cardiovascular injury or exacerbate existing cardiovascular disease cardiovascular sequela includes heart failure arrythmias disseminated intravascular dissemination dic and troponin elevation which may closely correlate with disease severity and the likelihood of inhospital death70 liu et al71 propose a mechanism whereby the virus which lowers hemoglobin hb levels72 binds to the porphyrin of heme and displaces iron thereby compromising the oxygencarrying capacity of red blood cells and exacerbating the hypoxemia since chloroquine and the experimental anticancer agent rrx001 also bind to porphyrins they may competitively interfere with binding by the virus rationale for continuation or discontinuation of cancer therapy preproofthe benefitrisk calculus that informs the decision whether and how to treat with anticancer therapy falls into a •gray zone– about which no consensus exists leading to a therapeutic dilemma on the one hand zhang et al73 in annals of oncology reported a strong association in patients of them with lung cancer between antineoplastic therapy in the past days and severe effects of covid19 hr4079 ci p0037 on this basis the authors recommend treatment interruption dose reduction or substitution of cytotoxic chemotherapy with nonimmunosuppressive options eg checkpoint inhibitors if available especially in the case of lung cancer patients that are already prone to develop respiratory infections and complications74 similarly heavily immunosuppressed patients such as those who have undergone hematopoietic stem cell transplantation are also particularly susceptible to viral respiratory infections these findings are supported by a nationwide analysis of data75 in china from covid19 patients of which were diagnosed with cancer this patient cohort experienced a higher incidence of severe events vs p and the administration of chemotherapy or surgery was found to have increased the risk of death andor intensive care unit admission even after adjusting for age sex and comorbidities odds ratio or ci “ p while these studies are limited by small sample sizes the data suggests that cancer predisposes to more severe disease therefore since inperson contact increases the risk of transmission several institutions have mandated realtime video or telephone interactions alternatively referred to as telehealth77 postponed surgeries biopsies endoscopies scans and routine investigations when possible and in line with esmo guidelines78 encouraged conversion from the intravenous to the oral route eg 5fluorouracil to capecitabine etoposide and vinorelbine on the other hand the immediate existential threat of progressive disease for which death is an impending imminent certainty rather than a remote possibility in the absence of treatment likely outweighs the theoretical risk of sarscov2 infection even in lower risk disease for example in situ or localized prostate breast and head and neck cancer delayed treatment is 0c preproofpotentially conducive to tumor development and progression and thus may unfavorably impact prognosis79 hanna et al have proposed a triage strategy80 which prioritizes treatment for those patients with imminent risk of early mortality from acute leukemias aggressive lymphomas metastatic germ cell tumors oncologic emergencies such as spinal cord compression chemoradiotherapeuticresponsive cancers such as head and neck cervical and anal cancers and neoadjuvant or adjuvant therapyresponsive tumor types such as stage iii colon cancer and deprioritizes visits for surveillance and survivorship however in the absence of a •one size fits all– consensus recommendation which is unlikely since cancer is so genetically diverse and heterogeneous the decisionmaking process and the subsequent treatment plan are individualized and to be determined tbd on casebycase basis taking into account multiple factors including the risk of cancer recurrence if therapy is delayed modified or interrupted the type of therapy eg surgery radiation chemotherapy checkpoint inhibitors and stem cell transplantation extent of comorbidities concomitant medications patient preferences physicianpatient relationship race age the number of cycles of therapy completed and treatment tolerance in terms of specific cancerrelated conditions asco makes the following heavily qualified recommendations81 \uf0b7 growth factor prophylaxis for neutropenia and neutropenic fever even at lower levels of risk as well as empiric antibiotics for acute care \uf0b7 erythropoietinstimulating agents for anemia prophylaxis and transfusion when necessary depending on the patient context and underlying comorbidities preproof treatment based on the high transmissibility of the virus82 the main nonpharmacologic countermeasures to mitigate or delay the impact of covid19 include rigorous hand hygiene use of facemasks respiratory etiquette ie coughing or sneeze into the upper sleeve or elbow not the hands flushing with the lid down to prevent bioaerosolization as well as quarantine stay at home policies and workplace and school closures which have upended the social cultural political and economic status quo no specific treatment or vaccine is currently available although promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir the mainstay of medical therapy includes symptomatic care such as supplemental oxygen antibiotics and hemodynamic and mechanical ventilatory support if indicated for septic shockmultiple an failure and respiratory failure respectively83 over active clinical treatment trials are underway84 these include vaccines as well as a number of different agents some with promising preliminary data as mentioned above and also those with potential anticancer activity which will hopefully serve a double purpose first to treat covid19 and second as an adjunct to bridge the time gap until the patient is recovered and the primary antineoplastic is startedrestarted as shown in table 0c preproof conclusions the alarming spread of the covid19 pandemic has disproportionately affected cancer patients an atrisk population both from the standpoint of increased disease severity and disruption to care which includes widespread suspension of clinical trials in the united states that are already fraught with barriers to enrollment and participation85 86because the symptoms of covid19 are nonspecific underlying symptoms from the cancer eg dyspnea cough fever fatigue diarrhea etc which overlap with those from the viral infection may obscure and delay the diagnosis hence if the covid19specific rapid reverse transcriptase polymerase chain reaction rtpcr test is not readily available andor in short supply which is currently the case diagnosis will depend on the maintenance of a high index of clinical suspicion especially in advanced cancer patients who check all the boxes for risk factors such as older age frailty disability immunosuppression generalized systemic inflammation and multiple comorbidities eg hypertension diabetes and cardiorenovascular diseases that predispose to severe disease and death preproofthese comorbidities are commonly treated with renin angiotensin system blockers such as angiotensinconverting enzyme inhibitors aceis or angiotensinreceptor blockers arbs which increase levels of ace2 the continued use of aceisarbs is the centerpiece of an intense debate because on the one hand sarscov2 coopts ace2 for target cell entry but on the other ace2 overexpression may counterbalance vasoconstriction and profibrotic processes and thereby reduce the incidence or mortality associated with covid19“associated ali or acute respiratory distress syndrome another controversy involves whether or not to continue cancer treatment given the high transmissibility potential of the virus however since no expert consensus recommendations have been issued to date and prognosis stage and responses to therapy are highly heterogeneous the riskbenefit tradeoff and subsequent treatment plan are highly individualized and context dependent currently the focus of treatment is infection control appropriate symptomatic care and oxygen therapy no approved medication or vaccine has been developed but promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir and several repurposed agents with antitumor properties are under investigation including thalidomide and rrx001 which may hopefully bridge the gap from the time covid is first diagnosed until the primary anticancer therapy is restarted finally multiple comparisons have been made between the allout mobilization efforts to combat covid19 with the massive scaleup of human and material resources that occurred during world war ii8788 in the words of winston churchill prime minister of great britain from whose intrepid fighting spirit iron will and intransigent defiance of tyranny galvanized the resolve of an entire nation to fight on in the face of seemingly impossible odds oncologists on the frontlines that have answered the call should •never worry about action only inaction– 0c preproof declaration of interests the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper references sitammagari k skandhan a dahlin awhat hospitalists need to know about covid19 medscape mar httpswwwmedscapecomviewarticle924596 meo sa alhowikan am alkhlaiwi t meo im halepoto dm iqbal m usmani am hajjar w ahmed n novel coronavirus 2019ncov prevalence biological and clinical characteristics comparison with sarscov and merscov eur rev med pharmacol sci feb24420122019 chan jf kok kh zhu z et al genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan emerg microbes infect kuba k imai y rao s et al a crucial role of angiotensin converting enzyme ace2 in sars coronavirusinduced lung injury nat med zhang h kang z gong h et al the digestive system is a potential route of 2019ncov infection a bioinformatics analysis based on singlecell transcriptomes [epub ahead of print] biorxiv de wit e van doremalen n falzarano d munster vj sars and mers recent insights into emerging coronaviruses nat rev microbiol “ w li et al angiotensinconverting enzyme is a functional receptor for the sars coronavirus nature hughes s covid19 and angiotensin drugs help or harm medscape march httpswwwmedscapecomviewarticle927542 yang x yu y xu j et al clinical course and outcomes of critically ill patients with sarscov2 pneumonia in wuhan china a singlecentered retrospective observational study [published online ahead of print feb ] [published correction appears in lancet respir med feb ] lancet respir med 2020s2213 gu j han b wang j covid19 gastrointestinal manifestations and potential fecaloral transmission [published online march ] gastroenterology ruiheng x chance missed but still there memoirs at the 10th anniversary of sars outbreak j thorac dis aug 5suppl s90“s93 jiang s don't rush to deploy covid19 vaccines and drugs without sufficient safety guarantees nature mar5797799321 doi 101038d41586020007519 peiris js guan y yuen ky severe acute respiratory syndrome nat med dec suppls8897 zheng h zhang m yang c et al elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid19 patients cell mol immunol2020 tognotti e lessons from the history of quarantine from plague to influenza a emerg infect dis “ httpswwwnihgovnewseventsnewsreleasesnihclinicaltrialinvestigationalvaccinecovid19begins httpswwwascoascocoronavirusinformationcareindividualscancerduringcovid19 onder g rezza g brusaferro s casefatality rate and characteristics of patients dying in relation to covid in italy published online march doi101001jama20204683 hanna tp evans ga booth cm cancer covid19 and the precautionary principle prioritizing treatment during a global pandemic nat rev clin oncol who advice on the use of masks in the community during home care and in healthcare settings in the context of the novel coronavirus 2019ncov outbreak jan rodriguezmorales aj macgregor k kanagarajah s patel d schlagenhauf p going global travel and the novel coronavirus travel med infect dis httpsdoi101016jtmaid2020101578 httpswwwnejmdoifull101056nejmc2004973 lu cw liu xf jia zf 2019ncov transmission through the ocular surface must not be ignored the lancet preproof 0c preproof holshue ml et al first case of novel coronavirus in the united states n engl j med yeo c kaushal s yeo d enteric involvement o
Colon_Cancer
" rectus sheath block rsb is known to attenuate postoperative pain and reduce perioperative opioidconsumption thus a retrospective study was performed to examine the effects of bilateral rectus sheath blockbrsb in cytoreductive surgery crs combined with hyperthermic intraperitoneal chemotherapy hipecmethods a total of patients undergoing crshipec at our hospital were included patient information andanaesthesiarelated indicators were collected from the electronic medical record emr system all subjects weredivided into the following two groups the g group general anaesthesia and the gr group rsb combined withgeneral anaesthesia patients in the gr group received ropivacaine for brsb before surgery the primaryoutcomes included the total amount of remifentanil and rocuronium the total consumption of dezocine aftersurgery the visual analogue scale vas score and the patientcontrolled intravenous analgesia pcia input dose at h t6 h t7 h t8 h t9 and h t10 after surgery other outcomes were also recorded such aspatient demographic data the intraoperative heart rate hr and mean arterial pressure map and postoperativecomplicationsresults compared with the g group the gr group showed a shorter time to tracheal extubation p adecreased total amount of remifentanil and rocuronium p and a reduced vas score pcia input dose andnumber of pcia boluses at h h and h after surgery p however at h and h after surgery therewere no differences in the vas score of pain at rest or during motion between the two groups p moreoverthe incidence of hypertension emergence agitation delayed recovery hypercapnia and nausea and vomiting waslower in the gr group than in the g group p there were no differences in the changes in map and hrduring the surgery between the two groups p no complications associated with nerve block occurred brsb could provide shortterm postoperative analgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complications it is an effective and safe procedure in crshipeckeywords cytoreductive surgery hyperthermic intraperitoneal chemotherapy rectus sheath block generalanaesthesia analgesia correspondence trmzltz126comdepartment of anesthesiology beijing shijitan hospital capital medicaluniversity beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc anesthesiology page of radical cytoreductive surgery crs combined withhyperthermic intraperitoneal chemotherapy hipec isconsidered a standard for the treatment of peritonealcancer such as rectal cancer ovarian cancer peritonealpseudomyxoma and peritoneal mesothelioma thistechnique could prolong the longterm survival of patients with a decreased recurrence rate although thepositive results of this treatment have been proven inprevious studies [“] because of the large peritonealsurface area involved in this kind of surgery crshipecis time consuming and complex which presents agreat challenge for the anaesthesiologist in terms of perioperative managementdue to the stable respiratory and circulatory supportgeneral anaesthesia is the preferred choice in this surgery however long periods of general anaesthesia leadto drug accumulation in the body followed by increasedanaesthesiarelated complications including delayed recovery respiratory inhibition and cognitive dysfunction consequently exploring better anaesthesia methodsfor this surgery is still a major concerna new approach called ultrasoundguided bilateral rectus sheath block brsb has been proven to amelioratepostoperative pain and reduce the consumption of morphine [“] nonetheless there have been no reportson the application of general anaesthesia combined withbrsb in patients undergoing crshipec based on theinformation presented above this retrospective observational study was conducted to examine the efficacy andsafety of brsb in patients treated with crs and hipecmethodssubjectsall patients who underwent crs and hipec at beijingshijitan hospital between august and december were retrieved from the institutional database inthis study the exclusion criteria were as follows laparoscopic surgery with crshipec intraoperativeblood loss volume greater than ml mechanicventilation required after surgery and use of analgesictechniques apart from brsb and general anaesthesiaaccording to this standard a total of patients wereincluded and divided into the following groups generalanaesthesia g group n and general anaesthesiacombined with posterior rsb gr group n anaesthesia methodgeneral anaesthesia was consistently induced in all patients with intravenous propofol mgkg sufentanil μgkg and rocuronium mgkg invasive arterialpressure and central venous pressure were monitored byradial artery puncture flotracvigileo® edwards lifesciences irvine ca usa and internaljugular veinsitetargeteffectpuncture respectively after anaesthesia induction anaesthesia was maintained with sevoflurane and remifenconcentration “ ngmltanilkeeping the bispectral index bis between and rocuronium mgkg wasintermittently used tomaintain muscle relaxation in the gr group before anaesthesia induction patients received brsb under ultrasound guidance the puncture site was placed at theouter edge of the bilateral rectus abdominis at the levelof the umbilicus fig a a total of ropivacaine ml was injected into each side the spindleshapedspread of ropivacaine was observed between the posterior sheath of the rectus abdominis and the rectus abdominis itself implying success of the procedure fig b c patientcontrolled intravenous analgesia pciawas applied in both groups after the surgery sufentanil μgkg palonosetron hydrochloride mg was diluted to ml the dose was mlh and asingle dose was mlh with a 15min lockout intervalafter the surgery all patients were sent to the surgicalintensive care unit sicu if the visual analogue scalevas score at rest after surgery was ‰¥ dezocine mgwas used as a rescue analgesicdata collectionall the indicators we needed were obtained from theemr system the records included patient demographicdata patient medical history american society of anesthesiologists asa grade and new york heart association nyha grade the hr and map were recordedat the time before brsb t1 the time of anaesthesiat2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgeryt5 in addition the duration of the surgery time totracheal extubation the time after skin closure totalamount of remifentanil and muscle relaxants total fluidvolume urine volume and the total volume of allogeneicerythrocytes and plasma infused during the surgery wereall recorded moreover after surgery the occurrence ofhypertension the systolic blood pressure dropped bymore than of baseline blood pressure beforeanesthesia or the sbp mmhg during surgery nausea and vomiting hypoxemia spo2 or pao2 mmhg hypercapnia paco2 mmhg and emergence agitation during the recovery period were recorded the recovery period was considered as the timefrom switching off inhalation anaesthetics remifentaniland muscle relaxant to recovery of the patients™ abilitiesto command movement orientation as well as conscious state when the recovery period of patients is beyond min it was considered as delayed recovery thevas score for pain at rest and during motion the pciainput dose and the number of boluses at h t6 ht7 h t8 h t9 and h t10 after surgery 0cwang bmc anesthesiology page of the incomplete datachemotherapy crshipec during the year from to in our hospital one hundred and six patientsreceived brsb seventeen patients were excluded because ofincluding patientsundergoing intraoperative haemorrhage blood ml and other patients received mechanic ventilationbecause of acute respiratory distress syndrome ardsallergic shock and cardiac insufficiency thus patients with brsb were eventually obtained finally patients without brsb were randomly selected to analysis in this study fig statistical analysisspss software was used for statistical analysisnormal distribution data were recorded as the mean ±standard deviation sd and analysed by independentsamples t test for comparison between the two groupsnonnormally distributed data are presented as themedian range and were analysed by kruskalwallistest chisquared test or fisher™s exact test was used forcategorical data a p value of was considered statistically significantresultscharacteristics of study populationin total patients were included in the study thebaseline demographic and surgical variables of patientsare presented in table there were no significant differences in age sex body mass index bmi basic diseases asa grade nyha grade total surgery time totalfluid volume urine volume total volume of allogeneicerythrocyte infusion or total volume of plasma p however the time to tracheal extubation was shorter inthe gr group than in the g group p the totalamount of both remifentanil and rocuronium used wasless in the gr group than in the g group p thus posterior rsb could reduce the use of remifentaniland rocuronium during surgerychanges in haemodynamic parametersthe changes in hr and map are presented in table there were no significant differences in hr or map atany point in time t1 to t5 between the two groupsp the results suggest that brsb did not affectthe haemodynamics ofthe patient undergoing crshipecpainrelated indicatorstable shows the postoperative vas score the pca input dose and the number of pca boluses at h t6 ht7 h t8 h t9 and h t10 after surgeryas well as the dose of dezocine used as a rescue analgesic from t6 to t8 compared with the g group thegr group showed significantly decreased vas scores offig ultrasoundguided brsbas well as the dose of dezocine used as a rescue analgesic were also recordedin addition brsbrelatedcomplications such as peritoneal punctureinternalan injury and systemic toxicity were all recordeda total of patients underwent cytoreductive surintraperitonealcombined withgeryhyperthermic 0cwang bmc anesthesiology page of fig flow chart showing patient consecutive enrolment and analysis abbreviations crshipec cytoreductive surgery and hyperthermicintraperitoneal chemotherapy ga general anesthesia brsb bilateral rectus sheath block ards acute respiratory distress syndrome vas visualanalogue scalepain at rest and during motion p however at h and h after surgery there were no significant differences in the vas scores of pain at rest and duringmotion between the two groups p from t6 tot10 the pcia input dose and the number of pca boluses were also obviously reduced in the gr group compared with the g group p in addition as arescue analgesic the dose of dezocine after surgery inthe gr group was significantly lower than that in the ggroup p postoperative adverse eventsadverse events that occurred in the sicu are presentedin table after surgery there were cases withhypertension cases of emergence agitation cases ofdelayed recovery cases of hypercapnia and cases ofnausea and vomiting in the g group fewer cases of allof these events occurred in the gr group p there were no differences in the incidence of hypoxemiabetween the two groups p there were no complications associated with nerve block in either group 0cwang bmc anesthesiology page of table demographic and surgical variables mean ± sdage ysex malefemalebmi kgm2medical historydiabetes mellitus n yesnohypertension n yesnocoronary heart disease n yesnoasa grade iiiiiinyha grade iiitotal surgery time mintime to tracheal extubation minremifentanil mgrocuronium mgtotal fluid volume mlurine volume mltotal volume of allogeneic erythrocyte infusion mltotal volume of plasma mlg group n ± ± ± ± ± ± ± ± ± ± gr group n ± ± ± ± ± ± ± ± ± ± p value asa american society of anesthesiologists bmi body mass index calculated as weight in kilograms divided by height in metres squared nyha new york heartassociation g general anaesthesia gr bilateral rectus sheath block combined with general anaesthesia before bilateral rectus sheath block t1 the time ofanaesthesia t2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgery t5discussionin this retrospective study we examined the efficacy andsafety of brsb combined with general anaesthesia in patients undergoing crshipec regarding efficacy theresults show that ultrasoundguided brsb significantlyreduced the total dose of remifentanil used during thesurgery and shortened the time to tracheal catheter extraction which is consistent with the findings of previous studies of other surgeries [ ] in addition rsbreduced the total dose of rocuronium in this studytable haemodynamic parameters in both groups mean ± sdindextimepointgn ± grn ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± pvaluemmhghrbpmt1t2t3t4t5t1t2t3t4t5map mean arterial pressure hr heart rate g general anaesthesia gr bilateralrectus sheath block combined with general anaesthesiawhich may be associated with the high concentration ofropivacaine used in the studyrsb also effectively relieved postoperative pain in thisstudy we found that the vas scores of pain at rest andduring motion were all lower in the gr group than inthe g group at h after surgery however at h and h after surgery there were no differences in the vasscores of pain at rest and during motion between thetwo groups suggesting that the analgesic effects of asingle brsb remained within h after surgery thisresult may be different from the findings of others cho reported that at h after surgerythere were no differences in the vas scores of painat rest and during motion between the rsb and nonrsb groups the discrepant results may be related todifferences in the concentration of ropivacaine andthe physical constitution of patients a high concentration can prolong the duration of action of a localanaesthetic in this study we selected not ropivacaine additionallythese patientsundergoing crshipec may have been adaptive topain furthermore compared with the control groupthe rsb group showed a reduced totalinfused doseof sufentanil as pcia number of pca boluses within h after surgery and total dose of dezocine used asa rescue analgesic after surgery these results furtherprove the role of rsb in providing shortterm postoperative analgesia 0cwang bmc anesthesiology page of table painrelated indicators in both groups median [range]vas score of pain at rest [median range]t6t7t8t9t10vas score of pain during motion [median range]t6t7t8t9t10total infused dose of pcia [ml median range]t6t7t8t9t10cumulative number of pcia boluses [median range]t6t7t8t9t10total dose of dezocine as a rescue analgesic mggn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± grn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± p value vas visual analogue scale pcia patientcontrolled intravenous analgesia g general anaesthesia gr bilateral rectus sheath block combined with generalanaesthesia the time at h after surgery t6 h after surgery t7 h after surgery t8 h after surgery t9 and h after surgery t10table postoperative adverse events in both groupsadverse eventsn gn grn hypertensionemergence agitation delayed recoveryhypoxemiahypercapnia nausea and vomiting peritoneal punctureinternal an injurysystemic toxicity g general anaesthesia gr bilateral rectus sheath block combined withgeneral anaesthesiawe also examined the safety of rsb during the surgery ultrasoundguided brsb had no significant effectson the haemodynamics of patients during surgery compared with general anaesthesia alone in terms of postoperative adverse events the results show that comparedwith the control group the rsb group showed a reducedincidence of hypertension emergence agitation delayedrecovery hypercapnia and nausea and vomiting whichmight be correlated with the decreased analgesic andmuscle relaxant doses no rsbrelated complications occurred in any patient these data indicate that rsbcould reduce the risk of complications associated withgeneral anaesthesia and is safe for patientsrsb an established technique has regained popularityin clinical applications [“] previous studies havedemonstrated that this technique could achieve relaxation of the anterior abdominal wall [ ] bashandyreported that anterior branches of the t7t12 thoracicnerve and the l1 lumbar nerve travelled through thepvalue“““ 0cwang bmc anesthesiology page of plane of the transverse abdominis muscle entered the rectus abdominis sheath and distributed on the surface of theskin the main process of rsb is to inject local anaesthetics between the rectus abdominis and the posteriorsheath of the rectus abdominis therefore rsb exerteda good effect in terms of perioperative analgesia for medianabdominal incisions a midline incision is required inthis kind of surgery thus based on these results rsbcould meet the need for analgesia in these patientsin addition for a long time epidural analgesia eawas thought to be an effective method for abdominalsurgery [“] studies have proved that epidural analgesia could maintain a good analgesic effect and reduceperioperative opioid consumption including in this typeof surgery [ ] however the safety of ea in crshipec remains controversial especially regarding effectson coagulation and circulatory function coagulationdysfunction and profound fluid loss are the main characteristics of patients with peritoneal cancer [ ]which might limit the administration of eaalthough epidural catheter is standard of care insolanki™s guideline in our hospital epidural catheter in not the standard of care we performed generalanesthesia combined with epidural anesthesia in somepatients to reduce the consumption of intravenous drugsand provide perfect analgesia but coagulation dysfunction and profound fluid loss are the main characteristicsof patients with peritoneal cancer in our previous studywe found that the mean arterial pressure of patientsundergoing epidural anesthesia was difficult to be maintained in the surgery besides there were many patientswith coagulation dysfunction before surgery who werenot suitable for the epidural anesthesia these results wefound in clinical were similar with others™ researcheskajdi and colleagues reported a case of epidural haematoma in their study godden found that the incidence of hypotension in the ea group was obviouslyhigher than that in the rsb group consequentlyrsb could be a better choice than ea in crshipecadditionally there are some limitations to this studyfirst all the data of this study were collected from theemr system as this was a retrospective study the findings are not as persuasive as those of a randomized controlled study we plan to conduct prospective studies toexplore the comprehensive influence of rsb in this surgery second we only examined the application of brsbestablished with a single injection which provides only ashortterm analgesic effect the efficacy of continuousanalgesia with brs catheters in crshipec remains unclear and needs further exploration third in our the results are initially presented according to the different aspects the primary outcome of this study is thetotal consumption of remifentanil during the surgeryother indicators were belonged to second outcomessin brsb could provide good postoperativeanalgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complicationsthis is an easily applicable and safe procedure in crshipecabbreviationsrsb rectus sheath block brsb bilateral rectus sheath blockcrs cytoreductive surgery hipec hyperthermic intraperitonealchemotherapy emr electronic medical record vas visual analogue scalepcia patientcontrolled intravenous analgesia hr heart rate map meanarterial pressure bis bispectral index sicu surgical intensive care unitasa american society of anesthesiologists nyha new york heartassociation spo2 pulse oximetry paco2 partial pressure of carbon dioxidepao2 oxygen partial pressure bmi body mass indexacknowledgementsi would like to express my heartfelt thanks to the staff of the informationdata center of beijing shijitan hospital affiliated to capital medical universityauthors™ contributionswsh study design data collection writing paper lpf gt data collectionand data analysis gl coordinated the study and manuscript revision ltzstudy design and manuscript revision all authors read and approved thefinal manuscript all authors ensure the accuracy of the manuscript andagree to take personal responsibility for their contributionsfundingno fundingavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethis study was approved by the ethics committee of beijing shijitan hospitalaffiliated to capital medical university approval code research ethicsno69 this study is retrospective only anonymous data sources were usedand informed consent was not requiredconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesklaver ce musters gd bemelman wa punt cj verwaal vj dijkgraaf mgaalbers ag van der bilt jd boerma d bremers aj adjuvanthyperthermic intraperitoneal chemotherapy hipec in patients with coloncancer at high risk of peritoneal carcinomatosis the colopec randomizedmulticentre trial bmc cancer 201515undefined428van oudheusden tr braam hj nienhuijs sw wiezer mj van ramshorst bluyer md lemmens ve de hingh ih cytoreduction and hyperthermicintraperitoneal chemotherapy a feasible and effective option for colorectalcancer patients after emergency surgery in the presence of peritonealcarcinomatosis ann surg oncol “passot g vaudoyer d villeneuve l kepenekian v beaujard ac bakrin ncotte e gilly fn glehen o what made hyperthermic intraperitonealchemotherapy an effective curative treatment for peritoneal surfacemalignancy a 25year experience with procedures j surg oncol“arjonasánchez a barrios p boldoroda e camps b carrascocampos jmartín vc garcíafadrique a gutiérrezcalvo a morales r ortegapérez g hipect4 multicentre randomized clinical trial to evaluate safety andefficacy of hyperthermic intraperitoneal chemotherapy hipec with 0cwang bmc anesthesiology page of huepenbecker sp cusworth se kuroki lm lu p samen cd woolfolk cdeterding r wan l helsten dl bottros m continuous epiduralinfusion in gynecologic oncology patients undergoing exploratorylaparotomy the new standard for decreased postoperative pain and opioiduse gynecol oncol “teoh da hutton mj else s walker a lee a mack la epidural analgesia aprospective analysis of perioperative coagulation in cytoreductive surgeryand hyperthermic intraperitoneal chemotherapy am j surg “schmidt c creutzenberg m piso p hobbhahn j bucher m perioperativeanaesthetic management of cytoreductive surgery with hyperthermicintraperitoneal chemotherapy anaesthesia “kajdi me beckschimmer b held u kofmehl r lehmann k ganter mtanaesthesia in patients undergoing cytoreductive surgery withhyperthermic intraperitoneal chemotherapy retrospective analysis of asingle centre threeyear experience world j surg oncol solanki sl mukherjee s agarwal v thota rs balakrishnan k shah sb desain garg r ambulkar rp bhorkar nm society of oncoanaesthesia andperioperative care consensus guidelines for perioperative management ofpatients for cytoreductive surgery and hyperthermic intraperitonealchemotherapy crshipec indian j anaesth “ godden ar marshall mj grice as daniels ir ultrasonography guidedrectus sheath catheters versus epidural analgesia for open colorectal cancersurgery in a single centre ann r coll surg engl “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsmitomycin c used during surgery for treatment of locally advancedcolorectal carcinoma bmc cancer baratti d kusamura s iusco d gimondi s pietrantonio f milione mguaglio m bonomi s grassi a virzì s hyperthermic intraperitonealchemotherapy hipec at the time of primary curative surgery in patientswith colorectal cancer at high risk for metachronous peritoneal metastasesann surg oncol “chua tc robertson g liauw w farrell r yan td morris dl intraoperativehyperthermic intraperitoneal chemotherapy after cytoreductive surgery inovarian cancer peritoneal carcinomatosis systematic review of currentresults j cancer res clin oncol “li y zhou yf liang h wang hq hao jh zhu zg wan ds qin lx cui szji jf chinese expert consensus on cytoreductive surgery andhyperthermic intraperitoneal chemotherapy for peritoneal malignanciesworld j gastroenterol “ willschke h bosenberg a marhofer p johnston s kettner sc wanzel o kaprals ultrasonographyguided rectus sheath block in paediatric anaesthesiaanew approach to an old technique br j anaesth “azemati s khosravi mb an assessment of the value of rectus sheath blockfor postlaparoscopic pain in gynecologic surgery j minim invasive gynecol“ dingeman rs barus lm chung hk clendenin dj lee cs tracy s johnsonvm dennett kv zurakowski d chen c ultrasonographyguided bilateralrectus sheath block vs local anesthetic infiltration after pediatric umbilicalhernia repair a prospective randomized clinical trial jama surg “ relland lm tobias jd martin d veneziano g beltran rj mckee c bhalla tultrasoundguided rectus sheath block caudal analgesia or surgical siteinfiltration for pediatric umbilical herniorrhaphy a prospective doubleblinded randomized comparison of three regional anesthetic techniques jpain res 201710undefined2629“ xu l hu z shen j pm mq efficacy of usguided transversus abdominisplane block and rectus sheath block with ropivacaine anddexmedetomidine in elderly highrisk patients minerva anestesiol “ cho s kim yj jeong k moon hs ultrasoundguided bilateral rectus sheathblock reduces early postoperative pain after laparoscopic gynecologicsurgery a randomized study j anesth “li t ye q wu d li j yu j doseresponse studies of ropivacaine in bloodflow of upper extremity after supraclavicular block a doubleblindrandomized controlled study bmc anesthesiol bell jc rylah bg chambers rw peet h mohamed f moran bjperioperative management of patients undergoing cytoreductive surgerycombined with heated intraperitoneal chemotherapy for peritoneal surfacemalignancy a multiinstitutional experience ann surg oncol “landmann a visoiu m malek mm development of a novel technique forbilateral rectus sheath nerve blocks under laparoscopicguidance j pediatrsurg “kumar a wilson ga engelhardt te ultrasound guided rectus sheathblockade compared to perioperative local anesthetic infiltration in infantsundergoing supraumbilical pyloromyotomy saudi journal of anaesthesia“ bashandy gm elkholy ah reducing postoperative opioid consumption byadding an ultrasoundguided rectus sheath block to multimodal analgesiafor abdominal cancer surgery with midline incision anesthesiol pain med201443e18263 dowidar aerm ezz haa shama aae eloraby ma postoperative analgesiaof ultrasound guided rectus sheath catheters versus continuous woundcatheters for colorectal surgery a randomized clinical trial ˜† egypt journalof anaesth “karaarslan e topal a avci o tuncer uzun s research on the efficacy of therectus sheath block method agri “ piccioni f casiraghi c fumagalli l kusamura s baratti d deraco m arientif langer m epidural analgesia for cytoreductive surgery withperitonectomy and heated intraperitoneal chemotherapy int j surg 16pt a99“ vesterandersen m lundstrøm lh møller mh the association betweenepidural analgesia and mortality in emergency abdominal surgery apopulationbased cohort study acta anaesthesiol scand httpsdoi101111aas13461 0c"
Colon_Cancer
" recent impressive advances in cancer immunotherapy have been largely derived from cellular immunity the role of humoral immunity in carcinogenesis has been less understood based on our previous observations we hypothesize that an immunoglobulin subtype igg4 plays an essential role in cancer immune evasionmethods the distribution abundance actions properties and possible mechanisms of igg4 were investigated with human cancer samples and animal tumor models with an extensive array of techniques both in vitro and in vivoresults in a cohort of patients with esophageal cancer we found that igg4 containing b lymphocytes and igg4 concentration were significantly increased in cancer tissue and igg4 concentrations increased in serum of patients with cancer both were positively related to increased cancer malignancy and poor prognoses that is more igg4 appeared to associate with more aggressive cancer growth we further found that igg4 regardless of its antigen specificity inhibited the classic immune reactions of antibody dependent cell mediated cytotoxicity antibody dependent cellular phagocytosis and complement dependent cytotoxicity against cancer cells in vitro and these effects were obtained through its fc fragment reacting to the fc fragments of cancer specific igg1 that has been bound to cancer antigens we also found that igg4 competed with igg1 in reacting to fc receptors of immune effector cells therefore locally increased igg4 in cancer microenvironment should inhibit antibody mediated anticancer responses and help cancer to evade local immune attack and indirectly promote cancer growth this hypothesis was verified in three different immune potent mouse models we found that local application of igg4 significantly accelerated growth of inoculated breast and colorectal cancers and carcinogen induced skin papilloma we also tested the antibody drug for cancer immunotherapy nivolumab which was igg4 in nature with a stabilizing s228p mutation and found that it significantly promoted cancer growth in mice this may provide an explanation to the newly appeared hyperprogressive disease sometimes associated with cancer immunotherapy there appears to be a previously unrecognized immune evasion mechanism with igg4 playing an essential role in cancer microenvironment with implications in cancer diagnosis and immunotherapyintroductionwhile new immune therapy for cancer focuses mostly on manipulating cellular immunity humoral immunity also holds great promise for cancer therapy1 igg4 is a unique antibody that has the lowest concentration among igg subtypes in healthy individuals and its function has not been well understood3“ igg4 was regarded as a ˜blocking antibody™ because of its reduced ability to trigger effector immune reactions6 therefore whatever molecules igg4 reacts to the subsequent immune reaction was subdued8 igg4 has a unique structure of fab arm exchange fae in which the two heavy and light chains of two different antibodies with different specificities are exchanged resulting in an asymmetric bispecific antibody with reduced ability to bind to antigen and to form immune complexes9 another unique feature of igg4 is that it can react to other iggs via its fc fragment and the significance of this property has not been well understood evidence suggests that fae and fc fc reactivity may involve the same molecular structure on igg4 molecule10 davies et al11 resolved the crystal structure of igg4 fc fragment revealing a unique molecular conformation supporting its fc binding property recent interests in igg4 related diseases unveiled a wide range of pathologies with a common phenomenon of often increased igg4 concentration in the serum and igg4 postive plasma cells in the affected ans accompanied by local inflammation and fibrosis but its pathogenic mechanism is still poorly understood13“to cite wang a0h xu a0q zhao a0c et a0al an immune evasion mechanism with igg4 playing an essential role in cancer and implication for immunotherapy for immunotherapy of cancer 20208e000661 101136jitc2020000661 –º additional material is published online only to view please visit the online http dx jitc hw qx cz and zz are joint first authorsaccepted july authors or their employers re use permitted under cc by nc no commercial re use see rights and permissions published by bmjfor numbered affiliations see end of correspondence toprofessor jiang gu qq comwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access the possible functions of igg4 in cancer and also in the immune system have not been well elucidated increases of igg4 positive plasma cells were reported in gastrocarcinoma16 extrahepatic cholangiocarcinoma17 and melanoma18 the above studies were performed on limited number of cases without convincing explanation of mechanism or significance wu 19 reported that serum igg4igg ratio could predict recurrence of hepatocellular carcinoma after surgery the most extensive study on igg4 and cancer was performed by karagiannis 20 who investigated the possible effect of cancer specific igg4 in inhibiting cancer immunity in melanomas and suggested competition between cancer specific igg4 and igg1 in binding to cancer antigens as the cause for the inhibition a recent report raised the concept of cancer educated b cells and toxic igg produced by these cells in facilitating lymph node metastasis for breast cancer in a mouse model21 we performed a multidimensional investigation of igg4 in a wide array of patients with cancer and tissues with both in vitro and in vivo experiments extensive new evidence led us to hypothesize that there is a potent humoral immune editing mechanism in cancer microenvironment with igg4 playing an essential role we propose that fc fc reaction could be the basic mechanism of this immune inhibition we validated this in three immune potent animal models this property was also found applicable to cancer immunotherapy drug nivolumab which was igg4 in nature our study points to a so far little appreciated mechanism of immune evasion in cancermaterials and methodskey resourcesdetailed information about key resources including antibodies biological samples chemicals assay kits cell lines and software are shown in online supplementary table experimental model and subject detailspatients and healthy volunteerswe collected tissue and blood samples from over patients with cancer in shantou university affiliated tumor hospital and the east guangdong provincial pathological consultation center details of the human samples are shown in online supplementary table immunohistochemistrydetails of the protocols and the antibodies are shown in online supplementary datasds techniquethe expressions of igg1 igg2 igg3 and igg4 in esophageal cancer were detected at the histological level the stain decolorize stain sds technology22 was performed on the same slide sequentially with four different antibodies to demonstrate the four antigens the distribution pattern abundance and relationship of the four antigens were processed with an image software photoshop to achieve an integrated figure the proximity of different cell types on the tissue sections was measured with an image analyzing software image pro plus v60 details of the protocol are presented in the online supplementary dataimmunofluorescence double stainingtwo antibodies from different species were incubated on the same tissue section primary antibodies were detected with goat antimouse 555antirabbit igg or goat antimouse 488antirabbit igg alexa fluor life sciences and immunoreactivity was visualized with a fluorescence microscope antigens were detected and demonstrated with two fluorescence signals in red and green and the blue signal of ' diamidino2 phenylindole dapi as for cell nuclei images were acquired with the evos fl fluorescence microscope life technologies usaigg4 immunohistochemistryto verify that igg4 could react to igg1 that had been immobilized to tissue sections we used human pancreas and brain and antibodies to insulin glucagon and neurofilament as models detailed protocol of this experiment is shown in the online supplementary dataimmunoglobulin preparationsfab and fc fragments of igg igg1 and igg4 were prepared with papain digestion in the presence of cysteine iggs were cleaved at a position above the hinge region by papain at °c for hours after purification with protein a affinity chromatography pure fab and fc fragments were isolated with elution buffer igg fc fragment was washed down from protein a column after centrifugation and concentration measurement igg preparations were collected and stored at °c before usewestern blotigg subclasses were resolved on sodium dodecyl sulfate polyacrylamide gels under reducing conditions and then transferred onto nitrocellulose membranes ge healthcare life sciences the membranes were blocked for hour with bovine serum albumin bsa in tris buffered saline with tween20 ph and then incubated with primary antibody biotin labeling kit anaspec overnight at °c it was then incubated with secondary antibody for hour at room temperature finally reaction density was measured with odyssey western at nm and nm detection channelserum igg4 and igg assessmentsera samples collected from patients with esophageal cancer and healthy volunteers were sent to golden field medical test company guangzhou china and roche immune turbidimetry method was used to wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cmeasure serum igg4 and total igg concentrations all serum samples were stored at ˆ’°c freezer immediately before analysis all quantitative data were treated statisticallymeasurement of igg4 concentrations in tumor and tumoradjacent normal tissues with elisaconcentrations of igg4 in pairs of esophageal cancer and adjacent normal tissues cm from the edge of the cancer mass were measured with elisa detailed protocol of this experiment is shown in the online supplementary datacell culture for fc receptor binding assaysu937 cell line was bought from procell life science technology china cl0239 and cells were cultured in rpmi gibco c22400500bt supplemented with fetal bovine serum gibco “ and uml penicillin streptomycin gibco “ at ×106ml in ml cell culture bottleprotein preparationsdetails of the protocols for protein preparation separation and papain digestion are shown in online supplementary datafluorescence activating cell sorter facs for fc receptor assaysdetails of the relevant protocols are shown in online supplementary dataadcc adcp and cdc teststhe classic antibody dependent cell mediated cytotoxicity adcc antibody dependent cellular phagocytosis adcp and complement dependent cytotoxicity cdc tests were performed based on previously reported protocols23“ non specific igg4 instead of cancer specific igg4 was used to inhibit these reactions igg1 was used as control details of the protocols are shown in online supplementary dataanimal modelsbreast cancer and colon cancer modelsbalbc mice were obtained from vital river technical beijing china mice aged between and weeks and weighed ± g were used in all experiments all mice were inoculated with 4t1 mouse breast cancer cells or ct26 mouse colon cancer cells subcutaneously under the left forearm × 4t1 cells per mouse to build cancer models the mice were divided into different groups and were treated with igg1 or igg4 derived from intravenous igg ivig ivig without igg4 nivolumab and fc of nivolumab respectively details of the protocols are shown in online supplementary datacarcinogeninduced skin tumor modelwe employed a well established carcinogen induced skin tumor model to study the effect of igg4 and ivig without igg4 in comparison with controls phosphate buffered open accesssaline pbs detailed protocol is shown in online supplementary dataquantification and statistical analysisdata were analyzed in graphpad prism all reported p values were derived from two sided comparisons with values of less than considered to be statistically significantresultsigg4 was significantly increased in the serum of patients with cancer and this increase was related to cancer stage and patient prognosiswe first measured the concentrations of igg1 igg2 igg3 and igg4 in sera of patients with esophageal cancer n82 igg4 was significantly increased in patients with cancer in comparison with normal healthy subjects n70 the concentration of igg4 was increased from about to the ratio of igg4iggtotal was also significantly increased the statistical significance of both reached p00001 the increase of igg4 was also positively correlated to the stages of cancer with late stage cancers having more obvious increases higher igg4 serum concentrations were associated with worse prognosis figure 1a“digg4containing lymphocytes and igg4 concentration were significantly increased in cancer microenvironment and this increase was associated with cancer cell infiltrationigg4 positive lymphocytes were significantly increased in cancer microenvironment in comparison with tissue more distant to the cancer mass igg4 positive lymphocytes were barely detectable in tumor adjacent normal tissue figure 1h on the same tissue sections employing the sds technique22 to simultaneously visualize the four subtypes of igg with four distinct colors we found that one plasma cell only contained one igg subtype that is igg1 igg2 igg3 and igg4 were all contained in their own plasma cell populations separately figure 1i the marked increase of igg4 containing plasma cells in comparison with other subtypes was clearly visualized on cancer tissue sections igg4 positive plasma cells appeared to accumulate more in tissues with extensive cancer cell infiltration than in other areas figure 1eh in addition igg4 positive cells are often in close proximity to cells containing igg1 and igg2 but not to igg3 this phenomenon was not seen among other igg subtypes apart from igg4 quantitative data of the proximity of different cell types are presented in online supplementary figure s2 with the multiple immunostaining method we also demonstrated that igg1 containing and igg4 containing lymphocytes were distinct from cd3 positive t lymphocytes in the same region of the cancer tissue figure 1j in addition we measured the concentrations of igg4 in cancer tissue and cancer adjacent normal tissue n46 pairs the average concentration of igg4 in cancer tissue was about four times higher than that in the adjacent wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access figure significant increase of igg4 and igg4iggtotal in serum and igg4 positive b lymphocytes in esophageal cancer a igg4 in serum of esophageal squamous cell cancer escc n82 was significantly higher when compared with healthy controls n70 p00001 b igg4iggtotal ratio in escc n82 was significantly higher than that in matching healthy adults n70 p00001 c igg4 in stage …³ n18 was significantly higher than those in stages …° and …± n16 p001 d igg4iggtotal in serum of stage iv escc n18 was significantly higher than those in stages …° and …± n16 p005 e scatter diagram of igg4 positive cell numbers in cancer cancer adjacent normal tissue adjacent and normal tissues igg4 positive lymphocytes in and around the esophageal cancer mass n110 are significantly more abundant than those in the adjacent normal tissue n60 and normal lymphoid tissues n63 p0001 for both increases of igg4 positive lymphocytes were most abundant in areas of cancer cell proliferation f the increase of igg4 positive cell numbers was related to the prognoses of the patients more igg4 positive cells were associated with worse outcome p005 g igg4 concentration in cancer tissue n46 was significantly higher than that in adjacent normal tissue n46 p001 h immunohistochemistry of igg4 in esophageal cancer tissues from left to right are igg4 in cancer tissues cancer adjacent tissue and normal lymphoid tissue tonsil it clearly demonstrates that igg4 positive lymphocytes red were markedly increased left in comparison with normal lymphoid tissue right and with tumor adjacent normal tissue middle scale bar µm i demonstration of four subpopulations of igg containing plasma cells with multiple immunostaining sds method in cancer each subclass has its own distribution pattern and one plasma cell only produces one subclass of igg igg1 yellow igg2 green igg3 purple igg4 red j on the same tissue section a triple immunostaining was performed with the sds method to demonstrate the distribution and relationship among cd3 positive t cells yellow igg1 positive b cells greens and igg4 positive b cells red each cell type has its own distribution and no overlap between different cell types is observed sds stain decolorize stainwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cnormal tissue and the difference between these two groups was statistically significant p001 figure 1gigg1 extracted from patients with cancer reacted to cancer cells of the same patients but igg4 extracted from the same patients did notwe extracted igg1 and igg4 from the serum of patients with esophageal cancer with breast cancer and with colon cancer with respective specific antibody columns we then labeled the antibodies with biotin and tested the reactivity of these extracted antibodies to cancer tissue sections of the same patients in all cases igg1 reacted to cancer cells from the same patients but igg4 did not figure 2a it appeared that the increased igg4 in cancer microenvironment and in the patient™s serum was not reactive to cancer antigens while igg1 extracted from the same patients reacted to the cancer antigensigg4 reacted to cancerspecific igg1 that was bound to cancer cellsalthough igg4 extracted from patients with cancer did not react to cancer antigen it did react to cancer specific igg1 that was bound to cancer antigen on tissue sections as shown in figure 2b when cancer specific igg1 that was not labeled with biotin was applied to cancer tissue sections followed by biotin labeled igg4 the cancer cells became positive while when the same biotin labeled igg4 was applied to the same cancer tissue section without prior application of igg1 it did not react this reaction of igg4 to cancer specific igg1 was not via the antigen specific variable region of igg4 as such igg4 was neither specifically against igg1 nor was it specifically against cancer antigen with its antigen recognizing fab variable region as shown in figure 2a the only explanation was that igg4 reacted to igg1 through its fc region this was validated by subsequent experiments with western blot analysis as shown in figure 3a“ein western blot igg4 was found to react to other iggs igg1 igg2 igg3 and igg4 via an fcfc mechanism and this reaction was across species but igg4 did not react to other ig subtypes igm ige iga or igdto test if and how igg4 could react to igg1 we performed western blot with igg4 from different sources extracted from patients with cancer from normal adults and commercially purchased igg4 was found to react to igg1 igg2 igg3 and igg4 at the molecular weight mw of about kd and this reaction was not seen when igg1 igg2 or igg3 was used as the antibody and igg4 as the target molecule running on the gel the above phenomenon was observed in western blot of both reducing and non reducing conditions figure 3a online supplementary figure s3 however human igg4 did not react to human igm iga igd or ige online supplementary figure s4 nevertheless we found that this reaction was across species that is human igg4 reacted to iggs of human mouse rabbit and goat online supplementary figure s5 we open accessigg4 extracted from a patient with cancer reacted figure to cancer bound igg1 and blocked antibody mediated cancer immunity a upper panel these photos serve as an example of the reactivity of igg1 and igg4 extracted from patients with cancer igg1 from the serum of a patient with breast cancer was labeled with biotin and stained a frozen cancer tissue section of the same patient cancer cells were positively stained by igg1 left the cancer cells were confirmed by their characteristic histopathology of he staining middle igg4 from serum of the same patient labeled with biotin and applied on the same cancer on a consecutive section was completely negative right lower panel another breast cancer positively stained by igg1 from the patient™s serum left the cancer cells were identified by positive immunostaining of cytokeratin ck on a consecutive section middle igg4 from the same patient was not reactive to the same cancer on a consecutive section right b the upper panel illustrates the principle of the experimental reactions and the middle and lower panels show staining results from two patients with breast cancer left igg1 from a patient with cancer positively reacted to frozen cancer tissue of the same patient brown cells middle igg4 from the same patient with cancer applied to consecutive sections but did not react to the same cancer right however when unlabeled igg1 was applied to the same cancer tissue section followed by biotin labeled igg4 the cancer cells were positively stained brown cellsfurther digested human igg4 and igg1 into fab and fc fragments with papain it was found that it was the fc fragment of igg4 reacting to fc of igg1 figure 3b“e this reaction was easy to occur as only min incubation wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access igg4 reacted to igg1 in western blot and tissue figure sections in an fc fc fashion a in western blot non cancer specific igg4 from a patient with breast cancer reacted to igg1 and igg4 from the same patient with cancer right panel arrows however when igg1 and igg4 were run on the gel and biotin labeled igg1 was used as the primary antibody no band was seen left panel these are the same antibodies used in figure a02ab providing support to explain the reaction between igg4 and igg1 seen on cancer tissue b western blot demonstrated that igg4 reacted with igg1 igg2 igg3 and igg4 c igg4 reacted with igg fc fragment but not with fab arm d igg4 reacted with igg1 fc fragment but not with fab arm e biotin labeled igg4 fc fragment reacted to igg1 and igg4 fc fragments but not with igg1 or igg4 fab biotin labeled igg4 fab did not react to igg1 or igg4 fc or fab these results demonstrate that it is the fc region of igg4 that reacted to fc of igg1resulted in a clear band therefore the positive reaction obtained by sequential applications with cancer specific igg1 followed by non cancer specific igg4 on cancer tissue figure 2b had to take place between the fc of igg4 and the fc of igg1 as shown in figure the fcfc reaction between igg4 and igg1 bound to tissue sections was further tested and validated with a number of antibodies and tissue types apart from cancerfollowing the same logic we tested the reactivity between the fc fragments of igg4 and igg1 already demonstrated in western blot on tissue sections we used igg1 primary antibodies to insulin and glucagon in normal human pancreas and antibody to neurofilament in human brain for this test the same principle was established with these normal tissues detailed results and figures are shown in online supplementary figure s6igg4 competed with igg1 to bind to fc receptors of pbmc and macrophageswe performed immunoglobulin and fc receptor binding assays with peripheral blood monocytes pbmc and with a human monocyte cell line u937 procell life science technology china cl0239 igg1 and igg4 were extracted from the serum of patients with cancer and pbmcs were isolated from the same human subjects the extracted and purchased igg1 and igg4 were labeled with biotin or fitc in the igg1 and igg4 binding assay we found that igg1 and igg4 competed with one another in binding to pbmc and this reaction could be completely blocked by fc receptor blocker this competition was concentration dependent that is as the concentration of igg4 increased more igg1 bound to pbmc was replaced the reverse was also true that is igg1 could also replace igg4 in this competition assay this phenomenon was demonstrated on cytospin slides of pbmc preparation online supplementary figure s7in addition flow cytometry was performed to examine the binding properties of igg1 and igg4 to fc receptors of monocytes the ability of igg1 and igg4 to bind to all three subtypes of fc gamma receptor fcγr”fcγr…° cd64 fcγr…± cd32 and fcγr…² cd16”was examined with corresponding antibodies we found that igg4 could compete with igg1 in binding to the fc receptor of monocytes u937 we further found that the binding force of igg1 was about twice as strong as that of igg4 for individual receptor subtypes igg1 could bind to all three receptor subtypes that is fcγr…° cd64 fcγr…± cd32 fcγr…² cd16 in contrast igg4 could only bind to fcγr…° cd64 although their binding sites were different igg4 could completely block igg1 we also found that it was necessary for a relatively high concentration of igg4 to be present in the solution in order to compete with igg1 in binding to fc receptors online supplementary figure s8igg4 inhibited the classic immune reactions of adcc adcp and cdc mediated via cancerspecific igg1 and effector immune cellscomplementswe first verified that non cancer specific igg4 indeed reacted to igg1 cetuximab used in adcc adcp and cdc figure 4a we then found that igg4 inhibited adcc elicited cytotoxicity with cancer specific igg1 antibody and pbmc figure 4b the igg4 used in our test was not directed against cancer antigen or to lymphocytes non cancer specific igg1 could also inhibit adcc but to a much lesser extent also reached statistical significance we obtained evidence to show that inhibition of adcc appeared to take place at the site of the cancer specific antibody that is igg4 reacted to the igg1 antibody bound to cancer cells figure 2b and the site of immune effector fc receptors the latter effect could wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen accessfigure non cancer specific igg4 inhibited classic adcc adcp and cdc reactions against cancer but had no direct effect on cancer cell growth a on western blot the chimeric antibody cetuximab igg1 against egfr was run on the gel and igg4 and igg1 at concentrations of and µgml were used as the primary antibodies igg4 reacted to cetuximab at a concentration dependent manner but igg1 did not react b left in a classic adcc experiment cetuximab igg1 was incubated with an egfr expressing lung cancer cell line a549 and then with pbmc from normal healthy adult cancer cell activity was significantly reduced n12 non cancer specific igg1 and hsa were used as controls showing that they had no direct effect on the cancer cells n12 middle when non cancer specific igg4 was added to the mixture the effect of cetuximab was significantly reversed demonstrating an inhibitory effect of igg4 in adcc n12 non cancer specific igg1 had a much smaller but also significant effect in inhibiting adcc action n12 right when fc receptor blocker was incubated with pbmc the effect of cytotoxicity was blocked c“e adcp was performed with a lung cancer cell line a549 expressing egfr as the targets human peripheral monocyte derived macrophages as the effector cells and the antibody cetuximab igg1 against egfr as the mediating antibody the tumor cells were stained with cfda se fluorescence probes green macrophages derived from pbmc were stained with dii fluorescent probes orange blue fluorescence is the nuclei stained with dapi d higher magnification of c the orange colored macrophages were in close contact with green tumor cells tumor debris ingested by macrophages appeared yellow in the cytoplasm of macrophages e bar chart showing the effect of adcp and its inhibition by igg4 f left in µgml rituximab mediated adcp model giemsa staining results of phagocytosis of raji cells by macrophages after the addition of µgml igg1 and igg4 respectively right igg4 significantly inhibited rituximab mediated adcp in phagocytosis by macrophage but igg1 could not inhibit the adcp effect scale bar30 µm p005 p001 p0001 g in a classic cdc experiment cetuximab anti egfr antibody was incubated with an egfr expressing lung cancer cell line a549 and then with complement co from serum of a normal healthy adult the cancer cell activity was significantly reduced h when non cancer specific igg4 was added to the mixture in the above cdc experiment the effect of cetuximab was significantly reversed i igg4 and igg1 were incubated with kyse150 for hours and no effect on cell growth was found adcc antibody dependent cell mediated cytotoxicity adcp antibody dependent cellular phagocytosis cdc complement dependent cytotoxicity cfse da carboxyfluorescein diacetate succinimidyl ester dapi ' diamidino2 phenylindole egfr epidermal growth factor receptor hsa human serum albumin pbmc peripheral blood mononuclear cellwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access be abolished with the addition of fc receptor blocker human trustain fcx biolegend china to the pbmcwe also performed an adcp experiment employing human monocyte derived macrophages and esophageal cancer cells cetuximab igg1 was used as the mediating antibody this was performed employing a coculture and cell counting method and fitc labeled antibody flow cytometry two models were employed and both methods showed that non cancer specific igg4 was able to reduce the effect of adcp mediated by cancer specific igg1 figure 4c“fin a classic cdc assay we used cancer cell line a549 atcc usa c4215 as the target cancer cells cetuximab as the cancer specific igg1 mediating antibody and human plasma as the source of complements and demonstrated the destructive effect on cancer cells we then used non cancer specific igg4 or igg1 to inhibit the effect we found that the cdc effect was partially inhibited by non cancer specific igg4 but not by igg1 figure 4ghfor comparison we added igg4 or igg1 at various concentrations in cancer cell culture for different periods of time and found no direct effect of these proteins on cancer cell growth figure 4iigg4 including nivolumab significantly accelerated breast cancer cell and colon cancer cell growth in two immune potent mouse models in vivothe above results point to a mechanism that igg4 plays an important role in local immune evasion by blocking immune responses mediated by cancer specific igg antibodies to further examine this mechanism mediated by such antibodies we performed in vivo studies to verify this hypothesis with immune competent mouse models in one model we injected non cancer specific igg4 into a location where breast cancer cells were inoculated subcutaneously in this group of mice cancer cell growth was significantly increased resulting in a much larger cancer mass by days in comparison with other groups injections of pbs or igg without igg4 figure 5ab as there is no direct effect of igg4 on cancer cell growth figure 4i these results unequivocally confirmed that igg4 can inhibit local immune reaction and thereby promote cancer growth in vivo through immune evasionin a separate but similar experiment of a colon cancer mouse model we injected antibody to programmed cell death1 pd1 nivolumab which is a widely used drug in cancer immune therapy and is also an igg4 isotype with s228p mutation which replaces a serine residu
Colon_Cancer
" trophoblast cell surface antigen trop2 is overexpressed in many squamous cell carcinomas andpromotes tumor development and invasion the association between trop2 expression and occurrence anddevelopment of oral squamous cell carcinoma oscc remains to be understoodmethods we investigated the role of trop2 in oscc patients using a combination of biophysical approaches atotal of oscc patient specimens with varying degrees of differentiation were subjected to hematoxylin andeosin staining immunohistochemistry kaplanmeier survival curve analysis and atomic force microscopy to analyzetrop2 expression morphology and mechanical properties of oscc tissuesresults trop2 was overexpressed in of poorly differentiated oscc samples high levels of trop2 wereassociated with survival rate lower than and patient age odds ratio [or] p confidence interval [ci “] tumor size or p ci [“] and tnm stageor p ci [“] average surface roughness of low medium and highly differentiatedoscc tissues were ± ± and ± nm respectively the pearson coefficient revealed anegative association between tumor stiffness and trop2 expression r ˆ’ p overexpression of trop2 negatively associated with patient survival degree of tumor differentiationand tissue mechanics taken together our findings demonstrated that trop2 may be an indicator of osccdifferentiation leading to the altered mechanical properties of oscc tissueskeywords oral squamous cell carcinoma trop2 tissue stiffness differentiation survival oral squamous cell carcinoma oscc is a commonsubtype of head and neck and other malignant tumors[ ] the past few decades have shown increased incidence of oscc that is expected to rise further in the future thereforeimperative to determineisit correspondence zhangkllzu163com baoping zhang shuting gao and ruiping li contributed equally to thiswork1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu chinafull list of author information is available at the end of the biological factors associated with the early diagnosis andtreatment of oscchuman trophoblast cell surface antigen trop2 alsocalled tumorassociated calcium signaltransduction2tacstd2 is a surface glycoprotein encoded by tacstd that has extracellular domains a single transmembrane domain and a short tail [ ] trop2 is overexpressed in many human cancers including ovarian [ ]gastric [ ] colorectal pancreatic and laryngealcancers inhibiting trop2 expression has shownpromise in clinical applications [ ] trop2 regulates the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang bmc cancer page of tumorigenic properties including cancer cell adhesion invasion and migration tang have recentlyshown that trop2 impacts growth and metastasis byactivating pi3kakt signaling this phenomenon hasalso been observed in gallbladder cancer amongtheinvolved intumorigenesis the role of catenin has been studiedextensively [ “] this has shed light on the biological functions of trop2 and its use as a prognostic biomarker for osccvarious biochemical mechanismsatomic force microscopy afm is a powerful toolthat generates surface topographical images with magnifications that range between macro and nanoscalesafm has been used to determine the mechanical properties of tumor tissues in a variety of cancers such asthose of the breast liver and lung parameters for tissue stress such as mechanical phenotypeindex correlate with cancer development and invasion advancements in technology used for determiningbiophysical properties have facilitated the nanolevelanalysis of tumor tissuesthis study aims at investigating the correlation between trop2 expression and clinicopathological characteristics of oscc we have demonstrated the tissuemorphology and mechanics of oscc samples duringtumor development using afm we believe our findingswill help develop trop2 in accurately diagnosing osccin tumors with different grades of differentiationmethodstissue preparationthe protocols in this study were approved by the researchethics committee of lanzhou university tumor sampleswere collected from patients after obtaining written informed consent a total of patients with oral squamous cell carcinoma oscc were registered atthesecond hospital of lanzhou university between january and march among these samples sampleseach showed high moderate and low levels of differentiation the experimental group comprised males and females aged “ years average years all patientswere diagnosed with oscc based on surgery andfig paraffin pathological sections of tissues a d g — 4fold b e h — 10fold c f i — 40fold 0czhang bmc cancer page of fig immunohistochemical staining was performed to detect the expression of trop2 at different stages of osccpathology patients did not undergo radiotherapy chemotherapy or immunotherapy before surgery pathologicalanalysis after tumor biopsy was performed by two experienced pathologists after which the diagnosis of other diseases including inflammation at other sites and secondarytumors were excluded cancer and cervical lymph nodetissues were collected after maxillofacial surgery all specimens were sampled from typical areas of the lesion andfixed with neutralbuffered formalin followed by conventional paraffin embedding among them and fig average optical density of trop2 poorly differentiated squamous cell carcinoma showed high expressionp 0czhang bmc cancer page of table correlation between trop2 expression and clinicopathological characteristicscharactersntrop2 expressionlow or nototalgendermalefemaleage‰¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 ‰ cm cmt2 ‰ cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvascular invasionnoyespearson x2p value highpatients exhibited no and cervical lymph node metastasesrespectively clinical tnm staging was performed according to the 7th edition tnm staging classification standardjointly developed by the international union for cancercontrol and american joint committee on cancer and world health anization guidelines hematoxylin and eosin he stainingoscc tissues were fixed overnight using neutralformalin solarbio beijing china paraffin embeddedsliced into 4μm thick sections dewaxed using xyleneand rehydrated using different concentrations of ethanol the sections were stained with hematoxylin for min and hydrochloric acidethanol and eosin for min followed by gradient dehydration transparentizationresin sealing solarbiobeijing china sections were visualized and imagedusing the olympus bx53 at magnifications of — and sealing and neutral 0czhang bmc cancer page of immunohistochemistryhe sections were subjected to the sp method to detecttrop2 expression the sections were incubated overnight with the primary antibody against trop2 abcam usa at °c followed by incubation withbiotinlabeled goat antirabbitigg abcamusa at °c for h the sections were then developed using dab beijing zhongshan golden bridgebiotechnology china dehydrated transparentizationand film and neutral resin sealed the prepared sections were visualized using microscopy olympusbx53 japanafmfixed tissues were placed in petri dishes containingphosphatebuffered saline all analyses for mechanical properties were performed using the biologicalatomic force microscope bioafm nanowizard iiibruker usa silicon afm probes from the pointprobe®constant of nmcoating nanoworld usa werecontrreflexused the spring constant ofthe probe was calibrated using builtin thermal vibration before measuringandthickness of μm afm was performed using theseries with a khzforcetheresonancefrequencyofcontact model and a scan rate of hzs in airforcedistance curves are generated when the probecontacts the tissue following whichthe structuremorphology and mechanical properties of samplesare measured at μms six random sites wereselected for each sample and each site was measured times we used the modified hertz contact modelto analyze forcedistance curves and calculateyoung™s modulus and roughness of oscc tissueswith varying differentiationstatistical analysisstatistical analyses were performed using spss statistical product and service solutions ibm forcespectrum data were expressed as mean ± standard errorand statistical comparisons were performed using oneway analysis of variance followed by the tukeykramerhsd test for pairwise comparisons pearson chisquaretest was used to analyze clinical features and trop2 expression based on the calculated odds ratios ors and confidence intervalci survival was evaluatedusing kaplanmeier curves and the difference was analyzed using the logrank test p was consideredstatistically significantfig trop2 total survival curve using kaplanmeier survival curves low blue line high green line 0czhang bmc cancer page of resultstissue morphology and trop2 expression across theclinical stages of oscctumor cells from poorly differentiated oscc samples exhibited characteristic atypia poor differentiation and irregular morphology fig howeverthe number volume atypia nuclear pyknosis andmitotic structures decreased in tumor cellsfromhighly differentiated oscc as compared to those inpoorly differentiated cells trop2 primarily localizedin the cytoplasm of tumor cells but not in adjacentnormal epithelial cells we observed that low differentiation and high malignancy of oscc was associated with higher trop2 expression fig theaverage optical density of trop2 among the lowmedium and highly differentiated oscc tissues were ± ± and ± respectively fig table trop2 expression risk factors with clinicopathological featurescharacteristicsntrop2 expressionlow or nototalgendermalefemaleage‰¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 ‰ cm cmt2 ‰ cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvscular invasionnoyesnote a well vs moderate b moderate vs poor c well vs poorp valueor cihigh 005a 0001b 0001c 0czhang bmc cancer page of association between trop2 expression and clinicalcharacteristics of osccwe analyzed the clinicopathological characteristics of patients with oscc with varying degree of differentiationdifferential expression of trop2 was associated with patient age tumor differentiation tumor size tnm stagepercutaneous nerveinvasiontable p patients with poorly differentiated tumors were more likely than patients with well and moderate differentiated tumors to have high trop2 expressionp however there was no association between theexpression of trop2 and patient gender tumor locationlymph node metastasis or distant metastases p and vascularfiltrationtrop2 expression and patient survivalusing kaplanmeier survival curves we observed that anincrease in trop2 expression negatively correlated withthe overall survival of patients fig and lowno oftrop2 expression group™s 3years survival rate was a for high expression group and 5years ratewere and respectively trop2 expression wasassociated with patient age p or ci[“] tumor differentiation well vs moderatep or ci [“] moderate vspoor p or ci [“]well vs poor p or ci [“] tumor size p or ci[“] tnm stage p or ci[“] vascular invasion p or ci [“] and peripheral nerve invasionp or table high trop2 expressionwas detected in older patients with low degree of differentiation larger tumor volume higher tnm staging andvascular and peripheral nerve invasion thereby resultingin lower overall survival thus trop2 may be a prognostic indicator for survival in oscc patientsfig surface morphology of oscc tissue sections via afm detection irregular morphology appeared in the low differentiation 0czhang bmc cancer page of surface morphology and roughness of oscc tissuesthe surface morphologies of oscc tissues with varying degrees of differentiation were analyzed directtopographical imaging using bioafm figure showsthe representative image from each tissue acquiredduring the cantileverbased afm nano indentationtest the tissue interface varied with tumor differentiationindicating that highly differentiated oscc tissues had a regular and flat morphology oscc tissueswith low differentiation exhibited an overall irregularmorphology with distinct modulation and loose tissueanization figure summarizes the roughness ofoscc tissues with varying differentiation the average surface roughness of low medium and highly differentiated oscc tissues were ± ± and ± nm respectively roughness ofthe tissue surface was enhanced with increasing differentiation of oscc tissuesyoung™s modulus of oscc tissueswe used bioafm to determine young™s modulusbased on the mechanical properties of oscc tissues with varying degrees of differentiation we randomly selected six contact points from each slice andeach contact point was measured times forcedistance curves were generated for each slice and thejpk data processing software version was usedto calculate young™s modulus figure shows theaverage variation in stiffness within individual tissuesin the range of “ kpa in the low differentiationsamples we observed low stiffness as compared tothat in high or medium differentiation samples p fig surface roughness results are express as mean ± sem nm 0czhang bmc cancer page of fig afm test average tissue stiffness young™s modulus e was thus confirmed to be a parameter of cell hardness for various cells and tissuepa p thus tissue differentiation was positively associated with its stiffness fig association between mechanical properties and trop2expression in osccthe pearson coefficient showed a negative associationbetween the stiffness of oscc tissues and trop2 expression fig r ˆ’ p thus we detectedan increase in stiffness with varying differentiation in thetumor samplesdiscussiontrop2 belongs to the family of genes involved in calcium signaling associated with tumorigenesis and foundin human trophoblast and chorionic cell lines studieshave shown that overexpression of trop2 is associatedwith tumorigenesis and malignancy [“]in thisstudy trop2 expression was observed to be a highlysensitive and specific marker of tongue squamous cellcarcinoma and tissue stiffness the relative thickness ofsamples helped accurately diagnose and determine thestaging of tongue squamous cell carcinomaimmunohistochemical analysis revealed that the expression of trop2 in poorly differentiated oscc tissueswas significantly higher than that in welldifferentiatedoscc tissues additionally trop2 upregulation wascorrelated with tumors of advanced tnm iii iv staging and poor differentiation than that in tumors withlow tnm i ii staging thus the abnormal expressionof trop2 may be associated with the occurrence anddevelopment of tongue malignancies furthermore hightrop2 expression predicted low survival as comparedto that in the tumors with low trop2 expression previous research has also demonstrated the correlation between shorter patient lifespan and high levels of trop2as compared to that in patients with laryngeal squamouscell carcinoma and low levels of trop2 trop2possesses sites for tyrosineserine phosphorylation thatregulate signal transduction or its expression and activity thereby rendering cancer cells resistant to apoptosis upregulated trop2 correlates with the poor prognosis of thyroid papillary carcinoma colon cancer liver cancer and other malignanciesthere have been an increasing number of studies on thebiological role of trop2 at the molecular level trop2induces the downregulationloss of pten thereby stimulating pi3kakt signaling and tumor development pten is a wellknown tumor suppressor that is a phosphatase and affects the pi3kpkbakt signaling axisduring the dephosphorylation of pip2 and pip3 pi3k signaling is important in regulating tumor cell proliferation migration and invasion [ ] thus pten is anegative regulator of cancer [ ] li have shownthat trop2 activates epithelialmesenchymal transitionvia pi3kakt signaling thereby promoting proliferationmigration and metastasis in gallbladder cancer similarly trop2 expression stimulates the proliferation migration and invasion of osteosarcoma cells hou demonstrated that trop2 regulates jak2stat3 signaling in glioblastoma cells 0czhang bmc cancer page of fig correlation analysis between changes in mechanical stiffness of oscc tissues and trop2 expression note changes have statisticalsignificance p and show a certain negative correlation r ˆ’ functional differentiation oftissues influences themicromorphology and mechanical stiffness of oscccells we detected low surface roughness on oscc tissues with loose structure reduced hardness and enhanced cell adhesion migration and invasion poorlydifferentiated oscc tissues are œsofter than highly differentiated oscc tissues pi3k is an important celladhesion molecule trop2 triggers the synthesis of proteins with homologous domains such as pleckstrinrac tiam and vav tiam and vav activate rac thatleads to reanization of the actin cytoskeleton cellrecognition and adhesion the underlying mechanisms involved in the alterationof micromechanical properties of oscc samples and occurrence development metastasis and invasion ofoscc tumors remain to be elucidated he staining isthe gold standard for tumor diagnosis with the development of biomechanics in the past two decades the mechanical properties of tissues need to be investigated based on biomedical and physical parametersin this study we have assayed the changes in mechanicalproperties at the micronanometer level using afm anddetermined the association between the tnm grademetastasis and stiffness of tumor samplesin we have demonstrated the association between differential expression of trop2 and patient agetumor differentiation tumor size tnm stage percutaneousnerve filtration and vascular invasion moreover high levelsof trop2 correlated with poor overall survival in patientshighly differentiated cancer tissues exhibited increasedsurface roughness and stiffness lastly high trop2 expression resulted in reduced tumor stiffness however thisstudy had some limitations first the cohort used in thisstudy was relatively small second we did not employ molecular methods of analysis such as western blotting orenzymelinked immunosorbent assay thus using a largerpatient cohort and multiple techniques in molecular andcell biology will help validate our findings and developtrop2 as a specific and efficient prognostic biomarker forosccthese findings could promote new methods for the earlyoscc diagnosis depend on the stage of cancer and developing screening methods with high sensitivity andspecificity more detailed studies are needed to determine the feasibility and therapeutic benefit of testing tissue stiffness in human diseaseabbreviationsoscc oral squamous cell carcinoma trop2 trophoblast cell surfaceantigen afm atomic force microscopyacknowledgementswe thank the individual who participated in this studyauthors™ contributionsbz sg and rpl are responsible for conception and design data wascollected by ytl rc jyc and ymg data was analyzed by ew and yh klzrevised the all authors have read and approved the manuscriptfundingthis work was supported by the fundamental research funds for thecentral universities no lzujbky2020cd03 baoping zhang doctoralmaster 0czhang bmc cancer page of students of the second hospital of lanzhou university sdkygg17 lan yangand key laboratory of mechanics on disaster and environment in westernchina the ministry of education of china no “ kailiang zhangavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatewritten informed consent was obtained from each participant before samplecollection the study was approved by the committee for ethical affairs ofschool of stomatology lanzhou universityconsent for publicationnot applicablecompeting intereststhe authors have no conflicts of interestauthor details1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu china 2institute of biomechanics andmedical engineering lanzhou university lanzhou chinareceived april accepted august referencesiyer s thankappan k balasubramanian d early detection of oral cancerscurrent status and future prospects curr opin otolaryngol head neck surg“caldeira pc soto aml de aguiar mcf martins cc tumor depth of invasionand prognosis of earlystage oral squamous cell carcinoma a metaanalysisoral dis online ahead of printkim y kim jh increasing incidence and improving survival of oral tonguesquamous cell carcinoma sci rep mcdougall ar tolcos m hooper sb cole tj wallace mj wallace trop2from development to disease dev dyn “guan gf zhang dj wen lj yu dj zhao y zhu l prognostic value oftrop2 in human nasopharyngeal carcinoma int j clin exp pathol “stewart d cristea m antibodydrug conjugates for ovarian cancer currentclinical development curr opin obstet gynecol “liu j yang d yin z gao m tong h su y a novel human monoclonaltrop2igg antibody inhibits ovarian cancer growth in vitro and in vivobiochem biophys res commun “zhao w jia l kuai x tang q huang x yang t the role andmolecular mechanism of trop2 induced epithelialmesenchymal transitionthrough mediated betacatenin in gastric cancer cancer med “zhao w jia l zhang m huang x qian p tang q the killing effect ofnovel bispecific trop2pdl1 cart cell targeted gastric cancer am jcancer res “jordheim lp chettab k crosperrial e matera el dumontet c unexpectedgrowthpromoting effect of oxaliplatin in excision repair crosscomplementation group transfected human colon cancer cellspharmacology ““ nishimura t mitsunaga m sawada r saruta m kobayashi h matsumoto n photoimmunotherapy targeting biliarypancreatic cancer withhumanized antitrop2 antibody cancer med “ wang xd wang q chen xl huang jf yin y da p trop2 inhibitionsuppresses the proliferation and invasion of laryngeal carcinoma cells viathe extracellular signalregulated kinasemitogenactivated protein kinasepathway mol med rep “ wanger tm dewitt s collins a maitland nj poghosyan z knauper vdifferential regulation of trop2 release by pkc isoforms through vesiclesand adam17 cell signal “tang g tang q jia l chen y lin l kuai x trop2 increasesgrowth and metastasis of human oral squamous cell carcinomathrough activation of the pi3kakt signaling pathway int j mol med“trerotola m li j alberti s languino lr trop2 inhibits prostate cancer celladhesion to fibronectin through the 1 integrinrack1 axis j cell physiol“li t su y yu x mouniir dsa masau jf wei x trop2 guaranteescardioprotective effects of cortical bonederived stem cells on myocardialischemiareperfusion injury cell transplant “stoyanova t goldstein as cai h drake jm huang j witte on regulatedproteolysis of trop2 drives epithelial hyperplasia and stem cell selfrenewalvia betacatenin signaling genes dev “sun x xing g zhang c lu k wang y he x knockdown of trop2 inhibitsproliferation and migration and induces apoptosis of endometrial cancercells via aktcatenin pathway cell biochem funct lee h jang y seo j nam jm char k nanopfunctionalized polymerplatform for controlling metastatic cancer cell adhesion shape and motilityacs nano “kruse sa smith ja lawrence aj dresner ma manduca a greenleaf jf tissue characterization using magnetic resonance elastographypreliminary results phys med biol “kaneko ts pejcic mr tehranzadeh j keyak jh relationships betweenmaterial properties and ct scan data of cortical bone with and withoutmetastatic lesions med eng phys “ goetz jg minguet s navarrolerida i lazcano jj samaniego r calvo e biomechanical remodeling of the microenvironment by stromalcaveolin1 favors tumor invasion and metastasis cell “edge sb compton cc compton the american joint committee on cancerthe 7th edition of the ajcc cancer staging manual and the future of tnmann surg oncol “ barnes l eveson jw reichart p sidransky d pathology genetics headand neck tumours lyon barness p “ zhang b li l li z liu y zhang h wang j carbon ionirradiated hepatomacells exhibit coupling interplay between apoptotic signaling andmorphological and mechanical remodeling sci rep yan jf huang gy a doublehertz model for adhesive contact betweencylinders under inclined forces philos trans a math phys eng sci kowalsky ca faber ms nath a dann he kelly vw liu l rapid fineconformational epitope mapping using comprehensive mutagenesis anddeep sequencing j biol chem “ zeng p chen mb zhou ln tang m liu cy lu ph impact of trop2expression on prognosis in solid tumors a systematic review and metaanalysis sci rep calvo a xiao n kang j best cj leiva i emmertbuck mr alterationsin gene expression profiles during prostate cancer progression functionalcorrelations to tumorigenicity and downregulation of selenoproteinp inmouse and human tumors cancer res “ju x jiao x ertel a casimiro mc di sante g deng s vsrc oncogeneinduces trop2 proteolytic activation via cyclin d1 cancer res “ cubas r li m chen c yao q trop2 a possible therapeutic target for latestage epithelial carcinomas biochim biophys acta “ zargari n mokhtari m evaluation of diagnostic utility ofimmunohistochemistry markers of trop2 and hbme1 in the diagnosis ofthyroid carcinoma eur thyroid j “ zhao p zhang z tnfα promotes colon cancer cell migration and invasionby upregulating trop2 oncol lett “sin stk li y liu m yuan yf ma s guan xy downregulation of trop2predicts poor prognosis of hepatocellular carcinoma patients hepatolcommun “ zhang y zhang r luo g ai k long noncoding rna snhg1 promotes cellproliferation through pi3kakt signaling pathway in pancreatic ductaladenocarcinoma j cancer “sai j owens p novitskiy sv hawkins oe vilgelm ae yang j pi3kinhibition reduces mammary tumor growth and facilitates antitumor immunityand antipd1 responses clin cancer res “ chen x pang b liang y xu sc xin t fan ht overexpression of zhang xr wang sy sun w wei c isoliquiritigenin inhibits proliferation andepcam and trop2 in pituitary adenomas int j clin exp pathol “metastasis of mkn28 gastric cancer cells by suppressing the pi3kaktmtor signaling pathway mol med rep “ 0czhang bmc cancer page of wise hm hermida ma leslie nr prostate cancer pi3k pten and prognosisclin sci lond “ yuan b zou m zhao y zhang k sun y peng x upregulation of mir130b3p activates the ptenpi3kaktnfκb pathway to defense againstmycoplasma gallisepticum hs strain infection of chicken int j mol sci pii e2172li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep “li x teng s zhang y zhang w zhang x xu k trop2 promotesproliferation migration and metastasis of gallbladder cancer cells byregulating pi3kakt pathway and inducing emt oncotarget “ gu qz nijiati a gao x tao kl li cd fan xp trop2 promotes cellproliferation and migration in osteosarcoma through pi3kakt signalingmol med rep “ hou j lv a deng q zhang g hu x cui h trop2 promotes theproliferation and metastasis of glioblastoma cells by activating the jak2stat3 signaling pathway oncol rep “ rivard n phosphatidylinositol 3kinase a key regulator in adherens junctionformation and function front biosci landmark ed “ pankova d jiang y chatzifrangkeskou m vendrell i buzzelli j ryan a rassf1a controls tissue stiffness and cancer stemlike cells in lungadenocarcinoma embo j 20193813e100532 wullkopf l west av leijnse n cox tr madsen cd oddershede lb cancer cells' ability to mechanically adjust to extracellular matrix stiffnesscorrelates with their invasive potential mol biol cell “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
Colon_Cancer
"introduction a diet low in fermentable oligosaccharides disaccharides monosaccharides and polyols fodmap is an effective way to reduce gut symptoms in people with irritable bowel syndrome ibs this diet reduces the intake of fermentable fibres leading to changes of the gut microbiota and insufficient fermentation in the large bowel resulting in reduced production of short chain fatty acids scfas such as butyrate which has unfavourable implications for gut health sleep and mental health this study will examine the effect of fibre fix a supplement containing a mix of dietary fibres on the human gut microbiome composition fermentative capacity sleep quality of life qol and mental health of people with ibs who consume a low fodmap diet lfdmethods and analysis a randomised double blind placebo controlled study design is proposed to examine whether fibre fix added to an existing lfd may help modulate gastrointestinal function improve markers of sleep mental health and promote qol in patients with ibs participants will provide stool and blood samples daily bowel symptoms diaries and day diet records additionally they will complete validated questionnaires relating to fodmap intake sleep mental health and qol before and after a week intervention gut health will be assessed via faecal microbiome composition faecal ph and scfa levels alteration of sleep will be recorded using an actigraphy device worn by all participants over the whole study multivariate analysis will be used to examine the gut microbiome and repeated measures analysis of variance anova will be used for dependent variables from questionnaires related to bowel symptoms stool type sleep mental health and qol to assess the differences between intervention and control groups after adjustment for confounding variablesethics and dissemination ethics approval was obtained from the human research ethics committee of edith cowan university yan results will be disseminated in peer review publications and conference presentations participants will be provided with a summary of findings once the study is completed if fibre fix is shown to result in favourable changes in gut microbial composition scfa production sleep and mental well being without exacerbating symptoms this will provide additional dietary management options for those with ibs following an lfdtrial registration number actrn12620000032954introductionirritable bowel syndrome ibs is one of the most frequently diagnosed functional gastrointestinal disorders affecting approximately of the global adult population1 diagnosis of ibs is challenging due to the subjective nature of digestive symptoms and is currently based on the rome iv criteria3 this functional disorder typically presents with recurrent abdominal pain with alterations in bowel habits namely stool consistency and frequency coexisting bloating flatulence and abdominal distention the syndrome is subtyped into four patterns ibs with predominant constipation or predominant diarrhoea mixed ibs and un subtyped4 due to the dominance of chronic symptoms and frequently present comorbidities somatic and psychological ibs imposes a heavy burden on individuals and communities both economically and socially5 of those employed patients with ibs reported absenteeism and presentism due to their syndrome7 in two independent studies it were estimated that yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0copen access ibs related absenteeism and presenteeism cost industry £“£ or to ‚¬“‚¬ annually9a diet that is low in fermentable oligosaccharides disaccharides monosaccharides and polyols fodmap10 alias a low fodmap diet lfd is an effective dietary intervention for ibs a blinded and placebo controlled trial found that approximately three quarters of patients with ibs benefit from an lf11 the lfd reduces food fibre compounds that are poorly absorbed in the small intestine rapidly fermentable in the proximal colon and thereby contribute to the gastroenterological symptoms the lfd includes a selective elimination diet for “ weeks followed by a reintroduction phase of fodmap containing foods followed by personalisation of a diet that minimises symptoms12 despite the positive effects of the lfd in reducing gut symptoms and improving quality of life qol in those with ibs it only treats the symptoms of ibs studies suggest potentially negative impacts of long term adherence to an lfd including nutritional inadequacy potential increased risk of gastrointestinal complications and imbalance of the gastrointestinal microbiome12 evidence from both animal and human studies has demonstrated that a low intake of dietary fibres can reduce microbiota diversity leading to increased cancer risk16“ reintroduction or restoration of dietary fibres to an lfd diet can be difficult with whole food due to the coexistence of a range of fibres in individual foods this study therefore reintroduces dietary fibre using a supplement however this process should be done gradually and continuous otherwise unwanted symptoms such as gas flatulence and cramps may impact adherenceresearch suggests a low fodmap intake rapidly and negatively changes the gut microbial community abundance and diversity15 in healthy people a week lfd resulted in an alteration of the gut microbiota reduced beneficial bacteria such as actinobacteria predominantly bifidobacterium and a lower overall total bacterial count22 after a week lfd bennet 23 observed an increased dysbiosis manifested as altered gut microbial fermentation leading to lower total short chain fatty acid scfa concentrations24 in patients with ibs additionally a randomised cross over trial comparing lfd with a standard australian diet15 found a marked reduction in butyrateproducing clostridium cluster xiva and cluster iv favourable mucus associated akkerkmansia muciniphila and an increase in mucus detrimental ruminococcus torques in another study bifidobacterium and faecalibacterium prausnitzii associated with butyrate production were significantly decreased following a week lfd25 taken together these results suggest the lack of fibre associated with lfd may explain the microbial changes human gut microbiota is able to recognise and degrade different forms of complex carbohydrate26“ a diet rich in dietary fibres with different extents of fermentability and solubility is recommended which means more varied and complex dietary fibres in the diet leads to a more dynamic diverse and stable gut microbiota29 various purified dietary fibres are capable of nourishing specific gut bacteria such as bifidobacteria f prausnitzii and eubacterium hallii26 “ dietary fibre improves the human gut microbiota by providing a substrate for fermentation and subsequently increases production of scfa it is well established in the literature that higher levels of scfa can be obtained from a higher intake of fermentable dietary fibres19 butyrate one of the major scfa throughout the colon provides the primary fuel for colonic cells to maintain growth and integrity and thereby improve gut health35“ furthermore research suggests that butyrate can positively affect circadian rhythm regulation38 and enhance sleep via interplay between gut and brain40 therefore this study will increase the amount of fibre in the diet of patients with ibs to restore the gut microbiome and its metabolite profile to potentially prevent increasing the risk of patient™s developing other more severe gastrointestinal diseasesthirty three per cent of patients with ibs reported they had sleep problems such as sleep fragmentation poor sleep quality reduced sleep time and frequent awakening41 disordered sleep or sleep disturbances are also recorded with a greater prevalence in ibs sufferers compared with healthy individuals43 despite unknown causal relationship between impacted sleep and ibs the close association between gastrointestinal symptoms and sleep disturbances has been identified by others45“ the gut microbiota is suggested to play a pivotal role and affect multiple mechanisms in this complex relationship between human sleep disturbances and gastrointestinal disease48 smith 50 found that gut microbial diversity was positively associated with total sleep time as well as sleep efficiency which also were positively correlated with phyla richness of bacteroidetes and firmicutes their findings indicate that an diet intervention represent a promising way to improve sleep by manipulating the gut microbiota to promote sleep related phyla and taxa in the human gut microbiome50 in addition it has been suggested that gut microbial composition could be altered by sleep fragmentation resulting in a reduction in actinobacteria and in bacteroidetes but a increase in firmicutes which is similar to the microbial profile of obese individuals51 many research studies have demonstrated the significant association between ibs and mental health even though the causation relationship is still unclear a meta analysis of guillaume fond et al53 concluded that patients with ibs were more likely to develop depression and anxiety than healthy volunteers groups whereas sibelli 54 found that depression and anxiety doubled the risk of ibs onset it is estimated that of patients with ibs have associated mental health conditions such as depression and anxiety55 patients with ibs have been observed with gut microbial alterations related to depression including greater rates of kynurenine a deleterious metabolite of tryptophan an elevated kynureninetryptophan ratio56 and declined lactobacillus and bifidobacterium which are also less abundant in patients with major yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0cdepressive disorder57 clostridia a major class within the phylum firmicutes appears at increased abundance in patients with ibs58 this link to an animal study showing abundance of clostridia were significantly higher after stress related stimuli in stress vulnerable rats compared with stress resilient rats60 demonstrating that gut microbial communities respond to stress differently among animals with distinct stress vulnerability furthermore the researchers suggest a bacilli to clostridia ratio can reflect stress effects with a higher value indicating less stress derived inflammatory reactions60some inflammatory markers in human blood are associated with both human gut health and mental health and provide a potential mechanism for the role of dietary fibre in mitigating mental health a randomised controlled trial in patients with serious depression demonstrated serum concentration of high sensitivity c reactive protein hs crp and scores of beck depression inventory questionnaire significantly decreased after taking a probiotic supplement lactobacillus spp and bifidobacterium bifidum61 additionally proinflammatory cytokines like tumor necrosis factor alpha tnfα interleukin il6 and il1β are able to cross the blood brain barrier bbb and their entry and following influences can have a negative effect on mental health62 the entry of the cytokines however can be reduced by improving the integrity of blood brain barrier the permeability of bbb can be decreased by the scfa butyrate which is produced in the gut via gut bacterial fermentation of fermentable carbohydrate residue escaping from small intestinal digestion63in summary a healthier gut microbiota altered by a dietary fibre intervention or supplement in patients with ibs may not only improve gut health but also sleep mental health and qol the objective of this research study is to determine in patients with diagnosed ibs and on an lfd whether fibre fix compared with a placebo control improves gut microbial composition faecal scfa levels sleep quality qol markers inflammation and of mental well being without exacerbation of ibs symptoms this study will be the first to explore the bidirectional relationship between dietary fibres supplement and sleep modulated by the gut microbiota in patients with ibs following the lfdmethods and analysisstudy designthe study is designed as a randomised double blinded placebo controlled trial with a week intervention period figure the total time required for participant involvement is weeks including a week baseline and a week interventionsample sizethe a priori sample size for the proposed study was determined based on the results obtained by mcorist 64 where a sample size of participants per group open accessfigure flow chart showing study design overviewwas required to detect a change in log scfa concentration of at power and level of significance allowing for a drop out rate the total sample size of subjects per group is required this sample size is sufficient to detect at least a medium between within group interaction effect cohen™s f025 in sleep improvement at power and significance level whereby the corresponding sample size requirement is participantsfifty eight people n58 with ibs on an lfd will be recruited participants must be between and years old and have been clinically diagnosed with ibs using the rome iv edition diagnostic criteria65 by a gastroenterologist or other medical professional participants will be on an lfd for month prior to the intervention additionally participants will need to be available to attend the local clinic visits and be willing to consume the fibre fix supplement or matched placeboparticipants will be excluded if they are current smokers pregnant or planning to become pregnant have a known diagnosis of other gastrointestinal illness eg inflammatory bowel disease malabsorption of any macronutrients bowel resection coeliac disease have had previous abdominal or gastrointestinal surgeries severe mental health and sleep related conditions eg insomnia renal or hepatic diseases and major medical illness currently use pharmaceutical agents that could modify or treat ibs eg probiotics antibiotics eluxadoline lubiprostone and linaclotide or sleep conditions follow other restrictive dietary patterns or therapies eg low carbohydrate ketopaleo diet take any prebiotics yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0copen access have any other disease condition or habit that may interfere with completion of studyrecruitmentparticipants will be recruited through networks of registered western australian based dietitians and gastroenterologists who will be provided with information flyers to promote the study to potential participants information flyers will be posted on websites including social media groups relating to ibs or fodmap a university webpage will advertise study informationallocation blinding and compliancea computer generated list of random numbers provided by a statistician will be used to randomly assign participants to either the intervention or control group the participants will receive a resealable snap lock bag labelled a or b containing sachets of either fibre fix or placebo both participant and researcher will be blinded from the group allocation bag labelling will be completed by an independent person the participants will be required to return unused sachets to calculate compliance an online daily checklist together with a weekly textemail reminder will be provided to participants to record time of consuming the intervention a daily tick listcalendar will be created for participants to follow consumption of of the sachets sachets during the week period will be considered compliantinterventionfibre fix consists of one soluble dietary fibre and one insoluble fermentable fibre which will be provided to participants in separate sachets with gradually increasing amount table after baseline data collection participants will be required to consume fibre fix as per the labels on the sachets one sachet per day for the first days and two for the remaining days according to the schedule table for participants™ convenience all sachets have been labelled with day and time am or pm on the package for example day am and will be provided orderly in resealable plastic bags the placebo sachet contains a combination of the same soluble dietary fibre and highly digestible fibre and will be delivered in the same way as the interventionprimary outcomesfaecal scfa and gut microbiotafaecal scfa levels and gut microbiota will be assessed through hours stool samples which will be obtained at baseline and at the end of the intervention participants will be provided with the stool collection kit including a portable cooler bag frozen icepacks and an instruction sheet all stool samples collected with the hours period will be pooled and homogenised if the number of individual samples is more than one on receipt stool samples will be immediately weighed and stored at ˆ’°c individual™s samples will be thawed at °c and kept at this temperature during homogenised and aliquoting for all planned analysis and re frozen at ˆ’°c until analysesthe concentrations of bacterial metabolites in faeces such as scfa acetate propionate and butyrate will be determined by gas chromatography66 in brief an acidified aqueous methanol solution will be used to extract scfa from faecal samples followed by separating scfa by gas chromatography with a fatty acid column using a thermo scientific tg wax column30 m x mm x μm the scfa level will be qualified via internal standardsmicrobial analyses will be performed at the wa human microbiome collaboration centre curtin university western australia dna will be extracted using the qiaamp powerfecal dna kit qiagen using qiacube extraction platform microbiome signatures are generated using the illumina miseq platform using uniquely barcoded 16s rrna gene primers 515806v4 for bacterial and its2 primers for fungal profiling pending on funding following pcr inhibition assessment of each dna extract pcr free ligation protocol is thereafter deployed for the process of library building samples will be sequenced to a depth of minimum reads which is sufficient to identify microbes to a genusspecies level quality control and mock community samples are included in the analysis from sample collection to sequence analysis sequence read quality is initially assessed with fastqc before demultiplexing and preprocessing by ghapv2 an in house tool cutadapt67 is used for removal of all non biological sequences dada268 is then used for quality filtering error correction amplicon sequence variants asvs picking a trained naïve bayes classifier then assigns asvs to genusspecies against a curated database of microbial reference sequences such as the ribosomal database project rdp69 or genome taxonomy database70 for fungal classification the unite database71 will be usedsecondary outcomesobjective measures of sleepparticipants will be provided with the readiband v5 readiband fatigue science canada a wrist activity monitor that has been validated and objectively assesses sleep using accelerometery72 compared against polysomnography”the gold standard of sleep measurement the wrist activity monitor does not require laboratory setup table grams of dietary fibre supplement in each sachet provided to participants during week interventional perioddayam pm \\\\yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0ctable definitions of sleep measures as extracted from the readiband fatigue science canada device based on dunican 72sleep measuresabbreviated measurement descriptionacronym unitsopen accesstime at lights outtalotime of dayhhmmsleep onset latencysolminutesmintime at sleep onsettasotime of dayhhmmtime at sleep onset variancesleep durationtasovminutessdminutesminminwake after sleep onsetfragmentation indexwasominutesminfifrequencynumbertime at waketawtime of dayhhmmtime in bedsleep efficiencytibseminutesminpercentage deriveddirectly measuredderiveddirectly measuredderivedderiveddirectly measureddirectly measureddirectly measuredderivedtime at sleep onset minus sleep onset latencynumber of minutes from time at lights out to time at sleep onsettime of day when the first epoch of sleep occurs between time at lights out and time at waketime at sleep onset consistency relative to mean time at sleep onsetnumber of minutes from time at sleep onset to time at wake minus number of minutes awake wasonumber of minutes awake after time at sleep onsetnumber of awakenings between time at sleep onset and time at waketime of day when awake with no further sleep durationthe total time spent in bed from time at lights out to time at wakesleep duration divided by time in bed multiplied by nor trained personnel72 moreover the readiband can automatically identify time at lights out using a proprietary algorithm which not only eases the burden of sleep diary but also avoids the potential bias from self reported data of recalling time for bed72 this technology has been widely applied to the sleep related research73“ participants will be required to wear the monitor on the non dominant wrist for hours per day during the study the monitor derived sleep measure data will be downloaded via the automated readiband sync software table subjective measures of sleepparticipants will be required to complete five validated questionnaires related to sleep at baseline and at the end of the interventionpittsburgh sleep quality indexthe pittsburgh sleep quality index psqi retrospectively assesses sleep quality and relevant disturbances over the previous month this self administrated questionnaire has been validated in a population based setting to measure sleep quality the summary score is calculated as the summation of items grouped into seven components ranging from better to worse a score indicates poor sleep quality77epworth sleepiness scalethe epworth sleepiness scale ess is a self rated eight item questionnaire designed to measure daytime sleepiness78 participants score each question from high chance of dozing to would never doze which yield a global score of ess ranging from to scores higher than nine reflect excessive daytime sleepiness and severe problems with daytime somnolence79insomnia severity indexinsomnia will be assessed through the self reported insomnia severity index isi comprising seven items participants are required to rate each question on a point likert scale as per their own experience over the past weeks the total score ranges from to and represents clinical insomnia when it is higher than sleep hygiene indexthe sleep hygiene index shi is a self reported instrument for assessing individual behaviours in sleep hygiene the items that comprise shi are rated on a point likert scale and produce a total score ranging from to with higher scores representing poorer status of sleep behavioural hygiene81restorative sleep questionnaire weekly versionthe restorative sleep questionnaire weekly version rsq w is composed of nine questions completed on restorative aspects of the sleep during the past week and whose reliability and validity has been published82 each item scales from one to five the first two items and the last item are reversed scored the total score ranging yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0copen access from to calculates as rsq w average score across completed itemsˆ’× the higher total scores indicate better restorative sleepmental health and qol assessmentthe condition of mental health and qol in both groups will be assessed using in total four validated questionnaires at baseline and at the end of the interventiondepression anxiety stress scalethe depression anxiety stress scale dass21 is a validated self reported questionnaire designed to measure three subscales which are depression anxiety and stress with seven items for each dimension83 higher scores are indicative of poorer mental condition and severity of symptoms but the dass21 is not a clinically diagnostic instrument nonetheless dass21 has broad applicability and free availability and has been validated among the general population and for patients with chronic disease84 scores above and in the three dimensions indicate severe depression anxiety and stress respectively in such instances participants will be referred to their medical practitioner for clinical carevisceral sensitivity indexthe visceral sensitivity index vsi is a validated self reported questionnaire and will be employed to measure gastrointestinal specific anxiety the total vsi score is generated from all items each defined on a point likert scale patients with a higher score will be regarded as experiencing severe gastrointestinal symptom specific anxiety86 ibs quality of lifefor assessment of participants™ qol ibs qol questionnaire will be used at the stages of baseline clinic prior to and after intervention the ibs specific questionnaire is a validated measurement tool generating one total and eight subscale scores with items covering dysphoria interference with activity body image health worry food avoidance social reaction sexual activity and relationships88who five wellbeing indexwho five well being index who5 is a validated self reported questionnaire consisting of five items that measures mental well being in relation to the past weeks responders rate each item on a point likert scale the result will be calculated by multiplying the raw total score ranging from to by four the higher scores represent those with a better imaginable well being condition89all questionnaires will be collated in the software qualtrics and administered online to reduce participant burdendemographic informationparticipants will complete a demographic questionnaire which requires personal information gender age nationality marital status area of residence mobile number email address smoking history alcohol consumption birth delivery mode dietary pattern and physical activityanthropometric measurementsparticipants™ height cm and weight kg will be measured to the nearest cm and kg respectively by an seca digital column scale seca usa where circumferences of waist and hips will be measured in accordance with international operating procedures for anthropometric assessment90 body mass index bmi and waisthip ratio will be calculated91 percentage of lean and fat mass will be obtained using the bod pod cosmed rome italy an air displacement plethysmograph using whole body densitometry to determine body composition fat vs lean fat mass92 and conducted following manufacturer protocols for measurement this will require subjects to fast for hours prior to testing and wear tightly fitted gym clothes for measurement blood pressure mm hg will be measured using an omron ia1b automated blood pressure device omron healthcare japan all measurements will be carried out at baseline and end of interventionblood biomarkersthe venous blood samples will be collected after an overnight fast at baseline and at the end of the intervention blood samples will be centrifuged and processed within half an hour after collection for separating plasma and serum and frozen at ˆ’°c after being aliquoted into ml vials analysis of fasting lipids glucose and glycated haemoglobin will be performed by a pathology laboratory in accordance with the protocols from the national association of testing laboratories the outcome measures of hs crp tnfα il6 and il1β will be analysed if funding is made availablegut symptomsparticipants will complete a bowel symptom questionnaire at baseline and postintervention to assess changes in symptom severity the questionnaire consists of gastrointestinal symptom rating scale for ibs gsrs ibs93 and the ibs severity scoring system ibs sss94 which have been validated in clinical trials the item gsrs ibs uses a point likert scale for severity of symptoms characteristic of ibs including abdominal pain diarrhoea constipation and bloating satiety this instrument is short and simple and will assist researchers to determine the specific symptoms encountered by patients93 the ibs sss point with a maximum of for each item is also used for classification of patients as remission mild “ moderate “ or severe participants will use a visual analogue scale to score each of the five questions regarding symptom severity which include pain severity and duration abdominal distention yan a0r et a0al bmj open gastro 20207e000448 101136bmjgast2020000448 0copen accessbowel dysfunction and qol for this study a reduction of more than points of ibs sss is defined as symptoms improvement13daily symptoms checklisteach participant will be provided with an online daily bowel symptom checklist to report the sachet consuming time and daily symptoms throughout the entire study period adapted from the not for profit international foundation for functional gastrointestinal disorders iffgd personal daily diary https aboutibs org symptom diary html the checkbox include bristol stool chart type time and amount of fibre added to meals bowel movement number of motions stress level and menstrual cycle adverse symptoms monitoring scoring symptoms “ will be reported by participants daily and include abdominal pain constipation diarrhoea bloating flatus eructation headache nausea and vomitingitems food recordsdietary intake will be assessed using a day weighed food record preintervention and postintervention this will be completed by participants via a free downloaded smart phone application research food diary xyris queensland australia participants will be provided with a set of scales propert supertex industries and instructed on the correct recording methods for weighed diet records the monash university comprehensive nutrition assessment questionnaire will be used to specifically quantify individuals™ fodmap intaketable schedule of primary and secondary endpoints that will be measured over the studystatistical analysesbaseline participant demographics and primary and secondary outcome variables will be described and compared for differences by group descriptive statistics in the form of mean±sd will be used to describe continuous variables and frequencies and proportions for nominal and ordinal variables all continuous outcome and demographic variables will be examined normality using the shapiro wilk test median±iqr range will be presented instead for non normal continuous variableschange in individual and total scfa levels and faecal ph will be examined using mixed model analysis of variance anova between groups and within groups covariates including gender and age will be entered into the model as confounders to analyse the gut microbiota profiles multivariate analysis primer7 and permanova primer e plymouth and various r packages will be used principal coordinates analysis will be deployed to visualise data distance based linear table study assessment schedulestudy itembaseline period week dietary interventionw1w2w3œ“œ“œ“œ“œ“œ“œ“œ“demographic informationbmi body fat waisthip ratioœ“gut symptoms questionnaire ibs sssgsrs ibs œ“pittsburgh sleep quality indexepworth sleepiness scalesleep hygiene indexinsomnia severity indexrestorative sleep questionnaire weekly versiondepression anxiety stress scalevisceral sensitivity indexibs quality of lifewho five well being indexmonash university comprehensive nutrition assessment questionnaireblood samplestool samplethree day diet record vi
Colon_Cancer
" excessive perioperative fluid administration may result in iatrogenic endothelial dysfunction andtissue edema transducing inflammatory markers into the bloodstream colloids remain longer in the circulationrequiring less volume to reach similar hemodynamic endpoints compared to crystalloids thus we tested thehypothesis that a goaldirected colloid regimen attenuates the inflammatory response compared to a goaldirectedcrystalloid regimemethods patients undergoing moderate to highrisk open abdominal surgery were randomly assigned to goaldirected lactated ringer™s solution n or a hydroxyethyl starch n fluid regimen our primaryoutcome was perioperative levels of pro and antiinflammatory cytokines secondary outcome was perioperativelevels of white blood cell count wbc creactive protein crp procalcitonin pct and lipopolysaccharidebindingprotein lbp measurements were performed preoperatively immediate postoperatively on postoperative day onetwo and fourresults the areas under the curve of interleukin il p il p il p and tumor necrosisfactor α p levels did not differ significantly between the groups wbc crp and pct values were alsocomparable lbp although significantly higher in the crystalloid group remained in the normal range patientsassigned to crystalloids received a median iqr amount of ml “ of crystalloid patients assigned tocolloids received ml “ of crystalloid and ml “ of colloid cytokine and inflammatory marker levels did not differ between goaldirected crystalloid and colloidadministration after moderate to highrisk abdominal surgerytrial registration clinicaltrialsgov nct00517127 registered 16th august correspondence barbarakabonmeduniwienacat1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austriafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cobradovic bmc anesthesiology page of is crucialintroductionvolume replacementin the perioperativeperiod and has great impact on postoperative outcome fluid restriction may cause hypotension and hypoperfusion leading to an dysfunction on the otherhand excessive fluid administration leads to destructionof the endothelial surface layer and consequently to tissue edema with harmful side effects [ ]fluidgdtbasedtherapygoaldirectedonoptimization of flowrelated hemodynamic parametersimproves clinical outcome in low to highrisk surgicalpatients compared to fixed fluid protocols [ ] specifically gdt enhances cardiac performance and gutmicrocirculation while avoiding iatrogenic hyperhydration [ ] in addition to hypervolemia the inflammatoryaggravatesdegradation of the endothelial barrier the socalled glycocalyx inflammation leads to cytokine release andmay thus worsen outcome for example high postoperative interleukin il levels are independently associatedwith postoperative complications response dueto surgicaltraumaso far in most previously performed gdt studieshemodynamic algorithms were based on colloid bolusadministration to improve hemodynamic variables colloids better maintain the intravascular oncotic pressure and provide a higher volume effect when used incase of hypovolemia goaldirected colloid administration reduces intraoperative fluid requirement and improves cardiac performance compared to crystalloids[ ] whether this translates into better outcomespecifically in a decreased postoperative inflammatoryresponse is still a matter of research the comparisonbetween colloid versus crystalloid based fluid regimenswas still lacking therefore we tested the primary hypothesis that perioperative levels of pro and antiinflammatory cytokines il il il and tumor necrosis factor alpha tnf α are reduced by goaldirectedcolloid versus crystalloid administration during the firstfour postoperative days in patients undergoing moderateto highrisk open abdominal surgery in addition wemeasured white blood cell wbc count creactive protein crp procalcitonin pct and lipopolysaccharidebinding protein lbp levelsmaterials and methodsthis prospective randomized controlled trial was conducted at the department of anesthesia intensive caremedicine and pain medicine medical university ofvienna vienna austria the institutional review boardof the medical university of vienna approved it as partof a large multicenter outcome trial evaluating the effectof goaldirected crystalloid and colloid on postoperativecombined morbidity and complications the ethicalcommittee of medical university of vienna viennaaustria provided ethical approval for this trial the trialwas conducted in accordance with the declaration ofhelsinki and good clinical practice and registered atclinicaltrialsgov nct00517127 and eudract “ a written informed consent was obtainedfrom all patients the authors have followed the applicable consort guidelinesfor this single center substudy consecutive eligiblepatients were included patients aged to yearsundergoing elective moderate to highrisk open abdominal surgery with american society of anesthesiologistsasa physical status iiii were included we excludedpatients with severe obesity body mass index bmi kgmˆ’ cardiac insufficiency ejection fraction ef coronary artery disease with angina severe chronicobstructive pulmonary disease autoimmune diseases coagulopathies renal insufficiency creatinine clearance mlminˆ’ or renal replacement therapy symptoms of infection or sepsis and preoperative crp higher than mgdlˆ’ allpatientsreceivedprotocolpreoperativelyantimicrobialprophylaxis using a single dose of a 2nd generationcephalosporine according to our clinicalstandardsanesthetic management wasstandardized standardmonitoring included electrocardiography ecg invasiveblood pressure surveillance pulse oximetry and esophageal core temperature monitoring a central venouscatheter was inserted when deemed clinically necessarywe used balanced anesthesia with sevoflurane none ofour patients received locoregional anesthesia accordingto patients™ requirements additional fentanyl and nondepolarizing neuromuscular blocking were administeredventilatory rate was adjusted to maintain endtidal carbon dioxide partial pressure etco2 of “ mmhgnormothermia was maintained with forced air warmingpatients were randomized to crystalloid lactatedringer™s solution or colloid hydroxyethyl starch voluven fresenius kabi germany grouprandomization was based on computergenerated codesto conceal allocation sealed opaque envelopes wereopened only shortly before induction of anesthesiaall patients were given “ mlkgˆ’ oflactatedringer™s solution during induction of anesthesia followedby “ mlkgˆ’ per hour for maintenance normalized toideal body weight ibw throughout surgery we calculated ibw according to the robinson formula thereafter the randomized fluid crystalloid or colloidwas esophageal dopplerguided cardiac q deltex medical group plc chichester uk according to a standardalgorithm this method is based on corrected aorticflow time ftc as well as stroke volume sv and allowsdistinguishing whether a patient is a fluid responder or 0cobradovic bmc anesthesiology page of not if mean arterial pressure map was below mmhg and no signs of hypovolemia were detected vasopressors were administratedpatients were transferred to postanesthetic care unitpacu or intensive care unit icu at the discretion ofthe attending anesthesiologist fluid management wasstandardized for the first postoperative hours in whichpatients received mlkgˆ’ ibw crystalloid per hourmeasurementsdemographic and morphometric data were recorded aswell as asa score medical history type of surgery andpreoperative laboratory values duration of anesthesiaand surgery were recorded we also recorded intraoperative fluid requirements estimated blood loss transfusion requirements and urinary output for evaluation ofanesthetic management the total amount of fentanylendtidal sevoflurane concentration core temperatureandnotedhemodynamic parameters such as map heart ratehr ftc sv and cardiac output co were recorded at10min intervals the application of phenylephrine usewas notedicu admission werepostoperativethe primary outcomes were the areas under the curveaucs of postoperative levels of pro and antiinflammatory cytokines il il il and tnf α andtheir differences between the crystalloid and the colloidgroup secondary outcomes were aucs of wbc crppct and lpb and their differences between the groupsall blood samples for parameteranalyses were obtainedbefore surgery as baseline values t0 immediately postoperatively t1 as well as on postoperative days onetwo and four t2 t3 and t4 respectively for analysisof il il il and tnf α blood samples were centrifuged within h at g for min and plasma wasimmediately stored at ˆ’ °c for later enzymelinkedimmunosorbent assay elisa analyses the serum concentrations of il il il and tnf α were determined according to the manufacturer™sinstructionshuman sil6 instant elisa human il8nap1 instant elisa and human sil10 instant elisaebioscience vienna austria wwwebiosciencecom human tnf α duoset rd systems minneapolis minnesota wwwrndsystemscom for that purpose opticaldensity was measured with a victor microplate readerat a wavelength of nm multiple testing of sampleson different plates revealed an intraassay variability of for il for il for il for tnf α andan interassay variability of for il for il for il and for tnf αfor investigation of wbc crp pct and lpb separateblood samples were obtained their analysis took placeimmediately after blood sampling as routine laboratoryanalysessample size calculation and statistical analysissample size calculations for our trial were based on thestudy of steppan and colleagues they observed amean standard deviation sd of pgmlˆ’ in il h after surgery in abdominal surgery patients assuming a similar coefficient of variation sdmean for each of the four cytokines primarily plannedfor evaluation in our study we calculated a total of patients in order to obtain a power to detect a reduction in any of the cytokines at an overall significance level with powergroups were primarily compared for balance in patients™ demographic data intraoperative characteristicsand postoperative variables absolute standardized differences asd were calculated for patients™ baseline covariates subsequent measurements ofintraoperativeparameters were first averaged within each patient andthen averaged among the patients in each treatmentgroup for descriptive analysis normal distribution wasassessed with qq plots and kolmogorowsmirnow testsnormally distributed variables were with unpaired twotailed ttests otherwise the in case of normally distributed values wilcoxon ranksum test was used for notnormally distributed continuous data paired comparisons between baseline data and postoperative data wereperformed with paired sample ttest or wilcoxonsignedrank test as applicable nominal data were analyzed with chisquare or fisher™s exact test for low expected cell counts data were presented as means ± sdmedians iqr or as numbers percentage as applicableadjustment for multiple testing was performed with thebonferroni method a p value was considered statistically significantanalysis was conducted with spss software version armonk ny ibm corp r for macintosh version321 r core team r a language and environmentfor statistical computing r foundation forstatistical computing vienna austria was used to calculate asdresultsa total of patients were included between november and october in the colloid group and in the crystalloid group fig at t0 all values weremeasured overall in the crystalloid group and inthe colloid group of the preplanned blood sampleswere collected and analyzedpatient™s baseline characteristics did not differ exceptfor height bmi with a slightly higher bmi in the colloidgroup type of surgery and crp also higher in the colloid group table duration of anesthesia and surgerywere comparable between both groups patients assignedto crystalloid administration received a median of ml “ crystalloids whereas patients assigned 0cobradovic bmc anesthesiology page of fig consort patient flow chartto the colloid group received ml “ ofcrystalloid solution and ml “ of colloidsblood loss transfusion requirements and urinary outputdid not differ between the groups anesthetic management map and hr did not differ between the groupsftc sv and co were significantly higher in the colloidgroup compared to the crystalloid group ftc ms“ versus ms “ p sv ml“ versus ml to p and co ± lminˆ’ versus ± lminˆ’ p thenumber of patients requiring vasopressor support wascomparable between groups the incidence of postoperative icu admissions did not differ in the crystalloid versus in the colloid group p table baseline values of il il il and tnf α in thecrystalloid group were comparable to values in the colloid group il pgmlˆ’ “ versus pgmlˆ’ “ p il pgmlˆ’ “ versus pgmlˆ’ “ p il pgmlˆ’ “ versus pgmlˆ’ “ p tnf α pgmlˆ’ “ versus pgmlˆ’ “ p immediatepostoperative values of il and were significantlyhigher compared to baseline values in both groupsp for all measurements while tnf α did notshow any significant increase in the crystalloid p and the colloid group p fig aucs of il il il and tnf α did not differ significantly between the groups table wbc values at baseline were glˆ’ “ in thecrystalloid versus glˆ’ “in the colloidgroup p crp pct and lbp baseline valueswere also comparable in both groupscrp mgdlˆ’ “ versus mgdlˆ’ “p pct ngmlˆ’ “ versus ngmlˆ’ “ p lbp mcglˆ’ “ versus mcglˆ’ “ p immediate postoperative values of wbc and pctwere significantly higher compared to the baseline valuesin both groups p for all measurements fig 0cobradovic bmc anesthesiology page of table baseline characteristicsage yrsweight kgheight cmbmi kgmˆ’ gender no menwomenasa score no iiiiiimedical history no pulmonary diseasecardiovascular diseasediabetes type idiabetes type iitype of surgery no colorectalliverpancreaticcrystalloidsn ± ± ± ± colloidsn ± ± ± ± asd preoperative laboratory valuescrp mgdlˆ’ ± ± patient characteristics data are presented as means ± sd or as counts for thecategorical outcomesabbreviations asd absolute standardized differences absolute difference inmeans or proportions divided by the pooled sd asd values of and represent small median and large differencesbmi body mass index m male f female asa american society ofanesthesiologists crp creactive protein sd standard deviationthe aucs for wbc crp pct and lbp for the timeperiods from t1 to t4 did not differ significantly between the groups fig table however lbpshowed significantly higher levels in the crystalloidgroup in the immediate postoperative period comparedto the colloid group mcglˆ’ “ versus mcglˆ’ “ p at all other postoperativetime points there were no significant differences betweenthe groups fig discussionthis trial is a substudy of a large multicenter randomized trial evaluating the effect of goaldirected crystalloidversus goaldirected colloid fluid administration on acomposite of serious complications after moderate tohighrisk open abdominal surgery the overall trial concluded that colloids did not decrease the composite ofmajorincomplicationsare ourresultsconcordance as they did not show any differences inperioperative pro and antiinflammatory markers between a crystalloid and a colloid fluid regimendespite multimodal care and enhanced recovery programs it still remains challenging to blunt the inflammatory response to surgery systemic inflammationafter abdominal surgery impairs outcome and thereforemany attempts have been made to alter the inflammatory response several factors influence the perioperative inflammatoryresponse such as the underlying disease type and invasiveness of surgery as well as type of anesthesia [“] themost important factor is the magnitude of surgical traumaand tissue damage which induce proliferation and activation of immune competent cells in turn triggering cytokine and inflammatory marker release so far veryfew trials have specifically investigated the influence offluid therapy and differences in terms of the type of fluidon the extent of inflammatory marker releaseto investigate the potential influence of goaldirected hydroxyethyl starch versus a lactated ringer™s solution fluid regimen on inflammatory response pro il il and tnf α and antiinflammatory cytokine il serum levels were measured during the perioperativeperiod additionally we measured wbc crp pct andlbpgenerally the most commonly measured biomarkersare crp and wbc if levels of crp are above mgdlˆ’ after postoperative day four a postoperative infection can be suspected crp levels in our studygroups increased on the first postoperative day droppingon the fourth postoperative day to nearly mgdlˆ’ inboth study groupswbcs are an imprecise marker to detect postoperativecomplications after major abdominal surgery amore sensitive parameter in predicting postoperativecomplications after major abdominal surgery is il surgical trauma and hypoperfusion of the colon aremain sources of il release in colorectal surgery noblett demonstrated that gdt during elective colorectal surgery significantly reduced il levels in comparison to a control group yates showed no differencesof il and il levels between goaldirected colloidand crystalloid fluid therapy during the first h in asubgroup of patients undergoing colorectal surgery although patients in the crystalloid group received significant more volume amount as compared to the colloid groupthere was no significant difference inhemodynamic variables our patients showed similar courses of il and il levels in the immediatepostoperative period in contrast to the trial of yateswho measured cytokine levels up to the first h aftersurgery we extended our measurement period to fourpostoperative days we showed comparable circulating 0cobradovic bmc anesthesiology page of table intraoperative dataduration of anesthesia minduration of surgery minfluid managementtotal fluid intake mlacrystalloid mlcolloid mlestimated blood loss mltransfusion yesno urinary output mlanesthesia managementfentanyl mcgtwa et sevoflurane core temperature °cicu admission yesno hemodynamictwa map mmhgtwa hr beatsminˆ’twa ftc mstwa sv mltwa co lminˆ’ phenylephrine yesno crystalloidsn ± ± “ “ “ “ “ [ ] “ ± ± ± “ “ ± colloidsn ± ± “ “ “ “ [ ] “ “ ± ± ± “ “ ± p value ° ° °° °°intraoperative data are presented as means ± sd medians iqr or as counts for the categorical outcomes means were compared with an unpaired twosided ttests ormannwhitneyu tests as appropriate medians with wilcoxon ranksum tests and counts with chisquare or fisher™s exact tests ° represents statisticalsignificance p abbreviations et end tidal icu intensive care unit twa time weighted average map mean arterial pressure hr heart rate ftc corrected flow time sv strokevolume co cardiac output sd standard deviationa total fluid intake includes baseline fluid boluses antibiotics analgesics and additional fluid administered at the discretion of attending anesthesiologistfig ad pro and antiinflammatory cytokines il il il and tnf α over time a il b il c il and d tnf α data arepresented as medians iqr abbreviations il “ interleukin tnf α “ tumor necrosis factor alpha pod “ postoperative day 0cobradovic bmc anesthesiology page of table areas under the curve of inflammatory markerspcrystalloidsn “ “ “colloidsn “ “ “ “ “ “ “ “auc il pgmlˆ’ 1dauc il pgmlˆ’ 1dauc il pgmlˆ’ 1dauc tnf α pgmlˆ’ 1d “auc wbc glˆ’1dauc crp mgdlˆ’ 1dauc pct ngmlˆ’ 1dauc lbp mcglˆ’ 1dtable areas under the curve of il il il and tnf α as well as wbccrp pct and lbp are presented as medians iqr medians were comparedwith wilcoxon ranksum testsabbreviations il interleukin tnf α tumor necrosis factor alpha wbc whiteblood cells pct procalcitonin lbp lipipopolysaccharidebinding protein “ “ “ “il and il levels between a gdt crystalloid and colloid administration these surrogates of inflammatoryresponse imply that gut perfusion during surgery waswell preserved with both types of fluid and suggest thatthe type of fluid might be of minor importance as longas the fluid is administered in a goaldirected fashionthe fact that tnf α levels in both groups remainedstable over the entire measured period further supportsour theory the course of tnf α levels during the perioperative period was in accordance with the study ofin which fluid therapy was guided withszakmanypicco versusin majorvenous pressurecentralabdominal surgery in patients at risk for postoperativecomplications as tnf α per se triggers glycocalyxdegradation we anticipate that tnf α did not influence glycocalyx shedding and thus possible fluid shifts inour study populationpct is an early predictive marker for systemic inflammation after abdominal surgery values above ngmlˆ’ are associated with postoperative complicationssuch as pneumonia or anastomotic leakage in ourstudy median pct levels did not exceed ngmlˆ’ atany measured time point these results are in concordance with our main study where infectious complications rate were held low and did not differ between thegroups moreover as pct production can also beinduced by tissue hypoperfusion we might assume thatgoaldirected fluid administration contributed to lowpct values by optimizing cardiac performance furthermore we measured lbp a prognostic markerfor bacterial infections patients in the crystalloidgroup showed significantly higher levels immediatelyafter surgery however the measured values remainedwithin the normal range therefore this difference ismost likely not to of clinical importancethe vascular endothelium is one of the earliest sitesinvolved in the inflammatory response syndrome an adequate perioperative fluid management has a major impact on the integrity of the glycocalyx with goaldirected fluid management individualized and time appropriate fluid resuscitation can be achieved enablingfig ad inflammatory markers wbc crp pct and lbp over time a wbc b crp c pct and d lbp data are presented as medians iqrabbreviations wbc “ white blood cells crp “ creactive protein pct “ procalcitonin lbp “ lipopolysaccharidebinding protein pod “postoperative day ˜… represents significant difference in lbp in the immediate postoperative period between the crystalloid and the colloidgroup p 0cobradovic bmc anesthesiology page of preservation of endothelial surface layer and sufficientan perfusion thus improving postoperative outcomesafter major surgery patients in the colloid group received significantly lessfluid ml confirming the previously publishedfluid sparing effect of colloids however clinical significance of this difference may be questionable during aperioperative period of nearly five hours further ourhemodynamic data showed significantly higher values ofsv and co with colloid administration though the absolute difference of ml in sv most likely has onlylimited clinical relevance it might very well be that thesignificant differences in sv and co are the result ofour number of patients included in this substudyassurrogatesfirst limitation of our study is that we measured inflammatory markers that reflect the inflammatory responseand not direct markers ofglycocalyx degradation like syndecan1 therefore wecannot draw any s about the preservation ofthe endothelial surface layer in our patients secondlywe did not control postoperative fluid management during the postoperative followup period a further limitation isthe time between patient enrolment andsubmission of our current results due to the fact thatthe main trial has been published recently a delay of oursubmission occurred nevertheless our results canstill be extrapolated to current clinical practicein summary goaldirected hydroxyethyl starch administration did not attenuate the inflammatory responseexpressed by cytokine levels of il il il and tnfα in patients undergoing moderate to highrisk open abdominal surgery wbc crp and ptc values did not differ between the different fluid regimes as wellabbreviationsasa american society of anesthesiologists asd absolute standardizeddifference auc area under the curve bmi body mass index co cardiacoutput crp creactive protein ecg electrocardiography ef ejectionfraction elisa enzymelinked immunosorbent assay etco2 endtidalcarbon dioxide ftc corrected flow time gdt goaldirected therapyhr heart rate ibw ideal body weight icu intensive care unitil interleukin iqr interquartile range lbp lipopolysaccharidebindingprotein map mean arterial pressure pacu postanesthetic care unitpct procalcitonin picco pulse contour cardiac output spss statisticalpackage for the social sciences sv stroke volume tnf α tumor necrosisfactor alpha twa time weighted average wbc white blood cell countacknowledgementsassistance with this we thank bianca tudor md department ofanesthesia intensive care medicine and pain medicine medical university ofvienna spitalgasse vienna austria for her support in laboratoryskills performing elisasauthors™ contributionsall authors have read and approved the manuscript mo patientrecruitment data acquistion and prepratation of the manuscript ak studyprotocol writing and preparation of the manuscript bk study protocolwriting and preparation of the manuscript statistical analyisis gr dataanalysis revision of the manuscript ok data analysis preparation of themanuscript oz patient recruitment data acquisition data managementab patient recruitment data acquisition revsion the manuscript cr dataacquistion revision of the manuscript as patient recruitment revision ofthe manuscript ef study protocol writing and preparation of the manuscriptfundingmedical university of viennapartially funded by fresenius kabi deltex medical provided oesophagealdoppler monitors and disposablesthe sponsors were not involved in protocol development data acquisitionor data analysisavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestbarbarakabonmeduniwienacatethics approval and consent to participatethe main trial was approved by the local ethics committee of the medicaluniversity of vienna in ek and was registered atclinicaltrialsgov nct00517127 and eudract “ a writteninformed consent was obtained from all patients prior to participationconsent for publicationnot applicablecompeting intereststhe authors declare no competing interestsauthor details1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austria2department of outcomes research and general anesthesiologyanesthesiology institute euclid avenue cleveland clinic cleveland ohusa 3department of anesthesiology and general intensive care franziskushospital nikolsdorfergasse vienna austria 4department ofgynecology klinik ottakring montleartstrasse vienna austria5department of surgery medical university of vienna spitalgasse vienna austriareceived july accepted august referencesbrandstrup b tonnesen h beierholgersen r effects of intravenousfluid restriction on postoperative complications comparison of twoperioperative fluid regimens a randomized assessorblinded multicentertrial ann surg “ myles ps bellomo r corcoran t restrictive versus liberal fluidtherapy for major abdominal surgery new engl j med “chappell d jacob m hofmannkiefer k conzen p rehm m a rationalapproach to perioperative fluid management anesthesiology “lowell ja schifferdecker c driscoll df benotti pn bistrian brpostoperative fluid overload not a benign problem crit care med “sun y chai f pan c romeiser jl gan tj effect of perioperative goaldirected hemodynamic therapy on postoperative recovery following majorabdominal surgerya systematic review and metaanalysis of randomizedcontrolled trials critical care calvovecino jm ripollesmelchor j mythen mg effect of goaldirected haemodynamic therapy on postoperative complications in lowmoderate risk surgical patients a multicentre randomised controlled trialfedora trial br j anaesth “noblett se snowden cp shenton bk han af randomized clinical trialassessing the effect of doppleroptimized fluid management on outcomeafter elective colorectal resection br j surg “ 0cobradovic bmc anesthesiology page of alphonsus cs rodseth rn the endothelial glycocalyx a review of thevascular barrier anaesthesia “ pearse rm harrison da macdonald n effect of a perioperativecardiac outputguided hemodynamic therapy algorithm on outcomesfollowing major gastrointestinal surgery a randomized clinical trial andsystematic review jama “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationskimberger o arnberger m brandt s goaldirected colloidadministration improves the microcirculation of healthy andperianastomotic colon anesthesiology “kolarova h ambruzova b svihalkova sindlerova l klinke a kubala lmodulation of endothelial glycocalyx structure under inflammatoryconditions mediat inflamm rettig tc verwijmeren l dijkstra im boerma d van de garde emnoordzij pg postoperative interleukin6 level and early detection ofcomplications after elective major abdominal surgery ann surg “ gan tj soppitt a maroof m goaldirected intraoperative fluidadministration reduces length of hospital stay after major surgeryanesthesiology “jacob m chappell d rehm m clinical update perioperative fluidmanagement lancet “feldheiser a pavlova v bonomo t balanced crystalloid comparedwith balanced colloid solution using a goaldirected haemodynamicalgorithm br j anaesth “ orbegozo cortes d gamarano barros t njimi h vincent jl crystalloidsversus colloids exploring differences in fluid requirements by systematicreview and metaregression anesth analg “kabon b sessler di kurz a effect of intraoperative goaldirectedbalanced crystalloid versus colloid administration on major postoperativemorbidity a randomized trial anesthesiology “ robinson jd lupkiewicz sm palenik l lopez lm ariet m determination ofideal body weight for drug dosage calculations am j hosp pharm “steppan j hofer s funke b sepsis and major abdominal surgery leadto flaking of the endothelial glycocalix j surg res “ wilmore dw from cuthbertson to fasttrack surgery years of progress inreducing stress in surgical patients ann surg “ desborough jp the stress response to trauma and surgery br j anaesth“ veenhof aa sietses c von blomberg bm the surgical stress responseand postoperative immune function after laparoscopic or conventional totalmesorectal excision in rectal cancer a randomized trial int j color dis “ gilliland he armstrong ma carabine u mcmurray tj the choice ofanesthetic maintenance technique influences the antiinflammatory cytokineresponse to abdominal surgery anesth analg “ wichmann mw huttl tp winter h immunological effects oflaparoscopic vs open colorectal surgery a prospective clinical study archsurg “ watt dg han pg mcmillan dc routine clinical markers of themagnitude of the systemic inflammatory respon
Colon_Cancer
ovarian cancer is one of the leading causes of common lethal gynecologic malignancy cortez in worldwide there were an estimated cases and deaths from ovariancancer bray because of the lack of an early diagnosis method and the absence ofspecific early warning symptoms patients with ovarian cancer are usually diagnosed at an advancedstage and have a poor prognosis scarlett and conejogarcia frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancerbased on histological origin ovarian tumors can becategorized into epithelial germ cell sex cord or stromal tumorsjayson around of primary ovarian tumors areof epithelial origin colombo ledermann so we mainly focus on evidence of epithelial ovarian cancer inthis review the world health anization who classifiedepithelial ovarian cancer eoc into the following types serousmucinous endometrioid clear celltransitional cell mixedepithelial undiï¬erentiated and unclassified ledermann according to architectural features eoc is also classifiedinto grades by the international federation of gynecologyand obstetrics figo system colombo in serouseoc figo grade is defined as lowgrade while figo grade and are combined as highgrade bodurka theclassification with histosubtypes and grades are with prognosticsignificance ledermann plussurgerycytoreductivethe current standardized treatmentoptimalfor ovarian cancerisplatinumbasedchemotherapy with the carboplatinpaclitaxel regimen bolton however with the development of chemotherapyresistant and refractory diseases the sensitivity of chemotherapyhas decreased lim and ledger therefore the longtermsurvival rate for ovarian cancer has decreased and the recurrencerate has increased lim and ledger hennessy reported that despite benefiting from firstline therapy of patients with advanced ovarian cancer stage iii or ivhave tumor relapse at a median of months from diagnosismoreover for patients with earlystage disease stage i or ii thelongterm survival rates years are “ in contrastpatients with advanced disease stage iii or iv had a “longterm survival rate therefore there is an urgent need tofind new targeted therapies to improve the treatment efficacy ofovarian cancer in recent years the tumor microenvironmenttme has been reported to play a vital role in the tumorigenesisof ovarian cancer and is considered a possible therapeutic targetfor ovarian cancer here we review the interactions betweenthe tme and ovarian cancer and various therapies targeting thetumor environmenttme in ovarian cancer‚ammatory cytokinesthe tme comprises the extracellular matrix ecm whichconsists of chemokinesintegrinsmatrix metalloproteinases mmps and other secreted moleculesand stromal cells including cancer cells cancer stem cellspericytes cancerassociated fibroblasts cafs endothelial cellsecs and immune cells figure hanahan and weinberg in this part we reviewed current findings on the impactof some key components above on ovarian cancer progressioncancerassociated fibroblastsfibroblasts which diï¬erentiate from mesenchymalderived cellsare part ofthe tme ishii they producevarious mmps tissue inhibitor of metalloproteinases timpsand most ofthe proteins comprising the ecm such ascollagens fibronectin and laminin kalluri and zeisberg erdogan and webb these fibroblasts in the tumor milieuare also called œcafs additionally cafs can transdiï¬erentiatefrom other cellssuch as pericytes epithelial cells andecs via exposure to plateletderived growth factor pdgftumorderived transforming growth factor tgf vascularendothelial growth factor vegf basic fibroblast growth factorbfgf mmps and reactive oxygen species ros cai yu y denton cafs are known to promote tumor progression via variousmechanisms cafs can enhance tumor cell proliferationinvasion and migration sjoberg showed that cafshighly expressed cxcl14 which was an important factor inpromoting cancer growth cafs also express the fibroblastactivation protein α fap yang™s study indicated that fapαenhanced the migration and invasion ability of ho8910pm cellsa highly metastatic ovarian cancer cell line additionally fapαincreased ho8910pm cell proliferationcafs promote immune inhibition and angiogenesis givel found that cafs increase the ltration offoxp3 regulatory t lymphocytes tregs at the tumor sitewhich exerts immune suppression eï¬ect in the tumor milieuadditionally in orimo™s research cafs have high expression ofstromal cellderived factor1 sdf1 released sdf1 promotesangiogenesis and tumor proliferation in a paracrine fashionorimo cafs also increase platinum resistance and acceleraterecurrence fauceglia™s study showed that cafs expressed thefap α by analyzing eoc tissues they found that theoverexpression of fap acted as a hallmark for platinumresistance additionally patients with fap stroma had ashortened recurrence compared to that of patients with fapstroma mhawechfauceglia several studies have indicated that cafs was a biomarker ofpoor prognosis in ovarian cancer yang mhawechfauceglia zhao givel in givel™s study of cafs in highgrade serous ovarian cancershgsoc the results showed that the expression of cxcl12and the ltration of cafs1 a subtype of cafs implied adismal prognosis givel despite that accumulatingstudies have demonstrated the protumor progression impact ofcafs it is worthy of notifying that there are diï¬erent subtypesof cafs with heterogenous function status recently hussain found caf subsets distinguished by the fapexpression level the faphigh caf subtype instead of thefaplow subtype was found to aggressively enhance tumorprogression and negatively ‚uence patient outcomes whichshed light on therapeutic strategies involving caf modulation toconsider caf status in patient selectioncafscrucialcell population in thetumormicroenvironment cafs promote the proliferation invasionand migration of cancer cells and stimulate angiogenesis bycoordinating with other cells a deeper understanding of cafsis needed to better understand how cafs aï¬ect the tumormicroenvironmentareaendothelial cellsendothelial cells ecs are components of the tme liningfor transporting oxygen andthe vessels ecs are crucialnutrientscloselyassociated with angiogenesisand arefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancerfigure cell components and functions in the tumor microenvironment tme cell components in the tme can be classified into cancer cells immune cells andstromal cells these cells actively interact with each other by molecules they secrete [including cytokines chemokines damageassociated molecular patternsdamps etc] and receptors they express such as histocompatibility complex class mhc molecules programmed cell death protein pd1 etc forming anevolving microenvironment on the continuous spectrum from antitumor to protumor effect different cell components can locate at distinct positions and the samegroup of cells may also be repolarized depending on signals in the tme the progression or regression of a single tumor site depends on the overall effect of thecomplex cellular and molecular regulating network in the tmecarmeliet and jain hanahan and weinberg as we all know angiogenesisis a complicated processaccommodated by angiogenesis activators and inhibitorsangiogenesis activators include vegf fgf2 pdgf tgfαand tgf tnfα prostaglandin e2 and interleukin il the angiogenesis inhibitors contain angiopoietin angsthrombospondin tsp1 and endostatin moreoverthesignaltransducing network of endothelial cells is associated withvegf fgf and angs signals cross and claessonwelsh ahmed and bicknell vegf is a protein family consisting of vegfa vegfbvegfc vegfd vegfe and plgf placental growthfactorregulated by the ischaemiahypoxiainducedgenes hifs epidermal growth factor egf and pdgfsemenza there are three receptors for vegf vegfreceptors vegfr1 vegfr2 and vegfr3 vegfr1 andvegfr2 are mainly expressed on ecs and are receptorsfor vegfa hagberg additionally vegfr1is also a receptor for vegfb and plgf vegfr3 is areceptornrp1and nrp2 are coreceptorsthe vegf family withthe binding affinity between vegfathe help of nrp1for vegfc and vegfd neuropilinsforitisferrara and adamisand plgf and vegfr2 increases similarly with the eï¬ect of nrp2 vegfc andvegfd have increased binding affinity with vegfr3 itis well known that the vegf family is implicated in theadjustment of angiogenesis and lymphangiogenesis tammelaand alitalo apte among them vegfafor angiogenesis while vegfc and vegfdis crucialregulatetammela and alitalo apte lymphangiogenesisangs also a protein family consisting of ang1 ang2 ang3and ang4 through combined with the receptors of angs œtiesthey perform diï¬erent functions in angiogenesis ang1 and can bind to tie2 and stimulate the tyrosine phosphorylationof tie2 on the contrary ang2 and can competitivecombined tie2 without stimulating tyrosine phosphorylationwhich stopping the signal transduction of angiogenesis sallinen lin sallinen however otherstudy indicated that with ang1 ang2 block the tie2 signalingwhile ang2 induce the tie2 signaling without ang1 yuan additionally ang1 promotes the maturation andstabilization of vessel while ang2 destabilize the stabilized vesseltse frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancervegf indicate poor clinical outcomes wimberger shen sopo wimberger foundthat by kaplanmeier analyses vegfr1 expression was closelyrelated to decreased overall survival os and progressionfreesurvival pfs wimberger sopo™ study discoveredthat vegfr1 vegfa and vegfd were highly expressedin omental metastases compared to expression in primaryovarian epithelial tumors interestingly patients with low vegfa expression were more likely to have a poor prognosis patientswith high vegfc expression were related to a short pfssopo the cd146 expression in the membrane of ecs promotesthe migration of ecs and angiogenesis cd146 is an endothelialbiomarker and the extracellular domain of cd146 directlyinteracts with vegfr2 yan™s study demonstrated that cd146can promote angiogenesis yan subsequentlyjiang™s reportindicates cd146 promotes the migration ofecs and the formation of microvasculature by enhancingvegfr2 phosphorylation and downstream signaling aktp38mapksnfκb activation jiang interestinglyzhou™s research indicated that the gene and protein levels ofcd146 and vegfra were increased in patients with eoccompared to those of noncancer patients zhou enhanced angs expression increase relapse and decreasesurvival time sallinen lin sallinen observed that patients with ovarian carcinoma hadhigher ang2 levels compared to those of patients with benignovarian tumors furthermore by analyzing the kaplan“meiercurves they found that increased ang2 levels ngml werea biomarker for poor recurrencefree survival sallinen subsequently they discovered that the expression levelsof ang1 and ang2 were and respectively higher inwomen with ovarian cancer than in normal women increasedang2 expression was significantly related to advanced stageand grade of cancer and relapse of ovarian cancer additionallyelevated ang2 expression is a predictor of poor os and short pfssallinen as an important part ofthe tumor microenvironmentecs are closely related to angiogenesis vegf and angs arecrucial regulators in angiogenesis both vegf and angs areassociated with poor clinical outcomes which provide possibletargets for treatmentcells myeloidderived suppressorimmune cellsimmune cells include macrophages dendritic cells dcsneutrophils mastcellsmdscs and lymphocytes hanahan and weinberg they play significant roles both in tumor progression and tumorsuppression participating in evolving processes of tumorigenesismetastasis and angiogenesis by producing various signalingmolecules such as egf vegf mmp9 ifns ils etcmacrophagesmacrophages are an essential population of immune cells thatparticipate in ‚ammation and tumourigenesis grivennikov among them macrophages residing in tumorsaretamstumorassociated macrophagestermedastams can derive from resident macrophages or ltratingmacrophages from bone marrow monocytes circulating in theblood ghosn depending on stimuli in the tme tams can present twomain phenotypes the antitumor m1 macrophages and protumor m2 macrophages sica grivennikov qian and pollard sica gupta when stimulated with interferongamma ifnγ bacteriallipopolysaccharide lps and granulocytemacrophagecolonystimulating factor gmcsf monocytes diï¬erentiated intom1 macrophages which can secrete il1 il12 tnfα andcxcl12 sica ramanathan and jagannathan m1 macrophages possess cytotoxicity tumor suppression andimmunestimulation functions galdiero when stimulated with cytokinesincluding il4 il10and il13 monocytes diï¬erentiated into m2 macrophagesleyvaillades van dalen in theimmune escape stage the tumor macroenvironment maintainsimmunosuppression due to the secretion of many growthfactors and cytokines such as il4 and il13 by cancer cellsthe immunosuppressive state accelerates monocytes to m2macrophages m2 macrophages in turn can promote tumrowth gordon roy and li in ovarian cancer tams are predominantly m2 macrophagesassociating with tumor invasion angiogenesis metastatic diseaseand early recurrence pollard reinartz yin they produce and secrete cytokines which haveimmunosuppressive eï¬ects such as il1r decoy il10 ccl17and ccl22 gordon via several mechanismstheysuppress adaptive immunity li noy and pollard firstly m2 macrophages can inhibit the proliferationof t cells and accelerate the immunosuppression of treg celltransport to tumors by producing the chemokine ccl22 li secondly m2 macrophages express the ligandreceptors for ctla4 and pd1 the activation of pd1 andctla4 inhibits cytotoxic function and regulates the cell cycleof t cells noy and pollard then m2 macrophagescan also inhibit the activation of t cells through the depletionof larginine which plays an essential role in t cell functiongaldiero arginase i arg1 a hallmark of m2macrophages is an larginine processing enzyme in the tmearg1 decomposes larginine into lornithine and urea thedepletion of larginine suppresses the reexpression of the cd3ζ chain which is internalized by antigen stimulation and t cellreceptor tcr signaling rodriguez aside from immune suppression m2 macrophages alsotake part in tissue repair ecm remodeling and angiogenesiswhich are processes involved in tumor progression as wellmantovani coï¬elt ruï¬ell finkernagel roy and li they canrestructure ecm and regulate ecm components by degradingecm via producing mmps serine proteases and cathepsinsruï¬ell which may facilitate tumor cell migrationinvasion and metastasis additionally they can secrete vegfa which is an angiogenic factor and produce proangiogeniccytokines such as il1 tnfα and upa urokinasetypeplasminogen activator roy and li in m2 macrophagesfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancerthere is a subtype expressing tie2 a tyrosine kinase receptorthe tie2 macrophages are involved in angiogenesis fagianiand christofori these tie2 macrophages recruited byccl3 ccl5 ccl8 and tie2ligand ang are consideredthe most important reason for tumor vascularization becausethe deficiency of this cell type restricts the angiogenic switchngambenjawong tams are plastic the simple dichotomy of m1m2macrophages cannot accountfor the complexity of tamheterogeneity ostuni transcriptome analysisuncovered a spectrum model of tams xue m1 andm2 macrophages can be regarded as two ends of a continuumwith wide ranges of functional states mantovani ostuni the subpopulations of tams in between thetwo ends can share features of both m1 and m2 types qian andpollard for example recently singhal foundthat tams could coexpress m1m2 markers together with tcell coinhibitory and costimulatory receptorsthe dynamic nature of the tme cellular environment gives abasis for the plasticity of tams macrophages present reversiblechanges in their functional phenotypes and distribution inresponse to diï¬erent microenvironmental stimuliincludingvarious cytokines and locally derived molecules which are tissueand tumorspecific stout okabe and medzhitov ostuni kim and bae therefore indiï¬erent histotypes of tumors zhang cassetta and diï¬erent microregions of the same tumormantovani kim and bae yang m there can be tams with diï¬erent extent of ltration andfunctional statusin ovarian cancer zhang found the densityand the cancerisletstroma ratio of tams vary amongserous mucinous endometrioid clear cell and undiï¬erentiatedhistotypes in the stroma and lumina of a small number of patientovarian tumor samples limited frequencies of inos expressingtams were found which were thought to be cytotoxic klimp in contrast in the malignant ascites of ovariancancer abundant tams can be found which are primarilym2like with protumor capacity gupta as thetumor grows stimuli in the tme alter resulting in changesin tam ltration and polarization in a tumor progressionleveldependent manner in ovarian cancer studies tam andm2 macrophage density were found to increase as cancer stageand ascites volume increased or as lymphatic invasion appearedzhang ke yuan gupta contrarily m1m2 ratio decreased as cancer stageincreased zhang despite expressing similar markers tams may not alwaysimplications in colon cancer studyhave similar functionaltams expressing pd1 presented weakened phagocytic potencyassociating with reduced survival gordon whilein early lung cancer study the pd1 tams did not aï¬ecttumorspecific t cell attack against tumor singhal this indicates the necessity of future studies focusing on tamfunctional status in the context of tumor tissue types and stagesof the disease this is especially true with ovarian cancer as it hasmany histotypes and high heterogeneityseveral studies revealed the prognostic value of tams inovarian cancer the m1m2 and m2tam ratio have beenreported to be positively associated with pfs and os while theoverall tam density in ovarian tumors indicated no prognosticsignificance lan zhang yuan m2 density in the ascites or tumor samples is associatedwith reduced relapsefree survival reinartz andpfs lan yuan however there is acontroversy in the relationship between m2 density alone andos lan reported a negative association betweenthe two factors while zhang found no significantrelevance this may be due to the diï¬erence ofincludedtumor histotypesdendritic cellsdcs capture endogenous or exogenousdendritic cellsantigens process them and presentthe antigenic peptidesto other immune cells banchereau acting asa bridge connecting the innate and the adaptive immunesystem timmerman and levy riboldi there are two main subtypes of dcs the conventional dccdc thatis specialized in antigen presentation and theplasmacytoid dc pdc that produces ifn upon antigenstimulation aside from activating lymphocytes and other myeloidcells labidigaly vu manh cdcscomprise “ of myeloid cells in most tumors pdcs arerare in mouse tumors butfound in most human tumorstang the initiating event ofimmune responses againstdcs play key roles in antitumor immunity because it isindispensable for t celltumorscasey dcs are responsible for tumor antigenrecognition which isthe tumorspecific adaptive immune response in both the animal ovariancancer model and human hgsoc patients dcs can sensedamageassociated molecular patterns damps released fromdead cancer cells such as doublestranded dna dsdnafragments and calreticulin an endoplasmic reticulum erchaperone eliciting th1 polarized immunity ding kasikova after capturing antigens dcs present peptides processedfrom those antigens to cd4 and cd8 t cells via majorhistocompatibility complex class ii mhc ii and mhc imolecules respectively which subsequently initiate a series of tcell activity dudek sabado this processhas been reported to be significant for tumor developmentprevention mackie galon besides t cell activation dcs are also crucialfor theaugmentation of cytotoxic t lymphocytes ctls population inthe tme it is reported that intratumoral cdcs are responsiblefor intratumoral ctl proliferation both in vivo and in vitrodiao and they are the only group of phagocytosingtumor myeloid cells that can stimulate cd8 t cell proliferationbroz as the major determinant of success intumor deterrent from the immune aspect budhu is to increase the functional tumorltrated ctl populationthe significance of cdcs in the tme for antitumor responsesis selfevidentfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancereï¬ective t cell activation by dcs require dc maturationa process happens after dc exposing to antigen characterizedby increased membrane expression of mhc and costimulatorymolecules cd80 cd86 cd40 bol bhatia alteration of chemokine receptors to favor dc lymph nodeln migration drakes and stiï¬ mature dcs producecytokines that favor th1 antitumor immunity truxova found in cohorts of hgsoc patients thattumorltrated mature lamp dcs is robustly associated with th1immune responses clinically favorable cytotoxic activities in thetme and favorable osthe process of dc maturation can be hampered by multiplefactors leaving dc immatured potentially developing into atolerogenic status and promote immune tolerance dhodapkar immature dcs express low levels of costimulatorymolecules and cytokines and mount limited immune activitiesdrakes and stiï¬ factors that lead to dc dysfunctionincluding the inhibition of dc maturation involve the immunemodulating molecules in the tme such as il6 il10 andvegftheactivation of oncogene stat3 in dcs the er stress responseand the abnormal intracellular lipid accumulation cubillosruiz tang devito thesefactors suppress dc functions by reducing the expression ofcostimulatory molecules and the secretion of pro‚ammatorycytokines inhibiting dc lymph node chemotaxis dampeningdc diï¬erentiation inducing tolerogenic phenotypes on dcs andshortening the lifespan of dcs tang tumorderived soluble mediators and exosomestolerogenic dcs suppresses antitumor immunity via severalmechanisms first they produce less pro‚ammatory cytokinesand induce immune suppressive cytokines labidigaly found in a cohort of ovarian cancer patients that intratumoural tolerogenic pdcs secreted fewer ifnα tnfα il6macrophage ‚ammatory protein1 and ccl5 while inducedil10 from cd4 t cells promoting immune tolerance in thesepatients second they harbor enzymes negatively regulatingt eï¬ector cell functions such as nitric oxide synthase nosand indoleamine 23dioxygenase ido casey ido is an enzyme catalyzing tryptophan degradation capableofsuppressing tumorltrated lymphocyte proliferationpromoting treg diï¬erentiationinducing t cell anergy andpromoting tumor angiogenesis as well as metastasis munn tanizaki munn and mellor in eocpatients there was significantly increased frequency of idodcs in tumor draining ln compared to the normal donorln besides in vitro study revealed ido significantly inhibitedproliferation oftumorassociated lymphocytes derived fromeoc patients qian many factors are aï¬ecting the actual dc functions andbehaviors which are with high plasticity contributing to eitherprotumor or antitumor eï¬ect tumor expressing moleculesare associated with mature dc ltration recently macgregor 2019a found higher surface expression of b7h4 ab7 family molecule was correlated with higher mature dccd11chladrhigh ltration in eoc patient sampleswhich may be associated with increased expression of cxcl17a monocyte and dc chemoattractant in those tumors thisgroup have also found that tumourtostroma ratio tsr whichrepresents the percentage of malignant cell component relativeto the stroma in the tumor tissue have an impact on ltrateddc phenotype high tsr was associated with elevated pdl1expression on mature dcs cd11chladrhigh ltrating inovarian tumor tissue macgregor 2019bdc functions can be regulated by their interactions withthe proximal milieu so diï¬erent locations of dcs may resultin diï¬erentfunction labidigaly discoveredthat in ovarian cancer patients tumor pdcs produced lesspro‚ammatory cytokinesfrom ascites orperipheral bloodthan pdcscan vary by diï¬erentalso dc performancetumordevelopment stage in an ovarian cancer mouse model atthe early stage tumor growth was prevented by ltrating dcsand dc depletion at this stage accelerated tumor expansion atthe advanced stage however dcs become immunosuppressivein the tme abrogating enduring activity of antitumor t cellsand dc depletion at this stage significantly delayed diseaseprogression scarlett similarly in a mouse model ofovarian cancer krempski also found progressivelygained immunosuppressive phenotype of ltrating dcs as thetumor progressed over time represented by gradually increasedpd1 expressionmore studies are favored in the future to reveal facts onhow dcs functions are regulated thereby providing clues fortherapeutic strategies in maintaining their antitumor potentialmyeloidderived suppressor cellsmyeloidderived suppressor cells mdscs are a heterogeneouspopulation of myeloid cells that coexpress the myeloid surfacemarkers gr1 and cd11b atretkhany and drutskaya mdscs consist of three phenotypes pmnmdsc mmdsc anda small group of cells that have myeloid colonyforming activityincluding myeloid progenitors and precursors gabrilovich pmnmdscs are similar to neutrophils in phenotypeand morphology and represent over of mdscs whilemmdscs are similar to monocyte gabrilovich studieshave confirmed that mdscs promote tumor progression byvarious mechanisms first mdscs are implicated in immunesuppression ostrandrosenberg and fenselau despitetheir involvementin the inhibition of many cells in theimmune system mdscs mainly target t cells we summarizedthe mechanisms involved in immune suppression mdscsaccelerate lymphocyte nutrient depletion rodriguez srivastava both larginine and lcysteine areessential amino acids that are important for t cell activationand function mdscs produce arg1 and depletion of largininethrough an arg1dependent manner rodriguez mdscs also sequester lcysteine srivastava thereforethe amount of ζchain in the tcr complex isdownregulated and the proliferation of antigenactivated tcells is suppressed mdscs disturb lymphocyte traffickingand viability hanson sakuishi galectin which is expressed in mdscs binds to tim3 onlymphocytes which induces the apoptosis of t cells sakuishi similarly mdscs express adam17 which canfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cyang tumor microenvironment in ovarian cancerdecrease the lselectin level on t cells and limit t cell recruitmentin lymph nodes hanson mdscs promotetreg cell activation and expansion gabrilovich mdscs stimulate cd4 t cells to translate into induced tregitreg cells and expand natural treg ntreg cells theseprocesses are associated with cd40cd40l interactions ifnγ il10 and tgf mdscs stimulate the generation ofoxidative stress oxidative stress is linked to ros and rnsreactive nitrogen species gabrilovich superoxide reactswith no and generates pnt peroxynitrite which nitratestcell receptors and limits the response of antigenmhccomplexes thus suppressing t cells directly pnt also nitratestcellspecific chemokines which decreases the combination ofantigenic peptides to mhc and limits the migration of t cellsmolon moreover mdscsfacilitate neovascularization throughdiï¬erent mechanisms hypoxia in tumors induces mdscs toproduce vegf fgf2 and mmp9 interestingly the activation ofstat3 in mdscs also stimulates neovascularization through il1 ccl2 and cxcl2 release bruno additionallythese factors stimulate invasion and metastasis by producingmmps ostrandrosenberg and fenselau mdsc is an important part of the tumor microenvironmentmdsc promote tumor progression by regulating immunesuppression and facilitating neovascularization moreover therelated to diï¬erentdiï¬erentfunctions and diï¬erentiation of mdsc neverthelessthemechanism is still not cleartumor microenvironmentisthe tmelymphocyteslymphocytes a major component ofinclude blymphocytes and t lymphocytes and mediate innate and adaptiveimmunity respectively sadelain b lymphocytesaccelerate tumor progression by producing protumorigeniccytokines and regulating the th1 th2 ratio quail and joyce t lymphocytes a major component of the tme are crucialfor adaptive immunity sadelain t cells developin the thymus before encountering the initial antigen t cellsare regarded as naïve tn cells after antigen encounter naïvetn cells are activated and start diï¬erentiation smithgarvin they proliferate rapidly and release ‚ammatorycytotoxic granules and cytokines which activate the immuneresponse according to the cytokine environment t cellsdiï¬erentiate into various subsets wang m due to the exclusive expression of cd4 or cd8 markersmature t cells are categorized into cd3cd4 cd3cd8t cells and cd4 treg cells kishton cd3cd4t cells are also called helper t cells th cells and regulateimmune responses by releasing cytokines that promote orinhibit ‚ammation joyce and pollard cd3cd4t cells can be divided into th1 and th2 cells among themth1 cells produce and release pro‚ammatory cytokines andassist cd3cd8 t cells in tumor rejection therefore th1cells are antitumorigenic however th2 cells release anti‚ammatory cytokines and promote tumor progression joyceand pollard quail and joyce cd3cd8 t cellscalled cytotoxic t lymphocytes ctls produce ‚ammatorycytokines and cell lytic molecules such as perforin and granzymewhich specifically recognize and destroy pathogeninfected ormalignant cells joyce and pollard zhang and bevan treg cells cd4cd25foxp3 also play a crucial rolein the immune response patsoukis duringdevelopmentin the thymus treg cells universally expressfoxp3 representing “ of cd4 t cells when respondingshow suppression tregto tcr and tgf treg cellscells protect hosts against autoimmune diseasesthroughinhibiting self and autoreactive cells de aquino additionally treg cells play a tumourigenic role mainly throughimmunosuppression monitoring lindau tregcells regulate the immune response through four mechanismsvignali facciabene secretingimmunosuppressive molecules treg cells suppress eï¬ector t cellfunctions by secreting cytokines such as il10 il35 and tgfadditionally il10 and tgf are reported as key mediatorsthat limit antitumor immunity and promote tumor progressionfacciabene interestingly these cytokines not onlyinhibit the function of eï¬ector cells but also promote dcpolarization to tolerogenic phenotypes additionally treg cellssecrete vegf which is also an immunosuppressive moleculevignali through vegf treg cells exert inhibitionand regulate the diï¬erentiation of dcs cytolysis treg cellsinduce the apoptosis of eï¬ector cells by secreting granzyme b andperforin vignali metabolic disruption severalmechanisms have been reported for the metabolic disruptionregulated by treg cells however it is still controversial tregcells deplete the local level of il2 which causes eï¬ector cellsto starve and results in the apoptosis of eï¬ector cells moreoverwith the expression of cd73 and cd39 treg cells catalyze atp toadenosine which inhibits the function of eï¬ector t cells deaglio vignali modulation of dc maturationand function ctla4 cytotoxic tlymphocyte antigen isexpressed on treg cells and cd80 and cd86 are expressedon dcs treg cells induce dcs through ctla4“cd80cd86interactions which induces the release of ido indoleamine23dioxygenase ido expression depletes essential tryptophanand inhibits the function
Colon_Cancer
" postmortem studies can provide important information for understanding new diseases and smallautopsy case series have already reported different findings in covid19 patientsmethods we evaluated whether some specific postmortem features are observed in these patients and if thesechanges are related to the presence of the virus in different ans complete macroscopic and microscopicautopsies were performed on different ans in covid19 nonsurvivors presence of sarscov2 was evaluatedwith immunohistochemistry ihc in lung samples and with realtime reversetranscription polymerase chainreaction rtpcr test in the lung and other ansresults pulmonary findings revealed earlystage diffuse alveolar damage dad in out of patients andmicrothrombi in small lung arteries in patients latestage dad atypical pneumocytes andor acute pneumoniawere also observed four lung infarcts two acute myocardial infarctions and one ischemic enteritis were observedthere was no evidence of myocarditis hepatitis or encephalitis kidney evaluation revealed the presence ofhemosiderin in tubules or pigmented casts in most patients spongiosis and vascular congestion were the mostfrequently encountered brain lesions no specific sarscov2 lesions were observed in any an ihc revealedpositive cells with a heterogeneous distribution in the lungs of of the patients rtpcr yielded a widedistribution of sarscov2 in different tissues with patients showing viral presence in all tested ans ie lungheart spleen liver colon kidney and brains in autopsies revealed a great heterogeneity of covid19associated an injury and theremarkable absence of any specific viral lesions even when rtpcr identified the presence of the virus in many anskeywords covid19 sarscov2 autopsy rtpcr immunohistochemistry correspondence isabellesalmonerasmeulbacbe1department of pathology erasme hospital universit libre de bruxellesulb route de lennik brussels belgium2centre universitaire inter rgional d™expertise en anatomie pathologiquehospitali¨re curepath chirec chu tivoli ulb rue de borfilet 12a jumet belgiumfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cremmelink critical care page of severeacuteincluding coronavirusesrespiratorysyndrome coronavirus sarscov and middle eastrespiratory syndrome coronavirus merscov causesevere acute respiratory failure which is associated withhigh mortality rates the novel sarscov2 strainexhibits phylogenetic similaritiesto sarscov andcauses coronavirus disease covid19 which hasresulted in more than deaths worldwide so faras the pandemic has progressed the pathophysiology ofthis viral infection has become clearer in particular ithas been shown that sarscov2 can directly alter cellfunction by a link to the angiotensin converting enzyme ace2 receptor which is almost ubiquitous in thehuman body nevertheless the mechanisms behind the high mortalityand severe an dysfunction associated with covid19remain poorly understood controversies exist regardingthe occurrence of fatal complications such as pulmonaryembolism or diffuse endothelial injury [ ] as well as onthe roles of direct viral cellular injury or concomitantcomorbidities in the fatality of this disease in this setting autopsy is of great importance to helpphysicians understand the biological characteristics andthe pathogenesis of covid19 most of the previously reported postmortem findings focused on lung morphologyand few data are available on complete postmortemanalyses of other ans [ ] the aim of this study wastherefore to investigate the presence of specific features ofviral injury as well as the distribution of the virus in different ans of patients who died from covid19methodsstudy designin this postmortem study we included the first adultpatients years who died in our hospital either in acovid19 unit or an intensive care unit from march with confirmed sarscov2 infection ie positivertpcr assay on nasopharyngeal swab andor bronchoalveolar lavage specimen exclusion criteria were lack offamily consent and a delay of more than days after deathbefore postmortem examination the study protocol wasapproved by the local ethics committee p2020218data collectionrelevantwe collected demographics comorbiditiesclinical dataincluding duration between symptomonset or hospitalization and death the results of chestcomputed tomography scan andif available microbiological tests and medical treatments eg hydroxychloroquine antivirals or antibiotics and use of ansupport acute respiratory distress syndrome ardsand acute kidney injury aki were defined accordingto standard definitions [ ]postmortem procedurethe belgian public health institute sciensano guidelines were integrated into our postmortem procedure the cadavers were kept in the refrigerator at °cand autopsies were performed to h after death toensure the safety of the autopsy team personal protective equipment consisted of two superposed disposablelatex gloves plastic sleeves ffp3 mask scrub hat clearface visor surgical gown plus plastic apron and rubberboots in the postmortem room dirty and clean circulations were used in the airlocks to allow decontaminationall analyses were performed at normal pressurespleen bone marrow kidney bladderusing standard surgical pathology processing completesets of tissue samples were collected for diagnosis andbiobanking the material was biobanked by biobanqueh´pital erasmeulb be_bera1 cub h´pital erasmebbmrieric the banked material consists of samplesper an including the trachea thyroid lymph nodesheartliverstomach colon and brain for the lungs we collected sixsamples per lobe ie a total of samples except fortwo patients who had undergone lobectomy for cancerand from whom only samples were taken for safetyreasons complete brain removal was not allowed butwith the help of a neurosurgeon in cases we used anew safe procedure with drills and protective devicesto avoid air dispersion to obtain between and samples from different brain regions as detailed inthe additional file additional material formalinfixed paraffinembedded ffpetissues underwentstandard processing to provide hematoxylin and eosinhestained sections special stains and immunohistochemistry ihc were used for lung masson™s trichromeperiodic acidschiff [pas] gomorigrocott anticmvihc antihsv ihc antipneumocystis j ihc and kidneypas masson™s trichromejones methenamine silversamplesmorphological analysismorphological analysis was performed on he stainedglass slides using the secundos digital platform tribvnhealth care chatillon francefor digital diagnosisafter the acquisition of whole slide digital scans — magnification using a nanozoomer ht slide scanner hamamatsu hamamatsu city japansarscov2 detection by immunohistochemistrysince no antibody against sarscov2 has been validatedfor ihc on ffpe tissues we selected an antisarsnucleocapsid protein antibody standard ihc was appliedas previously described to 4μmthick postmortem lungsections one sample for each lung lobe per patient to display sarsnucleocapsid protein invitrogen pa141098dilution on dako omnis agilent technologies 0cremmelink critical care page of santa clara ca usa using the envision flexdetection system according to the manufacturer™sprotocol the sections were counterstained withhematoxylin negative tissue controls were obtainedfrom patients who had an autopsy before the covid pandemic semiquantitative ihc evaluation wasperformed by two senior pathologists nd mr as follows negative ˆ’ between one and five positive cellsper whole slide scattered cells more than five cellsper whole slide but no foci isolated cells andwith foci more than cells in one — field sarscov2 detection by rrtpcrtotal nucleic acid was extracted from ffpe tissues usingthe maxwell rsc dna ffpe kit reference as1450promega corporation madison wi usa and thepromega maxwell extractor following the protocol described by the manufacturer onestep rtpcr assaysspecific for the amplification of sarscov2 e envelopeprotein gene were adapted from a published protocol briefly μl of rna ng was amplified in μl reaction mixture containing μl of taqman fastvirus 1step master mix life technologies μm ofeach forward acaggtacgttaatagttaatagcgt and reverse atattgcagcagtacgcacacaprimers and μm of probe famacactagccatccttactgcgcttcgbbq the amplification condition was °c for min for reverse transcriptionfollowed by °c for s and then cycles of °c for s and °c for s a clinical sample highly positivefor sarscov2 was diluted and used as a positive control in each analysis a clinical sample obtainedfrom a patient who was autopsied before the covid19pandemic was used as a negative control the quality ofthe rna from the samples showing negative results wasassessed by amplification of the human met rna according to a validated iso15189 accredited methodused as a routine diagnostic method in our laboratorystatistical analysisdata are reported as counts percentage or medians[interquartile ranges iqrs] all data were analyzedusing graphpad prism version graphpad software san diego ca usaresultsstudy cohortthe main characteristics of the study cohort malesout of median age [“] years are given intable the time period between the onset of symptoms and death ranged from to days median days and between admission and death from to days median days all except two patients had atleast one comorbidity including hypertension n diabetes n cerebrovascular disease n coronaryartery disease n and solid cancer n none ofthe patients had tested positive on admission for therespiratory syncytial virus or influenza a and b viruseseleven of the patients were treated with mechanicalventilation eleven patients died in the icu and on themedical ward the main causes of death were respiratoryfailure n and multiple an failure n laboratory data are reported in additional file table s1macroscopic findingsone patient had had a left pneumonectomy and onepatient a right bilobectomy the lungs were typicallyheavy and the lung parenchyma had a diffuse firmconsistency with redtan and patchy darkred areas ofhemorrhage thrombi were found in the large pulmonary arteries in patients and lung infarction in patientspleural adhesions associated with pleural effusions were observed in cases we observed cardiomegaly in and hepatomegaly in patients the kidneys were often enlargedwith a pale cortex and petechial aspect but no hemorrhageor infarct the gut had advanced postmortem autolysiswith no evidence of specific lesions except for one patientwho had ischemic enteritis in the patients for whombrain samples were available one had had a recently drainedsubdural hematoma and another a cerebral hemorrhagemicroscopic findingsfile as shown in figs and and additionaltable s2the main pulmonary findings includedearlystage diffuse alveolar damage dad which consisted ofinterstitial and intraalveolar edema withvariable amounts of hemorrhage and fibrin depositioninterstitial mononuclearhyaline membranes minimalinflammatoryii pneumocytehyperplasia microthrombi were noted in the smallpulmonary arteries in patients ten of the patientsalso had advanced dad lesions ie fibroblastic proliferation within the interstitium and in the alveolar spaces patients had evidence of acute pneumonia or bronchopneumonia had atypical pneumocytes and three hadsyncytial multinucleated giant cells we observed no viralinclusions or squamous metaplasiaand typeinfiltrateall the patients who survived more than weeksn had late dad lesions there was no relationship between the delay from onset of symptoms todeath orfrom hospitalization to death and thepresence of other histological lesions including bronchopneumonia pneumonia microthrombiischemiclesions pulmonary emboli or pulmonary infarct in of the patients who had not received mechanicalventilationthe delay between hospitalization anddeath was less than days in this group only casehad microthrombi the other patients had longer 0cremmelink critical care page of table characteristics of the study populationidct scancomorbiditiesagesexrrtpcrpostime todeathantemorteman failureardsakiposnegnegmfmfmcadcvddiabeteshypertensioncadcrfliver cirrhosiscopdcancerhypertensioncancercvdcopdcancerggoposmaggopospostreatmentscause of deathmechanicalventilationantibioticshydroxychloroquineantibioticscorticosteroidsmechanical ventilationhydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationhydroxychloroquineantibioticsmechanical ventilationecmorrthydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationecmohydroxychloroquineremdesivircorticosteroidsantiobioticshydroxychloroquineantibioticscardiogenicshockmofrespiratory failurerespiratory failurerespiratory failuremesentericischemiamofrespiratory failurerespiratory failureantibioticsseptic shockmofmechanical ventilationrrthydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationecmorrthydroxychloroquineoseltamivirantibioticshydroxychloroquineantibioticsrespiratory failurerespiratory failuresudden deathmechanical ventilationhydroxychloroquineantibioticsmofhydroxychloroquinerespiratory failurehydroxychloroquineantibioticsrespiratory failureardsakihypoxichepatitisardsakiardsardsakihypoxichepatitisardsakiardsakihypoxichepatitisakiardsakiardsakiardsardsakihypoxichepatitisardsardsakihypoxichepatitismhypertensioncrfbcposmnonebcposmfhypertensioncadcvdcrfdiabeteshypertensiondiabetesemphysemaposggoposmhypertensiondiabetesggobcposmmmfdiabetesliver cirrhosiscancerdiabeteshypertensioncaddiabetesdiabeteshypertensiondiabetesbcposggoposggobcggobcpospos 0cremmelink critical care page of table characteristics of the study population continuedidcomorbiditiesct scanagesexmggolpposrrtpcrtime todeathfmhypertensiondiabetesliver transplanthypertensioncvdggobcposggobclpposantemorteman failureardsakipulmonaryembolismardsakipulmonaryembolismardsakipulmonaryembolismtreatmentscause of deathmechanical ventilationrrthydroxychloroquineremdesivirantibioticsmechanical ventilationrrthydroxychloroquineantibioticsmechanical ventilationecmorrthydroxychloroquineantibioticsseptic shockmofseptic shockmofseptic shockmoftime to death time from admission to death days cause of death was reported by the attending physician m male f female rrtpcr reverse transcription realtime polymerase chain reaction used as diagnostic laboratory test neg negative pos positive cad coronary artery disease cvd cerebrovascular disease lp lobarpneumonia ggo groundglass opacity ma minor abnormalities bc bilateral consolidation copd chronic obstructive pulmonary disease crf chronic renal failureards acute respiratory distress syndrome aki acute kidney injury ecmo extracorporeal membrane oxygenation rrt renal replacement therapy mof multiplean failuredelays between hospitalization and death daysthey had no microthrombififteen patients had signs of chronic ischemic cardiomyopathy of different severities and patients had signsof acute myocardial infarction there was no evidence ofcontraction bands or myocarditis histological evaluationof the kidneys was limited because of moderate to severepostmortem autolysis occasional hemosiderin granuleswere observed in the tubular epithelium in patientsand pigmented casts in in the medulla edematousexpansion of the interstitial space without significant inflammation was observed in patients chronic renal lesions ie nodular mesangial expansion and arteriolarhyalinosis glomerulosclerosis or chronic pyelonephritisfig main histological findings green finding present gray finding absent black unavailable 0cremmelink critical care page of fig pulmonary histological findings a earlystage diffuse alveolar damage dad hyaline membrane he — magnification with a zoom ona giant cell — magnification b fibrin thrombi in a pulmonary artery he — magnification c latestage dad fibroblastic proliferationhe — magnification d latestage dad fibroblastic proliferation trichrome staining — magnification e acute pneumonia he — magnification f antisarscov immunohistochemistry ihcpositive cells — magnificationwere also observed no microthrombi were identifiedbut one patient had a thrombus in an interlobar arteryliver examination revealed congestive hepatopathyand steatosis but no patchy necrosis hepatitis or lobular lymphocytic infiltrate the histological changes in theabdominal ans including the esophagus stomachand colon are reported in additional file table s2most of the findings were related to chronic underlyingdiseases except for one case of ischemic enteritisbrain samplesshowed cerebral hemorrhage orhemorrhagic suffusion n ischemic necrosisn edema andor vascular congestion n anddiffuse or focal spongiosis n we found no evidence of viral encephalitis or vasculitis isolated neuronalnecrosis or perivascular lymphocytic infiltrationfocalsarscov2 detection in the lungs by ihcsarscov2 was identified by ihc in the lungs of ofthe patients fig howeverthere was largevariability in the distribution of sarscov2positivecells in the lung parenchymasarscov2 detection by rtpcrsarscov2 rna was detected in at least one anfrom every patient fig in the lung rtpcr waspositive in patients with threshold cycle ct valuesvarying from to viral rna was alsodetected in the heart n the liver n thebowel n the spleen n and the kidney n as well as in of the cerebral samples ct valuesfor nonpulmonary ans ranged from to eight patients had positive rtpcr in all tested ansabnormalitiesdiscussionthis postmortem study showed several histopathologicalin covid19 nonsurvivorshowever none of the findings was specific for direct viralinjury even though sarscov2 was detected in all examined ans using rtpcr we decided to performcomplete autopsies rather than other techniques such aspostmortem core biopsies so as to obtain a better overview of all ans especially the lungs we collected samples from each lobe this approach enabled us to 0cremmelink critical care page of fig detection of sarscov2 by immunohistochemistry ihc in ffpe post mortem lung samples of patients semiquantitative evaluationœˆ’ negative result œ scattered positive cells between and positive cellswhole slide œ positive isolated cells cellswhole slide butno foci œ foci of positive cells more than positive cells in one — field na not availabledocument the considerable heterogeneity of histologicallesions and of sarscov2 spread through the bodythe diagnosis of sarscov2related an injury ischallenging postmortem histologicalfindings wereheterogeneous and often associated with chronic underlying diseases in a previous autopsy study in covid19patients the authors reported that dad associatedwith viral pneumonia was almost impossible to distinguishfrom that caused by bacterial pneumonia no obviousintranuclear or intracytoplasmic viralinclusions wereidentified in another report desquamation of pneumocytes and hyaline membrane formation are frequentlydescribed in ards of many different causes especially inearlyphase ards the presence of multinucleatedcells with nuclear atypia is used to diagnose herpes virusinfection in daily practice as in previous reports [ ]we also observed the presence of multinucleated cellswithin lung alveoli in three patients however the significance of multinucleated cells is unclear and may not bespecific of sarscov2 infection finally some ofthe microscopic features of these patients are compatiblewith an changes secondary to shock or systemicinflammation and no histological finding could be specifically ascribed to sarscov2in the absence of typical postmortem viral featuresour results show that rtpcr is feasible on ffpe blocksand could be used in postmortem analyses to identifythe presence of sarscov2 in multiple ans and tounderstand the spread of the virus within the humanbody the discordant rtpcr and ihc results fordetection of sarscov2 in the lungs may be explainedby the different sensitivity of these assays which washigher for the rtpcr whereas lowlevel viral replication might not be detected by ihc moreover ihc wasbased on the only available antibodies which aretargeted against sarscov new antibodies againstsarscov2 need to be developed to improve theaccuracy of ihc in the analysis of tissue samples fromsuspected or confirmed covid19 patientsmost of the previous postmortem studies in covid19patients were conducted using needle biopsies and weretherefore rather limited in terms of sampling our completeautopsy analysis identified considerable heterogeneity ofsarscov2 spread through the human body and providesa more accurate description of macroscopic and microscopic an alterations as for previous coronavirusdiseases [ ] the lungs are the most affected ans incovid19 however dad findings werehighly 0cremmelink critical care page of fig molecular detection of sarscov2 rna in postmortem samples detection of sarscov2 by reverse transcription realtime polymerasechain reaction rtpcr in ffpe postmortem tissues of patients œ positive result œˆ’ negative result œna tissue not available nc noninformative test result due to lowquality rnaheterogeneous including both earlyonset and additionallate lesions this finding could be explained by the heterogeneity of the pulmonary injury including compliant lungsin the early phase and a more dense and nonrecruitablelung in the late phase as some patients died outsidethe icu without receiving mechanical ventilation we couldnot estimate lung compliance before death the heterogeneity could also reflect different treatments eg fluid administration or corticosteroids or different complications asan example half of the patients had concomitant acutepneumonia and it is difficult to conclude whether the dadreflected the natural timecourse of the viral disease or wassecondary to superimposed complications such as nosocomial infections in a recent report needle postmortem biopsies suggested that covid19 is not associated withdad but rather with an acute fibrinous and anizingpneumonia afop consequently requiring corticoid treatment a diagnosis of afop is based on the absence ofhyaline membranes and the presence of alveolar fibrin ballshowever hyaline membranes are heterogeneously distributed in the lung parenchyma with dad and complete lunganalysis not just biopsies are necessary to exclude theirpresence moreover afop may be a fibrinous variant ofdad the limitation of lung biopsy was also shown inanother study in which only of lung samples werepositive for sarscov2 using rtpcr when compared to almost in our series in addition we did notfind specific œendothelitis as previously reported in a smallcase series considering the heterogeneity of postmortem covid19 associated lesions molecular and ihcassessments are mandatory in the histological analysis ofcovid19 tissue samplespatients with covid19 often have altered coagulation and a prothrombotic status with the possible development of acute pulmonary embolism pe in ourstudy three patients had pe already diagnosed beforedeath four patients had pulmonary infarction in a previous study acute pe was considered as the main causeof death in four patients however the inclusion ofpatients who died before hospital admission and the lackof specific thromboprophylaxis during the hospital staymay account for the differences in the severity of pewhen compared to our study although we frequentlyobserved the presence of microthrombi in the lung parenchyma this feature is also reported in other forms ofards regardless of etiology [ ] as such whetherdiffuse pulmonary thrombosis is a main contributor ofthe fatal course of severe hypoxemia in covid19 0cremmelink critical care page of patients remains to be further studied in a systematicreview of pathologicalfindings in covid19 polak identified a timeline in the histopathologicalfindings in the lung with epithelial dad denudationand reactive pneumocytes atypia and vascular microvascular damage thrombi intraalveolar fibrin depositschanges present at all stages of the disease but fibroticchanges interstitialfibrous changes only appearingabout weeks after the onset of symptoms few patientshad fibrosis at early stages and in these cases it waslikely because of preexisting lung disease our resultsare consistent with those of polak except forthe lack of late fibrotic changes which may be related tothe use of antiinflammatory drugs at high doses fornearly all our patients we did not observe specific viral an injury such asmyocarditis hepatitis or encephalitis the cases ofœacute cardiac injury reported in covid19 clinicalstudies do not necessarily translate into myocarditisor acute myocardialischemia only two had acutemyocardial ischemia similar to data reported in septicpatients ie elevated troponin without overt cardiacischemia however using rtpcr we found thevirus in almost all the examined ans this suggeststhat the virus can bind to most cells probably via theace2 receptor which is ubiquitous but may notdirectly cause an injury as extrapulmonary directviral injury eg encephalitis hepatitis or myocarditishas only been reported in very few cases we suggest thatsarscov2 infection may be just the trigger for anoverwhelming host response which could secondarilyresult in covid19associated an dysfunction asrtpcr mightitremains unclear whether this represents active viral replication into the tissues or previous cellular infectionwithout clinically relevant significance just detect residual viral genomethis study has several limitations i we only includedpatients who had had a positive rtpcr on nasopharyngeal swab andor bronchoalveolar lavage to ensurethat only true positive cases were enrolled we decidednot to include three patients who had had thoracic ctscan findings suggestive of covid19 but had negativertpcr results this limitation in our study reflects thedifficulty of diagnosing covid19 on a clinical basis iithe sample size was relatively small and autopsies wereonly carried out from to h after death this delaydid not allow us to properly analyze the gastrointestinaltract and kidneys which showed signs of autolysis inparticular acute tubular injury in the proximal tubuleswas indistinguishable from autolysis iii we could notdetermine the timecourse andor sequence of anspread of the virus and no specific hypothesis regardinghow sarscov2 spreads eg hematogenously couldbe identified and iv the time to death differed frompatient to patient as did the course of the disease andtreatments received which limits a precise clinicalpathological correlation of histological findings related tocovid19 finally we did not evaluate specific mechanisms involved in the pathogenesis of an injurythese results underline the heterogeneity of an injuriesduring covid19 disease and the absence of specificsarscov2 lesions using rtpcr sarscov2 couldbe detected in all ans even those without evidentmicroscopic lesionssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s13054020032185additional file critical careautopsycovid additional materialprocedure to obtain brain samplesadditional file critical careautopsycovid additional table s1laboratory findings on the day of admissionadditional file critical careautopsycovid additional table s2detailed histological findings in all patientsabbreviationsace2 angiotensin converting enzyme afop acute fibrinous andanizing pneumonia aki acute kidney injury ards acute respiratorydistress syndrome covid19 coronavirus disease ct threshold cycledad diffuse alveolar damage ffpe formalinfixed paraffinembeddedhe hematoxylin and eosin ihc immunohistochemistry iqrs interquartileranges merscov middle east respiratory syndrome coronaviruspas periodic acidschiff pe pulmonary embolism rtpcr realtime reversetranscription polymerase chain reaction sarscov severe acute respiratorysyndrome coronavirusacknowledgmentsthe authors thank nathalie lijsen christophe valleys gees lacroixbarbara alexiou dominique penninck nicole haye and audrey verrellen fortechnical and logistic supports prof frdric schuind and dr djameleddineyahiacherif for neurosurgical procedure egor zindy diapath ulb forproofreading the paper and dr mariepaule van craynest for trainees™supervisionauthors™ contributionsis had the idea for and designed the study and had full access to all thedata in the study and takes responsibility for the integrity of the data andthe accuracy of the data analysis is ft jlv and cd drafted the paper mrcv ll pl mlr cm alt jcg lp rdm sd sr nd lp and od collected thedata mr nd and rdm did the analysis and all authors critically revised themanuscript for important intellectual content and gave final approval for theversion to be published all authors agree to be accountable for all aspectsof the work in ensuring that questions related to the accuracy or integrity ofany part of the work are appropriately investigated and resolvedfundingthis study received financial support from fonds y bo«l brussels belgiumfonds erasme pour la recherche mdicale brussels belgium and œappel  projet spcial covid19 ulb brussels belgium the cmmi is supported bythe european regional development fund and the walloon region ofbelgium walloniabiomed grant no project œcmmiulbsupport the center for microscopy and molecular imaging and its diapathdepartment cd is a senior research associate with the fnrs belgiannational fund for scientific research 0cremmelink critical care page of availability of data and materialsthe data that support the findings of this study are available from thecorresponding author on reasonable request participant data without namesand identifiers will be made available after approval from the correspondingauthor and local ethics committee the research team will provide an emailaddress for communication once the data are approved to be shared withothers the proposal with a detailed description of study objectives andstatistical analysis plan will be needed for the evaluation of the reasonabilityto request for our data additional materials may also be required during theprocess d'haene n melndez b blanchard o de n¨ve n lebrun l vancampenhout c design and validation of a genetargeted nextgeneration sequencing panel for routine diagnosis in gliomas cancersbasel corman vm landt o kaiser m molenkamp r meijer a chu dk detection of novel coronavirus 2019ncov by realtime rtpcr eurosurveill de hemptinne q remmelink m brimioulle s salmon i vincent jl ards aclinicopathological confrontation chest “ menter t haslbauer jd nienhold r savic s hopfer h deigendesch n ethics approval and consent to participatethe study protocol was approved by the local ethics committee erasmehospital p2020218 the ethical committee has waived the need for writteninformed consentpostmortem examination of covid19 patients reveals diffuse alveolardamage with severe capillary congestion and variegated findings of lungsand other ans suggesting vascular dysfunction histopathology epub ahead of print httpsdoi101111his14134franks tj chong py chui p galvin jr lourens rm reid ah lungpathology of severe acute respiratory syndrome sars a study of autopsy cases from singapore hum pathol “ nicholls jm poon ll lee kc ng wf lai st leung cy lungpathology of fatal severe acute respiratory syndrome lancet “ hwang d chamberlain d poutanen s low de asa sl butany j pulmonarypathology of severe acute re
Colon_Cancer
" the empirical dietary index for hyperinsulinemia edih score is a validated foodbased dietary scorethat assesses the ability of wholefood diets to predict plasma cpeptide concentrations although the edih hasbeen extensively applied and found to be predictive of risk of developing major chronic diseases its influence oncancer survival has not been evaluated we applied the edih score in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patterns after diagnosis and determine its influence on survivaloutcomesmethods we calculated edih scores to assess the insulinemic potential of postdiagnosis dietary patterns andexamined survival outcomes in a sample of stage iiii colorectal cancer patients in the nurses™ health studyand health professionals followup study cohorts multivariableadjusted cox regression was applied to computehazard ratios hr and confidence intervals ci for colorectal cancerspecific mortality and allcause mortalitywe also examined the influence of change in diet from pre to postdiagnosis period on mortalityresults during a median followup of years there were deaths which included colorectal cancerspecific deaths in the multivariableadjusted analyses colorectal cancer patients in the highest compared tolowest edih quintile had a greater risk of dying from colorectal cancer hr ci and a greater risk of allcause death hr 95ci compared to patients who consumed low insulinemicdiets from pre to postdiagnosis period patients who persistently consumed hyperinsulinemic diets were at higherrisk of colorectal cancer death hr151 95ci and allcause death hr 95ci our findings suggest that a hyperinsulinemic dietary pattern after diagnosis of colorectal cancer isassociated with poorer survival interventions with dietary patterns to reduce insulinemic activity and impactsurvivorship are warrantedkeywords colorectal cancer survival insulinemic dietary patterns insulin cpeptide correspondence fredtabungosumcedu1division of medical oncology department of internal medicine the ohiostate university college of medicine west 12th avenue 302b wisemanhallccc columbus oh usa2the ohio state university comprehensive cancer center arthur g jamescancer hospital and richard j solove research institute columbus oh usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctabung bmc cancer page of canceristhefourth most colorectalcommonlydiagnosed cancer in the united states while there ishigh potential for dietary patterns as a modifiable riskfactor for colorectal cancer development [ ] verylimited evidence exists among colorectal cancer survivors for example in a recent review we identified s published up to that reported on theassociation between dietary patterns and colorectalcancer development but only about five s onthe association between dietary patterns and outcomesamong colorectal cancer survivors [“] the evidenceshowed that the western dietary pattern often characterized by high intakes ofred andprocessed meats desserts and potatoesis associatedwith higher risk of allcause mortality but generally notwith colorectal cancerspecific mortality in patients withcolorectal cancer the prudent dietary pattern oftencharacterized by high intakes of fruits vegetables wholegrains and poultry showed similar results with inverseassociations for allcause mortality but no consistent association with colorectal cancerspecific mortality [“]higher adherence to other dietary patterns including themediterranean diet score dietary approaches to stophypertension meal plan american cancer society cancerprevention guidelines score healthy eating index scorewere generally associated with lower risk of allcausemortality but the associations were inconsistent acrossstudies [ ]refined grainschronic diseasesfurther research is therefore needed to clarify ifdietary patterns are importantfor colorectal cancerprognosis and if dietary changes can maximally impactoverall and cancerspecific survival biomarkerbaseddietary patterns may be helpfulin this regard forexample hyperinsulinemia and insulin resistance areconsidered important underlying mechanisms linkingpoor dietary patterns and lifestyle behaviors to the development of multipleincludingcolorectal cancer [“] studies have shown positiveassociations between circulating cpeptide concentrations a marker of beta cell secretory activity and colorectal cancer risk and prognosis [“] therefore adietary pattern associated with hyperinsulinemia may bemore predictive of outcomes following colorectal cancerdiagnosis than a dietary pattern not associated with thispathway we previously derived the empirical dietaryindex for hyperinsulinemia edih score to assess thepotential of dietary patterns to influence insulinemia which has been extensively applied in large cohortstudies and found to be predictive of nonfasting cpeptide concentrations [ ] longterm weight gain risk of developing colorectal cancer other digestive system cancers and other cancers however the influence of dietary insulinemic potentialon cancer survival outcomes has not yet been evaluatedthe objective of the current study is to apply the edihscore in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patternsafter diagnosis and determine its influence on survivaloutcomesmethodsstudy populationwe used data from the nurses™ health study nhs andthe health professionals followup study hpfs twoongoing cohorts in the united states hpfs was initiatedin and enrolled male health professionalsbetween the ages of and years nhs initiatedin enrolled registered female nurses aged to years fig data on medical lifestyleand other healthrelated factors was collected at baselineand have been updated every years thereafter ethicalapproval for our study was provided by the harvardth chan school of public health and those of participating registries as required and the institutional reviewboards of the brigham and women™s hospital studyparticipants provided consent by completing and submitting study questionnaires participants were free toterminate participation in the study at any timeassessment of diet and the empirical dietary index forhyperinsulinemia edih scorein both cohorts diet was assessed using a validated selfadministered food frequency questionnaire ffq thatassessed how often on average participants consumed astandard portion size of various foods in the past yearin the nhs diet was assessed in andevery years thereafter whereas in the hpfs diet wasassessed in and every years thereafter theedih score developed to empirically measure the insulinemic potential of whole diets using food groups hasbeen described in detail briefly thirtynine foodgroups were entered into stepwise linear regressionmodels to identify a dietary pattern most predictive ofplasma cpeptide levels the edih score represents aweighted sum of food groups with higher scores indicating hyperinsulinemic diets hyperinsulinemia andlower scores indicating low insulinemic diets the foodgroups contributing to lower edih scores are winecoffee fullfat dairy products whole fruit and green leafyvegetables whereas the food groups contributing to highedih scores arelowfat dairy products french frieslowenergy beverages cream soups processed meat redmeat margarine poultry nondark fish high tomatoesenergy beverage and eggs in the current study we calculated edih scores foreach participant based on the selfadministered ffqspostdiagnosis edih score was calculated based on the 0ctabung bmc cancer page of fig flow chart describing the flow of participants from the full cohorts to the final analytic sample in the nurses™ health study nhs andhealth professionals followup study hpfsfirst ffq returned at least months but not more than years after colorectal cancer diagnosis thus avoidingdiet assessment during active cancertherapy themedian time from diagnosisto postdiagnosis dietassessment was years prediagnosis edih score wascalculated based on the cumulative average of edihscores up to the last diet assessment before colorectalcancer diagnosis the median time from prediagnosisdiet assessment to diagnosis was yearspatients with colorectal cancer and mortality assessmentwhen a colorectal cancer diagnosis was reported duringthe previous years on the followup biennial questionnaires we requested permission to obtain hospital records and pathology reports blinded study physiciansthen reviewed these records and recorded data on tumorcharacteristics for nonrespondents the national deathindex was used to identify deaths and ascertain anydiagnosis of colorectal cancer that contributed to deathafter years of followup for disease diagnoses “ in nhs and to in hpfs we identified patients with pathologically confirmed colorectalcancer we excluded participants who died before in nhs or in hpfs had reported any cancerexcept nonmelanoma skin cancer before colorectalcancer diagnosis who died at diagnosis who did nothave prediagnosis diet or postdiagnosis diet patientswho did not complete a diet assessment between months and years after diagnosis or had diet assessedoutside of this period who had diabetes at colorectalcancer diagnosis and patients with stage iv or unknownstage at diagnosis therefore the current analysis included patients with stage i ii or iii colorectal cancerincluding participants from hpfs and from nhs fig deaths were ascertained throughreporting by family for persistent nonresponders wequeried the national death index with their names up 0ctabung bmc cancer page of to june for nhs and january for hpfs cause of death was assigned by blinded physicianscovariate assessmentboth cohorts assessed covariate data eg medical historylifestyle and health factorsthrough selfadministeredquestionnaires every years these factors included physical activity smoking habits alcohol intake multivitaminuse endoscopy status regular use of aspirin and othernonsteroidal antiinflammatory drugs nsaids familyhistory of colorectal cancer weight height menopausalstatus and postmenopausal hormone use only forwomenin both cohorts as previously described dietassessment was conducted every years [ ]statistical analysiswe categorized the edih score into quintiles withcohortspecific cutoffs then pooled the data for analysispersontime of followup was calculated from the dateof postdiagnosis diet assessment to death or to lastfollowup date january in hpfs or june innhs whichever was first we used the kaplanmeiermethod to generate survival curves by quintiles of edihscore and tested group differences highest vs lowestquintile using the logrank test for this test the edihscore was adjusted for total energy intake and bmi usingthe residual methodcox proportional hazards regression was used to calculate hazard ratios hrs of colorectal cancerspecificdeath or allcause death in edih quintiles quintile cutpoints were created separately by sex and applied in thepooled sample given that participants must survivefrom diagnosis until postdiagnosis diet assessment weused time since diagnosis as the underlying time scale toaccount for left truncation due to staggered entry thecox models were tested for the assumption of proportionality using timecovariate interaction terms andstratified by age sex and stage we fitted two models tothe data as follows model minimally adjusted modelincluded bmi demographic factors sex age at diagnosis and tumor characteristics stage subsite within thecolon grade of tumor differentiation model fullyadjusted model included all the covariates in model and postdiagnosisfactors packyears ofsmoking physical activity regular aspirin use pre topostdiagnosis weight change total alcohol intake andprediagnosis dietary pattern edih score testforlinear trend of risk across edih quintiles was performedusing the median postdiagnosis edih score in eachedih quintile as a continuous variable in the cox regression models and interpreting the pvalue of thisvariable as the pvalue for linear trendlifestyleto determine how changes in the insulinemic potential of diet before and after diagnosis influence survivalwe dichotomized pre and postdiagnosis edih scores atthe median and used to create a change variable withlow indicating a score below the median and high ascore above the median lowlow consistently lowdietary insulinemic potential before and after diagnosisie both scores below the median lowhigh patientsconsuming low insulinemic diets before diagnosis andmore hyperinsulinemic diets after diagnosis highlowpatients consuming hyperinsulinemic diets before diagnosis and then changed towards low insulinemic dietsafter diagnosis and highhigh patients who consistentlyconsumed hyperinsulinemic dietary patterns before andafter diagnosis we then applied these dietary patternchanges in multivariableadjusted cox models to examine risk of death from colorectal cancer and from othercauseschange preand postdiagnosisfrom postdiagnosis weightwe conducted exploratory subgroup analysesincategories of the following potential effect modifiers sexweightand prediagnosis edih score we categorized prediagnosisedih at the median median and ‰¥ median weightchange was calculated by subtracting prediagnosisweightthe continuousweight change variable was categorized as follows thosewho gained more than kg had a stable weight ˆ’ kg to kg or lost more than kg we also conducted subgroup analyses by time since diagnosis years ‰¥ years and age at diagnosis years ‰¥years tests of interaction between postdiagnosis edihscore and the potential effect modifiers were assessed byentering in the modelthe cross product of postdiagnosis edih score and the stratification variable andevaluated by the wald test all analyses were performedusing sas for unix all pvalues were two sidedresultscharacteristics of patients women with colorectal cancer after diagnosis are shown in table meanage at diagnosis was years and mean postdiagnosisbmi was kgm2 with of patients classified asoverweight or obese regarding disease stage hadstage i or ii and had stage iii disease during amedian followup of years there were allcausedeaths which included colorectal cancerspecificdeaths median overall survival by cancer stage was years for those with stage i disease years for thosewith stage ii and years for those with stage iiidisease fortyone percent of patients maintained astable weight between ˆ’ kg and kg between theprediagnosis and postdiagnosis period while lostmore than kg body weight and gained kgbody weight in the same period colorectal cancer patients with the most hyperinsulinemic dietary patterns 0ctabung bmc cancer page of table postdiagnosis characteristics of colorectal cancer patients by quintiles of postdiagnosis empirical dietary index forhyperinsulinemia edih score n characteristictotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile quintile ˆ’ to ˆ’ to n n quintile ˆ’ to ˆ’ n quintile ˆ’ to n quintile to n female age at diagnosisdage at diagnosis by sexdfemalemalecurrent smoker packyears of smokingbody mass index kgm2overweight bmi ‰¥ obese bmi ‰¥ physical activity methweekcphysical activity methweekcd by sex femalemale nonalcohol drinkers regular aspirin use location of cancer in the colon proximal colondistal colonrectumunspecifiedstage at diagnosis stage istage iistage iiimedian survival time yearsmedian survival time by staged years stage istage iistage iiiweight change categoriesd stable weight ˆ’ to kggained more than kglost more than kg avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intakecmetabolic equivalents from recreational and leisuretime activitiesdvalue is not age adjustedafter diagnosis quintile tended to have higher bodyweight and lower physical activity for example theaverage bmi among those classified in quintile was kgm2 and the average physical activity was methourweek compared with kgm2 and methourweek among those in quintile alsopatients consuming the most hyperinsulinemic dietarypatterns were less likely to have stage i disease and theyexperienced shorter survival times table patients consuming low insulinemic dietary patternshad higher intakes of wholegrains nuts vegetables wholefruits and coffee and lower intakes of refined grainscream soup eggs french fries butter margarine sugarsweetened beverages red meat and processed meat in 0ctabung bmc cancer page of table median 5th 95th percentile food and nutrient intake profiles of colorectal cancer patients by quintiles of postdiagnosisempirical dietary index for hyperinsulinemia edih scoretotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile n quintile n quintile n quintile n quintile n foods servingsweekprocessed meatred meat highenergy sugary beverages lowenergy sugary beverages margarinebutterfrench friesnondark fisheggslowfat dairycream souprefined grainstomatopoultrydark fishfullfat dairycoffeeteawhole fruitfruit juicepotatoesgreenleafy vegetablesdarkyellow vegetablesother vegetablesnuts total alcohol intake drinksweek whole grainsnutrient profile total carbohydrates gdtotal protein gd branchedchain amino acids gd total fat gdtotal fiber gd avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intaketerms of the nutrient profile resulting from this postdiagnosis dietary pattern patients consuming a lowinsulinemic dietary pattern had higher intakes of totalcarbohydrates and total fiber and lower intakes of total fattotal protein and branchedchain amino acids table kaplanmeier curves by quintiles of edih score areshown in fig with patients consuming a lowinsulinemic diet quintile experiencing better survivalfor colorectal cancerspecific and overall mortalitycompared to those consuming hyperinsulinemic dietsquintile in the multivariableadjusted analyses wefound that a hyperinsulinemic postdiagnosis dietarypattern was associated with higher risk of colorectalcancerspecific mortality and allcause mortality table 0ctabung bmc cancer page of fig kaplan“meier curves of a colorectal cancerspecific and b overall survival among patients with colorectal cancer by quintile of postdiagnosis empirical dietary index for hyperinsulinemia edih score logrank pvalues were calculated to test group differences quintile vs and adjusted for postdiagnosis total energy intake and postdiagnosis body mass indexcomparing colorectal cancer patients classified in the highestedih quintile to those in the lowest quintile there was a higher risk of colorectal cancerspecific death hr 95ci ptrend and a higher risk of allcause death hr 95ci ptrend afteraccounting for prediagnosis dietary insulinemic potentialamong other confounding variables table in relation to changes in the insulinemic potential ofthe diet before and after diagnosis patients whoconsumed a more hyperinsulinemic dietary patternconsistently before and after diagnosis were at higherrisk of dying from colorectal cancer hr ci and from other causes hr ci compared to patients who consistentlytable hazard ratios ci for colorectal cancerspecific and allcause mortality among patients with colorectal cancer byquintile of postdiagnosis edih scorestatistical modelcolorectal cancerspecific mortalityquintiles of the empirical dietary index for hyperinsulinemia edih scorequintile quintile quintile quintile ptrendquintile deathspatients aliveminimallyadjusted model reference fully adjusted model reference allcause mortalitydeathspatients aliveminimallyadjusted model reference fully adjusted model reference the minimallyadjusted models was adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stagegrade of tumor differentiation and location of primary tumor within the colon the fullyadjusted model was additionally adjusted for postdiagnosis physicalactivity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake andprediagnosis edih score 0ctabung bmc cancer page of fig hazard ratios for the association of change in dietary insulinemic potential between prediagnosis diet and postdiagnosis diet and risk ofdying form colorectal cancer crcsurvival and from all causes combined overall survival edih scores were dichotomized at the median lowlow the reference category represents participants who persistently consumed low insulinemic diets below the median edih from pre to postdiagnosis period lowhigh are those who changed from low insulinemic diets towards more hyperinsulinemic diets highlow represents thosewho changed from consuming hyperinsulinemic diets prior to diagnosis towards consuming low insulinemic diets after diagnosis whereas highhigh represents those who persistently consumed hyperinsulinemic diets prior to diagnosis and after diagnosis the number of deaths patientsalive in the four categories were as follows crcsurvival lowlow lowhigh highlow highhigh overallsurvival lowlow lowhigh highlow highhigh models were adjusted for age at diagnosis postdiagnosisbody mass index total energy intake sex race year of diagnosis cancer stage grade of tumor differentiation location of primary tumor withinthe colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake and prediagnosis edih scoreconsumed a low insulinemic dietary pattern before andafter diagnosis fig in subgroups of potential effect modifiers risk of colorectal cancerspecific mortality was significantly elevatedamong women and among those who lost body weightthose who were consuming a hyperinsulinemic dietarypattern before diagnosis and those younger than years for these subgroup analysesinteractions werestatistically significant only for sex in allcause mortalitytable discussionin the current study we showed that habitual consumption of hyperinsulinemic dietary patterns after colorectalcancer diagnosis or consumption of a hyperinsulinemicdietary pattern consistently before and after diagnosiswas associated with higher risk of dying from colorectalcancer and from all causes combinedthe insulinemic potential of diet was first estimatedby the insulin index which is based on a conceptsimilar to the more widely used glycemic index thatcharacterizes carbohydratecontaining foods accordingto their ability to raise blood glucose concentrationspostprandially compared with a reference food glucoseor white bread though carbohydrate content isone important factor influencing insulin response foodscan also stimulate insulin secretion in a carbohydrateindependent manner the insulin index directly quantifies the postprandial insulinemic potential of a food andtakes into account foods with a low or no carbohydratecontent it is important to understand that theinsulin index which was used in most previous studiesof dietary insulinemic potential and colorectal cancersurvival is conceptually and technically different fromthe edih and essentially uncorrelated spearman r ˆ’ the principle ofthe insulin index is how aparticularfood item stimulated insulin secretionindependent of underlying insulin resistance whereasthe edih is primarily driven by insulin resistance forcolorectal cancer the only other paper using the edihwas on cancer incidence both the insulin index and glycemic index assess thepostprandial shortterm effects of the diet unlike theedih score which predicts integrated insulin exposureie both fasting and nonfasting based on habituallongterm dietary intake postdiagnosis insulinindex and insulin load have been linked to higher risk ofdying from colorectal cancer [ ] higher dietary insulin load and insulin index after diagnosis of colorectalcancer were associated with increased risk of colorectalcancerspecific and overall mortality the association of postdiagnosis glycemic indices with colorectal 0ctabung bmc cancer page of table subgroup analyses of the association between dietary insulinemic potential and colorectal cancerspecific and allcausemortalitysubgroupptrendpinteractionquartile quartile quartile deathspatientsaliveedih quintilesquartile colorectal cancerspecific mortalitysexmenwomenweight change post minus prediagnosis weightstable weight ˆ’ to kgweight gain kgweight loss kgprediagnosis edih score median‰¥ medianage group at diagnosis years‰¥ yearstime since diagnosis years‰¥ yearsallcause mortalitysexmenwomenweight change post minus prediagnosis weightstable weight ˆ’ to kgweight gain kgweight loss kgprediagnosis edih score median‰¥ medianage group at diagnosis years‰¥ yearstime since diagnosis years‰¥ years ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref models were adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stage grade of tumordifferentiation and location of primary tumor within the colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirinuse weight change pre to postdiagnosis and postdiagnosis total alcohol intake and prediagnosis edih scorecancer prognosis has been inconsistent whereas onestudy found higher risk of colorectal cancer recurrenceand death associated with higher glycemic load but nothigher glycemic index another found no associationbetween glycemic load or glycemic index and colorectalcancer survival glycemic scores are primarilyreflective ofthe postprandial glucose responses ofcarbohydratecontaining foods whereas the edih scoredirectly reflects insulin increases induced by componentsof the dietary pattern that may or may not be contributing to calories eg coffee current study findingstherefore suggest that the direct effect of the diet on 0ctabung bmc cancer page of insulin may be more important than the effect of diet onglucose for colorectal cancer prognosis though theglycemic index is a measure of the shortterm postprandial effect of the diet on glucose concentrations it ispossible that such a habitual dietary pattern could overtime lead to sustained hyperinsulinemia and insulin resistance which could then mediate colorectal cancerprognosis however a previous study in these cohortsdid not observe an association between an overall lowcarbohydrate diet score and colorectal cancer or overallmortality although those who consumed a plantrichlowcarbohydrate diet which emphasized plant sourcesof fat and protein with moderate consumption of animalproducts had lower risk of colorectal cancerspecificmortality insulin is a growth factor and major regulator of cellmetabolism and its effects in target cells are mediatedby the insulin receptor a transmembrane protein withenzymatic activity evidence suggest that insulinstimulates growth mainly via its own receptor and notthe igf1 receptor and that in many cancer cells theinsulin receptor is overexpressed and the a isoformwhich has a predominant mitogenic effectis morerepresented than the b isoform the metabolicpathway stimulated by the activated insulin receptor toregulate glucose protein and lipid metabolism involvesthe pi3kakt pathwaycharacteristicsprovide a selective growth advantage to cancer cellswhen exposed to insulin therefore all conditions ofhyperinsulinemia both endogenousdiabetes metabolic syndrome obesity and exogenouseg hyperinsulinemic diets which also influence someof the endogenous conditions [ ] willincreasecancer risk and mortality theseegtypefor most ofalthough interactionsthe subgroupanalyses were not statistically significant some of thefindings merit some discussion the associations werestronger among women than among men which may berelated to severalfactors the larger sample size andstatistical power in our evaluation of women potentialconfounding with age as women were younger on average than men and a true biological interaction basedupon endocrine and associated metabolic factors wealso observed that there were worse outcomes amongpatients who lost weight than among those who maintained a stable weightto postdiagnosisperiod which may be consistent with complications ofprogressing disease leading to poor diet intakefrom premajor strengths of our study include the use of afoodbased edih score that is correlated with circulating cpeptide concentrations [ ] we had access tocomprehensive pre and postdiagnosis data on diet andimportant covariates which reduces the potential for residual confounding and recall bias our findings alsoaccounted for potential bias from staggered entry due todifferences between participants in the time betweendiagnosis and postdiagnosis diet assessment limitations to be considered in interpreting our findings include potential measurement error in the selfreporteddietary and lifestyle data though prior studies in thehpfs and nhs that evaluated the relative validity offfq data have shown reasonably good correlations between ffq and diet records [ ] though we adjusted for several potential confounding variables ahyperinsulinemic dietary pattern may be associated withother factors not included in the current study therefore we cannot completely rule out confounding byunmeasured variables given that we did not have information on cancer treatment which could influence dietary choices of cancer patients or modify the diet andsurvival association we adjusted all analyses by cancerstage at diagnosis which is the principal determinant ofcolorectal cancer treatmentin this large prospective study a higher edih scorereflecting higher insulinemic potential of the diet wasassociated with higher risk of death from colorectalcancer and from all causes taken together our resultssuggest that this association may be mediated partlythrough mechanisms involving hyperinsulinemia interventions with dietary patterns to reduce insulinemia mayenhance survivorship among colorectal cancer patientsabbreviationsbmi body mass index ci confidence interval edih empirical dietary indexfor hyperinsulinemia score ffq food frequency questionnairehpfs health professionals followup study hr hazard ratio methourweek metabolic equivalent hours per week nhs nurses™ health study nsaids nonsteroidal antiinflammatory drugs pi3kakt phosphatidylinositol kinaseprotein kinase b sas® statistical analysis software®acknowledgementswe would like to thank the participants and staff of the nurses™ health studyand health professionals followup study for their valuable contributions asw
Colon_Cancer
inanic arsenic ias is the chemical form of as commonlyfound in drinking water atsdr and in some foodscubadda chronic exposure to ias has been associatedwith risk of skin bladder lung and liver cancers iarc aswell as with other diseases naujokas including diabetes maull cardiovascular abhyankar moon saquib states respiratorysanchez and neurological diseases caito andaschner parvez humans and most other mammalian species have developed a mechanism for detoxifying iasthat involves the sequential conversion of ias to monomethylasmas and mas to dimethylas dmas thomas both methylation steps are catalyzed by orthologs of a singleenzyme arsenic oxidation state methyltransferase as3mtlin the methylation of ias by as3mt not onlyaddress correspondence to miroslav st½blo department of nutritionuniversity of north carolina at chapel hill chapel hill nc usa telephone email styblomeduncedu or beverly hkoller department of genetics university of north carolina at chapel hillchapel hill nc usa telephone emailbkolleremailuncedusupplemental material is available online 101289ehp6943this document was reviewed by the center for computational toxicology andexposure office of research and development us environmental protectionagency and approved for publication approval does not signify that thecontents reflect the views of the agency nor does mention of trade names orcommercial products constitute endorsement or recommendation for usethe authors declare they have no actual or potential competing financialinterestspublished august received february revised july accepted july note to readers with disabilities ehp strives to ensure that all content is accessible to all readers however some figures and supplementalmaterial published in ehp s may not conform to standards due tothe complexity of the information being presented if you need assistanceaccessing content please contact ehponlineniehsnihgov our staï¬will work with you to assess and meet your accessibility needs within working dayspromotes wholebody clearance of as drobn¡ hughes but also produces methylated intermediates that contain highly reactive and toxic trivalent as asiii watanabe andhirano masiii and dmasiii unlike their pentavalent counterparts masvand dmasv exceed ias in potency as cytotoxinsgenotoxins and enzyme inhibitors thomas hencemasiii and dmasiii may be critical determinants of toxic and carcinogenic eï¬ects associated with chronic exposure to ias alteredcapacity to methylate ias has been linked to an increased risk ofdiseases associated with ias exposure ahsan pierce vahter in most mammals the gene encoding as3mt is present asa single copy ˆ¼ kb from the gene encoding bloc1 relatedcomplex subunit borcs7 to date the methylation of ias isthe only known function of as3mt however recent studies haveidentified the as3mtborcs7 locus as conferring risk for schizophrenia duarte compared with healthy individualsexpression of borcs7 and of the humanspecific splicing variantof as3mt as3mtd2d3 has been found to be consistently higher inthe brains of patients with schizophrenia li notablyexpression levels of as3mt and borcs7 were found to be weaklycorrelated li suggesting that the two genes may sharetranscriptional regulatory elements although published datashowed that ias exposure can aï¬ect as3mt expression in micest½blo the role of ias exposure in the expression ofborcs7 or as3mtd2d3 which is thought to lack ias methylationactivity has never been studiedlaboratory studies of the mechanistic basis of iasassociateddiseases have been hindered by substantial diï¬erences between laboratory animals and humans in their capacity to metabolize anddetoxify ias rats unlike humans sequester significant portions ofingested ias in erythrocytes in the form of dmasiii lu mice the species most commonly used in mechanistic studies diï¬erfrom humans displaying very high rates of as methylation thisspecies diï¬erence is associated with faster rates of urinary clearanceof methylated metabolites and the predominance of dmas as themain urinary metabolite of ias in mice vahter interspeciesdiï¬erences in as metabolism may contribute to difficultiesenvironmental health perspectives august 0cencountered replicating some of the eï¬ects of ias exposure reportedin humans including the carcinogenic eï¬ects in laboratory micethus producing an ias methylation phenotype in mice that resembles the phenotype found in humans could create a better animalmodel to study the role of ias metabolism in adverse health eï¬ectsof chronic ias exposure to generate such a model we have humanized the entire borcs7as3mt locus in 129s6 mice by syntenicreplacement in this process the segment of mouse dna carryingthese two genes was removed and replaced with the syntenic regionof human dna in mouse embryonic stem es cells using homologous recombination this ensured that the junctions between thehuman and mouse dna are known to the base pair level mice weregenerated from the es cells with the expectation that they wouldexpress the human as3mt and borcs7 proteinsthe present study describes in detail the creation of thehumanized mouse strain expression of human as3mt andborcs7 in tissues of the humanized mice and metabolism ofias in these mice after a single dose and during a subchronic exposure to ias in drinking watermethodsrationale for humanizing the borcs7as3mt locuswe chose to excise the 61kb segment of mouse dna carrying theas3mt and borc7 genes and replaced it with the correspondingpromoter of as3mt abuts59kb segment of human dna the untranslated region of borc7 and the weak correlation inthe the expression pattern of the genes suggests possible shared transcriptional regulatory elements li in addition the current human transcript databases for example the university ofcalifornia santa cruz genome browser ucsc lists a putative readthrough borcs7as3mt transcript lacking the finalexon of borcs7 and the initial exon of as3mt this suggests thepossibility that at least some as3mt activity may be linked to transcription driven by the borcs7 promoter thus the inclusion ofborcs7 in the humanized region increases the likelihood that theelements directing the tissue species diï¬erences in the expressionof as3mt are included in the humanized regionassembly of borcs7as3mt displacerthe borcs7as3mt displacer construct was assembled using astandard recombineering approach figure the mouse arms ofhomology were derived from the 129s7ab22 bmq bac librarysource bioscience plc nottingham uk the short homologyarm was derived from bac bmq455l14 and the long arm ofhomology from bac bmq345l20 the segment of humangenomic dna containing the borcs7 and as3mt loci wasderived from the human tile path bac rp11753c18 bacpacresources center children™s hospital oakland research instituteoakland ca the single nucleotide polymorphisms snps previously associated with interindividual diï¬erences in ias metabolism apata or schizophrenia risk li which were included in the this borcs7as3mt segment aredescribed in table s1 a dna segment bp extendingfrom chr1946683032 to were deleted from the mousegenome and replaced with a 59261bp segment of human dnaextending from chr10102845563 to the haplotypeof the as3mt gene carried in the displacer construct is 1a wood the resistance marker gene used for selection of escells in which the displacer construct underwent genomic integration consisted of a phosphoglycerate kinase pgkneo cassetteflanked by mutant loxp sites the marker gene can be excised leaving only a nonfunctional lox site in its placeall polymerase chain reactions pcrs carried out during thedisplacer assembly used taq polymerase bio basic with an initial denaturation temperature of °c for s followed by cycles of denaturation for s at °c annealing for s at a°c and extension at °c with an extension time of min per bp of product length all redet recombination reactionswere carried out using a commercially available kit gene bridgescat no k001 all predesigned primers used during the construction of the displacer vector were from sigmaaldrich and arelisted in table s2 in descriptions of specific assays each of theseprimers is listed by the corresponding number as stated above the short arm of the displacer construct wasderived from the bmq455l15 bac library this was accomplished by first replacing the borcs7as3mt region of the bac withan ampicillin resistance marker by redet recombination theampicillin resistance marker was amplified from the pbluescriptii sk cloning vector stratagene primers and the homology arms referred to as the red arm primers and and the ds arm primers and respectively were generated by pcr amplification ofsegments of the bmq455l15 bac the homology arms werefused to the ampicillin resistance gene by overlap pcrabhuman locuscyp17a1borcs7as3mtmouse locuscyp17a1borcs7as3mt kb deletedcnnm2cnnm2cborcs7 as3mt displacerddisplaced mouse locuscyp17a1floxedneofloxedneoborcs7as3mtcnnm2homologousrecombinationborcs7as3mtcnnm2 kb insertedfigure scheme for humanization of the mouse borcs7as3mt locus a human borcs7as3mt locus showing the relative positions of the borcs7 andas3mt genes as well as the closest flanking genes b mouse borcs7as3mt locus with flanking genes c schematic of the borcs7as3mt displacer construct d humanized locus prior to cremediated marker excision the names of human genes are capitalizedenvironmental health perspectives august 0cwas used to insert the human borcs7as3mt segment and neomycin resistance cassette by redet recombination into thebmq354l20 bac carrying the short arm and ampicillin genesimultaneously displacing the ampicillin gene this reactionyielded the final borcs7as3mt displacer vector which wasdigested with noti to release the bac backbone before transfection into es cellsgeneration of mouse es cellsthe parental es cell line phnx43 used in these studies was generated as follows male and female 129s6svevtac mice taconicbioscience were mated females were checked every morning forcopulatory plugs indicative of successful mating three days laterfemales were euthanized by lethal carbon dioxide co2 exposurefollowed by physical euthanasia and the 35d embryos blastocysts were collected by flushing the uterus with co2independentmedium gibco cat no supplemented with bovine serum albumin sigmaaldrich cat no a3675 individualembryos were collected from the lavage fluid with a pipette andplaced in 2cm2 wells that had been seeded h earlier withprimary mouse embryo fibroblasts mefs gibco cat nogsc6005m blastocysts and mefs were cultured in gibcoknockout„¢ ko medium cat no supplementedwith esqualified fetal bovine serum fbs gibco cat no and 2mm lglutamine gibco cat no after “ d in culture when the inner cell mass of the embryosreached ˆ¼ mm in diameter the embryos were removed and disrupted with a pipette and placed in a new well seeded with mefsthis process of serial expansion of a single inner cell mass wascontinued until a single inner cell mass was expanded to at least colonies of cells cells were then further expanded by disruptionand exposure to trypsin for min at °c after expansionand cryopreservation in dimethyl sulfoxide sigmaaldrichcat no d2660 a sample of the cell line was subjected to karyotype analysis in karyologic inc rtp nc the cell line used inthis study had a xy normal male mouse karyotypeexpression of human borcs7as3mt in mouse es cellssinglecell suspensions of the parental es cell were prepared bytreatment with trypsin as described above in a volume of ml — cells were electroporated in the presence of lgof dna the apparatus used was a btx electro cell manipulator with cuvette btx cheshire analytical the settingsused were v lf r8 ohms after electroporation the cells were distributed onto seven 100mm tissue cultureplates corning cat no in gibco ko medium supplemented with esqualified fbs gibco cat no and mm lglutamine gibco cat no after hselection was initiated with the addition of geneticin„¢ final concentration mgml gibco cat no after d individual neomycin resistant colonies became visible cells fromˆ¼ colonies were pooled and used for rna isolation to confirmthat the human genes were expressed see details below once thiswas established individual colonies from the remaining plateswere transferred individually by pipette to a 96cell plate each colony was trypsinized and a portion used for pcr analysis to identify those in which the dna had integrated by homologousrecombination as described below the remaining cells wereallowed to grow and cultures carrying a targeted allele as determined by the assay detailed below were transferred sequentially to and 60cm2 tissue culture plates at this point some cellswere cryopreserved while a portion of each was used for karyotypeanalysis and to confirm the expression of human as3mt by probebased droplet digital pcr ddpcr using commercially availablethe displacer short arm together with the inserted ampicillingene was subcloned from the modified bmq455l15 bac into aplasmid vector by redet recombination the plasmid vectorwas prepared by ligation of four pcr products the vector backbone carrying a cole1 origin of replication and a spectinomycingene was derived by amplification of the pfloxxerx plasmid previously assembled in koller™s laboratory see table s3 primers and the bmq455l15 bac was used as atemplate for the other three pcrs us arm primers arm primers and andand short ds arm primers and all pcr productswere gel purified on a agarose gel using a commerciallyavailable kit qiagen cat no digested with bbsi nebcat no r3539s and mlui neb cat no r0198s and columnpurified qiagen cat no fifty nanograms of each pcrproduct was combined and ligated neb cat no m0202s in a20ll reaction for h at °c the ligation reaction was heatinactivated at °c for min and then ll of the reaction wastransformed into chemically competent e coli dh5afcellszymo research cat no t3002 according to the manufacturer™s directions and plated on luria broth agar plates supplemented with spectinomycin at lgml the resulting plasmidvector was digested with mlui and used for redet recombination with the modified bmq455l15 bac the resulting plasmidwas in turn digested with bbsi to release the displacer short armand ampicillin resistance gene flanked by the us and dshomology arms this bbsi restriction fragment was introducedinto the bmq345l20 bac by redrecombination to create abac vector in which the ampicillin resistance marker wasflanked by the displacer arms of homologythe segment of the human borcs7as3mt locus included inthe displacer vector was prepared for excision from the rp11753c18 bac in two steps in the first step a neomycin resistancegene was inserted into the bac upstream of the borcs7 gene toaccomplish this the neo gene flanked by mutant loxp sites wasligated to homology arms referred to as blackred and purplerespectively the neomycin resistance gene was excised from thevector psfiijt15neojtz17sfii previously assembled in koller™slaboratory see table s4 by digestion with sfii neb cat nor0123s the blackred homology arm was amplified primers and using the bmq455l15 bac as a templateand the purple homology arm was amplified primers and using the rp11753c18 bac as a template all fragments were gel purified as described above and the blackred andpurple homology arm pcrs were digested with bgli neb catno r0143s and then column purified as described above theloxpflanked neomycin resistance gene was ligated to the homology arms as described above in a 20ll reaction containing a totalof lg of dna with the three fragments at an equimolar ratiotwo microliters of the ligation reaction was used for a redetreaction with the rp11753c18 bac in the second step an ampicillin gene was inserted downstream of the as3mt gene in therp11753c18 bac carrying the neomycin resistance marker toaccomplish this homology arms referred to as bluegreen andyellow respectively were added to the ampicillin resistance genethe bmq455l14 bac was used as the template for the bluegreen pcr primers and pbluescript ii sk was used as the template for the ampicillinyellow pcr primers and the two pcr products were gel purifiedand then combined in an overlap pcr primers and the resulting pcr product was gel purified and ngwas used for a redet reaction with the rp11735c18 bac carrying the neomycin resistance cassetteapproximately ng of the modified rp11753c18 bacwas digested with mlui in a 20ll digest and ll of the digestenvironmental health perspectives august 0cprimers applied biosystems hs00960526 here a 20ll reaction mixture was pipetted into a dg8„¢ cartridge along with llof droplet generation oil for probes bio rad and loaded into theqx200 droplet generator bio rad according to the manufacturer™s instructions after droplet generation the droplets weretransferred to an eppendorf twintec® 96well plate and placed in ac100 touch thermal cycler bio rad using the manufacturer™srecommended cycling conditions after cycling the plate was readin the qx200 droplet reader bio rad quantasoft„¢ softwareversion a portion of each of the es colonies ˆ¼ cells was disruptedby incubation for min at °c in lysis buï¬er consisting of trisedta [ mm trisma base thermo fisher scientific cat nobp152 mm ethylenediaminetetraacetic acid edta thermofisher scientific cat no volvol triton„¢ x100millipore sigma cat no and mgml proteinase kroche cat no ] lysates from all colonies werescreened for homologous recombination by pcr using primersscreen f and see table s4 with gxl polymerase takarabio according to the manufacturer™s suggested protocol the pcrprogram used was °c initial denaturation for s followed by cycles with s denaturation at °c s annealing at °c minat °c and a final 10min extension at °c pcr products wereanalyzed by agarose gel electrophoresis agarose bio basiccat no d0012 in — trisacetateedta running buï¬er the sizeof the pcr product was determined by comparison with 2logladder neb cat no n3200ltwentyseven clones with homologous recombination wereidentified and were subjected to further analysis because of thishigh recombination frequency it was not necessary to use clustered regularly interspaced short palindromic repeats and crisprassociated protein 9crisprcas9 to stimulate recombinationevents rna was prepared from pools of the cells and examinedfor the expression of the human genes rna was also preparedfrom a number of the individual clones after expansion brieflycells were lysed directly in the culture dish and homogenized byrepeated pipetting rna was isolated using rna bee or stat60teltest according to the manufacturer™s instructions rna yieldand quality were assessed using a nanodrop thermofisher one to two micrograms total rna was reverse transcribedwith the high capacity reverse transcription kit appliedbiosystems expression of the human as3mt was examined byqualitative pcr qpcr on a c100 touch thermal cycler biorad using commercially available primers applied biosystemshs00960526generation of mice expressing human borcs7as3mtfor injection into blastocysts the es cells carrying the humanborcs7as3mt locus were again trypsinized to generate singlecell suspensions collection of recipient blastocysts blastocystinjection and transfer to pseudopregnant dams was carried out bythe unc chapel hill animal models core following proceduresdescribed by longenecker and kulkarni and by koller to maintain the mutation on the 129s6svevtac background the chimeric mice were bred to 129s6svevtac micetaconic bioscience the resulting f1 mice were identifiedby taq as wildtype wt homozygotes for the mouse as3mtborcs7 locus wtwt or heterozygous wths or homozygoushshs for the human as3mtborcs7 locus the primers usedwere common and endo for the endogenous locus and common anddisplaced for the displaced locus see table s2 the pcr assayswere carried out using taq polymerase bio basic with an initialdenaturation temperature of °c for s followed by cycles ofdenaturation for s at °c annealing for s at a °c andextension at °c for s with a final extension of min the f2generation”consisting of hshs homozygotes hswt heterozygotes and wtwt homozygotes”was produced by mating of f1hswt heterozygotes the hshs hswt and wtwt oï¬springfrom the f2 generation were housed together one litter per cagecunder controlled conditions with 12h lightdark cycle at ± and ± relative humidity the parental mice and mice in thef1 and f2 generations were fed purina labdiet 5v5r and drankdeionized water ad libitumall proceduresinvolving mice were approved by theuniversity of north carolina institutional animal care and usecommittee 0ecollection of tissues for mrna expression analysistissues for mrna expression analysis were collected from f2hswt mice both males and females measurement of rnaexpression in hswt mice which were heterozygous for thehuman and mouse gene allowed direct comparison of expressionin the same tissuerna sample minimizing the eï¬ects of physiological variation between animals or quality of sample whichcould result in subtle diï¬erences in rna quality and cdnaformationmice were euthanized by exposure to co2 followed by physical euthanasia the following tissues were collected whole brainparts of brain cortices pons medulla hippocampus cerebellumpituitary spinal cord adrenals liver spleen kidney duodenumjejunum colon stomach uterus ovaries testes heart and skinfrom back of neck neuronal and glial cells were also dissectedbrain parts and spinal cord were collected from male mice at dof age neuronal cells were isolated from embryonic day e17embryos and glial cells from postnatal day p1 pups all othertissues were collected from to 12wkold mice three mice wereused for each tissue blood was collected from lethally anesthetized mice via cardiac puncture with an edtacoated syringe justprior to thoracotomy to isolate mononuclear cells from blood ml of whole blood were layered onto ml of histopaque® sigma and centrifuged at room temperature for min with nobrakes at — g the upper layer was discarded and the interfacecontaining the mononuclear cells was transferred to a 15ml conical tube the cells were then washed twice with phosphatebuï¬ered saline and then lysed with rna beeneuronal cell isolation and culture brains were harvestedfrom eight e17 embryos and placed in a petri dish containinghanks™s balanced salt solution hbss gibco meninges wereremoved cortices dissected and placed in a new petri dish containing hbss cortices were transferred to a 15ml conical tube containing ml hbss and centrifuged at — g for min at °cthe tissue was digested with ml tryple express gibco catno and dnase i roche cat no for min at °c the digested cortices were centrifuged at — gfor min at °c the tryple dnase i was aspirated and thecortices were washed for min with ml fbs to inactivate thetrypsin the tissue was centrifuged again and resuspended in complete dulbecco™s modified eagle medium dmem gibco with fbs vwr glutamax gibco with penicillinstreptomycingibco and 2mercaptoethanol sigma the cortices were titurated times with a 10ml pipette and then times with aflamedtip pasteur pipette the cells were then passed through a100lm cell strainer into a clean conical tube cells were then pelleted by centrifugation at — g for min the cell pellet wasresuspended in ml complete dmem and plated in mm culture dishes coated with lgml poly dlysine sigma — cells per dish after h 1lm cytosine bdarabinofuranosidearac sigma cat no c6645 was added using a mediachange without exposing the cells to air the cells were harvestedfor rna isolation on day environmental health perspectives august 0cmixed glial cell culture brains were harvested from six p1pups and placed in a dish containing hbss meninges wereremoved cortices dissected and placed in a conical tube containing cold dmem cortices were centrifuged at — g for min at°c and media replaced with ll cold dmem the corticeswere titurated times with a flamedtip pasteur pipette coated inserum the cells were then passed through a 100lm cell strainerinto a fresh conical tube cells were pelleted by centrifugation at — g for min the cell pellet was resuspended in ml complete dmem and ml of the suspension was plated on three100mm culture plates the medium was changed h later andthen daily the cells were harvested for rna on day isolation of adrenal cortex and medulla to obtain samplesenriched for each of these two distinct functional regions of thegland the adrenal cortex was removed from medulla of male andfemale adrenal gland by microdissection enrichment was quantitated by qpcr analysis of genes known to be expressed in onlyone of these two regions chgb in the medulla and cyp11b1 inthe cortex the human protein atlas using appliedbiosystem taqman probes mm00483287 and mm01204952respectively rna was isolated from cells and tissues using themethod described for es cells however the tissues were first homogenized in stat60 in a desktop homogenizer fastprep mp bio using ceramic beadsanalysis of as3mt as3mtd2d3 borcs7 as3mt andborcs7 expression in collected tissues and cellsboth ddpcr and qpcr were used to detect and quantify as3mtas3mtd2d3 borcs7 as3mt and borcs7 mrna the specificmethod is indicated in each figure legend ddpcr is minimallyinfluenced by diï¬erences in the efficiency of the mouse and humanprimer sets quan and therefore provides a means ofdirect comparison of expression between the endogenous mouselocus and the humanized locus the distribution of the as3mttranscript in the tissues was compared with as3mt protein distribution described by the human protein atlas for the human tissues sybr green qpcr was used to compare rna expression inwhole brain cortex hippocampus pituitary cerebellum pons medulla spinal cord glial cells neurons adrenals ovaries testesheart kidney liver colon jejunum spleen and blood ddpcr wasused to assess rna expression in es cells whole brain liver adrenals spinal cord ovaries testes spleen and heart for all assays“ lg total rna was reverse transcribed with the high capacityreverse transcription kit applied biosystemsfor sybr green qpcrthe relative expression of fulllength as3mtthe d2d3 variant and the borcs7as3mtfusion transcript were assessed on a quantstudio6 flex„¢ realtime pcr system applied biosystems sybr green reactions were run using itaq universal sybr green supermixbio rad nm of each primer and ngll of cdnaprimers used for quantification of as3mt isoforms were d2d3fand d2d3r for the d2d3 isoform and fullf and fullr for thefulllength isoform see table s2 for analysis of borcs7as3mt readthrough transcripts expression in mouse tissues lgrna was reverse transcribed and sybr green quantitative pcrwas run using primers gaacagtcatcggatctacagga3and and itaq sybrgreen universal supermix bioradgaacagtcatcggatctacagga3for ddpcr absolute concentration of as3mt as3mt borcs7and borcs7 was acquired using the ddpcr supermix forprobes no dutp bio rad and taqman gene expressionassays as3mt mm00491075_m1 as3mt hs00960526_g1borcs7 mm01205060_m1 and borcs7 hs00376014_m1all from applied biosystems the ddpcr analysis was carriedout as described aboveevaluation of the metabolism of a single dose of iasthe metabolism of ias was examined in to 22wkold hshsmale n and female n f2 oï¬spring and their wtwtmale n and female n littermates the mice weregiven a single dose of ias sodium arsenite pure sigmaaldrich in deionized water diw by gavage lg askg ofbody weight immediately after dosing each mouse was placedin a metabolic cage mousecage and urine and feces were collected in 24h intervals for d during these d all mice drankdiw ad libitum the mice were fasted during the first h buthad free access to purified laboratory diet envigo teklad catno ain93g during the second and the third days our published data showed that ias content in this type of diet rangesfrom to ˆ¼ lg askg douillet huang murko the 24h urine and feces samples werefrozen in dry ice and stored at ˆ’c 0eevaluation of the metabolism of ias during subchronicexposureafter collection of urine and feces following the singledoseadministration the hshs and wtwt mice were again cagedtogether one litter per cage and maintained on the purified dietand diw for wk to allow for clearance of as from the bodywhich was confirmed by measuring total as tas levels inurine see the œresults section for details both wt andhshs mice now to 27wkold were then exposed to iassodium arsenite pure sigmaaldrich in drinking water lg asl for wk spot urine samples ˆ¼ to llwere collected weekly body weights were recorded before andafter the exposure after wk all mice were sacrificed by cervical dislocation without anesthesia and tissues were collectedincluding the liver kidneys pancreas spleen heart lung adrenals kidneys bladder visceral fat calf muscle testes or ovaries and cecum and colon all tissues were flashfrozen in dryice and stored at ˆ’c 0eanalysis of as species in urine feces and tissuesspeciation analysis of as was carried out in 24h urines and fecescollected after the single dose of ias and in spot urine samplescollected during subchronic ias exposure the speciation analysis was also performed in livers and kidneys collected after subchronic exposure at sacrifice ten percent homogenates of liverand kidney were prepared in icecold diw wtvol usingwheaton potterelvehjem“style tissue grinders with a ptfepestle and wheaton overhead stirrer apparatus dwk lifesciences feces were snap frozen in liquid nitrogen and pulverized to powder using a steel mortar and pestle placed in dry icethe powder was mixed with 2n ultrapure phosphoric acid thermo fisher and digested in a mars5 microwave cemcorp for h at °c this method eliminates the biologicalmatrix but does not alter as speciation currier the digestates were neutralized by sodium hydroxide sigmaaldrich to ph the urines tissue homogenates and neutralized digestates were treated with lcysteine sigmaaldrich at room temperature for h to reduce pentavalent asspecies to their trivalent counterparts prior to the analysiscurrier the cysteinetreated samples were analyzed by hydridegeneration atomic absorption spectrometrycoupled with a cryotrap hgctaas as previously describedcurrier hern¡ndezzavala this analysis determined the concentrations of ias mas and dmas tasconcentration in urine feces and tissues was calculated as thesum of ias mas and dmas for assessment of the efficiencyof ias metabolism after a single oral dose the amount of tasenvironmental health perspectives august 0cwas expressed as the percentage of the dose by comparing theamount of elemental as administered as ias to each mousewith the amount of tas in 24h urine and feces samples collected from that mousethe instrumental limits of detection lod for ias mas anddmas using this method are and pg as respectivelyhern¡ndezzavala an imputed value of was usedfor measurements below the lod hgctaas is also capableof detecting and quantifying trimethylarsine oxide tmasohern¡ndezzavala a product of ias metabolism byrat as3mt waters however because tmaso israrely detected in human urine and because tmaso was not aproduct of ias methylation by recombinant human as3mt inour published study ding we did not includetmaso analysis in this studystatistical analysisoneway analysis of variance with studentnewmankeuls ortukey™s multiple comparison posttest was used to assess diï¬erences in gene expression and in the concentrations and proportions of as species among wtwt and hshs mice student™s ttest was applied for comparison of mouse and human geneexpression in a single tissue of hswt mice and for comparisonof as species concentrations or proportions between male andfemale mice and between hshs and wtwt mice of the samesex the statistical analyses were performed using prism software graphpad software diï¬erences with p were considered statistically significantresultsexpression of borcs7 and as3mt in hswt miceexpression of as3mt and borcs7 were directly compared withthose of endogenous mouse genes in tissues collected from f2male hswt mice that carried one copy of the mouse locus andone copy of the human locus figure 2a using both conventionalqpcr and when appropriate ddpcr expression of humanas3mt and borcs7 was easily detected in all tissues expressingthe corresponding mouse genes although the level of expressionof the human and mouse genes diï¬ered figure 2bc notable inthis analysis was
Colon_Cancer
" excessive perioperative fluid administration may result in iatrogenic endothelial dysfunction andtissue edema transducing inflammatory markers into the bloodstream colloids remain longer in the circulationrequiring less volume to reach similar hemodynamic endpoints compared to crystalloids thus we tested thehypothesis that a goaldirected colloid regimen attenuates the inflammatory response compared to a goaldirectedcrystalloid regimemethods patients undergoing moderate to highrisk open abdominal surgery were randomly assigned to goaldirected lactated ringer™s solution n or a hydroxyethyl starch n fluid regimen our primaryoutcome was perioperative levels of pro and antiinflammatory cytokines secondary outcome was perioperativelevels of white blood cell count wbc creactive protein crp procalcitonin pct and lipopolysaccharidebindingprotein lbp measurements were performed preoperatively immediate postoperatively on postoperative day onetwo and fourresults the areas under the curve of interleukin il p il p il p and tumor necrosisfactor α p levels did not differ significantly between the groups wbc crp and pct values were alsocomparable lbp although significantly higher in the crystalloid group remained in the normal range patientsassigned to crystalloids received a median iqr amount of ml “ of crystalloid patients assigned tocolloids received ml “ of crystalloid and ml “ of colloid cytokine and inflammatory marker levels did not differ between goaldirected crystalloid and colloidadministration after moderate to highrisk abdominal surgerytrial registration clinicaltrialsgov nct00517127 registered 16th august correspondence barbarakabonmeduniwienacat1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austriafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cobradovic bmc anesthesiology page of is crucialintroductionvolume replacementin the perioperativeperiod and has great impact on postoperative outcome fluid restriction may cause hypotension and hypoperfusion leading to an dysfunction on the otherhand excessive fluid administration leads to destructionof the endothelial surface layer and consequently to tissue edema with harmful side effects [ ]fluidgdtbasedtherapygoaldirectedonoptimization of flowrelated hemodynamic parametersimproves clinical outcome in low to highrisk surgicalpatients compared to fixed fluid protocols [ ] specifically gdt enhances cardiac performance and gutmicrocirculation while avoiding iatrogenic hyperhydration [ ] in addition to hypervolemia the inflammatoryaggravatesdegradation of the endothelial barrier the socalled glycocalyx inflammation leads to cytokine release andmay thus worsen outcome for example high postoperative interleukin il levels are independently associatedwith postoperative complications response dueto surgicaltraumaso far in most previously performed gdt studieshemodynamic algorithms were based on colloid bolusadministration to improve hemodynamic variables colloids better maintain the intravascular oncotic pressure and provide a higher volume effect when used incase of hypovolemia goaldirected colloid administration reduces intraoperative fluid requirement and improves cardiac performance compared to crystalloids[ ] whether this translates into better outcomespecifically in a decreased postoperative inflammatoryresponse is still a matter of research the comparisonbetween colloid versus crystalloid based fluid regimenswas still lacking therefore we tested the primary hypothesis that perioperative levels of pro and antiinflammatory cytokines il il il and tumor necrosis factor alpha tnf α are reduced by goaldirectedcolloid versus crystalloid administration during the firstfour postoperative days in patients undergoing moderateto highrisk open abdominal surgery in addition wemeasured white blood cell wbc count creactive protein crp procalcitonin pct and lipopolysaccharidebinding protein lbp levelsmaterials and methodsthis prospective randomized controlled trial was conducted at the department of anesthesia intensive caremedicine and pain medicine medical university ofvienna vienna austria the institutional review boardof the medical university of vienna approved it as partof a large multicenter outcome trial evaluating the effectof goaldirected crystalloid and colloid on postoperativecombined morbidity and complications the ethicalcommittee of medical university of vienna viennaaustria provided ethical approval for this trial the trialwas conducted in accordance with the declaration ofhelsinki and good clinical practice and registered atclinicaltrialsgov nct00517127 and eudract “ a written informed consent was obtainedfrom all patients the authors have followed the applicable consort guidelinesfor this single center substudy consecutive eligiblepatients were included patients aged to yearsundergoing elective moderate to highrisk open abdominal surgery with american society of anesthesiologistsasa physical status iiii were included we excludedpatients with severe obesity body mass index bmi kgmˆ’ cardiac insufficiency ejection fraction ef coronary artery disease with angina severe chronicobstructive pulmonary disease autoimmune diseases coagulopathies renal insufficiency creatinine clearance mlminˆ’ or renal replacement therapy symptoms of infection or sepsis and preoperative crp higher than mgdlˆ’ allpatientsreceivedprotocolpreoperativelyantimicrobialprophylaxis using a single dose of a 2nd generationcephalosporine according to our clinicalstandardsanesthetic management wasstandardized standardmonitoring included electrocardiography ecg invasiveblood pressure surveillance pulse oximetry and esophageal core temperature monitoring a central venouscatheter was inserted when deemed clinically necessarywe used balanced anesthesia with sevoflurane none ofour patients received locoregional anesthesia accordingto patients™ requirements additional fentanyl and nondepolarizing neuromuscular blocking were administeredventilatory rate was adjusted to maintain endtidal carbon dioxide partial pressure etco2 of “ mmhgnormothermia was maintained with forced air warmingpatients were randomized to crystalloid lactatedringer™s solution or colloid hydroxyethyl starch voluven fresenius kabi germany grouprandomization was based on computergenerated codesto conceal allocation sealed opaque envelopes wereopened only shortly before induction of anesthesiaall patients were given “ mlkgˆ’ oflactatedringer™s solution during induction of anesthesia followedby “ mlkgˆ’ per hour for maintenance normalized toideal body weight ibw throughout surgery we calculated ibw according to the robinson formula thereafter the randomized fluid crystalloid or colloidwas esophageal dopplerguided cardiac q deltex medical group plc chichester uk according to a standardalgorithm this method is based on corrected aorticflow time ftc as well as stroke volume sv and allowsdistinguishing whether a patient is a fluid responder or 0cobradovic bmc anesthesiology page of not if mean arterial pressure map was below mmhg and no signs of hypovolemia were detected vasopressors were administratedpatients were transferred to postanesthetic care unitpacu or intensive care unit icu at the discretion ofthe attending anesthesiologist fluid management wasstandardized for the first postoperative hours in whichpatients received mlkgˆ’ ibw crystalloid per hourmeasurementsdemographic and morphometric data were recorded aswell as asa score medical history type of surgery andpreoperative laboratory values duration of anesthesiaand surgery were recorded we also recorded intraoperative fluid requirements estimated blood loss transfusion requirements and urinary output for evaluation ofanesthetic management the total amount of fentanylendtidal sevoflurane concentration core temperatureandnotedhemodynamic parameters such as map heart ratehr ftc sv and cardiac output co were recorded at10min intervals the application of phenylephrine usewas notedicu admission werepostoperativethe primary outcomes were the areas under the curveaucs of postoperative levels of pro and antiinflammatory cytokines il il il and tnf α andtheir differences between the crystalloid and the colloidgroup secondary outcomes were aucs of wbc crppct and lpb and their differences between the groupsall blood samples for parameteranalyses were obtainedbefore surgery as baseline values t0 immediately postoperatively t1 as well as on postoperative days onetwo and four t2 t3 and t4 respectively for analysisof il il il and tnf α blood samples were centrifuged within h at g for min and plasma wasimmediately stored at ˆ’ °c for later enzymelinkedimmunosorbent assay elisa analyses the serum concentrations of il il il and tnf α were determined according to the manufacturer™sinstructionshuman sil6 instant elisa human il8nap1 instant elisa and human sil10 instant elisaebioscience vienna austria wwwebiosciencecom human tnf α duoset rd systems minneapolis minnesota wwwrndsystemscom for that purpose opticaldensity was measured with a victor microplate readerat a wavelength of nm multiple testing of sampleson different plates revealed an intraassay variability of for il for il for il for tnf α andan interassay variability of for il for il for il and for tnf αfor investigation of wbc crp pct and lpb separateblood samples were obtained their analysis took placeimmediately after blood sampling as routine laboratoryanalysessample size calculation and statistical analysissample size calculations for our trial were based on thestudy of steppan and colleagues they observed amean standard deviation sd of pgmlˆ’ in il h after surgery in abdominal surgery patients assuming a similar coefficient of variation sdmean for each of the four cytokines primarily plannedfor evaluation in our study we calculated a total of patients in order to obtain a power to detect a reduction in any of the cytokines at an overall significance level with powergroups were primarily compared for balance in patients™ demographic data intraoperative characteristicsand postoperative variables absolute standardized differences asd were calculated for patients™ baseline covariates subsequent measurements ofintraoperativeparameters were first averaged within each patient andthen averaged among the patients in each treatmentgroup for descriptive analysis normal distribution wasassessed with qq plots and kolmogorowsmirnow testsnormally distributed variables were with unpaired twotailed ttests otherwise the in case of normally distributed values wilcoxon ranksum test was used for notnormally distributed continuous data paired comparisons between baseline data and postoperative data wereperformed with paired sample ttest or wilcoxonsignedrank test as applicable nominal data were analyzed with chisquare or fisher™s exact test for low expected cell counts data were presented as means ± sdmedians iqr or as numbers percentage as applicableadjustment for multiple testing was performed with thebonferroni method a p value was considered statistically significantanalysis was conducted with spss software version armonk ny ibm corp r for macintosh version321 r core team r a language and environmentfor statistical computing r foundation forstatistical computing vienna austria was used to calculate asdresultsa total of patients were included between november and october in the colloid group and in the crystalloid group fig at t0 all values weremeasured overall in the crystalloid group and inthe colloid group of the preplanned blood sampleswere collected and analyzedpatient™s baseline characteristics did not differ exceptfor height bmi with a slightly higher bmi in the colloidgroup type of surgery and crp also higher in the colloid group table duration of anesthesia and surgerywere comparable between both groups patients assignedto crystalloid administration received a median of ml “ crystalloids whereas patients assigned 0cobradovic bmc anesthesiology page of fig consort patient flow chartto the colloid group received ml “ ofcrystalloid solution and ml “ of colloidsblood loss transfusion requirements and urinary outputdid not differ between the groups anesthetic management map and hr did not differ between the groupsftc sv and co were significantly higher in the colloidgroup compared to the crystalloid group ftc ms“ versus ms “ p sv ml“ versus ml to p and co ± lminˆ’ versus ± lminˆ’ p thenumber of patients requiring vasopressor support wascomparable between groups the incidence of postoperative icu admissions did not differ in the crystalloid versus in the colloid group p table baseline values of il il il and tnf α in thecrystalloid group were comparable to values in the colloid group il pgmlˆ’ “ versus pgmlˆ’ “ p il pgmlˆ’ “ versus pgmlˆ’ “ p il pgmlˆ’ “ versus pgmlˆ’ “ p tnf α pgmlˆ’ “ versus pgmlˆ’ “ p immediatepostoperative values of il and were significantlyhigher compared to baseline values in both groupsp for all measurements while tnf α did notshow any significant increase in the crystalloid p and the colloid group p fig aucs of il il il and tnf α did not differ significantly between the groups table wbc values at baseline were glˆ’ “ in thecrystalloid versus glˆ’ “in the colloidgroup p crp pct and lbp baseline valueswere also comparable in both groupscrp mgdlˆ’ “ versus mgdlˆ’ “p pct ngmlˆ’ “ versus ngmlˆ’ “ p lbp mcglˆ’ “ versus mcglˆ’ “ p immediate postoperative values of wbc and pctwere significantly higher compared to the baseline valuesin both groups p for all measurements fig 0cobradovic bmc anesthesiology page of table baseline characteristicsage yrsweight kgheight cmbmi kgmˆ’ gender no menwomenasa score no iiiiiimedical history no pulmonary diseasecardiovascular diseasediabetes type idiabetes type iitype of surgery no colorectalliverpancreaticcrystalloidsn ± ± ± ± colloidsn ± ± ± ± asd preoperative laboratory valuescrp mgdlˆ’ ± ± patient characteristics data are presented as means ± sd or as counts for thecategorical outcomesabbreviations asd absolute standardized differences absolute difference inmeans or proportions divided by the pooled sd asd values of and represent small median and large differencesbmi body mass index m male f female asa american society ofanesthesiologists crp creactive protein sd standard deviationthe aucs for wbc crp pct and lbp for the timeperiods from t1 to t4 did not differ significantly between the groups fig table however lbpshowed significantly higher levels in the crystalloidgroup in the immediate postoperative period comparedto the colloid group mcglˆ’ “ versus mcglˆ’ “ p at all other postoperativetime points there were no significant differences betweenthe groups fig discussionthis trial is a substudy of a large multicenter randomized trial evaluating the effect of goaldirected crystalloidversus goaldirected colloid fluid administration on acomposite of serious complications after moderate tohighrisk open abdominal surgery the overall trial concluded that colloids did not decrease the composite ofmajorincomplicationsare ourresultsconcordance as they did not show any differences inperioperative pro and antiinflammatory markers between a crystalloid and a colloid fluid regimendespite multimodal care and enhanced recovery programs it still remains challenging to blunt the inflammatory response to surgery systemic inflammationafter abdominal surgery impairs outcome and thereforemany attempts have been made to alter the inflammatory response several factors influence the perioperative inflammatoryresponse such as the underlying disease type and invasiveness of surgery as well as type of anesthesia [“] themost important factor is the magnitude of surgical traumaand tissue damage which induce proliferation and activation of immune competent cells in turn triggering cytokine and inflammatory marker release so far veryfew trials have specifically investigated the influence offluid therapy and differences in terms of the type of fluidon the extent of inflammatory marker releaseto investigate the potential influence of goaldirected hydroxyethyl starch versus a lactated ringer™s solution fluid regimen on inflammatory response pro il il and tnf α and antiinflammatory cytokine il serum levels were measured during the perioperativeperiod additionally we measured wbc crp pct andlbpgenerally the most commonly measured biomarkersare crp and wbc if levels of crp are above mgdlˆ’ after postoperative day four a postoperative infection can be suspected crp levels in our studygroups increased on the first postoperative day droppingon the fourth postoperative day to nearly mgdlˆ’ inboth study groupswbcs are an imprecise marker to detect postoperativecomplications after major abdominal surgery amore sensitive parameter in predicting postoperativecomplications after major abdominal surgery is il surgical trauma and hypoperfusion of the colon aremain sources of il release in colorectal surgery noblett demonstrated that gdt during elective colorectal surgery significantly reduced il levels in comparison to a control group yates showed no differencesof il and il levels between goaldirected colloidand crystalloid fluid therapy during the first h in asubgroup of patients undergoing colorectal surgery although patients in the crystalloid group received significant more volume amount as compared to the colloid groupthere was no significant difference inhemodynamic variables our patients showed similar courses of il and il levels in the immediatepostoperative period in contrast to the trial of yateswho measured cytokine levels up to the first h aftersurgery we extended our measurement period to fourpostoperative days we showed comparable circulating 0cobradovic bmc anesthesiology page of table intraoperative dataduration of anesthesia minduration of surgery minfluid managementtotal fluid intake mlacrystalloid mlcolloid mlestimated blood loss mltransfusion yesno urinary output mlanesthesia managementfentanyl mcgtwa et sevoflurane core temperature °cicu admission yesno hemodynamictwa map mmhgtwa hr beatsminˆ’twa ftc mstwa sv mltwa co lminˆ’ phenylephrine yesno crystalloidsn ± ± “ “ “ “ “ [ ] “ ± ± ± “ “ ± colloidsn ± ± “ “ “ “ [ ] “ “ ± ± ± “ “ ± p value ° ° °° °°intraoperative data are presented as means ± sd medians iqr or as counts for the categorical outcomes means were compared with an unpaired twosided ttests ormannwhitneyu tests as appropriate medians with wilcoxon ranksum tests and counts with chisquare or fisher™s exact tests ° represents statisticalsignificance p abbreviations et end tidal icu intensive care unit twa time weighted average map mean arterial pressure hr heart rate ftc corrected flow time sv strokevolume co cardiac output sd standard deviationa total fluid intake includes baseline fluid boluses antibiotics analgesics and additional fluid administered at the discretion of attending anesthesiologistfig ad pro and antiinflammatory cytokines il il il and tnf α over time a il b il c il and d tnf α data arepresented as medians iqr abbreviations il “ interleukin tnf α “ tumor necrosis factor alpha pod “ postoperative day 0cobradovic bmc anesthesiology page of table areas under the curve of inflammatory markerspcrystalloidsn “ “ “colloidsn “ “ “ “ “ “ “ “auc il pgmlˆ’ 1dauc il pgmlˆ’ 1dauc il pgmlˆ’ 1dauc tnf α pgmlˆ’ 1d “auc wbc glˆ’1dauc crp mgdlˆ’ 1dauc pct ngmlˆ’ 1dauc lbp mcglˆ’ 1dtable areas under the curve of il il il and tnf α as well as wbccrp pct and lbp are presented as medians iqr medians were comparedwith wilcoxon ranksum testsabbreviations il interleukin tnf α tumor necrosis factor alpha wbc whiteblood cells pct procalcitonin lbp lipipopolysaccharidebinding protein “ “ “ “il and il levels between a gdt crystalloid and colloid administration these surrogates of inflammatoryresponse imply that gut perfusion during surgery waswell preserved with both types of fluid and suggest thatthe type of fluid might be of minor importance as longas the fluid is administered in a goaldirected fashionthe fact that tnf α levels in both groups remainedstable over the entire measured period further supportsour theory the course of tnf α levels during the perioperative period was in accordance with the study ofin which fluid therapy was guided withszakmanypicco versusin majorvenous pressurecentralabdominal surgery in patients at risk for postoperativecomplications as tnf α per se triggers glycocalyxdegradation we anticipate that tnf α did not influence glycocalyx shedding and thus possible fluid shifts inour study populationpct is an early predictive marker for systemic inflammation after abdominal surgery values above ngmlˆ’ are associated with postoperative complicationssuch as pneumonia or anastomotic leakage in ourstudy median pct levels did not exceed ngmlˆ’ atany measured time point these results are in concordance with our main study where infectious complications rate were held low and did not differ between thegroups moreover as pct production can also beinduced by tissue hypoperfusion we might assume thatgoaldirected fluid administration contributed to lowpct values by optimizing cardiac performance furthermore we measured lbp a prognostic markerfor bacterial infections patients in the crystalloidgroup showed significantly higher levels immediatelyafter surgery however the measured values remainedwithin the normal range therefore this difference ismost likely not to of clinical importancethe vascular endothelium is one of the earliest sitesinvolved in the inflammatory response syndrome an adequate perioperative fluid management has a major impact on the integrity of the glycocalyx with goaldirected fluid management individualized and time appropriate fluid resuscitation can be achieved enablingfig ad inflammatory markers wbc crp pct and lbp over time a wbc b crp c pct and d lbp data are presented as medians iqrabbreviations wbc “ white blood cells crp “ creactive protein pct “ procalcitonin lbp “ lipopolysaccharidebinding protein pod “postoperative day ˜… represents significant difference in lbp in the immediate postoperative period between the crystalloid and the colloidgroup p 0cobradovic bmc anesthesiology page of preservation of endothelial surface layer and sufficientan perfusion thus improving postoperative outcomesafter major surgery patients in the colloid group received significantly lessfluid ml confirming the previously publishedfluid sparing effect of colloids however clinical significance of this difference may be questionable during aperioperative period of nearly five hours further ourhemodynamic data showed significantly higher values ofsv and co with colloid administration though the absolute difference of ml in sv most likely has onlylimited clinical relevance it might very well be that thesignificant differences in sv and co are the result ofour number of patients included in this substudyassurrogatesfirst limitation of our study is that we measured inflammatory markers that reflect the inflammatory responseand not direct markers ofglycocalyx degradation like syndecan1 therefore wecannot draw any s about the preservation ofthe endothelial surface layer in our patients secondlywe did not control postoperative fluid management during the postoperative followup period a further limitation isthe time between patient enrolment andsubmission of our current results due to the fact thatthe main trial has been published recently a delay of oursubmission occurred nevertheless our results canstill be extrapolated to current clinical practicein summary goaldirected hydroxyethyl starch administration did not attenuate the inflammatory responseexpressed by cytokine levels of il il il and tnfα in patients undergoing moderate to highrisk open abdominal surgery wbc crp and ptc values did not differ between the different fluid regimes as wellabbreviationsasa american society of anesthesiologists asd absolute standardizeddifference auc area under the curve bmi body mass index co cardiacoutput crp creactive protein ecg electrocardiography ef ejectionfraction elisa enzymelinked immunosorbent assay etco2 endtidalcarbon dioxide ftc corrected flow time gdt goaldirected therapyhr heart rate ibw ideal body weight icu intensive care unitil interleukin iqr interquartile range lbp lipopolysaccharidebindingprotein map mean arterial pressure pacu postanesthetic care unitpct procalcitonin picco pulse contour cardiac output spss statisticalpackage for the social sciences sv stroke volume tnf α tumor necrosisfactor alpha twa time weighted average wbc white blood cell countacknowledgementsassistance with this we thank bianca tudor md department ofanesthesia intensive care medicine and pain medicine medical university ofvienna spitalgasse vienna austria for her support in laboratoryskills performing elisasauthors™ contributionsall authors have read and approved the manuscript mo patientrecruitment data acquistion and prepratation of the manuscript ak studyprotocol writing and preparation of the manuscript bk study protocolwriting and preparation of the manuscript statistical analyisis gr dataanalysis revision of the manuscript ok data analysis preparation of themanuscript oz patient recruitment data acquisition data managementab patient recruitment data acquisition revsion the manuscript cr dataacquistion revision of the manuscript as patient recruitment revision ofthe manuscript ef study protocol writing and preparation of the manuscriptfundingmedical university of viennapartially funded by fresenius kabi deltex medical provided oesophagealdoppler monitors and disposablesthe sponsors were not involved in protocol development data acquisitionor data analysisavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestbarbarakabonmeduniwienacatethics approval and consent to participatethe main trial was approved by the local ethics committee of the medicaluniversity of vienna in ek and was registered atclinicaltrialsgov nct00517127 and eudract “ a writteninformed consent was obtained from all patients prior to participationconsent for publicationnot applicablecompeting intereststhe authors declare no competing interestsauthor details1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austria2department of outcomes research and general anesthesiologyanesthesiology institute euclid avenue cleveland clinic cleveland ohusa 3department of anesthesiology and general intensive care franziskushospital nikolsdorfergasse vienna austria 4department ofgynecology klinik ottakring montleartstrasse vienna austria5department of surgery medical university of vienna spitalgasse vienna austriareceived july accepted august referencesbrandstrup b tonnesen h beierholgersen r effects of intravenousfluid restriction on postoperative complications comparison of twoperioperative fluid regimens a randomized assessorblinded multicentertrial ann surg “ myles ps bellomo r corcoran t restrictive versus liberal fluidtherapy for major abdominal surgery new engl j med “chappell d jacob m hofmannkiefer k conzen p rehm m a rationalapproach to perioperative fluid management anesthesiology “lowell ja schifferdecker c driscoll df benotti pn bistrian brpostoperative fluid overload not a benign problem crit care med “sun y chai f pan c romeiser jl gan tj effect of perioperative goaldirected hemodynamic therapy on postoperative recovery following majorabdominal surgerya systematic review and metaanalysis of randomizedcontrolled trials critical care calvovecino jm ripollesmelchor j mythen mg effect of goaldirected haemodynamic therapy on postoperative complications in lowmoderate risk surgical patients a multicentre randomised controlled trialfedora trial br j anaesth “noblett se snowden cp shenton bk han af randomized clinical trialassessing the effect of doppleroptimized fluid management on outcomeafter elective colorectal resection br j surg “ 0cobradovic bmc anesthesiology page of alphonsus cs rodseth rn the endothelial glycocalyx a review of thevascular barrier anaesthesia “ pearse rm harrison da macdonald n effect of a perioperativecardiac outputguided hemodynamic therapy algorithm on outcomesfollowing major gastrointestinal surgery a randomized clinical trial andsystematic review jama “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationskimberger o arnberger m brandt s goaldirected colloidadministration improves the microcirculation of healthy andperianastomotic colon anesthesiology “kolarova h ambruzova b svihalkova sindlerova l klinke a kubala lmodulation of endothelial glycocalyx structure under inflammatoryconditions mediat inflamm rettig tc verwijmeren l dijkstra im boerma d van de garde emnoordzij pg postoperative interleukin6 level and early detection ofcomplications after elective major abdominal surgery ann surg “ gan tj soppitt a maroof m goaldirected intraoperative fluidadministration reduces length of hospital stay after major surgeryanesthesiology “jacob m chappell d rehm m clinical update perioperative fluidmanagement lancet “feldheiser a pavlova v bonomo t balanced crystalloid comparedwith balanced colloid solution using a goaldirected haemodynamicalgorithm br j anaesth “ orbegozo cortes d gamarano barros t njimi h vincent jl crystalloidsversus colloids exploring differences in fluid requirements by systematicreview and metaregression anesth analg “kabon b sessler di kurz a effect of intraoperative goaldirectedbalanced crystalloid versus colloid administration on major postoperativemorbidity a randomized trial anesthesiology “ robinson jd lupkiewicz sm palenik l lopez lm ariet m determination ofideal body weight for drug dosage calculations am j hosp pharm “steppan j hofer s funke b sepsis and major abdominal surgery leadto flaking of the endothelial glycocalix j surg res “ wilmore dw from cuthbertson to fasttrack surgery years of progress inreducing stress in surgical patients ann surg “ desborough jp the stress response to trauma and surgery br j anaesth“ veenhof aa sietses c von blomberg bm the surgical stress responseand postoperative immune function after laparoscopic or conventional totalmesorectal excision in rectal cancer a randomized trial int j color dis “ gilliland he armstrong ma carabine u mcmurray tj the choice ofanesthetic maintenance technique influences the antiinflammatory cytokineresponse to abdominal surgery anesth analg “ wichmann mw huttl tp winter h immunological effects oflaparoscopic vs open colorectal surgery a prospective clinical study archsurg “ watt dg han pg mcmillan dc routine clinical markers of themagnitude of the systemic inflammatory respon
Colon_Cancer
" preproof 0c highlights f0b7 propolis made by bees from bioactive plant resins has antiviral activity f0b7 propolis potentially can interfere with host cell invasion by sarscov2 f0b7 propolis blocks proinflammatory pak1 a kinase highly expressed in covid19 patients propolis a resinous material produced by honey bees from plant exudates has long been used in traditional herbal medicine and is widely consumed as a health aid and immune system booster the covid19 pandemic has renewed interest in propolis products worldwide fortunately various aspects of the sarscov2 infection mechanism are potential targets for propolis compounds sarscov2 entry into host cells is characterized by viral spike protein f0b7 propolis is a safe widely consumed functional food with medicinal properties f0b7 standardized propolis has consistent properties for lab and clinical research preprooffactor in advanced covid19 disease propolis has also shown promise as an aid in the treatment of various of the comorbidities that are particularly dangerous in covid19 patients including induced lung inflammation fibrosis and immune system suppression propolis components have inhibitory effects on the ace2 tmprss2 and pak1 signaling pathways in addition antiviral activity has been proven in vitro and in vivo in preclinical studies propolis promoted α this immunoregulation involves monocytes and macrophages as well as jak2stat3 nfkb and inflammasome pathways reducing the risk of cytokine storm syndrome a major mortality interaction with cellular angiotensinconverting enzyme ace2 and serine protease tmprss2 this mechanism involves pak1 overexpression which is a kinase that mediates coronavirusimmunoregulation of proinflammatory cytokines including reduction in il6 il1 beta and tnfrespiratory diseases hypertension diabetes and cancer standardized propolis products with consistent bioactive properties are now available given the current emergency caused by the covid19 pandemic and limited therapeutic options propolis is presented as a promising and 0crelevant therapeutic option that is safe easy to administrate orally and is readily available as a natural supplement and functional food keywords propolis sarscov2 covid19 antiviral antiinflammatory pak1 blocker introduction the covid19 pandemic is of grave concern due its impact on human health and on the deadly disease most require more robust data in clinical trials before they can be widely and safely used isolation and stayathome measures do not effectively protect essential workers economy it is much more deadly than influenza and other types of diseases that recently have had worldwide impact forcing countries to take unusual measures such as limiting travel closing schools businesses and other locations where many people can come into contact with each other various public healthcare strategies have been adopted in an attempt to reduce the impact of the disease but with limited effectiveness as the virus continues to spread often through asymptomatic patients unfortunately tests to determine if people are infectious or were previously infected are not widely available often are costly and frequently do not provide timely and accurate results various therapeutic alternatives have been proposed and tested however preproofœnonessential professions return to the workplace they become more at risk for infection in this scenario any options that could help ameliorate disease progression and its consequences even marginally would be useful the world needs safe alternatives to help reduce the impact of this especially health care personnel who have become infected and are dying at alarming rates economic and other necessities limit how well and how long these isolation measures can be maintained especially in poor countries and in poor communities such as slums and favelas [ ] as populations gradually try to get back to normalcy reducing social distancing and people in natural products which have historically been widely used to help avoid and alleviate diseases are among the options being considered as an adjuvant treatment for sarscov2 infection because they are generally inexpensive widely available and rarely have undesirable side effects some have proven antiviral activity an important advantage of using natural remedies is that people who have other health problems or who have mild flurelated 0c infection infection by sarscov2 the virus that causes covid19 is characterized by binding symptoms but do not have the means or courage to visit an already overcrowded medical facility could take simple and inexpensive measures to help reduce the impact of infection with sarscov2 considering the large number of deaths and other types of damage that the covid19 pandemic is causing there is an urgent need to find treatments that have been approved as safe and potentially able to inhibit the new coronavirus reduce its infectivity andor alleviate the symptoms of infection [ ] along this line propolis and its components emerge as potential candidate materials that could help to reduce the pathophysiological consequences of sarscovfactors increased pak1 levels also suppress the adaptive immune response facilitating viral replication [ ] sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory impairment and pulmonary failure immunologicalinflammatory between viral spike proteins and angiotensinconverting enzyme ace2 activation of the spike protein is mediated through proteases such as tmprss2 which play important roles in the viral infection after entry followed by endocytosis coronavirus infection causes pak1 upregulation a kinase that mediates lung inflammation lung fibrosis and other critical mortality preproofphenomena such as cytokine release syndrome have been shown to be important in the spectrum of sarscov2 infection these mechanisms are associated with an dysfunction more than the viral load per se along this line a retrospective observational study found higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα in patients with severe inflammatory diseases by affecting various metabolic cycles recently several studies have shown that propolis extract and some of its components act against several important targets in the pathophysiological context of the disease caused by sarscov2 such as reducing tmprss2 expression and reducing ace2 anchorage which would otherwise facilitate entry of covid19 compared to individuals with mild disease there is considerable evidence that propolis can reduce and alleviate the symptoms of the virus into the cell this is in addition to immunomodulation of monocytes macrophages reducing production of and eliminating il1 beta and il6 reduction of the transcription factors 0cviral replication and antiinflammatory action [ ] propolis and its properties are particularly relevant to sarscov2 infection such as immune system fortification reduced nfkb and jak2 stat3 and blocking pak1 which determine inflammatory activities and fibrosis caused by covid19 various comorbidities have been associated with severe covid19 symptoms and a greater chance of patients requiring intensive care these include hypertension and diabetes also mortality rates of covid19 patients are much higher in those with cardiovascular disease chronic respiratory disease and diabetes [ ] there is considerable evidence that these conditions could be alleviated by treatment with propolis this includes research in animal models of diabetes [ ] hypertension [ ] and cardiovascular disease [ ] propolis has properties that preproofthese plant products to make propolis which they use to protect the colony the production and use of propolis by honey bees evolved to the point that these social bees have considerably fewer immune genes than solitary insect species bees in colonies that produce more propolis are healthier and live longer and propolis consumption by the bees augments their immune [ ] as this variability can affect their medicinal properties standardized propolis products have been developed to help meet the need for a product that does not vary in the main bioactive components and is safe with minimal interaction with pharmaceutical drugs and proven efficacy in clinical trials in recent decades it has been shown to have antimicrobial including the composition of propolis varies according to the plant species available in each region propolis is a product derived from resins and plant exudates plants defend themselves from pathogens mainly by producing phytochemicals many of which have been extracted and used in medicine plant defense substances collected by bees include phenols and terpenoids phytochemical compounds that show promise for the inhibition of coronavirus in humans include quercetin myricetin and caffeic acid all components of propolis honey bees and many other species of social bees recognize these antimicrobial properties and selectively collect and process response to bacterial challenge antiviral antiinflammatory immunomodulatory antioxidant and anticancer properties propolis has historically been widely used to alleviate various diseases [ ] it also has been considered among other natural alternatives as an adjuvant treatment for sarscov2 infection 0ccompared to the propolis that the bees make from these plant materials because it is generally inexpensive widely available and rarely causes undesirable side effects some types of propolis that are highly valued for their medicinal properties such as brazilian green propolis are mainly produced by bees from materials they collect from specific plants in this case baccharis dracunculifolia after the botanical origin of the propolis has been identified extracts of the plant can be made to develop useful products such as a medicinal mouthwash however the medicinal properties of these plant extracts are often inferior can be safely consumed these propolis products and the raw material for their manufacture are extensively exported by brazilian companies especially to asian countries including japan south among natural medicine alternatives propolis has been widely studied and is already extensively consumed in many countries [ ] for example propolis products such as throat sprays and extracts are produced by hundreds of companies in brazil and are sold as a health aid in nearly every pharmacy throughout the country demonstrating on a practical basis that they preproofin fact propolis has a wide spectrum of pharmacological properties and is a dietary supplement that is commonly consumed by both healthy and sick people as a preventative precaution and for treatment it is also used in veterinary medicine due its antibacterial antifungal antiviral korea and china [ ] the importance of propolis in chinese japanese russian and korean medicine is reflected in the number of patents for propolis containing products registered by including about by china and “ each for japan russia and korea since about new propolisrelated patents were applied for in the us patent office it is a key ingredient in traditional chinese medicine japanese scientists have isolated and patented various brazilian propolis components for cancer treatment demonstrating their usefulness why propolis may be a good fit for dealing with covid19 antiparasitic hepatoprotective and immunomodulatory activities in the wake of the coronavirus outbreak south korea has seen a boon in the use of functional foods according to their ministry of food and drug safety œhealth functional foods are nutrients that have been proven to be beneficial to health in march of this year in response to the coronavirus pandemic the ministry eased regulations for propolis which is considered a 0ccould help fight against the covid19 pandemic active site of this enzyme is a relevant target for drug discovery functional food and allowed new oral formulations however despite considerable evidence that propolis can reduce and alleviate disease symptoms its acceptance as a healthpromoting supplement in human medicine has been limited in many countries such as the usa because of a relevant criticism that propolis products are not standardized and vary in their components and biological activity in part this is because propolis varies with the species of plants available in each region from which the bees collect resins to produce it [ ] however standardized propolis products have recently become available to help fill the need for a product that does not vary in the main bioactive components and effectiveness [ ] one such option a standardized brazilian propolis extract blend has been tested for safety and effectiveness in clinical trials for treating kidney disease and diabetes denture stomatitis and burn patients therefore propolis as a nutraceutical or functional food should be considered as a resource that preproofcaffeic acid phenethyl ester cape galangin chrysin and caffeic acid substances found in several different types of propolis around the world appeared as potential drugs against this viral target table specifically cape was predicted to interact with sarscov2 mpro in a potential targets in order to inactive the virus and reduce the damage that it causes the main protease of coronavirus sarscov2 mpro 3chymotrypsinlike cysteine enzyme is essential similar study therefore although it will be necessary to run in vitro assays to evaluate the potential antisarscov2 effects of propolis andor its constituents these in silico results are along this line hashem evaluated various natural compounds with an in silico approach molecular docking to try to find useful options for treating sarscov2 infection curiously for coronavirus processing of polyproteins and for its life cycle and therefore inhibition of the some propolis compounds can potentially interact with sarscov2 mpro the research community has examined the genetic code of coronavirus and the mechanisms underlying the damages caused by sarscov2 to help search for drugs andor well boding propolis can interact with ace and tmprss2 potentially blocking or reducing sarscov2 invasion of the host cell 0c sarscov2 strongly binds to angiotensinconverting enzyme ace2 using this enzyme as a receptor for invasion and replication in the host cell [ ] causing damage and increasing interpersonal transmission [ ] consequently ace inhibitors have been considered as useful drug alternatives however potential deleterious effects on users of angiotensinconverting enzyme ace inhibitors and angiotensin receptor blockers arbs have tool against potential cardiovascular events inhibition of ace2 enzyme is an important target for treatment against sarscov2 myricetin caffeic acid phenethyl ester hesperetin and pinocembrin rutin interacts with zinc fingers of the active sites of ace2 a metalloprotease that presents the same zinc finger in ace1 emerged as a concern for treatment of covid19 patients an observational study involving patients did not confirm this suspicion and therefore these classes of drugs remain an important infection [ ] güler et al prepared an alcoholic extract of propolis and identified some hydroxycinnamic acids caffeic acid pcoumaric acid tcinnamic acid and cape the flavanons rutin and myricetin and the flavones hesperidin chrysin and pinocembrin using molecular docking evaluations they found that rutin had the highest binding energy to ace2 followed by preproofvarious characteristics including inhibition of ace they found strong inhibition for most of the propolis types they studied with higher than ace inhibition the best results were found in addition to the in silico evidence osés et al evaluated several types of propolis for with the propolis components catechin and pcoumaric acid ace2 and tmprss2 transmembrane serine protease on the surface of host cells are used by sarscov2 via interaction with spike glycoproteins in order to proceed with invasion and replication vardhan sahoo studied several molecules commonly found in medicinal herbs using molecular docking procedures with relevant targets such as rnadependent rna polymerase rdrp ace2 and spike glycoproteins and compared the resulting scores with those of hydroxychloroquine limonin was the most active compound however quercetin and kaempferol also propolis compounds gave high docking scores kaempferol was studied in prostate cancer models and the expression of tmprss2 was reduced showing a potential mechanism of action for an antitumoral effect kaempferol could be an important propolis component for use against covid19 since it is involved in the inhibition of tmprss2 0c potentially interacting with ace2 rdrp and spike glycoprotein sgp besides its antiviral activity table propolis blocks pak1 potentially avoiding lung fibrosis and restoring a normal immune response among the possible targets for controlling covid19 damage the major œpathogenic contributes to their suppression pak1 inhibitors can both help combat the virus and restore a normal immune response kinase pak1 is key it is an essential component in malaria and viral infections but it is also involved in a wide variety of other diseases and disease conditions including cancer inflammation and immunosuppression when abnormally activated consequences of pak1 activation include lung fibrosis which is an aggravating factor in covid19 pak1 is activated by rac xu et al demonstrated that caffeic acid and its ester cape components of propolis can inactivate rac consequently inhibiting pak1 the inactivation of pak1 directly or upstream can potentially attenuate coronavirus pathogenesis bcells and tcells are lymphocytes that produce specific antibodies against viruses and other intruders and pak1 preproofimprove the immune response its components increase neutralizing antibody titers activate phagocytosis and increase ifnγ levels and the number of lymphocytes an increase in ifnγ levels was also detected by shimizu et al who evaluated the mechanisms involved in the cape caffeic acid phenethyl ester is a potent inhibitor of activation of nfkb in myelomonocytic cells anse et al demonstrated that propolis cape quercetin hesperidin and some other propolis flavonoids can inhibit the cytokine production of th1 and th2 type t cells while increasing tgfbeta an important antiinflammatory cytokine moreover cape can attenuate oxidative stress and inflammation through downregulation of jak2stat3 signaling propolis from europe and temperate asia usually made by bees from resins collected from poplar trees has predominantly flavonoid compounds while green propolis from baccharis dracunculifolia a propolis exclusively found in brazil has various kinds of flavonoids and prenylated phenylpropanoids such as artepellin c baccharin and drupanin these and all other types of propolis can inactivate pak1 artepillin c selectively inhibits pak1 table some studies have shown that propolis can act as an immunostimulant with the ability to effects of some types of propolis in a herpes simplex animal model 0c as well as having an immunomodulatory effect in which cape inhibits il6 phosphorylation and stat3 which are important for proinflammatory th17 development besides the antiinflammatory effect of cape and kaempferol paulino et al evaluated the antiinflammatory effect of artepellin c in rat paw edema and in cell cultures demonstrating that the activity was at least in part mediated by prostaglandin e2 and no inhibition through nfkb modulation artepillin c is an important biomarker of brazilian green propolis botanical source baccharis dracunculifolia immune modulation is desirable since coronavirus infection dysregulates the immune response in the initial phases of infection which facilitates viral replication however in later stages of covid19 the body develops an exaggerated inflammatory response which can greatly damage the lungs and other ans propolis different from typical immunosuppressants can help avoid immunosuppression during the initial phases of disease and in later stages reduce an exaggerated host inflammatory response inhibiting excess il6 il2 and jak signaling cape a propolis component is also known as an immunemodulating agent and should be considered as an alternative to help reduce an exaggerated inflammatory response in a mouse model propolis had immunomodulatory action in vivo on tolllike receptor expression and on preproofreplication and infectivity potentially decreasing lung inflammation due to antiinflammatory properties while promoting immune system fortification these are useful properties that could there is ample evidence for interference of propolis andor its components with viral propolis has been tested against various viral disease anisms initial successes have prompted research to determine the most useful components which may be modified to produce more active and specific pharmaceuticals viruses that were controlled by propolis in animal models with suggestion for control in humans include influenza [ ] herpes simplex virus type and hiv [ ] shimizu et al evaluated three different types of propolis in ethanol extracts using a murine model of herpes simplex virus type despite the chemical differences proinflammatory cytokine production help minimize the symptoms and deleterious effects of covid19 figure figure propolis as an antiviral substance 0cdue to the different plant origins of the resins the bees used to produce the propolis baccharis dracunculifolia baccharis eriodata and myrceugenia euosma all three propolis extracts not only had direct antihsv1 effects but also stimulated immunological activity against intradermal hsv1 infection in mice antiviral activity of propolis has been reported for dna and rna viruses poliovirus herpes simplex virus and adenovirus in an in vitro model cultured cells the best results were obtained against poliovirus and herpes virus with inhibition of the latter at a propolis concentration of ugml the propolis components chrysine and kaempferol caused a concentrationdependent reduction of intracellular replication of herpesvirus strains when host cell monolayers were infected and subsequently cultured in a drugcontaining medium quercetin another propolis component had the same effect but only at the highest concentrations tested ugml against various human herpes simplex virus strains with a intracellular replication reduction of approximately while it reduced the infectivity of bovine herpes virus human adenovirus human coronavirus and bovine coronavirus about the reduction was in the preproofimpairment and pulmonary failure inflammatory response to infection sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory immunologicalinflammatory phenomena such as cytokine release syndrome have been shown to be important mortality factors in sarscov2 infection higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα are found in patients with severe covid compared to those of individuals with mild disease molecular mechanisms involved in this immune process are the targets of various synthetic medicines being tested in patients including ciclesonide hydroxy chloroquine ivermectin and ketorolac which are pak1 blockers pak1 raccdc42activated kinase is overexpressed in the lung in response to sarscov2 infection and is a critical mediator of the cytokine storm that frequently results in mortality the most critical cases of covid19 which require ventilatorassisted intensive care and often result in prolonged ventilator dependency and death are a result of an exaggerated case of rotavirus antiinflammatory and immunomodulatory properties of propolis 0cin hospitalized patients fortuitously propolis components are effective pak1 blockers table there is considerable evidence that propolis can reduce and alleviate the symptoms of inflammatory diseases and has immunomodulatory properties [ ] however these properties can vary according to the plant origin of the propolis as well as the extraction processsolvent used and the inflammatory protocol cell culture animal models induction by lipopolysaccharides when the propolis extracts are tested tests with animal models have shown that propolis can reduce the levels of il6 and tnfalpha which are key proinflammatory mediators and increase the levels of the regulatory cytokine il10 kaempferol a propolis component reduces il6 tnfalpha and vegf vascular endothelial growth factor via the preproofifnγ in serum fernandes et al found that propolis exerted a positive adjuvant effect on vaccines that were developed against canine coronavirus they assayed ifnγ which is an effective way to measure the cellular response induced by a vaccine in a mouse model propolis added as an adjuvant to inactivated swine herpesvirus type vaccine stimulated increased cellular propolis is considered a safe immunostimulant and a potent vaccine adjuvant propolis has been widely tested as a vaccine adjuvant because it induces an earlier immune response and provides a longer protection period it is also included as an adjuvant ingredient in traditional chinese medicine propolis flavonoids have potential as adjuvants enhancing igg il4 and propolis has potential as a vaccine adjuvant erknfkbcmycp21 pathway table tests on macrophage cell cultures also demonstrated that propolis inhibits the production of il1 beta an important component of the inflammasome inflammatory pathway in diseases such as rheumatoid arthritis lupus and other autoimmune diseases although the mechanisms of action are not well elucidated these propolis components have potential as complementary supplements in the preventive treatment of chronic inflammatory diseases and humoral responses increasing ifnγ [ ] propolis enhanced the immune response to inactivated porcine parvovirus vaccine in guinea pigs when added to a trichomonas vaginalis protein vaccine propolis increased the igg antibody response times in mice 0ccancer is considered a relevant comorbidity factor for covid19 cancer patients have a patients cancer patients compared to the protein alone propolis was also effective as an adjuvant in the immunization of cattle with bovine herpesvirus it improved the humoral and cellular responses in mice inoculated with inactivated virus vaccines propolis as an adjuvant gave a similar immune response increasing ifnγ levels to alum and freund™s adjuvant in mice vaccinated with an hiv1 polytope vaccine candidate with less risk of undesirable side effects comorbidities and evidence of how propolis can help reduce their impact in covid19 to risk a visit to a clinic or hospital to determine if they have cancer alternative therapies could help retard cancer or reduce the impact of cancer and cancer treatment in covid19 times higher risk of progressing to severe covid19 disease than patients without comorbidities also the hospital environment during the coronavirus pandemic can interfere with or delay the treatment that cancer patients should receive patients with symptoms may choose not preproofacid cape quercetin and chrysin propolis and its components normally have little impact on normal cells displaying differential cytotoxicity in liver cancer melanoma and breast cell carcinoma cell lines [ ] propolis enhances the activity of tumor various types including bladder blood brain breast colon head and neck kidney liver pancreas prostate and skin cancers propolis could help prevent cancer progression in various parts of the world it is considered an alternative therapy for cancer treatment propolis extracts have been found to inhibit tumor cell growth both in vitro and in vivo including inhibition of angiogenesis demonstrating potential for the development of new anticancer drugs various components of propolis have been shown to inhibit cancer cell growth including cinnamic propolis has potential as a complementary therapy for cancer it has shown efficacy against necrosis factor related apoptosis inducing ligand trail in cancer cells hypertension and cardiovascular disease 0c hypertension and cardiovascular disease are considered relevant comorbidities for covid19 propolis has demonstrated antihypertensive effects in rat models [ ] in cameroon it is used in popular medicine to treat various ailments including high blood pressure propolis has been widely used as a dietary supplement for its health benefits including cardiovascular protective effects [ ] in a human trial consumption of propolis improved critical blood parameters including hdl gsh and tbars levels demonstrating that obesity a natural antiobesity agent it could contribute to a reduced risk for cardiovascular disease obesity is a major comorbidity and predictor of increased mortality in covd19 patients obesity and sarscov2 both induce an inflammatory process exacerbating sarscov2 infection in the obese propolis reduced inflammation and prevented hyperlipidemia and metabolic syndromes in highly caloric diet induced obesity in mice body weight gain visceral adipose tissue liver and serum triglycerides cholesterol and nonesterified fatty acids were all reduced in the propolis fed mice [ ] caffeic acid phenethyl ester a propolis component is preproofplatelet aggregation was reduced by propolis in tests on human blood in vitro and in other in vitro tests caffeic acid phenethyl ester cape a wellstudied bioactive propolis component inhibits collagen induced platelet activation thromboembolism thrombosis and microthrombosis microthrombosis disseminated intravascular coagulation and consequent multian failure are common in severely affected covid19 patients with associated high mortality rates anticoagulants are sometimes prescribed to such patients because they can reduce mortality tang et al an elevated level of plasminogen activator inhibitor1 pai1 is a biomarker and risk factor for thrombosis and atherosclerosis [ ] various types of evidence demonstrate that propolis can reduce platelet aggregation and other thrombosisrelated parameters propolis decreased thrombotic tendencies in mice by suppressing lipopolysaccharideinduced increases in pai1 levels [ ] propolis downregulated plateletderived growth factor and platelet endothelial cell adhesion molecules in lowdensity lipoprotein knockout mice 0c old age the elderly are more often affected by chronic inflammation characterized by systemically increased levels of proinflammatory cytokines which can contribute to development of a cytokine storm a major cause of covid19 mortality propolis has antioxidant properties which could help retard o
Colon_Cancer
lipases are very versatile enzymes and produced the attention of the several industrial processes lipase can be achieved from several sources animal vegetable and microbiological the uses of microbial lipase market is estimated to be usd million in and it is projected to reach usd million by growing at a cagr of from microbial lipases ec catalyze the hydrolysis of long chain triglycerides the microbial origins of lipase enzymes are logically dynamic and proficient also have an extensive range of industrial uses with the manufacturing of altered molecules the unique lipase triacylglycerol acyl hydrolase enzymes catalyzed the hydrolysis esterification and alcoholysis reactions immobilization has made the use of microbial lipases accomplish its best performance and hence suitable for several reactions and need to enhance aroma to the immobilization processes immobilized enzymes depend on the immobilization technique and the carrier type the choice of the carrier concerns usually the biocompatibility chemical and thermal stability and insolubility under reaction conditions capability of easy rejuvenation and reusability as well as cost proficiency bacillus spp achromobacter spp alcaligenes spp arthrobacter spp pseudomonos spp of bacteria and penicillium spp fusarium spp aspergillus spp of fungi are screened large scale for lipase production lipases as multipurpose biological catalyst has given a favorable vision in meeting the needs for several industries such as biodiesel foods and drinks leather textile detergents pharmaceuticals and medicals this review represents a discussion on microbial sources of lipases immobilization methods increased productivity at market profitability and reduce logistical liability on the environment and userkeywords microbial lipase fatty acids triglycerides protein engineering biosensor food industry candida antarctica lipase b calbintroductionthe serine hydrolases are present in abundantly and known as lipase enzyme which belong to triacylglycerol ester hydrolase family ec they can catalyze the hydrolysis and synthesis of longchain triglycerides to fatty acids diacylglycerol monoacylglycerol and glycerol known as carboxylesterases [ ] besides hydrolysis activity they display interesterification esterification aminolysis and alcoholysis activity which are contributed correspondence pchandrabbaugmailcom food microbiology toxicology department of microbiology school for biomedical and pharmaceutical sciences babasaheb bhimrao ambedkar university a central university lucknow uttar pradesh indiafull list of author information is available at the end of the in wide range industries [ ] lipase synthesizes esters from glycerol and longchain fatty acids in nonaqueous medium the microbial lipases are more valuable comparison to derive from plants or animals due to their variety of catalytic activities available high yield production and simplicity of genetic manipulation absence of seasonal fluctuations regular supply more stability safer and more convenient and the growth rate of microanisms very high in economically media [ ] the bacterial isolates offer higher activities such as neutral or alkaline ph optima and the thermostability associated to yeasts bacterial strains such as pseudomonas alcaligenes p aeruginosa p fragi p fluorescens bj10 bacillus subtilis b nealsonii s2mt and some species of fungi are penicillium expansum trichoderma penicillium chrysogenum the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cchandra a0et a0al microb cell fact page of aspergillus niger produces lipases in higher quantities [“] the increasing awareness about animal health and quality of animal produce and increasing consumption of enzymemodified cheese emc and enzymemodified dairy ingredients emdi the lipase market has been extensively increased [ ] due to the more benefits of microbial lipases over animal and plant lipases are also motivating the market growth the request for microbial sources is projected to witness significant growth in the near future due to their wide range of food processing applications [ ] the microbial lipase market is projected to dominate due to cleaning agent segment through the forecast period the growth of industrial microbial lipases in the detergents industry is the innovative key factor to replacing harsh chlorine bleach with lipase and reduced the industrial as well as sewage pollution from fresh water [ ] the microbial lipases in the form of powder is projected to dominate the microbial lipase markets due to its stability easy to handle and easier for packaging and its transportation preferred by the consumers [ ] a0these are extensively applicable in several another industries such as dairy food and beverage animal feed cleaning biofuel pharmaceuticals textile cosmetic perfumery flavour industry biocatalytic resolution esters and amino acid derivatives fine chemicals production agrochemicals biosensor and bioremediation [“] additionally altering in the dietary patterns have led to augmented the consumption of dairy products in the region increasing in trepidations about superior hygiene in consciousness of personal hygiene contagious diseases and bleaching household industrial surfaces the manufacturers who operate on a global level and the rising in implementation of lipase enzymes drive the demand for microbial lipases in the region [ ]between the and the market scope of lipase is expected to reach million by globally at a cagr of the asia“pacific was the largest market for lipase consumption in [ ] and during the forecast period the asia“pacific market is estimated to grow at the highest cagr moreover the rising prospects in the developing markets such as india china and brazil are expected to enhance the market scope of lipases over the forecast period novozymes as denmark e i du pont de nemours and company genencor us koninklijke dsm nv netherlands and chr hansen holdings as denmark are the key industries reported for the consumption of lipases at worldwide httpwwwmarke tsand marke tscom due to the specific properties such as enantioselectivity regioselectivity and broad substrate specificity properties the lipase showing more interest between all the enzymes [ ] this present review focused on discussing the sources of microanisms immobilization methods and their potential applications of lipases including commercially availablehistorical inside or outside the cells enzymes are proteins and have ability of catalyzing the various chemical and biochemical reactions they are highly specific natural catalysts to the various types of substrates and operate under insignificant conditions of environmental factor such as temperature pressure ph with high conversion rates [ ] lipase was first discovered in pancreatic juice as an enzyme by claude bernard in which hydrolysed unsolvable oil droplets and transformed them to soluble products after that the productions of lipase have been observed in the bacteria bacillus prodigiosus b pyocyaneus and b fluorescens in and in the current scenario serratia marcescens pseudomonas aeruginosa and pseudomonas fluorescens species of bacteria have been detected for the production of lipases on large scale lipolase was the first commercial recombinant lipase industrialized from the fungus thermomycesl anugiwnosus and expressed in aspergillus oryzae in traditionally lipase has been achieved from the animal pancreas and was made applicable as digestive supplements in the form of crude or in purified grade it has been extensively used as biocatalytic procedures for the synthesis of several novel chemical compounds [“]definition of a0lipaseslipases ec are known as triacylglycerol acylhydrolase which acts on carboxylic ester bonds is the part of hydrolases family [ ] they do not require any cofactor and belongs to the class of serine hydrolases triglycerides hydrolyzed into diglycerides monoglycerides fatty acids and glycerol by using the lipases naturally fig a01a the carboxylic esters bonds can be hydrolyzed by esterases in addition to lipases [ ]the hydrolysis of ester bonds at the interface catalyzes by lipases between an unsolvable phase of substrate and aqueous phase where the enzymes keep on liquefied under natural conditions fig a0 1b however pseudomonas aeruginosa candida anatarctica b and burkholderia glumae possessed a lid but did not show interfacial activation [ ] esterification transesterification interesterification acidolysis alcoholysis and aminolysis conversion reaction takes place by lipases [ ]the presence of a lid and the interfacial activation are not the suitable criteria for to categorize a true lipase carboxylesterase simply defined that catalyzes the hydrolysis and synthesis of longchain acylglycerols 0cchandra a0et a0al microb cell fact page of fig a hydrolysis of triglyceride converts into glycerol and fatty acid b representation of a molecule of lipase with its featuresproperties and a0characteristics of a0lipasesthe molecular weight of lipases is in the range of “ a0kda and reported to be monomeric protein the position of the fatty acid in the glycerol backbone chain length of the fatty acid and its degree of unsaturation are the factors and the physical properties of lipases depend on it [ ] the sensory and nutritive values of given triglyceride also affected by these features several lipases catalyze a number of useful reactions such as esterification due to their activeness in anic solvents [ ] lipases displayed ph dependent activities generally at neutral ph or up to ph and lipases are stable chromobacterium viscosum a niger and rhizophus sp produced extracellular lipases are active at acidic ph and p nitroaeducens produced alkaline lipase and active at ph under certain experimental conditions lipases have capability to reversing the reactions which leads to esterification and interesterification in the absence of water [ ] for the expression of lipase activities the cofactors are not necessary but calcium is the divalent cation stimulates the activity [ ] co ni2 hg2 and sn2 inhibited the lipase activities drastically and zn2 mg2 edta and sds inhibited slightly the halflife values determined temperature stability profiles of lipases and lower temperature shows more stability [ ] according to the regionspecificity lipases divided into two groups and revealed with acyl glycerol substrate without display of regiospecificity only fatty acids are discharged from all three positions of glycerols in the first group of lipases [“] the fatty acids regiospecifically discharged from the positions of acylglycerols in the second group of lipase triacylglycerol hydrolysed by lipases and constructed 2monoacylglycerol and free fatty acids 3diacylglycerols in a arrhizus r delemar c cylindracea and p aeruginosa the partial stereospecificity have been detected in the hydrolysis of triacylglycerols [“] these enzymes may be used to extract optically pure esters and alcohols due to these properties at low water activity using the anic media offers an exceptional prospect over variation of the solvent so varying the properties of the solvents an enzyme™s specificity may be transformed any solvent may utilize a substantial influence on the catalytic properties of an enzyme due to the possession of soft structures and delicate [ ]kinetic model of a0lipolysisat the substratewater interface lipolysis arises so the michaelis“menten model cannot be described it in a homogeneous phase which is effective only for biocatalysis in which enzyme and substrate are soluble [ ] at an interface to describe the kinetics of lipolysis simple models has been proposed and be made up of two consecutive equilibrium [ ] the alterable adsorption of enzyme to the interface e†”e happens in the first equilibrium phase a single substrate molecule s 0cchandra a0et a0al microb cell fact page of binds by the adsorbed enzyme e in the formation of es complex as a result in the second phase of equilibrium [ ] for the enzyme“substrate complex to the michaelis menten equilibrium this latter equilibrium is equivalent ending with the discharge of the products and renovation of the enzyme in the e form the subsequent catalytic steps take place once the es complex is formed [ ] the adsorbed lipase in the vicinity of substrate concentration at the interface is at the surface concentration instead of volumetric concentration conventional in the atmosphere [ ] the rejuvenated lipase remnant adsorbed to the interface and is only unrestricted after a number of catalytic cycles in this model fig a0the activity of lipase is a utility of interfacial conformation the enzyme can be denatured as well as triggered or neutralized and the interface is a suitable spot for restraining lipolysis the directly interaction of lipase inhibitor with the enzyme and obstructs the activity of lipase on the other hand via the adsorption to the interphase or to the substrate molecules few compounds can postpone the lipolytic reaction [“]lipase inhibitors are grouped into two categoriesa synthetic lipase inhibitors including phosphonates boronic acids and fats analogues andb natural compounds βlactones and several botanical foodstuffs”plant extracts and metabolites chiefly polyphenols saponins as well as peptides and particular nutritive fibers lipases are essential enzymes for lipid absorption so the absorption of fat or obesity controlled by the lipase inhibition β lactones including orlistat are the natural compounds have the ability to inhibit the lipase activity [ ] over of total dietary fats the pancreatic lipase is responsible for the hydrolysis in several countries for the treatment of obesity orlistat is the registered drug fig lipase catalyzed different reactionslipase inhibitors from a0microbial sourcesfrom microanisms several metabolic products have potent pancreatic lipase pl inhibitory activity the several bacterial fungal and other marine species continued search of effective antiobesity agent screened to find new compounds with pl inhibitory activity [ ]lipstatinthe digestive activity of pancreatic lipases controls by the lipstatin is a βlactone molecule which also controls the absorption of fat in the small intestine lipstatin was first isolated from streptomyces toxytricini is a precursor for tetrahydrolipstatin also known as orlistat xenical and alli the only fdaapproved antiobesity medication for longterm use is a very potent inhibitor of pl [ ] lipase inhibitory activity was lost on opening of βlactone ring the catalytic hydrogenation product of lipstatin is crystalline tetrahydrolipstatin and generally known as orlistat is currently on the market as an antiobesity agent [ ]panclicinsstreptomyces sp nr produced panclicins is another class of potent pl inhibitors nformylalanyloxy or nformylglycyloxy substituent are two alkyl chains are found in panclicins too contains blactone structures panclicins a and b are alanine type while panclicins c d and e are glycine type of compounds the inhibitory activity was recognized to the amino acid moiety alaninecontaining compounds being two to three folds weaker than glycinecontaining compounds valilactonevalilactone first isolated from streptomyces albolongus mg147cf2 strain from shaken culture and jar fermentation valilactone potently inhibited hog pl with an ic50 of a0ngml it also influenced inhibitory activity of esterase from hog liver with an ic50 value of a0mgml ebelactonesebelactone a and b are two ebelactones were isolated from the fermentation broth of actinomycetes strain g7gl closely related to streptomyces aburaviensis both a and b revealed pl inhibitory activity with ic50 values of against hog pl are a0 ngml and a0 ngml respectively esterastinesterastin was isolated from actinomycetes streptomyces lavendulae md4c1 strain from the fermentation 0cchandra a0et a0al microb cell fact page of broth competitively esterastin introverted the hog pancreas lipase with ic50 value of a0ngml caulerpenynecaulerpenyne extracted and purified from an extract of caulerpa taxifolia competitively introverted the activity of lipase with ic50 values of a0mm and a0mm using creamed triolein and disseminated 4methylumbelliferyl oleate as substrates individually [ ] the inhibitory activity of caulerpenyne was independent of substrate concentration suggesting direct interaction but dependent on the lipase concentration with the lipase protein slightly than interacting with the substrate oral supervision of corn oil with caulerpenyne to rats demonstrated a reduced and hindered peak plasma triacylglycerol concentration signifying its potential as a lipid absorption inhibitor [ ]vibralactonevibralactone secreted from boreostereum virens microfungi is a scarce fused βlactonetype metabolite covalently but reversibly transforms the active site serine of the enzyme via acylation by the blactone the ic50 of the vibralactone was resolute to be a0mgml [ ]percyquininpercyquinin obtained from the cultures of basidiomycetes stereum complicatum st inhibited pl with an ic50 of a0mm is another βlactone metabolite in one study on βlactone class of compounds the stereochemistry 2s 3s of the βlactone ring was found to impart specificity for the pl while 2r 3r stereochemistry was accountable for inhibition of hmgcoa synthase sources for a0microbial lipasesmicrobial lipases found universal in nature and are commercially substantial due to the low manufacturing cost superior stability and more availability than animal and plant lipases naturally or recombinant microbial lipases are generally used in diverse bioengineering applications a wide diversity of microbial resources provides by nature microbes have more adaptation abilities and inhospitable atmospheres like dead sea antarctica alkaline lakes hot springs volcanic vents and contaminated soils which provides extraordinary potential for the lipases production with specific features [ ] an enormous spinoff with esteem to the enantioselectivity hydrolysis and the formation of carboxyl esters has produced ready availability the marine microfloras have more capabilities for the formation of enzymes and proteins active compounds mostly lipase fashioned extracellularly secretion from fungi and bacteria [ ]in numerous biocatalytic procedures candida antarctica lipase b calb is the most habitually used enzyme and have a more amount of patents candida rugosa lipase crl is another scientifically significant lipase from the yeast which is a mixture of different isoforms and is commercially accessible and this grounding is known as œgenerally recognized as safe gras and used in the food industry pla1s and pla2s from fusarium oxysporum t lanuginosus a niger and trichoderma reesei between the yeast and fungal phospholipases are used in the degumming of vegetable oils and commercialized while mostly used in the food industry are pla1s pla2s and plbs extracted from a oryzae and a niger [ ] due to their high transphosphatidylation and hydrolytic activities plds isolated from actinomycete strains are commercially available and used in several industrialized procedures mostly the bacterial genera for the production of lipases and phospholipases have been reconnoitered are pseudomonas bacillus and streptomyces followed by burkholderia chromobacterium achromobacter alcaligenes and arthrobacter some lipases producing microanisms reveal new sources and applications of industrial enzymes as shown in table a0bacterial lipaseslipase has been detected initially in b prodigiosus and b fluorescens presently serratia marcescens and p fluorescens observed today™s best lipase producing bacteria subsequently [“] the glycoproteins and lipoproteins are bacterial lipases in most of the bacteria the enzyme production is affected by the certain polysaccharides have been observed [“] some bacterial lipases are thermostable and most of the bacterial lipases are reported as constitutive and nonspecific in their substrate specificity [ ] achromobacter sp alcaligenes sp arthrobacter sp pseudomonas sp staphylococcus sp and chromobacterium sp have been exploited for the manufacturing of lipases between the bacteria fungal lipasessince ²s fungal lipases have been studied due to their affluence in thermal and ph stability substrate specificity and activity in anic solvents and downstream processing these lipases have been exploited the contemporary period machinery favors the procedure of batch fermentation and low cost extraction methods so the fungal lipases have assistances over bacteria major filamentous genera of fungi included are rhizopus aspergillus penicillium mucor ashbya geotrichum beauveria humicola rhizomucor fusarium acremonium alternaria eurotrium and ophiostoma for the production of 0cchandra a0et a0al microb cell fact page of table microbial source of a0lipase and a0their industrial applicationmicrobial sourcesfungal species fusarium solani nfccl yarrowia lipolytica aspergillus oryzae rhizomucor javanicus meih rhizomucor miehei geotrichum candidum and c antarctica candida antarctica candida rugosa candida lipolytica penicillium camembertii trichoderma lanuginosus penicillium roquefortii aspergillus niger meyerozyma guilliermondii a niger gzuf36 aspergillus flavus candida antarctica rhizomucor meihei rhizomucor meihei rhizomucor miehei candida tropicalis aspergillus oryzae penicillium abeanum rhizopus nodosus candida rugosa p chrysogenum rhizomucor meihei p chrysogenum thermomyces lanuginose m miehei c parapsilosis m miehei c antarctica m miehei rhizopus arrhizusbacterial species achromobacter sp hegn virgibacillus pantothenticus hegn pseudomonas mendocina acinetobacter radioresistens bacillus sp fh5 staphylococcus pasteuri p fluorescensapplicationsreferenceshalophilic lipase for biodiesel productiondegrades very efficiently hydrophobic and unusual substrates such as nalkanes oils fats and fatty acids as lowcost carbon sourcessaturated fatty acids synthesized faster cheese ripening flavour customized cheesenonhydrogenated solid fatscocoabutter equivalentsthrough biocatalytic processes preparation of chiral intermediates which synthesized the pharmaceutical compounds related to the elimination of bad cholesterol for the treatment of the alzheimer™s disease oils and fats enriched removal of size lubricants denim finishinghuman milk fat substitutecheese ripening fatty acid productionproduction of glycerolglycolipidssynthesis of saturated triacyl glyceridesproduced a lipase containing detergent ˜lipoprime®™production of characteristic flavor of blue cheese in dairy productsfaster cheese ripening flavor customized cheese dough stability and conditioningpromising feed lipase using cheese wheypotential of the enzyme in the synthesis of functional oilsfat stain elimination synthesis of pharmaceuticals polymers biodiesels biosurfactantspitch control in paper and pulp industry polycondensation ring opening polymerization of lactones carbonates in polymeras a biocatalyst in personal care products such as skin and suntan creams bath oils etcsurfactants for baking industry dairy products noodlesoils and fats enriched cocoa butter substitutes synthesis of bioactive moleculesdegradation of crude oil hydrocarbonsuse for docosahexaenoic acid enrichment of tuna oilleather processing and dehairing and fat removalactivated sludge treatment aerobic waste treatmentfood industry waste treatmentsurfactants for baking industry dairy products noodlesfood industry waste treatmentnonhydrogenated solid fatsused as aroma and fragrance in the food beverage and pharmaceutical industrieshydroxamic acids food additivesynthesis of short chain flavour thioester in solvent free mediumproduction of flavour esterstreatment of oily wastewaterdishwashinglaundry removal of fat strainused in detergent industryusing in oil degradationenantioselective transesterification of a racemate rs4methyl1heptyn4en3ol a component of the insecticide s2852 [ ] 0cchandra a0et a0al microb cell fact page of applicationsreferencesthe production of flavour estersused in leather processinginvolved in enantioselective degradation of aopp herbicidescommonly used detergents enhance the removal of oily stains from various types of fabricswaste water treatmentanic solventtolerant lipolytic enzymeanic solventtolerant lipase for biodiesel productionbaking industry for bread makingenhanced stability in methanolbiodegradation of oil and anics determination as chemical oxygen demand cod biodegradation of food wastewater from restaurantsfood processing and oil manufactureapplication in biocatalysisalkaline lipases able to removing fatty stains when used in a washing machinesolvay enzyme products applicable for is a nonionic andor anionic detergent formulationdetergent formulations containing alkaline lipase used in laundry detergent œtopdegrading “ of the fatty material in the waste water management table continuedmicrobial sources staphylococcus warneri and s xylosus bacillus sp brevundimonas sp qpt2 micrococcus sp bacillus cereus hss marinobacter lipolyticus haloarcula sp g41 bacillus subtilis geobacillus stearothermophilus pseudomonas aeruginosa hfe733 pseudomonas sp natronococcus sp p alcaligenes m1 pseudomonas plantarii chromobacterium viscosum acinetobacter sp bacillus thermocatenulatus lactobacillus casei lactobacillus paracasei lactobacillus rhamnosus and lactobacillus plantarumused in medical industrycheese industry for improvement of flavor penicillium roquefortii staphylococcus warneri s xylosus pseudomonas cepacia pseudomonas spcheese industry for cheese ripeningproduction of flavour estersbiodiesel fuel productionformation of ˆ’15deoxyspergualin in drug industry as antitumor antibiotic and immunosuppressive agentlipases [ ] other species such as candida rugosa candida antarctica t lanuginosus rhizomucor miehei pseudomonas mucor and geotrichum colletotrichum gloesporioides produced ul of lipase are the most productive strain identified from the brazilian savanna soil by using enrichment culture techniques [ ] a niger c rugosa h lanuginosa m miehei r arrhizus r delemar r japonicus r niveus and r oryzae are the principal manufacturers of these commercial lipases [“]purification of a0lipasesto get consistency of lipase from a large number of bacteria and fungi various novel purification technologies are available generally several steps are contains for the purification of lipases contingent upon the purity estimated for food application the extracellular microbial lipases from the culture broth eliminated by the centrifugation or filtration in the fermentation process and cells are became freed [ ] the ammonium sulphate precipitation ultrafiltration or extraction with anic solvents is concentrated the cellfree culture broth the gel filtration and affinity chromatography like several combination of numerous chromatographic approaches purified about of the using precipitation steps and then ammonium sulphate and ethanol a homogenous product produces is the final step of gel filtration the novel purification machineries such as the i membrane separation procedures ii immuno purification iii hydrophobic interaction chromatography using epoxyactivated spacer arm as a ligand and polyethylene glycol restrained on sepharose iv polyvinyl alcohol polymers as column chromatography stationary phases and v aqueous two phase systems are frequently engaged after these prepurification steps [ ] the enzyme recovery and fold purification outcomes are found acceptable using of hydrophobic interaction chromatography [ ] an 0cchandra a0et a0al microb cell fact page of acid resilient lipase has been filtered from crude profitable arrangements by size exclusion on biogelp100 and ion exchange on monoq from a niger fungi using the chromatography on hydroxyapatite octylsepharose and sephacryl s200 the lipase was purified to homogeneity from r japonicus nr400 substrates for a0lipasea chiral alcohol moiety possesses by the glycerides which is the natural substrate for lipases the lipases were mostly valuable for the resolution or asymmetrization of esters bearing a chiral alcohol moiety was assumed [“]methods for a0lipase assaydue to the wide substrate specificity of lipases a number of assay protocols are engaged for lipase assay at the lipid water interface the determination of lipase activity is the analytical of free lipase using various physiochemical approaches the determination activities can be carried as with all reactions catalyzed by enzymes and observing the vanishing of the substrate or by the product release for the determining of the hydrolytic activity several methods are presented such as titrimetry spectroscopy photometry fluorimetry and infrared chromatography radio activity interfacial tensiometry turbidimetry conductimetry immunochemistry and microscopy [ ] the triacylglycerol hydrolysis reaction catalyzed by lipases generally can be written asmultifold benefits such as increase in thermal and ionic stability are applicable using immobilized lipases which upturns its proficiency when the enzyme is immobilized it is easier to control reaction parameters like flow rate and substrates convenience [ ] for immobilization include large surface area low cost reusability good chemical mechanical and thermal stability and insolubility the desirable characteristics of solid supports used according to the interface among the enzyme and support the enzyme immobilization approaches can be classified like physical and chemical procedures the interactions among the enzymes and support are by weaker bonds like hydrogen bonds van derwalls exchanges which create these interactions adjustable in the physical method for the interface among the enzyme and support are stronger by covalent bonds the procedure created irrecoverable in chemical methods [ ]physical methodsadsorptionin the physical approaches of immobilization adsorption procedure the enzymes immobilized by van der waals bonds hydrophobic interactions hydrogen bonds and ionic bonds on the surface of the support the enzyme becomes adsorbed bound and the substrates used mostly for this procedure are cation and anion exchange resins activated carbon silica gel alumina triacylglycerols †’ diacylglycerols free fatty acids †’ monoacylglycerols free fatty acids†’ glycerols free fatty acidsthe activity of lipases can be
Colon_Cancer
nasopharyngeal carcinoma npc is an epithelial malignancy with high morbidity rates in the east and southeast asia the molecular mechanisms of npc remain largely unknown we explored the pathogenesis potential biomarkers and prognostic indicators of npcmethods we analyzed mrnas long noncoding rnas lncrnas and micrornas mirnas in the whole transcriptome sequencing dataset of our hospital five normal tissues vs five npc tissues and six microarray datasets normal tissues vs npc tissues downloaded from the gene expression omnibus gse12452 gse13597 gse95166 gse126683 and gse70970 gse43039 differential expression analyses gene ontology go enrichment kyoto encyclopedia of genes and genomes kegg analysis and gene set enrichment analysis gsea were conducted the lncrnamirnamrna competing endogenous rna cerna networks were constructed using the miranda and targetscan database and a protein“protein interaction ppi network of differentially expressed genes degs was built using search tool for the retrieval of interacting genes string software hub genes were identified using molecular complex detection mcode networkanalyzer and cytohubbaresults we identified mrnas 14mirnas and lncrnas as shared degs related to npc in seven datasets changes in npc were enriched in the chromosomal region sister chromatid segregation and nuclear chromosome segregation gsea indicated that the mitogenactivated protein kinase mapk pathway phosphatidylinositol3 oh kinaseprotein kinase b pi3kakt pathway apoptotic pathway and tumor necrosis factor tnf were involved in the initiation and development of npc finally hub genes were screened out via the ppi networks several degs and their biological processes pathways and interrelations were found in our current study by bioinformatics analyses our findings may offer insights into the biological mechanisms underlying npc and identify potential therapeutic targets for npccorrespondence chenchuanben2010126com yuanji xu and xinyi huang contributed equally to this work department of radiation oncology fujian medical university cancer hospital fujian cancer hospital no fuma road fuzhou fujian people™s republic of chinafull list of author information is available at the end of the the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cxu a0et a0al cancer cell int page of keywords nasopharyngeal carcinoma npc bioinformatics analysis gene expression omnibus geo differentially expressed genes degs gene ontology go competing endogenous rna cerna network nasopharyngeal carcinoma npc is an epithelial malignancy originating from the inner mucosal lining of the nasopharynx in there were an estimated new cases of npc worldwide accounting for of all cancer sites and deaths due to npc accounting for of all cancer sites the incidence of npc is geographically imbalanced with new cases mainly concentrated in east and southeast asia especially in south china [ ] the estimated agestandardized incidence rate of npc is per in china and only per in north america [ ] the oncogenesis and progression of npc are strongly associated with hereditary susceptibility environmental or random aspects and epsteinbarr virus ebv infection in the early stages of npc the main pathogenesis is related to ebv infection indeed the expression of ebvdna can be used for the monitoring and followup of npc patients and maybe a useful indicator for riskstratification strategies however despite the great advances in medical technology in recent years such as the application of intensitymodulated radiotherapy and optimized chemotherapy strategies the detection and treatment of npc remain challenging epidemiological investigations have shown that although the incidence and mortality rates of npc have been greatly reduced over the past decade even in endemic areas the survival rate of npc patients remains unsatisfactory due to local recurrence and distant metastasis especially in patients with advancedstage disease thus noninvasive cancerspecific biomarkers for early diagnosis and precision treatment are urgently requiredmicroarray and bioinformatics analyses have enabled researchers to screen the genetic alterations in npc and have proven to be convenient methods for identifying potential biomarkers in other diseases these analytic methods have uncovered several biomarkers with proven prognostic value and potential clinical applications in npc for example one study discovered a novel long noncoding rna lncrna named linc01385 involved in npc development and functional analysis demonstrated that linc01385 could serve as a therapeutic target in npc microarray and rnasequencing techniques have been used to identify differentially expressed genes degs and signaling pathways related to the oncogenesis and development of npc one study analyzed two microarray datasets to identify degs in normal tissue samples and npc tissue samples however the falsepositive rate for the two datasets was potentially high and the limited sample size may have led to unreliable results due to the substantial heterogeneity among the patients zhang et a0al investigated three microarray datasets to identify degs and hub genes that may serve as potential diagnostic biomarkers for npc both these studies analyzed only chip datasets and did not include sequencing data which would lead to offsets in the studies thus the precise molecular mechanisms and biological processes underlying npc remain largely unknown and must be urgently investigated to develop a precise curative treatment for npcin recent years competitive endogenous rna cerna has provided a new way to study the molecular mechanism of cancer he et a0al found that circgfra1 may serve as cerna to regulate gfra1 expression by sponging mi34a in triple negative breast cancer cerna is a transcript that can compete shared mirnas and regulate one another at the posttranscription level and the cerna networks link the function of mrnas to the function of micrornas mirnas lncrnas circular rnas and other rnas cerna can act as mirna sponges thereby affecting mirna expression cerna regulation network refers to the regulatory network with cerna participationtherefore in this study we aimed to explore the molecular pathogenesis potential biomarkers and prognostic indicators of npc by analyzing the full transcriptome sequencing data from fujian cancer hospital along with six microarray datasets acquired from the gene expression omnibus geo to identify degs between npc samples and normal tissue samples our findings may guide the precision treatment of npcmaterials and a0methodssample collection and a0preparationfresh nasopharyngeal tissues were collected from five npc patients who were treated in fujian cancer hospital between november and may normal nasopharyngeal tissues from five healthy donors were also collected all tissue samples were frozen using liquid nitrogen the five npc patients consisted of three men and two women with a median age of a0years the age and sex of five donors were matched to the npc patients two patients had stage iii npc while three patients had stage iva npc according to the 8th edition of the current international union against canceramerican joint committee on cancer guidelines for npc the 0cxu a0et a0al cancer cell int page of ethics committee of fujian cancer hospital approved the human tissue samples related to this work project ethics number sq201901801 the fresh tissue samples were removed from liquid nitrogen and subjected to total rna extraction using the trizol method the purity photometer® spectrophotometer implen ca usa of the extracted rna was determined using a nano the rna concentration was measured using the qubit® rna assay kit and a qubit® fluorometer life technologies ca usa and the rna integrity was evaluated using the rna nano assay kit of the bioanalyzer system agilent technologies ca usarna sequencinga total of a0μg rna from each tissue sample served as the somal rna was eliminated using epicentre ribozero„¢ input material for the rna sample arrangements riborrna removal kit epicentre usa the rrnafree residue was removed using ethanol precipitation next depleted rna by using the nebnext® ultra„¢ direcsequencing libraries were produced from the rrnational rna library prep kit for illumina® neb usa in brief fragmentations were implemented by bivalent cations below the high temperature in nebnext first strand synthesis reaction buffer5x the firststrand cdna thus obtained was compounded using a stochastic hexamer primer and mmulv reverse transcriptase rnase h then secondstrand cdna synthesis was carried out using dna polymerase i and rnase h in the reaction buffer dntps with dutp were substituted for dttp residual overhangs were turned into blunt ends through exonucleasepolymerase activities after the adenylation of the ² ends of the dna fragments the nebnext adapter carrying a hairpin loop structure was ligated to initiate hybridization to optimize the cdna fragments with a length of “ a0bp we purified the library fragments with the ampure xp system beckman coulter beverly usa next a0 μl user enzyme neb usa was applied with the sizeselected adaptorligated cdna at a0°c for a0min and then at a0°c for a0 min thereafter pcr was conducted using phusion highfidelity dna polymerase general pcr primers and index x primer finally the obtained products were refined ampure xp system and the library quality was evaluated on the agilent bioanalyzer system after the library was constructed qubit20 was used for preliminary quantification the library was diluted to a0ngμl then used the agilent system was used to determine the insert size of the library after the insert size was confirmed to be as expected qpcr was used to confirm the valid concentration a0 nm and accurate quantification of the library to ensure library quality when the library was deemed eligible varying libraries were pooled to meet the demands of valid concentration and enable offline data volume hiseq sequencing was conducted these wholetranscriptome sequencing data were termed the fjch datasetmicroarray datageo httpwwwncbinlmnihgovgeo is a public genomics dataset repository which collects highthroughput sequencing data chips and microarrays we downloaded the following six gene expression datasets from geo the mrna gene expression datasets gse12452 and gse13597 the mirna gene expression datasets gse43039 and gse70970 and the lncrna gene expression datasets gse95166 and gse126683 all six datasets were annotated using r software version via annotation documents all datasets were from the species homo sapiens and the dataset type was microarray expression profile details of every dataset study are provided in table a0identification of a0degsto identify degs between normal tissue samples and npc samples we analyzed the microarray data by using the limma package and a multivariate linear model of the adjusted tstatistic the cutoff criteria were as follows log fold change absolute value of log2 in table associated microarray datasets from a0the a0gene expression omnibus geo databasereferencepmidrecordtissue platformnormal cancerfjchdodd et al bose et al zheng et al unknownnpcnpc gse12452 gse13597npc gse126683 npcunknowm gse95166npcillumina hiseqtm2500miseqtm[hgu133_plus_2] affymetrix human genome u133 plus array[hgu133a] affymetrix human genome u133a arrayagilent045997 arraystar human lncrna microarray v3 probe name versionarraystar human lncrna microarray v20 agilent_033010 probe name versionlyu et al bruce et al gse43039 gse70970npcnpcccdtmmirna850version 4p14ncounter® human mirna assay v10 nanostring 0cxu a0et a0al cancer cell int page of the fold change of gene expression ‰¥ and adjusted p value ‰¤ enrichment analyses of a0npcgene ontology go is the main bioinformatics tool for gene annotation and analysis of the biological processes bps of genes and gene products which involves annotation of bps molecular functions mfs and cellular components ccs go analysis of degs was conducted using upsetr the kyoto encyclopedia of genes and genomes kegg is a bioinformatics database resource for determining the highlevel functions and uses of cells and anisms from their genomic information to investigate the functional and pathway enrichment in go and kegg we used upsetr to identify the modules involved in biological functions gene set enrichment analysis gsea is a knowledgebased method for the translation of genomewide expression profiles we analyzed pathways using gsea and identified each functional cluster using clusterprofiler the cutoff criteria were a false discovery rate and p value construction of a0the a0lncrna‘mirna‘mrna interaction networkthe lncrnamirna interactions were predicted using the mircode tool httpwwwmirco de which is described as œa map of putative mirna target sites in the long noncoding transcriptome three convenient online databases namely mirdb httpwwwmirdb mirtarbas httpmirta rbase mbcnctuedutw and targetscan httpwwwtarge tscan were used to predict the target mrnas of the mirnas data with five or more binding sites were retained we selected the mrnas at the intersection of the three databases as the predictive targets of mirnas for the construction of lncrna“mirna“mrna cerna networks two separate cerna networks were constructed using upregulated and downregulated rnas and these were visualized using cytoscape https cytos cape version a popular online bioinformatics database protein‘protein interaction network constructionthe protein“protein interaction ppi network was predicted using the search tool for the retrieval of interacting genes string httpstrin gdb version online database significant insights into the underlying mechanisms of npc can be provided by investigating the interactions between proteins all degs of mrnas were predicted using string and a comprehensive score over was regarded as statistically significant cytoscape version https cytos cape was used to visualize biological network and integrate the data the molecular complex detection mcode version algorithm of cytoscape was used for detecting densely connected regions in the ppi network which represented the most closely related gene sets among the degs networkanalyst https wwwnetwo rkana lystcafaces homexhtml a visual analysis platform for the networkbased metaanalysis of gene expression data was used to visualize the proportion of degs cytohubba a cytoscape plugin was used to filter out the top hub genes with the strongest connections to the other genes in the merged networkstatistical analysismost statistical analyses were conducted using the bioinformatics tools mentioned above and the version of r software is version differential expression levels of mrna mirna and lncrna were obtained using a twotailed student™s ttest for the identification of deg benjamini and hochberg false discovery rate method were performed to adjust pvalue functional and pathway enrichment analyses were analyzed by the hypergeometric test and bonferroni correction variables were expressed as mean ± standard deviation a p value was regarded as notably significantresultsdata collection and a0preprocessingto determine whether there was clustering or outliers in the sample set the differences between the clustering of the mrna fig a01a“c lncrna fig a01d“f and mirna fig a01g h expression matrixes of the npc and normal tissue samples in different datasets were examined using threedimensional principal component analysis pca the results showed that npc was well distinguished from the normal tissue samplesidentification of a0degs in a0npcto identify the degs in npc the mrna lncrna and mirna expression profiles were analyzed using the limma package the results showed that mrnas lncrnas and mirnas were dissimilarly expressed logfc ‰¥ adjusted p ‰¤ fig a0 between the npc and normal tissues of these mrnas lncrnas and mirnas were significantly upregulated while mrnas lncrnas and mirnas were significantly downregulated in total degs were shared among the three mrna datasets fig a02d differentially expressed lncrnas delncrnas were shared among the three lncrna datasets fig a02h and differentially expressed mirnas demirnas were shared among the two mirna datasets fig a02k those degs may provide new insight into the biological mechanisms of npc and serve as 0cxu a0et a0al cancer cell int page of fig principal component analysis pca showing the clustering of mrna long noncoding rna lncrna and microrna mirna expression matrices in different samples and different datasets a“c pca of mrna expression between the nasopharyngeal carcinoma npc cluster and normal tissue cluster in the fjch a gse12452 b and gse13597 c datasets the purple dots represent the npc samples and the blue dots represent the normal tissue control samples d“f pca of lncrna expression between the npc cluster and normal cluster in the fjch d gse95166 e and gse126683 f datasets the blue dots represent the npc samples and the red dots represent the normal tissue control samples g h pca of mirna expression between the npc cluster and normal cluster in the gse70970 g and gse43039 h datasets the green dots represent the npc samples and the blue dots represent the normal tissue control samplespotential therapeutic targets for npc functional roles of delncrnas shared among the three lncrna datasets are provided in additional file a0 table a0s1 and functional roles of demirnas shared among the two mirna datasets are provided in additional file a0 table a0s2construction of a0the a0cerna networkto explore the role of mirnas and corresponding target mrnas as well as corresponding lncrnas in npc we predicted the target mrnas of the demirnas and lncrnas that may have interrelations with mirnas the results may help to better explain the 0cxu a0et a0al cancer cell int page of fig volcano plots of the distributions of degs in different datasets a“c volcano plots of the distributions of demrnas in the fjch a gse12452 b and gse13597 c datasets e“g volcano plots of the distributions of delncrnas in the fjch e gse126683 f and gse95166 g datasets i j volcano plots of the distributions of demirnas in the gse43039 i and gse70970 j datasets differentially expressed genes degs were those with a fold change of and a pvalue of in the mrna expression matrix lncrna expression matrix and mirna expression matrix upregulated degs are mapped as red spots and downregulated degs are mapped as green spots genes without notable variation are labelled as black spots d venn diagram of the degs among the mrna expression datasets fjch gse12452 and gse13597 h venn diagram of the degs among the lncrna expression datasets fjch gse43039 and gse70970 k venn diagram of the degs among the mirna expression datasets gse43039 and gse70970critical regulatory functions of mirnas mrnas and lncrnas the interaction of upregulated and downregulated mirnas with delncrnas was predicted based on mircode the prediction of target mrnas of upregulated and downregulated mirnas was performed using three databases mirdb mirtarbas and targetscan lncrnas mirnas and mrnas were included in the upregulated and downregulated lncrnamirnamrna cerna networks respectively fig a0 3a b the blue red and green nodes represent mirnas lncrnas and mirnas respectively additional files tables s3 s4 shows the details of the interactions of the upregulated and downregulated mirnas and mrnas respectively additional file a0 tables s5 and s6 shows the details of the interactions of the upregulated and downregulated mirnas and lncrnas respectivelygo and a0kegg analyses of a0degsto further analyze the possible functions of the degs we conducted biological analyses by using clusterprofiler and upsetr the results suggested that the degs were significantly enriched in go and kegg terms the go analysis showed that the following bps were notably enriched among the degs chromosome segregation nuclear chromosome segregation sister chromatid segregation mitotic sister chromatid segregation negative regulation of chromosome anization fig a0 4a the following mfs were largely enriched in atpase activity protein serinethreonine kinase activity atpase activity coupled tubulin binding catalytic activity acting on dna dna dependent atpase activity dna helicase activity and singlestranded dna dependent atpase activity fig a04b finally the following ccs were found to be largely enriched in the chromosomal region condensed chromosome chromosome centromeric region 0cxu a0et a0al cancer cell int page of fig interaction networks of mrnamirnalncrna in nasopharyngeal carcinoma npc a a cerna network of upregulated genes b a cerna network of downregulated genes the blue red and green nodes represent predictive mirnas predictive long noncoding rnas lncrnas and predictive micrornas mirnas respectivelyfig upsetr plots showing the distributions of the gene ontology go annotations associated with the differentially expressed genes degs in nasopharyngeal carcinoma npc a biological processes b molecular functions c cellular components d upsetr plot showing the distribution of pathways associated with the degs in npc based on kyoto encyclopedia of genes and genomes kegg analysiscondensed chromosome centromeric region nuclear chromosome telomeric region and condensed chromosome kinetochore fig a04c the kegg pathway analysis suggested that degs in npc were largely enriched in the cell cycle dna replication and small cell lung cancer fig a0 4d the results suggested that chromosomal 0cxu a0et a0al cancer cell int page of dysfunction was closely related to the development of npcgsea of a0npc‘related genesto explore the biological functions of the degs involved in npc gsea was applied the mrna expression profile of the fjch dataset was subjected to gsea by means of clusterprofiler the analysis showed that the following biological pathways were overrepresented in the npc tissues as compared to the normal tissues the mitogenactivated protein kinase mapk signaling pathway the phosphatidylinositol3 oh kinaseprotein kinase b pi3kakt signaling pathway fig a0 5a the apoptotic pathway and the tumor necrosis factor tnf signaling pathway fig a0 5b the pathways found our study were involved with cancer progression metastasis and apoptosisppi network analysis of a0degsthe string database was used version to explore the ppi network based on the correlations among the degs in npc the obtained data were then examined using cytoscape software the ppi network of degs was constructed using mcode to obtain the vital gene module the networkanalyzer plugin was applied to further analyze the ppi network according to the scores the cytohubba plugin was used to analyze the hub genes associated with npc and the following genes with the top grades were deemed to be hub genes nusap1 racgap1 prc1 kif4a top2a pbk kif2c tpx2 cenpu oip5 ttk mad2l1 ndc80 birc5 melk cenpf foxm1 tyms cdk1 and cep55 fig a0 those genes may contribute to the investigation of biological mechanisms and uncover underlying therapeutic targets for npcfig gene set enrichment analysis gsea of the gene expression profiles of the fjch dataset a gsea shows that the mitogenactivated protein kinase mapk pathway and the phosphatidylinositol 3kinaseprotein kinase b pi3kakt pathway are concentrated in nasopharyngeal carcinoma npc b gsea reveals that the apoptosis pathway and the tumor necrosis factor tnf pathway are concentrated in npc 0cxu a0et a0al cancer cell int page of fig proteinprotein interaction ppi networks a a ppi network of differentially expressed genes degs constructed using string software b most relevant gene sets in the ppi network extracted using mcode c further analysis of degs using networkanalyzer d the top hub genes with the most correlations identified using cytohubbago and a0kegg analyses of a0hub genesto analyze the functions of the top hub genes we again conducted biological analyses by using clusterprofiler and upsetr the results suggested that the hub genes were significantly enriched in go and kegg terms go analysis showed that changes in the following bps of hub genes were notably enriched in chromosome segregation nuclear chromosome segregation sister chromatid segregation mitotic sister chromatid segregation microtubule cytoskeleton anization involved in mitosis and regulation of chromosome segregation fig a07a in addition the changes in the following mfs were mainly enriched in protein serinethreonine kinase activity tubulin binding microtubule binding and protein cterminus binding fig a0 7b finally changes in the following ccs of degs were enriched in the chromosomal region spindle condensed chromosome chromosome centromeric region kinetochore microtubule midbody condensed chromosome centromeric region condensed chromosome kinetochore and mitotic spindle fig a0 7c kegg pathway analysis indicated that the degs in npc were mainly enriched in the cell cycle cellular senescence oocyte meiosis progesteronemediated oocyte maturation and platinum drug resistance fig a07d enrichment analyses of the hub genes were similar to the results of the analyses of the degs hence the findings obviously 0cxu a0et a0al cancer cell int page of fig upsetr plots showing the distributions of the gene ontology go annotations associated with the hub genes of nasopharyngeal carcinoma npc in the case of a biological processes b molecular functions and c cellular components and d upsetr plot showing the distribution of pathways associated with the hub genes of npc based on kyoto encyclopedia of genes and genomes kegg analysissuggested that chromosomal dysfunction was a vital contributor to the tumorigenesis of npcdiscussionin this work we performed a comprehensive analysis of the full transcriptome sequencing dataset of fujian cancer hospital and six microarray datasets downloaded from the geo repository to uncover degs between npc tissues and normal nasopharyngeal tissues we identified differentially expressed mrnas demrnas demirnas and delncrnas among the seven datasets and constructed a lncrnamirnamrna network of npc go enrichment analysis kegg enrichment analysis and gsea proved that the enriched components and pathways among the degs associated with npc were inseparable from the chromosome dysfunction mapk signaling pathway and pi3kakt signaling pathway discovered in npc we also identified the top hub genes in the ppi network related to npc and the results of the enrichment analysis of the hub genes were similar to those of the degsstudies have shown that lncrnamirnamrna networks play significant roles in the development and progression of tumors by constructing visual networks we can see the interaction between degs of different molecular types the lncrnamirnamrna network constructed in our study indicated that in npc mrnas could be regulated by lncrnas via corresponding mirnas li et a0al identified mirnas including highly expressed mirnas and lowly expressed mirnas from the serum of npc patients with different radiosensitivity these mirnas were found to have remarkable differences between the patients fold change ‰¥ or ‰¤ and p the highly expressed mirna hsamir6088 and the lowly expressed mirna hsalet7f13p from the above study were also found in our cerna networks we also identified hsamir29a3p and hsamir103a3p as demirnas which were recently found to act as circulating biomarkers of npc with fairly good diagnostic accuracy for detecting npc as compared with controls area under the curve the radioresistant npc cne2ir cell 0cxu a0et a0al cancer cell int page of line has been shown to overexpress jun guo et a0al identified junrelated mirnas by using mirdip software including hsamir200b3p hsamir1395p hsamir200c3p hsamir95p and hsamir92b3p thus jun could promote tumorigenesis and tumor development qing et a0 al found that inhibiting cjun expression could enhance radiosensitivity and induce cell cycle arrest and apoptosis the above results show that cerna networks can offer insights into the complex regulation patterns of npc and potentially facilitate the individualized treatment of npcgo analyses of the bps of the degs associated with npc showed that the negative regulation of chromosome segregation nuclear chromosome segregation sister chromatid segregation mitotic sister chromatid segregation and negative regulation of chromosome anization were closely associated with the oncogenesis of npc among the cc annotations chromosomal region condensed chromosome chromosome centromeric region condensed chromosome centromeric region condensed chromosome kinetochore and nuclear chromosome telomeric region were notably related to npc several studies have reported on the chromosomal aberrations involved in the carcinogenesis of npc including chromosomal gains or losses [ ] loss of heterozygosity chromosomal rearrangements and chromosomal imbalances in one study loss of heterozygosity on 3p was observed in “ of primary npc specimens and almost of precancerous lesions tan et a0al hypothesized that apoptosis induced by oxidative stress may lead to cadmediated chromosomal breakage after incorrect dna repair cells that survive apoptosis may carry chromosomal rearrangements leading to the tumorigenesis of npc to investigate common genetic variations in npc natasya et a0al screened out cases of npc in the malaysian population by the comparative genomic hybridization cgh technology and the results showed chromosomal changes in all npc cases enrichment analyses of the hub genes identified in our study were greatly compliant with the results of the enrichment analyses of the degs thus the above findings clearly implicate chromosomal dysfunction as an important contributor to the carcinogenesis of npcgsea showed that the mapk signaling pathway pi3kakt signaling pathway apoptotic pathway and tnf signaling pathway were the top four pathways associated with npc the enriched pathways identified in our investigation are related to tumor progression metastasis and
Colon_Cancer
" although rnabinding proteins play an essential role in a variety of different tumours there are stilllimited efforts made to systematically analyse the role of rnabinding proteins rbps in the survival of colorectalcancer crc patientsmethods analysis of crc transcriptome data collected from the tcga database was conducted and rbps wereextracted from crc r software was applied to analyse the differentially expressed genes degs of rbps to identifyrelated pathways and perform functional annotation of rbp degs gene ontology go function and kyotoencyclopedia of genes and genomes kegg pathway enrichment analyses were carried out using the database forannotation visualization and integrated discovery proteinprotein interactions ppis of these degs were analysedbased on the search tool for the retrieval of interacting genes string database and visualized by cytoscapesoftware based on the cox regression analysis of the prognostic value of rbps from the ppi network with survivaltime the rbps related to survival were identified and a prognostic model was constructed to verify the model thedata stored in the tcga database were designated as the training set while the chip data obtained from the geodatabase were treated as the test set then both survival analysis and roc curve verification were conductedfinally the risk curves and nomograms of the two groups were generated to predict the survival periodresults among rbp degs genes were upregulated while were downregulated of which twelve rbpsnop14 mrps23 mak16 tdrd6 pop1 tdrd5 tdrd7 ppargc1a lin28b celf4 lrrfip2 msi2 with prognosticvalue were obtaineds the twelve identified genes may be promising predictors of crc and play an essential role in thepathogenesis of crc however further investigation of the underlying mechanism is neededkeywords colorectal cancer crc rnabinding protein rbp prognostic model construction survival analysisintroductionas a significant class of cellular proteins rnabindingproteins rbps can interact with rna by recognizingspecial rnabinding domains and are widely involved inmultiple posttranscriptional regulatory processes suchas rna shearing transport sequence editing intracellular localization and translation control it is estimatedthat there are up to different proteins that have the correspondence lgzhyd1962163comdepartment of neurology the first affiliated hospital of harbin medicaluniversity you zheng street harbin heilongjiang provincepeople™s republic of chinapotential to bind rna in the human genome rbpsare characterized by the presence of an rnabindingdomain rbd that contains “ residues and usuallyadopts an α topology found in single or multiple copies these domains usually bind to rna depending onthe exact sequence or structure to date rbps havebeen reported to be associated with various human diseases such as spinal muscular atrophy and myotonicdystrophy there are various rbps involved intumourigenesis src associated with kda mitosissam68 is a member of the star signal transductionand rna metabolism activation family of rbps it is the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cfan world of surgical oncology page of involved in several steps of mrna metabolism such astranscription alternative splicing and nuclear export inaddition sam68 is associated with the signal transduction pathways required for the response of cells to stimuli cell cycle transition and viral infection tarbp2is overexpressed in metastatic cells and metastatichuman breast tumours and its abnormal activation canpromote the progression of breast carcinomas by affecting the stability of its target mrna colorectal cancer crc which includes colon andrectal cancer is a common digestive tract tumour themolecular pathogenesis of crc is a complex multistepprocess involving multiple acquired genetic and epigenetic abnormalities some rbps are known to be associated with colorectal cancer according to some studiesmuscleblindlike mbnl1 an rbp implicated in developmental control can significantly suppress crc cellmetastasis in vitro mbnl1 destabilizes snail transcriptsand thus inhibits the epithelialmesenchymal transitionemt of crc cells through the snailecadherin axisin vitro ras oncogenearecommonly seen in colon cancer activation mutationsinteractionsin this study an analysis was conducted of rbprelated genes in crc patients through differential geneexpression and protein moleculeinaddition a prognostic model was adopted to identifytwelve genes associated with the survival of crc patients we verified the model and performed survivalanalysis and risk assessment these results will help elucidate the underlying mechanism related to the survivalof crc at the molecular level thus providing a new direction for the prognosis of crc and clinical treatmentmethodsdata sourcethe fpkm transcriptome data of crc were obtainedfrom the tcga database website portalgdccancergov the total number of samples is of whichthere are samples in the tumour group and samples in the normal group then the rbp gene was obtained from the goa database website wwwebiacukgoa combined with the crc transcriptome sequencing map crc rbps were obtained the data ongene expression gse17536 in colorectal patients wereobtained from the geo database website wwwncbinlmnihgovgeo involving a total of cases allthe data were publicly available online this study requiresno experiments to be conducted by any author on humansor animals the flowchart of it is shown in fig between the two groups with the adjusted p andlogfc go and kegg pathway analysis of degsgo analysis represents a common method applied toconduct largescale functional enrichment study genefunctions can be categorized into biological processesbp molecular functions mf and cellular componentscc kegg is known as a commonly used databasewhere a large amount of data on genomes biologicalpathways diseases chemicals and drugsstoredthrough go and kegg analysis of degs barplot andbubble were drawn respectively all of the go and pathway terms were ranked by their ˆ’log10 q valueisproteinprotein interaction ppi networkthe search tool for the retrieval of interacting genesstring database stringdb is designedto analyse the ppi information degs were input intothe string database to obtain ppi information subsequently the cytoscape software was applied to visualizethe ppi network the cytoscape plugin mcode wasused to obtain the most relevant subnetwork moduleand then the hub genes ofthe four modules wereenriched for go and kegg analysisconstruction and analysis of prognostic modelscox regression analysis was conducted on the prognosticvalue of rbps from the ppi network with survivaltime the rbps related to survival were identified and aforest map was generated then the samples of thetcga database were designated as the training set andthe samples of the geo database were treated as the testset to construct the best prognostic model based on thetraining set twelve survivalrelated genes were identified by the model based on which the correlation coefficient of each gene was obtained then the risk score ofeach patient in the training set and test set was calculated according to gene expression in additionthepatients were classified into highrisk and lowriskgroups by the median value of the risk score the patients in the training set and the test set were categorized into either the highrisk group or lowrisk groupa survival analysis was conducted an roc curve wasgenerated and then the risk curves were constructed forthe training and test sets furthermore with univariateand multivariate analyses nomograms based on thegenes obtained from the prognostic model were generated to predict the length of survival for the patientsdata processing of differentially expressed genes degsthe rbps were analysed using r software to identify thedifference between the tumour group and the samplegroup wilcoxon test was carried out to identify degsresultsidentification of rbps degstranscriptome sequencing data of rbps of crcwas obtained from the tcga database the differential 0cfan world of surgical oncology page of fig flowchart of systemic analysis of rnabinding protein in patients with crcexpression analysis was conducted to find out that therewere upregulated genes and downregulatedgenes based on which volcano and heat maps weredrawn as shown in fig functional enrichment analyses of degsthe up and downregulated genes of degs were analysed for go function and kegg pathway enrichmentwhile both barplot and bubble were plotted theenriched go terms were divided into cc bp and mfrelevantontologies the top mostitems wereselected as shown in fig with regard to the upregulated genome the results of go analysis indicated thatdegs were mainly enriched in bps including ncrnametabolic process ncrna processing ribonucleoproteincomplex biogenesis and ribosome biogenesis and so oncc analysis revealed that the degs were significantlyenriched in preribosome tutp complex smallsubunitprocessome and cytoplasmic ribonucleoprotein granuleand so on as for the mf the degs were enriched in 0cfan world of surgical oncology page of fig volcano and heat map of rnabinding protein degs a volcano map b heat map red nodes represent upregulated genes and greennodes represent downregulated genescatalytic activity thus influencing rna and ribonucleaseactivityin the downregulated genome bp analysisdemonstrated that the degs were significantly enrichedas reflected in the regulation of translation rna splicing the regulation of cellular amide metabolic processand so on cc analysis showed that the degs weresignificantly enriched in cytoplasmic ribonucleoproteingranule ribonucleoprotein granule cytoplasmic stressgranule etc as for the mf the degs were enriched intranslation regulator activity mrna ²utr bindingand so on regarding the results of kegg pathwayanalysis as shown in fig the degs in the upregulatedgenome were primarily enriched in the pathways inribosome biogenesis in eukaryotes and rna transportetcthe degs werelargely enriched in the pathways in spliceosome andrna transport etcin the downregulated genomeppi network constructionthe protein interactions among the degs were predicted using string tools a total of nodes and edges in the ppi network were obtained as shownin fig 5a then cytoscape software was applied to drawa network diagram of genes as shown in fig 5bbesides four key subnetworks with the mcode plugin were extracted go was performed table andkegg enrichment analysis was conducted table onthe genes of the four subnetworks respectively finallythe four subnetworks were visualized as shown in fig5c“e the number of hub genes in these subnetworksis and respectivelyconstruction and analysis of prognostic modelscox regression analysis was carried out of the prognostic value of rbps interacting with survival time rbps related to survival were screened and a forestmap was drawn as shown in fig 6a then a prognosticmodel was constructed for the rbps related to prognosis and a prognostic marker gene comprised of rbps was established these twelve genes are nucleolarprotein nop14 mitochondrial ribosomal proteins23 mrps23 mak16 homolog mak16tudordomaincontaining tdrd6 processing of precursor pop1 tudor domaincontaining tdrd5 tudordomaincontaining tdrd7 peroxisome proliferatoractivated receptor1alphappargc1alin28 homolog b lin28b cugbpelavlike family member celf4 leucinerich repeatflightlessinteracting protein lrrfip2 and musashirnabinding protein msi2 then the corresponding forest map was drawn for these twelve genes asshown in fig 6b among them tdrd5 elf4 andlrrfip2 are classed as highrisk genes while the rest isclassed as lowrisk genes based on the establishedmodel the risk value of each patient was calculatedaccording to the median value the patients in thecoactivatamma 0cfan world of surgical oncology page of fig the gene ontology analyses of rnabinding protein degs a barplot shows go functional enrichment analysis predicted upregulateddegs including biological process cellular components and molecular functions the colour indicates the significance of the p value b bubbleshows go functional enrichment analysis predicted upregulated degs the size of the circle represents the number of genes enriched in theentry and the colour indicates the significance of the p value c barplot shows go functional enrichment analysis predicted downregulated degsd bubble shows go functional enrichment analysis predicted downregulated degstraining set and the test set were divided into either ahighrisk group or a lowrisk group among them thenumber of patients in the training set as well as thehighrisk group was the number of patients in thelowrisk group was in the test set the number ofpatients in the highrisk group was and that of patients in the lowrisk group was according to theresults the patients with highrisk scores had a shortersurvival time as shown in fig 6c d finally in terms ofsurvival prediction the roc curve showed a relativelydecent performance as shown in fig 6e f the aucvalue in the training set was and the auc valuein the test set was then the risk curves wereplotted for the training and test sets as shown in fig which reveals that their abscissas are the same theywere divided into high and lowrisk groups by the median value the patients were ranked by risk value inascending order the risk value of patients from left toright increased on a continued basis as did the risk offatalitythen independent prognostic analysis was conductedof univariate and multivariate for the training and testsets as shown in fig 8a“d according to the results ofsinglefactor independent prognosis analysisfor thetraining and test sets age and tumour stage can betreated as independent prognostic factor for the survivalof colorectal patients p in the multivariate independent prognostic analysis age and stage can be takenas independent prognostic factor for crc in the test setp for the training set however only stage can 0cfan world of surgical oncology page of fig the kegg pathway enrichment analyses of rnabinding protein degs a barplot shows kegg pathway analysis predicted upregulateddegs the colour indicates the significance of the p value b bubble shows kegg pathway analysis predicted upregulated degs the size of thecircle represents the number of genes enriched in the entry and the colour indicates the significance of the p value c barplot shows keggpathway analysis predicted downregulated degs d bubble shows kegg pathway analysis predicted downregulated degsbe taken as independent prognostic factors for crc p not age p finally nomograms were plotted for these rbpprognostic genes in the training set to predict the survival time of the patients as shown in fig 8e the rnaexpression of rbps was applied as parameters todraw the point line in nomograms the scores wereadded to obtain the total score which can be used topredictthe 1year 2year and 3year survival ratesamong crc patientsdiscussionas one of the most common malignant tumours crc ischaracterized by a high recurrence rate and poor prognosis especially in developed countries it is the thirdmost common cancer among males and ranks secondamong females [ ] to date various methods havebeen applied to predict biomarkers of crc prognosis rbps can regulate mrna stability and contributeto cancerassociated pathways in this paper therbps of crc were analysed through a series of analyses marker genes related to the prognosis of crcwere identifiedtudor domaincontaining tdrd refers to a family ofevolutionarily conserved proteins in general piwi andtdrd proteins are recognized as the major influencingfactors in pirna biogenesis and the development ofgerm cells in a previous study it was found thatmethyl lysinebound tdrds are primarily involved inhistone modification and chromatin remodelling whilemethyl argininebound tdrds are usually associatedwith rna metabolism alternative splicing small rna 0cfan world of surgical oncology page of fig rnabinding proteins degs are used to construct proteinprotein interaction networks and subnetworks a ppi interaction network mapobtained from string website b cytoscape visualizes the genes of the interacting ppi network red nodes represent upregulated genes whileblue nodes refer to downregulated genes c four mcode modules visualization d“g four most significant mcode components form theppi networkpathways and germ cell development [ ] tdrdshave now been detected in various cancers tdrd9 ishighly expressed in a subset of nonsmall cell lung carcinomas and derived cell lines through hypomethylationof its cpg island tdrd1 is closely associated witherg overexpression in primary prostate cancer according to the findings by jiang tdrdgenesphf20l1 arib4b setdb1 lbr tdrkhtdrd10 and tdrd5 showed high levels of amplification in more than of tcga breast cancer datasetstdrd5 has significant prognostic value for hepatocellular carcinoma hcc patients with higher tdrd5expression exhibit significantly poorer overall survivalthan patients with low tdrd5 expression an earlystudy revealed that tdrd5 was expressed in normalgastric and colonic mucosal tissues suggesting the possibility that the tdrd5 gene is modified in crc tdrd6 is capable of differentiating irradiated prostatecancer patients into early and late relapse groups inaddition tdrd7 may play a certain role in the migration of tumour cells in an analysis of crc mo discovered not only frameshift mutations butalso intratumoural heterogeneity of tdrd1 tdrd5and tdrd9 which in combination might alter tdrdgene functions and affectthe tumorigenesis of highmicrosatellite instability crc in our study it was foundthat tdrd5 tdrd6 and tdrd7 are differentiallyexpressed in crc and further studies on the role ofthese three genes in colon cancer are neededpop1 is a component of ribonuclease p which is aribonucleoprotein complex that generates mature trnamolecules by cleaving their ² end s[ ] in additionit is a component of the mrp ribonuclease complexwhich cleaves prerrna sequences in a previousstudy pop1 was found to be enriched in human prostate cancer celllines suggesting that it may besuitable as a potential marker for the diagnosis andprognosis of prostate cancer in addition pop1 is upregulated in crc and applicable as a prognostic factor forcrc nevertheless there is still no relevant research onthe mechanism of pop1 in crc so further studies arenecessaryppargc1a also known as pgc1α is a transcriptionalcoactivator of genes encoding proteins responsible forthe regulation of mitochondrial biogenesis and function d™errico discovered that in the presenceof bax pgc1αinduced ros accumulation is one of themain apoptosisdriving factors in crc cells they also 0cfan world of surgical oncology page of table the go function enrichment analysis of four most significant mcode componentsontologyidsubnetwork descriptioncountp valuepadjustbpbpbpccccccmfmfmfgo0042254ribosome biogenesisgo0016072rrna metabolic processgo0006364rrna processinggo0030684preribosomego0034455tutp complexgo0032040smallsubunit processomego0140098catalytic activity acting on rnago0003724rna helicase activitygo0030515snorna bindingsubnetwork bpbpbpccccccmfmfmfgo0000377rna splicing via transesterification reactions with bulged adenosine as nucleophilego0000398 mrna splicing via spliceosomego0000375rna splicing via transesterification reactionsgo0071013catalytic step spliceosomego0000974prp19 complexgo0005682 u5 snrnpgo0090079translation regulator activity nucleic acid bindinggo0003743translation initiation factor activitygo0008135translation factor activity rna bindingsubnetwork bpbpbpccccccmfmfmfgo0000460 maturation of 58s rrnago0034427 nucleartranscribed mrna catabolic process exonucleolytic ²²go0043629ncrna polyadenylationgo1905354exoribonuclease complexgo0000176 nuclear exosome rnase complexgo0000178exosome rnase complexgo0017091go0000175go0016896aurich element binding²²exoribonuclease activityexoribonuclease activity producing ²phosphomonoesterssubnetwork bpbpbpccccccgo0051028 mrna transportgo0050657 nucleic acid transportgo0050658rna transportgo0000346transcription export complexgo0016607 nuclear speckgo0000784 nuclear chromosome telomeric region157eˆ’730eˆ’221eˆ’503eˆ’251eˆ’516eˆ’842eˆ’262eˆ’160eˆ’737eˆ’737eˆ’871eˆ’697eˆ’203eˆ’310eˆ’282eˆ’154eˆ’166eˆ’504eˆ’622eˆ’147eˆ’217eˆ’150eˆ’103eˆ’707eˆ’129eˆ’154eˆ’264eˆ’113eˆ’113eˆ’569eˆ’235eˆ’506eˆ’118eˆ’238eˆ’171eˆ’426eˆ’584eˆ’690eˆ’108eˆ’438eˆ’103eˆ’103eˆ’103eˆ’662eˆ’966eˆ’983eˆ’223eˆ’437eˆ’437eˆ’524eˆ’323eˆ’367eˆ’369eˆ’127eˆ’582eˆ’818eˆ’818eˆ’818eˆ’190eˆ’223eˆ’223eˆ’911eˆ’188eˆ’found that pgc1α induced mitochondrial proliferationand activation in human intestinal cancer cells shin demonstrated that pgc1α overexpression waseffective in upregulating the proliferation of hek293and ct26 cells in addition its overexpression was correlated with an enhancement of tumourigenesis in acasecontrol study heterozygous carriers of rs3774921 inpgc1α showed an increased risk of crc pgc1αplays an essential role in the pathogenesis of colon cancer in a clinical study the expression of pgc1α wasassessed in crc patients using realtime quantitativepcr and the mrna level of pgc1α was found to bedecreased in the tumours of most patients however immunohistochemistry has also been performed to 0cfan world of surgical oncology page of table the kegg function enrichment analysis of four most significant mcode componentslistsubnetwork1descriptionribosome biogenesis in eukaryotesidhsa03008countsubnetwork2subnetwork3hsa03040hsa03013hsa03015hsa03010hsa03018spliceosomerna transportmrna surveillance pathwayribosomerna degradationp value184eˆ’408eˆ’100eˆ’547eˆ’padjust368eˆ’285eˆ’351eˆ’128eˆ’475eˆ’475eˆ’detect the expression of pgc1α the results revealedthat of the crc samples were positive whileno or weak pgc1α expression was detected in the nucleiof normal mucosa cells pgc1α expression is demonstrated to be related to lymph node metastasis thus itcan serve as a possible prognostic marker our results also show that pgc1α can be used as an independent prognostic factor for crcit is thought that lrrfip2 functions as an activator ofthe canonical wnt signalling pathway which is associated with dvl3 a factor upstream of ctnnb1betacatenin it positively regulates tolllike receptor tlrsignalling in response to agonists probably by competing with the negative flii regulator for myd88 bindingwhich plays a crucial role in the progression of coloncancer [ ] in this study lrrfip2 was identified asa candidate gene for alternative splicing in colon andprostate cancer there were three splice variants thatdiffered in their inclusion or skipping of exons andor these exons contain five predicted putative serinephosphorylation sites and one putative oglycosylationsite and could modulate lrrfip2 protein function as a familial hereditary disease hereditary nonpolyposisis mainly caused by dnacrc lynch syndromefig rnabinding protein dges are used to construct prognostic models survival analysis and verification of geo data sets a the prognosticrelated rbps shown in the forest map red indicates highrisk genes and green denotes lowrisk genes b the rbps obtained byconstructing the prognostic model shown in the forest map c survival analysis curve of training set red indicates patients in the highrisk groupblue denotes patients in the lowrisk group d survival analysis curve of test set e roc curve of training set f roc curve of test set 0cfan world of surgical oncology page of fig risk curve of training and test sets a the risk score distribution of training set b the distribution of survival status for training set c intraining set the heat map of rbps for the high and lowrisk groups d the risk score distribution of test set e the survival status distributionfor test set f in test set the heat map of rbps for the high and lowrisk groupsmismatch mismatch repair in lynch syndrome morakand colleagues discovered a paracentric inversion onchromosome 3p222 between the dna mismatch repairgene mlh1 and the downstream lrrfip2 gene transcribed in the antisense direction this generates twonew stable fusion transcripts thus removing the mlh1gene and protein function in another study conducted on a lynch syndrome family it was found thatthe mlh1itga9 fusion allele caused loss of heterozygosity loh in five genes including lrrfip2 which resulted in the loss of mismatch repair capabilities thus lrrfip2 may play a critical role in the pathogenesis of crccelf4 is responsible for encoding a protein with threedomains that bind an rna recognition motif andregulate premrna alternative splicing some studiesshowed that celf4 was hypermethylated in endometrialcancer methylated celf4 may be suitable for endometrial cancer screening of cervical smears further research is still needed to determine the role of celf4 intumoursas a member of the musashi family msi2 belongs tothe family of drosophila melanogaster rnabindingproteins it has been identified as a critical regulator ofhaematopoietic stem cell hsc selfrenewal and fatedetermination [ ] in this study msi2 was found tobe a central component in an unknown oncogenic pathway to promote intestinal transformation via the pdkaktmtorc1 axis msi2 is highly expressed in avariety of cancers including hcc and lung cancer recent studies on colon cancer celllines havesuggested that both usp10 and msi2 proteins areupregulated in addition ubiquitinspecific protease usp10 could stabilize the oncogenic factor msi2through deubiquitination the expression of msi2was detected in crc and control specimens from patients by the tissue microarray technique and immunohistochemical staining msi2 was highly expressed in of crc samples in addition high msi2expression was related to liver metastasis in crc patients in other cancers guo found that msi2expression was markedly increased in both pancreatic 0cfan world of surgical oncology page of fig independent prognosis analysis and prediction of and years of nomograms of crc patients in the training and test sets a singlefactor prognosis analysis of training set b multifactor prognosis analysis of training set c singlefactor prognosis analysis of test set d multifactor prognostic analysis of test set e the nomograms for predicting 1year 2year and 3year survival probability of patients with crc fortraining setlines and humanductal adenocarcinoma pdac cellpdac specimens and high msi2 expression was associated with poor prognosis of pdac high expressionof msi2 mrna is associated with decreased survival inacute myeloid leukaemia furthermore msi2 mayact as a prognostic biomarker in patients with cervicalcancer bladder cancer and oesophageal squamous cell carcinoma it was also found that its expression is upregulated in crc which makesitapplicable as a prognostic marker gene for crclin28 an oncofoetal rnabinding protein modulatesstem cell maintenance somatic reprogramming metabolism anismal growthtissue development andtumourigenesis two paralogues of lin28 were included lin28a and lin28b it is well established thatlin28a and lin28b inhibit let7 family mirnas andderepress let7 targets including ras pi3kakt mychmga2 and igf2bps thus promoting oncogenesis in liver cancer stem cells fang found thatoverexpression of msi2 resulted in the upregulation oflin28a stemness and chemotherapeutic drug resistance induced by msi2 overexpression were dramaticallyreduced by lin28a knockdown moreover msi2 andlin28a levels positively correlated with the clinical severity and prognosis in hcc patients king found that lin28b overexpression is associatedwith reduced survival time and increased probability oftumour recurrence in patients constitutive lin28b expression promotes not only tumorigenesis but alsolgr5 and prom1 expression in colonic epithelial cells in addition lin28b promotes the proliferationcolony formation and tumourigenesis of colon cancercells by increasing bcl2 expression a clinicalstudy found that lin28a and lin28b were overexpressed in oesophageal cancer cells especially on the invasive front high expression of lin28a and lin28bcorrelated significantly with lymph node metastasis andpoor prognosis hu found that gastric adenocarcinoma gac patient survival time was negativelycorrelated with the lin28b expression level wherebyhigher lin28b expression correlated with shortersurvival time in pdac patients high lin28b expression was significantly correlated with high levels oflymphatic metastasis distant metastasis and a poor 0cfan world of surgical oncology page of prognosis in addition patients with increased lin28bhad markedly reduced overall survival compared tothose with low lin28b in hcc and oral squamouscell carcinoma oscc thus lin28b is highlyexpressed in crc and plays an important role in itspathogenesisindicating that it is suitable as a targetgene for crc prognosisnop14 is a stressresponsive gene required for 18srrna maturation and 40s ribosome production as indicated by zhou nop14 in pancreaticcancer cells promotes motility proliferation and metastatic capacity according to the findings by du nop14 induced tumour invasion and metastasisby improving the stability of mutp53 mrna by inhibiting the wntcatenin pathways nop14 suppressesbreast cancer in addition nop14 can reducemelanoma cell proliferation and metastasis by regulating the wntbcatenin signalling pathway inclinical studies of patients with ovarian cancer downregulation of nop14 was associated with a significantly worse survival rate this study showedthatthe expression of nop14 was upregulated incrc but its role in pathogenesis requires further research and confirmationthe mrps23 gene which is responsible for encodinga 28s subunit protein has been found to be overexpressed in breast cancer uterine cervical cancer hcc colorectal cancer and uterine leiomyoma as revealed by gao inhibitingmrps23 could lead to a significant reduction in breastcancer metastasis by inhibiting the emt phenotype pu found that high mrps23 levels can predict poorclinical outcomes in hcc although the expressionof mrps23 is increased in crc its specific pathogenesisremains unclearmak16 encodes a ribosomal protein and plays an important role in ribosome biogenesis throughout the cellcycle in this study it was found that mutations inmak16 can induce cell cycle arrest at g1 phase duringwhich the cell synthesizes mrna and proteins in preparation for cell division at present there is still nostudy of the role of mak16 in the pathogenesis of tumours which requires further research to confirmin this paper a discussion was conducted about therole of the identified genes in tumours althoughsome genes were found irrelevant to the pathogenesis ofcrc their biological functions and changes in their expression in crc suggest that they may play a role incrc to some extent and further experiments need to beconducted for verification this is also a limitation ofour study more research is needed to explore the pathogenesis of crcthe above genes are related to the prognosis of crcmore research especially experimental studies is neededto verify the specific function of each gene our findingsmay improve the understanding of the incidence andprognosis of crc thus providing a reference for furtherimprovement of the diagnosis and tr
Colon_Cancer
hepatocellular carcinoma hcc is a high mortality disease the fifth most general cancer worldwide and the second leading to cancer‘related deaths with more than new patients diagnosed each year first the high expression of centromere m cenpm in mammary gland tissue of b‘catenin transformed mice was identifiedmaterials and methods in our study we evaluated the expression of cenpm in hepatocellular carcinoma based on data obtained from an online database multivariate analysis showed that the expression of cenpm and m classifica‘tion was an independent prognostic factor for patients with hepatocellular carcinomaresults survival analysis showed that patients with high cenpm had a worse prognosis than patients with low cenpm p a multivariate cox regression hazard model showed that b cells cd8 t cells macrophages and dendritic cells infiltrated by immune cells were statistically significant in liver cancer p using the network the most frequently changed neighbor genes of cenpm were shown and the most common change was rad21 our study found that the expression of cenpm was significantly increased in patients with hepatocellu‘lar carcinoma and it was related to a variety of clinical characteristics its correlation with the level of immune infiltra‘tion and poor prognosis so cenpm can be used as a useful prognosis for patients™ markers and hcckeywords hepatocellular carcinoma centromere protein m data mining prognosis hepatocellular carcinoma hcc a high mortality disease which is the fifth most general cancer in the world and the second most common lead to cancerrelated deaths with over new patients diagnosed each year [ ] viral hepatitis and nonalcoholic steatohepatitis are the most common causes of cirrhosis and approximately of cases develop to hcc due to the recurrence of hcc the prognosis of hcc remains discouraging and the 5year overall survival rate which correspondence wawang123soutlookcomdepartment of infectious diseases union hospital tongji medical college huazhong university of science and technology wuhan chinais only to despite the rapid development of advanced medical technology there are still no useful curable strategies for hcc patients byeno et a0al reported that based on longterm survival data serum opn and dkk1 levels in patients with liver cancer can be deemed as novel biomarkers that show prognostic useful for liver cancer other serum markers such as alphafetoprotein afp and alkaline phosphatase alp or akp are proverbially used in clinical but they lack sufficient sensitivity and specificity therefore finding useful biomarkers is indispensable for diagnosis and treatment for hcc patientsposttranscriptional modifications are essential for tumorigenesis and development centromere protein m cenpm otherwise called pane1 cenpm and the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cwu a0and yang cancer cell int page of c22orf18 which encodes a kinetic protein binds to spindle microtubules to regulate chromosomal separation during cell division expression of the pane1 gene was found preferentially in immune cells involving tumor tissues and tumor derived cell lines and leukemias and lymphomas brickner et a0 al found highly expressed in b lineage chronic lymphocytic leukemia bcll cells and resting cd19 b cells may be a potential therapeutic target for bcll bierie et a0al also demonstrated that human cenpm transcript crna was detected only in a0vivo or in a0vitro in activated b cells and t cells these studies suggested cenpm may play critical role in tumor immune response and may be deemed to therapeutic target for immunotherapy however the role of cenpm in hcc prognostic remains unclear in our study we evaluated the expression of cenpm in hcc based on data from an online database to further understand the biological pathway of cenpm related to the pathogenesis of hcc in addition we also analyzed the connection between cenpm expression and clinical features as well as the correlation of its expression with immune infiltration level in hcc comes an online tumor infiltrating immune cells analysis toolmaterials and a0methodsdata collectioninformation on rnasequencing data tissues workflow type htseqcounts and comparative clinical data patients data format bcr xml were identified and got from the level standardized fpkm of the tcgahcc cohort use boxplots to imagine expression differences for discrete variables the clinical factors included gender stage age grade tphase mphase nphase survival status and number of days of survival data analysis were checked by r version and r bioconductor software packagesgsea enrichmentgene set enrichment analysis gsea created a list of all gene permutations related to cenpm expression the samples were then divided into a high cenpm group and a low cenpm group as training sets to distinguish potential functions and use gsea to clarify significant survival differences genome replacement is performed multiple times with each exam the degree of expression of cenpm was used as a phenotypic marker normalized enrichment scores nes and nominal pvalues have been used to classify the pathways of enrichment in each phenotypeimmune infiltrates analysistimer is a comprehensive database for the systematic study of immune infiltration in various malignant tumor types the abundance of immune infiltrates cd8 t cells b cells cd4 t cells macrophages neutrophils and dendritic cells was evaluated by our statistical methods and has been estimated using pathology methods evaluated it the network also enables users to explore the clinical relevance of one or more tumor immune subpopulations and has the flexibility to correct multiple covariates in a multivariate cox proportional hazard model meanwhile we contrast the differential level of cenpm between tumors and normal on all tcga tumorsualcan and a0c‘bioportal analysisualcan is a userfriendly intelligent network asset for analyzing discovering cancer data and indepth analysis of tcga gene expression information one of the highlights of the portal is that it allows users to found between biomarkers or computer approval of potential genes of interest and to evaluate genes in different clinical subgroups such as gender age race tumor grade etc expression cbioportal is an online free asset that can visualize analyze and download largescale cancer transcription datasets the portal included cancer studies the tab biological interaction network of cenpm and its coexpressed genes was got and neighboring genes with altered frequencies were containedtargetscan analysistargetscan is a web for predicting potential biological targets of mirnas targetscanhuman deems that the match to human ²utr and its orthologs is estimate by a ucsc genomewide adjustment as an alternative they are ranked according to their predicted conservative positioning possibilities funrich is a tool designed to process varieties of geneprotein datasets in spite of the anism and used for functional enrichment analysis we used funrich tools for mirna enrichment analysis including analysis of biological pathways biological processes bp cellular components cc and molecular functions mfstatistical analysisscatter plots and paired plots visualize the differences between normal and tumor samples use delete ways to handle disappeared data and if any individual value is disappeared the data will exclude the full sample the relationship between clinical factors and cenpm was used by logistic regression wilcoxon rank sum test and kruskal test multivariate cox analysis was used to assess the effect of cenpm expression on survival and other clinical factors such as age gender stage distant metastasis benjamini“hochberg™s means of converting p values to fdr 0cwu a0and yang cancer cell int page of resultspatients™ characteristicsthe tcga database contains patients the clinical and pathological properties of these samples are shown in table a0 the middle age at diagnosis in tcga was a0 years old range “ a0 years and median finally contact for subjects was a0 months range “ a0months meanwhile followup for subjects conformed alive and death patients our study cohort included female and table tcga hepatic carcinoma patient characteristicsclinical characteristicsage at diagnosis yearfutime monthgender female malestage i ii iii iv nagrade g1 g2 g3 g4 nat‘classification t1 t2 t3 t4 tx nam‘classification m0 m1 mxn‘classification n0 n1 nx nastatus alive deathdata express as mean min“maxtotal “ “ male patients stage i was located in patients stage ii in stage iii in and stage iv in tumor stage was found t1 in patients t2 in t3 in and t4 in node stage contained n0 in and n1 in of cases had distant metastases all the subjects were adenomas or adenocarcinomascenpm expression and a0clinical factorsscatter plot showing difference in cenpm expression among normal and tumor samples p we then use paired plot to demonstrated the cenpm expression between normal and tumor from the same patients and the results was significant difference p fig a01a b the outcomes suggested that the expression of cenpm was significant difference the expression of cenpm correlated significantly with the patient grade p clinical stage p and tclassification p fig a01d“f univariate analysis utilizing logistic regression uncovered that cenpm expression as a clearcut ward variable was related to poor prognostic clinicopathologic factors table a0 cenpm expression in hcc as appreciably connected with grade or ci “ g1 vs g3 stage or ci “ i vs iii and tclassification or ci “ t1 vs t3 indicated that patients with low cenpm expression are inclined to advance to a further advanced stage than those with high cenpm expressionsurvival and a0multivariate analysissurvival analysis found that hcc with cenpmhigh had a worse outcome than that with cenpmlow p fig a0 1c the univariate analysis suggested that cenpm linked essentially to stage hr ci “ p and tclassification hr ci “ p table a0 multivariate analysis showed that the expression of cenpm hr p and m classification hr p were independent prognostic factors for patients with hcc table a0gsea analysisto identify useful pathways that may be differentially initiated in liver cancer we performed a gsea analysis between low and high cenpm expression datasets we chose the most abundant signaling pathway depending on the standardized enrichment score nes table a0 the results showed that cenpm high expression differentially enriched cell cycle dna replication rna degradation certain cancers phagocytosis p53 signaling pathway and purine metabolism fig a0 0cwu a0and yang cancer cell int page of fig cenpm expression and the association among clinicopathologic factors a the scatter plot showed the difference of cenpm expression between normal and tumor samples p b paired plot to demonstrated the cenpm expression between normal and tumor from the same patients and the results was significant difference p c survival analysis p d grade e stage f t‘stagetable cenpm expression associated with a0pathological characteristics logistic regressionclinical clinical characteristicstotal nage vs ‰¤ gender female vs malegrade g1 vs g3stage i vs iiit‘stage t1 vs t3odds ratio in a0cenpm expression “ “ “ “ “p‘valuecategorical dependent variable greater or less than the median expression levelimmune infiltrates related to a0cenpm in a0hccthe correlation between cenpm liver cancer in expression and the abundance of immune infiltrates was statistically significant p fig a0 3a a multivariate cox proportional hazard model showed that bcells cd8 t cells macrophages and dendritic cells infiltrated by immune cells were statistically significant in liver cancer p indicating that these immune cells significantly affect the prognosis it is worth further research and exploration table a0 at the same time the expression of cenpm was also statistically significant p finally we compared cenpm expression between various tumors and normal tissues the results showed that cenpm was overexpressed in various cancers p fig a03bassociations survival table a and a0 clinicopathologic characteristics in a0 tcga patients using cox regression b multivariate survival model after a0variable selectionwith a0overall clinicopathologic variablehr cip‘valuea age continuous gender female vs male stage iiiiiiiv grade g1g2g3g4 t‘classification t1t2t3t4 distant metastasis m0m1mx lymph nodes n0n1nx cenpm expression high vs lowb distant metastasis m0m1mx cenpm expression high vs low “ “ “ “ “ “ “ “ “ “ualcan and a0c‘bioportal analysis in a0hccin the age subgroup normal age “ a0years normal age “ a0years normal age “ a0years and normal age “ a0 years among patients with liver cancer cenpm has substantially higher transcription levels than healthy individuals analysis in the weight subgroup gender subgroup ethnicity subgroup tumor grade subgroups analysis also showed significantly higher cenpm in hcc patients fig a0 in order to determine the 0cwu a0and yang cancer cell int page of table gene sets enriched in a0phenotype highgene set namekegg_cell_cyclekegg_dna_replicationkegg_rna_degradationkegg_bladder_cancerkegg_non_small_cell_lung_cancerkegg_thyroid_cancerkegg_fc_gamma_r_mediated_phagocytosiskegg_p53_signaling_pathway kegg_purine_metabolismsizenesnom p‘valfdr q‘valnes normalized enrichment score nom nominal fdr false discovery rate gene sets with nom pval and fdr qval are considered as significantbiological interaction network of cenpm in liver cancer we used the network in the network tab in cbioportal showing the most frequently changed neighbor genes in cenpm and the most common change was rad21 fig a0 and table a0mirnas related to a0cenpmaccording to the online database the top of the mirna families are hsamir13075p hsamir449b3p and hsamir67785p related to the gene cenpm the conserved sites of the mirna family that are widely conserved in vertebrates fig a06a using the funrich database to explore the function of the identified mirnas bp are significantly enriched in the regulation of nucleobases signal transduction cell communication transport regulation of gene expression and anogenesis cc are mainly concentrated in the nucleus cytoplasm golgi apparatus endosome actin cytoskeleton and early endosome the mf are mainly transcription factor activity transcription regulation activity protein serine gtpase activity and ubiquitinspecific protease activity rich biological pathways in the erbb receptor signaling network trail signaling pathway glypican pathway and syndecan1 mediated signaling events and signal transduction events mediated by hepatocyte growth factor receptor cmet fig a06b“ediscussionin this work we performed a detailed assessment of cenpm expression in hepatocellular carcinoma based on the tcga database and explored its relationship with clinicopathological features survival function immune infiltration and expression differences understanding whether higher expression biomarkers in tumors are directly related to hepatocellular carcinoma can help us understand the mechanism of the observed clinical survival patterns in our findings the significant expression of cenpm suggests that cenpm may play an important cenpm is an role in regulating cancer progression this should draw attention to current views on the improvement of liver cancer and may reveal potential biomarkers or indicators to determine prognosisindispensable centromere protein involved in centromere assembly which regulates mitochondrial protein assembly and chromosome segregation huang et a0 al cloned and identified the cdna sequence of porcine pane1 and found that porcine pane1 gene was expressed differently in seven different tissues with the highest expression in lymph nodes and the lowest expression in kidney until now the expression of cenpm and its potential prognostic effect on hepatocellular carcinoma has not yet been investigated our outcomes showed that the expression of cenpm in hepatocellular carcinoma was related to advanced clinical pathologic factors grade clinical stage tclassification survival time and poor prognosis univariate analysis uncovered that cenpm expression as a clearcut ward variable was related to poor prognostic clinicopathologic factors and mclassification may play an indispensable role in the inclined to advance to a further advanced stage the univariate and multivariate analysis also suggested cenpm still remained freely connected with os and recommended that cenpm may act as a potential prognostic biomarker of prognosis and therapeutic target in hepatocellular carcinoma but more researches needed to conduct for further study in addition we further analyzed various clinicopathological features of hcc samples using the ualcan database and all of them showed high transcription of cenpmto identify differential signaling pathways in liver cancer gsea analysis results show that cell cycle dna replication rna degradation some cancers phagocytosis p53 signaling pathway and purine metabolism are differentially enriched in cenpm high expression phenotype cenpm may influence cell cycle dna replication rna degradation then controls the begins and development 0cwu a0and yang cancer cell int page of fig enrichment plots from gene set enrichment analysis gseaof cancer cells kim et a0al was identified cenpm as a key gene that mediates the anticancer effect of garlic and cisplatin on bladder cancer and showed that patients with low cenpm expressed better progressionfree survival than patients without high expression studies also found the cenpm genes encode a human minor histocompatibility antigen expressed by tumor cells [ ] yu et a0al found cenpm could as afprelated diagnostic biomarkers in hcc and validate the results using quantitative realtime pcr our study for the first time investigated the cenpm mrna expression and its prognostic significance in hepatocellular carcinoma chen et a0al demonstrated that lhx6 can inhibit the proliferation invasion and migration p53 signaling pathways during hepatocarcinogenesis qin et a0 al found that p53stabilizing and activating rna can strengthen the interaction between hnrnp k and p53 which ultimately leads to the accumulation and transactivation of p53 so cenpm may play a role via p53 signaling pathway and more researches needed to conduct in the future 0cwu a0and yang cancer cell int page of fig immune infiltrates correlation with cenpm in hcc a correlation between cenpm in hcc expression and abundance of immune infiltrates p b cenpm expression between various tumor and normal tissuetable multivariate survival model analysis based on a0timer online toolclinicopathologic variablecoefhr cip‘value sigagegender malerace blackrace whitestage iistage iiistage ivpurityb cellscd cellcd4 t cellsmacrophages “ˆ’ “ “ˆ’ “ “ “ “ “ˆ’ “ˆ’ “ˆ’ “ “neutrphilsdendriticcenpmpvalue significant codes ‰¤ ‰¤ ‰¤ ‰¤ · ˆ’ “ “ “·previous studies demonstrated that human cenpm transcript crna was only detected in activated b and tcells either in a0vivo or in a0vitro these studies suggested cenpm may play important role in tumor immune response so we used an online tool to analysis immune infiltrates correlation with cenpm in hcc multivariable cox proportional hazard model showed that b cells cd8 t cells macrophages and dendritic cells of immune infiltrates statistically significant p in hcc indicating that these immune cells significantly affecting the prognosis a latest study showed cd8 cd68 and foxp3 immune cells were associated with hcc particularly in the invasive margin macrophages not only promote the proliferation colony formation and migration of hcc cells but also maintain tumor growth and metastasis by secreting hepatocyte growth factor hgf pang et a0 al proposed that fusion of dendritic cells dc with tumor cells can effectively activate antitumor immunity in the body and affect tumor progression these studies indicate that cenpm may play an important role in tumor immune response and can be a good therapeutic target for immunotherapy 0cwu a0and yang cancer cell int page of fig boxplot showing relative expression of cenpm in subgroups of patients with hcc ualcanto determine the biological interaction network of cenpm in liver cancer we applied the most frequently changed neighbor genes of cenpm on the network tab in cbioportal and the most frequent change was rad21 rad21 is a nuclear phosphoprotein which becomes hyperphosphorylated in cell cycle m phase one study found that depletion of rad21 resulted in reduced levels of h3k27me3 at the hoxa7 and hoxa9 promoters resulting in enhanced selfrenewal of hematopoietic stem and progenitor cells hspc recent studies have shown that removing rad21 in a lacking pds5 can rescue the phenotype observed only in the absence of pds5 our study may provide information on adhesion kinetics in replication fork studies in patients with liver cancer our study also used the targetscan online tool to distinguish cenpmrelated mirnas to check the function of the identified mirnas bioenrichment was performed through the funrich database it is rich in erbb receptor signaling network trail signaling pathway glypican pathway syndecan1 mediated signaling events and biological pathways of hepatocyte growth factor receptor cmet signaling events studies have reported that selective cmet inhibitors have antitumor activity in hcc and have acceptable safety and tolerability in child“pugh a liver function patients a recent study found that abnormal hgfcmet upregulation and activation are often observed in bladder cancer studies have also found that metastasis associated with colon cancer macc1 regulates pdl1 expression and tumor immunity in gastric cancer gc cells through the cmetaktmtor pathway we hypothesized that cenpm may regulate the expression of cmet leading to the occurrence of hcc and more related research fig the network for cenpm and the most frequently altered neighbor genestable the type and a0frequency of a0cenpm neighbor gene alterations in a0hcc cbioportalgene symbolrad21rps27ahctf1nuf2pmf1amplification homozygous deletionmutation total alteration 0cwu a0and yang cancer cell int page of fig enrichment analysis of the mirna altered in the cenpm in hcc funrich and targetscan a conserved sites for mirna families broadly conserved among vertebrates b cellular components c kegg pathway analysis d biological processes e molecular functionsis needed to date this study demonstrates for the first time the important role of cenpm in the prognosis of hepatocellular carcinoma however future clinical trials are needed to validate these results and promote the use of cenpm in the prognostic evaluation of hepatocellular carcinomasour study found that the expression of cenpm was significantly increased in patients with hepatocellular carcinoma and was related to a variety of clinical features its correlation with the level of immune infiltration and poor prognosis so cenpm may become a useful biomarker for the prognosis of patients with liver cancerkegg kyoto encyclopedia of genes and genomes bp biological processes cc cellular components mf molecular functions os over survivalacknowledgementsnot applicableauthors™ contributionswzh designed and analyzed the research study wzh wrote and revised the manuscript ydl and wzh collected the data and all authors contributed to final manuscript all authors read and approved the final manuscriptfundingthis work is not supported by grantsavailability of data and materialsrna‘seq data and corresponding clinical data were acquired from the data portal for tcga https porta lgdccance rgovethics approval and consent to participatenot applicableabbreviationshcc hepatocellular carcinoma cenpm centromere protein m gsea gene set enrichment analysis tcga cancer genome atlas go gene ontology consent for publicationnot applicable 0cwu a0and yang cancer cell int page of competing intereststhe authors declare that they have no competing interestsreceived january accepted august references torre la bray f siegel rl ferlay j lortet‘tieulent j jemal a global cancer statistics ca cancer j clin “tang y wang h ma l et al diffusion‘weighted imaging of hepatocellular carcinomas a retrospective analysis of correlation between appar‘ent diffusion coefficients and histological grade abdominal radiol “ coskun m hepatocellular carcinoma in the cirrhotic liver evaluation using computed tomography and magnetic resonance imaging exp clin transplant 201715suppl lang h sotiropoulos gc brokalaki ei et al survival and recurrence rates after resection for hepatocellular carcinoma in noncirrhotic livers j am coll surg “jiao y fu z li y meng l liu y high eif2b5 mrna expression and its prognostic significance in liver cancer a study based on the tcga and geo database cancer manag res “ byeon h lee sd hong ek et al long‘term prognostic impact of osteo‘ pontin and dickkopf‘related protein in patients with hepatocellular carcinoma after hepatectomy pathol res pract “shen y bu l li r et al screening effective differential expression genes for hepatic carcinoma with metastasis in the peripheral blood mononu‘clear cells by rna‘seq oncotarget “ renou jp bierie b miyoshi k cui y djiane j reichenstein m shani m hennighausen l identification of genes differentially expressed in mouse mammary epithelium transformed by an activated beta‘catenin onco‘gene “ bierie b edwin m melenhorst j et al the proliferation associated nuclear element pane1 is conserved between mammals and fish and preferentially expressed in activated lymphoid cells gene expr patterns “ brickner ag the pane1 gene encodes a novel human minor histocom‘patibility antigen that is selectively expressed in b‘lymphoid cells and b‘cll blood “ kruppa j jung k automated multigroup outlier identification in molecu‘lar highthroughput data using bagplots and gemplots bmc bioinf li t fan j wang b et al timer a web server for comprehensive analysis of tumor‘infiltrating immune cells cancer res 20177721e108“e110110 https doi1011580008‘5472can‘‘ chandrashekar ds bashel b balasubramanya sah creighton cj rodri‘guez ip chakravarthi bvsk varambally s ualcan a portal for facilitat‘ing tumor subgroup gene expression and survival analyses neoplasia “ https doi101016jneo201705002 gao et al sci signal cerami et al cancer discov when publishing results based on cbioportal https doi1011582159‘ agarwal v bell gw nam j bartel dp predicting effective microrna target sites in mammalian mrnas elife 20154e05005 https doi107554elife pathan m keerthikumar s ang cs gangoda l quek cy williamson na mouradov d sieber om simpson rj salim a bacic a funrich an open access standalone functional enrichment and interaction network analysis tool proteomics “ https doi101002pmic20140 foltz dr jansen le black be bailey ao yates jr iii cleveland dw the human cenp‘a centromeric nucleosome‘associated complex nat cell biol “ huang h deng h yang y et al molecular characterization and associa‘tion analysis of porcine pane1 gene mol biol rep “ kim wt seo sp byun yj et al the anticancer effects of garlic extracts on bladder cancer compared to cisplatin a common mechanism of action via centromere protein m am j chin med “ yu z wang r chen f et al five novel oncogenic signatures could be uti‘lized as afp‘related diagnostic biomarkers for hepatocellular carcinoma based on next‘generation sequencing dig dis sci “ chen hq zhao j li y et al epigenetic inactivation of lhx6 mediated microcystin‘lr induced hepatocarcinogenesis via the wntβ‘catenin and p53 signaling pathways environ pollut 2019252pt a216“ qin g tu x li h et al lncrna pstar promotes p53 signaling by inhibit‘ing hnrnp k desumoylation and suppresses hepatocellular carcinoma hepatology https doi101002hep30793 ihling c naughton b zhang y et al observational study of pd‘l1 tgf‘β and immune cell infiltrates in hepatocellular carcinoma front med laus‘anne dong n shi x wang s et al m2 macrophages mediate sorafenib resistance by secreting hgf in a feed‘forward manner in hepatocellular carcinoma br j cancer “ pang yb he j cui by et al a potential antitumor effect of dendritic cells fused with cancer stem cells in hepatocellular carcinoma stem cells int janco jmt lamichhane p karyampudi l knutson kl tumor‘infiltrating dendritic cells in cancer pathogenesis j immunol “ fisher jb peterson j reimer m et al the cohesin subunit rad21 is a negative regulator of hematopoietic self‘renewal through epigenetic repression of hoxa7 and hoxa9 leukemia carvajal‘maldonado d byrum ak jackson j et al perturbing cohesin dynamics drives mre11 nuclease‘dependent replication fork slowing nucleic acids res “ bouattour m raymond e qin s et al recent developments of c‘met as a therapeutic target in hepatocellular carcinoma hepatology “ sim wj iyengar pv lama d et al c‘met activation leads to the establish‘ment of a tgfβ‘receptor regulatory network in bladder cancer progres‘sion nat commun tong g cheng b li j et al macc1 regulates pdl1 expression and tumor immunity through the c‘metaktmtor pathway in gastric cancer cells cancer med “publisher™s notespringer nature remains neutral with regard to jurisdictional claims in pub‘lished maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your field¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold open access which fosters wider collaboration and increased citations maximum visibility for your research over 100m website views per year ¢ at bmc research is always in progresslearn more biomedcentralcomsubmissionsready to submit your research choose bmc and benefit from 0c'
Colon_Cancer
"it is well understood that the level of molecular oxygen o2 in tissue is a very important factor impacting both physiology and pathological processes as well as responsiveness to some treatments data on o2 in tissue could be effectively utilized to enhance precision medicine however the nature of the data that can be obtained using existing clinically applicable techniques is often misunderstood and this can confound the effective use of the information attempts to make clinical measurements of o2 in tissues will inevitably provide data that are aggregated over time and space and therefore will not fully represent the inherent heterogeneity of o2 in tissues additionally the nature of existing techniques to measure o2 may result in uneven sampling of the volume of interest and therefore may not provide accurate information on the œaverage o2 in the measured volume by recognizing the potential limitations of the o2 measurements one can focus on the important and useful information that can be obtained from these techniques the most valuable clinical characterizations of oxygen are likely to be derived from a series of measurements that provide data about factors that can change levels of o2 which then can be exploited both diagnostically and therapeutically the clinical utility of such data ultimately needs to be verified by careful studies of outcomes related to the measured changes in levels of o2k e y w o r d sclinical measures of oxygen oxygen in tissues partial pressure1department of radiology dartmouth medical school hanover nh usa2department of medicine section of radiation oncology dartmouthhitchcock medical center lebanon nh usa3thayer school of engineering dartmouth college hanover nh usa4department radiation and cellular oncology university of chicago chicago il usa5department of surgery dartmouthhitchcock medical center lebanon nh usa6louvain drug research institute universit catholique de louvain brussels belgium7department radiation oncology university medical center university of freiburg freiburg germany8german cancer center consortium dktk partner site freiburg german cancer research center dkfz heidelberg germanycorrespondenceann barry flood clinepr llc river road lyme nh usaemail annbarryflooddartmouthedufunding informationmajor funding for this work was from the national institutes of health national cancer institute ppg grant p01ca190193 r01 ca p30 ca023108 and national institute of biomedical imaging and bioengineering p41 this is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided the original work is properly cited the authors physiological reports published by wiley periodicals llc on behalf of the physiological society and the american physiological societyphysiological reports 20208e14541 1014814phy214541 wileyonlinelibrarycom phy2 of 0c of introduction the overall goal of this review is to facilitate clinically effective use of measurements of molecular oxygen o2 in tissues with the explicit intent of improving clinical care that is improving the accuracy and effectiveness of diagnoses treatments and prognoses for individual patients this review focusses especially on improving personalized medicine and outcomes of care by carefully considering the basis and validity of clinical measurements of o2 in tissues and how those measurements can be used to advance diagnosis and therapy while measurements of o2 in tissues have been recognized as an important factor in the clinical evaluation and treatment of many diseases especially cancer busk overgaard horsman a0 and pathologies involving ischemia such as in peripheral vascular disease and wound healing insufficient attention often has been paid to the meaning of the values that have been obtained note this review is derived in part from a series of recent papers on this topic flood et a0al a0 swartz flood swartz vaupel instead all too often when a measurement technique has indicated that the level of o2 in a given tissue is œx that is is some specific quantitative number for the o2 in the tissue researchers and clinicians alike assume that œx is a reliable accurate representation of the œtrue oxygenation status of the tissue this approach ignores the complexity and dynamics of o2 in living biological systems the reality is that any o2 measurement has been taken at only one point in time of a distribution in the subvolume that was interrogated by the method while the o2 is in fact varying with time and across space in the tissue and is unlikely to be uniform in the volume that is being interrogatedin this review we focus on the biologicalclinical meaningfulness of o2 measurements made in living anisms while recognizing that tissue o2 is in constant flux we emphasize that to obtain maximum clinical utility of the measurements it is necessary to consider the goal of the measurements and the limitations of the data that are obtained we particularly focus on the clinical value of making repeated measurements of o2 especially in association with strategiesevents that potentially change o2 levels what are oxygen levels physical concepts and terminology for in tissues reporting on oxygen levels in tissuesthe level of molecular oxygen that is o2 is usually reported as partial pressure of oxygen po2 or concentration of oxygen [o2] or co2 these terms have physically rigorous meanings that can usefully be extended to describe gases such as o2 that are dissolved in liquids or solids including tissues partial pressure is the pressure exerted by oxygen in a mixture of gases while concentration is the content of oxygen in the gas mixture or solid partial pressure is commonly expressed in mmhg and these units are sometimes referred to as torr or kpa si unit used in the eu while concentration of o2 is commonly expressed in ml of o2 per a0ml for example in bloodhowever the solubility of oxygen varies greatly in different media bennett swartz brown koenig a0 jordan et a0al a0 and this affects the relationship between po2 and [o2] the transport of o2 across lipid membranes is known to depend on both diffusion and solubility in the bilayer and to be affected by changes in the physical state and by the lipid composition especially the content of cholesterol and unsaturated fatty acids for example because o2 partitions preferentially into lipophilic media such as membranes the solubility of o2 in membranes is about four times greater than in aqueous solutions m¶ller et a0 al a0 this difference has significant consequences for physical and chemical interactions involving molecular oxygen in biological systems because these interactions depend on the number of oxygen molecules that are present and their rate of diffusiondoes it matter clinically to know whether the technique is reporting [o2] or po2 while these measures are not identical there is a known relationship between them according to the ideal gas law po2 is directly proportional to concentration assuming the volume and temperature are constant that ispv nrtwhere p a0 a0pressure po2 v a0 a0volume n a0 a0amount of substance [o2] r a0 a0ideal gas constant t a0 a0temperatureit is less straightforward in biological systems if the solubility of oxygen in each component of tissue is known and this is not always fairly readily derived experimentally and po2 can be measured then it is relatively straightforward to calculate [o2] conversely if the component in which [o2] is measured and the solubility of o2 in that compartment is sufficiently known then it should be feasible to determine po2 however it often is not feasible to measure these parameters readilybecause of the biological complexities in assessing o2 in tissues each reported measure of o2 level in a tissue can be better considered as an average value average is used here in its more colloquial usage rather than as a statistically defined term because different techniques output their measurement of o2 using differing methods pertinent to that technique each technique gathers information from a particular volume of tissue irrespective of whether that volume is well characterized which we refer to hereafter as the œinterrogated swartz 0cvolume the sampling of data within the interrogated volume is then used to produce a measure based on a sort of average o2 within that volume characterizing that œaverage measure is made difficult both by the imprecision of knowing the exact volume queried but also because the detection of o2 in that volume may be affected by factors such as its distance from the detector or for optical techniques different rates of scattering that also may not be well characterized hence we conclude that it is important to bear in mind that the measurements of o2 in vivo are fundamentally based on a sort of œaverage within the interrogated volumebecause of all of these challenges in obtaining precise measurements of relevant parameters necessary to assess whether the data obtained by any technique is truly measuring either po2 or [o2] and because of the imprecisions of knowing the volume being assessed and the tissues within that volume it is more realistic to acknowledge the complexity of these issues for in vivo measurements in tissues by using a less precise term for measures of molecular oxygen in tissues such as œo2 levels which is the convention we follow in this review we also argue that while it is important to recognize the biological imprecisions in these measures there are still many clinically viable uses of this information such as assessing change in o2 levelsnote too within this paper while focusing on the uncertainties due to sampling issues of each technique and variations due to biological factors we are not taking into consideration further uncertainties due to inevitable instrumental noise variations in the placement of the detector etc expected levels of o2 in tissues table a0 presents some illustrative data on the o2 levels in various tissues both in normal states and as altered by some diseases these measures are presented here as reported in the literature the first column presents the median po2 obtained using the eppendorf electrode or comparable polarographic techniques to measure o2 levels in patients also presented are two other indications of o2 levels the hypoxic fraction the percentage of measurements in a given type of tissue that is below a defined œhypoxic level in this case a0mmhg and the range of po2 values found experimentally these data illustrate both the variation in median o2 levels between types of tissues and how they may vary with physiology or disease for example intertissue in general the median o2 levels are lower in skeletal muscle and heart compared to the spleen intratissue the median o2 in skeletal muscle at rest is higher than with exercise while in contrast there is almost no variation between the normal spleen and with hodgkin's disease the data in column illustrate the wide range that any given measurement can have in the œsame type of tissue that is almost all tissues range from of to a0mmhg even when their median value is quite different see again spleen vs bone however these very high values in the upper range may include experimental artifacts due to the measurement being taken within or very close to an arteriole for example a vessel feeding the microcirculatory bedtable a0 presents the same types of information for a more detailed analysis of changes in an important pathology cancer where o2 levels have been an especially important focus for informing clinical treatment and prognosis to give the reader a sense of how well supported the numbers are the data in table a0 have been ordered by the number of patients included in each rowof interest here all seven cancer types with at least patients studied have a fairly consistent and fairly hypoxic median o2 level a0mmhg similarly all but soft tissue sarcoma have a similar hypoxic fraction sarcoma appears to be about half that glioma appears to be an outlier on the range glioma had no patient whose o2 level was above while all others as was true for the tissues in table a0 have at least one measurement in the upper 80s or 90s these occasional high readings are not surprising since it is plausible that randomly some readings will have been obtained in or very near to arteriolesfeeding microvessels in contrast to the first seven cancers the types of cancers with fewer than patients appear to be more varied in their o2 levels but this is possibly due to being based on few patientsnevertheless even though as noted elsewhere in this review the data presented are not unconditionally œabsolute values of o2 levels as they are sometimes referred to eg koch a0 macnab gagnon gagnon minchinton fry a0 nevertheless as argued in this review they can provide very useful data as long as their limitations are recognized by researchers and clinicians heterogeneity of distributions of levels of oxygen in tissues examples and causesheterogeneity of distributions of oxygen values in the tissues of interest exists over many dimensions including time and space and over a wide range of scales harrison vaupel a0 tables a0 and focus on intertissue and intratissue variation in overall levels of o2 figures a0 and illustrate heterogeneity using more refined data points to illustrate the skewed nature of the data particularly for malignancies the data in figure a0 are based on multiple measurements made in a series of patients using a computerized polarographic microsensor technique which enables direct assessment of the o2 levels with an o2sensitive needle electrode subject to the limitations of providing true absolute values as discussed swartz 0c of table oxygenation status of anstissuesantissuekidneycortexouter medullainner medullaliverpancreasspleennormalin hypersplenismhodgkin's diseasemyocardiumsubepicardialsubendocardialmucosaoralrectallarge bowelbreastnormalfibrocystic diseaseprostateuterine cervixsubcutisbonecorticalhematopoietic marrowadipose marrowskeletal musclerestingexercisehypovolemic shockpaodskinthermoneutral conditionscritical limb ischemialimbs venous diseasebraingray matterwhite mattermedian po2 mmhghf po2 range mmhgreferences““““““““““““““nanana““nanana“““““““““““““““““““““““““““““nag¼nther aum¼ller kunke vaupel and thews samesamekallinowski and buhr 1995akoong vaupel wendling thom and fischer wendling vaupel fischer and br¼nner samewinbury howe and weiss moss for bothkallinowski and buhr 1995asamesamevaupel schlenger knoop and h¶ckel vaupel and harrison samevaupel and kelleher h¶ckel schlenger knoop and vaupel samespencer et a0al a0samesamelandgraf and ehrly jung kessler pindur sternitzky and franke harrison and vaupel landgraf schultehuermann vallbracht and ehrly carreau et a0al a0harrison and vaupel clyne ramsden chant and webster vaupel samecontinuesswartz 0c of table continuedantissueretinamedian po2 mmhghf napo2 range mmhg“referenceshogeboom van buggenum van der heijde tangelder and reichertthoen linsenmeier and zhang white adipose tissuenonobeseobesenana““pasarica et a0al a0 hodson lempesis van meijel manolopoulos and goossens abbreviations arterial hf25 hypoxic fraction ‰¡ fraction of po2 values ‰¤ a0mmhg na information not available paod peripheral arterial occlusive diseaseelsewhere in the paper oxygen was measured along several electrode tracks in each individual during a given measurement session from near the tumor surface up to tissue depths of a0 a0 a0mm in breast cancers and in cancers of the uterine cervix each row in figure a0 presents a type of tissue breast and uterine cervix with comparisons of o2 levels made in normal tissue green versus malignancies prior to treatment of these ans red the summary measures median po2 values parallel to data reported in tables a0 and are included in the text boxesnote that the data in figure a0 are not normally distributed the distribution of o2 levels made in normal breast tissue is the closest approximation to a normal distribution comparing the two distributions for malignancies we see that breast cancer is more highly skewed than cervical cancer although they have the same median thus using a single measure such as the median could overlook potentially important clinical informationsimilarly comparing the distributions for the normal tissues the normal cervix had a substantial number of o2 measurements within a hypoxic range defined as ‰¤ a0 mmhg while there were no hypoxic measurements in the normal breast while figure a0 does not differentiate the measurements made per patient it illustrates why several authors report the hypoxic fraction when trying to capture a meaningful overall number to characterize a tissue finally these data underscore why it is important to understand what is well captured by a given measure of o2 level”and what is missed or obscuredanother example of heterogeneity is presented in figure a0 these o2 measurements were taken in a breast cancer patient using epr oximetry with carlo erba ink as the o2 sensor flood et a0al a0 jeong et a0al a0 the data are presented as line widths because the epr oximetry technique using india ink as a sensor undoubtably gathers data from volumes too large to have homogenous oxygen levels however because this sensor remains in the same place in the tissue it still can provide very useful indications of changes over time andor the impact of interventions such as breathing enriched oxygen the data were taken in 30min sessions during which epr spectra were collected continuously but the period was divided into three 10min periods differing by the gas mixture the patient was breathing room air red baseline o2 delivered by a nonrebreather mask green followed by again breathing room air blue recovery the sessions were repeated approximately weekly throughout the period of radiation therapy thus mimicking a clinical course of radiation therapy although there was no attempt to impact therapy in this studythe data for this particular patient illustrate that the o2 levels responded to the patient's breathing an hyperoxic gas mixture and then returned rapidly to the baseline level after the 10min period in this patient there appeared to be some variation across the weeks of treatment with the final levels for each period baseline o2 and recovery all being slightly higher than at the beginning of radiation therapy based on nonoverlapping standard deviations of the first and last measurements causes of heterogeneity in normal tissuesthere are spatial variances in oxygen levels in normal tissues due to the longitudinal gradient in oxygen as the blood passes through the microcirculatory bed decreasing from the arterial inlet to the outlet of the microvessels erickson after the oxygen leaves the microvascular networks the partial pressure of o2 decreases due to radial gradients that is o2 diffuses through the tissues as it gets further from the vessels due to o2 being consumed by the cells as a result there are variations in o2 levels from cell to cell according to their distance from the microvessel within the cells o2 decreases in a microspatially complex manner as it is intracellularly consumed with most of the consumption occurring in the mitochondria there is growing evidence that diffusion of o2 into the cell may be constrained that is that o2 does not freely and rapidly flow into cells across the membrane and therefore there are gradients from outside to inside of cells khan et a0al a0 kurokawa et a0al a0 pias a0these variations of o2 levels that is gradients between and within cells cannot currently be measured as detailed below in discussing temporal variations even if such measurements of spatial heterogeneity could be made they would still be inadequate to understand the full complexity of heterogeneity of o2 for example in some normal tissues there is additional significant macroscopic heterogeneity of o2 swartz 0c of table pretherapeutic oxygenation status of human tumorstumor type ordered by no of patientscervix cancerhead and neck cancerprostate cancersoft tissue sarcomabreast cancerglioblastomavulvar cancermedian po2 mmhg“no of patientsrectal cancerlung cancermalignant melanoma metastaticnonhodgkin's lymphomapancreas cancerbrain metastasesnahf po2 range mmhg“““““““““““““liver metastasesrenal cell carcinomagall bladder cancerbile duct cancerabbreviations hf25 hypoxic fraction ‰¡ fraction of po2 values ‰¤ a0mmhg na information not available““““nanananareferencesvaupel et a0al a0vaupel vaupel data synopsesthese ref apply to allabove the linevaupel thews mayer h¶ckel and h¶ckel vaupel mayer and h¶ckel stone et a0al a0kallinowski and buhr 1995a mattern kallinowski herfarth and volm falk ward and bleehen le et a0al a0lartigau et a0al a0powell et a0al a0koong graffman bjork ederoth and ihse rampling cruickshank lewis fitzsimmons and workman kallinowski and buhr 1995a 1995blawrentschuk et a0al a0graffman et a0al a0graffman et a0al a0over space because of their physiology as a consequence of substantial differences in vascularity blood flow and oxygen consumption eg macroscopic heterogeneity between gray and white matter of the brain between subepicardial and subendocardial layers of the myocardium and between renal cortex and renal inner medullathere also are temporal changes in o2 levels in normal tissues griffith a0 moreover the supply of o2 can vary periodically due to rhythmic changes in microcirculatory blood flow which is reflected at all levels from the inflow arteries to the microcirculation finally within the microcirculation there are variations in microvascular flow due to regional regulation in response to varying metabolic demands kimura et a0al a0 important differences in o2 solubility across tissues affecting the relationship between po2 and [o2] were discussed earlier impact of pathology on heterogeneity of o2 levelsthe presence of pathology often significantly increases the amount and extent of oxygen heterogeneity both spatially and temporally vaupel harrison vaupel mayer a0 the presence of pathology often impacts the structuremorphology of the vessels in tumors there often is a significant amount of neoangiogenesis which results in much less ordered and less functional blood vessels busk et a0al a0 the resulting vessels are much less efficient in delivering blood and also tend to be much more prone to leak leakage from these vessels can cause increases in the interstitial pressure which can reduce the effectiveness of the microcirculation due to reduction of the perfusion pressure within the tumor capillaries fukumura duda munn jain a0pathological changes also can result in altered consumption of o2 in malignant tumors o2 consumption is likely to decrease due to poor oxygen delivery andor because of a switch to glycolysis due to metabolic reprogramming ie the warburg effect as a consequence of hif1α overexpression upregulation of oncogenes downregulation of suppressor genes and activation of certain signaling pathways vaupel multhoff a0 vaupel schmidberger mayer a0 pathology can also impact the integrity of the blood vessels for example tumor growth may physically impinge on the integrity of the blood vessels and the swartz 0c of figure distributions of multiple o2 levels made in patients with normal versus malignant tissue breast and cervix figure adapted from vaupel mayer a02017b p figure repeated o2 level measurementsa during each measurement session and over a0days breast cancer patient measured in skin and superficial breast tissue within the radiation field during a course of radiation therapy figure adapted from flood et a0al a0 p afor carlo erba ink epr line width increases with increasing o2 level but the relationship is nonlinear and can be impacted by several factors therefore the data are given here as line widthmetabolic abnormalities in diabetes can impact the structure of blood vessels causing either microangiopathy andor macroangiopathy the results of these processes can produce very significant local variations in the availability of fully functional vascular structures resulting in locally hypoxic regionsswartz 0c of pathology also can impact temporal changes of oxygen and the response to treatment the presence of pathology especially cancer eg acute and cycling hypoxia in cancers and peripheral vascular disease can result in significantly greater variability in o2 levels braun lanzen dewhirst a0 these include shortterm changes especially associated with the structural abnormalities of the microcirculation resulting in increased local variability in flow and longterm changes that develop over time such as those due to disease progression and responses to therapy baudelet et a0al a0 kimura et a0al a0 konerding fait gaumann a0 matsumoto there also is a potential for pathologies to interrelate with each other for example anemic hypoxia can develop in tumors due to the underlying systemic anemia of the patient vaupel mayer a0in addition to these underlying effects of pathology on tissue oxygen levels any applied therapeutic interventions are very likely to induce changes for example cell killing due to radiation or chemotherapy will alter oxygen consumption patterns these same therapies will also affect the o2 supplying vasculature via both antiangiogenic effects and”possibly”normalization of vessel structure jain a0 the effects of therapies will generally vary both spatially and temporally reiterating the complexity of meaningfully characterizing tissue oxygen levels analysis of the ability of clinically available techniques to directly measure levels of o2 in tissues andor resolve the heterogeneity of o2 distributions in tissuesalthough many techniques are often thought to measure actual o2 in tissues only a few actually have the potential to make o2 measurements directly in the tissues of interest springett swartz a0 tatum techniques that can potentially assess o2 directly in tissues include epr epel et a0al a0 swartz et a0al a0 swartz the eppendorf electrode vaupel h¶ckel mayer a0 some optical methods based on direct measurements of target molecules in tissues for example phosphorescence quenching of optical sensors placed directly in tissues or as part of a physical probe such as the œoxylite wen et a0al a0 and nmr relaxation techniques colliez et a0al a0two other types of measurements assess o2 in the vascular system blood gases do this directly while optical methods that measure both hemoglobin saturation and total hemoglobin especially near infrared spectroscopy [nirs] scheeren schober schwarte a0 provide a plausible link to the po2 in the bloodhowever the techniques most often used clinically to characterize tissue oxygenation do not in fact measure o2 directly instead they measure œindirect parameters that can be plausibly linked to actual o2 levels but only under appropriatedefined circumstances this latter group of techniques includes positron emission tomography pet imaging of glucose derivatives neveu et a0al a0 pet imaging of drugs that localize in hypoxic tissues tran laser doppler flow measures of metabolites that may be affected by o2 levels for example lactate and redox intermediates and several magnetic resonance imagingnuclear magnetic resonance mrinmr blood oxygenation level dependent bold imaging baudelet gallez a0 and mri egeland et a0al a0 note if their basis is understood and the data considered accordingly these can all provide clinically and physiologically useful information even though they do not provide direct information on the amount of o2 in the tissues direct measures of o2 in targeted tissues that potentially can be used in human subjectsthese are techniques that while they have the capability of providing direct quantitative measurements of o2 in homogeneous media cannot provide such data in tissues in vivo because the volumes that they sense are larger than the volumes of homogeneity of o2 in actively metabolizing tissues consequently all in vivo measurements of o2 are inherently averages of the actual oxygen content in that volume even neglecting the need to include measures of heterogeneity inside cells based on the usual volume of cells and assuming that differences are sought for aggregates of ‰¤ cells for a measurement of heterogeneity sensed within a a0mm diameter volume the spatial resolution needed to appropriately characterize o2 levels in this volume becomes million voxels the measuring techniques may not even provide a welldefined averaged po2 value within the volume that they sense for example sensors for the signal that is being measuredin the next sections we review the characteristics of each technique that can directly measure o2 levels in tissues we also briefly remark on the volumes they measure and how the measures obtained can be useful clinically see also ortezprado dunn vasconez castillo visco epr oximetry using appropriate particulate paramagnetic materials epr oximetry can provide direct measurements of o2 that is the epr signal is directly proportional to the amount of o2 epel bowman mailer halpern a0 swartz vaupel swartz 0cthe because each multisite sensor senses a volume that is much larger than capillary networks these techniques provide a volume averaged sampling of all compartments within the tissues the time resolution of the techniques can be milliseconds or shorterthe measured parameter of an epr spectrum that indicates the amount of o2 present is the line width of the observed resonance peak there usually is a fixed relationship between the line width and the amount of o2 with the relationship being specific for each type of paramagnetic material for example carbon charcoal or phthalocyanine particulates using particulate oximetric materials measurements can be continuous over any span of time and can be repeated indefinitely see example in figure a0 the method requires that the sensing material be injected or implanted in one or more regions of interest but thereafter all measurements can be made entirely noninvasively importantly the measurements can be carried out in a clinical setting and can fit into the workflow needed for patient careinitial clinical epr measurements of oxygen in tissues have used india ink as the oxygen sensor swartz et a0al a0 the carbon ps are the components that respond to oxygen lan beghein charlier gallez a0 after injection of “ a0µl of the suspension through a small needle the carbon ps disperse nonuniformly through the local region as small extracellular aggregates they are often engulfed by macrophages the resulting epr spectra in the region probed by the resonator ie the surface coil used for signal detection are a composite of the oxygendependent line widths from each of the ps in reality because of the relatively broad lines from the india ink ps the range of œoxygen levels that are likely to be present in the tissue and the limited number of ps in each subregion it is a challenge to resolve directly even the major groups of similar line widths therefore using the observed line width to provide a quantitative measure of oxygen would seem to have modest utility in itselfthe other method of clinical epr oximetry is based on the use of microcrystalline probes eg lipc lincbuo encapsulated in biocompatible polymers swartz et a0al a0 clinical measurements currently are being performed using the œoxychip which consists of oxygen sensitive microcrystals of lithium octanbutoxynaphthalocyanine lincbuo embedded in polydimethylsiloxane pdms hou khan gohain kuppusamy kuppusamy a0 hou et a0al a0 jarvis et a0al a0 the dimensions currently used in humans are cylinders that are a0 mm long with a diameter of a0mm the epr signal from the sensor oxychip reflects the po2 within the pdms which itself reflects an average of the po2 in contact with the external surface of the cylinder the dimensions of the oxy
Colon_Cancer
during the covid19 pandemic emergency departments have noted a significant decrease in strokepatients we performed a timely analysis of the bavarian telestroke tempis œworking diagnosis databasemethods twelve hospitals from the tempis network were selected data collected for january through april in years through were extracted and analyzed for presumed and definite ischemic stroke is amongst otherdisorders in addition recommendations for intravenous thrombolysis rtpa and endovascular thrombectomy evtwere noted and mobility data of the region analyzed if statistically valid groupcomparison was tested with fisher™sexact test considering unpaired observations and apvalue was considered significantresults upon lockdown in midmarch we observed a significant reduction in recommendations for rtpa compared to the preceding three years [“] vs p¼ recommendations for evt werep¼ reflecting its increasing importance following the covid19 lockdown midmarch the number ofevt decreased back to levels in “ [“] vs p¼ absolute numbers of issignificantly higher in january to midmarch compared to “ [“] vs decreased in parallel to mobility datas the reduced stroke incidence during the covid19 pandemic may in part be explained by patientavoidance to seek emergency stroke care and may have an association to population mobility increasing mobilitymay induce a rebound effect and may conflict with a potential second covid19 wave telemedical networks maybe ideal databases to study such effects in nearreal timekeywordstelestroke covid19 lockdown stroke thrombolysisdate received may date accepted june introductionimplementation of social distancing to combat theimpact of corona virus pandemic sequelae has emergedas the major strategy to contain the spread of infectiongiven the lack of specific treatments for covid19 andlimited intensive care resources1 major concerns forstroke neurologistsin this extraordinary scenarioinclude the following a rapid specific managementof cases of acute stroke with possible covid19from initiation of the stroke call in the preclinical setting through the ambulance system emergency department and hospital stroke department and in the1department of neurology university of regensburg bezirksklinikumregensburg tempis telemedical stroke center regensburg germany2cts herdecke germany3department of neurology tempis telemedical stroke centeracademic teaching hospital of the university of munich mu¨nchen klinikharlaching munich germanycorresponding authorfelix schlachetzki md department of neurology university ofregensburg center for vascular neurology and intensive care tempistelemedical stroke center bezirksklinikum regensburguniversit‚¬atsstr84 regensburg germanyemails felixschlachetzkiklinikuniregensburgde 0c of telemedicine and telecare bthe factneuroradiological department when needed to aid instroke diagnosis and treatmentthatpatients with mild stroke symptoms or transient ischemic attacks tias may be reluctant to request hospitaladmission for acute stroke23 and c that covid19itself is associated with severe stroke syndromes this issuggested in a recent case series of covid19 patientsfrom wuhan china focusing on neurological symptoms that described cerebrovascular events in of cases especially in elderly patients and in thosewith more severe infections also authors of a secondcase series reported unusual cases of young covid19patients yrs with large vessel stroke and otherauthors reported three stroke patients with coagulopathy and antiphospholipid antibodies in the context ofsevere covid infections4“the number ofin contrast several stroke departments in germanyincluding our own the usa and china have noted asignificant drop in the number of stroke patient admissions during the corona pandemic7 data on this phenomenon are still scarce howeverin a descriptivereport by morelli from piacenza lombardyitaly covering the period february appearance ofthe first sarscov2 patient recorded in italy to march stroke admissionsdecreased from an average of with largevessel occlusions lvos to two tias one lvoand three lacunar strokes8 using a commercial neuroimaging database with the rapid software platform kasangra and hamilton observed a decrease in stroke imaging procedures with the nadirfollowing the first statewide stayathome order in theusa9 the decrease was observed in all age sex andstroke severity subgroups within all participatinghospitals which processed overall patientsbetween july and april cardiologistsin france observed a similar significant drop in admissions to nine intensive cardiac care units after initiationof social distancing and selfquarantine in midmarch overall there are scarce data available on theimpact of the covid19 infection itself on cardiovascular morbidity including cerebral stroke11aims and hypothesisthe primary aim of this study was to evaluate the effectof the covid19 pandemic lockdown on stroke consultations and treatment recommendations using theacute consultant database of the telestroke networktempis12 we focused on data collected during thefirst four months of which included the emergence of the corona virus pandemic in southeasternbavaria through the first two months of social distancingregion shutdown we compared these data withcomparable data collected during the same months inthe years “methodsdata from daily consultations at clinics withoutneurology departmentsin the telestroke networktempis form the basis of this study the consultationstook place between january and april in the years“ all data were pseudonymized weextracted the actual working diagnoses based on telemedical consultation and neuroimaging results mainlycerebral computed tomography two major databaseswere used to calculate the population within these districts wwwdestatisde and experiencearcgiscomexperience478220a4c454480e823b17327b2bf1d4pagepage_1 this retrospective study was approvedby the local ethics committee of the university ofregensburg and performed in accordance with guidelines of the declaration of helsinkimobility data available at wwwapplecomcovid19mobility were extracted these data were generated from the relative request volume for directionsin munich germany compared with a base volumeon january to observe the relationship ofmobility and the reported stroke decline in piacenzawe also extracted mobility data from milan close topiacenza italy8the major ˜working diagnostic groups™ were asfollows a ischemic stroke b tia c intracranialhaemorrhage d epileptic seizure e migraine andf other disorder including facial palsy headacheand brain tumour also included were cases in whichthere were recommendations for iv thrombolysis ivrtpa or endovascular therapy evt thrombectomyfor lvoexploratory descriptive summary statistics withmean values and standard deviations were appliedin an analysis of data covering january through aprilin years “ in comparison with data coveringthe same period in counts are presented as agraphic display showing incidences standardized to15day periods if statistically valid especially percentage of recommendations for iv thrombolysis andthrombectomy groupcomparison was tested withfisher™s exact test considering unpaired observationsa pvalue was considered significantresultsthere were telemedical consultations during thespecific time frames investigated and the population inthe geographical areas covered by these rural hospitals is most hospitals reside in areas with ahigh number of covid19 cases figure 1a the 0cschlachetzki number of covid19“positive cases in the whole ofbavaria rose from five at the end of february to cases on april the public lockdownwas initiated on march however the recommendation of personal quarantine for people who hadtravelled to northern italy was broadcast earlier on march in munich applevr mobility trends demonstrated a decrease in walking activity in midmarch to “ “ to “ of the baseline levelin milan lombardy italy on march walking activity began to decrease soon reaching “ of baselineactivity and remaining fairly constantthereafterfigure 1boverall consultations were analysed and excluded being nonacute consultations within thenetwork ie followup examinations statistically significant changes in the number of recommendations foriv thrombolysis were observed in figure 1cwhile in “ iv thrombolysis was recommendedin of consultations with suspected ischemicstroke of the frequency of this recommendation decreased to of in p¼ no differences in the number of ivthrombolysis recommendations were observed duringthe time period covering january to march in “ vs in notfigure a incidence of new covid19 infections in bavaria on april red dots indicate network hospitals and green andyellow squares depict the two academic stroke centres that alternate weekly for the tempis consult service modified with permission from the bavarian state office for health and food safety httpwwwlglbayerndegesundheitinfektionsschutzinfektionskrankheiten_a_zcoronaviruskarte_coronavirus b mobility data according to covid19 mobility trends reports apple thedata reflect requests for routing in apple maps for munich which resides in the centre of the tempis network and for milan nearpiacenza where the first decline in the number of strokes was reported morelli 8 horizontal dotted line indicates reportedreduced stroke activity in piacenza c recommendations absolute numbers for application of iv thrombolysis and thrombectomyvertical dashed line indicates the official beginning of lockdown in bavaria time and patient numbers on yaxis are standardized to15day periods xaxis in each month to compensate for shorter february and longer january and march months j1¼ january first half j2 “ january second half f¼ february m¼ march a¼ april d working diagnoses of the telestrokeconsultations vertical dashed line indicates the official beginning of lockdown in bavaria time and patient numbers on yaxis arestandardized to 15day periods xaxis in each month to compensate for shorter february and longer january and march months j1¼ january first half j2 “ january second half f¼ february m¼ march a¼ april 0c of telemedicine and telecare significant no trend in fewer recommendations forevt was observed between march and april in compared with the same time periods in “ of vs “ of however in the preceding time frame january to march significantly more recommendations for thrombectomy were made comparedwith “ of vs of in “ p¼ the data reflect the development of consultationsand treatment recommendations for lvo in the network from onward the number of recommendations for evt steadily rose with increasing evidencefor recanalization even in later time windows andincreasing employment of computed tomography angiography in the tempis network table shows thedevelopment of consultations in up to the end ofthe study including the lockdown period it shows adrop in the number of consultations and more importantly fewer recommendations for iv thrombolysisand evt which suggests fewer incidences of ischemicstroke severities table figure 1dalthough bavaria is the state with the highestnumber of covid19 cases in germany especially inour region we only performed five telestroke consultations for the network hospitals in which possiblecovid19 infection was discussed including a singlepatient with stroke symptoms and feverdiscussionthe tempis telestroke working data confirm thecurrent observation of a low stroke incidence insoutheastern bavaria with relative proportions of theworking diagnosis remaining similar the number ofcases of disabling stroke from intracranial haemorrhage and ischemic stroke requiring iv rtpa or evtalso diminished challenging the theory that onlypatient avoidance to call for emergency treatment isresponsible for this phenomenon this study also demonstrates the potential and importance of telestrokenetworks in the current covid19 pandemic313the observation of fewer stroke cases during thecovid19 pandemic seems to contradict two essentialassumptions with regard to stroke risk a sarcov is a strong risk factor for stroke and b physicalinactivity in a lockdown setting may increase the riskof stroke especially among elderly persons firstsarcov2 may induce hypercoagulability and highlevels of creactive protein ddimer and interleukin6placing patients at risk to develop thrombotic complications14 in a series of intensive care unit patientsin the netherlands reported by klok only threestrokes complicated the course of covid19 whereasthe majority of complications included pulmonarythrombosiscatheterassociatedembolism n¼ and peripheral venous thrombosisn¼ andobservations in case series that concurrent covid infection complicates or triggers unusual ischemicstroke may well prevail but case control studies focusing on this phenomenon are urgently needed to affirmor deny the assertion5 second physical inactivity has aprofound effect on atrial fibrillation obesity diabetesmellitus management and hypertension among othersand contradicts current recommendations on mid andlongterm stroke prevention16 a recent study in consecutive patients with nonstsegment elevationacute coronary syndromes acss and optical coherence tomography of the culprit lesion reported bykato found that the combination of greaterphysical activity outdoor acs onset and high bodymass index had a significant effect on the incidence ofcoronary plaque erosion17 interestingly mobility datasuch as those provided by the apple mobility databasevr demonstrated a parallel reduction in incidencesof stroke and acs in three published papers8“ inaddition to oursour data confirm the observation from morelli who termed the phrase ˜baffling case of ischemicstroke disappearance™ these authors also discuss thatthis effect cannot be totally explained merely by thereluctance of patients to call for help in a stroke emergency because the number of cases presenting withsevere stroke requiring evt and the number of generalconsultations in tempis also decreased an analysisbased on a large database associated with the application of rapid software in acute stroke by kansagra is in line with our observation that also severestroke patients diminished during the early lockdownphase9 the number of ischemic core volumes “ml and greater than ml were observed to decreaseby and respectively core volumes “ ml decreased by and and very smallcore infarct volumes measuring “ ml decreased the decrease in the number of very small infarctvolumes may well be explained by the generally proposed hesitation to seek emergency care while thereduction in large ischemic core volumes is morelikely due to fewer lvos as observed in our studywith a sharp decline in iv thrombolysis and thrombectomy recommendationsanother explanation may be a concurrent low infection rate with other viruses that can trigger atherosclerosis and plaque rupture resulting in neuro andcardiovascular morbidity18 the lockdown not onlyreduces physical activity strict social distancing anduse of facial masks should also lead to low rates ofexposure to and transmission of other common virusesand allergens that by themselves appear to triggerstroke19 additional studies with detailed analyses of 0cschlachetzki stekcarberauqsniatadhtnomehtfoshtgneltnereffidrofstnemtsudajtuohtiwtubsdoirepkeewotnidedvdiilirpa“yraunajrofsnoitatlusnocforebmunlatotlebatrpa“rpa“ram“ram“bef“bef“naj“naj“sopdvocilatotairavabnisesacnoitaiveddradnatsdnaseulavnaemni“morfatadwohssipmetkrowtenekortsnoitatlusnoceetl] 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06[snp p¡ 8a 06[¡snp§p§§§§ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[  8a 06[§ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[lsisyobmorhtvi 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06 06[ 8a 06[ 8a 06 06[ytilibadaerrettebrofldoblynoerasrebmunldobskcattaicmehcsitneisnartatiamotamehlarudbushdsegahrromehidonhcarabushasegahrromeahlainarcartnihciigndeebllainarcartnsnsn¼¼¼¼bp§pcpp§§ ¡iids 06“naemsnoitatlusnocekortsicmehcsids 06“naemds 06“naemymotcebmorhtds 06“naemds 06“naema tids 06“naemh cids 06“naemhashdsbcieniargimeruziessrehto 0c of telemedicine and telecare symptom onsettodoor times stroke severity neuroimaging and inflammatory markers are needed tounderstand the reason for the reduced number of revascularization therapies requested during the covid19pandemiclimitations of the studyanalysis of daily working diagnoses in the tempistelestroke network has the advantage of being highlytimely yet it lacks specificity because the final diagnosismay differ from the initial one this may be compensated by the creation of a large common database fortelestroke networks that incorporates corrections forthe actual population covered analyses of otherstrokerelated databases such as the one associatedwith rapid software healthcare provider databasesand common stroke registries for quality control thedecrease in the number of thrombectomy recommendations in our cohort midmarch did not reach statistical significance when compared with the sameperiod in years through because rates forthis procedure increased according with levels of evidence2021 in agreement with this development thrombectomy recommendations by tempis neurologists in prior to the covid19 pandemic occurred morefrequently than in previous yearssour study using the tempis telestroke database confirms lower incidences of ischemic stroke and otheracute neurological disorders requiring consultationsuch as intracerebral haemorrhage seizure disorderand migraine next to a reluctance within the population to seek immediate medical assistance for acutestroke the covid19 lockdown which resulted inless physical activity and fewer other common infections may also be responsible for the fewer numberof patients with severe stroke especially those withintracranial haemorrhage and those eligible for recanalization therapies if lockdownassociated factors areindeed responsible for a lower stroke incidence we mayexpect a rebound effect following the lockdown periodwith an increased incidence of stroke as well as ofmyocardial infarcts and traumatic brain injuries aspatients™ frailty may have increased during the lockdown analyses of large stroke databases may revealfurther insights into this phenomenon however telestroke networks such as tempis may be ideal tools tomonitor stroke occurrence in real timeacknowledgmentsthe authors acknowledge all consulting neurologists intempis and colleaguesin badin partner hospitalsebersbergburglengenfeldreichenhalleggenfeldenerding freising kelheim mu¨ hldorf rotthalmu¨ nstervilsbiburg dingolfing and zwiesel the authors like tothank jo ann elison ma elsdfor editing thispaper for english grammar and languagedeclaration of conflicting intereststhe authors declared no potential conflicts of interest withrespect to the research authorship andor publication of thisarticleanonymized data are available on requestfundingthe authors received no financial support for the researchauthorship andor publication of this articleorcid idfelix schlachetzkiorcidorg0000000161672597references jawaid a protecting older adults during social distancing science khosravani h rajendram p notario l protectedcode stroke hyperacute stroke management during thecoronavirus disease covid19 pandemic stroke “ markus hs and brainin m covid19 and stroke a global world stroke organization perspective int jstroke “ mao l jin h wang m neurologic manifestationsof hospitalized patients with coronavirus disease inwuhan china jama neurol “ oxley tj mocco j majidi s largevessel stroke asa presenting feature of covid19 in the young n engl jmed e60 zhang y xiao m zhang s coagulopathy andantiphospholipid antibodies in patients with covid19n engl j med e38 zhao j rudd a and liu r challenges and potentialsolutions of stroke care during the coronavirus disease covid19 outbreak stroke “ morelli n rota e terracciano c the baffling caseofischemic stroke disappearance from the casualtydepartment in the covid19 era eur neurol “ kansagra ap goyal ms hamilton s collateraleffect of covid19 on stroke evaluation in the unitedstates n englnejmc2014816 online ahead of printj meddoi huet f prieur c schurtz g one train may hideanother acute cardiovascular diseases could be neglectedbecause of the covid19 pandemic arch cardiovasc dis “ bansal m cardiovascular disease and covid19diabetes metab syndr “ audebert hj schenkel j heuschmann pu effectsof the implementation of a telemedical stroke networkthe telemedic pilot project for integrative stroke care 0cschlachetzki tempis in bavaria germany lancet neurol “ patel uk malik p demasi m multidisciplinaryapproach and outcomes of teleneurology a reviewcureus e4410 terpos e ntanasisstathopoulos i elalamy i hematological findings and complications of covid am j hematol “ klok fa kruip m van der meer njm incidenceof thrombotic complications in critically ill icu patientswith covid19 thromb res “ kyu hh bachman vf alexander lt physicalactivity and risk of breast cancer colon cancer diabetesischemic heart disease and ischemic stroke eventsaystematic review and doseresponse metaanalysis forthe global burden of disease study bmj i3857 kato a minami y katsura a physical exertion asa trigger of acute coronary syndrome caused by plaqueerosion j thromb thrombolysis “ grau aj urbanek c and palm f common infectionsand the risk of stroke nat rev neurol “ pagliano p spera am ascione t infections causing stroke or strokelike syndromes infection “ campbell bcv donnan ga lees kr endovascular stent thrombectomy the new standardof care for large vessel ischaemic stroke lancet neurol “ vinny pw vishnu vy and padma srivastava mvthrombectomy to hours after stroke n engl jmed 0c'
Colon_Cancer
" oral administration is the most common way to deliver drugs to the systemic circulation or targetans orally administered drugs are absorbed in the intestine and metabolized in the intestine and liver in theearly stages of drug development it is important to predict firstpass metabolism accurately to select candidatedrugs with high bioavailability the caco2 cell line derived from colorectal cancer is widely used as an intestinalmodel to assess drug membrane permeability however because the expression of major drugmetabolizingenzymes such as cytochrome p450 cyp is extremely low in caco2 cells it is difficult to predict intestinalmetabolism which is a significant factor in predicting oral drug bioavailability previously we constructed a mouseartificial chromosome vector carrying the cyp cyp2c9 cyp2c19 cyp2d6 and cyp3a4 and p450 oxidoreductasepor 4cypsmac genes and increased cyp expression and metabolic activity in hepg2 cells via transfer of thisvectorresults in the current study to improve the caco2 cell assay model by taking metabolism into account weattempted to increase cyp expression by transferring the 4cypsmac into caco2 cells the caco2 cells carryingthe 4cypsmac showed higher cyp mrna expression and activity in addition high metabolic activity availabilityfor permeation test and the potential to assess drug“drug interactions were confirmeds the established caco2 cells with the 4cypsmac are expected to enable more accurate prediction ofthe absorption and metabolism in the human intestine than parental caco2 cells the mammalian artificialchromosome vector system would provide useful models for drug developmentkeywords mammalian artificial chromosome chromosome transfer cytochrome p450 intestinal metabolismcaco2 cell correspondence kazukitottoriuacjp1division of genome and cellular functions department of molecular andcellular biology school of life science faculty of medicine tottoriuniversity nishicho yonago tottori japan2chromosome engineering research center cerc tottori university nishicho yonago tottori japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cohta bmc biotechnology page of bioavailability is an important area of concern in drugdevelopment poor oral bioavailability has led to drugwithdrawal oral drug bioavailability is often limited bymetabolizing enzymes and efflux transporters in the gut the caco2 cell line derived from human colon carcinoma is a commonly used model for estimating the intestinal absorption of new drug candidates althoughcaco2 cells express a variety of efflux and uptake transporters they have an absence or low levels of cytochrome p450 cyp isoforms such as cyp3a4 andcyp2c that are typically expressed in the human intestinal epithelium therefore caco2 cells are of limited use in evaluating the role of metabolism inintestinal absorption after oral administration to predict the intestinal absorption of drugs more accurately itis necessary to modify caco2 cells to increase their expression of cyp isoformssome studies reported that cyp3amediated metabolism in caco2 cells was enhanced by transfection withboth cyp3a4 and cyp oxidoreductase por [ ] treatment with 1α25dihydroxyvitamin d3 [ ] or the combination of transfection with cyp3a4 and treatment withboth sodium butyrate and 12otetradecanoylphorbol13acetate in contrast few studies aimed at enhancingmultiple cyp isoforms in caco2 cells have been performed honkakoski and his collaborators created caco2cell lines expressing nuclear receptors pregnane x receptor and constitutive androstane receptor [“] thesenuclear receptors upregulated the expression of somecyp isoforms in caco2 cells but cyp activities remainedvery low in the absence of 1α25dihydroxyvitamin d3therefore a new approach is needed to introduce multiple cyp isoforms in caco2 cellsmammalian artificial chromosome ac vectors derived from native chromosomes have several advantagesover conventional vectors acs segregate freelyfrom host chromosomes through a set of cell divisionsand are adapted to carry multiple target genes with a desired copy number and mbsized genomic regions withendogenous regulatory elements furthermore acs carrying genes of interest can be transferred into varioustarget celllines via microcellmediated chromosometransfer mmct considering these advantages acshave been used to generate several model cells for pharmacokinetic and toxicokinetic studies previously a lack of cyp3a4 expression in the caco2cell line was addressed through the introduction of exogenous cyp3a4 and por which is a coenzyme ofcyps via a human artificial chromosome hac vectorderived from human chromosome [ ] the haccarrying cyp3a4 and por genes conferred sufficientcyp3a activity to parental caco2 cells to be useful forpredicting the intestinal extraction ratio in humansrecently a mouse artificial chromosome mac vectorconstructed from native mouse chromosome wasused to increase the activity of multiple cyps in hepg2cells which are a liver cancer cell line typically exhibiting low cyp activity in this study four cyp genescyp3a4 cyp2c9 cyp2c19 cyp2d6 and a pene were loaded on the mac 4cypsmac and transferred to hepg2 cells tchepg2 to make the cellsmore suitable as a model to evaluate drug“drug interactions ddis and hepatotoxicity in the initial screeningof candidate drugs the expression and activity ofcyps in tchepg2 were comparable to those in humanhepatocytes and this expression was sustained after along culture period because of the stability of the macin human cells regarding the assessment of ddisthe activity of cyps in tchepg2 was reduced in a concentration and timedependent manner by specific inhibitors which reflects the conditions in primary humanhepatocytes furthermore metabolic toxicity of aflatoxinb1 which is converted to its active metabolite viacyp3a4 and exerts hepatotoxicity through dna damage was clearly recapitulated in tchepg2 cells rather than parental hepg2 cells this study suggestedthat tchepg2 can provide a useful model to assess notonly hepatic metabolism but also cypmediated hepatotoxicity during the early stages of drug development andthe system using the mac can improve the existingcellbased modelin the current study we aimed to utilize previouslyconstructed 4cypsmac to generate a novel caco2 cellline with increased activity of multiple major cyps the4cypsmac was transferred to caco2 cells via mmctto establish caco2 cells carrying the 4cypsmac andthe caco2 4cypsmac cells were examined to determine whether they exhibited sufficient cyp activity foruse in initial drug screeningresultsmmct and analyses of acquired clonescho cells carrying a mac vector with cyp2c9cyp2c19 cyp2d6 cyp3a4 por and gfp genes wereprepared 4cypsmac fig 1a using cho cells asdonor cells and caco2 cells as recipient cells weattempted to generate caco2 cells carrying the 4cypsmac via mmct fig 1a after selection four drugresistant gfppositive clones were obtained caco24cypsmac fig 1b to examine whether the cypand por genes were introduced into the obtainedclones genomic pcr analyses were performed chocells with the 4cypsmac and caco2 cells were usedas positive and negative controls respectively consequently a band of the desired size was observed for eachprimer set in the candidate clones fig 1c next achromosome specimen was prepared from the acquired 0cohta bmc biotechnology page of fig introduction of the 4cypsmac into caco2 cells a transfer of the 4cypsmac into caco2 cells the structure of the mac carrying fourcyps and por is shown at the top a cag promoter was placed upstream of each gene a schematic view of the transfer of the 4cypsmac tocaco2 cells is shown at the bottom the 4cypsmac was transferred from cho cells to caco2 cells through the mmct method b an image ofgfp fluorescence in parental caco2 cells and caco2 4cypsmac cells the gfp fluorescence indicates the presence of the 4cypsmac in thecaco2 cells the white bars indicate a distance of μm c results of genomic pcr analyses to detect four cyp and por transgenes on the macin caco2 cells donor cho cells and caco2 cells are positive and negative controls respectively d images of fish analyses of caco2 cellscarrying the 4cypsmac red and green signals indicate the mac and transgenes respectively the arrow shows the 4cypsmac and the insetshows an enlarged image of the 4cypsmacclones and fish analysis was performed to check thekaryotype fish analysis revealed that a single copy of the4cypsmac existed in the candidate clones fig 1drtqpcr analysis was performed to examine themrna expression level of the introduced cyps and porin the obtained clones compared with that in parentalcaco2 cells the gene expression level was particularlyhigh in the caco2 4cypsmac and caco2 4cypsmac clones fig among the introduced genespor expression was only slightly enhanced in theseclones basal expression of por in parental caco2 cells ishigh as observed in a previous study caco2 4cypsmac showed high expression of the majority of genescompared with caco2 4cypsmac the caco2 cellline consists of a heterogeneous population of cells therefore difference of the gene expression levels between obtained clones may partly depend on the population into which the 4cypsmac has been introducedalthough the other two clones obtained by mmct stillshowed higher expression levels than the parental caco2cells the level of increase was moderate therefore we selected caco2 4cypsmac and caco2 4cypsmac clones for further analyses to evaluate the availability asan improved model system these results suggest that wesuccessfully transferred the 4cypsmac to caco2 cellsand the genes on the mac were highly expressedmonolayer formation of caco2 4cypsmac cellswe seeded caco2 4cypsmac and caco2 4cypsmac cells at a concentration of × cellswell 0cohta bmc biotechnology page of fig gene expression analyses of caco2 cells with the 4cypsmac the expression levels of the four cyps and por in caco2 cells with the4cypsmac the relative expression levels of the four cyps and por genes of the parental caco2 cells and acquired clones were analyzedthrough rtqpcr gapdh was used for normalization mean ± se n in a millicell 24well cell culture insert plate the caco 4cypsmac cells spread across the entire membrane and formed a cell layer while the caco2 4cypsmac cells did not spread and there were gaps in thecell layer fig 3a caco2 4cypsmac appeared toaggregate and form multiple layers rather than spreadand form a single layer it was reported that multilayeredareas appeared in the cell population for late passagecells the teer value was measured using amillicellers fig 3b the teer value is an index oftight junction formation and when the value is almostconstant it is considered that a cell layer has formedwith the exception of the caco2 4cypsmac clonethe caco2 cells and caco2 4cypsmac cellsshowed an increase in teer value untilit plateauedafter d the caco2 cells and caco2 4cypsmac showed almost equivalent teer values because monolayer formation is essential for the permeation test thesubsequenttests were performed using the caco24cypmac cellsculture timedependent change in gene expressiontotal rna was extracted from caco2 cells andcaco2 4cypsmac cells on the 4th 11th and22nd days after seeding we compared the expressionlevel of each gene on each day and confirmed thatthe expression levels of the four cyps and por increased in both the caco2 cells and caco2 4cypsmac cells fig 3c the expression levels of allgenes analyzed were significantly higher in the caco24cypsmac cells on the 22nd day than those inparental caco2 cells the gene expression levelincreased depending on the culture time and the geneexpression levels of the caco2 4cypsmac cellsestablished in the current study were higher thanthose of parental caco2 cells with the exceptions ofcyp3a4 and cyp2d6 the expression levels in parental caco2 cells were higher in all cases until day the parental caco2 cell line appears to have higherpotential to enhance the expression of cyp2c9 andcyp2c19 during differentiation 0cohta bmc biotechnology page of fig monolayer formation assay a images of bright and gfp fluorescence from cells seeded on the membrane of a millicell24 plate in caco24cypsmac cells did not spread throughout the membrane and did not form a cell layer but in caco2 4cypsmac there were no gapsbetween cells and they formed a cell layer the white bars indicate a distance of μm b transepithelial electrical resistance teer values ofcaco2 cells caco2 4cypsmac and caco2 4cypsmac cells c culture timedependent change in gene expression the relative expressionlevel was evaluated in caco2 and caco2 4cypsmac mean ± se n the expression levels in caco2 and caco2 4cypsmac at day were compared with those in humanadult intestine additional file figure s1 in caco24cypsmac the expression levels of cyp2c9 andcyp2c19 were comparable and that of cyp2d6 washigher than in human adult intestine although cyp3a4expression was significantly enhanced in caco2 4cypsmac compared with that in parental caco2 cellsthe expression was stilllower than in human adultintestinecyp metabolic activity measurementa p450glo assay with each specific substrate wasemployed to measure the metabolic activity of cyps inthe caco2 4cypsmac cells which had high geneexpression levels as confirmed through rtqpcr analysis the activities of all four cyps were higher in thecaco2 4cypsmac clone than in caco2 cellsfig 4a the resultsthe introducedindicate that4cypsmac expressed functional cyps and increasedthe total activity of each cyp in the caco2 cells theenhancements in the rates of metabolic activity ofcyp2c9 cyp2c19 and cyp2d6 were generally correlated with those of mrna expression however therewas a gap between the enhancement of the rate ofcyp3a4 mrna expression and that of metabolic activity this may have been because the parental caco2 originally had extremely low expression of cyp3a4mdz permeability testa permeation test was conducted using midazolammdz a cyp3a substrate to examine whether the cellsreflected the behavior of small intestinal epithelial cellsin terms of mdz permeation the penetration test wasperformed on day after cell seeding when the teervalue plateaued we measured the amount of ²ohmdz in each of the donor side apical recipient sidebasal and intracellularly the amounts of ²oh mdz 0cohta bmc biotechnology page of fig activity of each cyp in the caco2 4cypsmac cells a the metabolic activity of each cyp in caco2 4cypsmac cells the relative activityfor each cyp was measured by comparing the parental caco2 cells and the caco2 4cypsmac mean ± se n b permeability test usingmdz the permeability test was performed d after seeding caco2 cells and caco2 4cypsmac whereby μm mdz was added to theapical side and after min the apical intracellular and basal supernatants were collected the ²oh mdz in the supernatant was measuredthrough lcmsms c cyp3a4 inhibition test ketoconazole an inhibitor of cyp3a4 was added to the caco2 4cypsmac and incubated for h a luminescent substrate was measured to detect cyp3a4 activity with different concentrations of ketoconazolein alllayers of the caco2 4cypsmac cells werehigher than in those of caco2 cells fig 4b moreoverer was calculated using eq and the results were and for caco2 and caco2 4cypsmac respectively er indicates the rate of metabolism during cellpermeation cyp3a4 was scarcely expressed in parentalcaco2 cells so the er value was extremely low howevercaco2 4cypsmac cells showed an er of which was higher than in the caco2 cells and mdz wasmetabolized by cyp3a4 when passing through the cellsinhibition testto determine the availability of the established clone forthe assessment of ddis we added ketoconazole an inhibitor of cyp3a4 to the caco2 4cypsmac cells andexamined whether the metabolic activity was reduced ketoconazole at and μm was addedand cells were incubated at °c for h followed by themeasurement of metabolic activity the metabolic activityof cyp3a4 decreased as the inhibitor concentration increased fig 4c the activity of cyp3a4 in caco2 4cypsmac appeared to be sufficient for the inhibition test compared with that in parental caco2 cells in addition to thepermeation test for cyp3a4 inhibition of cyp3a4™s function by ketoconazole in caco2 4cypsmac cells was alsoconfirmed this suggests that the established cells could beused for ddi testing of drugs that are substrates ofcyp3a4 therefore the caco2 4cypsmac cells moreaccurately reflect the behavior of cyp3a4 substrates in human epithelial cells than parental caco2 cellsdiscussionin the current study we introduced four cyps and porinto caco2 cells to increase their drug metabolic 0cohta bmc biotechnology page of abilities which are typically low this study was intendedto establish a better human cell model to more preciselyevaluate the behavior of drugs in the small intestine the4cypsmac was successfully introduced into the caco2cells and successfully increased cyp activityin contrastin this studythe gene expression and activity of cyps in tchepg2 carrying the 4cypsmac are either comparableto or higher than those in primary human hepatocytes to the other cypscyp3a4 mrna expression was still low in caco2 carrying the 4cypsmac compared with the level in human adult intestine despite the significant enhancementof mrna expression regarding the genes on the4cypsmac each is present as a single copy becausethe nature of gene regulation is supposed to differ between hepg2 and caco2 changing copy number of thecyp3a4 gene may further optimize the expression profile of caco2 cells to that of human intestinethe established caco2 4cypsmac cells with particularly high gene expression showed high activity in all cypsin the future we will conduct metabolic tests inhibitiontests and permeation tests using drugs that are substratesfor other cyps and investigate whether the caco2 4cypsmac cells reflect the behavior of drugs in small intestinal epithelial cells the cyp expression level in the humansmall intestine is reported to be approximately forcyp3a4 approximately for cyp2c9 approximately for cyp2c19 and approximately for cyp2d6 it will be necessary to evaluate whether the proportion ofcyp expression in the caco2 4cypsmac is close tothat of the human small intestineif cyp metabolic capacity is guaranteed in the established clones the established cell line can be used asnew human small intestine model cells in recent yearssmall intestine model cells prepared from induced pluripotent stem cells have been reported but such cells areconsidered difficult to use for screening large quantitiesof drug candidate compounds however caco2cells are easy to handle therefore it is possible to usecypmodified caco2 cells to test large numbers of candidate compounds as a first screeningwako osaka japan supplemented with fetal bovine serum fbs and μgml g418 parental caco2cells atcc® htb37„¢ atcc manassas va usawere maintained in dulbecco™s modified eagle™s mediumdmem wako supplemented with fbs memnonessential amino acids gibco thermo fisher scientific waltham ma usa m hepes gibco mmsodium pyruvate gibco mm glutamax gibcoand penicillinstreptomycin wako caco2 cells withthe 4cypsmac were maintained in the above mediumsupplemented with μgml g418 these cells werecultured at °c in co2microcellmediated chromosome transfertransfer of 4cypsmac from cho cells to caco2 cellswas performed using a standard procedure brieflydonor cho cells were cultured in f12 medium supplemented with fbs and μgml colcemid after h microcells were isolated through centrifugation withdmem containing cytochalasin b and filtration thenmicrocells suspended in phytohemagglutinin p phapdmem were poured onto caco2 cells in a 6cm dishand incubated for min the cells were treated withpolyethylene glycol peg solution g of peg1000 ml of dmem ml of dimethyl sulfoxide for minfollowed by washing with dmem after h of recoveryculture cells were seeded in a 24well collagencoatedplate corning ny usa and maintained with selectionmedium h after seeding thereafter the medium waschanged twice a week to obtain drugresistant clonesbecause the mac carries a gfp gene gfppositiveclones were selected from the drugresistant clonesgenomic pcr analyseswe extracted genomic dna from cell lines using a genomic dna extraction kit with dnasefree rnase gentra systems minneapolis mn usa the primers forthe genomic pcr are listed in additional file tables1 they amplified each gene region on the 4cypsmac we used kod fx takara otsu japan in accordance with the manufacturer™s instructionsthe mammalian artificial chromosome vector systemwould provide useful models for drug development theestablished caco2 cells with the 4cypsmac are expected to more accurately predict absorption and metabolism in the human intestine than parental caco2 cellsmethodscell culturechinese hamster ovary cho cells jcrb0218 jcrbcell bank nibiohn osaka japan carrying the 4cypsmac were maintained in ham™s f12 nutrient mixturefish analysestrypsinized cells were incubated for min in m kcland fixed with methanol and acetic acid and thenslides were prepared using standard methods fish analyses were performed using the fixed metaphase of each cellhybrid using digoxigeninlabeled roche germany dna[mouse cot1 dna invitrogen carlsbad ca usa] andbiotinlabeled dna [pac 4cypspor] essentially as described previously chromosomal dna was counterstained using dapi sigmaaldrich st louis mo usaimages were captured using an axioimagerz2 fluorescencemicroscope carl zeiss germany 0cohta bmc biotechnology page of rtqpcrwe extracted mrna using the rneasy mini kit qiagen germany and synthesized firststrand cdna usingthe high capacity cdna reverse transcription kit applied biosystems foster city ca usa the primersare listed in additional file table s1 for rtqpcranalysis tb green premix ex taq takara was usedand relative mrna expression was evaluated throughthe δδct method gapdh was used for normalizationculture timedependent expression level change of fourcypscaco2 cells and caco2 4cypsmac were seededin a 6cm dish at a concentration of × cellswell cells were lysed using trizol invitrogen causa on days and after seeding and rnawas extracted and purified using an rneasy mini kitqiagen thereafter cdna synthesis was performedusing the highcapacity cdna reverse transcriptionkit applied biosystemsactivity test of the four cypswe tested the metabolic activity ofthe four cypsusing the p450glo„¢ assay promega madison wiusa the luminogenic substrates used for the testwere luciferinipa cyp3a4 luciferinme egecyp2d6 luciferinh cyp2c9 and luciferinhege cyp2c19 cells wereseeded in 48wellcollagencoated plates corning at a density of × cellswell after h the medium was changedand h later we added transport medium tm containing substrate tm was prepared using hanks™ balanced salt solution hbss with mm nahco3 mm glucose and mm hepes which was adjusted to ph after incubation we added detectionreagent and measured the luminescence using an infiniteandcyp2c19 required h of incubation while cyp2d6and cyp3a4 required h after the measurementthe cells were washed with pbs dissolved in lysisbuffer and diluted fivefold the same amount of celltiter glo promega was added to μl of the lysateand luminescence measurement was performed tonormalize data to the number of viable cellswako cyp2c9f500 platereadermidazolam mdz permeability testthe obtained clones were assessed in an mdz permeation test each cell was seeded on a 24well cell cultureinsert plate millipore billerica ma usa at a concentration of × cellswell the medium was changedonce a week after seeding and every d after the secondweek transepithelial electrical resistance teer wasmeasured using millicellers millipore before mediumexchange the test was performed d after seedingfor the test tm ph prepared by adding mmnahco3 mm glucose and mm hepes tohbss at ph was used the donor side solutionwas prepared by dissolving μm mdz in tm ph the acceptor side solution was prepared by adding fbs to tm ph on the test day themedium was removed from the culture insert seededwith the cells and the cells were rinsed twice withtm ph tm ph and tm ph wereadded to the apical and basal chambers respectivelyand cells were incubated at °c for min the testwas started by adding the donor side solution to thedonor side chamber and the acceptor side solution tothe acceptor side chamber thirty minutes after thestart of the test the solution in the donor side andacceptor side chambers was collected moreover tomeasure the amount of mdz and ²hydroxy mdz²oh mdz in the cells after the test the cultureinsert was quickly rinsed three times with icecoldtm ph the membrane was cut using a cutterand μl of icecold tm ph was added cellswere detached from the membrane through sonicationand a cell suspension was used as a sample thesesamples were deproteinized and stored at ˆ’ °cuntil measurementlcmsms was used for the measurement of mdzand ²oh mdz in the samples qtrap5500 sciexframingham ma usa and a prominence uflc system shimadzu kyoto japan were combined for measurement the hplc conditions and msms conditionsare shown in additional file table s2 quantitativeanalysis was performed in multiple reaction monitoringmode mass transitions mz were †’ formdz †’ for ²oh mdz and †’ for ²oh mdz d4 data were analyzed usinganalyst software sciexequation formula to calculate extraction ratio erer ¼metabolite donorþreceiverþintracellularpþparent receiverþintracellularððþ þ pmetabolite donorþreceiverþintracellularðþtokyo chemicalinhibition testketoconazoleindustry tokyojapan was used as an inhibitor against cyp3a4 andchanges in metabolic activity were measured using ap450glo assay with luciferinipa cells were seededin a 48well collagencoated plate at × cellswell and the medium was changed after d thenext daythe medium was collected cells werewashed twice with pbs and then μl of tm ph containing ketoconazole tokyo chemical industry at and μm was added toeach set of three wells tm was adjusted to ph byadding mm nahco3 mm glucose and mm 0cohta bmc biotechnology page of received april accepted august referencesbenet l wu c hebert m wacher v intestinal drug metabolism andantitransport processes a potential paradigm shift in oral drug delivery jcontrol release “xie f ding x zhang qy an update on the role of intestinal cytochromep450 enzymes in drug disposition acta pharm sin b “takenaka t kazuki k harada n kuze j chiba m iwao t matsunaga t abes oshimura m kazuki y development of caco2 cells coexpressingcyp3a4 and nadphcytochrome p450 reductase using a human artificialchromosome for the prediction of intestinal extraction ratio of cyp3a4substrates drug metab pharmacokinet “hu m li y davitt cm huang sm thummel k penman bw crespi cltransport and metabolic characterization of caco2 cells expressing cyp3a4and cyp3a4 plus oxidoreductase pharm res “schmiedlinren p thummel ke fisher jm paine mf lown ks watkins pbexpression of enzymatically active cyp3a4 by caco2 cells grown onextracellular matrixcoated permeable supports in the presence of1alpha25dihydroxyvitamin d3 mol pharmacol “fan j liu s du y morrison j shipman r pang ks upregulation oftransporters and enzymes by the vitamin d receptor ligands 1alpha25dihydroxyvitamin d3 and vitamin d analogs in the caco2 cell monolayer jpharmacol exp ther “cummins cl mangravite lm benet lz characterizing the expression ofcyp3a4 and efflux transporters pgp mrp1 and mrp2 in cyp3a4transfected caco2 cells after induction with sodium butyrate and thephorbol ester 12otetradecanoylphorbol13acetate pharm res “korjamo t honkakoski p toppinen mr niva s reinisalo m palmgren jjmonkkonen j absorption properties and pglycoprotein activity of modifiedcaco2 cell lines eur j pharm sci “korjamo t monkkonen j uusitalo j turpeinen m pelkonen o honkakoskip metabolic and efflux properties of caco2 cells stably transfected withnuclear receptors pharm res “kublbeck j hakkarainen jj petsalo a vellonen ks tolonen a reponen pforsberg mm honkakoski p genetically modified caco2 cells withimproved cytochrome p450 metabolic capacity j pharm sci “mes to hbss the cells were preincubated for h at °c and 1000fold diluted cyp3a4 substrate wasadded one hour later μl of the supernatant wascollected from the well mixed with μl of detectionreagent and the luminescence value was measuredusing an infinite f500 plate reader wakosupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12896020006378additional file figure s1 comparison of gene expression betweenhuman small intestine and day culture of caco2 and caco2 4cypsmac table s1 primer sequences for genomic pcr and rtqpcrtable s2 lcmsms analysis conditions mdz ²oh mdzabbreviationscyp cytochrome p450 ac artificial chromosome mmct microcellmediated chromosome transfer por p450 oxidoreductase hac humanartificial chromosome mac mouse artificial chromosome ddi drug“druginteraction cho chinese hamster ovary mdz midazolamteer transepithelial electrical resistance er extraction ratioacknowledgmentswe thank satoru iwado at tottori university for technical assistance with theexperiments we also thank dr hiroyuki kugoh dr masaharu hiratsuka drhiroyuki satofuka and dr takahito ohira at tottori university for criticaldiscussions this research was partly performed at the tottori bio frontiermanaged by tottori prefecture we thank edanz group wwwedanzeditingcomac for editing a draft of this manuscriptauthors™ contributionsall authors conceived and designed the experiments yo and kka performedthe experiments yo sa kko and yk wrote the paper mo and yksupervised the study all authors read and approved the final manuscriptfundingthis work was supported in part by the basis for supporting innovative drugdiscovery and life science research binds from the japan agency formedical research and development amed under grant numberjp18am0301009 ykavailability of data and materialsthe data and materials used andor analyzed during the current study areavailable from the corresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting interestsdr mitsuo oshimura is ceo and a shareholder of trans chromosomics incdr satoshi abe is a member of trans chromosomics inc and the otherauthors declare no conflict of interestauthor details1division of genome and cellular functions department of molecular andcellular biology school of life science faculty of medicine tottoriuniversity nishicho yonago tottori japan 2chromosomeengineering research center cerc tottori university nishicho yonagotottori japan 3trans chromosomics inc nishicho yonagotottori japan 4laboratory of biopharmaceutics meijipharmaceutical university noshio kiyose tokyo japan oshimura m uno n kazuki y katoh m inoue t a pathway fromchromosome transfer to engineering resulting in human and mouseartificial chromosomes for a variety of applications to biomedicalchallenges chromosom res “satoh d abe s kobayashi k nakajima y oshimura m kazuki y human andmouse artificial chromosome technologies for studies of pharmacokineticsand toxicokinetics drug metab pharmacokinet “kazuki y hoshiya h takiguchi m abe s iida y osaki m katoh m hiratsukam shirayoshi y hiramatsu k ueno e kajitani n yoshino t kazuki k ishiharac takehara s tsuji s ejima f toyoda a saka
Colon_Cancer
purpose pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer lapc prevents surgical resection this study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationmethods in this phase iii trial patients with lapc were randomised to gemcitabinenab paclitaxel plus arm a n24 or minus arm b n13 pamrevlumab those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria resection rates progression free and overall survival were evaluatedresults eighteen patients in arm a and seven in arm b completed six cycles of therapy with similar toxicity patterns in arms a and b carbohydrate antigen “ response as defined by ‰¥ decline from baseline occurred in and respectively sixteen per cent of patients were radiographically downstaged by national comprehensive cancer network criteria in arm a and in arm b positron emission tomography normalised in vs of patients in arm a vs arm b respectively and correlated with surgical exploration eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in arm a vs arm b p01193 respectively postoperative complication rates were not different between armss neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with lapc without added toxicity this combination merits evaluation in a larger patient cohortintroductionpancreatic cancer is currently the third leading cause of cancer death in the usa1 and by it will likely become the second leading cause of cancer related death after key questionswhat is already known about this subject –º pamrevlumab is anti ctgf1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseasewhat does this study add –º this study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerhow might this impact clinical practice –º this study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged os curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 however only of patients have disease amenable to upfront curative resection at the time of diagnosis4 approximately “ of patients are diagnosed with locally advanced disease5 determined surgically unresectable per national comprehensive cancer network nccn guidelines6 patients with locally advanced pancreatic cancer lapc have a prognosis similar to those with metastatic disease with a historical median overall survival os of picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access“ months with recent trials demonstrating median os of months7 recent single institution retrospective studies have reported the potential for resection of lapc with neoadjuvant therapy irrespective of imaging findings with promising results8 however these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation current prospective trials have documented resection rates of lapc in the range of to therefore novel approaches are needed to improve patient outcomesthe tumour biology inherent to pancreatic ductal adenocarcinoma pdac significantly contributes to the poor outcomes seen in this disease notably pdac exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor ctgf expression12 ctgf appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition this leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 this stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticspamrevlumab is a human monoclonal antibody that targets ctgf preclinical studies showed that ctgf overexpression is associated with both desmoplasia and gemcitabine resistance in the kpc pancreatic cancer mouse model14 when pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of xiap an antiapoptotic protein15 when tested in patients with advanced pancreatic cancer stage iv and locally advanced stage iii treated with gemcitabine and erlotinib in a phase iii study n75 pamrevlumab displayed multiple favourable outcomes16we hypothesised that through inhibition of the downstream effects of ctgf overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of pdac tumours with this in mind this novel phase iii randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in lapc with special emphasis on surgical eligibility and safetymethodsstudy designthis was a phase iii randomised trial of safety and efficacy in patients with lapc who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy the randomisation was preplanned and blinded to the investigator the study was approved by individual institutional review boards at nine us institutions and conducted according to the declaration of helsinki the trial was registered at clinicaltrials gov as nct eligibilitykey protocol eligibility requirements included biopsy proven diagnosis of pdac radiographic staging consistent with locally advanced unresectable disease as defined nccn guidelines v2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per response evaluation criteria in solid tumors recist v11 eastern cooperative oncology group ecog performance status of or adequate haematological renal and hepatic function no prior therapy for pdac and no concomitant cancer diagnosis within the past yearsstudy schemaeligible patients were randomised to arm a or arm b to receive a total of six treatment cycles “ weeks of therapy figure patients in arm a received pamrevlumab mgkg by intravenous infusion on days and of each day cycle with an additional dose given on day in the first cycle patients in both arms a and b received gemcitabine mgm2 by intravenous infusion on days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on days and of each day cycle doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care soc15 patients remained on therapy for six treatment cycles “ weeks unless they had disease progression an intolerable adverse event ae or toxicity withdrew consent or were withdrawn at the investigator™s discretion all patients were followed for drug toxicity until days after the last drug dose patients undergoing surgery were followed for days following hospital discharge for surgical complications ctgf levels were obtained prior to treatment from all patients plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug after all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy postoperative complications including day readmissions and day mortality were notedresponse assessmentpatients were evaluated for response by the following measures carbohydrate antigen ca “ measured at baseline first day of each cycle and end of treatment eot recist v11 read based on full body ct imaging high resolution dual phase fine cut ct imaging at baseline and every weeks thereafter fluorodeoxyglucose fdg positron emission tomography pet imaging and nccn v2 resectability criteria at baseline and eotpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessfigure patient flow and surgery outcomes in arm a four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery in arm a four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive sma encasement in arm b one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement sma superior mesenteric arterysurgical assessmentsubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol pp defined criteria given that patients included in the trial were determined to be initially unresectable by radiographic imaging and nccn criteria objective criteria were developed to standardise attempts at surgical resectionpatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma ca “ level by ‰¥ at eot compared with baseline reduction in fdg pet maximum standardised uptake value suvmax by ‰¥ at eot compared with baseline radiological tumour response per recist of partial response pr or complete response cr at eot or met the definition of resectable or borderline resectable per nccn guidelines subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on ct scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein pvsplenic vein thrombosis pancreatitis or decline in performance status to a karnofsky score ‰¤ or picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes the final decision regarding whether resection was to be performed was made by the treating surgeonendpointssafety endpoints included serious adverse events sae during neoadjuvant therapy and surgical complications postresection the efficacy endpoints included surgical eligibility r0 resection r0r1 resection median os progression free survival pfs and year survival rate all patients were followed and data analysis was stratified by pp population and intention to treat itt cohortstatistical considerationsthe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the χ² test exact cis for the point estimates as well as the treatment difference were obtained from the sas proc freq procedure with the exact option the two treatment arms were compared using the cochran mantel haentzel test controlling for baseline factors tnm stage ecog ca “ pet suvmax 0copen accesssuperior mesenteric artery sma involvement coeliac abutment and so on as prespecified in the protocol all cause mortality was used in determining os which was analysed by the kaplan meier method survival status was updated within month before the data cut off date data from patients who were alive at the cut off date were censored for survival analysis all statistical tests were performed at the significance level of α005 using two sided testsresultspatient characteristics and dispositionthirty seven patients were randomised to study treatment to arm a pamrevlumabgemcitabinenab paclitaxel and to arm b gemcitabinenab paclitaxel alone patient characteristics at baseline are summarised in table all patients enrolled were unresectable by nccn criteria patients had tumour arterial involvement sma encasement ° coeliac abutment table patient characteristicsbaseline demographics “ years “ years ‰¥ years median male femaleage group sex bmi kgm2 mean sd median min maxecog grade grade tnm stage t3 n0 m0 t3 n1 m0 t4 n0 m0 t4 n1 m0 t4 nx m0location of the tumour in the pancreas non resectability per nccn criterion head body tail median tumour size mm ° sma encasement any coeliac abutment inferior vena cava invasion or encasement unreconstructible smvportal occlusion aortic invasion and encasementarm agnppn24 arm bgnpn13 totaln37 to to to · ok as isnot mutually exclusivebmi body mass index ecog eastern cooperative oncology group g gemcitabine n number of subjects nccn national comprehensive cancer network np nab paclitaxel p pamrevlumab pv portal vein sma superior mesenteric artery smv superior mesenteric veinpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible pvsuperior mesenteric vein smv occlusion a higher percentage of patients with sma encasement ° were randomised to arm a vs arm b patient disposition is summarised in figure twenty four patients in arm a received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles six patients discontinued treatment early due to progressive disease three patients aes two patients or physician decision one patient thirteen patients in arm b received gemcitabinenab paclitaxel patients completed six treatment cycles six patients discontinued treatment early due to progressive disease two patients aes two patients or patientphysician decision two patientssafetysaes are summarised in table forty one per cent of patients had a treatment emergent sae arm a arm b no individual toxicity category occurred with frequency except systemic infection patients there was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapytable summary of treatment emergent serious adverse eventssystem organ classpreferred term ascites nausea pancreatitis vomiting device occlusion drug withdrawal syndrome feverno of patients with any treatment emergent saeblood and lymphatic disorders haemolytic uremic syndrome lymphadenopathycardiac disorders cardiac failure supraventricular tachycardiagastrointestinal disorders general disorders and administrative site conditions hepatobiliary disorders infections sepsis cellulitis urinary tract infectioninjury poisoning and procedural complications respiratory thoracic and mediastinal disorders skin and subcutaneous disorders cholangitis hyperbilirubinaemia craniocerebral injury pneumonitis pulmonary embolism rasharm an24n arm bn13n overalln37n · ok as ispicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessresponse to therapyin arm a had ‰¥ ca “ decline at eot response by recist pr ‰¥ decline in pet suvmax and were radiographically downstaged by nccn criteria during the treatment period the median ca “ decline was patients were non secretors seven out of patients had best objective recist response crpr some patients had ˜exceptional™ responses defined as normalisation or ‰¥ decline of ca “ patients or normalisation pet suvmax in in arm b had ‰¥ ca “ decline at eot response by recist pr ‰¥ decline in pet suvmax and were radiographically downstaged by nccn criteria four out of patients had best objective recist response cr pr in arm b of patients had an œexceptional ca “ response and had an ˜exceptional™ pet response as defined by either ‰¥ normalized ca response normalized suv max andorradiographic downstaging post therapy completion surgical evaluationoverall of the total study patients were eligible for surgical exploration using protocol defined criteria arm a arm b p00019 resection was completed in of the patients arm a arm b p01193 details of the nine resected patients are shown in table in arm a of the patients were eligible for surgical exploration in the itt population and of the patients were eligible in the pp population patients who completed six cycles of treatment in arm a out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined eight out of patients in arm a were resected r0 r1 the remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively in arm b of the patients were eligible for surgical exploration in the itt population and were eligible in the pp population of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionpredictors of resectionhigh ca “ response ‰¥ decline andor normalisation was contributive to surgical eligibility vs p03 normalisation versus non normalisation of pet suvmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection all nine successful resections were identified by one or both of these criteria table summary of resected patientssitesubject idtreatmentarmresponse to treatmentnccnbaselinenccnend of treatmentresection status“““““““““aaaaaaaab unresectablecoeliacunresectablesma smvunresectablecoeliacunresectablecoeliacunresectablesmvunresectablesmaunresectablesma smv coeliacunresectablesmaunresectablecoeliacunresectablecoeliacunresectablesma smvunresectablecoeliacborderline resectableunresectablesmvunresectablesmaunresectablecoeliacunresectablesmaunresectablecoeliacr0r1r0r0r1r1r1r0r0protocol defined criteria ca “ decrease fdg pet suvmax decrease ‰¥ recist v11 response pr or cr nccn resectable or borderline resectable criteriaca carbohydrate antigen cr complete response fdg fluorodeoxyglucose nccn national comprehensive cancer network pet positron emission tomography pr partial response recist response evaluation criteria in solid tumors sma superior mesenteric artery smv superior mesenteric vein suvmax maximum standardised uptake valuepicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cconversely radiographic features of response did not correlate with operative potential neither recist response nor radiographic downstaging per nccn criteria statistically correlated with completed resectionsurgical complicationspostoperative complications were summarised according to the clavien dindo classification posthoc analysis ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in arm a grade ii there was one episode of clinically significant pancreatic leak in each arm grade iiia no reoperations and no day or day surgical mortality were noted one patient in arm b had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively grade iiib no wound complications or superficial site infections were noted in either group four out of patients and out of patients in arm a and b respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalas of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months pfs was months ci to and months ci to in arm a and arm b respectively one year survival and median os were and months ci open accessto in arm a and and months ci nr in arm b the median os for all patients who were eligible for surgical exploration arm a arm b vs ineligible arm a arm b was months ci nr vs months ci to p00766 the median os for resected arm a arm b vs non resected patients arm a arm b was not reached ci nr vs months ci to p00141 figure discussionthe treatment of lapc with neoadjuvant therapy remains challenging and there is no established soc several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 the combination of more active regimens delivered over an extended period and surgeons™ comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates a meta analysis of studies using folfirinox has demonstrated resection rates ranging from to in lapc17 one of the larger studies including patients with lapc reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen folfirinox or gemcitabine based18 recently a single institution and single arm prospective study of neoadjuvant folfirinox and losartan with selective use of radiation in patients with lapc reported an r0 resection rate of figure overall survival resected vs non resected patientspicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access however the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease these retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of lapcthe anti ctgf mechanism of action with respect to gemcitabine based therapy a recent large scale prospective trial of patients with lapc treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation more recently the la pact trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with lapc were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigator™s choice11 last although folfirinox has been the most studied induction combination chemotherapy regimen in this population recent randomised data from european patients who received neoadjuvant folfirinox versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to r0r1 to resection rate vs p0135 or os vs months p0268given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a ˜softening™ of tumours by endoscopic ultrasound elastography21 this stromal depletion also translated into a decrease of suv uptake on pet22 in the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsthe protocol specified therapeutic response criteria ca “ pet suvmax recist and nccn criteria were used as criteria to determine eligibility for surgical exploration in lapc this is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards nccn criteria for example by nccn conversion alone ie converted from unresectable to borderline resectable only of patients in arm a would have been eligible for surgical exploration however by protocol criteria of patients in arm a were eligible for surgical exploration a higher percentage of patients were eligible for surgical exploration by the above criteria in arm a vs arm b vs respectivelyoverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size of the nine subjects that were successfully resected in this trial only one was converted by nccn criteria to borderline resectable prior to surgical exploration despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti ctgf mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients this hypothesis needs to be confirmed and patients should be stratified by coeliac andor sma involvementthe most common predictive factors for eligibility for surgical exploration and resection were ca “ decline and pet suv max response which are indicators of tumour response to treatment the combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success both the ability of ca “ response and the inability of radiographic response recist and nccn criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials in the mpact study both ca “ and pet response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 recent surgical series of patients with borderline resectable and lapc have also corroborated their impact in the localised setting25 correlation of clinical response with plasma levels of endogenous ctgf and pamrevlumab exposure as shown in the prior study by picozzi et al16 may provide added prognostic and predictive insightwith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel in addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone pamrevlumab is well tolerated and considered safe compared with the soc drugs for patients with pdac these observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in lapc in addition there were no signals of increased surgical morbidity or wound healing problems with ctgf blockade by pamrevlumab in fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationfinally while survival data are not yet mature both patients who were eligible for surgery and those that picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cwere ultimately resected had longer pfs and os highlighting the importance of surgical resection of the tumour therefore more investigation into newer agents targeting lapc and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseasein this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in lapc with prespecified criteria for surgical exploration the use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates further evaluation of this drug combination in the neoadjuvant treatment setting for lapc is warranted and a larger phase iii trial with resection and survival endpoints is ongoingcontributors fibrogen inc was the study sponsor that designed the study in consultation with the principal investigator vp and surgical co investigator fgr all authors except those of the sponsor contributed patients to the study fibrogen was responsible for data collection and analysis all authors reviewed the manuscript and signed off on its accuracyfunding the study was funded by fibrogen inc san francisco cadisclaimer the corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the bmj publishing group ltd and its licensees to permit this article if accepted to be published in esmo open editions and any other bmjpgl products to exploit all subsidiary rights as set out in our licencecompeting interests mc mz sp ek and ec are employees of fibrogen and hold stock andor stock options in fibrogenpatient consent for publication not requiredprovenance and peer review not commissioned externally peer revieweddata availability statement data are available on reasonable request all data relevant to the study are included in the article or uploaded as supplementary information data will be available as presented in this manuscriptopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idewa a0carrier http orcid org references american cancer society cancer facts figures available httpswww cancer org content dam cancer org research cancer facts and statistics annual cancer facts and figures cancer facts and figures pdf rahib l smith bd aizenberg r et a0al projecting cancer incidence and deaths to the unexpected burden of thyroid liver and pancreas cancers in the united states cancer r
Colon_Cancer
laparoscopic surgery for rectal cancer is commonly performed in china however compared with open surgerythe effectiveness of laparoscopic surgery especially the longterm survival has not been sufficiently provedmethods data of eligible patients with nonmetastatic rectal cancer at nanfang hospital of southern medical university andguangdong provincial hospital of chinese medicine between and were retrospectively reviewed longterm survival outcomes and shortterm surgical safety were analysed with propensity score matching between groupsresults of cases collated from two institutes matched pairs were analysed after propensity score matching the estimated blood loss during laparoscopic surgery was significantly less than that during open surgery p¼ and the operativetime and hospital stay were shorter in the laparoscopic group both p the postoperative complications rate was inthe laparoscopic group and in the open group p¼ no significant difference was observed between the laparoscopicgroup and the open group in the 5year overall survival rate vs p¼ 5year relapsefree survival rate vs p¼ or 5year cancerspecific survival rate vs p¼ an elevated carcinoembryonic antigen harvested lymph nodes and perineural invasion were independent prognostic factors affecting overall survival and relapsefreesurvivals our findings suggest that open surgery should still be the priority recommendation but laparoscopic surgery isalso an acceptable treatment for nonmetastatic rectal cancerkey words laparoscopic surgery open surgery propensity score matching rectal cancersubmitted february revised april accepted june vc the authors published by oxford university press and sixth affiliated hospital of sun yatsen universitythis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0c kl tan introductioncolorectal cancer is the second most commonly diagnosedcancer and the fifth leading cause of cancerrelated death forboth sexes in china in a multidisciplinary approach thatcombines chemotherapy with radiotherapy for the treatmentof colorectal cancer surgery remains the major approach thefirst successful use of laparoscopy in colorectal surgery waspublished in by jacobs laparoscopic surgery hasbeen performed widely in colon cancer all over the world andseveral randomized“controlled trials have demonstrated thatlaparoscopic surgery for colon cancer is safe and feasible withbetter shortterm outcomes including a decrease in postoperative pain a shorter hospital stay and earlier recoveryand equivalent longterm results compared to open surgery[“] however laparoscopic surgery for rectal cancer is morearduous than that for colon cancer so the early clinical trialsexcluded rectal cancer [“] although a few clinical trialshave shown the advantages of laparoscopic rectalcancer resection compared with open surgery [“] both the acosogz6051 and alacart trials did not support the use of laparoscopic surgery for rectal cancer [ ] it is still controversialwhether laparoscopic surgery is suitable for rectal cancer especially for low rectal cancer therefore we conducted thisretrospective cohort study to compare longterm survival outcomes and shortterm surgical safety between laparoscopicand open surgery for nonmetastatic rectal cancer in thechinese population propensity score matching psm was performed for the study designpatients and methodsstudy designall consecutive eligible patients with rectal cancer were confirmed from the department of general surgery of nanfanghospital of southern medical university and the department ofproctology of guangdong provincial hospital of chinesemedicine between january and december these twocenters were members of the southern chinese laparoscopiccolorectal surgery study group demographic clinical pathologic and imaging features together with the management andoutcomes were carefully reviewed written informed consentwas acquired from patients preceding the surgical proceduresthis study was approved by the ethical committee of nanfanghospital and guangdong provincial hospital of chinesemedicine no ze2019052“study subjectsinclusion criteria were patients with clinical stage i“iii rectalcancer who underwentrectal cancerexclusion criteria were patients with i combined operationsextending to the surrounding an ii multiple cancers iiiemergency operation iv conversion to open surgery or vpatients who received neoadjuvant therapyradical surgery forall included cases were classified into two groups based onthe surgical approach which was either laparoscopic or opensurgery the surgical approach was decided by the individualcolorectal surgeon based on a combined assessment of clinicalendoscopic and imaging featuresdata collectiondata were collected in a prospectively maintained databasefrom clinical report forms the demographic and clinicopathological data included age gender body mass index bmi preoperative carcinoembryonic antigen cealocationoperative time estimated blood loss surgical procedure protective ileostomy tumor grade tumor stage and hospital staypreoperative cea was defined as cea measured closest to theoperation time tumor location was divided into the followingthree sections upper rectum above cm from the anal vergemiddle rectum “ cm from the anal verge and lower rectumbelow cm from the anal verge surgical procedures consistedof three categories low anterior resection abdominoperinealtumorfigure flow diagram of patient disposition 0claparoscopic vs open surgery for rectal cancer table baseline characteristics of the study populationcharacteristictotal cohortmatched cohortlaparoscopic groupn¼ open groupn¼ pvaluelaparoscopic groupn¼ open groupn ¼ pvalueage years mean sdgender n malefemalebmi kgm2 mean sdpreoperative cea n 14 ngml ngmltumor location n upper rectummiddle rectumlower rectumtumor stage n iiiiii sd standard deviation bmi body mass index cea carcinoembryonic antigentable operative and pathological results in matched cohorts variablesurgical procedure n low anterior resectionabdominoperineal resectionhartmann™s procedureprotective ileostomy n operative time min median iqrintraoperative blood loss ml median iqrhospital stay day median iqrtumor grade n wellmoderatepoorothersharvested lymph nodes n 15lymphovascular invasion n perineural invasion n tumor deposits n postoperative complications n wound infectionileusurinary dysfunctionanastomosis leakageintraabdominal bleedingpneumoniacardiac eventreoperation n mortality n iqr interquartile rangelaparoscopic group n¼ open group n ¼ “ “ “ “ “ “ pvalueresection and hartmann™s procedure tumor grade was dividedinto three types well differentiated moderately differentiatedand poorly differentiated including signet or mucinous adenocarcinoma tumor stage was based on the final pathologic report and preoperative imaging examinationoutcome measurementsthe primary endpoint of this study was overall survival osrelapsefree survival rfs and cancerspecific survival cssos was defined as the time from operation to death from any 0c kl tan figure survival curve after laparoscopic surgery vs open surgery in matchedcohortscause or the last followup rfs was defined as the time fromoperation to identified recurrence or any cause of death csswas defined as the time from operation to death due to rectalcancer the last followup was january ileus urinary dysfunctionthe secondary endpoints were operative time estimated bloodloss hospital stay reoperation postoperative complications andmortality postoperative complications were defined as woundinfectionleakageintraabdominal bleeding pneumonia and cardiac eventsintraabdominal bleeding was defined in this study as bleeding requiring transfusion or reoperation all complications within daysafter surgery were recorded postoperative mortality was traditionally defined as any death occurring within days after surgeryanastomoticstatistical analysisdata are presented as mean standard deviation or medianwith interquartile range iqr for quantitative variables withparanormal distribution and numbers with percentages for categorical variables quantitative variables were compared usingthe student™s ttest or mann“whitney u test categorical variables were analysed using the chisquare test or fisher™s exacttest the estimates of the differences in age gender bmi preoperative cea level tumor location and tumor stage between thetwo groups were performed using psm [ ]survival rates were calculated by using the kaplan“meiermethod and comparisons between groups were performed withthe logrank test to identify the prognostic factors univariateand multivariate analyses were performed using the cox proportional hazards regression model and the results were presented as hazard ratios hrs with confidence intervalscis only factors with p in the univariate analysis wereevaluated in subsequent multivariate analysis using forwardstepwise selection for os and rfs a p was regarded asstatistically significant all statistical analyses were carried outwith ibmvr spssvr statistics version resultsbaseline characteristicsbetween january and december eligiblepatients were collected from hospitals in china of patients cases were excluded among the remaining cases underwent laparoscopic surgery and underwent open surgery after psm of pairs ofpatients were successfully matched figure baseline characteristics are outlined in table before psm there were differences in age and preoperative cea between the two groups afterpsm all variables were well balancedshortterm surgical outcomesthe perioperative and pathological results in matched cohortsare presented in table the estimated blood loss during laparoscopic surgery was significantly less than that during opensurgery p¼ in the laparoscopic group the operative timeand hospital stay were shorter than in the open groupp the incidence of postoperative complications was in the laparoscopic group and in the open groupp¼ in the open group the most common complicationswere wound infection and pneumonia followed byanastomosis leakage whereas in the laparoscopic groupthe most common complication was anastomosis leakage followed by pneumonia longterm survival outcomesin the matched cohorts the median followup period was months in the laparoscopic group iqr “ monthsand months in the open group iqr “ monthsduring the followup patients died among whom diedfrom rectal cancer and had locoregional recurrence or distantmetastasis no significant difference was observed between thelaparoscopic group and the open group in 5year os vs p¼ 5year rfs vs p¼ or 5yearcss vs p¼ figure subgroup analyses for os were conducted for gender agebmi tumor location and tumor stage compared with open surgery male patients or those with an intermediate bmi to who underwent laparoscopic surgery tended to show worseos figure 0claparoscopic vs open surgery for rectal cancer figure subgroup analysis of overall survival in matched cohortstable multivariate analysis for os and rfs in matched cohortsvariableosrfspreoperative cea vs 14 ngmlnumber of harvested lymph nodes 15 vs perineural invasion yes vs nohr cipvalue“““hr cipvalue“““os overall survival rfs relapsefree survival cea carcinoembryonic antigen hr hazard ratio ci confidence intervalprognostic factors for longterm survivalprognostic factors affecting survival are presented in table univariate analyses revealed that an elevated cea ngml harvested lymph nodes perineural invasion and tumordeposits were associated with poor os and that an elevatedcea harvested lymph nodes perineural invasion and lymphovascular invasion were associated with poor rfs data notshown the surgical approach laparoscopic vs open was notassociated with os hr ci “ and rfs hr ci “ multivariate analyses testified that an elevated cea harvested lymph nodes and perineural invasionwere independent factors affecting os and rfs table discussionlaparoscopic surgery for rectal cancer is commonly performedin many countries nevertheless the evidence for laparoscopicsurgery for rectal cancer is insufficient this study focused onthe longterm survival outcomes and surgical safety of patientswho underwentlaparoscopic or open surgery for nonmetastatic rectal cancer in the chinese population in this twocenter study psm was performed to make selection balance between patients treated with laparoscopic and open surgery thesix factors of age gender bmi preoperative cea level tumor location and tumor stage were used as described in the protocolthe baseline characteristics were ideally balanced between thelaparoscopic and open groupssome studies have reported similar postoperative complications and mortality between laparoscopic surgery and opensurgery for rectal cancer [ ] and other studies have reportedfewer postoperative complications after laparoscopic surgerythan after open surgery [ ] in our study there were no significant differences in postoperative complications includingwound infectionileus urinarytract infection anastomosisleakageintraabdominal bleeding pneumonia and cardiacevent between the two groups the longer operative time is often considered a disadvantage of laparoscopic surgery according to some previous reports [ ] in contrast our studyshowed that the operative time of laparoscopic surgery wasshorter than that of open surgery the clasicc trial and colorii trial both showed that hospital stay was significantly shorterin the laparoscopic group [ ] similarly our study alsoshowed that the hospital stay for laparoscopic surgery wasshorter than for open surgerywith regard to longterm survival no largescale clinical trials have demonstrated a statistically significant difference between laparoscopic and open surgery for rectal cancer thecolor ii trial indicated no statistically significant differences indfs and os between laparoscopic and open surgeries inthe corean study dfs in laparoscopic surgery is noninferiorcompared to that in open surgery for mid or low rectal cancer consistently with previous studies os rfs and css didnot differ in both groups in our study interestingly subgroupanalyses for os showed that male and intermediate bmi to kgm2 subgroups were associated with unfavorable outcomes in the laparoscopicsurgery group vs the opensurgerygroup chinese male populations have a narrow pelvis whichmight affect the visualization of and access to the deep pelvic 0c kl tan anatomy during laparoscopic surgery kitano foundthat laparoscopic surgery might affect longterm outcomes inthe highbmi kgm2 subgroup in the current study thebmi subgroup unfavorable for laparoscopic surgery that weidentified was intermediate bmi not high bmi it might be dueto lower bmi in the chinese population compared to that in thewestern population and the small proportion of patientswith high bmi in our cohort further evaluation will be neededto determine which subgroups of patients require additional attention when undergoing laparoscopic surgerylymphovascularwe evaluated several possible prognostic factors that mayinfluence survival in patients with rectal cancer including tumor location tumor stage tumor grade surgical approach preoperative cea levelinvasion perineuralinvasion and tumor deposits [“] as expected our studyshowed that perineural invasion was the significant prognosticfactor affecting os and rfs perineural invasion refers to the invasion of cancer cells into any of the layers of the nerve sheatha higher grade of perineural invasion was related to local recurrence and metastasis in distant ans such as the liver lungand peritoneum all patients in this study underwent radical surgery with lymphnode dissection a minimum of harvested lymph nodes is recommended to ensure adequatestaging and oncologic resection for colorectal cancer themore lymph nodes harvested the better the prognosis [ ]in this study the average number of harvested lymph nodeswas we found that patients with 15 harvested lymphnodes had better os and rfs than those with harvestedlymph nodes several studies have shown that elevated preoperative cea was a poor prognostic factor in colorectal cancer[“] in our study we also found that patients with an elevated preoperative cea had poorer os and rfsour study has several limitations first a selection biasexisted due to its retrospective design to reduce this the twogroups were matched carefully using psm second the statistical power is insufficient because the number of patients enrolled may not be sufficient after matching third data aboutadjuvant therapy after surgery were not collected which mightbe different between both groups and thus have influenced survival outcomes fourth the exclusion of converted cases mayintroduce a bias in favor of laparoscopic surgery finally thebowelrecovery data could not be exactly assessed due to thelack of records in this retrospective study therefore further research with a large population is still awaitedanydifferencesinin our study revealed the benefit of laparoscopicsurgery on shortterm outcomes including less blood lossshorter operative time and shorter hospital stay we did notfindcomplicationslaparoscopic surgery was similar to open surgery in terms ofos rfs and css for patients however male patients and thosewith an intermediate bmi in the laparoscopic group tended toshow worse os than those in the open group findings fromthis study suggest that open surgery should still be the priorityrecommendation but laparoscopic surgery is also an acceptabletreatment for nonmetastatic rectal cancer in the chinesepopulationpostoperativeauthors™ contributionsklt hjd zqc and tym collected and analysed the dataklt hl and rsx performed statistical analysis klt andhjd drafted the manuscript gxl and xhf performed theprocedure conceived of and designed the study and criticallyrevised all the intellectual content of the manuscript allauthors read and approved the final manuscriptfundingthis work was supported by clinical research of guangdongprovincial hospital of chinese medicine [no yn10101902]and a scientific research project of guangdong provincialacademy of chinese medical sciences [no yn2018ml11]acknowledgementsthe authors thank the patients and their families for making this retrospective study possible we also thank all theinvestigators and staff who contributed to the patientfollowup and data collection in nanfang hospital ofsouthern medical university and guangdong provincialhospital of chinese medicineconflicts of interestthe authors declare that there is no conflict of interests inthis studyreferences feng rm zong yn cao sm current cancer situation inchina good or bad news from the global cancerstatistics cancer commun “jacobs m verdeja jc goldstein hs minimally invasive colonresection laparoscopic colectomy surg laparosc endosc “ hewett pj allardyce ra bagshaw pf shortterm outcomes of the australasian randomized clinical study comparing laparoscopic and conventional open surgical treatmentsfor colon cancer the alccas trial ann surg “ buunen m veldkamp r hop wc survival after laparoscopic surgery versus open surgery for colon cancer longterm outcome of a randomised clinical trial lancet oncol “ clinical outcomes of surgical therapy study group a comparison of laparoscopically assisted and open colectomy forcolon cancer n engl j med “ bonjer hj haglind e jeekel i laparoscopic surgery versusopen surgery for colon cancer shortterm outcomes of arandomised trial lancet oncol “ fleshman j sargent dj green e laparoscopic colectomyfor cancer is not inferior to open surgery based on 5year datafrom the cost study group trial ann surg “ van der pas mh haglind e cuesta ma laparoscopic versus open surgery for rectal cancer color ii shortterm outcomes of a randomised phase trial lancet oncol “ kang sb park jw jeong sy open versus laparoscopicsurgery for mid or low rectal cancer after neoadjuvant chemoradiotherapy corean trial shortterm outcomes of anopenlabel randomised controlled trial lancet oncol “ zhou zx zhao ly lin t longterm oncologic outcomesof laparoscopic vs open surgery for stages ii and iii rectal 0ccancer a retrospective cohort study world j gastroenterol“iii colon cancer jcog0404 a phase randomised controlledtrial lancet gastroenterol hepatol “laparoscopic vs open surgery for rectal cancer fleshman j branda m sargent dj effect of laparoscopicassisted resection vs open resection of stage ii or iii rectalcancer on pathologic outcomes the acosog z6051 randomized clinical trial jama “ stevenson ar solomon mj lumley jw effect oflaparoscopicassisted resection vs open resection on pathological outcomes in rectal cancer the alacart randomizedclinical trial jama “ austin pc an introduction to propensity score methods forreducing the effects of confounding in observational studiesmultivariate behav res “ wang h chen x liu h laparoscopyassisted colectomyas an oncologically safe alternative for patients with stage t4colon cancer a propensitymatched cohort study bmc cancer bonjer hj deijen cl abis ga a randomized trial of laparoscopic versus open surgery for rectal cancer n engl j med“ lujan j valero g biondo s laparoscopic versus opensurgery for rectal cancer results of a prospective multicentreanalysis of patients surg endosc “ landi f vallribera f rivera jp morbidity after laparoscopic and open rectal cancer surgery a comparative analysisof morbidity in octogenarians and younger patientscolorectal dis “ toda s kuroyanagi h laparoscopic surgery for rectal cancercurrent status and future perspective asian j endosc surg“ guillou pj quirke p thorpe h shortterm endpoints ofconventionalinpatients with colorectal cancer mrc clasicc trial multicentre randomised controlled trial lancet “laparoscopicassisted surgeryversus jeong sy park jw nam bh open versus laparoscopicsurgery for midrectal or lowrectal cancer after neoadjuvantchemoradiotherapy corean trial survival outcomes of anopenlabel noninferiorityrandomised controlled triallancet oncol “ kitano s inomata m mizusawa j survival outcomes following laparoscopic versus open d3 dissection for stage ii or mehrkhani f nasiri s donboli k prognostic factors insurvival of colorectal cancer patients after surgery colorectaldis “ wiratkapun s kraemer m seowchoen f high preoperative serum carcinoembryonic antigen predicts metastatic recurrence in potentially curative colonic cancer results of afiveyear study dis colon rectum “ huang qs qin hb xiao j association of tumor differentiation and prognosis in patients with rectal cancer undergoingneoadjuvant chemoradiation therapy gastroenterol rep “ liebig c ayala g wilks ja perineural invasion is an independent predictor of outcome in colorectal cancer j clin oncol“ ueno h shirouzu k eishi y study group for perineuralinvasion projected by the japanese society for cancer of thecolon and rectum jsccr characterization of perineural invasion as a component of colorectal cancer staging am j surgpathol “ amin mb edge sb greene fl eds ajcc cancer stagingmanual 8th edn new york springer “ rosenberg r engel j bruns c the prognostic value oflymph node ratio in a populationbased collective of colorectal cancer patients ann surg “ sjo oh merok ma svindland a prognostic impact oflymph node harvest and lymph node ratio in patients withcolon cancer dis colon rectum “ tarantino i warschkow r worni m elevated preoperative cea is associated with worse survival in stage iiii rectalcancer patients br j cancer “ huang sh tsai ws you jf preoperative carcinoembryonic antigen as a poor prognostic factor in stage iiii colorectal cancer after curativeintent resection a propensity scorematching analysis ann surg oncol “ konishi t shimada y hsu m association of preoperativeand postoperative serum carcinoembryonic antigen and colon cancer outcome jama oncol “ 0c'
Colon_Cancer
four to nine percent of the sequences™ transcription are long noncoding rnas lncrnas inmammalian genomes canzio ji lncrna was regarded as the noise ofgenome transcription and did not have biological functions at first however an increasing numberof studies have reported that lncrna is widely robinson involved in chromosomeedited bylei dengcentral south university chinareviewed byhao linuniversity of electronic science andtechnology of china chinainner mongolia university chinajuan wangcorrespondencenan dudunan05aliyuncomganfeng xiexiegfaliyuncom these authors share first authorshipspecialty sectionthis was submitted tomolecular medicinea section of the frontiers in cell and developmentalbiologyreceived june accepted june published august citationliu z zhang y han x li c yang xgao j xie g and du n identifying cancerrelated lncrnasbased on a convolutional neuralnetwork front cell dev biol 103389fcell202000637frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnasgenomicimprintingchromatin modificationsilencingtranscriptional activationinterference andnuclear transport cheng 2018a recently it has beenproven to be associated with many kinds of cancerstranscriptionalthe secondary structure spliced form and subcellularlocalization of most lncrnas are conserved karner which is very important for lncrna to execute functionshowever compared to the functions of micrornas mirnasand proteins the function oflncrna is more difficult todetermine according to the position of lncrna in the genomerelative to proteincoding genes it can be divided into five typessense antisense bidirectional intronic and intergenicmany researchers have found lncrnas play an important rolein cancers avgeris cheng 2018b zhao and neurodegenerative diseases peng and zhao as other biological molecules zhang t bai cheng 2019a liang although manyresearchers have verified many associations between lncrnasand cancers by biological experiments compared with ourknowledge about diseaserelated genes we still do not knowenough about diseaserelated lncrnas considering the timeand money cost of finding diseaserelated lncrnas more andmore researchers tend to use computational methods to identifydiseaserelated lncrnas these methods could be divided intothree categories machine learning methods network methodsand other methodsmachine learning methods build models based on thesimilarities of diseases orlncrnas and their biologicalcharacteristics cheng cheng 2019b zeng zou lan developed thelncrna“disease association prediction ldap which is amethod based on bagging support vector machine svm toidentify lncrna“disease associations they used similarities oflncrnas and diseases as the features yu developedcollaborative filtering naive bayesian classifier cfnbc based onnaive bayesian they integrated mirna“lncrna associationsmirna“disease associations and lncrna“disease associationsto infer more lncrna“disease associations considering thediscriminative contributions of the similarity association andinteraction relationships among lncrnas disease and mirnasxuan 2019a developed a dual convolutional neuralnetwork cnn with attention mechanisms to predict diseaserelated lncrnasnetwork methods are the most common way to identifyassociations between diseases and lncrnas nowadays gu yu zhang j kuang wang l liu thiskind of method would build one or multiple networks toinfer new information wang l built a lncrna“mirna“disease interactive network and used their novel methodœldlmd to predict associations between lncrnas and diseasessumathipala used a multilevel network topologywhich includes lncrna“protein protein“protein interactionprotein“disease relationship to use network diï¬usion algorithmto predict diseaserelated lncrnas the graph convolutionalnetwork gcn and cnn were used on a lncrna“mirna“disease network by xuan 2019b deng builtlncrna similarity network disease similarity network mirnasimilarity network and their associations then they calculatedthe metapath and feature vector for each lncrna“disease pair inthe heterogeneous information networkother methods may borrow the feature extraction methodor similarity conjecture of network methods but the core ofthis method is matrix decomposition or matrix completionlu developed the geometric matrix completionlncrna“disease association gmclda which is a methodbased on geometric matrix completion they calculated diseasesimilarity based on disease ontology do and calculatedthe gaussian interaction profile kernel similarity for lncrnasthen they inferred diseaserelated lncrnas based on theassociation patterns among functionally similar lncrnas andsimilar diseases wang y proposed a weightedmatrix factorization to capture the interintraassociationsbetween diï¬erent types of nodes then they approximated thelncrna“disease association matrix using the optimized matricesand weights to predict diseaserelated lncrnas localityconstrained linear coding label propagation latent dirichletallocation llclplda was developed by xie firstly localconstraint features of lncrnas and diseases wereextracted by localityconstrained linear coding llc thenthey predicted diseaserelated lncrnas by label propagationlp strategyhowever previous methods did not consider the regulatingtarget gene expression of lncrna which is an important functionof lncrna and plays an important role in associations betweenlncrnas and diseases in addition deep learning methods arean important tool and have shown their power in bioinformaticschen lv wei wu zhao 2019abc therefore in this paper we used thisinformation as features of lncrna in addition the expressionof lncrna in diï¬erent tissues were also used as the featuresof lncrna then the deep belief network dbn was used toencode and the cnn was used to classifymethodsfeature extractiontissue expression specificity of long noncodingrnacompared with proteincoding geneslncrna shows strongtissue specificity the specificity of lncrnas in diï¬erent kindsof tissues and cell types has been proven by many biologicalexperiments the diï¬erent expression also plays an importantrole in essential cellular processes sasaki testedthe expression of lncrnas in diï¬erent tissues and found lncrnas exhibited tissuespecific expression and oflncrnas were only expressed in one discrete tissue thereforethe expression of lncrnas in diï¬erent tissues were used asthe featureswe obtained the expression of lncrnas in diï¬erenttissues which included adipose adrenal breast colon heartkidney liver lung lymph node ovary placenta prostate testisand thyroidtherefore the dimension of each lncrna™s expression featureis ˆ— frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnastherefore the dimension of each lncrna™s target gene featureis ˆ— deep belief networkthe dbn can eï¬ectively learn complex dependencies betweenvariables zhao 2019d the dbn contains many layers ofhidden variables which can eï¬ectively learn the internal featurerepresentation of the data and can also be used as an eï¬ectivenonlinear dimensionality reduction methodwhen the observable variables are known the joint posteriorprobabilities of the hidden variables are no longer independentof each other so it is difficult to accurately estimate the posteriorprobabilities of all hidden variables the posterior probability ofearly dbn is generally approximated by monte carlo methodbut its efficiency is relatively low which makes its parameterlearning difficult in order to eï¬ectively train the dbn weconvert the sigmoid belief network of each layer to a restrictedboltzmann machine rbm the advantage of this is that theposterior probabilities of the hidden variables are independentof each other which makes it easy to sample in this way thedbn can be regarded as being stacked from top to bottom bymultiple rbms and the hidden layer of the lth rbm is used asthe observable layer of the l 1th rbm further the dbn canbe trained quickly by layerbylayer training that is starting fromthe bottom layer and training only one layer at a time until thelast layer the specific layerbylayer training process is to trainthe rbm of each layer in turn from bottom to top assuming wehave trained the rbm in the first l1 layer we can calculate theconditional probability of the bottomup hidden variablesphihiˆ’ σ bi wihiˆ’where bi is the bias of ith layer of rbm wi is the connectionweight hi is the ith layer of rbmthe process of training dbn is as followsfigure the number of target genes for each long noncoding rnalncrnafigure the distribution of the number of target genes lncrna longnoncoding rnareversetarget gene of long noncoding rnaquantitativechainreaction qrtpcr and western blot were used to testthe diï¬erentexpression genes after knocking down oroverexpressing lncrnastranscriptasepolymerasewe obtained target genes of lncrna from lncrna2targetinput train dataset ˆvn learning rate λjiang as we can see in figure there are kinds of lncrnasone lncrna has more than target genes then we drawthe distribution of the number of target genes correspondingto lncrnaasshown in figure most ofthe target genes arecorresponding to less than five lncrnas therefore if we usedthem to be the features of lncrnas the features would be sparsetherefore we only select the most common target genes to bethe features the genes which are corresponding to more thanfive lncrnas were selected as the features of lncrnas there are kinds of genes then we need to encode these genesf [g1 g2 · · · g45]where g1 denotes the first gene of these genes and f denotesthe feature of lncrna for each lncrna if g1 is the target geneof it then g1 otherwise g1 output weight matrix wl bias al and blfor l 1linitialization wi al bi sample from train dataset ˆh0for i lˆ’sample hi based on phi ˆhiˆ’endset hi1as the train sample to train lth layer ofrbmendsince the dimension of expression feature and target genefeature are diï¬erent we should reduce the dimension of targetgene feature and make it the same as the expression feature™stherefore in this paper two layers of rbm were used to builda dbn modelthe number of nodes oftheand respectively sigmoid function was used astwo layers was thefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu activation functionσ x eˆ’xtherefore the dimension of final features is ˆ— f cid20 g1 g2 · · · g13e1 e2 · · · e13 cid21a method to identify cancerrelated lncrnasconvolutional neural networkthe power of cnn in dealing with bioinformatic problems hasbeen proven by many researchers we selected cnn as theclassifier based on two reasons the dimension of features is ˆ— which can be regarded as an image the outstandingperformance of cnn in image classificationthere are five layers in our cnn model the structure of cnnis shown as table where g1 g2 · · · g13 denotes target gene feature after dbnand e1 e2 · · · e13 denotes the expression of lncrnas in diï¬erent tissuestable the structure of convolutional neural network cnnlayersparameterconvolutional layerpooling layerconvolutional layerpooling layerfully connected layeroutputfilter kernel size activation function tanhpool size activation function tanhfilter kernel size activation function tanhpool size activation function tanhunits activation function tanhunits activation function sigmoidwork framefigure shows the work frame of our method œdbn“cnnthere are three steps of our methods firstly we should extractfeatures of lncrnas there are two parts of features expressionfeature and target gene feature then dbn was used to encodethe target gene feature after encoding the two kinds of featureswere combined together finally cnn was used to classifyresultsdata descriptionthe known associations between lncrna and diseases wereobtained from lncrnadisease database bao wetotally obtained kinds of cancerrelated lncrnas the numberof their corresponding lncrnas is shown as figure as shown in figure people™s understanding of cancerrelated lncrnas varies widely we have known more than lncrnas for some cancers but few lncrnas are known for somecancers to better build our model we only selected cancerswhich have more than related lncrnas therefore kindsof cancers were selectedfigure work frame of deep belief network dbn“convolutional neural network cnn lncrna long noncoding rnafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnasfigure the number of long noncoding rnas lncrnas for each cancertable the performance of deep belief network dbn“convolutional neuralnetwork cnn in cancerscancerarea undercurve aucarea under precisioncurve auprcervical cancerbreast cancercolorectal cancerstomach cancerurinary bladder cancerlung cancerovarian cancerthyroid cancerprostate cancerliver cancerpancreatic cancerovarian epithelial cancergallbladder cancerendometrial cancercolon canceresophageal cancerthetargetgenes oflncrnas were obtained fromlncrna2target database we have discussed about this insection target gene of long noncoding rnafigure the receiver operating characteristic roc curves of the threemethods dbn deep belief network cnn convolutional neural network pcaprincipal component analysisfigure the area under the precision“recall curve aupr of the threemethods dbn deep belief network cnn convolutional neural network pcaprincipal component analysisthe expression oftissues wasobtained from noncodev5 zhao we only usedhuman datalncrnas in diï¬erentthe performance of deep beliefnetwork“convolutional neural networkwe did 10cross validation on each cancer area under the curveauc cheng dao zhang and areaunder the precision“recall curve aupr were used to evaluatethe performance of dbn“cnn the results are shown in table as we can see in table the performance of dbn“cnn isquite diï¬erent in diï¬erent cancers this may be caused by thediï¬erent sample sizes the average auc is and aupr is comparison experimentsto verify the superior of dbn“cnn we compared it with similarmethods since the main function of dbn is to reduce dimensionprincipal component analysis pca has the same functiontherefore instead of using dbn to encode we used pca thistime and cnn was used to classify the features after pca we callthis method pca“cnn in addition we also used the deep neuralnetwork dnn to replace cnn so this comparison method wascalled dbn“dnnwe used these three methods to test on cancers andsummarized the results to get a final auc and aupr for eachmethod the receiver operating characteristic roc curves areshown in figure as shown in figure the blue curve denotes the results ofdbn“cnn the red and black curves denote pca“cnn anddbn“dnn respectively as we can see dbn“cnn performedbest among these three methods the auc of dbn“cnn is which is better than and for pca“cnn anddbn“dnn respectivelyfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnasas shown in figure the aupr of dbn“cnn is the highestwith the least standard errorcase studyliu found down syndrome cell adhesion molecule antisense rna dscamas1 is associated with breast cancerby constructing two suppression subtracted cdna librariesmartensuzunova reported the associationbetween h19 and bladder cancer they also pointed out that h19could be the biomarker of bladder cancershi measured the expression level of lncrnasloc554202 in breast cancer tissues and found that loc554202was significantly increased compared with normal control andassociated with advanced pathologic stage and tumor sizeconclusionsincreasing evidence has shown the relationship between lncrnasand cancers lncrnas could be the biomarkers to help diagnosecancer and also help researchers understand the mechanismof cancers compared with people™s knowledge of diseaserelated protein coding genes we knew few about diseaserelated lncrnas however the biological experiments for findingdiseaserelated lncrnas are timeconsuming and expensivetherefore in this paper we proposed a novel method foridentifying cancerrelated lncrnas we called this methodœdbn“cnn which is a fusion of dbn and cnn two kindsof features were used based on the biological background sincelncrnas have tissuespecific expression and the expression ofcancer tissues is diï¬erent from normal tissues the expressionoftissues could provide importantin diï¬erentlncrnasreferencesavgeris m tsilimantou a levis p k tokas t sideris d c stravodimosk loss of gas5 tumour suppressor lncrna an independentmolecular cancer biomarker for shortterm relapse and progression in bladdercancer patients br j cancer “ 101038s4141601803206bai y dai x ye t zhang p yan x gong x plncrnadba repository of plant lncrnas and lncrnarbp protein interactions currbioinform “ bao z yang z huang z zhou y cui q and dong d lncrnadisease an updated database of long noncoding rnaassociateddiseases nucleic acids res d1034“d1037 101093nargky905canzio d nwakeze c l horta a rajkumar s m coï¬ey e l duï¬y ee antisense lncrna transcription mediates dna demethylationto drive stochastic protocadherin α promoter choice cell “653e15 101016jcell201903008chen x shi w and deng l prediction of disease comorbidity usinghetesim scores based on multiple heterogeneous networks curr gene ther “ cheng l computational and biological methods for gene therapy currgene ther cheng l hu y sun j zhou m and jiang q 2018a dincrna afor exploring disease“comprehensive webbased bioinformaticsassociations 101093bioinformaticsbty002ncrna functionbioinformaticstoolkitandcheng l jiang y ju h sun j peng j zhou m 2018busingcrossontologyinfacrontsimilaritiescalculatingtermtheirexecutelncrnasinformation for us to identify cancerrelated lncrnas inadditionregulation function byinteracting with their target genes therefore the target genesof lncrnas can also be the features of lncrnas to encode thefeatures dbn was used to reduce the dimension finally cnnwas used to identify real cancerrelated lncrnas based on thefinal featureto verify the eï¬ectiveness of our method we compareddbn“cnn with pca“cnn and dbn“dnn since pca canalso reduce the dimension of features and dnn can also doclassification the results showed that dbn“cnn performedbest finally case studies have been done to verify the accuracy ofour results we found potential lncrnas for kinds of cancerswhich can be a kind of guidance for researchers finding novelcancerrelated lncrnasdata availability statementthe datasets presented in this study can be found in onlinerepositoryrepositoriesrepositories theandnumbersbethesupplementary materialaccessionnamesfoundcantheofinauthor contributionsnd and gx designed the research zl performed the researchand wrote the manuscript yz and xh acquired the dataand reviewed and edited the manuscript cl xy and jganalyzed the data all authors reviewed the manuscript andprovided commentsinformation flow by a random walk bmc genomics 19suppl 101186s1286401743386cheng l yang h zhao h pei x shi h sun j 2019a metsigdisa manually curated resource for the metabolic signatures of diseases briefbioinform “ 101093bibbbx103cheng l zhao h wang p zhou w luo m li t 2019bcomputational methods for identifying similar diseases molecular therapynucleic acids “ 101016jomtn201909019dao f y lv h zulfiqar h yang h su w gao h acomputational platform to identify origins of replication sites in eukaryotesbrief bioinform 101093bibbbaa017 [epub ahead of print]deng l li w and zhang j ldah2v exploring metapaths acrossmultiple networks for lncrnadisease association prediction ieeeacmtransac comput biol bioinform 101109tcbb20192946257 [epubahead of print]gu c liao b li x cai l li z li k global network randomwalk for predicting potential human lncrnadisease associations sci rep 101038s4159801712763zjij tangj xia kjandjiang rtumorigenesis microenvironment currbioinformlncrna in“jiang q wang j wu x ma r zhang t jin s lncrna2targeta database for diï¬erentially expressed genes after lncrna knockdown oroverexpression nucleic acids res d193“d196 101093nargku1173karner h webb ch carmona s liu y lin b erhard m functional conservation of lncrna jpx despite sequence and structuraldivergence j mol biol “ 101016jjmb201909002frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnaskuang l zhao h wang l xuan z and pei t a novel approachbased on point cut set to predict associations of diseases and lncrnas currbioinform “ lan w li m zhao k liu j wu fx pan y ldap a webserver for lncrnadisease association prediction bioinformatics “ 101093bioinformaticsbtw639liang c changlu q he z tongze f and xue z gutmdisorder acomprehensive database for dysbiosis of the gut microbiota in disorders andinterventions nucleic acids res liu d rudland p sibson d and barraclough r identification ofmrnas diï¬erentiallyexpressed between benign and malignant breast tumourcells br j cancer “ 101038sjbjc6600456liu x hong z liu j lin y alfonso rp zou q computational methods for identifying the critical nodes in biologicalnetworks brief bioinform “ 101093bibbbz011lu c yang m li m li y wu f and wang j predicting humanlncrnadisease associations based on geometric matrix completion ieee jbiomed health inform 101109jbhi20192958389 [epub ahead of print] protein function predictionto deep learning proteomics 19e1900119lv z b ao c y and zou qfrom traditionalclassifier 101002pmic201900119martensuzunova e s böttcher r croce c m jenster g visakorpi t andcalin g a long noncoding rna in prostate bladder and kidneycancer eur urol “ 101016jeururo201312003peng j and zhao t reduction in tom1 expression exacerbatesalzheimer™s disease proc natl acad sci usa “ 101073pnas1917589117robinson e k covarrubias s and carpenter s the how and why oflncrna function an innate immune perspective biochim biophys acta generegul mech 101016jbbagrm2019194419sasaki y t sano mideue t kin t asai k and hirose t identification and characterization of human noncoding rnas withtissuespecific expression biochem biophys res commun “ 101016jbbrc200704034sumathipala m maiorino e weiss s t and sharma ashi y lu j zhou j tan x he y ding j long noncodingrna loc554202 regulates proliferation and migration in breast cancer cellsbiochem biophys res commun “ 101016jbbrc201402144network diï¬usion approach to predictlncrna disease associationsusing multitype biological networks lion front physiol 103389fphys201900888wang l xuan z zhou s kuang l and pei t a novel modelassociations based on the lncrna“for predicting lncrnadiseasemirnadisease interactive network curr bioinformwang y yu g wang j fu g guo m and domeniconi c weightedmatrix factorization on multirelational data for lncrnadisease associationprediction methods “ 101016jymeth201906015wei l su r wang b li x zou q and gao x integrationof deep feature representations and handcrafted featuresto improvethe prediction of n 6methyladenosine sites neurocomputing “ 101016jneucom201804082wu b zhang h lin l wang h gao y zhao l a similarity searching system for biological phenotype images using deepconvolutional encoderdecoder architecture curr bioinform “ xie g huang s luo y ma l lin z and sun y llclplda a novelmodel for predicting lncrna“disease associations mol genet genomics “ 101007s00438019015908xuan p cao y zhang t kong r and zhang z2019a dualconvolutional neural networks with attention mechanisms based methodfor predicting diseaserelated lncrna genes front genet 103389fgene201900416xuan p pan s zhang t liu y and sun h 2019b graph convolutionalnetwork and convolutional neural network based method for predictinglncrnadisease associations cells 103390cells8091012yu g fu g lu c ren y and wang j brwlda birandomwalks for predicting lncrnadisease associations oncotarget “ 1018632oncotarget19588yu j xuan z feng x zou q and wang l a novel collaborativefiltering model for lncrnadisease association prediction based on the naïvebayesian classifier bmc bioinform 101186s1285901929850zeng x x wang w deng g s bing j x and zou q prediction ofpotential diseaseassociated micrornas by using neural networks mol thernucleic acids “ 101016jomtn201904010zhangand deng lj zhang z chen zintegratinglncrnadisease associationieeeacm transac comput biol bioinform “multiple heterogeneous networks for novelinference 101109tcbb20172701379zhang t tan p wang l jin n li y zhang l rnalocate aresource for rna subcellular localizations nucleic acids res d135“d138 101093nargkw728zhang z m tan j x wang f dao f y zhang z y and linh early diagnosis of hepatocellular carcinoma using machinelearning method front bioeng biotechnol 103389fbioe2020zhao t cheng l zang t and hu y 2019a peptidemajor histocompatibilitycomplex class i binding prediction based on deep learning with novel featurefront genet 103389fgene201901191and cheng lidentifyingalzheimer™s diseaserelated proteins by lrrgd bmc bioinform 101186s1285901931247zhao t hu y zang t2019bzhao t hu y zang t and cheng l mrtfb regulates the expressionof nomo1 in colon proc natl acad sci usa 101073pnas2000499117zhao t hu y zang t and wang y 2019c integrate gwas eqtland mqtl data to identify alzheimer™s diseaserelated genes front genet 103389fgene201901021zhao t wang d hu y zhang n zang t and wang y 2019d identifyingalzheimer™s diseaserelated mirna based on semiclustering curr gene ther “ zhao y li h fang s kang y wu w hao y noncode an informative and valuable data source of long noncoding rnas nucleicacids res d203“d208 101093nargkv1252zou q xing p wei l and liu b gene2vec gene subsequenceembedding for prediction of mammalian n6methyladenosine sites frommrna rna “ 101261rna069112118conflict of interest the authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestcopyright liu zhang han li yang gao xie and du this is an openaccess distributed under the terms of the creative commons attribution license ccby the use distribution or reproduction in other forums is permitted providedthe original authors and the copyright owners are credited and that the originalpublication in this is cited in accordance with accepted academic practiceno use distribution or reproduction is permitted which does not comply with thesetermsfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0c'
Colon_Cancer
biochemically interleukin6 belongs to the class of fourhelical cytokines the cytokine can be synthesised and secreted by many cells it acts via a cell surfaceexpressed interleukin6 receptor which is not signalling competent this receptor when complexed with interleukin6 associates with the signalling receptor glycoprotein kda gp130 which becomes dimerised and initiates intracellular signalling via the janus kinasesignal transducer and activator of transcription and rat sarcoma proto oncogenemitogenactivated protein kinasephosphoinositide3 kinase pathways physiologically interleukin6 is involved in the regulation of haematopoiesis and the coordination of the innate and acquired immune systems additionally interleukin6 plays an important role in the regulation of metabolism in neural development and survival and in the development and maintenance of various cancers although interleukin6 is mostly regarded as a proinflammatory cytokine there are numerous examples of protective and regenerative functions of this cytokine this review will explain the molecular mechanisms of the in part opposing activities of the cytokine interleukin6keywords gp130 sgp130fc il6 il6r sil6r transsignalling adam17invited reviewersversion aug faculty reviews are review s written by the prestigious members of faculty opinions the s are commissioned and peer reviewed before publication to ensure that the final published version is comprehensive and accessible the reviewers who approved the final version are listed with their names and affiliations elke roeb justus liebig university giessen germany hana alg¼l technical university of munich munich germany jacqueline bromberg department of medicine memorial sloan kettering cancer center new york usaany comments on the can be found at the end of the a0page of 0cf1000research 9faculty rev1013 last updated aug corresponding author stefan rosejohn rosejohnbiochemunikieldeauthor roles rosejohn s conceptualization data curation formal analysis funding acquisition investigation methodology project administration resources supervision writing “ original draft preparation writing “ review editingcompeting interests stefan rosejohn has acted as a consultant and speaker for abbvie amgen janssen chugai roche genentech roche pfizer eli lilly and sanofi he also declares that he is an inventor on patents owned by conaris research institute which develops the sgp130fc protein olamkicept and he has stock ownership in conarisgrant information the work of stefan rosejohn has been supported by grants of the deutsche forschungsgemeinschaft bonn germany under the grant numbers crc841 project c1 and crc877 project a1 and by the german excellence cluster inflammation at interfaces the funders had no role in study design data collection and analysis decision to publish or preparation of the manuscriptcopyright rosejohn s this is an open access distributed under the terms of the creative commons attribution license which permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedhow to cite this rosejohn s interleukin6 signalling in health and disease [version peer review approved] f1000research 9faculty rev1013 1012688f1000research260581first published aug 9faculty rev1013 1012688f1000research260581 a0page of 0cintroductioninterleukin6 il6 is considered one of the most prominent proinflammatory cytokines1 blockade of il6 by the neutralising monoclonal antibody tocilizumab has been approved in more than countries for the treatment of patients with autoimmune disorders such as rheumatoid arthritis2 additionally the cytokine storm sometimes encountered when cancer patients are treated with chimeric antigen receptor car tcells3 could be effectively treated with the antibody tocilizumab leading to us food and drug administration fda approval of the drug for this condition even more recently it has been recognised that many patients experience a similar cytokine storm upon infection with sarscov2 covid19 virus4 and that these patients could also be treated with tocilizumab5 these new data led to a rekindled general interest in the cytokine il6il6 was initially discovered and cloned in the kishimoto laboratory as a bcell stimulatory factor6 immediately after the molecular cloning it was evident that il6 was identical to hepatocyte stimulating factor7 hybridomaplasmacytoma growth factor8 interferon 29 and kda protein10 this already indicated the pleiotropic nature of the cytokine later on it was also recognised that il6 shows profound activities in the brain1112 in the regulation of metabolism1314 in the response of the body to exercise15 and in the development and maintenance of various cancers16this review gives a short overview of the complex biology of il6 and explains how one cytokine can have extremely different biologic effects on different cells and in different physiologic states of the human body17the interleukin6 receptor complexthe fourhelical cytokine il6 figure on cells binds to a membranebound il6 receptor il6r and the complex of il6 and il6r associates with a second receptor protein glycoprotein kda gp130 which dimerises and initiates intracellular signalling via the janus kinase jaksignal transducer and activator of transcription stat and rat sarcoma proto oncogene rasmitogenactivated protein kinase and phosphoinositide3 kinase pathways figure importantly il6 exhibits only a measurable affinity to the il6r but not to gp130 and the il6r does not bind on its own to gp130 it is only the complex of il6 and il6r that binds to gp130 and induces its dimerisation figure all cells in the body express gp130 but only a few cells such as hepatocytes and some leukocytes express il6r it follows that cells that express only gp130 but not il6r cannot be stimulated by il61noteworthy gp130 is a component of the receptor complexes of the socalled gp130 cytokine family which besides il6 comprises il11 ciliary neurotrophic factor cntf cardiotrophin1 ct1 cardiotrophinlike cytokine clc leukaemia inhibitory factor lif oncostatin m osm and il27 for details please refer to recent reviews1920it has however been noticed that the membranebound il6r can be cleaved by the membranebound metalloprotease a f1000research 9faculty rev1013 last updated aug figure fourhelical topology of the interleukin6 il6 protein il6 belongs to the family of fourhelical cytokines the figure shows the four helices with the connecting loops the a“b and the c“d loops are long enough to reach the length of a helix whereas the b“c loop is short consequently il6 has an upupdowndown topology meaning that helices a and b point upwards whereas helices c and d point downwards this topology is common to most cytokines such as il2 il4 il7 il11 il15 leukaemia inhibitory factor oncostatin m growth hormone leptin and many othersdisintegrin and metalloprotease adam17 to generate a soluble il6r sil6r21 to a minor extent the human”but not the murine”sil6r can be generated by translation from a differentially spliced mrna22 intriguingly the sil6r can still bind il6 and the complex of il6 and sil6r can associate with gp130 and induce signalling even on cells that lack the membranebound il6r23 this process has been named il6 transsignalling figure strikingly following this paradigm il6 can in the presence of sil6r stimulate any cell in the body since all cells express gp13017interestingly most il6rexpressing cells including hepatocytes express far more gp130 than il6r molecules therefore stimulation of such cells with il6 alone will only lead to engagement of few gp130 molecules whereas stimulation with the complex of il6 and sil6r will stimulate all cellular gp130 proteins a threshold for a given response might not be reached with il6 stimulation but only with stimulation of all gp130 molecules via il6 transsignalling this might be an explanation for the observed differences in signalling between transsignalling and classical signalling that lead to different phenotypes25molecular tools to elucidate the functions of interleukin6the concept of transsignalling has been corroborated by the use of two designer proteins the first such protein consists of il6 covalently fused to the sil6r via a flexible peptide linker which allowed the placement of il6 il6 page of 0cf1000research 9faculty rev1013 last updated aug figure stimulation of target cells by interleukin6 il6 il6 orange first binds to the il6 receptor il6r red the complex of il6 and il6r associates with glycoprotein kda gp130 blue which dimerises and leads to intracellular signalling it is important to note that il6 and il6r alone exhibit no measurable affinity to gp130 only the complex of il6 and il6r binds to and activates gp130 therefore il6 cannot stimulate cells that do not express il6r signalling occurs via the signal transducer and activator of transcription stat 1stat3 yamaguchi sarcoma viral oncogene homolog yesyesassociated protein yap phosphoinositide3 kinase pi3kakt and rat sarcoma proto oncogene rasmitogenactivated protein kinase mapk pathways jak janus kinaseat the correct distance to reach the il6 binding site of the sil6r this protein was called hyperil6 figure 3a26 this protein was shown to stimulate gp130expressing cells in vitro and in vivo and it was shown that liver regeneration27 stimulation of neural cells28 and expansion of hematopoietic cells29 was far more efficient in the presence of hyperil6 as compared to il6 alone30turned out while hyperil6 demonstrated only the biologic potential of il6 transsignalling these experiments did not prove that this process occurred in vivo a second soluble protein was designed which consisted of the entire extracellular portion of gp130 covalently fused to the fc region of human igg1 figure 3b the resulting protein named soluble gp130fc sgp130fc to exhibit similar properties as membranebound gp130 it did not bind il6 or il6r alone but it bound with high affinity the complex of il6 and sil6r3132 consequently the sgp130 protein in vitro and in vivo specifically inhibited il6 transsignalling without compromising il6 signalling via the membranebound il6r ie classic signalling32 the sgp130fc protein could be used to define il6mediated biologic responses which were dependent on classic or transsignalling this was accomplished by comparing the treatment of animals with sgp130fc or with neutralising antibodies against il6 or il6r which blocked all il6 signalling figure 3c d using animal models of human inflammatory diseases or inflammationassociated cancer it turned out inflammationassociated cancers were mainly driven by il6 transsignalling whereas regenerative and protective activities of il6 were mediated by classic il6 signalling via the membranebound il6r figure that autoimmune disorders and levels in physiologic and pathophysiologic functions of interleukin6under homeostatic conditions il6 the circulation are as low as “ pgml but during inflammatory states these levels can rise more than 1000fold and under extreme conditions leading to sepsis il6 levels in the µgml range have been reported33 il6 is produced by myeloid cells upon tolllike receptor stimulation together with the cytokines il1 and tumor necrosis factor α tnfα which via a feedforward loop lead to an immense amplification of il6 production during inflammatory conditions34 there is perhaps no other protein in the human body whose level can go up by six orders of magnitude this lets us conclude that il6 is the major alarm signal to infection inflammation and possibly cancer35the human body in response in however under normal conditions il6 plays an important role in ancellular homeostasis mice in which the il6 gene has been ablated il6 knockout mice become obese late in life13 cannot regenerate their liver upon hepatectomy36 and show no signs of osteoporosis upon ovariectomy37 indicating roles for il6 in body weight regulation liver physiology and bone metabolism in pathophysiologic states however there are marked differences between il6 knockout mice and wildtype mice il6 knockout mice are completely protected in animal models of rheumatoid arthritis38 and multiple sclerosis39 indicating a key role for il6 in these autoimmune disorderswith the help of the sgp130fc protein and of neutralising monoclonal antibodies it was possible to selectively block il6 transsignalling or to block all il6 signalling respectively page of 0cf1000research 9faculty rev1013 last updated aug tumour progression was induced by tumourinfiltrating myeloid cells which stimulated the neoplastic cells via il6 transsignalling47 selective blockade of this pathway by the sgp130fc protein blocked progression of pancreatic intraepithelial neoplasias to pancreatic ductal adenocarcinomas47 indicating a prominent role for il6 transsignalling in the development of pancreatic cancer in the murine apcmin model of colon cancer it was established that the genetic deletion of adam17 which is responsible for generating not only sil6r but also soluble tnfα and soluble ligands of the epidermal growth factor receptor egfr resulted in completely abrogated tumour development16 moreover the formation of neoplasias stimulated adam17 on macrophages leading to egfr ligand cleavage and subsequent egfr stimulation these macrophages now produced il6 and sil6r which led to the outgrowth of the tumours again selective blockade of the il6 transsignalling pathway by the sgp130fc protein blocked tumour development in the apcmin model and an additional mouse model of colon cancer16 this was highly reminiscent of a study in liver cancer in which it was shown that the egfr expressed in macrophages but not egfr in hepatocytes was involved in the development of hepatocellular carcinoma48 apparently macrophage activation may be an important step in the initiation and progression of tumours via the il6 transsignalling pathway20 figure therapeutic targeting of interleukin6 activitytherapeutic targeting of the proinflammatory cytokine tnfα was introduced as an efficient strategy to treat patients with autoimmune disorders such as rheumatoid arthritis and inflammatory bowel disease49 subsequently blockade of the biologic activity of the cytokine il6 was shown to be an efficient treatment for patients with rheumatoid arthritis and other autoimmune diseases2 and it was shown that blocking il6 activity was more efficient than blocking tnfα in a monotherapy trial50 blockade of il6 activity with the il6r neutralising monoclonal antibody tocilizumab was also highly effective in the treatment of patients with car t cellinduced severe cytokine release syndrome51 in patients with severe covid19 disease the administration of tocilizumab resulted in a marked improvement of the condition in the majority of patients the fever subsided creactive protein decreased and oxygen intake could be lowered no obvious adverse reactions were observed these preliminary data indicated that tocilizumab is a candidate for effective treatment of covid19 patients552 interestingly treatment of covid19 patients with the il6r neutralising monoclonal antibody sarilumab resulted in no significant difference in clinical improvement and mortality53summarythe discovery that the proinflammatory activities of il6 are mediated by il6 transsignalling whereas the protective and regenerative activities of il6 rely on classic signalling via the membranebound il6r suggested that the sgp130fc protein might be an ideal candidate for a more specific mode of cytokine blockade as opposed to global cytokine inhibition20 it was shown in appropriate animal models that blockade of il6 transsignalling was indeed superior to global il6 blockade in a bone healing model4445 in a sepsis model42 in abdominal page of figure designer proteins to probe for modes of interleukin il6 signalling a hyperil6 is a fusion protein between il6 and soluble il6 receptor sil6r b sgp130fc is a fusion protein of the extracellular portion of glycoprotein kda gp130 and the constant part of a human immunoglobulin g1 igg1 antibody c il6 can signal via the membranebound il6r classical signalling and via the sil6r transsignalling hyperil6 can be used to mimic il6 transsignalling b the sg130fc protein does not interfere with classical il6 signalling but it specifically blocks il6 transsignallingusing this approach it was shown that classic il6 signalling via the membranebound il6r was responsible for the defence of the body against bacteria4041 intestinal regeneration upon polymicrobial sepsis42 prevention of aortic rupture in animal models of abdominal aortic aneurysm43 and healing of bone fractures4445 important processes are severely compromised under blockade of global il6 activity46 it has been hypothesised that the same might apply for the treatment of covid19 patients46 figure indicating that these besides being the major alarm signal in the human body il6 plays a dominant role in various types of cancer one important reason could be that il6 via stimulation of the stat3 pathway is a prominent growth factor of many cancer cells the following scenario has been worked out in pancreatic cancer47 it was noted that in the krasg12d model the massive activation of the stat3 pathway which led to 0cf1000research 9faculty rev1013 last updated aug figure pro and antiinflammatory activities of interleukin6 il6 left antiinflammatory and protective activities of the cytokine il6 are associated with signalling via the membranebound il6 receptor il6r right proinflammatory activities of the cytokine il6 are associated with signalling via the soluble il6r sil6r the membranebound metalloprotease a disintegrin and metalloprotease adam17 orchestrates the pro and antiinflammatory activities of il6 treg regulatory t cellin patients with is presently ongoing aortic aneurysm models43 and in bacterial infection models4041 the sgp130fc protein was expressed and purified according to gmp regulations phase i clinical trials were successfully performed with healthy individuals and a phase ii clinical trial inflammatory bowel disease54 the future will tell whether this elegant therapeutic approach which was successfully tested in many animal models leads to a novel paradigm in cytokineblocking therapies in patients with autoimmune disorders46 similarly blockade of transsignalling while leaving classical signalling intact may prove to be beneficial for patients experiencing œcytokine storms from covid19 or car tcell therapies finally we suggest that malignancies promoted by high levels of transsignalling could be contained by this therapeutic modalityinterleukin6 il6r abbreviationsadam17 a disintegrin and metalloprotease egfr epidermal growth factor receptor gp130 glycoprotein kda il6 interleukin6 receptor ras rat sarcoma proto oncogene sgp130fc soluble gp130fc fusion protein which under the name of olamkicept is in phase ii clinical trials sil6r soluble il6r stat signal transducer and activator of transcription tnfα tumor necrosis factor α yap yesassociated protein yes yamaguchi sarcoma viral oncogene homologacknowledgementsi thank all past and current colleagues of our laboratory for many helpful discussionsreferencesfaculty opinions recommended kishimoto t interleukin6 from basic science to medicine40 years in immunology annu rev immunol “ pubmed abstract publisher full text tanaka t narazaki m ogata a et al a new era for the treatment of inflammatory autoimmune diseases by interleukin6 blockade strategy semin immunol “ pubmed abstract publisher full text faculty opinions recommendation teachey dt lacey sf shaw pa et al identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor tcell therapy for acute lymphoblastic leukemia cancer discov “ pubmed abstract publisher full text free full text page of 0c moore jb june ch cytokine release syndrome in severe covid19 science “ pubmed abstract publisher full text faculty opinions recommendation xu x han m li t et al effective treatment of severe covid19 patients with tocilizumab proc natl acad sci u s a “ pubmed abstract publisher full text free full text faculty opinions recommendation hirano t taga t yamasaki k et al molecular cloning of the cdnas for interleukin6b cell stimulatory factor and its receptor ann n y acad sci discussion pubmed abstract publisher full text gauldie j richards c harnish d et al interferon beta 2bcell stimulatory factor type shares identity with monocytederived hepatocytestimulating factor and regulates the major acute phase protein response in liver cells proc natl acad sci u s a “ pubmed abstract publisher full text free full text brakenhoff jp de groot er evers rf et al molecular cloning and expression of hybridoma growth factor in escherichia coli j immunol “ pubmed abstract zilberstein a ruggieri r korn jh et al structure and expression of cdna and genes for human interferonbeta2 a distinct species inducible by growthstimulatory cytokines embo j “ pubmed abstract free full text haegeman g content j volckaert g et al structural analysis of the sequence coding for an inducible 26kda protein in human fibroblasts eur j biochem “ pubmed abstract publisher full text rothaug m beckerpauly c rosejohn s the role of interleukin6 signaling in nervous tissue biochim biophys acta pt a “ pubmed abstract publisher full text willis ef macdonald kpa nguyen qh et al repopulating microglia promote brain repair in an il6dependent manner cell 833846e16 pubmed abstract publisher full text wallenius v wallenius k ahr©n b et al interleukin6deficient mice develop matureonset obesity nat med “ pubmed abstract publisher full text findeisen m allen tl henstridge dc et al treatment of type diabetes with the designer cytokine ic7fc nature “ pubmed abstract publisher full text faculty opinions recommendation pedersen bk febbraio ma muscles exercise and obesity skeletal muscle as a secretory an nat rev endocrinol “ pubmed abstract publisher full text faculty opinions recommendation schmidt s schumacher n schwarz j et al adam17 is required for egfrinduced intestinal tumors via il6 transsignaling j exp med “ pubmed abstract publisher full text free full text rosejohn s the biology of interleukin6 in the 21st century semin immunol pubmed abstract publisher full text schaper f rosejohn s interleukin6 biology signaling and strategies of blockade cytokine growth factor rev “ pubmed abstract publisher full text jones sa jenkins bj recent insights into targeting the il6 cytokine family in inflammatory diseases and cancer nat rev immunol “ pubmed abstract publisher full text faculty opinions recommendation garbers c heink s korn t et al interleukin6 designing specific therapeutics for a complex cytokine nat rev drug discov “ pubmed abstract publisher full text m¼llberg j schooltink h stoyan t et al the soluble interleukin6 receptor is generated by shedding eur j immunol “ pubmed abstract publisher full text lust ja donovan ka kline mp et al isolation of an mrna encoding a soluble form of the human interleukin6 receptor cytokine “ pubmed abstract publisher full text mackiewicz a schooltink h heinrich pc et al complex of soluble human il6receptoril6 upregulates expression of acutephase proteins j immunol “ pubmed abstract rosejohn s heinrich pc soluble receptors for cytokines and growth factors generation and biological function biochem j 300pt “ pubmed abstract publisher full text free full text rosejohn s the soluble interleukin receptor advanced therapeutic options in inflammation clin pharmacol ther “ pubmed abstract publisher full text fischer m goldschmitt j peschel c et al i a bioactive designer cytokine for human hematopoietic progenitor cell expansion nat biotechnol f1000research 9faculty rev1013 last updated aug “ pubmed abstract publisher full text galun e zeira e pappo o et al liver regeneration induced by a designer human il6sil6r fusion protein reverses severe hepatocellular injury faseb j “ pubmed abstract publisher full text m¤rz p otten u rosejohn s neural activities of il6type cytokines often depend on soluble cytokine receptors eur j neurosci “ pubmed abstract publisher full text audet j miller cl rosejohn s et al distinct role of gp130 activation in promoting selfrenewal divisions by mitogenically stimulated murine hematopoietic stem cells proc natl acad sci u s a “ pubmed abstract publisher full text free full text rosejohn s interleukin6 family cytokines cold spring harb perspect biol a028415 pubmed abstract publisher full text free full text horsten u schmitzvan de leur h m¼llberg j et al the membrane distal half of gp130 is responsible for the formation of a ternary complex with il6 and the il6 receptor febs lett “ pubmed abstract publisher full text jostock t m¼llberg j ozbek s et al soluble gp130 is the natural inhibitor of soluble interleukin6 receptor transsignaling responses eur j biochem “ pubmed abstract publisher full text waage a brandtzaeg p halstensen a et al the complex pattern of cytokines in serum from patients with meningococcal septic shock association between interleukin interleukin and fatal outcome j exp med “ pubmed abstract publisher full text free full text tanaka t narazaki m kishimoto t il6 in inflammation immunity and disease cold spring harb perspect biol a016295 pubmed abstract publisher full text free full text faculty opinions recommendation rosejohn s il6 transsignaling via the soluble il6 receptor importance for the proinflammatory activities of il6 int j biol sci “ pubmed abstract publisher full text free full text cressman de greenbaum le deangelis ra et al liver failure and defective hepatocyte regeneration in interleukin6deficient mice science “ pubmed abstract publisher full text poli v balena r fattori e et al interleukin6 deficient mice are protected from bone loss caused by estrogen depletion embo j “ pubmed abstract publisher full text free full text alonzi t fattori e lazzaro d et al interleukin is required for the development of collageninduced arthritis j exp med “ pubmed abstract publisher full text free full text okuda y sakoda s bernard cc et al il6deficient mice are resistant to the induction of experimental autoimmune encephalomyelitis provoked by myelin oligodendrocyte glycoprotein int immunol “ pubmed abstract publisher full text hoge j yan i j¤nner n et al il6 controls the innate immune response against listeria monocytogenes via classical il6 signaling j immunol “ pubmed abstract publisher full text sodenkamp j waetzig gh scheller j et al therapeutic targeting of interleukin6 transsignaling does not affect the outcome of experimental tuberculosis immunobiology “ pubmed abstract publisher full text barkhausen t tschernig t rosenstiel p et al selective blockade of interleukin6 transsignaling improves survival in a murine polymicrobial sepsis model crit care med “ pubmed abstract publisher full text paige e cl©ment m lareyre f et al interleukin6 receptor signaling and abdominal aortic aneurysm growth rates circ genom precis med e002413 pubmed abstract publisher full text free full text kaiser k prystaz k vikman a et al pharmacological inhibition of il6 transsignaling improves compromised fracture healing after severe trauma naunyn schmiedebergs arch pharmacol “ pubmed abstract publisher full text free full text prystaz k kaiser k kovtun a et al distinct effects of il6 classic and transsignaling in bone fracture healing am j pathol “ pubmed abstract publisher full text magro g sarscov2 and covid19 is interleukin6 il6 the ˜culprit lesion™ of ards onset what is there besides tocilizumab sgp130fc cytokine x pubmed abstract publisher full text free full text faculty opinions recommendation lesina m kurkowski mu ludes k et al stat3socs3 activation by il6 transsignaling promotes progression of pancreatic intraepithelial page of 0cf1000research 9faculty rev1013 last updated aug neoplasia and development of pancreatic cancer cancer cell “ pubmed abstract publisher full text faculty opinions recommendation lanaya h natarajan a komposch k et al egfr has a tumourpromoting role in liver macrophages during hepatocellular carcinoma formation nat cell biol “ pubmed abstract publisher full text free full text faculty opinions recommendation sacre sm andreakos e taylor p et al molecular therapeutic targets in rheumatoid arthritis expert rev mol med “ pubmed abstract publisher full text gabay c emery p van vollenhoven r et al tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis adacta a randomised doubleblind controlled phase trial lancet “ pubmed abstract publisher full text faculty opinions recommendation le rq li l yuan w et al fda approval summary tocilizumab for treatment of chimeric antigen receptor t cellinduced severe or lifethreatening cytokine release syndrome oncologist “ pubmed abstract publisher full text free full text faculty opinions recommendation campochiaro c dellatorre e cavalli g et al efficacy and safety of tocilizumab in severe covid19 patients a singlecentre retrospective cohort study eur j intern med “ pubmed abstract publisher full text free full text faculty opinions recommendation dellatorre e campochiaro c cavalli g et al interleukin6 blockade with sarilumab in severe covid19 pneumonia with systemic hyperinflammation an openlabel cohort study ann rheum dis pubmed abstract publisher full text faculty opinions recommendation safety and efficacy of tj301 iv in participants with active ulcerative colitis clinicaltrials reference sourcepage of 0cf1000research 9faculty rev1013 last updated aug open peer reviewcurrent peer review status editorial note on the review processfaculty reviews are review s written by the prestigious members of faculty opinions the s are commissioned and peer reviewed before publication to ensure that the final published version is comprehensive and accessible the reviewers who approved the final version are listed with their names and affiliationsthe reviewers who approved this areversion jacqueline bromberg department of medicine memorial sloan kettering cancer center new york ny usa competing interests no competing interests were disclosedhana alg¼l comprehensive cancer center munich university hospital klinikum rechts der isar mildredscheelchair of tumor metabolism technical university of munich munich
Colon_Cancer
ulcerative colitis is a type of ‚ammatory bowel disease thatcan potentially lead to cancer e age of onset of colitis istypically “ years old and it can seriously threaten thequality of life of patients e immunopathogenesis andimmunosuppressive treatment of colitis are currently theresearch topics of significant interest e research goals areto diagnose and treat colitis in order to prevent exacerbationof the disease e drugs used to treat colitis in the clinicoften have adverse eï¬ects after their longterm applicationanother crucial area of colitis research is focused on thediscovery of functional foods that can prevent colitis withoutside eï¬ects natural plants including aegle marmeloslinn also have the intervention eï¬ect on colitis a recentstudy has shown that the intestinal flora is closely related tocolitis and that the intestinal flora participates in the mucosalimmune response bacteria are an important promoter of‚ammatory bowel disease e symptoms of colitis can bealleviated by regulating the intestinal flora preventing floralimbalance and increasing the number of probiotics yak yoghurt is a natural fermented food that is rich innutrients and is common in the minority areas of theqinghai tibet plateau previous research has suggested thatyak yoghurt exerts various physiological activities such asantioxidationimmunitycholesterolreductionand 0cevidencebased complementary and alternative medicineenhancement e qinghai tibet plateau has a specificclimate and unique environment for the fermentation of yakyoghurt additionally the availability of yak milk and specialtibetan fermentation utensils eg certain fermentationmicroanisms can make the flavor and quality of yakyoghurt diï¬er greatly from ordinary fermented milk aprior study on the intestinal physiological activity of lacticacid bacterial species in yak yoghurt showed that the lacticacidproducing bacteria isolated from yak yoghurt hadantioxidant and constipation preventing eï¬ects in this studythe potential eï¬ects of lactobacillusplantarum ys4 lpys4 on oxazolidoneinduced colitiswere investigated for the first time e findings provide apossible foundation for further development of lpys4especially its application in functional food or medicineratio � solution massnormal group were daub treated with ml of ethanolwith those in the remaining four groups being daub treated with ml of oxazolidonesolvent � after treatment for days the mice wereanesthetized en the blunt head of a silicone tube wasinserted into the intestinal tract from the anus of the mouse at adepth of cm e mice in normal group were administeredwith ml of ethanol solution while those in theremaining four groups were administered with ml of oxazolidone solution mass ratio � solvent � ethanoltwenty seconds later the catheters were removed and the micewere lifted up by their tails for half a minute on the last dayof treatment day all the mice were sacrificed by decapitation and their plasma samples and colon tissues were collected e length and weight of the colon were documentedexperiment animal materials materials and methodsand reagentsexperimental strain the strain was isolated from yak yoghurt in the yushu area of qinghai province china by ourteam it was named lpys4 and stored in china center fortype culture collection cctcc wuhan china nom2016750 e negative control strain lb was purchasedfrom the cctcc no ab fifty male balbc mice weeks old were purchasedfrom the experimental animal center of chongqingmedical university certificate no syxk yu “oxazolone was purchased from sigmaaldrich co llcusa il2 il10 et1 sp ss and vip serum cytokine kitswere purchased from biolegend inc usa gsh sodmpo and mda kits were purchased from nanjing jiancheng bioengineering institute nanjing china trizol reagent oligodt18 rnase dntp mlv primer bca proteinquantitative kit aps temed sdspage pvdf membrane first antibody and second antibody were purchasedfrom ermo fisher scientific inc usa instruments and equipment imark microplate readerwas purchased from biorad usa steponeplus pcrinstrument was purchased from ermo fisher scientificinc usa tanon chemiluminescence imager waspurchased from tanon science and technology co ltdchina sas v91 statistical software package was purchasedfrom sas institute inc usalb animal grouping and intervention a total of balbcmale mice were assigned to five groups model groupnormal group lactobacillus bulgaricustreatmentgroup and highdose lpys4h and lowdoses lpys4l treatment groups and there were mice in each groupe mice in lb lpys4h and lpys4l groups were fedwith — — and — cfukg ml livingbacteria physiological saline of each corresponding straindaily by oral gavage for consecutive days and normal andmodel groups were fed with ml physiological salineafter days of treatment the abdomens of all mice wereshaved with an area of cm — cm e mouse abdomens in detection of endothelin1 et1 substance p spsomatostatin ss and vasoactive intestinal peptide vipconcentrations in serum samples e whole blood samplesof mice were allowed to clot at room temperature for h andthen centrifuged at rpmmin for min after collecting the serum samples the concentrations of et1 sssp and vip were detected using commercial kits determination of interleukin2 il2 and interleukin10il10 levels in serum samples e mouse serum sampleswere prepared according to section en the serumlevels of il2 and il10 cytokines were assessed usingcommercial kits determination of glutathione gsh malondialdehydemda myeloperoxidase mpo and superoxide dismutasesod activities in colon tissues a mixture of g colontissue and ml normal saline was prepared at weightratio after homogenizing the mixture the activities of gshmda mpo and sod in colon tissues were evaluated usingcommercial kits pathological observation of he staining e lesionsite — — cm of colon was cut with a scalpel etrimmed tissue and corresponding label were placed in neutral formalin solution for h e colon tissue wasdehydrated embedded sliced dewaxed stained and thendehydrated transparent and sealed finally the pathologicalstate of colon tissue was observed under a microscopebx43 olympus tokyo japan qpcr assay total rna was isolated using rnazol andthen diluted to the final concentration of μgμl for cnasynthesis μl of the diluted rna extract was taken andcdna was prepared using a reverse transcriptase kit enthe cdna template μl was added into μl of sybrgreen pcr master mix and μl of forward primer andreverse primer each table qpcr amplification wascarried out for cycles under the reaction conditions of95oc s °c s °c s and °c s followed by 0cevidencebased complementary and alternative medicineckitinosenosnnossequencegene nametable sequences of primers used in this studyforward ²agagagatcgggttcaca3²reverse ²cacagaactgagggtaca3²forward ²tcgtccaacttctgggctctt3²reverse ²ccttctcttcctcccctctcttc3²forward ²tcagccatcacagtgttccc3²reverse ²atagcccgcatagcgtatcag3²forward ²catagcccaggtaaagcacaat3²reverse ²gaacactccagaatcgtcaactcforward ²tcagggactacgctgcgaaag3²reverse ²aagagctggcagaccgactca3²forward ²tgcaccaccaactgcttag3²reverse ²gatgcagggatgatgttc3²Î´ctdetection gene δctgapdh measured according to the following equation ˆ’δct �cycle under the reaction conditions of °c s and°c s gapdh was used as internal control for thisdetermination and the relative mrna expression was²gapdhscf western blotting mg of tissue samples was mixedwith μl of pmsf and ml of ripa and then homogenized at rmin 4oc for min protein quantificationwas conducted using the bca protein quantitative kit andthe protein samples were diluted to μgml en thediluted protein and sample buï¬er were mixed at heatedat 100oc for min and icebathed for min subsequentlyacrylamide starting buï¬er resolving buï¬er temed aps and diï¬erentiated water were mixed in specific proportions in order to prepare sdspage separation glue andconcentration glue e prestained samples and proteinladder were placed into the sample hole of the rubber sheetrespectively and then the proteincontaining sdspageglue was subjected to vertical gel electrophoresis for minafter activation with methanol for min the pvdf wereblocked with skimmed milk in — tbst solution for hen the blocked pvdf membranes were rinsed with — tbst followed by incubation with the primary antibodyat °c for h after washing with — tbst for times thesecondary antibody was incubated at °c for h lastly theprotein bands were visualized by supersignal west picoplus chemiluminescent substrate and the images werecaptured using a chemiluminescence imager multiple comparisons were conducted using onewayanova followed by tukey™s test statistical analysis e average value of three experimental results was determined and the statistical softwaresas was used to analyze whether there was a significantdiï¬erence between each group at the level of p longest in normal group ± cm while being the results eï¬ect of lpys4 on colon parameters e experimentalresults demonstrated that the length of mouse colon was thepared to those in the remaining four groups in contrast thethe ratio of colon weightlength was the highest in normalpgml and the lowest concentrations were found for sslpys4h group and these eï¬ects were better than those of eï¬ect of lpys4 on the serum contents of et1 sp ss andvip in mice it can be seen from figure that the serumml was decreased in normal mice compared to that of theremaining four groups e colitis model mice exhibited theopposite results in which the highest concentrations wereserum concentrations of ss and vip in colitis mice andmarkedly decrease those of et1 and sp more importantlythe eï¬ects were better after treatment with lpys4hshortest in the colitis model group ± cm similarlygroup ± while being the lowest in the colitis modelgroup ± figure it was found that lpys4 couldsignificantly p attenuate the decline in colon lengthand weightlength ratio induced by colitis ± cm and± for lpys4l group ± cm and ± forlb ± cm and ± concentrations of ss ± pgml and vip± pgml in normal mice were increased comlevel of et1 ± pgml and sp ± pgobserved for et1 ± pgml and sp ± ± pgml and vip ± pgml interestingly lpys4 could significantly p improve the± ± ± and ± pglb ± ± ± and ±± pgml in colitis model mice was signifigroups p following the treatment with lpys4 anhigher in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml and the serum levels of il10 werelower in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml p gsh ± μmolmg and sod ± μmolhighest while those of mpo ± mumg and mda± nmolmg were the lowest among the five± μmolmg and sod ± μmolgprotwere significantly reduced p while those of mpo± mumg and mda ± nmolmg wereremarkably increased p in the model group mice eï¬ect of lpys4 on the serum concentrations of il2 andil10 in mice it can be seen from figure that the serumcontentand il10± pgml eï¬ect of lpys4 on the activities of mpo sod gsh andmda in mouse colon as shown in figure the activities ofincrease in il2 and a decrease in il10 cytokine level wereobserved notably the serum levels of il2 were markedlyml and the eï¬ects of lpys4 were better than those ofgroups after induced by oxazolidone the levels of gshcantly reduced and raised compared to the remaining fourgprot in the colon tissue of the normal group were the pgmlofil2 0cevidencebased complementary and alternative medicinebacmice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgfigure ac colon length and colon weightcolon length of each group of mice data are presented as the mean± standard deviationa“d diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4land mda levels p such eï¬ect was stronger comparedto lbtreated mice ± μmolmg ± μmolgprot ± mumg and ± nmolmg more± μmolgprot in colon tissue and markedly decreased those of mpo ± mumg and mda± nmolmg when compared to colitis model groupand the goblet cells were increased compared with the modelgroup among them lpys4h had the most obvious eï¬ecton improving colon tissue which indicated that lpys4 couldreduce the colon injury caused by dss and the high efficiencyeï¬ect was also enhanced with the increase of concentrationlpys4 could markedly attenuate the decline in gsh andsod levels and prevented colitisinduced increase in mpo± μmolmgspecifically treatment with lpys4h significantly increasedthesodlevelsandof pathological observation as shown in figure in thenormal group the epithelial cells of colon mucosa were intactthe ‚ammatory cells were normal without ltration andthe goblet cells were arranged orderly without congestionand edema in the model group the epithelial cells of colontissue were obviously damagedthe intestinal wall wasthickened and edema ‚ammatory cell ltration andgoblet cells were reduced after treatment with lb and lpys4 congestion edema and cell ltration were alleviated eï¬ect of lpys4 on mrna and protein expression inmouse colon as shown in figures and colitis inductioncould lead to the upregulated mrna and protein expressionof inos in mouse colon but downregulated the relativeexpression of ckit enos nnos and scf treatment withlpys4h could increase the relative expression of nnosenos ckit and scf and decrease that of inos in the colontissues of colitis mice such eï¬ects were stronger than thoseof lpys4l or lb treatment group discussione ratio of colon weightlength is employed as a vitalstandard for assessing colitis in vivo e colon length ofnormalmodellblpys4llpys4hadccb0020406080100normalmodellblpys4llpys4hcolon length cmcolon lengthadccb00100200300400500normalmodellblpys4llpys4hcolon weightcolon lengthcolon weightcolon length 0cevidencebased complementary and alternative medicineabfigure a et1 b ss c sp and d vip serum levels of each group of mice data are presented as the mean± standard deviationa“d diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4lmice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgdcfigure a il2 and b il10 serum levels of each group of mice data are presented as the mean± standard deviation a“e diï¬erentletters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4l mice treatedwith a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentration oflactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgbadabbc0050100150200250normalmodellblpys4llpys4het1 level pgmlet1aedcb00100200300400500600700normalmodellblpys4llpys4hss level pgmlssvipdabbc00100200300400500600700normalmodellblpys4llpys4hsp level pgmlspaedcb00100200300400500600700normalmodellblpys4llpys4hvip level pgmlvipadccb050100150200250300normalmodellblpys4llpys4hil2 level pgmlil2eabcd02004006008001000normalmodellblpys4llpys4hil10 level pgmlil10 0cevidencebased complementary and alternative medicineabfigure a mpo b no c gsh and d mda colon tissue levels of each group of mice data are presented as the mean± standarddeviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent testlpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a highconcentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgcdcolitis mice was shorter on average than that of control miceand the ratio of colon weightlength was lower in colitis micethan in control mice howeverit appeared thattreatment with lpys4 could attenuate the decline in colonlength and weightlength ratio induced by colitisa prior study has shown that vasoconstriction of theendothelin can lead to colonic mucosa erosion and ulceration which in turn can exacerbate the progression of colitis ss however can reduce gastrointestinal ‚ammationby suppressing the production of gastric acid and othergastrointestinal fluids us a decrease in ss level can inducethe secretion of gastrointestinal fluids thus aggravatingcolitis excessive accumulation of sp can induce colitisbut after antagonizing it has been shown to relieve colitis invivo vip inhibits no production by regulating thetranscriptional activity of inos in the colon tissue thusprotecting the intestinal mucosa besides vip can also ‚uence certain immune aspects of colitis in this studylpys4 inhibited colitis by downregulating the levels of etand sp and upregulating those of ss and vipil2 is an eï¬ector cytokine produced by cells whichhas been closely associated with colitis cells regulate the‚ammatory response that causes colitis and il2 is involved in the suppression of ‚ammatory process andseverity reduction of colitis by ‚uencing cells il is another cytokine produced by treg cells with immunoinhibitory eï¬ects which plays a significant role in thedevelopment of colitis ‚ammatory bowel diseaseibd is a chronic refractory intestinal ‚ammatory diseasemainly including ulcerative colitis uc and crohn™s diseasecd although the etiology of ibd is still unclear theimbalance between and has been recognized as themain cause of mucosal damage in ibd under the action ofdiï¬erentiation factor il10 regulatory t cells derived fromintestinal mucosa associated lymphoid tissue can correct deviation by secreting high level of il10 andmedium level of tgfβ so as to achieve the purpose oftreating ‚ammatory bowel disease to a certain extent it was observed that lpys4 could increase the level of il2thus regulating immunity and alleviating colitis and lpeabcd00100200300400normalmodellblpys4llpys4hmpo level mumgmpoadccb00100200300400normalmodellblpys4llpys4hsod level μmolgprotsodaedcb0020406080100120normalmodellblpys4llpys4hsgh level μmolmggshdabbc000510152025normalmodellblpys4llpys4hmda level nmolmgmda 0cevidencebased complementary and alternative medicinefigure observation of colon pathology in mice by he stainingys4 could inhibit the colitis and reduce the secretion of ile aggregation of neutrophils began to decline afterintestinal ‚ammation and a large amount of them enteredinto the circulation and migrated to tissues at the sametime free radicals such as reactive oxygen species and reactive nitrogen species gathered in large quantities which inturn led to damage and toxicity of colon tissue and furtheraggravated colitis after colitis the levels of sod andgsh were reduced in colon tissue while those of mda andmpo were elevated our findings also indicated that colitiscould lead to decrease in gsh and sod levels as well asincrease in mda and mpo levels [ ] in addition lpys4 attenuated colitis by inhibiting the transcriptional responses to oxidative stressnos can be divided into nnos enos and inos it hasbeen reported that no produced by enos plays a key rolein response to colonic tissue damage and excessive nogenerated by inos promotes colitis damage enoscontrols the production of no to keep the colonic tissue ina normal state which plays an important role in reducingcolitisinduced colonic injury e presence of excessiveno aggravates colon damage nnos can also controlthe level of no in tissue and protect the tissue from beingdamaged by excessive no in this study lpys4upregulated the expression of enos and nnos in the colonand downregulated that ofthereby attenuatingcolitisinosulcerative colitis not only shows hematochezia anddiarrhea but also presents colonic motility disorders it hasbeen proved that interstitial cells of cajal icc are related tocolonic motility dysfunction and directly participate in theprogression of colitis as a specific marker of gastrointestinalicc ckit is a transmembrane glycoprotein specificallyexpressed on icc cell membrane ckit gene located onchromosome 4q1213 belongs to protooncogene and itsproduct is tyrosine kinase type iii as a receptor of scfckit can regulate the proliferation and diï¬erentiation ofhematopoietic stem cells through a series of signalingpathways [ ] scf exerts a direct eï¬ect on ‚ammatorybowel disease by regulating the function and number oficc scf can interact with its ligand ckit and the dysregulation of scfkit signaling pathway may decrease theproliferation and diï¬erentiation of icc thus exacerbatingcolitis [ ] e abnormal expression of scfckitsignaling pathway can also change the physiologicalfunction of icc weaken gastrointestinal motility andaggravate intestinal dysfunction in the present studylpys4 could inhibit colitis by regulating the expressionlevels of scf and ckiticc autophagy regulation has become a new target forthe treatment of intestinal motility disorder in ulcerativecolitis because the drug treatment of colitis often hasside eï¬ects and once stopped it is easy to relapse ereforethe use of natural harmless substances through the regulation of icc prevention and treatment of colitis canmaintain longterm health a study has shown thataurantii fructusimmaturus and atractylodis macrocephalae rhizoma can inhibit the autophagy of cajal stromalcells induced by glutamate which may play an inhibitoryrole in colitis meanwhile there is also a study showingthat lactic acid bacteria can regulate icc thus regulatingnormallblpys4llpys4hmodel 0cevidencebased complementary and alternative medicineacbdthe mean± standard deviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestlyfigure eï¬ect of lpys4 on a nnos b enos c inos d ckit and e scf mrna expression in mouse colon data are presented asesignificantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillusbulgaricus — cfukgintestinal function and protecting the intestine isstudy also confirmed that the lpys4 can regulate the scfckitsignaling pathway and the scfckit signalingpathway is an important icc regulatory pathway erefore the lpys4 may also inhibit the colon by regulating iccaedcb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupnnosadccb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupscfaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineacbdmean± standard deviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly sigfigure af eï¬ect of lpys4 on nnos enos inos ckit and scf protein expression in mouse colon data are presented as thenificantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillusbulgaricus — cfukgef conclusionin this study oxazolone was used to induce colitis in balbcmice and the inhibitory eï¬ects of lpys4 on colitis weredetected rough the observation of colon tissues and serum samples of mice it was found that lpys4 treatmentcould alleviate colitis by restoring the levels of ‚ammatoryindicators closer to those measured in healthy control miceis work suggests that lpys4 is superiorquality lactic acidbacteria with a potential role in colitis treatment and provides a foundation for further research and developmentabbreviationslpys4 lactobacillus plantarum ys4lbqpcr quantitative polymerase chain reactionhelactobacillus bulgaricushematoxylineosinnormalmodellblpys4llpys4hckitenosinosnnosscfβactinadcbcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupnnosaedcb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosadccb0010203040506070normalmodellblpys4llpys4hrelative to multiple of model groupscfadccb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineendothelin1substance psomatostatinvasoactive intestinal peptideinterleukin2interleukin10et1spssvipil2il10mpo myeloperoxidasesodgshmda malondialdehydeenosnnos neuronal nitric oxide synthaseinducible nitric oxide synthaseinosscfstem cell factorsuperoxide dismutaseglutathioneendothelial nitric oxide synthasedata availabilityno data were used to support this studyconflicts of intereste authors of this manuscript state that they do not haveconflicts of interest to declareauthors™ contributionsruokun yi and fang tan contributed equally to this workruokun yi and fang tan performed the majority of theexperiments and wrote the manuscript huayi suo wenfengli xianrong zhou and jianfei mu contributed to the dataanalysis xin zhao and peng xie designed and supervised thestudy and read the final manuscriptacknowledgmentsis research was funded by national key rd program ofchina 2018yfd0502300 children™s research institute ofnational center for schooling development programmeand chongqing university of education csdp19fs01103theand technology research program ofchongqing municipal education commission kjzdk201901601 and research project of chongqing universityof education ky2015tbzc chinasciencereferences r k yi f tan w liao et al œisolation and identification oflactobacillus plantarum hfy05 from natural fermented yakyogurt and its eï¬ect on alcoholic liver injury in mice microanisms vol no p j liu f tan x h liu et al œexploring the antioxidanteï¬ects and periodic regulation of cancer cells by polyphenolsproduced by the fermentation of grape skin by lactobacillusplantarum kfy02 biomolecules vol no p t s olson b k reuter k g e scott et al œe primarydefectfrom a nonhematopoietic source journal of experimental medicinevol no pp “ ileitis originatesin experimental b manandhar k r paudel b sharma and r karkiœphytochemical profile and pharmacological activity of aeglemarmelos linn journal of integrative medicine vol no pp “ x zhou h liu j zhang et al œprotective eï¬ect of lactobacillus fermentum cqpc04 on dextran sulfate sodiuminduced colitis in mice is associated with modulation of thenuclear factorκb signaling pathway journal of dairy science vol no pp “ m c arrieta k madsen j doyle and j meddings œreducing small intestinal permeability attenuates colitis in theil10 genedeficient mouse gut vol no pp “ h suo x zhao y qian et al œerapeutic eï¬ect of activatedcarboninduced constipation mice with lactobacillus fermentum suo on treatment international journal of molecular sciences vol no pp “ y qian h suo m du et al œpreventive eï¬ect of lactobacillus fermentum lee on activated carboninduced constipation in mice experimental and eerapeutic medicinevol no pp “ x zhao y qian h suo et al œpreventive eï¬ect of lactobacillus fermentum zhao on activated carboninduced constipation in mice journal of nutritional science andvitaminology vol no pp “ x long y pan and x zhao œprophylactic eï¬ect ofkudingcha polyphenols on oxazolone induced colitis throughits antioxidant capacities food science and human wellnessvol no pp “ k zhu g huang j xie x zhou j mu and x zhaoœpreventive eï¬ect of flavonoids from wushan shencha malus doumeri leaves on ccl induced liver injury foodscience nutrition vol no pp “ x zhao j zhang s yi et al œlactobacillus plantarumcqpc02 prevents obesity in mice through the pparα signaling pathway biomolecules vol p w strober i j fuss and r s blumberg œe immunologyofmucosalmodels ofinflammation annual review of immunology vol no pp “ jl song y qian gj li and x zhao œanti‚ammatoryeï¬ects of kudingcha methanol extract ilex kudingcha cjtseng in dextran sulfate sodiuminduced ulcerative colitismolecular medicine reports vol no pp “ y qian x zhao jl song et al œinhibitory eï¬ects of resistant starch rs3 as a carrier for stachyose on dextransulfate sodiuminduced ulcerative colitis in c57bl6 miceexperimental and eerapeutic medicine vol no pp “ x y chen x zhao h w wang et al œprevent eï¬ects oflactobacillus fermentum hy01 on dextran sulfate sodiuminduced colitis in mice nutrients vol no p y qian a l lei x j liu et al œinhibitory eï¬ects oflactobacillus plantarum ys2 in dextran sulfate sodiuminduced c57bl6j mice colitis science and technology of foodindustry vol no pp “ s chen x zhao p sun j qian y shi and r wangœpreventive eï¬ect of gardenia jasminoides on hclethanolinduced gastric injury in mice journal of pharmacologicalsciences vol no pp “ l xie z h xing r x jiang et al œeï¬ect of sanpi decotionon the expression of il2 and il10 of mice with ulcerativecolitis chinese journal of experimental traditional medicalformulae vol no pp “ l hang s kumar a m blum j f urban m c fantini andj v weinstock œheligmosomoides polygyrus bakeri infection decreases smad7 expression in intestinal cd4 t cells 0cevidencebased complementary and alternative medicinewhich allows tgfβ to induce il10producing regulatorytcells that block colitis ee journal of immunology vol no pp “ j zhang q li y wei et al œprocess design of the antioxidant shuidouchi and its eï¬ect on preventing dextransulfate sodium dssinduced colitis in mice via antioxidantactivity applied sciences vol no p c fiocchi œ‚ammatory bowel disease new insights intomechanisms of ‚ammation and increasingly customizedapproaches to diagnosis and therapy current opinion ingastroenterology vol no pp j zhang r yi y qian p sun x zhao and z yangœlactobacillus plantarum cqpc06 activity prevents dextransulfate sodiuminduced colitis by regulating the il8 pathway journal of food science vol no pp “ j zhang x chen jl song et al œpreventive eï¬ects oflactobacillus plantarum cqpc07 on colitis induced bydextran sodium sulfate in mice food science and technologyresearch vol no pp “ lh chen jl song y qian x zhao hy suo and j liœincreased preventive eï¬ect on colon carcinogenesis by use ofresistant starch rs3 as the carrier for polysaccharide ofinternationallarimichthysjournal of molecular sciences vol no pp “ x feng j zhang y qian et al œpreventative eï¬ects oflactobacillus plantarum ys3 on oxazoloneinduced balbccolitis in mice applied biological chemistry vol no pp “ swimming bladdercrocea j m wang m li r tang et al œeï¬ect of yiqi jianpitongbian recipe on icc and scfckit signaling pathway incolon tissue of slow transport type constipation rats chinesearchives of traditional chinese medicine vol no pp “ j feng j gao s zhou et al œrole of stem cell factor in theregulation of icc proliferation and detrusor contraction inrats with an underactive bladder molecular medicine reports vol no pp “ c lu h lu x huang et al œcolonic transit disordermediated by downregulation of interstitial cells of cajalanoctamin1 in dextran sodium sulfateinduced colitis micejournal of neurogastroenterology and motility vol no pp “ y c dai l zheng y l zhang et al œjianpi qingchangdecoction regulates intestinal motility of dextran sulfate sodiuminduced colitis through reducing autophagy of interstitial cells of cajal world journal of gastroenterologyvol no pp “ s yan y z yue m m sun et al œsuppressive eï¬ect ofaurantii fructus immaturus and atractylodis macrocephalaerhizoma on glutamic acidinduced autophagy of interstitialcells of cajal journal of integrative medicine vol no pp “ c li sp nie kx zhu et al œeï¬ect oflactobacillusplantarumncu116 on loperamideinduced constipation inmice international journal of food sciences and nutritionvol no pp “ r yadak m breur a
Colon_Cancer
the introduction of combined conventional cytostatics and pathway‘specific inhibitors has opened new treatment options for several cancer types including hematologic neoplasia such as leukaemias as the detailed understanding of the combination‘induced molecular effects is often lacking the identification of combination‘induced molecular mechanisms bears significant value for the further development of interventional approachesmethods combined application of conventional cytostatic agents cytarabine and dexamethasone with the pi3k‘inhibitor idelalisib was analysed on cell‘biologic parameters in two acute pro‘b lymphoblastic leukaemia b‘all cell lines in particular for comparative characterisation of the molecular signatures induced by the combined and mono application whole transcriptome sequencing was performed emphasis was placed on pathways and genes exclu‘sively regulated by drug combinationsresults idelalisib cytostatics combinations changed pathway activation for eg œretinoblastoma in cancer œtgf‘b signalling œcell cycle and œdna‘damage response to a greater extent than the two cytostatics alone analyses of the top‘ regulated genes revealed that both combinations induce characteristic gene expression changes a specific set of genes was exclusively deregulated by the drug combinations matching the combina‘tion‘specific anti‘proliferative cell‘biologic effects the addition of idelalisib suggests minor synergistic effects which are rather to be classified as additivekeywords pik3‘inhibition acute lymphoblastic leukaemia idelalisib cytostatics drug combinations cytarabine dexamethasonecorrespondence hugomuruaescobarmeduni‘rostockde department of medicine clinic iii ‘ hematologyoncologypalliative care rostock university medical center rostock germanyfull list of author information is available at the end of the acute lymphoblastic leukaemia all is a malignant disease which is characterized by a clonal proliferation of lymphoid progenitor cells most commonly of bcells all affects children as well as elderly individuals the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0csklarz a0et a0al cancer cell int page of with a significantly different outcome while children are reported to have a longterm survival probability of approximately [ ] in adults relapsefree survival is lower than patients showing mixedlineage leukaemia mll rearrangements display even lower survival rates [“]all therapy is currently dominated by the application of cytostatic agents according to the current clinical practice guidelines however severe side effects development of drug resistance and relapse limit the therapeutic success the introduction of pathway specific tyrosine kinase inhibitors tki such as imatinib and immunotherapeutics such as the anticd20 antibody rituximab have advanced curative treatment in chronic leukaemia the 3kinase pi3k represents a key molecule within the b cell receptor bcr pathway and different tkis are currently evaluated to target this kinasephosphatidylinositol45bisphosphate idelalisib idel is a selective pi3k pathway inhibitor targeting the δ subunit [ ] mono and combined administration of idel were approved for the treatment of indolent nonhodgkin lymphoma and chronic lymphocytic leukaemia [“] however postmarketing surveillance suggested increased mortality caused by infections as a side effect of idel the molecular mechanisms leading to this observed side effect are not yet fully understood [ ]idel preferentially targets the deltasubunit pi3kδp110δ of the pi3k kinase which plays a key role in signal transduction cell proliferation and survival energy metabolism cellular motility and cell cycle progression the kinase is highly activated in several tumour types of different origins [ ] consequently pi3k is targeted in several novel therapeutic approaches [ ] table a0the introduction of tkis such as idel enabled the evaluation of new drug combinations potentially featuring lower doses of the individual drugs and thereby reducing side effects and drug resistance in turn understanding the respective combination modes of action is critical for a rational selection of the best candidate drugs introduction of nextgeneration sequencing such as whole genome and exome sequencing and rnasequencing provided profound knowledge of the disease acting molecular mechanisms especially rnasequencing has been of considerable value as mrna allowed to characterise drug combination action as well as drug combination induced effects therefore in the present study the cellbiological and molecular effects of the pi3kinhibitor idel in mono and combined drug application with the conventionally used cytostatics cytarabine arac and dexamethasone dex on proball cells were investigated cellbiological assays analysing cell proliferation metabolism and apoptosis induction revealed combinationspecific table pathway analysis of a0 rs411 and a0 sem exposed to a0 arac dex and a0 idel and a0 two drugs combined arac idel dex idelpathway regulation arac idel vs arac vs idelcell lineretinoblastoma in cancer uptgf‘beta signaling pathway downtgf‘beta signaling pathway upsids susceptibility pathway uptnf‘alpha signaling pathway uppathway regulation dex idel vs dex vs idelproteasome degradation downcytoplasmic ribosomal proteins updex idel micrornas in cardiomyocyte hypertrophy upectoderm differentiation upretinoblastoma in cancer downcell cycle downdna replication downg1 to s cell cycle downdna damage response down ranking positions of the pathways and amount of corresponding genes in parentheses are representedaracpathway ranking position corresponding genesarac idel dex idelidel “““ ““ “ “ rs411semcell liners411sem 0csklarz a0et a0al cancer cell int page of enhanced antiproliferative effects of the combined drug applications comparative whole transcriptome sequencing analyses identified pathways and gene signatures specifically regulated by the respective drug combinations within the top20 modulated pathways the œretinoblastoma in cancer œtgfb signalling œcell cycle and œdnadamage response were predominantly affected by the combination in order to identify key player genes using these pathways the top20 modulated were analysed revealing a gene set exclusively regulated by the drug combination in both cell lines this gene set featured cyp3a4 steap1 slitrk1 ackr3 and ccl25 some of these genes are reported to be deregulated in leukaemic cells thus exclusively regulation by drug combination may explain the rather additive effectsresultsidel enhances the a0anti‘proliferative and a0anti‘metabolic effect of a0arac and a0dexin rs411 cells enhanced effects on proliferation inhibition were observed for combinations of arac idel ± and dex idel ± compared to the respective mono drug applications arac ± dex ± idel ± control fig a01a comparison of cell count and metabolic activity wst1assay fig a0 1b revealed a reduction in cell numbers while barely decreasing metabolic activity for arac and arac idel ± and ± in sem cells all combinations arac dex ± arac idel ± dex idel ± resulted in an enhanced antiproliferative effect compared to the respective mono applications arac ± dex ± or idel ± fig a0 1d akin to rs411 the incubation with arac ± and arac idel ± resulted in a decreased metabolic activity fig a01e the observed reduction of metabolism did not match the observed reduction in cell numbersidel boosts the a0apoptotic effect of a0dex in a0rs411incubation with dex idel resulted in a significantly higher apoptosis rate ± compared to the respective mono substance application dex ± idel ± control ± in rs411 cells fig a0 1c in sem only the combination arac dex resulted in an increased amount of early and late apoptotic cells ± compared to the respective mono substances arac ± dex ± control ± fig a01f in fig a01g are exemplarily the plots of the flow cytometry analysis additionally all plots are shown in the supplementary file additional file a0 facs plotsin summary biological assays revealed enhanced antiproliferative effects triggered by combined application of idel with arac and dex respectively therefore we further investigated drug exposure induced effects on gene and pathway regulation by rnasequencing for all drug combinations and mono applicationscombined drug application of a0idel with a0arac and a0dex induces enhanced changes in a0gene expressiondrug combination induces an a0enhanced amount of a0regulated genesin rs411 genes were differentially expressed by arac idel exposure compared to the respective control cells while mono application of arac modulated and idel genes see additional file a0 supplement tables thereby an overlap of genes in all three conditions was identified fig a02aalso dex idel exposure modulated more genes compared to dex or idel here an overlap of genes was detected by both mono and as well as combined drug application fig a02bin sem the arac idel combination led akin to rs411 to a stronger gene modulation compared to arac and idel thereby genes were modulated by both the mono and the combined drug application fig a0 2c moreover dex idel exposure resulted in a higher amount of regulated genes in comparison to their respective mono applications dex idel all three conditions showed an overlap of genes in totalin summary for both cell lines and both drug combinations the total number of genes modulated by these combinations exceeds the absolute number of the respective mono application especially the conditions arac vs arac idel and dex vs dex idel showed a high overlap of modulated genes heatmaps of the top100 regulated genes are shown in the additional file a0 supplement figureshowever the comparison of the up and downregulated genes revealed a higher amount of upregulated genes by arac idel exposure in both cell lines moreover dex idel led nearly to a similar amount of up and downregulated genes in both cell linesdrug combinations induced stronger changes of a0gene expression levelsin rs411 we observed a higher range of gene expression level changes by arac idel ˆ’ to in comparison to arac ˆ’ to and idel ˆ’ to an enhanced range was also observed by exposure with dex idel ˆ’ to in contrast to the respective mono application dex ˆ’ to idel ˆ’ to 0csklarz a0et a0al cancer cell int page of srmesa] sll ec dedees[ tnuoc llecd] sllec dedees [ tnuoc llecproliferationb] lortnoc[ ytivitca cilobateme] lortnoc[ ytivitca cilobatemmetabolismapoptosisc]evitisop ipvnixenna[ sllec degamadf]evitisop ipvnixenna[ sllec degamadgieddoiimudporpiaracdexidelaracideldexidelsrmesannexin v fitc fig pro‘b all cell lines rs411 a“c and sem d“f exposed with arac dex and idel and two drugs combined aracdex aracidel dexidel for h influence of mono and combined application on a d proliferation cell count b e proliferation and metabolism wst‘ proliferation assay and c f apoptosis annexin vpi‘ staining a pairwise students t‘test compared to control cells and single compounds reveals significance p ‰¤ p ‰¤ p ‰¤ [n ‰¥ ] g plots of apoptotic annexin v fitc and propidium iodide‘ and necrotic cells annexin v fitc and propidium iodide detected by flow cytometry analysis at h data are representative of three independent experiments further plots are represented in the additional file facs plots 0csklarz a0et a0al cancer cell int page of arac vs idel vs aracideldex vs idel vs dexidelasrcmesbdfig pro‘b all cell lines rs411 a b and sem c d exposed with arac vs idel vs aracidel and dex vs idel vs dexidel venn‘diagrams represent the differential expressed genes deg of each sample and there overlap among these samplesas with rs411 a higher range was observed in sem cells by combined drug incubation with arac idel ˆ’ to compared with mono application arac ˆ’ to idel ˆ’ to similar effects were detected for dex idel ˆ’ to exposure dex ˆ’ to idel ˆ’ to arac idel and a0dex idel modulated combination specific gene setsin rs411 arac idel exposure led to a set of genes which were exclusively regulated by this combination but not by dex idel in contrast dex idel led to selective modulation of genes which were only regulated by this combined drug application see additional file a0 supplement tablesin sem arac idel led to an exclusively regulation of genes which were not modulated by the other drug combination exposure to dex idel resulted led to a set of exclusively effected genes which were not regulated by arac idel see additional file a0 supplement tablesin summary the combined application of idel with arac or dex resulted in regulation of an exclusively gene set and also in higher gene expression levels further the specific combinations induce characteristic expression changes 0csklarz a0et a0al cancer cell int page of combined drug application of a0idel with a0arac or a0dex leads to a0combination specific pathway modulationa combination of arac idel or dex idel led to specifically enhanced pathway modulation compared to the respective mono applications absolute numbers of genes included in the respective pathways were increased in the following we focussed on pathways that did not rank within the top30 deregulated pathways within the mono applications but ranked in the top20 pathways during combined setup overview in table a0 a complete listing table in the additional file a0 xlsfile section œadditive pathways genesarac idel modulates tgf‘beta signalling in a0rs411 and a0sem and a0further three cell line specific pathwaysin rs411 cells the combination of arac idel led to upregulated genes corresponding to the œretinoblastoma in cancer pathway while the respective mono applications modulated only four arac respectively two idel genes respectively accordingly the pathway ranked on position of the top modulated pathways arac position idel position further œtgfbeta signalling showed more downregulated genes after arac idel exposure compared to the mono applications arac genes idel genes combined pathway ranking on position arac position idel position in sem with respect to the modulation of the œtgfbeta signalling pathway a higher number of downregulated genes was found by arac idel in comparison to the respective mono applications arac idel this pathway ranked on position for the combined application arac position idel nafurther œtnfalpha signalling was found on position of the top upregulated pathways arac position idel na modulating combination specific genes compared to arac genes and idel genes accordingly tgfalpha signalling was found to be a major target of idel combination induced pathway modulationfurther upregulation of genes corresponding to the œsids susceptibility pathway was observed by arac idel exposure compared to the respective mono applications arac genes idel genes this pathway was found at position of the list of the top20 regulated pathways arac idel nadex idel exposure induces extensive œcytoplasmic ribosomal proteins œretinoblastoma in a0cancer and a0œcell cycle pathway deregulationwhile arac idel modulated four different pathways in total in both cell lines the induced observed modulation by the addition of idel to dex resulted in a high number of combination specific pathway deregulations in total nine different pathways were modulated by dex idel in both cell lines interestingly eight of nine pathways were found to be significantly deregulated in sem upregulated downregulatedthereby the total number of modulated genes exceeded the number of genes modulated by the respective mono application in pathways such as œcytoplasmic ribosomal proteins upregulated œretinoblastoma in cancer and œcell cycle both downregulated while genes belonging to the œcytoplasmic ribosomal proteins pathway were found upregulated by dex idel only five genes were found upregulated by dex and by idel a similar pattern was observed for the œretinoblastoma in cancer pathway dex idel downregulation of genes vs dex genes vs idel gene as well as for the œcell cycle pathway dex idel genes vs dex genes vs idel genesfurther pathways showing a similar combination specific enhanced modulation were pathways such as œmicrornas in cardiomyocyte hypertrophy œectoderm differentiation œdna replication œg1 to s cell cycle and œdna damage response in sem cells and the œproteasome degradation pathways in rs411 detailed listing in additional file a0 xlsfile section œadditive pathways genesin summary the additional application of idel enhances the observed effects of arac and dexidel combination modulated pathways showed characteristic gene deregulationsdex idel as well as arac idel combination induced specific gene modulation not found in either of both mono applications in total arac idel led to an exclusive modulation of genes annotated with the four drug combination specific pathways log foldchange ˆ’ to the dex idel combination induced exclusive modulation of in total of genes within the drug combination specific modulated pathways log foldchange ˆ’ to exemplarily dex idel exposure led to exclusive modulation of the cell division cycle a cdc25a log foldchange ˆ’ cell division cycle cdc6 log foldchange ˆ’ and myosin lightchain kinase gene mylk log foldchange genesa detailed listing of all affected genes of both cell lines can be found in the additional file a0 xlsfile section œadditive pathways genesidel combination specific pathways show enhanced gene expression regulationas described above the addition of idel to arac or dex led to exclusive gene regulations as well as increased 0csklarz a0et a0al cancer cell int page of log gene numbers belonging to to the top deregulated pathways additionally the respective combinations led to enhanced log foldchanges for a set of specific genes summary is given in the additional file a0 supplement tables thereby the respective range in the combinations exceeded the respective mono application detailed listing in the additional file a0 xlsfile section œadditive pathways genes as mentioned before arac idel led to a drug combination specific pathway modulation of four pathways further investigation revealed a deregulation of genes by arac idel log foldchange range ˆ’ to while arac deregulates genes log foldchange range ˆ’ to and idel genes log foldchange range ˆ’ to incubation with dex idel led to a deregulation of nine drug combination specific modulated pathways from these pathways dex idel deregulated genes log foldchange range ˆ’ to while dex deregulated genes log foldchange range ˆ’ to and idel none exemplarily genes such as aristaless related homeobox arx and zinc finger and btb domain containing zbtb16 were upregulated by dex application arx log foldchange zbtb16 log foldchange and stronger deregulated by the drug combination dex idel arx log foldchange zbtb16 log foldchange for a detailed comparison of the combined and mono application induced expression changes see additional file a0 xlsfile section œadditive pathways genes arac exposure led to an upregulation of distalless homeobox a0 dlx2 log foldchange while the addition of idel induced a log foldchange for the combined applicationtop‘ drug combination modulated genes revealed combination specific modes of a0actionto further explore combination specific acting mechanisms the top20 deregulated genes pathway independent log foldchanges combined drug exposure were compared to the corresponding expression values of the respective mono applications additional file a0 xlsfile section œrs411top20 genes aidi and œsemtop20 genes aidi akin to the observed exclusive gene modulations within the top ranking pathways comparable effects were found when analysing the general pathway independent top20 deregulated genesarac idel modulates histone genes predominantlyarac idel exposure led in both cell lines to a downregulation of different histones thereby hist1h2bo was found the only histone which ranked within the top downregulated genes for both cell linesin rs411 twelve histones belong to the general top downregulated genes thereby the observed log log foldchange of the histones hist1h1e hist1h2ah hist1h1d hist1h2bm and hist1h3b were found stronger deregulated given the observed log foldchanges compared to both respective mono applications range log foldchange arac idel ˆ’ to ˆ’ vs arac ˆ’ to ˆ’ vs idel ˆ’ to ˆ’ further the histones hist1h3i and hist1h3f were exclusively found to be downregulated by arac and arac idel while not being affected by idel interestingly both histones were more affected by arac idel log foldchange ˆ’ and ˆ’ compared to arac log foldchange ˆ’ and ˆ’ further the histones foldchange ˆ’ hist1h2aj hist1h2bf log foldchange ˆ’ hist12ad log foldchange foldchange ˆ’ and ˆ’ hist1h3g hist1h2bo log foldchange ˆ’ were found to be exclusively downregulated by the arac idelin sem histones as hist1h2bb log foldchange ˆ’ and also hist1h2bo log foldchange ˆ’ were only affected by arac idel while histones as hist1h4b hist1h2be hist1h4j hist1h2bg and hist1h3a were downregulated by arac and stronger affected by arac idel log foldchanges are detailed listed in the additional file a0 xlsfile section œsemtop20 genes aidiin addition to the mentioned histones both cell lines showed a specific pattern of the remaining combination specific top20 deregulated genes in rs411 downregulation of the genes small nucleolar rna snora12 log foldchange ˆ’ nucleoside diphosphate kinase nme1nme2 log foldchange ˆ’ and eukaryotic translation initiation factor 5alike eif5al1 log foldchange ˆ’ and an upregulation of camp responsive element binding protein like creb3l3 log foldchange and transmembrane and immunoglobulin domain containing tmigd2 log foldchange were foundin sem cytochrome p450 family subfamily a member cyp3a4 log foldchange ˆ’ six transmembrane epithelial antigen of the prostate steap1 log foldchange ˆ’ and potassium voltagegated channel subfamily j member kcnj1 log foldchange ˆ’ represents genes which were found only downregulated by the combination arac ideldex idel leads to a0regulation of a0similar top‘ modulated genes in a0both a0cell linesin contrast the exposure with dex idel induced a higher number of genes commonly deregulated in both cell lines in both cell lines two genes were found upregulated as well as eight downregulated ranking within the top20 deregulated genesto arac idel 0csklarz a0et a0al cancer cell int page of as ring finger protein rnf175 was downregulated in both cell lines by dex and dex idel exposure in rs411 rnf175 was significantly more affected by the drug combination dex log foldchange ˆ’ dex idel log foldchange ˆ’ in sem rnf175 showed a similar expression level with dex idel exposure log foldchange ˆ’ as with dex log foldchange ˆ’ also zbtb16 described above olfactory receptor family subfamily a member orc7a5 and olfactory receptor family subfamily c member or7c1 were upregulated and more affected by the drug combination in both cell linesadditionally some genes were exclusively deregulated by drug combination in rs411 leucinerich repeatcontaining protein slitrk1 log foldchange ˆ’ and matrilin matn4 log foldchange ˆ’ were only downregulated by the drug combination dex idel in sem atypical chemokine receptor ackr3 log foldchange ˆ’ and c“c motif chemokine ligand ccl25 log foldchange ˆ’ were also only downregulated by dex idel in both cell lines the top20 upregulated genes did not contain any gene which was exclusively regulated by the drug combination dex idel the here reported genes comprise a short overview and more genes are listed in the additional file a0 xlsfile section œrs411top20 genes aidi œsemtop20 genes aididiscussionthe combined application of idel and arac or dex resulted in enhanced antiproliferative effects depending on the addressed cell line the combination led to enhanced antiproliferative effects on the cellular level characteristic gene regulation and expression thereby the specific exclusively regulated genes and pathways were identified in both mllpositive proball cell lines the focus here is on mono and combined therapy of maximum two agents in cell lines and adds insights into the previously gained knowledge of expression profiling as well as fusion gene detection in patients with ball using standard treatment regimen containing arac and dex [ ]addition of a0idel to a0arac results in a0pronounced anti‘proliferative effects independent from a0arac‘sensitivitya different aracsensitivity characterises both cell lines while a0µm arac exposure reduces rs411 proliferation to approx half sem proliferation is inhibited to nearly by the 100fold lower concentration the addition of idel induced in both cell lines independently from their characteristic aracsensitivity an enhanced decrease of proliferation interestingly arac exposure led to an increase of metabolic activity in both cell lines while the addition of idel leads to a proportional ratio of remaining cells and corresponding metabolic activity further the addition of idel initiates an increase of the number of regulated genes and stronger modulated gene expression levels further the addition of idel led to a modulation of genes belonging to the œtgfbeta signalling pathway in both cell lines this pathway is an essential regulator of proliferation differentiation migration and cell survival additionally several genes regulating cellular key processes as proliferation and cell cycle were found regulated explicitly by the addition of idel exemplarily various histones with direct effect on dnapackaging were found downregulated and thus influence dnareplication further the elongation factor eif5al1 was found exclusively downregulated by arac idel in rs411 both mechanisms show a specific enhancement of cell division critical checkpoints which could be mediators of the observed cellular responsegenes snora12 nme1nme2 cyp3a4 and steap1 were exclusively downregulated by arac idel these genes are described to be found overexpressed in cancer of different origins while snora12 is found upregulated in lung cancer nme1nme2 upregulation is described to promote the survival of aml cells steap1 overexpression is detected in different cancer types and was associated with a poorer prognosis for aml multiple myeloma diffuse large b cell lymphoma and colorectal cancer accordingly the observed exclusively downregulation potentially represents a molecular mechanism resulting in the enhanced antiproliferative effects of arac idel further we detected a selective downregulation of cyp3a4 cyp3a4a290g polymorphism that resulted in overexpression was found in many acute myeloid leukaemia aml samples additionally an overexpression in breast cancer had been detected cyp3a4 is responsible for the detoxification of more than of the drugs on the other hand we discovered an exclusive downregulation of kcnj1 this gene is reported to inhibit proliferation and metastasis in renal cell carcinoma currently data of kcnj1 for leukaemia is missing the examined downregulation of these genes by arac idel may contribute to the more potent effect of the drug combination in comparison to the respective mono applicationwhile the previous represents the loss of prooncologic cellular features also gain of function modulations were observed leading to the enhanced antiproliferative molecular mechanism exemplarily creb3l3 was found exclusively upregulated while dlx2 expression was found stronger upregulated by the combination creb3l3 overexpression suppresses proliferation in hepatoma 0csklarz a0et a0al cancer cell int page of cells and has been described to be downregulated in hepatocellular carcinoma dlx2 is reported to be downregulated in paediatric precursor balls carrying mllrearrangement and may be induced during metabolic stressinduced necrosis these functional gene modulations represent candidate mechanisms mediating the observed enhanced antiproliferative effects in the all cell linesinterestingly in all similar cell biological effects were observed showing that the addition of pi3k or mtor inhibitors to arac was able to induce enhanced antiproliferative effects in a0vitro lately comparative observations were described for the combination of the pi3kδ inhibitor puquitinib with arac in an aml xenograft model addition of a0idel to a0dex leads to a0enhanced anti‘proliferative effects in a0glucocorticoid‘sensitive and a0‘resistant cellsthe addition of idel to dex resulted in an enhanced antiproliferative effect and higher apoptosis rates in glucocorticoid gcresistant and “sensitive proball cell lines both cell lines are characterized by a translocation between chromosome hsa4 and hsa11 in general the presence of this translocation is associated with gcresistance and often observed in cases of relapses however only sem cells established from a 5yearsold girl at relapse show reduced sensitivity to dex while rs411 established from a 32yearold woman at relapse are considered as highly sensitive while in sem a0µm dex exposure inhibited the rate of proliferation to approx a half rs411 proliferation was found decreased to nearly by the 1000fold diluted concentration the addition of idel to dex led to a strongly dexsensitizing effect on both cell lines sem proliferation was further reduced to approx while rs411 proliferation was reduced to nearly additionally the observed apoptosis inducing effect of dex was found increased by the addition of idel in both cell linesakin to the effects observed for arac idel the addition of idel to dex induced specific pathways modulations and also induced exclusive gene expression influencing key regulators such as cell cycle and dnareplication thereby key mediato
Colon_Cancer
subcutaneous hydration and medicationsinfusions effectiveness safety acceptability asystematic review of systematic reviews one e0237572 101371 pone0237572editor jose´ das neves university of portoportugalreceived november accepted july published august copyright broadhurst this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation filesfunding this project was partially supported viaan unrestricted project grant provided by bectondickinson canada a portion of the work of dswas funded as salary by the sponsor grant db asa former employee of medical pharmacies duringthe conduct of this research reports the researchas work independent of her employer with noemployer contributions to the research db is soleobjectiveto synthesize the current evidence for subcutaneous hydration and medication infusionsfrom systematic reviews and to assess their methodological qualityintroductionperipheral intravascular cannulacatheter insertion is a common invasive procedure foradministering fluids and medications venous depletion is a growing concern for severalpatient populations subcutaneous access for the administration of isotonic solutions andmedications is an alternative however vascular access assessment and planning guidelines rarely consider this routemethodssystematic review of systematic reviews prospero crd42018046504 we searched databases published in english language from to june identifying subcutaneousinfusions an alternate route for fluids or medication methodological quality was evaluatedusing amstar criteria and data for mechanisms of infusion and outcomes related toeffectiveness safety efficiency and acceptability extracted the johanna briggs institute™sgrades of recommendation informed the strength of recommendationresultsthe search yielded potential systematic reviews were excluded through and duplicate screen of the remaining s were excluded and were includedoverall evidence is strong for recommending subcutaneous hydration infusions for olderadults weak for pediatric patients and inconclusive for palliative patients there is strongevidence for medications weak evidence supporting medications however there areeight medications with inconclusive evidence to make a recommendation and four medications not appropriate for subcutaneous delivery one 101371 pone0237572 august one 0cproprietor of infusion excellence consulting whoseservices are unrelated to this research bg ownsand is employed as senior consultant and directorof education by clinical pharmacy partners aneducational and consulting firm for infusiontherapy providers and clinicians the funders hadno role in study conception design data collectionand analysis decision to publish or preparation ofthe manuscript the specific roles of these authorsare articulated in the ˜author contributions™ sectioncompeting interests db reports personal andconsulting fees ie travel and honoraria and nonfinancial support from 3m including researchgrant in unrelated field angiodynamicscardiomed medical supplies covalon freseniusand excelsior medical db™s former employermedical pharmacies provides infusion serviceshowever db affirms research was conductedindependent of her employer™s servicesproductsand of her current consulting business mc reportsgriffith university has received unrestrictedinvestigator initiated research or educational grantson mc™s behalf from product manufacturersbaxter becton dickinson and company centurionmedical products and entrotech lifesciencesunrelated to this research ds reports no conflictsbg reports personal and consulting fees ieconsultancy travel and honoraria from nutrishareand covalon bg affirms no relationshipspresenting competing interests or influences thisdoes not alter our adherence to one policieson sharing data and materialssubcutaneous hydration and medication infusionssubcutaneous access should be considered alongside intravenous therapy for hydration inolder adults and several medications there are additional benefits in terms of ease of useand costeffectiveness of this mode inclusion of subcutaneous access in clinical guidelinesmay promote uptake of this route to help preserve vessel health of vulnerable patients further highquality research is needed to inform subcutaneous infusion therapy in a variety ofpopulations including pediatrics and palliative care and medications and clarifying themechanism of deliveryintroductioninfusion therapy is a common treatment modality to deliver medications and fluids in theacute and home care settings and is gaining prevalence in the longterm care setting traditionally these parenteral therapies have been delivered via the intravenous route howevervenous depletion is a growing concern with an increasing aging population and patients withlongterm complex comorbidities [“] the problems of venous depletion are compoundedby unnecessary peripheral venipuncture which add to patient physical and psychologicaltrauma compromised intravenousrelated outcomes suboptimal use of healthcare resourcesand increased costs [ “] additionally infusion therapy is moving beyond the boundariesof the acute care and home care sector to hospices and longterm care facilities recentlypublished vascular access planning tools are either oriented primarily to the acute care settingor do not address the option of subcutaneous access [ ]the infusion therapy standards of practice recommend consideration of subcutaneousaccess for the administration of isotonic solutions and for continuous opioid and other infusion therapiesmedications eg immunoglobulin therapy subcutaneous access isachieved through the placement of a small catheter in the subcutaneous tissue with the infusate absorbed from this space into the circulatory system this route has the advantages ofrequiring a small catheter and less technical insertion skills than that used for intravenousaccess with its ease of application lending to its use in multiple settings additionallytime to place the catheter is less and fewer complications are likely resulting in cost benefits[ ] although practiced since there has been a slow uptake in part due to a lack offamiliarity with the technique among physicians and healthcare professionals and perceivedsuboptimal outcomes eg hypovolemic shock due to inappropriate use of hydration solutions [ ] in our scoping literature search to prepare for a systematic review of primarystudies numerous systematic reviews were identified however each either had a narrowscope addressing one treatment eg fisher and colleagues™ review of iron overload management or provided limited literature search or limited evidence eg duemsnoreiga andblasco™s review of subcutaneous fluid and drug deliverygiven the world™s aging population health care systems rapidly changing diversity in practice setting and complexity of care and patient health conditions the uptake and use of subcutaneous continuous infusions may help address these issues and challenges the aim of ourstudy is to determine the effectiveness safety acceptability and efficiency related to the use ofsubcutaneous infusion sci as an alternate route to intravenous for the management of conditions or treatments such as dehydration and palliation for children and adults in all care settings through a synthesis of systematic reviews of studies a secondary objective is to identify one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsthe mechanisms of subcutaneous fluid and drug delivery that facilitated achieving these outcomes this will provide an uptodate and rigorous review of subcutaneous infusion therapymaterial and methodswe adopted the systematic review of systematic reviews methodology from the joannabriggs institute™s to conduct this review the study protocol was registered with theprospero database registration number crd42018046504 and we report the reviewaccording to the preferred reporting items for systematic review and metaanalysisprisma guidelines s1 table eligibility criteriawe considered all types of systematic reviews which included the following characteristics¢ a clearly stated set of objectives with an explicit reproducible methodology¢ a systematic search that attempts to identify all studies that would meet the eligibilitycriteria¢ an assessment of the validity of the findings of the included studies eg assessment of riskof bias and confidence in cumulative estimates and¢ systematic presentation and synthesis of the characteristics and findings of the includedstudies metaanalyses where some of the primary studies in the reviews were duplicated we did not exclude thereviews as they had different aims and objectives and thus added to the overall understandingof sci to meet the aims of this current systematic review we did however only include the primary studies™ findings once in our analysis and synthesis expert opinionconsensus andbench research s and editorialscorrespondence were excluded no restrictionsregarding age gender diagnosis geographical location or healthcare setting were appliedwe included reviews that assessed interventions that used subcutaneous infusion for aduration of around hours or more as an alternate route for fluid or medication therapy subcutaneous infusion is defined as the delivery of fluids or medication into the subcutaneousspace for absorption into the circulation via œperfusion diffusion balance between hydrostaticosmotic pressure and lymphatic drainage p118 an infusion of around hoursduration was determined from considering the studies in a review by caccialanza where the range of infusion was hours to greater than days during the full textscreening numerous intermittent sc insulin vs continuous sc insulin systematic reviewswere identified and excluded because they did not meet our inclusion criteria of sci as analternate route reviews that included other routes as comparators such as intravenous andintraosseous were excluded if data on subcutaneous infusions could not be extractedseparatelyoutcome measuresthe primary outcomes of interest investigated included¢ effectiveness defined as clinical response to therapy eg cureimproved clinical failure orno change completion of therapy within prescribed time frame¢ safety defined as medication or vascular access adverse event eg abscess erythema bruising electrolyte imbalance edema infection pain fluid overload vascular collapse and one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsroute failure survival status eg died of underlying condition other causes lost to followup or status unknown and complications related to treatment eg unplanned hospitalreadmission related to treatment¢ acceptability defined as patient andor health care provider preference satisfaction or perceived benefits of subcutaneous therapy¢ efficiency defined as healthcare resource utilization including costs of infusion therapy supplies and treatment timesecondary outcomes included indications for subcutaneous infusion therapy medicationsolution type infusion rates volumes and duration subcutaneous access sites dwell timesand infusion control devices usedsearch strategya systematic search was conducted november and updated in june of reviews from as recommended by aromataris from the following databases excerpta medica database embase pubmed cumulative index to nursing and allied health literaturecinahl cochrane database of systematic reviews joanna briggs institute of systematicreviews and database of s of reviews of effects dare search terms included combinations of medical subject headings mesh and key word terms [˜subcutaneous infusion™or hypodermoclysis or ˜subcutaneous therapy™] and the term ˜systematic review™ with theassistance of a university librarian study resources limited inclusion to only english languageas listed in s2 table one author of a relevant was contacted to establish the status ofthe review however the review had not been completedthe title and of each were scanned independently by one reviewer ds andfull copies of s of potentially eligible reviews were obtained full texts of these reviewswere then screened independently by two reviewers mc and db against the review selectioncriteria disagreements were resolved by discussion between these reviewers and with consultation by a fourth reviewer and subject expert bgdata extraction and quality appraisaldata were extracted and assessed independently by the reviewers in pairs [bg db] [mcds] with anomalies reconciled by agreement data were obtained primarily from the systematic reviews although primary studies were consulted for critical missing data eg missinginfusion properties data extracted included authoryear aim design and number of studiesincluded search strategy population interventioncomparator quality appraisal of primarystudies infusion characteristics outcomes as previously defined key findings study limitations funding and smethodological rigour of each review was independently assessed by four reviewers in pairsas above using the amstar tool this is a 16item tool used to appraise the quality ofsystematic reviews and is frequently used in cochrane overviews a score of overall confidence high moderate low and critically low was assigned by the reviewers to depict the accuracy and comprehensiveness of the data summary and critical methodological flaws thisassessment informed the final grading of the recommendationsdata analysis and synthesisdue to the different interventions outcomes and outcome measures a metaanalysis was notfeasible quantitative outcome data are provided and synthesized where possible only data one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsfrom randomised controlled trials rcts and prospective cohort studies were included todetermine subcutaneous hydration outcomes however reviews exploring medications alsoincluded retrospective studies and case reports the jbi grades of recommendation were usedto derive the grading score to inform the strength of recommendation for the interventionjoanna briggs institute levels of evidence and grades of recommendation working party data synthesis for hydration and medications are provided as a final summary ofrecommendations grade a indicates the intervention™s desirable effects outweigh undesirableeffects with adequate supporting evidence a weak recommendation grade b depictsdesirable effects outweigh undesirable effects although this is not as clear or evidence not ofhigh quality where there was insufficient evidence we determined that a recommendation isinconclusiveresultsthe search strategy yielded potential systematic reviews fig of which wereexcluded through and duplicate screen of the remaining s were excludedafter full text examination against inclusion criteria in the full text screening for reasonsdescribed as above as listed in s3 tablecharacteristics of included studiestable details the systematic reviews of subcutaneous infusion of medications andor fluidswith focused on medications only on fluids and on both types of therapies the publication dates of reviews ranged between and [ ] most reviews focused onadult populations generally or specifically for example pregnant women and adults withamyotrophic lateral sclerosis als two reviews however are specifically paediatric [ ]eight all ages [ “] two focused on older adults and paediatrics [ ] andone where age was not reported the systematic reviews incorporated a diversity of study designs and quality levels fromcase studies to systematic reviews and randomised controlled trials however only reviewsincluded only randomised controlled trials [ “] similarly sample sizes varieddepending on the number of sci studies included in the review synthesise of findings withvery few metaanalyses performed due to varied study designs outcomes and sizemethodological qualitythe amstar quality scores of the included reviews are described in s4 table while reviews achieved an overall confidence rating of high sixteen of the reviews had critical methodological weaknesses studies with moderate low and studies rated as criticallylow the main deficiencies noted were failure to report a priori protocol study design selection full details of excluded studies funding sources of primary studies and results reflectingrisk of bias assessment the cochrane risk of bias tool or an adaptation was the most commonly used tool by systematic review investigators to determine quality of the primary rctstudies n while only provided a narrative discussion of quality most reviewauthors noted that results should be interpreted with caution due to study limitationsmechanisms of subcutaneous accesslimited data were reported on the materials and techniques used to achieve subcutaneousaccess for hydration or medication therapy table describes the characteristics of sc devicedesign and dwelltime subcutaneous anatomical sites and insertion techniques and infusion one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsfig prisma flow chart the diagram details our literature search and screening process for selection of systematic reviews included in thesystematic review adapted from moher d liberati a tetzlaff j altman dg the prisma group preferred reporting items for systematicreviews and metaanalyses the prisma statement med e1000097 101371 pmed1000097101371 pone0237572g001delivery systems in relation to studies reporting on hydration therapy no studies comparedthe safety and efficacy of the various characteristics including mode of delivery the findingsfor hydration and medication sci are presented separatelysubcutaneous hydrations5 table describes the indications and contraindications for sci for hydration as described inthe studies the most common indicator was mild to moderate dehydration with the mostcommon contraindication being rapid high fluid volume requirements one 101371 pone0237572 august one 0ctnatropmiyllacinilcmumnmiidecneirepxedahohwstneitaphtlaehdnaefilfoytilauqsuhtgivinoseacimetsyslatipsohnehwnoitpecrepfostroperdesaercnievahyamemohotdegnahcgividesabelihwseacimetsysdnalacolseidutsgicsdesabdellorneevahyamseiduts¢™stneitapnitnemevorpmi¢gicsnostneitaperomderreferpdnadetarelotdnacirtaidepybgivinahtsnoitcejnigicsdesabemoh¢stneitaptludamuresggielbatpeccaseveihcayparehttnemecalpergics¢sleveldnaytienegoretehlaitnatsbusseidutsssorcanosirapmocsnoitatimilllacigoodohtemsaibfoksiretaredomecnediveytilauqwol¢mrettrohsnoylnoatadloq¢puwollofnotcefferettebaevahyamlanoitnevnocnahtloqyparehtdesisehtnysseidutsylno¢suoenatucbussuounitnoc¢icsottnavelerenositrocordyhfonoisufnicsotviderapmocstcron¢derosnopsyrtsudni¢daehotdaehecnereffidehtniecnacifingislacitsitatssawerehthguohtla¢aenpsydecuderdnafoesuehthtiwdwmnillarevoehtsnilcycatsorpehtteemtondidtceffenoitarudpuwolloftrohsllasedulcnisisylanaatemsnoitnevretnifosepyt¢¢caidracgenoitcnufcaidracerusserplairtathgirxednievorpmiyamlanitsorpertcs¢gnitarehtgnitagitsevnirofairetircfoytilauqllacigoodohtemytilauqwolseiduts“ytilauqetaredomytilauqhgihrnsgnittesnodesabairetirc”stcrnerdlihcdnastludatrohocborenarhcocyranibnodesabseidutsydobitnayramirphtiwseicneicifedgnisuselbairavonsey“segnareziselpmassesabatadotpuetadyraunajotelbacilppaseidutsseidutslatotweiveremoctuo]noitatic[snoitacidemfonoisufnicsseidutsginilubolgonummiinassahobalslairtlacinilcevitcepsorp¢¢strohocevitcepsorter¢strohocytefasdnassenevitceffevisusrevcs][saibfoksirhgih”muidem”seidutslanoitavresboelacsawattoeltsacwenseidutslanoitavresborofytilauqriafstcrnielpmasycneiciffusniylujotpuetadsuounitnoc“egnarezisrnsgnittesdnaegacsenositrocordyhstcrretnecitlumlacinilclebalnepo¢ydutsrevossorc¢noisufnissenevitceffestnemtaertlootborenarhcoc“stcrlanerdahtiwstneitapsesabataddedulcniseidutsdiocitrococugl][lafonlagnitroperdnanoitcelessisongaidsraey“sehcraesdnahicsottnavelerycaciffelinitsorpertfoksirwolborenarhcocegaegaeziselpmassesabatadstcrtcrnilcycatsorp][senrabsaiblairetrayranomuplotpusetadnoisnetrepyhrebmetpesytefasdnasnoitatimilnoisulcnocllarevos™rohtualootlasiarppaytilauqnoitalupopygetartshcraesforebmundnangisednoitnevretnimairohtuadaelsweivercitametsysfoscitsiretcarahcelbatsubcutaneous hydration and medication infusionsdeunitnocelbaileraedivorptonnacdnataienmatekfotceffeylekilemoctuoylekilfonoitacidniottnavudanajsaesodynawolsiecnediveytilauqwol¢ehtfonoitacidnielbaileron¢stceffeebnacniaprecnacnisdioipostroperoslatubnoitacidemssenderdnanoitatirrietissertemfodohserhtldetroperstneveesrevda¢ehtotdetaleryltsomevitingocdnasnoitanicullahrofdetroperecnabrutsidienmatekfosesodrehgihebotthguohttcrnidetroperydutsehtotdetalerylbissopstneveesrevdasuoiresowt¢gurdgestneveesrevda¢dedivorpborraelcnudahniaprecnaclairtlacinilcpuienmateksraeynaemegaeracevitaillapgnittespuobecalpeziselpmasotsetadceddnenajignnnigebdesiversretsigerweiverenarhcoctcr¢ytefasdnassenevitceffeicssdioipoottnavudajtcrlootborenarhcocyrotcarferhtiwstneitaptludalsupsesabatadottnavelerseidutsnasaienmatek][lleb one 101371 pone0237572 august one 0cdnadetimilsawsemoctuomretgnolfognitroper¢evisulcnocnisawecnedivefoytilauqllarevotaslairtoteudwolyrevotwolesicerpmisetamitseemoctuoborsuoiresyrevrosuoiresesuairetircnoisulcxeevitcirtserfokcaldnastnemerusaemloqdetadilavnonfoatadfognitroperaimeassalahthtiwelpoepsiofdnehwrojamrodetacidniartnocetauqedanifoesuroetuorsuonevartnirosrotalehcnorilarorehtorosuoenatucbusrehtieybdehsilbatseehtsniamerhtobcaidracesreverottnemtaertnorioteudnoitcnufsydtnemtaertofddeifisnetni¢daolrevohtiwderapmocenorpirefeddetaertstneitapnidnaofdenorpirefeddenibmochtiwhtiwderapmocofddnahtiwdetaertstneitapnienolaofddesaercnierastneveesrevda¢ecnerehdasevorpmiyparehtofdfoicsotderapmocnoitalehclarofiniatrecnu¢foksirehtsecuderyparehtnoitalehclarofiniatrecnu¢ofdfoicsotderapmocstceffeesrevdasuoiresytilatromsecuderyparehtofdfoicsotderapmocnoitalehclarofiniatrecnu¢otdesulootbordetadilavon¢elbaliavaivdehcraessesabatadylno¢niaprecnacyrotcarfertnemtaertrofnoitpollamsfonoitatimilgehtiwseidutsforebmunwefseziselpmasllamsesreviddnastcrdednilblseigoodohtem“egnarseidutsseidutsfoytilauqssessaelbaivaebyamenmateki¢“ecnediveehtfoytilauqehtcirtaideaprofeziselpmasicsrofonoitaredisnocasedulcni“egnarstludascirtaidepseidutsydutslebalnepolairtdellortnocnustroperesacweiverevitcepsortertcr¢¢¢¢¢atadlevelhgihdetimli¢erasegasoddednemmocer¢loottnemssessaonhtiwnerdlihcdnastludasesabatadicsottnavelerseidutstnemtaertienmatek][ualderbsnoitatimilnoisulcnocllarevos™rohtualootlasiarppaytilauqnoitalupopygetartshcraesforebmundnangisednoitnevretnimairohtuadaelgnitarotelbacilppaseidutsseidutslatotweiveremoctuo]noitatic[deunitnocelbatihgkgmotmorfnoissucsiddetroperelpmasniapdetalerrecnacrnsetadstludaytefasdnassenevitceffeweivernidaolrevonorignitaertrof”ksirwol”ksirraelcnutnednepednoisufsnartlacidemrehtoenorpirefedderapmocnidedulcnislairticsylno¢detacidnillitssienorpirefed¢lootborenarhcochtiwnerdlihcdnastludalsupsesabatadseidutstcrsusrevenorpirefed][rehsifrofpunidnaspuehtrofsraeydnanaemhtiwsrydnaydutsydutsyadcinilctneitaptuognittescirtaideapertneceraclatipsohelpmasaimeassalahtdnasesabatadnienolaofddnaenola“egnaregaezisseirtsigerlairtslairtesehtfosryydutsrnydutsotpusetadsuoenatucbusdetropernisryfonaemhtiwhcramofdfonoisufniienmaxoirrefseddnaytefasofdssenevitceffesubcutaneous hydration and medication infusionsdeunitnocdnaytidilavehtniecnedifnoc¢lduohsyparehtpmupfoesu¢siecnedivefoytilibicudorperhcraeserehtotdetimilebwolgnittesfoytefasgnidragersnrecnoc¢tsisrepyparehtsmrahfoelacstnemssessalanoitanadnasetisretnecsgnitarsaibfoksirllarevomargorplatanireptneitaptuoehtmorfsmetidetcelesdessessasaibnoitirttaytilauqretsammcelgnissgnittes±egareva“egnarrneziselpmas”muidem“hgih”wolesabataddnanoitcetedecnamrofrepsnoitcartnocmreterp“setadtrohocevitcepsortertrohocevitcepsorpseiresesacdezimodnarnonlairttcr¢¢¢¢¢ssenevitceffednaytefassisylocotrofpmupnoitcelesgnidnuofnochtiwnemowtnangerpsesabatadseidutsybenilatubretfoics][teduagegaeziselpmasgnittes“egnarcinorhccinilctneitaptuolatipsohcirtaidepscinilcertnecitlumertneceraclootborenarhcocrostnecselodanerdlihclsupsesabatad“ksirhgihraelcnuesaesidllecelkcishtiw”wolraelcnustludadnasrevigeracriehtlacidemrehtodnasesabatadstcrseidutsotecnerehdaevorpmitcrnoitnevretninoitacidemotsnoitnevretninoitacidem][nitrofaimeassalahtyraurbefyparehttnednepednoisufsnartnonotpusetadnoitalehclarohtiwssenevitcefferotnednepednoisufsnartseirtsigerlairtofdfoicsderapmocyparehtnoitalehcnori one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsehtetacilpmocseirtnuocstluserfonoitazilarenegtnereffidnistcudorpgicshtiwloqhdevorpmiotsanoisulcnocmrifon¢emohsawtirehtehwtahtesreptnemtaertdetadtuosiseidutsredolehtnistsocfonoitamitse¢wolhtiwderewoprednudnahtiwelpoepnitceffelaicifenebdengisedylrooperewseiduts¢foecnedivetnacifingison¢llasedulcnisisylanaatem¢enietsyclytecafostifenebon¢snoitnevretnifosepytshtnomtalavivrusnidnastnapicitrapfosrebmunnoitarudtrohsfosla”thgieemitforeptrohsdetareloteradebircsedstceffeataddetimilsuhtskeewstneitapybllewylbanosaerrevostceffeedisrolavivrusnoefildnasuoiresyllaitnetoparevodemrofreperewseiduts¢edisehtfoemoshguohtla¢repregnolanacsnoitacilpmocignnetaerhtecnediveytilauqdetimliderohtuadnaderosnopsstroperdesimodnarnonyrtsudniseidutsllahtiwniaprecnacfotnemtaertehtnistifenihpronerpuberehwyasottluciffidecnediveytilauqwol¢¢¢¢ehtotdetalerylniamruccohtiwmetsysyreviledgurdnoitartsinmdaisuonevartnierevestoneraohwsmotpmyssuonevartnieriuqerothguonegnitaertnielortnatropmietaredomhtiwstneitapnaevahyamlinitsorpertcs¢nilcycatsorpdetimillebduohsicssngisedsisemerepyhelbatcartniotydutsytilauqwoloteud¢eromotevisnopsernutnemtaertlanoitnevnocenihpronerpublaugnilbus¢feilerniapfotesnoretsaflamredbusotderapmocenihpronerpubdeunitnoccseraseidutsrehtofiraelcnu¢tneitap”seiduts¢icsrosnoitcejnivirevocsrofecnereferpsdioipognortsfeilerniapfonoitarudralimisnisecnereffidtnacifingisonsea¢¢emitdnaemohtacsesuacebgnivasnoitcafsitasecneirepxetneitapdnaecnerehdaecneulfnilliwtnemtaertderreferptneitap¢viseliforpytefasdnaycaciffeelbarapmocdewohsyduts¢acpcsehtfoseicilopecirptnereffidesodnoitutitsbuscsehtlwo“cimonocehtlaehgnirusaemfosdohtemesrevidgicsdnagivineewtebseahtiwwolecnedivefoytilauq¢niecnereffidfoecnediveelttil¢snoitcefnignitroperdnanoitutitsbusecnarusnidnahtlaehlanoitansatluciffidnoitartsinmdaviidnaseirtnuocfosmetsysetaluclacotdesusdohtemowtneewtebsecnereffidrojam¢dnacsneewtebsnosirapmoc¢etaredomlanoitavresbowefmorf“egnarlwo“lanoitavresbodetropertahtseidutsetaredom“tcrtnemssessarneziselpmasycneicifedlicnuochsidewsedargroyramirphtiwstneitaplygoonhcethtlaehnoenummiyradnocessesabatadrnsetadgnitarsnoitatimilnoisulcnocllarevos™rohtualootlasiarppaytilauqnoitalupopygetartshcraesforebmundnangisednoitnevretnimairohtuadaeldeunitnocelbattubertnecitlumsgnittesdnadehsilbupeganaematadrnscificepsdehsilbupnuenilesabemoctuoairetirccitsongaidksiretauqedapuwolloffossenetelpmocdnasecnereffidgnidnilbdnasraeynaemegaeziselpmasrngnittesseidutslatnemirepxeisauqrostcr¢ssenevitceffetnemlaecnocksirraelcnusisorelcslaretal“setadicsobecalpsvtnemtaerthcaorppaborenarhcoccihportoymahtiwstludasesabatadottnavelerseidutstnadixoitnacs][llerroelacstniopdadajehtdesueziselpmaslairttnavelerrofstnemssessalanoitiddahppinoisnetrepyhdetcatnocerewnoitacollafotnemlaecnoclyranomupyramirpseinapmocgurdobecalpsvlinitsorpertniseugolanastissenevitceffe][othcaorppaenarhcoccihtapoidihtiwstludasesabataddeidutsstcrfoenoronilcycatsorpnayahtomarapdetroperecnedivefolevelaesuanhtiwnemowtnangerpesabatadseidutsedimarpolcotemcsnnamhcier”iiievel”iiileveleziselpmasroetadonsetadgnitimovdnaralohcselgoogdehctamdezimodnarnonevitcepsorter¢otnortesnadnodnadnaaesuantaert][“egnarrngnittessnoitcirtseregaugnalesacevitcepsorter¢seiresydutstrohocssenevitceffegnitimovairetirc“setadrofksirhgihotraelcnuniaprecnacsecruoserrehtoicsytefasdnassenevitceffe][nesnahborenarhcochtiwnerdlihcdnastludasesabatadottnavelerstcrenihpronerpubtdimhcswol”e“egnarnaemegaroilanmodbaevitceleyregrusytimertxelatipsohgnittesytefasytilibatpeccavisvcsacpeziselpmas“setadicsottnavelergenoitartsinmdaiborenarhcocstludarnsesabatadstcrseidutsnoitacidem][renotsegaynaegarngnittesotelbacilppaseidutsseidutslatotweiveremoctuo]noitatic[seidutsnilubolgonummicsnamgnlilanoitavresboseidutstcr¢¢cimonocehtlaeh¢seidutsycneiciffeytefasssenevitceffevisusrev][emmarf one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsdeunitnocsaibfoksirregralrofylisaedeveihcaotderapmocdiulffosemuovlsisihtfitubsetuorrehtonahtsuoessoartniehtelbissoptonerasetuorsuoenatucbusdnasevitanretlaelbaiveraohwstneitaprofelbatiustubdetardyhedylerevestonsessoldiulfgniognonehwekatnilaroybtemebtonnacebyametuorsuoenatucbus¢eraatadytefasdetimli¢tcrderosnopsyrtsudni¢elbaliavagnittesdeehtnitahttseggussdiulfcserapmocseidutson¢vitsniagaenolalaitnetopevahslairtgnitsixe¢ynapmoclacituecamrahpaybderosnopssaibfhrgnicudorpotroirefninonerasdiulfcshtiwnoitardyhedetaredomenilrofsemitnaemrekciuqotdlimfosesacniivitnerapretaergdnatnemecalpnoitcafsitasrevigerachcihwsnoitcaeretisnoisufnidecneirepxestneitaptsom¢rehtruftuohtiwdevorpmisuoiresonnoitnevretnistneveesrevdalufsseccusdetroperseiduts¢csfhrhtiwnoitardyherenolasdiulfsraey”htmcirtaideapsrynaemhtiwsraeytonsetaddetimilelgoogfodeepsdnaesufoesaeytilibailersdohtemnoitresnihgihraelcnutaslairtderewopfitsebsisseccasuonevartnisaibfoksirhgihraelcnustluda“stcregatnangilamnonniylralucitrapedometismsinahcemmrofniotsnoitalupopybemuovdnalycneuqerfecnedivetsuboron¢ehttroperrednuseiduts¢ecitcarpsinoitardyhlaicifitrahcihwfoyticuapagnitaercdedivorpybecnadiugdesabecnediveecitcarpothcihwemmargorplatsop“eziselpmas”wolnsrotcodfoyevrus”muidemevitaillapsgnittesega“hgihytinummocredolctlgnittesllaweivercitametsyslairtdellortnoctcrevitcepsorpyevrusevitcepsorterevitarapmocevitpircsed¢¢¢¢¢¢¢noitarudsnoitacolepytdnaytitnauqsllikslasiarppalacitircnoitalupopssenllidecnavdasetadsesabatadseidutssmsinahcemics][tabrofgnitarotelbacilppaseidutsseidutslatotweiveremoctuo]noitatic[cdhsisylcomredopyhsdiulffonoisufnicssnoitatimilnoisulcnocllarevos™rohtualootlasiarppaytilauqnoitalupopygetartshcraesforebmundnangisednoitnevretnimairohtuadaeldeunitnocelbatsnoitadnemmocerevitinifeddetsissayllacidemfoesumrofniotecnedivedetimli¢ehtnitifenebtnacifingison¢eracevitaillapninoitardyhstneitapksirraelcnuhgihscpksirhgihtcrksirwolscprofelacseracevitaillapgnittesrnegakcnrilootborenarhcocetadpusihtrofstcreziselpmashcramrecnacsetadydutsevitarapmocevitcepsorpscptcr¢¢dnaloqnoitardyhefilfohtgnelrofelacsytilauqdrofxodecnavdahtiwstneitaptludasesabatadicsottnavelerseidutsdetsissayllacidem][doogecnedivefoytilauqdetimli¢otecnedivetneiciffus¢yletauqedafokcaloteudlarehpireptahtedulcnocyltsomdahstcrllascirtaideapdnasetebaiddnastsilecnereferstcrsseccalaretnerapborenarhcochtiwstneitapstludaredolsesabatadtnavelerseidutstnereffidfonoitresni][rekroopdnaseiresesacesacdnatrohocytilauqseidutslortnoctrohoclaudividnibeziselpmasdnalacidemrehtoytilauqwolgnidulcniycnegremelatipsohsgnittesseirtsigerslairttcrtnemtrapedrnsetadtolipertnecitlumseiresesactcr¢¢¢noitardyhedetaredomslevelmbeccirtaideaplsupesabatadseidutsrofivisvics][rakiram one 101371 pone0237572 august one 0chtiwlyamedirohcmuissatopotdeddaylefasebnoituacstseggusecnedivedetimli¢icsrofdetiusyllaediebyamcdh¢noitardyhedfotnemtaertehtodsecnalabmicilobatemsadetcerrocebotevahtonyletaidemminodesabetuorrehtiegnisuylralimisdebrosbasidiufl¢stcrgnitaertrofnoitponasicdh¢stneitapnehwnoitardyhedekatnilaroetauqedanievahstceffeesrevdasnoituosleerfetylortcelediova¢etomorpotesadinorulayh¢sniamernoitprosbadevlosernufonoitaredisnocasedulcniecnediveehtfoytilauqehtroopsa“egnaregarngnittesseidutselbaliavafoytirojam¢etylortcelehtiwcdh¢ytilauqroopfoeraefassisdiulfignniatnocnoissucsiddetropereziselpmas“setadloottnemssessaonnoitardyhedhtiwstludaesabatadseidutsytefasnoitardyhcs][nohcorseiresstroperesa
Colon_Cancer
n6methyladenosine m6a regulators are involved in the progression of various cancers via regulating m6amodification however the potential role and mechanism of the m6a modification in osteosarcoma remains obscurein this study wtap was found to be highly expressed in osteosarcoma tissue and it was an independent prognosticfactor for overall survival in osteosarcoma functionally wtap as an oncogene was involved in the proliferation andmetastasis of osteosarcoma in vitro and vivo mechanistically m6a dot blot rnaseq and meripseq meripqrtpcrand luciferase reporter assays showed that hmbox1 was identified as the target gene of wtap which regulatedhmbox1 stability depending on m6a modification at the ²utr of hmbox1 mrna in addition hmbox1 expressionwas downregulated in osteosarcoma and was an independent prognostic factor for overall survival in osteosarcomapatients silenced hmbox1 evidently attenuated shwtapmediated suppression on osteosarcoma growth andmetastasis in vivo and vitro finally wtaphmbox1 regulated osteosarcoma growth and metastasis via pi3kaktpathway in this study demonstrated the critical role of the wtapmediated m6a modification in theprogression of osteosarcoma which could provide novel insights into osteosarcoma treatmentintroductionosteosarcoma is a primary malignant bone tumor thatis common among childhood and adolescents worldwide1despite the improvements including multiagent chemotherapy with surgery in recent years the 5year survival rate is for localized osteosarcoma and is for recurrent and metastatic osteosarcoma23 thereforea better understanding of molecular mechanism isurgent for developing novel therapeutic strategies forosteosarcomacorrespondence jinglei miao miaojinglei126com orjinsong li jinsongli_csu163com1department of orthopaedics the third xiangya hospital of central southuniversity tongzipo rd changsha hunan china2shanghai key laboratory of regulatory biology institute of biomedicalsciences and school of life sciences east china normal university shanghai chinafull list of author information is available at the end of the edited by a stephanouitn6methyladenosine m6a is the prominent dynamicmrna modification which is involved in various biological process by regulating mrna translocation translation and stability4is catalyzed by m6a writermethyltransferase removed by erasers rna demethylases and recognized by m6a readers involving in variousbiological progression5“ the formation of m6a is catalyzed by a methyltransferase mettl3 and mettl14form a core catalytic complex of methyltransferases that isstabilized by wtap8 recently mettl16910 mettl5zcchc411 and zc3h1312 were showed to play a criticalrole in compositing methyltransferase complex andfacilitating m6a methylation13 the eraser alkbh5 andfto has m6a demethylation activity to remove the m6amodification the reader proteins are from yth familyheterogeneous nuclear ribonucleoprotein hnrp familyand insulinlike growth factor mrnabinding protein the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cchen cell death and disease page of isfamily they recognize the m6a modification to adjustrna metabolisms14dynamic and reversible m6a regulation was reported tobe involved in various physiological and pathologicalprocesses including stem cell differentiation cardiachomeostasis adipogenesis neuronal disorders and spermatogenesis15“ recent years compelling evidence hasrevealed that m6a modification plays an important role inthe tumorigenesis of various cancers20“ for examplemettl3 was reported to play key role in the progressionof colorectal carcinoma25 bladder cancer2627 gastriccancer28 and pancreatic cancer29 ythdf2 was involvedthe progression of acute myeloid leukemia aml viaregulating hematopoietic stem cells hscs activity4 ftowas also reported to play a crucial role in the progressionof melanoma30 breast cancer31 gastric cancer32 andintrahepatic cholangiocarcinoma nevertheless the role ofm6a modification in osteosarcomastill poorlycharacterizedwtap a wilms™ tumor wt1 associated protein33has been reported to be critical in the biological processesincluding g2m transition and premrna splicing34“in addition accumulated studies identified the importantrole of wtap in the progression of various cancers37 forexample wtap function as oncogene in cholangiocarcinoma38 acute myeloid leukemia39 colon cancer40 ovariancancer41 and diffuse large bcelllymphoma42 recentstudies reported that wtap is strictly connected to afunctional m6a methylation complex43 here we revealedthe increased expression of wtap in osteosarcoma tissuewhich was associated with clinicopathological features andpoor prognosis in osteosarcoma patients wtap functionas an oncogenic gene and it promotes the m6a modification and progression of osteosarcoma mechanistically wtap induced the growth and metastasis ofosteosarcoma via downregulating hmbox1 expression inm6adependent manner further investigations demonstrated that wtaphmbox1 regulated osteosarcomagrowth and metastasis via pi3kakt pathway overallthese results imply that wtaphmbox1 may be animportant mechanism of osteosarcoma progression andserve as a novel therapeutically target of osteosarcomamaterials and methodsclinical samples and ethicsonehundredandfour paired fresh osteosarcoma metastatic os sample and nonmetastatic os sampleand normal tissues were obtained from patients withoutradiotherapy andor chemotherapy at the third xiangyahospital of central south university and then immediately frozen in ˆ’ °c for rna and protein extraction orformalxed and paraffinembedded for further usedinformed consent was obtained from each patient or theirguardians and the study were approved by the ethicsofficial of the cell death differentiation associationcommittee of the third xiangya hospital of centralsouth universitycell culture transfection and infectionthe human hfob119 cells and osteosarcoma cell linessjsa1 mg63 hos u2os and 143b were obtainedfrom the cell bank of the chinese academy of sciencesshanghai china the osteosarcoma cells were culturedin dmem with fbs gibco grand island ny usathe hfob119 cells were cultured in dmemf12 df with fbsthe sequences of shrna cloned into plko1 vectorand then shwtapplko1 or shhmbox1plko1 werecotransfected with packing and pax2 plasmids into293tf cells fortyeight hours after transfectionthelentivirus was collected and infected hos and u2os cellsfor h the μgml puromycin was used for screeninginfected cells the primers were listed in table s1western blotproteintechantiythdf2antipi3k ab191606 abcamthe proteins were obtained from cells and tissues beinglysed with ripa buffer and then separated by sdspage the antiwtap ab195380 abcam antihmbox1161231apab220163abcamantippi3kab182651 abcam akt 9272s cell signaling paktser473and antigapdhab181602 abcam were used for pvdf membrane incubating overnight at °c the protein expression was detected by incubating with antirabbit secondary antibodies4051s cellsignalingrnaseq analysis and qrtpcrtotal rnas were extracted from osteosarcoma tissueand cells using trizol thermofisher usa for rnaseqanalysis the library construction and illumina sequencingusing the illumina truseq rna sample prep kit sandiego ca usa for qpcr validation the cdna wasobtained using first strand cdna synthesis kittoyobo finally the mrna expression was detectedusing sybr green biorad california usa all primers were in table s1rna m6a dot blot assay and rna m6a quantificationtotal rnas were extracted from osteosarcoma cells bytrizol thermofisher usa and then nanodrop3000was used for detecting rna quality the m6a content wasdetected by epiquik m6a rna methylation quantification kit colorimetric epigentek usathe polya rnas and ng were spottedonto a nylon membrane ge healthcare for rna m6adot blot assay and then incubated with m6a antibody noabe572 merck millipore usa at °c overnight them6a dots was analyzed using an imaging system biorad usa 0cchen cell death and disease page of meripseq and meripqrtpcrtotal rnas were extracted from osteosarcoma cells bytrizol the dnafree fragmented rnas were incubatedwith magnetic dynabeads bounded antim6a antibodyabcam usa to enrichment the mrna with m6a andthen beads were treated with proteinase k and rna wasextracted for meripseq or validation by qrtpcr theprimers are in table s1cell proliferation assaysthe cck8 cell counting kit8 c0038 beyotimebiotechnology china was used for cell proliferationability44colony formation assaysthe cells were seeded into 6well plates with a density × cells per wells and cultured in dmem medium for weeks and then paraformaldehyde pfa was usedto fix the colonies and crystal violet was used to stain asprevious described45woundhealing assayosteosarcoma cells were cultured for h to reach confluency and then a straight artificial wound wasscraped with a μl pipette tip the cell migration abilitywas measured by photographing the distance at and h45transwell invasion assaysthe transwell chamber bd science bedford mausa was used for invasion assays as previously described1 in brief × osteosarcoma cells were seeded intothe upper chambers and incubated for h the invasivecells were counted and quantified for cellinvasion asprevious described45luciferase reporter assaysthe ²utr of hmbox1 was cloned into pmiglovector promega usa the putative m6a sites bases awere mutated into bases c in ²utr using sitedirectedmutagenesis kit thermo usa the wt and mutplasmids were transfected in osteosarcoma cells and thenthe dualluciferase assay kit promega was used fordetecting the luciferase activity44animal experimentsnude mice weeks male were used for tumor modelall animal care and handling procedures were approvedby the institutional animal care and use committee ofthe third xiangya hospital of central south universitychangsha china for the subcutaneous tumor model ×shwtapshhmbox1 u2os cells seeded into mice via subcutaneous injection tumor volume and tumor weightshhmbox1 orshncshwtap orofficial of the cell death differentiation associationwere measured to analyze tumor growth as previousdescribed46 for orthotopic xenografttumor modelshnc shwtap shhmbox1 or shwtapshhmbox1u2os cells were labeled with a luminescent dye and gfpand injected into the cavity of the tibia of mice thirtydays after injection tumor growth was detected for themetastasis model the cells were injected into the tail veinand the lung metastasis were detected days afterinjection using in vivo bioluminescence imaging systemimmunohistochemistryimmunohistochemistry ihc analysis was performedusing antiwtap ab195380 abcam antihmbox1161231ap proteintech as pervious described26bioinformatics analysisthe geo dataset gse87624 and gse46705 weredownloaded and analyzed by r version httpswwwrproject the different expressed gene wascalculated with edger package and identified by thethreshold criteria of log2 foldchange fc ‰¥ and adjp go and kegg analysis was performed toinvestigate the potential role of genes protein“proteininteraction ppi network was analyzed by the stringdatabasestatistical analysisthe spss spss inc chicago il usa was usedfor the statistical analyses using anova or student™s ttest in this study kaplan“meier analysis was used forsurvival the correlation between wtap and hmbox1were analyzed using pearson analysis statistical significance was defined p resultselevated wtap is associated with poor prognosis ofosteosarcoma patientsto reveal the important role of m6a modification inosteosarcoma we explored the expression levels of m6arelative genes in osteosarcoma tissue and normalbone tissue gse87624 normal and osteosarcomaas shown in figs 1a and s1a we examined the expressionof m6arelated genes in geo database and found thatwtap rbm15 ythdf1 and ythdf2 were differentlyexpressed in tumor tissue compared with control tissuethe m6a writers were reported to play key role on theprogression of various cancers so wtap was selected forfurther analysis next we detected the expression ofwtap in pairs of osteosarcoma tissue and the corresponding paratumor tissues from the third xiangyahospital of central south university txhcsu consistent with the geo results the significant higher mrnaand protein levels of wtap in osteosarcoma was detectedusing the qpcr and wb analysis fig 1b c moreover 0cchen cell death and disease page of fig the expression levels of wtap in osteosarcoma tissue and cell lines a wtap expression in osteosarcoma tissue compared with normalbone tissue from gse87624 b qpcr assay and c western blot assay revealed the wtap expression in osteosarcoma tissue and the correspondingparatumor tissues from txhcsu d the kaplan“meier survival analysis e univariate and multivariate survival analysis f establishment of the overallsurvival nomogram for osteosarcoma patients g timedependent roc analysis h the mrna expression levels of wtap in osteosarcoma cell linesand hfob119 cell line using qpcrthe osteosarcoma patients with high wtap showed pooroverall survival in txhcsu fig 1d and the univariateand multivariate cox analysis showed that wtap andmetastasis were the independent prognostic factor foroverall survival in osteosarcoma patients fig 1e theprognostic nomogram wasthepredictusedtoprobabilities of overall survival rates of osteosarcomapatients and the calibration curves showed has a goodconsistency between the prediction and the actual observation fig 1f in addition the years roc curve aucof wtap in osteosarcoma patients from txhcsu was fig 1g besides the overexpression of wtap wasofficial of the cell death differentiation association 0cchen cell death and disease page of table the association of wtap expression andclinicopathologic characteristics of osteosarcoma patientswtap acts as an oncogene that promoted cell proliferation migration and invasion in osteosarcomacharacteristics case number wtap expressionp valuehmbox1 is potential target of wtap in osteosarcomagendermalefemaleage‰¤tumor size‰¤ cm cmmetastasisyesnotnmiiiiiiivhighn lown p p p p p associated with tumor size metastasis and tnm stagetable and fig s1b finally the higher expression ofwtap was also detected in osteosarcoma cell lines sjsa mg63 hos u2os and 143b compared with that inthe human osteoblast hfob119 cell fig 1h collectively these results demonstrated that wtap is highlyexpressed in osteosarcoma and is correlated with its poorprognosissilenced wtap significantly represses osteosarcomaprogression in vitroto generateto further clarify the role of wtap in osteosarcomashwtap1 andwe next used shrna lentivirusshwtap2stable wtapknockdownosteosarcoma cells and analyzed the role of wtap oncells migrationinvasion and proliferation of osteosarcoma the shwtap1 and shwtap2 significantlyknockdown the expression levels of wtap in osteosarcoma cells fig 2a the cck8 assay and colonyformation assay showed that wtap deficiency significantly reduced proliferative capacity of osteosarcomacells fig 2b c the transwellinvasion and woundhealing assays showed that the invasion and migrationability of the osteosarcoma were significant reduced bysilenced wtap fig 2d e these results determine thatofficial of the cell death differentiation associationto reveal the potential mechanism by which wtapregulates the progression of osteosarcoma we performedrnaseq m6aseq in shwtapshnc u2os cell andclipseq to revealthe potential genes regulated bywtapmediated m6a modification the rnaseq resultsrevealed downregulated degs and upregulateddegs with logfc and adjp in shwtapgroup compared with shnc group fig s2a geneontology go showed that degs were enriched in neutrophil activation cell cycle and so on fig s2b kegganalysis showed that degs were enriched in metabolismrelated signal pathway fig s2c the m6aseq analysisidentified differentially m6a modification genes inwtapsilenced u2os cell compared with normal u2oscells table s2 the clipseq from gse46705 revealed wtaptargeted rna in wtap overexpressed cellusing parclip technology and then we obtained theoverlapped genes in rnaseq m6aseq and clipseq asshown in fig 3a genes were overlapped in three groupsincluding two upregulated gene slc3a2 casp7 andfour downregulated gene abr hs6st1 cd59 andhmbox1 consistent with these results slc3a2 andcasp7 were significantly upregulated and abr hs6st1cd59 and hmbox1 were significantly downregulated inosteosarcoma tissues from geo gse87624 databasefig s3a these results were also confirmed in theosteosarcoma tissues from our txhcsu data fig s3band then we evaluated the regulation of wtap on thesix candidates in osteosarcoma cells using qpcr amongwhich hmbox1 was the most significantly upregulatedgene in shwtap osteosarcoma cells fig 3b and wasselected for further analysis consistent with the mrnaexpressionthe protein level of hmbox1 was alsoremarkably increased by silenced wtap fig 3c wenext evaluated the relationship between wtap andhmbox1 expression in gse87624 and txhcsu as ourspeculation hmbox1 expression was negatively correlated with wtap expression r ˆ’ in gse87624and r ˆ’ in txhcsu fig 3d moreover weanalyzed the relationship between hmbox1 expressionclinicopathological features in osteosarcoma patientsfrom txhcsu hmbox1 expression was significantlyreduced with tumor size and metastasis table and figs3c and the survival analysis results showed that thepatients with low level hmbox1 had poor overall survival in osteosarcoma fig 3e moreover the univariateand multivariate analysis demonstrated hmbox1 as anindependent prognosticsurvivalp in osteosarcoma patients fig 3f we alsorevealedinof hmbox1expressionfactorfor overallthelower 0cchen cell death and disease page of fig silenced wtap significantly repressed osteosarcoma progression in vitro a the shwtap1 and shwtap2 downregulated wtapexpression b cck8 assay revealed that silenced wtap reduced the cell proliferation ability in osteosarcoma c colony formation assay showed thatsilenced wtap decreased the cloning number of osteosarcoma cells d transwell invasion and e woundhealing assay revealed the inhibition ofsilenced wtap on osteosarcoma cell invasion and migrationosteosarcoma cell lines sjsa1 mg63 hos u2os and143b than that in the human osteoblast hfob119 cellline fig 3g in these results indicated thathmbox1 is a potential target of wtap and is related topoor prognosis of osteosarcoma patientswtap regulates hmbox1 expression via m6a modificationin osteosarcomaas meripseq data revealed different m6a modificationof hmbox1 in nc and wtapsilenced cells we nextanalyzed whether wtap regulated the hmbox1official of the cell death differentiation association 0cchen cell death and disease page of fig hmbox1 is a potential target of wtap a the venn diagram was generated from differentially expressed genes in rnaseq m6aseq andclipseq in gse46705 the expression of the overlapped genes in hos and u2os cells with silenced wtap using b qpcr assay and c western blotassay d correlation analysis of wtap and hmbox1 in osteosarcoma from gse87624 and txhcsu e the kaplan“meier survival analysis f univariateand multivariate survival analyses g wtap hmbox1 expression in osteosarcoma cell lines and human osteoblast hfob119 cell lineexpression in an m6adependent manner using m6a dotblot and rna methylation quantification assay asexpected m6a levels were substantially decreased inwtapknockdown osteosarcoma cells compared controlosteosarcoma cells fig 4a moreover meripqpcrassay showed that hmbox1 was effectively enriched bym6aspecific antibody and enriched hmbox1 wasremarkably decreased by in wtapknockdown cells fig4b therefore we supposed that wtap could regulatem6a levels of hmbox1 according to the m6a rnaseqthe ²utr ofresulthmbox1 and the sramp httpwwwcuilabcnsramppredicted three very high confidence m6a sites at the ²utr of hmbox1 fig s4 to further prove the directedthe m6a modification was attarget role of wtap on hmbox1 with m6a modification we cloned the hmbox1 ²utr containing these m6a sites into pmirglo vectors and then we mutant thebases a into bases c in the predicted m6a sites fig3f in fig 3g the luciferase activity of hmbox1 wassignificantly increased by silenced wtap however theluciferase activity of mut hmbox1 did not affected bysilenced wtap in both hos and u2os cells fig 4cythdf2 is a well know m6a reader and plays animportant role in the progression of several cancers viaregulating mrna degradation figure 1s showed thatythdf2 was upregulated in osteosarcoma tissues ingse87624 in fig 4e ythdf2 evidently upregulated inosteosarcoma tissue and cells we next shed light on theofficial of the cell death differentiation association 0cchen cell death and disease page of table the association of hmbox1 expression andclinicopathologic characteristics of osteosarcoma patientscharacteristics case number hmbox1 expressionp valuehighn lown gendermalefemaleage‰¤tumor size‰¤ cm cmmetastasisyesnotnmiiiiiiivp p p p p expression of ythdf2 in osteosarcoma and the role ofythdf2 on hmbox1 expression in osteosarcoma disappointedly silenced ythdf2 showed no effects onhmbox1 expression in osteosarcoma cells fig 4f suggesting that wtap regulated m6amediated hmbox1expression in ythdf2independent mannertogether these data suggested that wtaprepressedhmbox1 expression via regulating m6a modification ofhmbox1 at its ²utrhmbox1 is involved in wtapmediated osteosarcomaproliferation and metastasis in vitrowe next explored whether wtap promoted osteosarcoma progression by regulating hmbox1 expressionas shown in fig 5a hmbox1 expression was evidentlyincreased by wtap knockdown and was decreased byhmbox1 knockdown in osteosarcoma cells the cck8results showed that the proliferation levels were increasedby shhmbox1 in wtapsilenced hos and u2os cellsfig 5b consistent with the cck8 results silencedhmbox1 also alleviated shwtapmediated repressionof cell proliferation in colony formation assay fig 5cthe similar effects of hmbox1 were also observed inwtapsilenced osteosarcoma cell using woundhealingand transwell invasion assays fig 5d and e in additionofficial of the cell death differentiation associationwestern blot results showed that silenced wtap evidently repressed the expression of mesenchymal markerscadherin and vimentin and induced the expression ofepithelial marker ecadherin which was attenuated byshhmbox1 in osteosarcoma cells fig 5f these datasuggested wtap promotes osteosarcoma cells proliferation and metastasis via repressing hmbox1 expressionhmbox1 inhibits osteosarcoma growth and metastasisin vivowe next verified the role of hmbox1 in vivo byinjecting shnc shwtap shhmbox1 and shwtapshhmbox1 u2os cells to induce subcutaneous osteosarcoma mice model orthotopic xenograft tumor modeland tail vein metastasis model the expression levels ofhmbox1 was significantly upregulated by silencedwtap in subcutaneous osteosarcoma tissue figs 6a ands5 and s6 moreover silenced wtap repressed thetumor size and tumor weight in subcutaneous osteosarcoma mice which was rescued by silenced hmbox1fig 6b we next used a luminescent dye and gfplabeled u2os cells to build an orthotopic xenografttumor model the bioluminescence imaging showed thatthe wtap knockdown reduced the proliferation ofu2os cells in situ while silenced hmbox1 alleviatedthis repression fig 6c to further detect the role ofwtap and hmbox1 on the metastatic ability ofosteosarcoma in vivo u2os cells were injected into nudemice via the tail vein the bioluminescence imagingshowed that silenced hmbox1 alleviated the repressionof silenced wtap on osteosarcoma metastasis fig 6dtaken together these results suggest that hmbox1 isinvolved in wtapmediated tumor growth and metastasis of osteosarcomawtaphmbox1 regulates the proliferation and metastasisof osteosarcoma partly by pi3kakt pathwaythe kegg results identified that pi3kakt pathwayscould be regulated by wtap fig s2b pi3kakt signaling pathway promotes the growth migration andinvasion of various cancers including osteosarcoma4748we hypothesized that wtaphmbox1 was involved inthe progression of osteosarcoma via regulating pi3kaktsignaling pathway as shown in fig 7a phosphopi3k andphosphoakt were evidently repressed by shwtap andinduced by shhmbox1 and shhmbox1 significantlyattenuatedandphosphoakt in both hos and u2os cells ly294002 api3kakt pathwaysreducedshhmbox1induced cell proliferation migration andinvasion in both hos and u2os cells fig 7b cthereforethese results imply that wtaphmbox1regulates the proliferation and metastasis of osteosarcomapartly via pi3kakt pathway fig shwtaprepressedphosphopi3kinhibitorremarkably 0cchen cell death and disease page of fig hmbox1 is negative correlation wtap expression and is associated to the poor prognosis of osteosarcoma a the m6a level in hosand u2os cells with silenced wtap b meripqpcr assay followed by qrtpcr revealed the hmbox1 m6a modification c wildtype or m6a sitemutant hmbox1 were cloned in pmirglo d luciferase reporter assays revealed the target role of wtap on the ²utr of hmbox1 e ythdf2expression in osteosarcoma tissue and cells f the effects of silenced ythdf2 on hmbox1 expression in osteosarcoma cellsdiscussionin the past several years m6a modification is considered as a pervasive internal modification of mrna andplays critical roles in the progression of a variety of humandiseases including cancers20 however the underlyinginvolvement of m6a regulators in osteosarcoma progression is still unclear in the present study we focused onthe role and underlying mechanism of wtap and itmediated m6a modification in the progression andmetastasis of osteosarcoma in this study wtap wasfirstly identified to upregulated which was associated withpoor prognosis of osteosarcoma functionally wtappromoted the growth and metastasis of osteosarcomain vitro and vivo mechanistically hmbox1 was identified as the target gene of wtap and it was regulated bywtap with m6a modification at the ²utr finallywtaphmbox1 regulated osteosarcoma growth andmetastasis in a pi3kaktdependent patternactually wtap was reported as an oncogene in various cancers37“ recent studies reported that wtapis strictly connected to a functional m6a methylationcomplex43 howeverfew study demonstrated theimportant role of wtap as a m6a regulator here weconcluded that wtap is not only upregulated but alsoplays key role on the m6a modification in osteosarcomanotably the aberrant of m6a modification is related toofficial of the cell death differentiation association 0cchen cell death and disease page of fig hmbox1 as a tumor suppressor was involved in wtapmediated progression a hmbox1 expression in osteosarcoma cell with shwtapand shhmbox1 knockdown of hmbox1 effectively alleviated wtapdependent b cell proliferation c colony formation d transwell invasion ande woundhealing assay f the protein expression level of emt transition related protein p various biological processes via regulating mrna stability splicing and translation next we shed light on thedownstream mrna that modified by wtapmediatedm6a modification by combining the data from rnaseqm6aseqshowed thathmbox1 is a potential target gene of wtap subsequently meripqpcr western blotand luciferasereporter assay results revealed that wtap repressedand clipseq theresultshmbox1 expressed with wtapdependent m6a modthe ²utr of hmbox1 thus wtapification atinvolved in tumorigenesis depending on its role of m6amodification although ythdf2 is a wellknown m6areader in several cancers we found that ythdf2 showedno effects on hmbox1 expression suggesting thatwtap regulated m6amediated hmbox1 expression inythdf2independent manner and the m6a readerofficial of the cell death differentiation association 0cchen cell death and disease page of fig silenced hmbox1 attenuated shwtaprepressed osteosarcoma growth and metastasis in vivo a the expression levels of hmbox1 inosteosarcoma mice models b knockdown of hmbox1 effectively alleviated shwtaprepressed osteosarcoma growth in mice c the orthotopicxenograft tumor and d lung metastasis were detected using a vivo bioluminescence imaging system representative images and a histogram areshown n each groupwhich was involved in the m6a modification of hmbox1need be further investigatedhmbox1 a homeobox containing protein49 wasreported to be a transcriptional repressor involving in thebiological processes in bone marrowderived stromacells50 nk cells5152 and vascular endothelial cells53recent studies demonstrated the dysregulated hmbox1in various cancers for example high expression ofhmbox1 was observed in gastric cancer and it wasassociated with the poor prognosis of gastric cancer54moreover hmbox1 also observed as tumor suppressorin ovarian cancer55 and cervical cancer56 hmbox1repressed the progression of ovarian cancer via regulatingcell proliferation and apoptosis55 hmbox1 repressedliver cancer progression via regulating autophagy as wellas and immune escape57 consistently hmbox1 isofficial of the cell death differentiation association 0cchen cell death and disease page of fig wtap hmbox1 was involved the progression of osteosarcoma via pi3kakt pathway a western blot analysis for the expression ofppi3k pi3k pakt and akt in osteosarcoma cells b cell proliferation c colony formation transwell invasion and woundhealing assay ofosteosarcoma cell treated with μm akt inhibitor ly294002 abcam ab120243 for hunclear how hmbox1 as a transcriptional repressorregulates pi3kakt pathway nonethelesssilencedhmbox1 only partly alleviated wtapmediated osprogressionthere are other potentialmolecular mechanisms regulated by wtapmediatedepigenetic modulation in osit means thatin summary we identified the elevated wtap inosteosarcoma and which is associated with poor clinicaloutcome and serves as an independent prognostic factorfor osteosarcoma patients wtap dramatically promotedosteosarcoma proliferation and metastasis via regulatinghmbox1 mrna stability in a m6adependent mannerwtaphmbox1 regulated osteosarcoma growth andmetastasis via pi3kakt pathway altogether our resultsdetermined wtap hmbox1 as a potential therapeutictarget for osteosarcomaacknowledgementsthis study was supported by national natural science foundation of chinagrant nos national natural science foundation of china grantnos natural science foundation of hunan province grant nos2018jj2617 natural science foundation of hunan province grant nos2016jj3176 national key research and development program of china no2016yfc1201800 natural science foundation of hunan province no2018sk2090author details1department of orthopaedics the third xiangya hospital of central southuniversity tongzipo rd changsha hunan china 2shanghai keylaboratory of regulatory biology institute of biomedical sciences and schoolof life sciences east china normal university shanghai china 3fourgynecological wards ningbo women and children™s hospital ningbozhejiang china 4the second xiangya hospital of central southuniversity changsha china 5school of basic medical science central southuniversity changsha china 6department of anatomy histology andembryology changsha medical university changsha chinafig a schematic model illustrating wtapmediated m6a regulationin osteosarcomaevidentlydownregulated and closely related to the poor prognosisof osteosarcoma in the present study in addition silencedhmbox1alleviated wtapknockdownmediated repression of osteosarcoma progression whichimplied the import roles of hmbox1 in wtapdrivenosteosarcoma development finally we analyzed thedownstream pathway of wtaphmbox1 in osteosarcoma pi3kakt pathway was reported to an important role in the progression of various cancers includingosteosarcoma58 we found that pi3kakt pathway we
Colon_Cancer
during the last decade green synthesized cerium oxide nanops ceo2 nps attracted remarkable interest in various fields of science and technology this review explores the vast array of biological resources such as plants microbes and other biological products being used in synthesis of ceo2 nps it also discusses their biosynthetic mechanism current understandings and trends in the green synthesis of ceo2 nps novel therapies based on green synthesized ceo2 nps are illustrated in particular their antimicrobial potential along with attempts of their mechanistic elucidation overall the main objective of this review is to provide a rational insight of the major accomplishments of ceo2 nps as novel therapeutics agents for a wide range of microbial pathogens and combating other diseases keywords nanotechnology green synthesis cerium oxide nanops antimicrobial infections biomedicalintroductionnanotechnology has got a remarkable interest in every field of science and technology and is presently considered among one of the leading research avenues it has a multitude of applications in the field of electronics imaging industry and healthcare14 mostly in healthcare it has been exploited in diseases diagnostics treatment delivery and formulations of novel drugs14 it exploits nano size structures with size ranges from “ nm known as nanop nps these nano scale entities have unique physiochemical properties and have been utilized in various fields of physics biology and chemistry5among other nps cerium oxide ceo2 nps have been mostly exploited due to their unique surface chemistry high stability and biocompatibility67 it is mostly used in the fabrication of sensors cells catalysis therapeutics agents drug delivery careers and antiparasitic ointments figure presently ceo2 nps is mostly synthesized via two methods such as physical and chemical79 however these methods utilize toxic reducing solvents posing several threats to the biodiversity and ecosystem moreover the nps obtained with such approaches are toxic and unstable making them less efficient910 thus recently a safe less toxic method has been used by researchers known as green synthesis this method utilizes various biological resources such as plants microbes or any other biological derivative1115 these biological extracts have a rich source of phytochemicals international of nanomedicine “ nadeem this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomterms php and incorporate the creative commons attribution “ non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cnadeem dovepresslatter is extensively utilized for its biomedical pharmacological and food applications due to their safe and biocompatible nature9 moreover features like high yield everlasting stability and better morphologies can be obtained using a greener approach79green synthesis from plantsgreen syntheses of ceo2 nps have been reported using plant extracts microbial and other biological derivatives plants in this regard have been the most efficient source due to their abundance safe nature and rich source of reducing and stabilizing agents2629 various parts of plants such as leaves flower and stem have been used for the synthesis of ceo2 nps163031 till date the majority of green synthesis studies have been conducted on leaves extracts as it is a rich source of metabolites11163233 a broad variety of metabolitesphytochemicals in plant extracts such as ketones carboxylic acids phenols and ascorbic acid are used as reduction and stabilizing agents figure plants based ceo2 nps are produced through a simple approach in which bulk metal salt is mixed with the extract and the reaction completes in minutes to a few hours in ordinary lab conditions282934 the metallic salt solution is reduced into respective nanops via the phytochemicals whose synthesis is confirmed firstly through color change from colorless to yellowish brownish or whitish and then characterized through various spectroscopic and imaging techniques162935leaf extract of moringa oleifera l was used to synthesize ceo2 nps with spherical morphologies and size of nm the synthesized nps showed potential antimicrobial and wound healing properties36 gloriosa superba leaf extract was used as a reducing and stabilizing agent in synthesis of ceo2 nps and has shown potential antibacterial properties37 hibiscus sabdariffa natural extract yielded crystalline ceo2 with a diameter of nm30 spherical shaped nanops of size nm synthesized from gel extract of medicinally important plant aloe barbadensis38 the resultant ceo2 nps showed high antioxidant potential green synthesis of ceo2 nanops was demonstrated using jatropha curcus leaf extract and a monodispersed shape of “ nm10 spherical shaped cerium oxide nanops are synthesized using leaf extract of oleo europaea with a size of nm having high antimicrobial activity against both gramnegative and positive strains of bacteria16 origanum majorana extracts were used to synthesize ceo2 nps having pseudo photocatalytic activity having high figure general applications of ceo2 nanopssuch asketones amines enzymes and phenols which are believed to be responsible for the reduction and stabilization of bulk salts into respective nanops nps1619to date various applications of green synthesized ceo2 nps have been reported such as antimicrobial anti cancer antilarvicidal photocatalysis and antioxidant therapies162022 among other biomedical applications the antimicrobial potential is certainly the most exploited previously it has been reported that ceo2 nps to display their antimicrobial actions through various mechanisms9 but mostly ceo2 nps kill microbes via triggering the production of an excess of reactive oxygen species in cells916 however further studies need to be conducted to fully elucidate the complete mechanism of action here in this review we aim to focus on the following topics arrays of biological resources have been exploited to date for the synthesis of ceo2 nps moreover the synthesis mechanisms along their biomedical applications are discussed with special emphasis on the antimicrobial activitysynthesis of ceo2 npsnanops are synthesized through various physicochemical methods5 however both methods require toxic solvents high temperature and pressure which pose threats to the environment2325 moreover higher cost laborious downstream processing lesser biocompatibility instability and low yield make them further inefficient710 there is a growing need to fabricate nanostructures which have the potential to solve these problems59 presently researchers have exploited the green method to overcome all these challenges5 for instance plants microbes and other biological products have been used as reducing and or stabilizing agents in the fabrication of ecofriendly nps25 ceo2 nps have also been synthesized using various physical chemical and biological methods9 the submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure biosynthesis reduction stabilization and characterization of ceo2 nanopsspherical shape nm ftir confirmed that the reduction is attributed to the presence of different phenolic and flavonoids compounds in the extract18 ceo2 was synthesized using rubia cordifolia leaf fusions spectroscopic and microscopic analysis revealed hexagonal shaped nps having a size of nm the biogenic ceo2 nps also showed excellent anticancer potential33 nano rod size ranges from “ nm ceo2 nps resulted when pedalium murex l was added to the aqueous solution of salt at room temperature having high antibacterial activity32 china rose petal was used as a robust bio template for the facile fabrication of novel ceria nano sheet with a size of about nm39 the deviation in size and morphology noticed among the reported studies might be due to the different influence of reaction temperature ph time concentration of salt precursor or plants extracts and part of the plant being used13272840 moreover plants based ceo2 np™s showed excellent stability at diverse conditions for instance green mediated ceria np™s remain stable at liquid solution changes were observed1336 similarly biogenic ceo2 np™s also showed high thermal stability at high temperature and remained stable for a longer period of time which indicates their long durability and everlasting stability27283341 until now various plants have been used in the biogenic synthesis of ceo2 nps and are shown in table physiochemical and no green synthesis from microbesmicrobes also have an intrinsic potential to synthesize nanops as they are a rich source of secondary metabolites23 among other nanops ceo2 nps with various shapes and sizes have been synthesized in recent years from microbes table green synthesis of ceo2 from microbial species is a simple reliable costeffective and ecofriendly approach42 microbial metabolites such as enzymes proteins and heterocyclic derivatives play a crucial role in reducing and stabilizing of ceo2 bulk salt into respective nps4243 moreover microbiogenic ceo2 nps exhibited improved stability water dispensability and showed high fluorescent properties and were less agglomerated43aspergillus niger extract yielded cubic fluorite nps with spherical morphology and an average size of nm ftir analysis revealed the presence of an hydroxyl group carboxylic group and phenol group which are supposed to be involved in the reduction of nps21 curvularia lunata extract has also been used to synthesize spherical shaped ceo2 nps with size ranges from “ nm color change from white to yellow brown indicated initial reaction the nps were tested against microbial pathogens and showed excellent antibacterial potential spherical shaped ceo2 nps of size ranges from “ nm were made using fusarium solani extract which showed effective growth inhibition and biofilm formation of pathogenic bacterial strains42 shadab ali khan observed the biosynthesis of spherical shaped “ nm ceo2 nps by using a thermophilic fungus humicola capping agent43 the resultant nps were characterized by uv xps pl spectroscopy tem ftir and xrd moreover these nps showed excellent potential in treatment of neurodegenerative disorders such as alzheimer™s and parkinson™s diseases bacterial extract has also been exploited in the fabrication of ceo2 nps for instance bacillus shaped nps with an average size of nm the bacterial mediated nps also showed excellent antioxidant potential in vitro44 despite all these applications the microbial route of synthesis has certain shortcomings such as the high probability of pathogenicity laborious culturing and contamination issues however it offers a lot of promise in the field of nanotechnology and could become a leading avenue in subtilis extract yielded spherical international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable ceo2 nanops made from various plants speciesplant namepartcharacterizationshapesize nmftir groupoh ceoh hoh ocoleafxrd xps tem ftir uv vissphericalflower hrtem sem xrd xps eds ftirceohleafleafleafsem xrd ftir tgasphericalxrd sem tem uv vis ftir tgasphericaluvvis psa ftir xrd xps hrtemrtesphericalco oh hhoh ch nooh ceoseedxrd uvvis ftir fesem tgasphericalceo oceo ch co []acalypha indicaleafxrd sem tem eds ftiraloe barbadensisleafxrd tem ftir psasphericalspherical“ oh ceoh ceoce“ch co ch cf ch cclaloe veraleafftir xps hrtemspherical““ch “ccrubia cordifolialeafuv vis xrd xps sem ftir edaxhexagonalprosopis farctaaerialuv“vis pxrd tem fesem ftirsphericalchina rosepetalfesem fetem afm xrd xpsnanosheetcentella asiatica“uvvis dls sem hrtem edssphericaloh ceooh ceo and no__ walnutshellleafxrd sem tem eds ftiruvvis xrd xps fesem tem hrtem eds uvdrs ftirsphericalspherical“ceo and oh“ceoc oh chstempxrd sem tem ftir plflaky“oh coo and chs nogloriosa superbahibiscus sabdariffaoliveoleo europaeaprosopis jujliflorasalvia macrosiphon boissazadirachta indicaeuphorbia tirucallipetroselinum crispummoringa oleiferaref[][][][][][][][][][][][][][][][][][][][]lemon grassgrassxrd pl tem saed““leucas asperaleafpxrd sem uvvis tem saedmicrosphere“uvvis ir xrd sem temspherical“peeluv vis ftir xrd hrtemspherical watermelonfruit juicecarrageenanpxrd ftir uvvis semirregularpxrd ftir fesem uv“vis and tga dtaspherical___ceo ceoceceo co co ch oh“so3 ceo hoh c“o []continuedsubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem table continued plant namepartcharacterizationshapeleafxrd fesem tem ftirsphericalxrd fesem eds vsmspherical“size nmftir group__ceratonia siliquastevia rebaudianasalvadora persicamorus nigraannona muricatajusticia adhatodas nopxrd ftir uvvis tem fesem edsspherical“0h ch cc ceo co cfruitfruittem xrd uvvisxrd ftir uvdrs fesemirregular_ ___leafxrd sem tem ftir uvdrssticklike“oh ch co no cc co cncjatropha curcusleafxrd tem uvvismonodispersed ˆ’_origanum majoranapedalium murexelaeagnus angustifoliaeuphorbia amygdaloidesleaftem fesem xrd ftirsphericaloh ch coleafxrd ftir uvvis drs semnanorod“oh ch co co cn cn ceoleafxrd sem tem ftirspherical“ch cotem sem xrd uvvisspherical“orangepeelxrd tem ftir uvviscubic structure“coh or corpiper betleleafxrd ftir sem eds xps tem“nh no cnref[][][][][][][][][][][][][]nanomedicine but is yet to be explored particularly these microbial based nps can be used in designing novel fertilizers fabricating sterile surfaces polymers and medical accessories moreover these biogenic nps can also be exploited in disease management drug synthesis and deliverytable ceo2 nanops synthesized from various fungus speciess nomicrobe namecurvularia lunatacharacterizationshapesize nmftir grouptgdta xrd raman pl ftir uvvis and temspherical“_humicola spuvvis xps pls tem ftir xrdspherical“ceo ceocefusarium solaniftir pl tgdta fesem xrd edax tem xps saed clsmspherical“oh cn cocaspergillus nigeruvvis ftir xps xrd tgdta pl temspherical“hoh oco ce oref[][][][]international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepressgreen synthesis from biological productsapart from the synthesis of nanomaterial from eukaryotes and prokaryotes anisms nps have also synthesized from biological derivatives23 they also play a crucial role in the reduction and stabilization of nps2325 in contrast to plants and the microbial approach bioproduct based ceo2 are much safer scalable and have shown excellent biocompatibility4547 for instance egg white protein was used in order to synthesize ceo2 nps having size ranges from “ nm with spherical morphologies48 these nps were characterized and confirmed by uv™s ftir tgadta and pxrd ftir analysis revealed that the phenol ether hydroxyl and amide groups were responsible for the reduction of these nps it also showed a good in vitro cytotoxicity effect towards human periodontal fibroblasts cells agarose is a natural matrix and has been used as a stabilizing and capping agent for ceo2 nps nps obtained have spherical morphologies with a diameter of nm nps were characterized using various methods including uv fesem ftir tgadta pxrd and tgadta techniques as revealed by ftir analysis it was found that the hydroxyl ether phenol and amide groups were involved in biosynthesis45 starch has also been exploited as a novel source for the synthesis of nanoceria with the results revealing spherical shape nps with a diameter of nm12spherical shaped ceo2 nps with a size of “ nm were synthesized from dextran the resultant nps exhibited strong anticancer potential46 gum tragacanth reported by darroudi 49 was used in the biosynthesis of ceo2 nps these nps were monodispersed shape with an average size range from “ nm the ceo2nps exhibited very low cytotoxic effects on neuro2a cell lines making them suitable candidates for various biomedical and pharmacological applications49 some other biological products which have been used for synthesis of ceo2 nps are listed in table despite their biological applications these biogenic nps could be used as a promising candidate in diseases treatment drug delivery and packing food some other products have also been explored for the synthesis of ceo2 nps which are shown in table biological activity of greenmediated cerium oxide nanopsantimicrobial activity of greenmediated cerium oxide nanopsin the last few years nanotechnologybased therapies have been exploited in disease diagnostics treatment and table biomediated ceo2 nanops from different sources of biological productss nonamecharacterizationshapeegg proteinuvvis fesem ftir tgadta pxrdsphericalsize nm“honeyuvvis fesem ftir tgadta eds pxrdsphericalagaroseuvvis fesem ftir tgadta pxrdsphericalstarchdextranpolyethylene glycoluvvis pxrd temtem dls xps uvvishrtem tem uvvis slsdlschitosanxrd hrtem ftir tgadta uvvispectindls fesem edss xrd ftir nmr pl fesem edss uvvissphericalsphericalsphericalsphericalspherical“ ‰¤ ftir groupoh ceo ceoce ch nooh ceo ceoce ceoc nooh ceo ceoce no____oh ch co och3 coc no ceotannic acidftir xps xrd hrtem uvvisiblepolycrystalline _ref[][][][][][][][][]submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure schematic representation of antibacterial activity of ceo2 nanops cell wall disruption cell membrane disintegration free radicals productions loss of protein peptides dna fragmentation vital enzymes inhibition loss of cellular fluids and disruption in electron transportformulations of novel drugs1 for instance the antimicrobial potential of nps has been mostly exploited and has showed substantial outcomes2324 presently ceo2 nps have attracted great interest as an antimicrobial agent in particular against bacterial pathogens151650 the exact mechanism of killing microbes is yet not clearly elucidated however it is proposed that ceo2 nps mostly kill microbes via a massive production of reactive oxygen species ros in cells as shown in figure the bactericidal potential of ceo2 nps is attributed to strong electrostatic properties distinctive morphologies small size and low band energy1652 due to strong electrostatic potential ceo2 nps interact with membrane proteins thiols groups which results in protein denaturation membrane impermeability eventually leads to microbial death4253 figure exposure to ceo2 nps kills microbes via membrane collapse by attachment with mesosomes malfunctioning of cellular compartments and bio anic molecules which ultimately lead to abnormal metabolism and physiology1642 similarly green mediated nps killpathogens in a similar fashion and various biological species have been exploited and tested against a wide variety of microbes1316 table however biogenic ceo2 nps unique morphologies small size and biocompatible nature were found to be more effcient and have the potential to range of pathogenic bacterial species1113151642 moreover it also has the potential to kill both grampositive and gramnegative bacteria but due to structural complexity of gramnegative bacteria™s membranes it is more sensitive against grampositive species1321374754 in antimicrobial in electrostatics between nps and the bacterial wall plant species and wall compositiondifference to differences treat a wide activity the is due international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable various microbes tested against biogenic ceo2 nanopss nosourcemicrobes testedolea europaeafungus aspergillus flavus fusarium solani and aspergillus niger bacterial sps staphylococcus aureus escherichia coli pseudomonas aeruginosa and klebsiella pneumoniamoringa oleiferas aureus and e colicurvularia lunatastaphylococcus aureus streptococcus pneumoniae and bacillus subtilis pseudomonas aeruginosa proteus vulgaris and klebsiella pneumoniaeleucas asperaklebsiella aerogenes escherichia coli pseudomonas desmolyticum and staphylococcus aureusacalypha indicaescherichia coli and staphylococcus aureusannona muricataenterococcusfaecalis staphylococcus aureus klebsiella pneumonia and escherichia coligloriosa superbastaphylococcus aureus and streptococcus pneumonia ecoli proteus vulgaris klebsiella pneumonia shigella dysenteriae and pseudomonas aeruginosaaspergillus nigerstreptococcus pneumoniae bacillus subtilis proteus vulgaris and escherichia colifusarium solanistaphylococcus aureus pseudomonas aeruginosa escherichia coli and klebsiella pneumoniajusticia adhatodastaphylococcus aureus and escherichia colieuphorbia amygdaloidesp acidilacticipectine coli and b subtilisref[][][][][][][][][][][][]resistance to confer due to rapid evolution of the bacterial genome bacteria have evolved to antimicrobial agents5556 thus in quest of a new treatment biogenic ceo2 nps has shown promising results in treating multi drug resistance bacteria and could be a promising candidate against such refractory pathogenesis26 ceo2 nps along other conjugates have been amalgamated with various anic and inanic hybrids to enhance the antimicrobial response1746 similarly biomediated ceo2 nps kills fungi by producing a mass number of free radicals and ros which causes distorted structure and physiology and leads to fungus death16 however a few studies have only been conducted on fungi despite the increasing knowledge on the antimicrobial activity of ceo2 nps much remains unknown about their exact mechanism of encountering bacteria toxicity in vivo studies and environmental concerns which needs to be addressed moreover the low band energy potential of ceo2 nps could be used in fabricating sterile surfaces at hospital or lab settings and will diminish nosocomial and other acquired infectionsother potential biomedical applicationsbeside antimicrobial therapies ceo2 nps have also been used in management of other ailments3557 for instance siliqua showed high biogenic ceo2 nps have been mostly used in treatment of various cancers such as osteosarcoma colon cervical and breast cancers1820274652 results indicated that these nps exhibited strong anticancer potential and could be used as a chemotherapeutic agent thanks to their minimal toxicity capacity to induce apoptosis andor necrosis in cancer cells46 ceo2 nps synthesized from origanum majorana and ceratonia antioxidant activity183157 results showed higher expression of antioxidant enzymes which in turn eradicated free radicals and improved cellular functions31 furthermore antioxidant potential was higher when compared to commercial synthetic antioxidants18 ceo2 nps synthesized from fruit extract of morus nigra exhibited excellent antidiabetic activity on l6 cell lines the treatment was dosage dependent and nps with lesser size resulted in higher uptake of glucose in vitro35 though biogenic ceo2 nps have shown excellent pharmacological potential however the mechanism of action minimum inhibitory concentration and best possible delivery system should need to be determined moreover cytotoxicity and genotoxicity should be tested in vivo models to evaluate the compatibility in both in vivo and in vitro modelssubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem to their unique conclusions and future prospectsin this paper we have reviewed the recent trends and understandings of biogenic ceo2 nps and their pharmacological applications various sources such as plants microbes and other biological products have been discussed with the mechanism of synthesis and their biomedical applications due surface morphologies crystal small nature and biocompatible nature biogenic ceo2 nps have got phenomenal interest in biomedical and other fields for instance it has been used in treating various cancers antimicrobial and antioxidant therapies in particular the green synthesized nanops have shown significant antimicrobial potential against a wide range of bacterial species the mechanism of combating such pathogens have also been elucidated and supposed to be due to the mass production of reactive oxygen species and deactivation of scavenging enzymes the ros impedes the membranes disrupts the cellular compartments and disintegrates the bio anic molecules and hampers the associated functions and ultimately causes death it has also shown promising results against multidrug bacteria and could be a potential antimicrobial agent in future against such refractory pathogens however further studies should conduct in vivo models to reveal the full mechanism alongside any sideeffects moreover it has also shown excellent anticancer and antioxidant potential in vitro setups but their toxicity and dosage are yet unknown which needs to be addressed despite their role in various therapies their mechanism of synthesis needs to be optimized whereas in vivo evaluation as well as toxicity should be further screenedabbreviationsceo2 nps cerium oxide nanops dga differential thermal analysis dsl dynamic light scattering eds energydispersive xray spectroscopy fesem field emission scanning electron microscopy ftir fourier transform infrared spectroscopy hrtem highresolution transmission electron microscopy pl photoluminescence pxrd powder xray diffraction ros reactive oxygen species sls static light scattering tem transmission electron microscopy tga thermal gravimetric analysis uvvis uvvisible spectroscopy xps xray photoelectron spectrometry xrd xray diffractiondisclosurethe authors report no conflicts of interest for this workreferences kubik t boguniakubik k sugisaka m nanotechnology on duty in medical applications curr pharm biotechnol “ doi1021741389201053167248 bhushan b springer handbook of nanotechnology springer jianrong c yuqing m nongyue h et al nanotechnology and biosensors biotechnol adv “ doi101016j biotechadv200403004 smith dm simon jk baker jr jr jr applications of nanotechnology for immunology nat rev immunol “ doi101038nri3488 mohanraj v chen y nanopsa review trop j pharm res “ das s dowding jm klump ke cerium oxide nanops applications and prospects in nanomedicine nanomedicine “ doi102217nnm13133 he l su y lanhong j et al recent advances of cerium oxide nanops in synthesis luminescence and biomedical studies a review j rare earths “ doi101016s1002 walkey c das s seal s catalytic properties and biomedical applications of cerium oxide nanops environ sci “ doi101039c4en00138a rajeshkumar s naik p synthesis and biomedical applications of cerium oxide nanops“a review biotechnol rep “ doi101016jbtre201711008 magudieshwaran r ishii j raja kcn et al green and chemical synthesized ceo2 nanops for photocatalytic indoor air pollutant degradation mater lett “ doi101016jmatlet arunachalam t karpagasundaram m rajarathinam n ultrasound assisted green synthesis of cerium oxide nanops using prosopis juliflora leaf e
Colon_Cancer
" bridge to surgery bts using a selfexpandable metallic stent sems for the treatment of obstructivecolorectal cancer improves the patient™s quality of life this study aimed to examine prognostic factors ofobstructive colorectal cancermethods we analyzed stage iiiii resectable colon cancer cases cur a retrospectively registered between january and december overall patients with cur a obstructive colorectal cancer were evaluated ofthem underwent emergency surgery es group and of them after bts with sems placement bts group wecompared surgical results and prognoses between the two groupsresults a total of patients underwent endoscopic sems placement which technical success of andmorbidity rate of primary anastomosis rates were in es and in bts p postoperativecomplication in es and in bts p pathological findings of lymphatic invasion in es and in bts p venous invasion were in es and in bts p and recurrence of in esand in bts the 3year overall survival was significantly different between two groups es 868bts bts is worse than es logrank test p venous invasion independently predicted worsened recurrencefreeand overall survivals the vascular invasiveness was correlated with tumor progression after sems placement and thesurvival rate was lower in bts sems potentially worsens prognostic outcomes in stage ii“iii obstructive colorectalcancerkeywords bowel obstruction colorectal cancer selfexpandable metallic stent colorectal cancer crc remains the leading cause ofcancerrelated deaths worldwide because several patientsare initially diagnosed during advanced stages correspondence ohtakmedkindaiacjp1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japanfull list of author information is available at the end of the approximately “ of patients with crc were diagnosed with acute colonic obstruction [“] severe malignancy with bowel obstruction needs urgent surgicalintervention which includes primary lesion resectionand stoma creation leading to increased morbidity andmortality and a potential failure to achieve completeoncological resection [ ]an endoscopic procedure with selfexpandable metallic stent sems is an acceptable bridge to surgery bts the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cohta bmc surgery page of treatment for acute colonic obstruction [“] preoperative sems placement provides an opportunity to perform medical resuscitation comorbidity optimizationbowel preparation tumor staging and observation ofproximal lesions the procedure prevents highriskemergency surgeries and increase oncological resectionand primary anastomosis rates [ ] after the inclusion of colonic sems placement as bts in the coverageof the national health insurance in japan several physicians joined the colonic stent safety procedure researchgroup and developed skills to provide safe treatmentthe largest multicenter prospective study demonstratedthe feasibility and safety of sems placement as bts inpatients with malignant colorectal obstruction the oncological safety and minimalinvasiveness ofthis procedure have confirmed that sems placement asa bridge to elective surgery is not recommended as astandard treatment for symptomatic leftsided malignantcolonic obstruction [ ] several studies reportedthat prognostic factors of malignant colonic obstructionin sems placement had oncological disadvantages compared with those in emergency surgery es [ ] incontrast several trials showed that sems placement as abridge to elective surgery did not improve the survivalrates [“] how sems placement worsens prognosticoutcomes remains unclear [ ]this study aimed to evaluate the induction of curativesurgery in patients with malignant colorectal obstructionafter a sems placement and its longterm results andprognostic factors postoperatively compared to patientswithout sems placement we demonstrated prognosticfactors and overall survival os and recurrencefreesurvival rfs rates for curative surgery after a semsplacementmethodspatientsmedical records of patients who underwent primarycolorectal resection at higashiosaka city medical centerbetween january and december werereviewed all participants provided written informedconsent oralintake and symptoms before and aftersems placement were assessed in table using thecolorectal obstruction scoring system cross from to we recruited patients with all class oftable the colorectal obstruction scoring system crosspatient™s symptom and their condition of an oral intakesolid meal low residue and full diet without symptomcrosscross as stent insertion candidate from we excluded patients with cross and based on updatedstent insertion guideline malignant colorectal obstruction was diagnosed through clinical examinationcross radiography and computed tomography surgery was performed using three approaches es comprised laparotomylymph node dissection as possibleand primary anastomosis on the same day between and bts after sems placement comprised standbylaparoscopy d3 lymph node dissection and primaryanastomosis since january overall patientswith stage iiiii cur a obstructive colorectal cancerwere evaluated of them underwent emergency surgery as es group and of them after bts with semsplacement as bts group we compared surgical resultsand prognoses between the two groupssems devices and the procedurepatients were endoscopically treated with placement ofan uncovered wallflex enteral colonic stent boston scientific corporation natick ma usa or nitis enteralcolonic uncovered stent taewoong inc gimpo southkorea placements were performed as presented in thepreintroduction publicity announcement placement details were mentioned on the website as a brief guideline obstruction structures were determined using aguide wire and a contrast tube was inserted into theproximal colorectal lumen obstructions were measuredusing contrast agents and then the endoscopist determined the number size and type of stent pathologicalbiopsies were recommended after sems locations andintraluminal or extraluminal marking using an endoscopic clip were recommended via visual recognition ofthe endoscopist dilatation of the colonic obstructionbefore sems placement was generally not allowedhistological findingsparaffinembedded specimens were obtained from a cohort of patients diagnosed by the union for international cancer control stage ii“iiisurvival definitionsos was defined as the duration from surgery to anydeath or last followup diagnosis of recurrence was calculated based on recist according to the chemotherapy criteria rfs was defined as the durationfrom surgery to any recurrence includes local recurrenceor distant metastasissolid meal low residue and full diet with symptomliquid or enteral nutrient intakeno oral intakerequiring continuous decompressionstatistical analysisstudent™s ttest and wilcoxon test for continuous variables and the χ2 and fisher™s exact tests for categoricalvariables were conducted survival curves were generated using the kaplan“meier method and compared 0cohta bmc surgery page of table baseline characteristics and outcomes of endoscopicsems placementtable comparison of baseline characteristics in patientsundergoing emergency surgery and bridge to surgerybts n es n pmalefemalemedian range “ “genderagelocationmalefemalemedian rangececumascendingtransversedescendingsigmoidrectumlength of obstructionmedian range cmtechnical successprocedurestentingmorbiditymortalityclinical successthrough the scopethrough the wirewall flex cmnitis cmoverall cda iiicda vcrossbts n “ “ a clavien“dindo classificationusing a logrank test univariate and multivariate survival analyses were performed using the cox proportional hazards regression model all statistical analysesused jmp version sas institute cary nc or statistical scripting language r httpwwwrprojectpvalues of ‰¤ twosided were considered statistically significant this prognostic study complied withthe reporting recommendationsfor tumor markerprognostic studies resultsa total of patients underwent endoscopic semsplacement which was technically safe for malignantcolorectal obstruction with the technical success rate of the clinical success rate was and the patient™ssymptoms and oral intake dramatically improved afterthe sems placement shown in table a total of patients were reviewed and patients underwentes and bts respectively as shown in table baselineclinical characteristics were balanced between the twogroups moreover cases of patients underwent es on the same day as in open surgery the median waiting period for surgery was days for bts thegenderagelocationtype of operationcecumascendingtransversedescendingsigmoidrectumstandbyemergency “ “duration tooperationmedian range dayssurgical procedurelaparotomy laparoscopy timemedian range minblood lossmedian range ml“ ““ “ before afterstoma creation “morbidity30day complication cda iii anastomosticleakagehospital stayaclavien“dindo classificationmedian range days “ “primary anastomosis ratios were in es and in bts p postoperative complication rateswere in es and in bts p postoperative hospital stay was shorter in bts days comp patients withpared to esobstructive crc showed significantinpostoperative complication rate and hospital stay withsems placement operative procedures were dramatically changed and the primary anastomosis rate improved after the sems placementimprovement daysthe pathological tissue type accounted for of differentiated types shown in table tumordepth was similar between the two groups lymphatic vesselinvasion ratios were in es and in bts p and venous invasion ratioswere in es and in bts p recurrence rates were cases in es and cases in bts nodenegative patients stage iimorewhereas nodepositive patients stage iii more frequently had liver metastasis in the kaplan“meier survival analysis in fig 1a the 3year rfs waslung metastasisfrequentlyhad 0cohta bmc surgery page of table comparison of pathological characteristics of emergency surgery and bridge to surgerypt factortotal lymph nodespn factorhistologicallymphatic invasionvenous invasionsurgical clearancet4b t4a t3median rangen0 n1 n2 n3tub1 tub2 othersly v cur a b csignificantly different between the two groups es bts which was significantly low inbts than that in es logrank test p the3year os rate was also significantly different between thetwo groups es bts p shown in fig 1b the relationship between lymph node metastasis and sems placementwas also evaluated the pathological nodenegativestage ii 3year rfs rate was not different betweenthe two groups es bts as shown infig 2athe pathological nodepositivestage iii 3year rfs rate was different between thetwo groups es bts as shown in fig 2bthe stage ii 3year os rate was not different between thetwo groups es bts as shown in fig 2cwhereas stage iii 3year os rate was different between thetwo groups es bts as shown in fig 2dthese results suggestthat vascular invasiveness andpathological nodepositive status were correlated withtumor progression after sems placementthein contrastthuses n “ bts n “ p “survival rate was affected by poor prognosis in the btsgroupresults of adjusted multiple cox proportional hazard regression for rfs and os in all stages and stage iii diseaseare presented in table after adjusting for possible confounders venous invasion and bts independently predicted poor rfs in all stages and venous invasionindependently predicted poor rfs in stage iii disease venous invasion and bts were also significantly associatedwith os in stage iii diseasediscussionacute colonic obstruction requires emergent surgicalintervention a mandatory conventional treatment skillemergent surgicalis associated with highmorbidity mortality and stoma creation rates affectingthe quality of life of patients malignant colorectal obstruction is not only an intestinal obstruction but alsoan advanced stage crc their prognosis was poorerthan that in patients with nonocclusive disease becausetreatmentfig kaplan“meier survival curves in patients undergoing emergency surgery vs bridge to surgery a recurrencefree survival b overall survival 0cohta bmc surgery page of fig kaplan“meier survival curves in patients undergoing emergency surgery vs bridge to surgery a rfs nodenegative patients b rfs nodepositive patients c os nodenegative patients d os nodepositive patientsof highly invasiveness and distant metastasis [ ]chen revealed that the prognosis in patients withperforation associated with obstruction was poor early intervention in the clinical setting before the colonic perforation has been established endoscopic placement of colonic stents improves the high decompressioneffect and reduces clinical symptoms high postoperative complication rates were correlatedwith poor prognosis in patients with cancer in severalans [“] reducing complication rates can improve the prognosis our results showed high clinicalsuccess rate after sems placement and high primarysurgicalinterventions howeveranastomosis rate stentrelated complications requiredemergentthe stentplacement is safe and feasible in this study moreoverthe laparoscopic rate was high and postoperative complication rate was clinical results including shortterm outcomesin bts after sems were verifiedthrough a metaanalysis [ ]the prognosis was poor in patients with stent perforation and increased local recurrence rate after the colonic stent placementthe longtermprognosis in patients with colorectal obstruction afterbts was not different compared with that in patients however 0cohta bmc surgery page of table multivariate analysis of recurrencefree survival at all stages and stage iiipvaluevariablesrecurrence free suvivalall stages hazard ratio ± sd cistage iii hazard ratio ± sd cipvaluees vs semspt factor t3t4pn factor n0n1n2“verous invasion v0“1v2“lymphatic invasion ly0“1ly2“hr ± hr ˆ’ ± ˆ’ ˆ’hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ overall suvivales vs semspt factor t3t4pn factor n0n1n2“verous invasion v0“1v2“lymphatic invasion ly0“1ly2“hr ± hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ without obstruction [“] according to the europeansociety of gastrointestinal endoscopy clinical guidelinethat considers the risk of perforation due to colorectalstents only limited uses are allowed therefore colorectalstent placement is not a standard treatment [“]the prognostic outcomes of bts in this study were significantly worse than those of es particularly in lymphnodepositive patientslymphatic and venous invasionseemed to be a significant prognostic factor althoughreduced postoperative complication rate would improve the prognosis our results were contradictoryafter the stent replacement these results suggestedthat stent placement leads to poor prognosis a concern that colonic stents may be associated with adverse effects of mechanical expansion also exists mechanical expansion may be associated with thegrowth of solid tumors particularly lymphatic andvenous invasion [ ]we found that recurrence and os were associatedwith high vascular invasion after a colonic stent placement venous invasion was an independent factor for recurrence and prognosis the ck20 mrna level anepithelial marker is significantly increased in peripheralblood serum suggesting stent deployment into the vasculature alliteratively ki67 level associated withcellular proliferation and p27 gene assisting cell cycleprogression were measured using specimens obtainedbefore and after sems insertion next the ki67 leveldecreased in the specimen after an sems placementcompared with that before and cell proliferation wassuppressed the prognostic nutritionalindex andserum albumin levels were significantly decreased afterstenting suggesting its disadvantage as bts theduration from stent placement to surgery was daysoncological and nutritional factors might change in theblood and contribute to poor prognosis during the waiting period mechanical expansion of the replacement hr ± ˆ’ hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ hr ± hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ should be minimized to prevent perforation and molecular cytological factors to improve the materials expansion and establishment of new mechanism are necessaryin colorectal obstruction [ ]thisisafirstretrospectivethese findings should be considered in light of severallimitationsnonrandomized small sample sized study from a single institution thereby the heterogeneity of the surgical strategy may have affected the prognostic factors secondalthough validated endoscopic procedures were validated stent devices used in this study had differentlengths types and thickness and obtained from differentvendors lastly we performed stent placement in the patients with cross and who are not indicated forstent insertion until to investigate the oncological longterm prognosis ofcolonic sems placement as a bridge to elective surgerylarge sample size and prospective randomized controlledstudies are warranted to develop a treatment strategy forcrc with obstructionvascular invasiveness was correlated with tumor progression after a sems placement and os and rfs rateswere lower in bts sems placement potentially worsensprognostic outcomes in stage ii“iii malignant colorectalobstructionabbreviationsbts bridge to surgery crc colorectal cancer cross colorectal obstructionscoring system es emergency surgery esge european society ofgastrointestinal endoscopy jsccr japanese society for cancer of the colonand rectum recist response evaluation criteria in solid tumors version os overall survival rfs recurrencefree survival sems selfexpandablemetallic stent wses world society of emergency surgeryacknowledgementsthe authors thank for contribution as endoscopic technical adviser kenkonishi md phd from department of surgery hyogo prefecturenishinomiya hospital 0cohta bmc surgery page of authors™ contributionsall authors have read and approved the manuscript ko protocolproject development data collection and management andmanuscript writingediting mi protocolproject developmentmanagement and manuscript writingediting mu protocolprojectdevelopment and data collection and management jm se jm and ikdata collection and management yt and sn data collection ki andtn data analysis and manuscript writingediting mt and ty dataanalysis and managementfundingauthors have no grant support and no financial relationship for this studyavailability of data and materialsthe datasets used and analyzed during this study are available from thecorresponding author upon reasonable requestethics approval and consent to participateall procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional researchcommittee and with the helsinki declaration and its later amendmentsor with comparable ethical standards all participants or their guardians haveprovided their written informed consent and that the study protocol wasapproved by higashiosaka city medical center ethical committee on humanresearch assignment number “consent for publicationno applicablecompeting intereststhe authors declare that they have no conflict of interest to discloseauthor details1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japan 3thoracic surgeryhigashiosaka city medical center osaka japan 4digestive surgery kawasakimedical school okayama japan 5gastroenterology higashiosaka citymedical center osaka japanreceived june accepted august referencesjemal a bray f center mm ferlay j ward e forman d global cancerstatistics ca cancer j clin “ pubmed pmid epub eng winner m mooney sj hershman dl feingold dl allendorf jd wright jd incidence and predictors of bowel obstruction in elderly patients withstage iv colon cancer a populationbased cohort study jama surg “ pubmed pmid pubmed central pmcid pmc45 epub engjullumstro e wibe a lydersen s edna th colon cancer incidencepresentation treatment and outcomes over years color dis “ pubmed pmid epub engcheynel n cortet m lepage c benoit l faivre j bouvier am trends infrequency and management of obstructing colorectal cancers in a welldefined population dis colon rectum “ pubmed pmid epub engcuffy m abir f audisio ra longo we colorectal cancer presenting assurgical emergencies surg oncol ““ pubmed pmid epub eng mcardle cs hole dj emergency presentation of colorectal cancer isassociated with poor 5year survival br j surg “ pubmedpmid epub eng mainar a tejero e maynar m ferral h castanedazuniga w colorectalobstruction treatment with metallic stents radiology “pubmed pmid epub engzhang y shi j shi b song cy xie wf chen yx comparison of efficacybetween uncovered and covered selfexpanding metallic stents inmalignant large bowel obstruction a systematic review and metaanalysiscolor dis 2012147e367“ pubmed pmid epub engtilney hs lovegrove re purkayastha s sains ps westonpetrides gk darziaw comparison of colonic stenting and open surgery for malignantlarge bowel obstruction surg endosc “ pubmed pmid epub engsaito s yoshida s isayama h matsuzawa t kuwai t maetani i aprospective multicenter study on selfexpandable metallic stents as a bridgeto surgery for malignant colorectal obstruction in japan efficacy and safetyin patients surg endosc “ pubmed pmid epub engsaida y sumiyama y nagao j uramatsu m longterm prognosis ofpreoperative bridge to surgery expandable metallic stent insertion forobstructive colorectal cancer comparison with emergency operation discolon rectum suppls44“ pubmed pmid epub engtomita m saito s makimoto s yoshida s isayama h yamada t selfexpandable metallic stenting as a bridge to surgery for malignant colorectalobstruction pooled analysis of patients from two prospectivemulticenter series surg endosc “ pubmed pmid pubmed central pmcid pmc6342866 epub eng chen hs sheenchen sm obstruction and perforation in colorectaladenocarcinoma an analysis of prognosis and current trends surgery “ pubmed pmid epub eng huang x lv b zhang s meng l preoperative colonic stents versusemergency surgery for acute leftsided malignant colonic obstruction ametaanalysis j gastrointest surg “ pubmed pmid epub engkim hj huh jw kang ws kim ch lim sw joo ye oncologic safetyof stent as bridge to surgery compared to emergency radical surgery forleftsided colorectal cancer obstruction surg endosc “pubmed pmid epub engshigeta k baba h yamafuji k kaneda h katsura h kubochi k outcomesfor patients with obstructing colorectal cancers treated with onestagesurgery using transanal drainage tubes j gastrointest surg “ pubmed pmid epub engvan hooft je bemelman wa oldenburg b marinelli aw lutke holzik mfgrubben mj colonic stenting versus emergency surgery for acute leftsided malignant colonic obstruction a multicentre randomised trial lancetoncol “ pubmed pmid epub engvan hooft je fockens p marinelli aw timmer r van berkel am bossuyt pm early closure of a multicenter randomized clinical trial of endoscopicstenting versus surgery for stage iv leftsided colorectal cancer endoscopy“ pubmed pmid epub engsloothaak da van den berg mw dijkgraaf mg fockens p tanis pj vanhooft je oncological outcome of malignant colonic obstruction inthe dutch stentin trial br j surg “ pubmed pmid epub eng gorissen kj tuynman jb fryer e wang l uberoi r jones om localrecurrence after stenting for obstructing leftsided colonic cancer br j surg“ pubmed pmid epub eng ormando vm palma r fugazza a repici a colonic stents for malignantbowel obstruction current status and future prospects expert rev meddevices “ pubmed pmid epub englauro a binetti m vaccari s cervellera m tonini v obstructing leftsidedcolonic cancer is endoscopic stenting a bridge to surgery or a bridge tonowhere digestive diseases and sciences pubmed pmid epub engschwartz lh litière s de vries e ford r gwyther s mandrekar s recist 11update and clarification from the recist committee europeanjournal of cancer oxford england p “ pubmed pmid pubmed central pmcid pmc5737828 epub eng mcshane lm altman dg sauerbrei w taube se gion m clark gm reportingrecommendations for tumour marker prognostic studies remark eur jcancer “ pubmed pmid epub eng hashiguchi y muro k saito y ito y ajioka y hamaguchi t japanesesociety for cancer of the colon and rectum jsccr guidelines for thetreatment of colorectal cancer int j clin oncol pubmed pmid epub eng cortet m grimault a cheynel n lepage c bouvier am faivre j patterns ofrecurrence of obstructing colon cancers after surgery for cure a population 0cohta bmc surgery page of based study color dis “ pubmed pmid epub eng nojiri t maeda h takeuchi y funakoshi y kimura t maekura r predictive value of btype natriuretic peptide for postoperative atrialfibrillation following pulmonary resection for lung cancer eur jcardiothorac surg “ pubmed pmid epub engpubmed pmid pubmed central pmcid pmc4128744 epub engkaragiannis gs poutahidis t erdman se kirsch r riddell rh diamandis epcancerassociated fibroblasts drive the progression of metastasis throughboth paracrine and mechanical pressure on cancer tissue mol cancer res“ pubmed pmid pubmed central pmcidpmc4399759 epub eng nojiri t inoue m yamamoto k maeda h takeuchi y funakoshi y b maruthachalam k lash ge shenton bk han af tumour celldissemination following endoscopic stent insertion br j surg “ pubmed pmid epub eng matsuda a miyashita m matsumoto s sakurazawa n kawano y yamahatsuk colonic stentinduced mechanical compression may suppresscancer cell proliferation in malignant large bowel obstruction surg endosc“ pubmed pmid epub eng haraguchi n ikeda m miyake m yamada t sakakibara y mita e colonic stenting as a bridge to surgery for obstructive colorectal canceradvantages and disadvantages surg today “ pubmedpmid epub eng zhu z li b liao w lv n chen y shu x novel predictive nomogram foridentifying difficult guidewire insertion in patients with malignant colorectalobstruction and sphincterotomeassisted guidewire insertion for improvingthe success rate of selfexpandable metal stent insertion front oncol pubmed pmid pubmed central pmcid pmc7237730epub eng miyasako y kuwai t ishaq s tao k konishi h miura r newlydeveloped selfexpandable nitis md colonic metal stent for malignantcolonic obstruction world j gastrointest surg “ pubmedpmid pubmed central pmcid pmc7215972 epub engpublisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationstype natriuretic peptide as a predictor of postoperative cardiopulmonarycomplications in elderly patients undergoing pulmonary resection for lungcancer ann thorac surg “ pubmed pmid epub eng cowie mr struthers ad wood da coats aj thompson sg poolewilsonpa value of natriuretic peptides in assessment of patients withpossible new heart failure in primary care lancet “pubmed pmid epub eng cuthbertson bh card g croal bl mcneilly j hillis gs the utility of btypenatriuretic peptide in predicting postoperative cardiac events and mortalityin patients undergoing major emergency noncardiac surgery anaesthesia“ pubmed pmid epub engsabbagh c browet f diouf m cosse c brehant o bartoli e isstenting as a bridge to surgery an oncologically safe strategy for themanagement of acute leftsided malignant colonic obstruction acomparative study with a propensity score analysis ann surg “ pubmed pmid epub engtung kl cheung hy ng lw chung cc li mk endolaparoscopic approachversus conventional open surgery in the treatment of obstructing leftsidedcolon cancer longterm followup of a randomized trial asian j endoscsurg “ pubmed pmid epub eng gianotti l tamini n nespoli l rota m bolzonaro e frego r aprospective evaluation of shortterm and longterm results from colonicstenting for palliation or as a bridge to elective operation versus immediatesurgery for largebowel obstruction surg endosc “pubmed pmid epub eng yang sy park yy han yd cho ms hur h min bs oncologicoutcomes of selfexpandable metallic stent as a bridge to surgery andsafety and feasibility of minimally invasive surgery for acute malignantcolonic obstruction ann surg oncol “ pubmed pmid epub engvan hooft je van halsema ee vanbiervliet g beetstan rg dewitt jmdonnellan f selfexpandable metal stents for obstructing colonic andextracolonic cancer european society of gastrointestinal endoscopy esgeclinical guideline endoscopy “ pubmed pmid epub eng ansaloni l andersson re bazzoli f catena f cennamo v di saverio s guidelenines in the management of obstructing cancer of the leftcolon consensus conference of the world society of emergency surgerywses and peritoneum and surgery pns society world j emerg surg pubmed pmid pubmed central pmcid pmc3022691epub eng arezzo a balague c targarona e bhi f giraudo g ghezzo l colonic stenting as a bridge to surgery versus emergency surgery formalignant colonic obstruction results of a multicentre randomisedcontrolled trial esco trial surg endosc “ pubmedpmid epub eng amelung fj burghgraef ta tanis pj van hooft je ter b f siersema pd critical appraisal of oncological safety of stent as bridge to surgery inleftsided obstructing colon cancer a systematic review and metaanalysiscrit rev oncol hematol “ pubmed pmid epub eng domingo s puertolas s graciavilla l puertolas ja mechanical comparativeanalysis of stents for colorectal obstruction minim invasive ther alliedtechnol “ pubmed pmid epub engsuzuki n saunders bp thomasgibson s akle c marshall m halligan scolorectal stenting for malignant and benign disease outcomes incolorectal stenting dis colon rectum “ pubmed pmid epub eng voutouri c mpekris f papageis p odysseos ad stylianopoulos t roleof constitutive behavior and tumorhost mechanical interactions in thestate of stress and growth of solid tumors plos one 201498e104717 0c"
Colon_Cancer
"circadian clocks have important physiological and behavioral functions in humans and other anisms whichenable anisms to anticipate and respond to periodic environmental changes disturbances in circadian rhythmsimpair sleep metabolism and behavior people with jet lag night workers and shift workers are vulnerable tocircadian misalignment in addition non24h cycles influence circadian rhythms and cause misalignment anddisorders in different species since these periods are beyond the entrainment ranges in certain special conditionseg on submarines and commercial ships non24h watch schedules are often employed which have also beendemonstrated to be deleterious to circadian rhythms personnel working under such conditions suffer fromcircadian misalignment with their onwatch hours leading to increased health risks and decreased cognitiveperformance in this review we summarize the research progress and knowledge concerning circadian rhythms onsubmarines and other environments in which non24h watch schedules are employedkeywords circadian rhythm circadian clock entrainment range metabolism alertness submarine most anisms living on this planet possess circadianclocks that orchestrate their daily physiological and behavioral rhythms to the 24h rotation period of the earth however under special conditions in which thecycling periods do not extend over h the circadiansystems may be challenged in the laboratory manualnon24h conditions are established to study the basicfeatures of circadian rhythms although such conditionsare very rare in nature however in human societies asubstantial group of people lives or works under non24h schedules the non24h conditions were observedto lead to a decrease in adaptability to the environment correspondence guojinhumailsysueducn1key laboratory of gene engineering of the ministry of education state keylaboratory of biocontrol school of life sciences sun yatsen universityguangzhou chinafull list of author information is available at the end of the in all tested anisms across different kingdomscluding bacteria fungi plants and animalsinthe circadian clocks are well conserved across speciesthus we will first introduce the knowledge derived fromcircadian studies in model anisms and the next focuson summarizing studies in humans the former partwould help to elucidate circadian misalignment in circadian rhythms metabolism sleep and the cognitive andbehavioral consequencescircadian clock and circadian rhythmscircadian clocks govern life processes at multiple layersincluding the molecular cellular anism and integrallevels at the anism level circadian clocks are hierarchically composed of master clocks located in thepacemakers and peripheral clocks located in other tissues in mammals the pacemaker is the suprachiasmaticnucleus scn in the anterior of the hypothalamus inbirds and reptiles the eyes pineal gland or scn are the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cguo military medical research page of range circadian rhythms can be adjusted to match theenvironmental periods a process that is termed entrainment or synchronization circadian rhythms are both robust and flexible within a certain range tcycles thatdeviate from the natural 24h cycles can also inducerhythms with the same periodicity as the environmentin this scenario the endogenous circadian periodicitymay be masked by the non24h tcycles the factorsthat can entrain circadian rhythms are known as zeitgebers synchronizers or time givers [ ]to experimentally determine the entrainment rangesanisms are exposed to a series of fixed tcycles radually changing tcycles in addition the entrainment range to an environmental cue can be inferredby the phaseresponse curve prc information of thespecies given that according to the prc of a speciesthe maximum phase delay and maximum phase advanceare x hours and y hours respectively the entrainmentrange of this species to the indicated cue would rangefrom h “ y to h x hours prc representsthe nonparametric effects of zeitgebers on entrainmenthowever parametric components are also involved indetermining the entrainment range the parametricmodel assumes that light changes the velocity of the circadian rhythms for entrainment while the nonparametric model emphasizes the phase shift theentrainmentranges differ between specieshumans have a circadian clock with an endogenousperiod τ of approximately h [ ] with upper andlower limits of and h respectively in conjack bean canavalia ensiformis displayed leaftrastmovement rhythmicity synchronized to lightdark ld but not ld suggesting that the lower entrainment limit is between and h fig 2a somespecies such as bakery mold neurospora crassa andhouse sparrow passer domesticus show very wide entrainment ranges in banding rhythms of conidia releaseconidiation fig 2b and locomotor rhythms respectively [ ]circadian rhythms run free when the t cycles are beyond the entrainment ranges under ld squirrelmonkeyscore bodytemperature cbt rhythm with a period of hsaimiri displaycebidaeacircadian pacemakers depending on the species [“]the peripheral clocks have their own circadian rhythmsbutthey can also be synchronized by pacemakersthrough the nervous and endocrine pathways at the molecular level circadian rhythms are generated by a set of core circadian genes which constitutetranscriptiontranslational negative feedback loops despite differences in circadian clock genes the regulatory mechanisms are highly conserved across kingdoms[ ] in mammals including humans positive circadianelements include brain and muscle arntlike protein bmal12 and clock and the negative elements include period per “ and cryptochromecry reverbs ˆ’α and and retinoic acidreceptor rarrelated orphan receptor ror ˆ’α ˆ’and Î are additional regulators in mammalian circadiancircuits which act as ancillary loops to regulate bmail1expression reverb proteins inhibit while ror proteins activate the transcription of bmal1 [ ] at thetranscriptomic level circadian clocks regulate the expression of approximately “ of proteinencodinggenes in mice [ ] via the output pathways circadianclocks control most of the physiological and behavioralprocesses in mammals circadian clocks govern thecircadiandiurnal rhythms in sleepwake cycles feedingfasting control metabolism hormone secretion and immunity function fig although circadian rhythms are endogenous they areto the influence of environmental geneticsubjectphysiological and pathological factors fig [ ] ithas been demonstrated that disruptions in circadianrhythms impair adaptability in a variety of anisms in humans compromised circadian rhythms causechronic health consequences including an increased riskof gastrointestinal illness loss of bone mineral densitycoronary artery disease endocrine disruption metabolicsyndrome and cancer moreover disrupted circadianrhythms also affect emotions cognition and performance fig [“]entrainment range of circadian rhythmsthe cycling periods of the environment such as lightand temperature are defined as tcycles in a certainfig circadian rhythm disturbance and their consequences 0cguo military medical research page of fig circadian rhythms of different anisms in non24h conditions a leaf movement alternatively opening and closing rhythms ofcanavalia ensiformis c ensiformis b conidiation banding rhythms of n crassa neurospora is inoculated in a long and hollow glass tube withmedium inside which is called a race tube in the race tube neurospora grows towards the other end and releases conidiation bands coloredorange the release of conidiation is controlled by the circadian clock only the left part of the race tubes is shown c rectal temperature rhythmsof c saimiri neurospora conidiation rhythms are entrained in ld ld and ld while canavalia leaf movement rhythms andsubmarine crew rectal temperature rhythms were not entrained in ld therefore they have different entrainment ranges panel a was drawnaccording to the study by panel b was drawn according to the study by and panel c was drawn according to the study by instead of h suggesting the occurrence of a nearlyfreerunning cbt rhythm fig 2c an approachhas been proposed to measure the freerunning periodbased on the displayed periods under non24h cyclesbeyond the entrainment ranges [ ] importantly therhythms beyond the entrainment ranges should be calledsuperposition or superimposition [ ] superposition may result in a period different from the one measured in a constant condition despite the different entrainment ranges differentanisms exhibit rhythms with endogenous periodsafter transfer to constant conditions suggestingthe endogenous characteristics of the sustainability ofcircadian rhythms in a study the pregnant mice werekept in ld or ld and their progenies werealso raised in these tcycles until they reached puberty the progeniesshowed freerunning periodssoon after they were placed in constant conditions the sole recorded exception was that hydrodictyon maintains a period of h in the constantdark for at least days after having been retrievedfrom the ld condition different physiological and behavioral rhythms mayalso have different entrainment ranges within one species or even within individuals suggesting the existence of independent oscillators in addition to affectingthe pacemaker of the circadian clock zeitgebers alsobypass it directly impacting the overt rhythms whichcausesinternal desynchronization between differentphysiological processes as a consequence when t cyclesare out of the entrainment ranges eg the sleepwakecycles and the cbtthe molecular mechanisms determining the entrainment range have not been elucidated the only clue regarding this mechanism was that in mice clock mutantsexhibit a wider entrainment range ld to ld compared to wildtype mice suggesting that circadianclock genes play critical roles in regulating entrainment mutants with less robust rhythms may be moreprone to environmental entrainment although such passive rhythms have no anticipating property unlike circadian rhythmscompromised adaptive fitness in non24h cyclingconditionsthe circadian clocks derive from the past longterm evolution which confers the fitness of the cycling environment and it is plausible that lives on earth cannotadapt to non24h cycling conditions in a short periodof time two strategies were employed to test this hypothesis one strategy is to study the effects of abnormalcycles of environmental cues on anisms and theother strategy is to compare the competitiveness of mutants with different endogenous circadian period lengthsor with no circadian rhythmicity the adaptive fitness ofa number of model anisms has been investigatedconcerning the circadian clock for the environmenttable all the available evidence suggests that living undercircumstances with cycling periods beyond the entrainment ranges is harmful to reproductive fitness cyanobacteriaendogenouscircadian periods were mixed and after a month in culturethe strain with a period resonating with thepossessingstrainsdifferent 0cguo military medical research table selected studies changes in circadian rhythms and adaptation of model anisms under non24h conditionsspeciescyanobacteria synechococcus elongates ld ld adaptabilitydecreased growth competence and adaptabilitynon24h periods hpage of references[ ]bakery mold neurospora crassatemperature cycles t12conidiation rhythms ran freeld ld ld conidiation rhythms were entrainedld ld ld conidiation rhythms were entrainedtomatold ld significantly decreased growththale cress arabidopsis thalianald ld decreased leaf chlorophyll content decreased photosyntheticcarbon fixation decreased vegetative growth and survivalld ld the mutants showed a strong positive correlation in competitionunder the tcycle growth conditionsjack bean canavalia ensiformisld ld ld leaf movement rhythms were entrained under ld ran freeunder ld and ld fruit fly drosophila melanogasterld ld reduced life spanblowfly phormia terraenovaet26 t28decreased life spans under t26 and t28house sparrow passer domesticusld ld ld locomotion rhythms were at least partially entrained orshowed superpositioncommon chaffinch fringilla coelebsld ld ld ld activity rhythms were entrained to ranges from ld to ld mouse mus musculustemperature cycles t20 superposition of luciferase rhythms in lung and scn tissuespartially entrainednile grass rat arvicanthis niloticusrat rattus rattust22“feeding cycles t20 t28locomotion rhythms were entrained between t and t decreased food intake and weight gain squirrel monkey cebidae saimiristudies under asymmetric tcycles are not includedl denotes œlight d denotes œdark ld xy denotes cycling conditions with light for x hours and dark for y hours alternatively scn suprachiasmatic nucleuscore body temperature ran free lower amplitudeld ld environment prevailed while the strain with a perioddifferent from the environment failed in the competition [ ] several models have been proposedto explain the clockcontrolled fitness in cyanobacteria including the limited resource model diffusiblefactor model celltocell communication model andthe escapefromlight hypothesis which may alsohelp to characterize the enhanced fitness by the circadian clock in other anisms [ ] the escapefromlight hypothesis postulates that in the early stageof evolutionthe circadian clock acts to segregatelightsensitive reactions temporally to nighttime egcell division and dna damage repair to avoid thedeleterious effects of radiation and oxidation fromsunlight [ ]the growth ofgrowth and flowing time were extensively affected although the results varied in different species a comparison oftomato under different ldconditions demonstrated thattomato grows mostquickly in ld but very slowly in either ld or ld similar experiments have been conducted in a variety of species including house sparrowjack bean canavalia ensiormis n crassa and squirrelmonkey saimiri sciureus which indicate that light regimens with periods that deviated dramatically from hhampered growth or survival [ ] in a previousstudy several plants were tested in a series of ld cyclesranging from ld s5 s to ld for instance soybean soja max limper under ld min1 minshowed a markedly chlorotic phenotype and a notablereduction in size contained numerous spots of dead tissue and showed a tendency to die prematurely ofcourse the day length also contributes to the comprehensive influences on plantsit has been demonstrated that in many anismsnon24h cycles have extensive negative or deleteriouseffects on survival and competitive fitness lesion of thescn in antelope ground squirrels ammospermophilusleucurus led to the loss of circadian rhythmicity in locomotor activity and an increased ratio of loss caused bypredation the tau mutation in the enzyme caseinkinase 1ε ck1ε had a dramatic influence on circadianperiods in mice with the heterozygous mice showing atwohour shorter period while the homozygous miceshowed a fourhour shorter period in the seminaturalenvironment the relative frequency of the tau allele decreased to from the initial half in months due todisadvantages in both adult survival and the recruitmentof juveniles into the cohorts in addition to fitness misalignment of circadianrhythms with non24h cycling conditions impairs manyphysiological and metabolic processes moreover the 0cguo military medical research page of disruption of circadian rhythms leads to abnormalities incognition in animals the circadian control of cognitionand behavior has been demonstrated in many animalspecies disruption of mouse circadian rhythms by ldcycles led to extensive ramifications in metabolism brainfunction and behavior for instance in ld miceandchanges in emotionality which were consistent withchanges in neural architectureincluding shorter dendritic length and reduced complexity of neurons in themedial prefrontal cortex exhibited decreased cognitiveflexibilityin addition non24h cycles shorten life spans in manytested anisms for instance pittendrigh and minisraised drosophila under the conditions of t h andt h and the life spans of drosophila in both conditions were considerably shorter than those lived in t h a hypothesis proposed that when the cycling zeitgeberperiod is close to a demultiplicative period of the freerunning period the oscillator may also be entrainedwhich is known as frequency demultiplication however frequency demultiplication is more likely torepresent a passive response to cycling environmentalstimuli than circadian oscillations [ ]effects of non24h conditions on human healthand performancenon24h regimens affect circadian rhythms and sleepkleitman and richardson were the first persons who recorded their experiences under a 28h condition in acavern of mammoth national park in kentucky usain the constant dark of mammoth cave the sleepwakecycles and body temperature of richardson adapted tothe 28h cycles but kleitman failed in forced t21and t27 schedules simulated in spitzbergen the bodytemperatures in of subjects adapted immediatelywhile the excretory rhythms adapted in only three subjects suggesting the occurrence of desynchronization[ ] even periods with a slight difference from hcan be harmful to the growth or fitness of an individualfor instance a study of the sleepwake rhythms andsleep time in mars lander phoenixin which participants lived and worked on a 2465h schedule whichwas in accordance with the daily cycling period of marsrevealed inadaptation in the sleepwake rhythms in someof the subjects and loss of sleep in almost all of the subjects since the thirteenth century the 4h on8h off watchschedule has been employed in maritime crews which isdramatically different from a 24h day in the militarythe watch schedule of the us board vessel crews shiftedin the last century from the 4h on8h off routine tothe 6h on12h off routine to accommodate the threeshift sections [ ] approximately of enlistedmen serving aboard us navy submarines stand watchon the latter schedule in contrast most officers andsome senior enlisted men stand watch on a foursectionrotation ie h on and h off in studies of the circadian rhythms of submarine crewmembers some physiological variables including bodytemperature serum or urine melatonin cortisol andurine catecholamine have been used as hallmarks [“] in a 4h on8h off schedule the oral temperatureof the subjects exhibited an 24h period instead of a12h period kelly investigated circadianrhythms of salivary melatonin in crew members during a prolonged voyage on a trident nuclear submarineand found that under 18h duty cycles the endogenousmelatonin rhythm of the crew members showed an average period of hin non24h cycles the circadian parameters including period amplitude and phase are all subject tochange during a prolonged voyage under a 4h on8h off schedule altered circadian rhythms in cbt occurred among the watchers the mostcommonphenomenon was the dampened amplitude one subject even showed a freerunning pattern naitoh found that a 6h on12h off routine duringa nuclear submarine patrol caused a loss of 24hrhythmicity in oral temperature which was due notonly to a decreased circadian amplitude but also to adispersion of the time of the peak a different watchtime in three shift groups who used alternative 4hon8h off regimens induced changes in the phase ofrectalandadrenaline excretion noradrenalinetemperatureexcretionchanges in circadian variables suggest that under non24h schedules circadian rhythms are prone to alteration and desynchronization may occur as a consequence in schaefer analyzed the rhythms ofbody temperature pulse rate and respiration rate of crew members under two different schedules an 18hwatch schedule 6h on12h off and a 24h schedule the results showed that crew members using theformer schedule but not the latter one exhibit superposition of 18h and 24h periods in all tested variables asimilar superposition was also observed in other studies[ ] as the schedule continues the impact oncircadian rhythms becomes more pronounced naitoh revealed that in a longterm voyage the groupsynchronization in oral temperature rhythmicity disappeared after approximately daysearly in it was noticed that sleep insufficiencywas caused by a 6h on12h off schedule in the submarine crew in schaefer measured theparameters of cbt heart rate and respiration rate every h for subjects living under different schedules some ofwhom employed a 6h on12h off the roster other 0cguo military medical research page of subjects employed the normal 24h schedule theformer schedule caused superposition on the rhythmsshowing both 24h and 18h periods sleep insufficiencywas also recorded in these subjects a survey of submarine crew members indicated that over ofthem had medical complaints and most complaintswere about sleeping difficulty however prevalentstudies have demonstrated that under non24h conditions both the quantity and quality of sleep are altered [ ] in real missions the sleep time ofthe submarine crew varies in a wide range extendingfrom h to h which may be influenced by manyenvironmental factors almost all studies indicated that non24h scheduleselicit misalignment with the environmental cycles anddeficiency in sleep quantity due to split sleep patternswhich means that sleep is separated into two or moreparts during a day usually both sleep quantity and sleepquality at night are better than those in the daytime [“] in contrast kosmadopoulos reportedthat a split sleep pattern showed no detrimental effectson sleep quality and performance suggesting the necessity of conducting more extensive and systematicanalysesmelatonin regulates sleep in a circadian fashionfor subjects living in an 18h routine or a combinedroutine of h and hthe melatonin levels ofthese subjects showed periods between h and h suggesting thatinfree running occurred tothethiscyclesleepwakeendogenouscontrastphysiological parametersincluding melatonin andtemperature have considerably narrower entrainmentcausedesynchronization in physiology psychology and behavior at the systemic level discrepancy mayrangesin submarines apart from non24h adherence to arotating watch roster other physical and chemicalenvironmental cues in the cabin also influence the physiology and behavior of the crew members these cues include confinement deleterious atmospheres sunlightdeprivation and insufficient lighting hypercarbia noiseelectromagneticlightspectrum and intensity high temperature and humidityfig [ “] the effects of many of thesecues on human circadian rhythms have not been fullyelucidatedradiationradiationionizingmoreover in a real mission jet lag may further contribute to the effects of circadian desynchronizationcondon found that the sleep length of watchkeepers was shorter when they crossed four to five timezones eastbound compared to crossing the time zoneswestboundshowedslower adaptation on eastbound travel this findingmay be explained by the fact that the human freerunning period is h which facilitates westboundadaptation a 3day shift regimen is widely employedin submariners which may cause frequent shiftssuch as social jet lagsubjectiveand thealertnessfig disturbed circadian clock and impaired physiology and behavior in the submarine crew some connections between the components arenot shown 0cguo military medical research page of non24h regimens lead to changes in metabolismthe circadian clock governs metabolism by modulating the expression and function of essential geneswithin severalimportant metabolic pathways in atemporal fashion conversely misalignment in circadian systems disturbs metabolic homeostasis [ ]the homeostasis of metabolism is also tightly associated with sleep [ ] in mice decreased food intake and weight were observed in mice raised underld or ld suggesting that non24h cyclingconditions may influence metabolic homeostasis in an anchoredsleep study subjects were exposed toa schedule that enabled them to sleep for h fixedfrom to and another h unfixed duringthe day the subjects showed altered acrophases inincludingbody temperature and urinary variablespotassium and sodium suggesting thattheirmetabolisms were affectedthe level ofsleep consistentlyinsulin is under the control of thecircadian clock which peaks at nightin humans ma found that saliva insulin dramatically increased late at night and early in the morning in subjects under a schedule of h for work andrest and h forthe insulinlevels and insulin glucose ratios were increased inmice under ld conditions metabolic syndrome is a constellation associated with metabolicriskincluding abdominal obesity abnormal bloodlipid levels hypertension and impaired fasting glucose kang and song reported that submarine crew members had higher risks of metabolicsyndrome and changed levels oflow highdensitylipoprotein cholesterol and impaired fasting glucoseunder a non24h onwatch schedule taken together these data suggest that metabolism may bedisturbed which might be largely attributed to thenon24h schedulesin recent years the involvement of the gut microbiome in animal and human metabolism has beenincreasingly recognized changes in the category andthe abundance of gut and oral microbiota also display a circadian pattern [“] changes in alimentary tract microbiota account for alterations in hostmetabolism relative to the fecal microbiomethesalivary microbiome has rarely been studied despiteits higher accessibility the oral microbiome containsbacteria viruses and fungi which associate and formbiofilms environmental changes might convert themto be pathogenic and trigger intestinal diseases orother inflammatory diseases recently ma found that disruption of the composition of theoral microbiome occurred in subjects living under anon24h schedule some bacterial species showedspecific change patterns eg tenericutes and sr1corresponding to sleep deprivation and circadiandesynchronization respectivelynon24h regimens affect mood cognition andperformancefatigue is an important cause of accidents and incidents at sea one requirement of safe submarineoperations is to ensure that sailors work at their highest level of readiness the non24h onwatch schedules cause misalignment in circadian rhythms whichin turnleads to sleep deprivation [ ] impairments in circadian rhythms and sleep have extensivephysiological and cognitive effects including increasedfatigue and changes in mood vigilance and workproductivity figs and it is critical to pay closeattention to the circadian rhythms and performancein submarineas mooreede pointed outœthere should be some global concern aboutthehealth and performance of these men since they arethe ones with their fingers directly on the nuclearbutton crewsnaitoh assessed the mood changes in agroup of nuclear submariners by using subjectivequestionnaires of thayer™s activation mood œactivity ma and mood œhappiness mh and revealedthat during a 10week submerged patrol the 24hrhythmicities of mood œactivity and mood œhappiness werecontrolthe results from moodperiodscale questionnaires also indicated that compared tothe patrol period significantly more feelings of œactivity and œhappiness were observed in the postpatrol period abolished compared to thein another studyscheduletheresults ofin humansthe circadian nadir trough of cbtand alertness is late at night and early in the morning and changes in body temperature reflect the dynamics of metabolism which is also one ofthedeterminants of alertness [ “] under the 4hthe letteron8h offcancellation test and vector testfrom watchkeepers demonstrated in the case of the former thatthe œbest time is forenoon while in the latter it islate afternoon the slowest performance occurred either at the start of watches beginning immediatelyafter wakening or near in the morning which isthe nadir of alertness under submarine watchroutines both misalignments in circadian rhythmsand decreases in sleep length and quality imposenegative effects on mood cognition and performance[ ] during the existingwatch schedules used in canadian forces were evaluated revealing thatthe cognitive efficacy in thesubmarine crew dramatically decreased to a dangerously low level showed that ma the effects of 0cguo military medical research page of under a 4h on8h off routine the alertness of thesubjects decreased dramatically in the morning aschronic sleep deprivation may occur during non24h cyclesit is unclear whether misaligned circadianrhythms or sleep loss contributed more to the decreased alertnessthe fatigue avoidance scheduling toolfast„¢program has been developed to optimize the scheduling and performance of the air force consideration and integration of moresuch asindividual differences might help to improve theprediction accuracy and practicability in not only theair force but also for personnel working under conditions with non24h periodsincluding submarinecrew members factorstheseand most ofto date many studies have documented the impairment of non24h schedules on circadian rhythms andcognitionstudies have beenconducted at the physiologicallevel by contrast themolecular mechanisms governing the effects of disruptedcircadian rhythms have not been elucidated which maythethuslimitphysiologicalofcountermeasuresunderstandingtheofdevelopmentindepthandthechangesin militaryand commercialcountermeasures conclusions and perspectivescircadian disruption profoundly affects health andperformancevesselcrews during a 90day voyage which is the longestvoyage recorded to date disturbance in circadianrhythms was reported in many chinese crew members most of the crew members experienced sleepdisordersflagging dysphoria and faintness somecrew members attempted to overcome these conditions by chewing dry peppers or daubing mentholcamphorate on their temples in maritime missions circadian misalignment causes serious consequences including high personnel turnover “per voyage and requirements to train the new entrant howeverthe question remains of how we canovercome circadian misalignment and avoid deleterious consequences the entrainment range needs tobe considered if the environmental period is withinor close to the entrainment rangeit may be usefulto usecircadianrhythms if the environmental period is far beyondthe entrainment range it may be useful to constructa manmade 24hperiod microenvironmentisalso possible to combine both approaches someefforts have been made to lessen the deleterious effects of disrupted circadian rhythms and to improvehealth and performance which primarily involvedcountermeasuresto adjusttheitoptimization of the watch schedules and modifications of the zeitgebersoptimized watch schedules may improve circadianrhythms and performance duplessis compared the impacts of different watch schedules 6hon8h off and a compressed close6 h watch schedule and the wrist activity results showed that sleepduration was longer under the 6h on8h off schedule sleep discontinuity appears to be considerablyless severe on the 6h on12h off watch schedulethan that of the 4h on8h off watch schedule incontrast the 6h on12h off schedule allowed morecontinuous sleep which might be explained bythe fact that t12 is shorter than t18 compared tohuman frp the best approach is to use a 24h roster instead of a non24h roster a 24h schedule wasused durin
Colon_Cancer
" national economies are increasingly facing the challenge of having to finance the prevention andtreatment of human diseases and of having to compensate for the resulting loss of economic production physicalinactivity is demonstrably closely related to the risk of developing certain disease group physical inactivity results indirect and indirect burdens that the present study intends to quantify in hungary for the period between and methods based on the data of the hungarian public finances this study determines the direct and indirect costsincurred by hungary due to illnesses and through the par method it quantifies the financial burden of physicalinactivity incurred by the hungarian treasuryresults the total financial burden of illnesses in hungary showed a decreasing tendency from to eventhough the year saw an increase in costs compared to similarly while total public expenditure on illnessesassociated with physical inactivity increased by when compared to the total amount attributable to medicalconditions stemming from physical inactivity still showed a decrease of billion huf in the overall period the biggesteconomic burden is posed by cardiovascular diseases hypertension and type diabetess the increase in the economic burden associated with physical inactivity can be attributed to thecombined effect of two factors changes in total expenditure on specific disease groups which showed an increase inthe period under review and changes in the physical activity levels of the hungarian population which showed animprovement over the period under review initiatives in hungary aimed at encouraging an active lifestyle fromchildhood onwards should be continued since “ beyond the initial impact that has already been felt to some extent inrecent years these initiatives will come to their full fruition in the coming decadeskeywords physical inactivity economic burden parmethod direct costs indirect burden population attributable risk the fundamental change towards a more sedentary lifestyle has a serious impact on people™s health physical inactivity is one of the most important global issues of thetwentyfirst centuryleading to an increased risk ofchronic diseases such as type diabetes cardiovascular correspondence davidpaaretkptehu1university of pecs faculty of health sciences pecs hungaryfull list of author information is available at the end of the disease certain types of cancer rectal colon breastobesity and osteoporosis these diseases may even become the cause of death the world health anizationwho has also identified these medical conditions as themost burdensome noncommunicable diseases of today™sdeveloped world regular moderate physical activity reduces the risk of the most common of these diseases andcontributes to an increased sense of wellbeing [ ] acinactivity ranks as thecording to the who physical the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0c¡cs bmc public health 20suppl page of fourth most significant mortality factor in the world with million deaths a year worldwide [ ]another study suggests that there is a lower likelihoodof health problems among people engaging in regularphysical activity than among those leading a sedentarylifestyle furthermore there is convincing evidence thatregular physical activity increases life expectancy and reduces the likelihood of developing coronary and cardiovascular problems of suffering a stroke or developingcolon cancer inactive and sedentary lifestyles directly affect metabolism bone mineral composition and magnify the healtheffects of cardiovascular disease furthermore thereis epidemiological evidence to suggest that a sedentarylifestyle increases the risk of cancer obesity metabolicand psychosocial problems according to oecd data the average life expectancyof hungarians at birth in was years which is years below the oecd average actually one of the lowest on the list for men this value is years forwomen years both showing an increasing trend in recent years the hungarian government has made anumber of efforts to bring about significant changes in theinactive lifestyle of the hungarian population these include measures to increase the number of physical education lessons and to improve the conditions in pe lessonsat school also the development and construction of sportsfacilitiesincreased funding for sports associations andeven the use of corporate tax incentives for sporting purposes while improving the conditions alone does notresult in a change in the attitudes of the population towards sport it is certainly a prerequisite procedures that quantify the burden on the hungarianeconomy resulting from physicalinactivity are one ofthe ways of measuring the effectiveness of state intervention [ ] this study aims to contribute to this bodyof research and proposes to analyse a longer timespectrummethodsto analyze the economic burden of physical inactivity weneed to start with the burden of diseases on the nationaleconomy as physical inactivity plays a vital role in the onset of several diseases and leads to various causes of deathat a national level diseases have direct and indirect costsdirect costs of diseases include treatments medications sickpay allowances and associated ancilliary coststhat are directly related to the illness the direct costs inhungary are mainly financed by the national health insurance fund nhif since it is called the national health insurance fund administration nhifa but we must not disregard the cost of sick leave and private costs outside of nhifnhifa financing the latterof which are directly borne by members of societyamong the indirect burdens we include items that constitute a loss to the economy or to society as a result ofthe loss of work caused by a disease there was a significant change in this area during the research periodwhile in and there was a longterm loss ofproduction only in jobs on the skillsshortage list or invery special cases by the number of job vacanciesin hungary reached thousand while by july thisnumber rose to thousand people which is of theworkforce our calculations were based on the following assumptions in and in a labor marketcharacterised by an oversupply of labor a frictional unemployment of months groupbased performance expectations and the market of goods and services beingoversupplied people on average worked days a yearand loss was calculated based on gdp per capita thestudy that inspired our calculations had a similarcalculation but we replaced its assumptions with theabovementioned assumptions and we broadened andtightened formulas and corrected data that had becomefact since then however when calculating the results we had to change the assumptions about the labormarket from the previouslyoutlined conditions as by hungarian labor market had become characterizedwith overdemand therefore we had to increase frictiontime as well monthsanother economic burden is presenteesim which isthe term used for the phenomenon when a sick individual goes to work which results in poorer performanceand thus loss of productionour main goalin this research was to quantify theeconomic burden of diseases for the years and and more specifically the costs to thenational economy directly attributable to physicalinactivity in the market years and during our research we treated as relevant secondarydata eurobarometer and and nhifnhifa data from and [“]in the course of our resreach we examined the typesof medical conditions related to physical inactivity andtheir possible complications the factual data for whichwas obtained from nhif and nhifawith the help of par method population attributable risk the most commonly used method in international research we were able to obtain quantitativemeasurements that were used uniformly in the analysisof data for all three yearspar ¼ pexp 02 rrˆ’¾ ¾ pexp 02 rrˆ’°°¾ 02 wherepexp prevalence refers to the section of the populationwhere a given risk is present 0c¡cs bmc public health 20suppl page of rr relative risk describes the risk associated with asedentary lifestylewhen using the index it is necessary to break downthe population into physically active and inactive sections and then by determining the relative risk rate wecan estimate the number and cost of illnesses stemmingfrom a physically inactive lifestyle the physical activity indicators ofthe hungarianpopulation showed fluctuations during the period underreview the situation was the worst in when wesaw of the population as physically inactive in ouropinion the health protection effect does not manifestitself in the case of those who never excercise or only doso “ times a month by this figure dropped to and at the same time the ratio of those engaging inexercise at least times per week increased threefold inthe following years a more negative trend was observed as the rate of inactive people rose to although this is still significantly better than the basefigure for fig with the help of metaanalysis we calculated the relative risk ratio rr an indicator which is prevalent ininternational literature in order to estimate the futureexpenditure stemming from physical inactivity for all affected disease groups such as cardiovascular diseasestroke hypertension colon cancertype diabetesosteoporosis depression gastrointestinal complicationsobesity high triglyceride diseases and deliberate selfharm [“]the rr is the proportion of the applicaple diseasesamong people with inactive lifestyles divided by the proportion of the applicable diseases among people with active lifestyles on the basis of the rr values it is possibleto quantify the par indicator by disease group for eachyear table in order to allow the data to be compared over timethe data on the burden of illnesses stemming from physicalinactivity for and was recalculated to prices while the total amount of burden imposedby illnesses was recalculated to prices using thefig the ratio of physical activity and inactivity in hungary in “ sourcespecial eurobarometer special eurobarometer specialeurobarometer 0c¡cs bmc public health 20suppl page of table the cumulative relative risk rate and par values for theexamined disease types in “disease typesheart and coronary diseasespar par rrpar strokehypertensioncolon cancertype diabetesosteoporosisdepressiongastrointestinal complicationsobesityhigh triglyceridesdeliberate selfharmsource katzmarzyk aldoori ewing andersen schuch domestic producer and consumer price index of thehungarian central statistical office hcso the economic burden ofresultsat pricesillnessesamounted to more than billion forints huf in of which the direct burden was billion forintsdirect costs accounted for of the burden of illnessesand the billion huf sickness benefit represented justover of total direct costs indirect burden representeda significantly lower percentage amounting to over billion huf the economic burden imposed by sicknessin was of hungary™s gdpby the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden of illnesses that year less than of which amounting to billion huf was forsickness allowance expenditues indirect burdens decreased to billion huf the burden of sicknessamounted to of the gdp in by the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden that year and of it weresick allowances amounting to billion forints indirectburdens fell to billion huf the burden of sicknessdecreased to of the gdp in by the economic burden of illnesses increasedcompared to but it was still below the initial figure huf billion and it decreased in comparison with the gdp the share of direct costs dropped significantly to within which the sickness benefitrepresented to the value of billion forints atthe same time indirect burdens increased significantlyto billion huf all in all the burden of sickness decreased to of the gdp in between and the economic burden of diseases fell by billion huf which is a total decrease of corresponding to an average annual decrease of and in the meantime the country™s gdp increasedsignificantly altogether at current prices obviously the decrease is due to a number of reasons butthe effect of the increase in physical activity is an important factor among them table in the years examined in the case of disease groupslinked to physical inactivity the burden of illnesses onthe state budget excluding sickness allowance amounted to billion huf and billion hufrespectively of which the lowest value was in however only a part of these can be directly linked tophysical inactivity as many other risk factors play a rolein the development of these diseases as regards therelative weight of each disease group cardiovascular disease is the biggest burden followed by hypertension atthe same time type diabetes was only ranked the fifthfor costs in the first year but by it became thethird largest item only slightly behind high blood pressure expenditure on stroke obesity and deliberate selfharm were almost negligible compared to other diseasegroups table based on the results it can be stated that in theexpenditures in the state budgetfor the diseasegroups examined drastically decreased by approximately billion huf compared to the initial starting positionof billion huf but by the expenditures hadsurpassed the base total from by more than billion huf compared to only type two diabetesand osteoporosis showed an increase and respectively compared to although the latter is dueto the relatively low total expenditure for all other disease groups the level of expenditure declined in absoluteterms resulting in a significant decrease of billionhuf in total expenditurehowever in the case of the picture is more varied if we examine the relative position of certain diseasegroups compared to type diabetes showed themost significant increase to the tune of more than billion huf the other diseases lag behind in terms ofexpenditure cardiovascular diseases and colon cancerare next with an increase of “ billion forints inaddition there is an increase in the costs associated withosteoporosis stagnation or decrease was observed forthe other disease groups but this could not compensatefor the increase in the costs of the aforementioned diseases the most significant drop in expenditure is observed in hypertension billion huf and hightriglyceride diseases billion huf table focusing on the direct burden of physical inactivitywe can conclude that “ of the total expenditure ofthe disease groups is directly attributable to physical 0c¡cs bmc public health 20suppl page of table economic burdens of diseases in hungary “ in huf million in real terms in direct costs statefinancedeconomic burdens of diseasesin hungary “medicationmedical aidsgeneral practitioner servicesdental servicesoutpatient carect mrimedical centers exluding vd clinicsdialysishome careinpatient carehighcost medical procedurespatient transportspa treatmentsgovernmental health careexpendituresick leavedisability rehabilitation treatmentcharged tonhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifnhifanhifnhifain totalprivate costsprivate health care expenditureindirect costsexpenditure associated withsick leavehealth insurance managementand other costsfriction due to sickness leading toloss of productionof which reduced pay due to sickpay and sick leaveof which tax loss for the statefriction due to disability leadingto loss of productionindividualemployernhifaemployer individualstateindividualstatesocietypresenteeism costsin totalemployerinactivity the major part is the cost of cardiovasculardiseases and hypertension and these were closelyfollowed by type diabetes by due to the factthat the total expenditure for stroke obesity and deliberate selfharm was also insignificant compared to otherdisease groups their expenditure related to physical inactivity is insignificant in the case of deliberate selfharm the costs cannot be measured even in the order ofone hundred thousand forints table compared to the decrease for the year ofthe direct costs stemming from physical inactivity is larger in proportion than the decrease of total expenditurethis is true of each disease group and for those twogroups type diabetes and osteoporosis where therewas an actual increase in costs the increase was less forrespectivelyphysical inactivityrelated expenses than for overall expenses the largest drop in monetary terms can be observed in the case of hypertension and cardiovasculardiseases over billion huf but there was a decreaseof and billion hufin hightriglyceriderelated diseases and colon cancer howeverpercentagewise nhif achieved the highest cost reduction for high triglycerides and colon cancer closely followed by a decrease in stroke expenditure and deliberate selfharm although inthe last two categories the low sum total spent alsomakes this decrease appear larger percentagewise thanwould be the case with larger totalscompared to the expenditure related to physicalinactivity in shows a decrease of billion huf 0ctotal amountinactivitytotal amountinactivitytotal amountinactivitycardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotal¡cs bmc public health 20suppl page of table total cost incured by nhifa nhif of those disease groups that are associated with physical inactivity and costs directlyattributtable to physical inactivity itself in terms of prices million hufdisease typesat the level of the individual disease groups the amountsvary the most significant decline in absolute terms is inthe high blood pressure and high triglyceriderelated illness groups however the burden of type diabetes increased significantly and there was an increase in coloncancer and osteoporosis disease groups the direction andextent of the changes are mostly comparable to the totalexpenditure amounts at the overall level of the diseasegroups although the changes in the physical inactivity ratenaturally lead to differences in the specific values this isso much so that the total expenditure amounts increasedat the level of all disease groups by but overall theexpenditure related to physical inactivity shows a decreaseof table discussionwe can clearly conclude similarly to other international researches [ “] that physical activityand forms of recreational exercise have a protectiveeffect eg a preventive effect against certain types ofchronic diseases cardiovascularlocomotor disordersdiabetes and certain types of tumors the decreasein physical inactivity has a positive effect on productivity as fewer people avail themselves of sick leave atable changes in total expenditure as reported by nhifa nhif and changes in expenditure directly stemming from physicalinactivity compared to the base level expenditure in in real terms adjusted to prices million hufdisease typescardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotaltotal amountˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ ˆ’ˆ’ˆ’ ˆ’inactivityˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ total amountˆ’ ˆ’ˆ’ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’inactivityˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ˆ’ ˆ’ ˆ’ 0c¡cs bmc public health 20suppl page of study of economic development over the past centuryhas concluded that the advancement of the population™s health status is responsible for about “ ofeconomic growth [“]in our comparative study we used four samplingpoints between and to demonstrate the burden of diseases at the level of the national economy forthe various loadcarriers in the period under review theeconomic burden decreased significantly overall from of the gdp to the weight of indirect burden increased however as in the currently demanddominated labormarket it is more difficult to replacelost workforce in the period of analysis the number ofemployees in hungary increased with which increased the amount of sick leave and number of sicknessdays but their gdp contribution was significantly higheralthough associated costs and burdens increased innominal terms they decreased in relation to the gdpa large part of diseases™ burdens are borne by the stateand society followed by households andemployers the proportions are similar to ding in european countries included hungary although we estimate that the burdens on employers arehigher and the burdens on households are lower inhungarysince the physical activity rate of the hungarianpopulation has been fluctuating but overall there is animproving tendency which is also apparent in the savings potential of the examined expenditures categoriescompared to the gdp the amount of spending dependsheavily apart from the physical inactivity rate on thenumber of employees as well as those people who arenot employed can not have sick leavefor exampleoverall government spending depends on the budget ofthe country which is connected to the overall economicsituationcardiovascular diseases accounts for most of the costof physical inactivity in hungary which coincides withmattli in switzerland however of directinactivityto depression inswitzerland while nhifa™s depression costs account foronly “ in hungaryattributablecostsarein hungary a number of measures have recently beentaken in order to integrate physical activity and sportinto people™s daily lives such measures include theintroduction of everyday physical education in schoolsor the extensive development of sports infrastructure however the effects of these measures will have amore pronounced effect in the long run several studieshave shown that high physical activity in childhood isnot yet measurable in terms of economic returns lessfrequent use of health care and a lower cost associatedwith using them as some effects “ such as the high costof sports injuries high rates of childhood illness “ haveresearch data confirm the facta negative bearing on the rate of return on investmenthowever a longterm change of attitude and opennessto physical activity at later stages in life are where thesemeasures bear a profit so any effort to support childinterhood sports is rewarding [ ] in additionnationalthatthoseparents who are themselves engaged in sport or currently do so are more likely to prefer sporting activitiesamong their children it is important to draw attentionto the fact even minimal physical activity has a healthimproving effect at any stage of life that is whysport and health policies at all times should promoterecreational activities for all ages not just young peoplewe would like to expand the scope of our current research if we could also examine how the patient numbers varied each year by disease group unfortunatelythe data was not available this would be of particularinterest for the year as the expenditure on illnessesshowed a significant increase in real terms compared to which may be due to the fact that more patientsreceived care and treatment but may also be due an increase of the normative provision per person by the government possibly to provide better quality careif we posit based on the eurobarometer data that thephysical activity rate improved compared to wecould also assume that fewer people were treated for theanalysed medical conditions in which would basically have a downward effect on total expenditure at thesame time however the picture is somewhat shaded bythe fact that even if the attitude of the population towards regular physical activity has changed in the lastfew years it is not certain that the number of illnesseswould decrease significantly in such a short period asthe negative effects of a sedentary lifestyle led for decades would not be easily offset by a few years ofexcerciseladen lifestyle this is especially true forolder age groups that is to say a reduction in the number of patients is not realised yet in patient care at thesame time the use of rapidlydeveloping medical technologies is also increasing the financial burden on thebudget as the higher costs of new technologies makemedical care per patient more expensive on the otherhandit should not be fotten that healing can bemade more effective and can lead to higher returns onhuman capitalthe study examined the development and compositionof direct and indirect burdens of disease in hungary andthe costs of physical inactivity to the state budget theseburdens fell in the examined periode which was associated with an increase of gdphowever there was an increase in the economic burinactivity which can beden associated with physical 0c¡cs bmc public health 20suppl page of attributed to the combined effect of two factors changesin total expenditure on specific disease groups whichshowed an increase in the period under review andchanges in the physical activity levels of the hungarianpopulation which showed an improvement over theperiod under review initiatives in hungary aimed at encouraging an active lifestyle from childhood onwardsshould be continued since “ beyond the initial impactthat has already been felt to some extent in recent years these initiatives will come to their full fruition in thecoming decadesabbreviationsct computed tomography gdp gross domestic product hcso hungariancentral statistical office huf hungarian forint mri magnetic resonanceimaging nhif national health insurance fund nhifa national healthinsurance fund administration oecd anisation for economic cooperation and development par population attributable risk rr relativerisk vd veneral disease who world health anizationacknowledgementsthe authors acknowledge to the nhifa™s colleagues for their help incollecting the dataset especially to mr zsolt kiss director general to drmihaly palosi head of department to petra fadgyasfreyler head ofdepartment and to valentina beitl analistthe authors would like to express their special thanks to prof attila fabianformer vice state secretary for his cooperative help during the datacollectionabout this supplementthis has been published as part of bmc public health volume supplement level and determinants of physical activity in the v4countries part the full contents of the supplement are available online athttpsbmcpublichealthbiomedcentralcomssupplementsvolume20supplement1authors™ contributionspa was the leader of the complete research coordinated the different coauthors™ work systematized the dataset summarised the literature related tothe relative risk ratios of illnesses calculated the par indices and contributedto the s dp has made calculations of par indices the direct costs ofphysical inactivity in the nhifa budget and contributed to the sfrom its results ms has made calculations of the total burdens direct andindirect of illnesses in hungary and contributed to the s from itsresults mh and psz summarised the related literature to the section ak and tsz have revised the results and contributed to the sall authors read and approved the final manuscriptfundingthe research was carried out and the publication costs funded by thesupport of hrdop36216201700003 cooperative research network ineconomy of sport recreation and health the authors declare that thefunding body does not have any role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe data of the state financed direct costs that support the findings of thisstudy are available from national health insurance fund administration butrestrictions apply to the availability of these data which were used underlicense for the current study and so are not publicly available data arehowever available from the authors upon reasonable request and withpermission of national health insurance fundthe datasets of the private ind indirect costs used and analysed during thecurrent study are available from the corresponding author on reasonablerequestethics approval and consent to participatethe ethical approval was granted for the study by ethics committee ofuniversity of p©cs nr participants were informed about theresearch aim and methods before signing the informed consent form theinvestigation conforms to the principles outlined in the declaration ofhelsinkiconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1university of pecs faculty of health sciences pecs hungary 2university ofphysical education budapest hungary 3corvinus university of budapestcorvinus business school budapest hungaryreceived march accepted march published august referencessebestyen a boncz i molnar a korosi l kovi r kriszbacher i olah apentek m sandor j relationship between surgical intervention type and days mortality of elderly femoral neck fracture in the prsence of differentcomorbidities value health 2009123a66kruk j health and economic costs of physical inactivity asian pac j cancerprev “reiner m niermann c jekauc d woll a longterm health benefits ofphysical activitya systematic review of longitudinal studies bmc publichealth pratt m norris j lobelo f roux l wang g the cost of physical inactivitymoving into the 21st century br j sports med “ who global recommendations on physical activity for health switzerlandgeneva who blair sn cheng y holder js is physical activity or physical fitness moreimportant in defining health benefits med sci sports exerc s379“tremblay ms colley rc saunders tj healy gn owen n physiological andhealth implications of a sedentary lifestyle appl physiol nutr metab “rishiraj n inactivity a bad œhabit costing our productive lifestyle int j physmed rehabil oecd health status in edited by development ofecoa ¡cs p h©cz r pa¡r d stocker m a fitts©g m©rt©ke a fizikai inaktivit¡snemzetgazdas¡gi terhei magyarorsz¡gon k¶zgazdas¡gi szemle “ gabnai z m¼ller a b¡cs z b¡ba ©b the economic burden of physicalinactivity at national level [a fizikai inaktivit¡s nemzetgazdas¡gi terhei]eg©szs©gfejleszt©s health dev “ acs p stocker m fuge k paar d olah a kovacs a economic and publichealth benefits the result of increased regular physical activity eur j integrmed “ hcso in hcs o editor edn stadat time series of annual data labour market distribution of job vacancies koll¡nyi z imecs o az eg©szs©g“befektet©s budapest demosmagyarorsz¡g special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan powell ke population attributable risk of physical inactivity phys actcardiovasc health “katzmarzyk pt gledhill n shephard rj the economic burden of physicalinactivity in canada cmaj “ 0c¡cs bmc public health 20suppl page of aldoori wh giovannucci el rimm eb wing al willett wc use ofacetaminophen and nonsteroidal antiinflammatory drugs a prospectivestudy and the risk of symptomatic diverticular disease in men arch fammed “ andersen lb schnohr p schroll m hein ho allcause mortality associatedwith physical activity during leisure time work sports and c
Colon_Cancer
" in head and neck cancer hnc the relationship between a delay in starting radiotherapy rt andthe outcome is unclear the aim of the present study was to determine the impact of the amount of time beforetreatment intervention tti and the growth kinetics of individual tumors on treatment outcomes and survivalmethods two hundred sixtytwo hnc patients with primary tumors treated with definitive chemo rt wereretrospectively analyzed the tti was defined as the time interval between the date of histopathologic diagnosisand the first day of the rt course volumetric data on tumors were obtained from diagnostic and rt planningcomputer tomography ct scans in order to calculate the tumor growth kinetic parametersresults no significant association between locoregional control or causespecific hazards and tti was found thelog hazard for locoregional recurrence linearly increased during the first days of waiting for rt although this wasnot significant the median tumor volume relative increase rate and tumor volume doubling time was 32dayand days respectively and neither had any impact on locoregional control or causespecific hazards the association between a delay in starting rt and the outcome is complex and does not harm allpatients waiting for rt further research into imagingderived kinetic data on individual tumors is warranted inorder to identify patients at an increased risk of adverse outcomes due to a delay in starting rtkeywords head and neck cancer radiotherapy waiting time treatment delay outcome with new cases and deaths reported in head and neck cancer hnc is the eighth mostcommon and most lethal cancer in men worldwide in addition to surgery and systemic therapy radiotherapy rt is one of the cornerstones for treatment of thiscancer owing to the rising cancer incidence rate in ageing populations and the widening list of indications forirradiation the demands for rt have increased dramatically over the past decades [ ] the increasing complexity of pretreatment diagnostics and rt technology correspondence pstrojanonkoisi1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia4chair of oncology faculty of medicine university of ljubljana ljubljanasloveniafull list of author information is available at the end of the has led to delays in treatment decisionmaking and thereduction in linear accelerator throughputthat hasresulted in a significant imbalance between the demandfor rt and the availability of rt capacities in manypublically funded health systems this is also the case inslovenia [ ]due to obvious ethical reservations the only way tostudy the impact of delays in starting rt on treatmentoutcomes are retrospective observational analyses of cohorts from different institutions or countries intuitively one would expect that the prolongation of thetime taken before treatment intervention tti is harmful to patients both the likelihood of tumor growth andthe acquisition of a metastatic phenotype increases as afunction of time furthermore advanced tumors aremore difficult to treat than smaller tumors the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cžumer radiation oncology page of indeed a systematic review of pertinent literature fromthe period “ by chen showed an increasein the risk of local hnc recurrence of for everymonth of delay in definitive rt however certainstudies included in chen™s metaanalysis and also somemore recent reports negated the association betweenthe delay in definitive rt and the increased risk of treatment failure [“] several different biases inherent inretrospective analyses either related to the quality ofdiagnostic procedures and treatment or to the inhomogeneity of the studied population as well as a selectionbias ie patients with fasttumor progression or ahigher burden of symptoms receive priority in treatment and significant variability in the kinetics of individual hnc cases may abolish the effect of tti inoutcomes [“] however if patients with advancedor fastgrowing tumors have to wait longer the magnitude of this effect may be overestimated furthermore no compelling relationship between treatmentdelay and prognosis was found in some other cancertypes [“]in order to determine what would be an acceptabletti in hnc patients treated with definitive rt or concurrent chemoradiation we aimed to analyze the impactof tti and growth kinetics of individual tumors on theoccurrence of localregional failure distant metastasisand survival in the present study of a cohort of slovenepatients with hncmethodsin a retrospective study patients with oral cavity oropharyngeal hypopharyngeal or laryngeal squamous cellcarcinoma scc who were treated with curativeintentdefinitive rt with or without concurrent chemotherapybetween january and december at the institute of oncology ljubljana slovenia were includedpatients with t1n0 or t2n0 glottic cancer were left outof this cohort the “ period was chosen because of fluctuations in the waiting time for irradiationas a result of intensive renovations and expansion in thedepartment of radiotherapy that took place over thistime span from patients™ medical and rt charts wecollected information on clinical gender age onset ofsymptoms date and type of disease recurrence anddeath tumor histology site of origin tnm stage andtreatment characteristics rt technique regimen anddose duration of rt type of concurrent chemotherapy[cct] and the number of cycles administered thetnm stage was determined according to the 7th editionof the uicc classification systemfor analysis of the impact of tumor growth kinetics ontreatment outcomes the volumes ml of primary tumors and neck nodal metastases as marked on diagnostic and rt planning computer tomography ct scanswere compared patients with the same basic clinicaldisease and treatment characteristics as indicated abovebut with both sets of ct scans available were selectedfor this part of the study diagnostic ct scans were performed through the acquisition of mm thin ct sections whereas planning ct scans had a slice thicknessof mm both with intravenous iodine contrast enhancements sets with extensive artefacts were excluded forthe purposes of rt planning patients were positionedsupine on the flat tabletop and a fivefixation pointthermoplastic mask was used lymph nodes were considered positive if the smallest diameter was more than cm andor the necrotic center or extracapsular extension was seen if available segmentation was guided bymagnetic resonance imaging mri sets and the resulting contours around the primary tumors and metastaticneck nodes represented a consensus between two radiation oncologists and a radiologist volumes of primarytumors and metastatic neck nodes were separately calculated by a computer software program used for rt planning xio computerized medical systems inc stlouis usa eclipse varian medical systems inc paloalto usa the end points in this part of the study werechanges in the tumornodal volume and tnm stage thecalculation of the primary tumor volume doubling timeand their impact on the treatment outcomestatisticsthe study protocol was approved by the republic ofslovenia national medical ethics committee no “ for retrospective studies a written consentis deemed unnecessary according to national regulationsbasic descriptive statistics were reported with meansstandard deviations and ranges for numerical variablesand as percentages for categorical variables in patientswith two simultaneous hncs some characteristics werereported in regards to patients while others were reported in regards to tumorsthe survival curves were computed using the kaplanmeier estimator and the aalenjohansen estimator wasused to estimate the cumulative probabilities of competing risks the effect of covariates was analyzed using amultiple cox regression analysis with all the analysesthe data were censored at a fiveyear followupwhen focused on the survival of patients the analyseswere completed with patients as the units and the timewas measured from the first day of therapy until deaththe overall survival os regardless of the cause ofdeath and the absolute risk cumulative probability ofdying due to index cancer were reported in the cox regression only the index cancer deaths causespecifichazard csh were considered to be events of interestin the analyses where locoregional control lrc wasof primary interest the calculations were performed in 0cžumer radiation oncology page of regards to tumors excluding the nonresponders to rtie those with residual local or regional tumors at “ weeks after rt completion for the latter group weconcluded that it is the radioresistance of tumor cellsthat are responsible for the persistence of the diseaseafter therapy and not that patients had to wait for rtthe followup time was calculated from the last day oftherapy the estimated cumulative probability of localandor regional recurrence distant metastases lrcprobability of being still alive and without local andorregional recurrence and diseasefree survivaldfsprobability of being still alive and without events locoregional and distant failure and deaths were the events ofinterest were reported all the analyses were conductedin regards to the tumors as independent units this assumption was checked in the sensitivity analysis andallowed for gamma frailtythe assumptions of the cox regression were checkeda nonlinear effect a spline with degrees of freedomwas allowed for the numerical variables and the proportional hazards assumption was tested using schoenfeldresidualsthe tti was defined as the time interval between thedate of histopathologic diagnosis and the first day of thert course tumor growth kinetics was expressed as thetumor volume relative increase rate per day and as thetumor volume doubling time in daysthe tumor volume relative increase rate per day was 13 calculated as 12v t 2ðþþv t 1ðt2 ˆ’ t1where vt1 gross tumor volume at time t1 ieon diagnostic ct scans and vt2 gross tumor volume at time t2 ie on planning ct scans it was reported as the percentage increase was reported as per day the tumor volume doubling time was calculated asðv t 2ðv t 1ðln 2ð þ t ˆ’ t þþlnþsince a onetoone relationship existed between thetwo only the tumor volume relative increase rate whichrequires no extrapolation was considered for modellingall analyses were performed using r statistical software version and a pvalue below was considered statistically significantresultsimpact of time to treatment initiationbetween and patients with oral cavity oropharyngeal hypopharyngeal or laryngeal primarysccs were treated with definitive chemo radiotherapywith curative intent there were men and women aged “ years mean the majority oftumors originated in the oropharynx tumors in patients and were tnm stage iv tumors in patients the tti ranges from to days with mean days the distribution of the tti is only slightlyasymmetric with median and interquartile rangeiqr “ fig patients were irradiated with dimensionaltechnique or 3dimensionalconformal isocentric technique to the median rt dose gy iqr “ delivered in gy dailyfractions iqr “ concurrently to rt patientsreceived chemotherapy consisted of platinbased monochemotherapy patients or mitomycinecbleomycin combination patients the characteristics of patients and their tumors are shown in table treatment outcome and survivalclinical andor radiologic assessment at “ weeksposttherapy confirmed disease persistence locally andor regionally in cases ie in patients and thesepatients were excluded from further analyses of lrcand dfs thus patients with tumors were analyzed and during followup a local andor regional relapse was recorded in cases with a mediantime from treatment completion of months range “ months the two and fiveyear probability of localrecurrence after the end of treatment was and respectively whereas the regional relapse was and respectively after threeyears posttherapy we only observed a small increase ie inlocal recurrence probability whereas the probability ofregional relapse was stable after the third year followupthe probability of occurrence of distant metastases at years was and at years it was at the second and fifthyear followup the lrc was confidence interval [ci] and ci respectively and the dfs was ci and ci respectivelyin the fifth year posttreatment out of patients had died the index cancer was the reason in of the cases during the first years posttherapythe probability of cancerrelated death steeply increasedfrom zero to but later the changes were less pronounced at the second and fifth year posttreatment theos independent of the cause of death was ci and ci respectivelyeffect of covariatesin the univariate analysis the following parameters weretested for lrc and csh age gender stage of diseasetype of treatment rt vs rt cct and tti for rtas continuous variable results are presented in table 0cžumer radiation oncology page of fig distribution of ttitable clinical characteristics of patients and their tumorscharacteristicsgenderno femalemaleage yearsatumor locationboral cavityoropharynxhypopharynxlarynxtumor stagebstage istage iistage iiistage ivtreatmentbradiotherapyconcurrent chemoradiotherapyrt duration daysatime to treatment interventiona ± “ ± “ ± “the same parameters were also included in the multivariate model table the occurrence of locoregional recurrence was significantly related to the diseasestage p whereas the relationship with agewas only of marginal significance p higherage had a lower hazard no significant associationwith the type of treatment could be found p table the log hazard for locoregional recurrenceseemed to linearly increase during the first days oftable effect of covariates on locoregional control and indexcancerspecific hazard n patients with tumors anunivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherap cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt““““““““““““ a mean ± sd rangeb eleven patients had two simultaneous primary tumorsci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapy 0cžumer radiation oncology page of table impact of tti on locoregional control and indexcancerspecific hazard n patients with tumors amultivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt““““““““““““ ci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapywaiting for rt although the association between thehazard and tti wasregardless ofwhether we made an allowance for nonlinearity ornot fig 2ainsignificantthe hazard of dying due to the index cancer ie cshwas found to be favorably associated with a lower disease stage p and the addition of cct to rtp whereas age gender and tti did not reachthe level of statistical significance table fig 2bassumptions and sensitivity analysisproportional hazards for all included variables and thelinearity assumption for age were analyzed and presented graphically some indication of nonproportionalhazards could be found in gender but allowing for nonproportional hazards did not change the interpretationof the other covariates as a part of the sensitivity analysis the assumption of the dependence of the tumorsbelonging to the same individual was relaxed but nochanges in the interpretation of the results could be observed as an additional confounder the timedependentcovariate œduration of rt was considered but its effectdid not prove to be importantimpact of tumor growth kineticsdiagnostic and planning ct scans median interval days range “ days were available from patientsfive of these patients had two primary tumors the majority of patients were males with a meanage of years range “ and the patients hadprimary tumors located in the oropharynx table when two ct sets were compared the original tstage of the primary tumor was increased ie upgradedin two cases and the nodal stage was increased in six patients due to the limited number of npositive casesn only volumes of primary tumors werecompared between the two ct sets for the calculationof the tumor volume relative increase rate per dayand tumor doubling time the absolute increase intumor volume ranged from ˆ’ to cm3 per daymedian no increase in volume was observed infive patients ct scans in these five patients were takenfig trend of the hazard for locoregional recurrence a and for index cancerspecific death b 0cžumer radiation oncology page of table characteristics of patients with available diagnostic andplanning ct scanscharacteristicsgenderno femalemaleage yearsatumor locationboropharynxhypopharynxlarynxstagebstage istage iistage iiistage ivtherapybradiotherapyconcurrent chemoradiotherapytumor volume relative increase rate dayatumor volume doubling time daysaa mean ± sd rangeb five patients had two simultaneous primary tumors “ “ “from days to days apart median days the median tumor volume relative increase rate was perday median range “ and the mediantumor volume doubling time was days range “days no difference was observed when these two parameters were compared between different tumor stagestreatment outcome and survivalamong the tumors the time taken for local andorregional relapse and the occurrence of distant metastaseswere assessed for tumors in which treatment resultedin clinicalradiological eradication of the disease at “ weeks posttherapy local andor regional relapse wasrecorded in nine cases with a median time fromtreatment completion of months range “ monthsthe two and fiveyear probability of local recurrenceafter the end of the treatment was and respectively whereas for the regional relapse it was and respectively at years posttherapy only anincrease in the local recurrence probability of wasobserved whereas the probability of regional relapseremained stable the two and fiveyear probability ofdistant metastases was at two and years thelrc was ci and ci respectively and the dfs was ci and ci respectivelyin the yearperiod after the start of treatment outof patients died the index cancer was thereason in cases at two and years posttreatment the os rates were ci and ci respectivelyeventseffect of covariatesdue to the low number of events local andor regionalrecurrence ninecancerspecific deaths events only univariate regression models were fittedthe following covariates were tested in the modelsoverall disease stage initial tumor volume tumor doubling time and relative increase rate day of primarytumor volume measures of tumor kinetics did not showany impact on lrc or csh only an inverse relationshipbetween the initial primary tumor volume and csh wasobserved hr for index cancerspecific death perevery cm3 increase in the volume of primary tumorstable there was no difference in the value of dfsbetween patients with a primary tumor volume relativeincrease rate 1day and 1day p fig discussionwhile it is intuitively anticipated and confirmed by theresults of the metaanalysis that a delay in starting rtwould have a negative impact on the treatment of patients with hnc this association was not confirmed inour study we only found a statistically insignificantupward trend in the risk of locoregional recurrence forthe first days of rt delay in addition the differencesin the growth kinetics between individual tumors whichwere studied in a smaller group of patients were considerable but did not appear to be of significance for theprediction of treatment outcomesobviously the relationship between tti and diseaseprognosis in patients with hnc is more complex than itmight seem at first glance the first negative impact ofwaiting for treatment to begin is the risk that the tumorwill increase in size andor metastasize during this timemaking it harder to treat or resulting in it becoming untreatable [ ] however the time for a tumor to growis only one of the factors that determines prognosis andthe absence of a statistically significant association between tti and the treatment outcome in our study andis thus not surprising [“]many other reportsmoreover no obvious methodological differences couldbe found between these negative studies and the positive studies that confirmed the association between ttiand treatment outcomes [“] in both groups thereare individual studies that have similar sample sizestumor sitestage mix and periods covered speaking infavor of comparable quality of diagnostics therapy andstatistics across the studies 0cžumer radiation oncology page of table effect of stage and tumor kinetics on locoregional control and overall survivalparameterstnm stageiv vs iiiivtuper cm3vtu relative increase rateper dayvtu relative increase rate‰¤ vs 1dayvtu volume of primary tumorlocoregional controlhr ci““““pvaluecause specific hazardhr ci““““pvalueon the contrary in more recently reported analyses ofthe national cancer registries data an adverse effect ofwaiting for radiotherapy was clearly established [“]however the results from this type of analysis should betaken with caution not only due to the limitations inherent in the tumor registry data unmeasured confounding selection biasincomplete data and codingerrors but also because the effect of delaying rt oncancerspecific outcomes was not evaluated in additionpatients irradiated in postoperative and definitive settings were not analyzed separately overall the researchmethodology and interpretation used in these studieswere criticized and the magnitude of the effect that theysupposedly demonstrated was questioned in the present study a linear increase in the log hazardfor locoregional recurrence was found during the first days of waiting for rt although it was not statisticallysignificant it is possible that unknown confounders thatwere not accounted in our analysis eg tumor growth kinetics reduced the statistical power of the tti the resultsfrom fortin and naghavi who reported on theincreased risk of locoregional failure with tti daysand days respectively [ ] are the most comparable to our own however optimal ttithresholdsfig impact of primary tumor volume relative increase rate to diseasefree survival in patients with no residual disease at “ weeks posttherapy 0cžumer radiation oncology page of identified in different studies showed considerable variations pointing to the uncertainty of such calculations andtheir dependence on the characteristics of the analyzedpopulation [ “] the possible role of classical prognostic factors such as location of primary tumor diseasestage and addition of cct is not expected to be relevant inthis respect as in our and other similar studies the statistical significance of tti was verified by multivariateanalysisthe effect of tti on treatment outcomes however isnot only conditioned by the duration of waiting for rtbut also on the rate of tumor growth in hnc a vast heterogeneity in tumor cell kinetics has been observed conditioned by the local milieu from which it arises whichdiffers from patient to patient [ ] historicallydifferent methods were explored to evaluate tumor cellkinetics but did not succeed in providing clinically relevant kinetic parameters [ ] more recently a comparison of two sets of imaging data acquired at twodifferent time points was successfully employed for thispurpose usually volumetric data are extracted from diagnostic and rtplanning ct scans for the calculation ofdifferent parameters reflecting the rate of tumor growtheg tumor volume doubling time or absolutepercentagetumor volume increase per day despite some differencesin the calculation methodology across studies a large variation in the individual values of kinetic parameters wasseen in all of them including ours indicating that all ofthe studied populations represent a unique mix of slowand fast growing tumors [“] among our patientsfive had no measurable increase in primary tumor volumeeven when the interval between ct scans was up to days the inverse relationship between kinetic parametersdetermined by the comparison of two ct datasets andtreatment outcomes was implied or even confirmed inseveral smaller studies pointing to potential clinical utilityof imagingderived kinetic data [“] in our grouphowever no such association was found a small numberof patients and “ in contrast to other studies “ the inclusion of different tumor sites could contribute to the negative result as well as differences in the changes in kineticproperties triggered by rtccr in individual tumors tumor radiosensitivity may also influence the effect oftti on outcome while to some extent it may be evaluated before rt begins eg by molecular profiling identification of hypoxic cells and the determination of hpvstatus in oropharyngeal primary tumors in daily clinicalpractice it is usually not considered when planning treatment [ ] in order to diminish the impact of intrinsic tumor radiosensitivity the patients in our study withresidual disease at “ weeks postrt were excludedfrom the analysis of lrc we hypothesized that the inability to achieve a complete tumor response to chemoradiation was due to the radiobiological characteristicsof the disease and not the delay in starting rt howeverthe exclusion of these patients from the analysis did notaffect the end resultsour study has weaknesses which are mostly due to itsretrospective nature however for obvious ethical reasons this is an inevitable feature of studying delays inthe initiation of cancer therapy we are aware that thereis always some doubt as to the accuracy of the diagnosticprocedures the staging the quality of planning and theimplementation of rt in the case of retrospective research nonetheless the same restraint exists in the caseof other similar studies and even more so in the case ofanalyses of cancer registry datasets an important feature of our data set is that no patients were lost duringthe followup and in our opinion is free from manyhidden biases that larger studies inevitably bring withsince almost half of our patients had oropharyngeal carcinoma missing information regarding the p16 or human papillomavirus hpv tumor status could be ofimportance howeverin the cohort of oropharyngealcancer patients treated at our institution between and only had a hpvrelated tumor thus we reasonably assume that the impact of p16hpv tumor status on the study results was negligible inaddition in hpvpositive cases perni found nosignificant association between tumor growth velocitycalculated from serial pretreatment ct scanslocaland distant control or os and the same was reportedby chu who measured metabolic growth velocityusing pretreatment petct scans [ ]other drawbacks to consider include technical limitationsin ct scan acquisition the accuracy of presentation of actual tumor volume on ct images and the precision of thedelineation of gross tumor volume to minimize errors inthe estimation of comparative tumor volumes only highquality pairs of ct images were selected for the analysis oftumor kinetics in addition physical findings documentedin clinical records and when available diagnostic mriswere used for this purpose and labelled tumor volumes represented a consensus between two experienced radiationoncologists and a radiologist all dedicated to hnc management like many other studies [“ “] the comorbidity burden was not registered in our patientsalthough it should be taken into account when assessingsurvival outcomes finallylags in the prebiopsyperiod were not addressed in our study including patientdelay and delays in referral and diagnostics which may addsignificantly to the total tti these are also costly and potentially fatal but can be successfully reduced by effectivecoordination between providers [“]sthe relationship between tti and treatment outcomesis multifaceted so the controversy of published results is 0cžumer radiation oncology page of not surprising in this study we found that delays in theonset of rt do not harm all patients as tti is a problem in many public health systems further research iswarranted and should focus on two areas evaluatinglarge population surveys with highquality data andtreatmentrelated outcomes not just os and the prognostic relevance of imagingderived kinetic data of individualtumors which appeared promising in severalsmaller and statistically underpowered studies in orderto obtain a tool to identify patients at increased risk oftreatment failure due to delays in starting rt in a situation without clear knowledge to whom waiting for irradiation is harmful the only possible recommendationcould be that the waiting time for rt should be œasshort as reasonably achievable asara abbreviationshnc head and neck cancer rt radiotherapy tti time to treatmentintervention scc squamous cell carcinoma cct concurrent chemotherapyct computer tomography mri magnetic resonance imaging os overallsurvival csh causespecific hazard lrc locoregional control dfms distantmetastasisfree survival dfs diseasefree survival iqr interquartile rangeci confidence interval hpv human papillomavirusacknowledgementsthis study was financially supported by the slovenian research agencyprogram no p30307authors™ contributionsstudy concepts žumer b strojan p study design žumer b pohar perme mstrojan p data acquisition all authors quality control of data strojan p dataanalysis and interpretation pohar perme m strojan p statistical analysispohar perme m manuscript preparation all authors manuscript editingstrojan p manuscript review žumer b pohar perme m strojan p the authors read and approved the final manuscriptfundingthis study was financially supported by the slovenian research agencyprogram no p3“availability of data and materialsthe datasets analyzed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study protocol was approved by the republic of slovenia national medicalethics committee no “ all patients gave consent for using theirdata for study purposes at the start of their treatment for retrospective studies awritten consent is deemed unnecessary according to national regulationsconsent for publicationthe republic of slovenia national medical ethics committee approved thestudy which was conducted in accordance with the ethical standards laiddown in an appropriate version of the declaration of helsinki theneed for consent was waived by the republic of slovenia national medicalethics committeecompeting intereststhe authors declare that they have no competing interestsauthor details1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia 2institute of biostatistics and medicalinformatics faculty of medicine university of ljubljana ljubljana slovenia3department of radiology institute of oncology ljubljana ljubljanaslovenia 4chair of oncology faculty of medicine university of ljubljanaljubljana sloveniareceived may accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin “ world health anization who life expecta
Colon_Cancer
malignant mesothelioma is an aggressive cancer associated with asbestos exposure with median survival time of to months following diagnosis given that mesothelial cells also line the peritoneum and pericardium malignant mesothelioma can present in unusual sites and in patients with nonrespiratory complaints a 73yearold male presented to the emergency department for worsening intermittent diffuse abdominal pain for the past months with associated unintentional 40pound weight loss early satiety and diarrhea he denied exposure to asbestos computed tomography imaging revealed multiple masses concerning for malignancy including the primary retroperitoneal mass a mass involving the terminal ileum and a mass in the right upper lung esophagogastroduodenoscopy demonstrated significant mass effect within the stomach without signs of endoluminal infiltration computed tomography“guided biopsy of the retroperitoneal abdominal and intramuscular paraspinal masses was performed stage iv epithelioid mesothelioma was confirmed when hematoxylin and eosin staining revealed pleomorphic malignancy nuclei containing a vesicular chromatin pattern and prominent nucleoli and immunohistochemical staining was positive for ck oscar cytokeratin gata3 calretinin ema and ck56 he was started on cisplatin pemetrexed and bevacizumab but developed severe abdominal pain with pneumoperitoneum and bowel perforation month later and expired shortly thereafter to our knowledge this represents a highly atypical presentation of malignant mesothelioma considering the involvement of the retroperitoneum with diffuse lesions in the abdominopelvic cavity and thorax sparing the lung pleurae this case also calls attention to the occurrence of malignant mesothelioma in patients without known asbestos exposure and the crucial role of pathology in diagnosing atypical presentationskeywordsepithelioid mesothelioma neoplasm asbestos esophagogastroduodenoscopy retroperitonealintroductionmalignant mesothelioma mm is a rare cancer originating from mesothelial cells forming the linings of the pleura to of mm cases12 peritoneum to of cases23 and pericardium of cases456 typically the pathophysiology of mm is believed to occur in patients with asbestos exposure leading to chronic inflammation of the pleura67 subsequently mm presents in a patient with shortness of breath with findings on imaging of nodular thickening of the pleura pleural effusion or a mass7 in cases involving the peritoneum a more common initial presentation would be ascites with computed tomography ct findings including irregular thickening of the peritoneum lymph node enlargement and possible metastasis to the chest table regardless of location published guidelines detail the importance of first looking at the histology of a biopsy prior to immunohistochemical ihc and molecular testing to confirm suspicion of mm from the histology mm is classified into epithelioid sarcomatoid or biphasic mixed with epithelioid mm remaining the most common with its polygonal cells resembling reactive mesothelial cells other histologic features include scalloped cell borders with increased cytoplasm prominent and enlarged nucleoli and nuclear atypia10 the presence of a solid mass separate from the pleura and peritoneum with histologic features of mm eliminates the requirement for stromal invasion for the diagnosis910immunohistochemical testing should use markers of mesothelial origin such as cytokeratin markers and alternative tumor markers to narrow the differential diagnosis based on histology10 this will vary by histology and location 1mercyone des moines medical center des moines ia usa2des moines university des moines ia usareceived may revised june accepted july corresponding authordustin j uhlenhopp mercyone des moines medical center 6th avenue des moines ia usa email duhlenhoppmercydesmoinescreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c of investigative medicine high impact case reportstable typical characteristics of malignant abdominal mesothelioma1819characteristicspercentage of casespathological subtypes epithelial sarcomatous mixedasbestos exposure male femalepresenting symptoms ascitesa abdominal pain asthenia weight loss anorexia fevera diarrhea vomitingact scan findings ascites abdominal mass peritoneal thickening mesenterial thickeningadditional laboratory findings thrombocytosis 000mm3 anemia male gdl female gdlmaletofemale ratiomedian overall survivalaverage age of onset months yearsabbreviation ct computed tomographyamost significant symptoms on presentation associated with deathof the mm epithelioid markers are needed to rule out carcinomas and peritoneal markers are needed to rule out adenocarcinomas and cancers causing peritoneal carcinomatosis810 key markers for mesothelioma are positive ihc staining for calretinin d240 podoplanin and cytokeratin in this case report we present an unusual case of mm with metastatic disease in a patient with no known asbestos exposure and a history of abdominal pain and weight loss found to have multiple lesions and diffuse lymphadenopathy on imagingcase descriptiona 73yearold male presented to the emergency department with worsening intermittent diffuse abdominal pain and 40pound weight loss over the past months associated with increased flatulence early satiety and frequent exive nonbloody diarrhea the patient described the pain as cramping that typically worsened in severity at night he denied feverchills dyspnea cough epistaxis hematemesis hemoptysis hematochezia and melena the patient was a longtime science instructor at a local community college prior to transitioning to the swedish importexport business with no significant exposure history including asbestos he had a prior history of right middle cerebral artery stroke and was a former smoker with a 60packyear smoking historyinitial testing revealed normal electrolytes and liver function panel elevated lactic acid mmoll leukocytosis white blood cell 12100mm3 and anemia hematocrit abdominal ct with oral and intravenous iv contrast revealed a homogenous retroperitoneal mass measuring — — cm with displacement of the gastroesophageal junction and lesser curvature of stomach as well as a soft tissue mass involving the terminal ileum measuring — — cm both were concerning for neoplasm additional findings included diffuse lymphadenopathy in the retroperitoneum likely signifying metastatic disease no evidence of obstruction was noted see figure esophagogastroduodenoscopy confirmed concerns for extrinsic compression of gastroesophageal junction and fundus of stomach suggesting an underlying mass no endoluminal gastric masslesions were appreciated see figure colonoscopy was attempted but scope was not able to advance past the sigmoid colon safely due to colonic stricturecomputed tomography chest with iv contrast identified moderate diffuse emphysema a right upper lung lobe spiculated mass measuring cm and indeterminate small left upper and lower lobe nodules see figure diffuse retrocrural and gastroesophageal lymphadenopathy was noted in addition to the retroperitoneal lymphadenopathygiven the concern for lymphoma versus alternative neoplastic disease ctguided biopsy was performed on the retroperitoneal abdominal mass and an intramuscular paraspinal mass hematoxylin and eosin staining revealed pleomorphic malignancy nuclei containing a vesicular chromatin pattern and prominent nucleoli see figure cells were predominantly polygonal with occasional spindling noted ihc staining was positive for ck oscar cytokeratin gata3 calretinin ema and ck56 staining was negative for pax8 tte1 p40 p63 d240 wt1 moc31 cd34 mari cd45 dog desmin cd117 sox10 actin ca9 and myogenthese findings were suggestive of epithelioid mm with metastasis to the terminal ileum and lung a diagnosis that was confirmed by mayo clinic laboratories noting this is a particularly atypical presentation of mesotheliomathe patient opted for treatment of stage iv mesothelioma with plans to pursue a 21day cycle of cisplatin pemetrexed and bevacizumab for up to cycles however he presented to the emergency department days after starting the second cycle with severe abdominal pain of hours duration he was found to have a moderate pneumoperitoneum with bowel perforation worsening intraabdominal mesothelioma and several small bilateral acute renal parenchymal infarcts the patient and his family opted for comfort care and he passed the following day 0cuhlenhopp et al figure abdominal computed tomography with oral and intravenous contrast demonstrating a homogenous retroperitoneal neoplastic mass measuring — — cm green arrows on left image with displacement of the gastroesophageal junction and lesser curvature of stomach a soft tissue mass involving the terminal ileum measuring — — cm and a 17cm midmesentery mass are also noted green arrows on right imagefigure esophagogastroduodenoscopy revealed extrinsic compression of the fundus suggesting an underlying mass or lesion no endoluminal gastric masseslesions were appreciated computed tomography“guided biopsy later revealed this to be epithelioid mesothelioma originating from the retroperitoneal spacediscussionthe diagnosis of mm carries a poor prognosis and requires careful review by pathology our case details the workup necessary to diagnosis mm in a patient with an atypical presentation of intermittent abdominal pain found to have figure computed tomography chest with intravenous contrast identified moderate diffuse emphysema a right upper lung lobe spiculated mass measuring cm green arrows and indeterminate small left upper and lower lobe nodules as this did not involve the pleura as is traditionally seen this was felt to be a metastasis of the primary retroperitoneal mass with metastasis to lungs and ileumsignificant metastatic disease prior to identification of a primary malignancyreview of the case reveals the importance of tissue analysis and the continued public health concern of mesothelioma 0c of investigative medicine high impact case reportsongoing research is investigating whether these genetic changes have a role in disease development in cases without asbestos exposure610 in the case of malignant peritoneal mesothelioma there is a higher fraction of mm without an identifiable environmental exposure which raises the question whether the disease process and role of genetics vary by the location of mm14 in the future these will hopefully lead to the development of immunotherapies that are able to target these pathways and improved disease prognosis6known asbestos exposure was not a factor in our case and should not be used to rule out mesothelioma10 over of mm cases are attributable to asbestos exposure with of malignant peritoneal mesothelioma attributed to asbestos1516 recent studies detail that despite the restriction of asbestos products in the united states for over years the role of asbestos in chronic inflammation and dna damage will continue to cause malignancy615 in the united states alone there were approximately new mm cases in with current estimates anticipating a peak incidence of mm worldwide in given the continued use of asbestos worldwide and time to disease development6715 this case highlights the importance of considering mesothelioma in the differential diagnosis of abdominal pain regardless of asbestos exposure historydeclaration of conflicting intereststhe authors declared no potential conflicts of interest with respect to the research authorship andor publication of this fundingthe authors received no financial support for the research authorship andor publication of this ethics approvalour institution does not require ethical approval for reporting individual cases or case seriesinformed consentverbal informed consent was obtained from the patient for their anonymized information to be published in this orcid idsdustin j uhlenhopp orcid0000000315092203ann saliares orcid0000000281484435tagore sunkara orcid0000000195369027references robinson bm malignant pleural mesothelioma an epidemiological perspective ann cardiothorac surg doi103978jissn2225319x20121104 hui m harshavardhana kr uppin sg pericardial mesothelioma presenting as chronic constrictive pericarditis a series of three cases from a single institution indian j pathol microbiol doi104103ijpmijpm_711_17figure hematoxylin and eosin stain of the retroperitoneal mass — malignant mesothelioma that is somewhat pleomorphic but mostly epithelioid polygonal cells with some spindling are seen cells have pleomorphic nuclei occasionally multinucleated with moderate amount of eosinophilic cytoplasm nuclei have a vesicular chromatin pattern with distinct nucleoli there is no obvious glandular or squamous differentiationmm carries a median survival time of to months following diagnosis7 this poor prognosis is partially due to the fact that over of cases are not discovered until the distant metastasis stage11 while most cases of epithelioid mm have an improved prognosis compared with sarcomatoid mm it can possess pleomorphic features that indicate highly aggressive cancer and shortened expected survival time71012 treatment consists of chemotherapy radiation and surgery although no therapy is curative7in our case ihc staining had an important role in developing the diagnosis of epithelioid mm with pleomorphic features it distinguished the disease from reactive mesothelial hyperplasia and other malignancies with possibly better prognoses10 the right lung lobe mass is not the typical presentation for mm involving the thorax and complicates the clinical picture the presentation of the retroperitoneal mass coupled with diffuse metastatic lesions and atypical involvement of the thorax make this patient™s presentation particularly unique calretinin gata3 and ck56 were positive in the tissue obtained from the retroperitoneal and paraspinal muscle masses which implicated a mesothelial origin for the malignancy calretinin is a strong indicator of epithelioid mm and is useful in distinguishing mm from adenocarcinoma10 recent reports estimate a positive staining for gata3 in mesothelioma in of cases and note a presence in epithelioid mm in one third of cases1013 another important ihc markers for mm is d240 which was negative in our patientwhile not completed in our patient other markers present in mm include the deletion of p16 occurring in of epithelioid mm and the presence of a bap1 mutation101214 0cuhlenhopp et al lev½ m boubl­kov¡ l b¼chler t Å¡imÅ¡a j treatment of malignant peritoneal mesothelioma klin onkol doi1014735amko2019333 sardar mr kuntz c patel t et al primary pericardial mesothelioma unique case and literature review tex heart inst j mutsaers se the mesothelial cell int j biochem cell biol napolitano a carbone m malignant mesothelioma time to translate trends cancer bibby ac tsim s kanellakis n et al malignant pleural mesothelioma an update on investigation diagnosis and treatment eur respir rev doi10118316000617 liang yf zheng gq chen yf song h yin wj zhang l ct differentiation of diffuse malignant peritoneal mesothelioma and peritoneal carcinomatosis j gastroenterol hepatol gill rr imaging of mesothelioma in a tannapfel ed malignant mesothelioma recent results in cancer research vol springer husain an colby tv ordonez ng et al guidelines for pathologic diagnosis of malignant mesothelioma update of the consensus statement from the international mesothelioma interest group arch pathol lab med howlader n noone am krapcho m et al seer cancer statistics review accessed february seercancergovcsr1975_2016 arif q husain an malignant mesothelioma diagnosis arch pathol lab med miettinen m mccue pa sarlomorikala m et al gata3 a multispecific but potentially useful marker in surgical pathology a systematic analysis of epithelial and nonepithelial tumors am j surg pathol attanoos rl churg a galateausalle f gibbs ar roggli vl malignant mesothelioma and its nonasbestos causes arch pathol lab med craighead je epidemiology of mesothelioma and historical background in a tannapfel ed malignant mesothelioma recent results in cancer research vol springer spirtas r heineman ef bernstein l et al malignant mesothelioma attributable risk of asbestos exposure occup environ med bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin doi103322caac21492 manzini vde p recchia l cafferata m et al malignant peritoneal mesothelioma a multicenter study on cases ann oncol doi101093annoncmdp shih ca ho sp tsay fw lai kh hsu pi diffuse malignant peritoneal mesothelioma kaohsiung j med sci doi101016jkjms201305003 0c'
Colon_Cancer
of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified dtpa complexes21 the original substance of the modified dtpa dtpamod was synthesized in tomsk polytechnic university preparation of colloid solution dtpamod was produced using the following method a sample of modified dtpa with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of nahco3 solution by heating to a0°c after that the volume was adjusted with the same solvent up to the mark in order to reduce the p size the container with suspension was heated in water to a0°c and treated with ultrasound for a0min 1tomsk polytechnic university lenina avenue tomsk russia 2tomsk national research medical center russian academy of sciences kooperativny street tomsk russia email sadkintpuruscientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorfigure a0 the general scheme for the synthesis of 99mtcdtpamodwhich reduced the average p radius up to a0nm the general scheme for the synthesis of 99mtcdtpamod is shown in fig a0the second type of colloids is iron nanops coated with a carbon shell of fec fig a03a these ps were obtained from the institute of metal physics urb ras ekaterinburg russia in order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates adt onto the surface of these ps has been developed an effective method for the synthesis of adt followed by their application onto the carbon surface of ps was developed at the tomsk polytechnic university22 the general scheme for the synthesis of fec ps and their subsequent interaction with 99mtc is shown in fig a03bin the third type of colloids technetium99m was adsorbed on aluminum oxide powder a powder of lowtemperature cubic modification of gammaoxide al2o3 prepared from aluminum hydroxide powder aloh3 by its calcination in a muffle furnace was used the substance was synthesized in tomsk polytechnic universitya reducing agent”tin ii chloride dihydrate was used in order to obtain complexes of 99mtc with colloidsgelatin powdered ph eur uspnf pure pharma grade cas number was purchased from applichem gmbh darmstadt germanymethods method for preparation of 99mtc labeled nanocells the introduction of the radioactive label 99mtc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent sncl2ˆ™2h2o “ a0mgml in different ratios and then adding a a0ml of eluate of 99mtc “ a0mbqml to the mixtures the mixtures were incubated for a0min at a temperature of “ a0°c the preparation is ready for use after cooling at room temperature the reducing agent sncl2ˆ™2h2o was used as a hydrochloric acid solution the amount of a0g of tin chloride ii is added to the vial and a0ml of a0m hydrochloric acid hcl scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cfigure a0 a carbon encapsulated iron nanop b the general scheme for the synthesis of fec psis then added for its preparation after dissolution the volume is adjusted with distilled water to a0ml dissolution was carried out in an inert gas argon mediumdetermination of the size of 99mtc labeled colloidal ps the determination of the size of the labeled nanocolloids was carried out by spectroscopy on a nanophox p size analyzer œsympatec gmbh germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity calculations of the yield of products with different p sizes were determined according to the formulas given belowc220 avc ˆ’ a1avc c100 a1 ˆ’ a2a1 c50 a2 ˆ’ a3a2where avc is the activity of the initial suspension prior to filtration a1 is the activity measured after filtration through a a0nm filter a2 is the activity after filtration through a0nm filter a3 is the activity measured after filtration through a0nm filterin parallel determination of the radiochemical purity rcp of preparations by thin layer chromatography method was carried outthin‘layer chromatography tlc procedure to determine radiochemical purity of 99mtc“nanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip sorbfil russia — a0 cm to determine scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0camount of sncl2ˆ™2h2o mga[sn99mtc]a atcviia table change in relative activities of the complex [sn99mtc] and 99mtc viipertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min in this system pertechnetate migrated with the front of the mobile phase rf to determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min in this system the 99mtc“nanocolloid migrated with the front of the mobile phase rf stability the stability of 99mtc“nanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mtc“nanocolloid following by incubation at a0°c for a0h at different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using tlc24determination of the functional suitability of preparations for scintigraphic detection of sln a study to assess the functional suitability of new nanocolloid rps was performed in series of experiments involving white wistar male rats weighing “ a0g injection of rp in a dose of “ a0mbq was performed between the first and second fingers of the rat™s hind paw the animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame” a0s in a — pixel matrix static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of — with a set of pulses scintigraphy of animals was performed on an ecam signature gamma camera siemens germany the results of scintigraphic studies determined the percentage of accumulation of rp in the lymph node and the injection site the maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the œeuropean convention for the protection of vertebrates used for experiments or other scientific purposes strasbourg the experimental protocols were approved by cancer research institute biomedical ethics committee protocol number all invasive manipulations with animals were performed using inhalation or drug anesthesiastatistical analysis all mean values are expressed as idg ± sd data were analyzed statistically using methods of general statistics with a commercially available software package œstatistics for windows statsoft inc version results and discussionto carry out the labeling of colloids 99mtc extracted from a standard generator in the form of pertechnetate ions contained in a nacl solution was used it has a higher degree of oxidation vii in this chemical form and is not prone to complex formation a reducing agent”tin ii chloride dihydrate widely used for the preparation of various compounds labeled with 99mtc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 as a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess sncl2·2h2o or the additional formation of a complex of reduced 99mtc with tin26 all this required preliminary experimental studies to establish the necessary and sufficient amount of sncl2·2h2o in the reaction mixtureduring the experiments it was found that the optimal concentration of sn ii in the composition of the reaction mixture when it interacts with 99mtc should be in the range of “ a0mgml table a0it was found that when the eluate with the preliminarily reduced 99mtc vii was added to the nanops the sn ii concentration introduced in the rp was csn a0mgml almost the entire amount of 99mtc has time to enter the composition of the largesize complex with tin even before its mixing with nanops this means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the sn ii solution into the reaction mixture in this connection the reduction of 99mtc with tin ii was carried out in the presence of the selected substance in this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex the technique of applying of the 99mtc label to the surface of nanosized ps is given in the previous sectionas a result of the studies reagent compositions and conditions for obtaining three nanocolloid rps were determined table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of “ a0nmproceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mtc vii in the obtained preparations is “ preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in fig a0scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0ccomposition of the preparation per a0mldtpamod a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg n fec a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg n al2o3 a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg n colloid yield “ a0nm rcp ± ± ± table composition of reagents for production of technocium99 a0m nanocolloidsfigure a0 distribution of the preparation in the rat when the preparation is administered [al2o3 99mtc sn ii] a immediately after the administration of the drug b a0min after the administration c a0min after the administrationcomposition of preparations per a0mlal2o3 a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg g a0mg n dtpamod a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg g a0mg n fec a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg g a0mg n yield of colloid “ a0nm rcp ± ± ± table indicators of rcp and the yield of a colloid with a fraction of “ a0nm after the introduction of gelatin in the reagentsscintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mtc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed gelatin g was introduced into the reaction mixture in this connection to increase the œmobility of the labeled ps and increase the speed of their movement through the lymph system matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mtc the results of the experiments showed that the addition of gelatin “ a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of “ a0nm table a0in addition the size of these ps was determined on a nanophox p analyzer the obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in fig a0 a b c the average p size diameter is and a0nm respectivelystability test showed that complex 99mtc“nanocolloid was stable in normal serum at least for a0h radiochemical purity of the tracer at the end of the experiment was ± a study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes fig a0 table a0 displays the al2o3 99mtc dtpamod 99mtc fec 99mtc biodistribution data at different time points postinjectionthe level of accumulation of preparations in the lymph nodes is “ of the total injected activityconclusionas a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid rps were determined an experimental dependence of the change in the content of 99mtc vii impurities on the concentration of tin ii was established and its minimum amount a0mgml was determined to reach a rhp greater than in this case the yield of the target colloid with p sizes of ± a0nm is “ preliminary tests of the developed preparations on experimental animals showed that accumulation of rp in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition “ a0mgml in addition there was an increase in the yield of the colloid scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 change in the density of the distribution of the number of ps from their size in radiopharmaceuticals a œ99mtcal2o3 b œ99mtcfec c œ99mtcdtpamodwith p sizes of “ a0nm to “ with radiochemical purity of the preparations of “ repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cstomachtime h99mtcal2o399mtcdtpamod99mtcfec ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± figure a0 distribution of the preparation in the rat with injection of suspension [al2o3 99mtc sn ii gelatin] a immediately after the administration of the preparation b a0min after the administration c a0min after the administration d a0min after the administration the numbers indicate ”lymph node ”site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± bloodmlheart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± table biodistribution data up to a0h after injection of “ a0mbq of 99mtc in healthy male rats data represent the average value n accumulation in the sln thus the level of accumulation of rp œ99mtcdtpamod and rp œ99mtcfecadt in the sln is and respectively at the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityreceived march accepted july references jakobsen j k sentinel node biopsy in urooncology a history of the development of a promising concept urol oncol “ weixler b et al sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival a prospective singlecenter trial world j surg 101007s0026 beasley g m et al sentinel lymph node biopsy for recurrent elanoma a multicenter study ann surg oncol moser j et al sentinel node biopsy in melanoma a singlecentre experience with consecutive patients br j dermatol 101245s1043 “ buda a et al optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer comparison of realtime fluorescence with indocyanine green and methylene blue int j gynecol cancer “ scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0c“ sahbai s et al pericervical injection of 99mtcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in spectct clin nucl med “ hoogendam j p et al 99mtcnanocolloid spectmri fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer j nucl med “ stoffels i leyh j p¶ppel t schadendorf d klode j evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies prospective randomized clinical trial to compare icg99mtcnanocolloid hybrid tracer versus 99mtcnanocolloid eur j nucl med mol imaging “ beisani m et al initial experience in sentinel lymph node detection in pancreatic cancer rev esp med nucl imagen mol schubert t uphoff j henke r p wawroschek f winter a reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer verification in consideration of the european guidelines bmc urol “ jaukovic l et al lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions hell j nucl med “ subramanian s pandey u shah s rangarajan v samuel g an indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection nucl med commun “ ruizdom­nguez j m ibarzservio l garc­ade manuel g calaf peris© o intraoperative injection of 99mtcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery actas urol “ schauer a j specific developments in sentinel node labling using 99mtccolloids in the sentinel lymph node concept springer berlin wang y et al gasphase chemistry of technetium carbonyl complexes chem phys “ o™connor m k et al comparison of tc99m maraciclatide and tc99m sestamibi molecular breast imaging in patients with wang j zhang r evaluation of 99mtcmibi in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer ejnmmi res br j radiol costa p et al scintigraphic imaging with technetium99mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats acta cir bras “ vera d r wallace a m hoh c k mattrey r f a synthetic macromolecule for sentinel node detection 99mtcdtpamannosyldextran j nucl med “ hoh c k wallace a m vera d r preclinical studies of [99mtc]dtpamannosyldextran nucl med biol “ skuridin v et al modified dtpa moleculebased nanocolloid radiopharmaceuticals j radioanal nucl chem “ filimonov v d et al unusually stable versatile and pure arenediazonium tosylates their preparation structures and synthetic applicability lett “ lukasz k thin layer chromatography in drug analysis “ crc press london skuridin v et al radiopharmaceutical drug based on aluminum oxide indian j sci technol 1017485 ijst2015v8i36 sazonova s i et al synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging iran j nucl med “ skuridin v s et al synthesis and biological characterization of 99mtclabeled ciprofloxacin pharm chem j “ acknowledgementsthis work was financially supported by ministry of education and science of the russian federation rfmefi57514x0034author contributionsvs conducting experimental research analysis and interpretation of the data final approval for manuscript publication vs development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication en development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication es development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication ar conducting experimental research analysis and interpretation of the data final approval for manuscript publication nv conducting experimental research analysis and interpretation of the data final approval for manuscript publication rz conducting tests of the functional suitability of drugs preparation of the section evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and figures „– final approval of the manuscript for publication of the manuscriptcompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to vsreprints and permissions information is available at wwwnaturecomreprintspublisher™s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0copen access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate if changes were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0c'
Colon_Cancer
" vibrio cholerae are oxidasepositive bacteria that are classified into various serotypes based on the osurface antigen v cholerae serotypes are divided into two main groups the o1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v choleraenovc can cause choleralike diarrhea a pubmed search revealed that only cases of necrotizing fasciitis causedby novc have been recorded in the scientific literature to date we report the case of a japanese woman whodeveloped necrotizing fasciitis caused by novc after traveling to taiwan and returning to japancase presentation a 63yearold woman visited our hospital because she had experienced left knee pain for thepast days she had a history of colon cancer stage iv t3n3 m1a and had received chemotherapy she hadvisited taiwan days previously where she had received a massage she was diagnosed with septic shock owingto necrotizing fasciitis she underwent fasciotomy and received intensive care she recovered from the septic shockhowever after weeks she required an aboveknee amputation for necrosis and infection her condition improvedand she was discharged after weeks in the hospitals with the increase in tourism it is important for clinicians to check patients™ travel history cliniciansshould be alert to the possibility of necrotizing fasciitis in patients with risk factors necrotizing fasciitis caused bynovc is severe and requires early fasciotomy and debridement followed by intensive postoperative carekeywords necrotizing fasciitis vibrio cholerae taiwan massage septic shock polymyxin b correspondence kei610805gmailcom1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctsuruta bmc infectious diseases page of vibrio cholerae are curved gramnegative rod gnrbacteria that are oxidase positive they are classified intovarious serotypes based on the o surface antigen vcholerae serotypes are divided into two main groups theo1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v cholerae novccan cause choleralike diarrhea novc are found as autochthonous microbes in coastal and marine environments outbreaks of choleralike illness caused bynovc have been reported in the united states o141and o75 former czechoslovakia o37 sudan o37peru o10 o12 and mexico o14 [“] moreovernovc can cause a range of extraintestinal infectionsincluding bacteremia meningitis pneumonia peritonitischolangitis salpingitis and softtissue infection seafood including oysters fishes shrimps clams musselsand apple snail is the most common source of infection a pubmed search revealed that only cases of necrotizing fasciitis caused by novc have beenreported in the scientific literature to date we reportthe case of a patient who developed necrotizing fasciitisand septic shock caused by novc which necessitatedan aboveknee amputation of her left legcase presentationa 63yearold woman visited minaminara general hospital in nara japan because she had experienced leftknee pain for days prior to her visit she had been diagnosed with colon cancer stage iv t3n3 m1a years and months previously and had undergone surgery and received chemotherapy her most recent doseof chemotherapy was administered days before herinitial consultation she had visited taiwan days previously where she had received a massage after themassage she developed gradually worsening pain in herlower left leg on presentation she was able to walkunaided and she reported her history of colon cancerand recent travel as we suspected that the pain in herleg could be due to necrotizing fasciitis we requestedmagnetic resonance imaging mri of her left lowerleg the images showed a swollen soleus muscle andposterior tibial muscle and the t2weighted imageshowed hyperintensity of the muscle tissue fig after the mri our patient™s condition deteriorated andthe following vital signs were observed blood pressurebp mmhg heart rate beatsmin respiratory rate breathsmin and temperature °cthe results of arterial blood gas analysis were as folˆ’ mmhglows ph paco2 mmhg hco3base excess be ˆ’ meql and lactate mmoll the patient™s laboratory test results were as followscreactive protein crp mgdl blood ureanitrogen bun mgdl creatinine mgdlprocalcitonin ngml nterminal probrain natriuretic peptide ntprobnp pgml and fibrinfibrinogen degradation products fdp μgmlinitiatedadministeredlowvenovenoushemodiafiltrationsurgery her blood pressure wasintravenous infusion of meropenem and noradrenaline wasand the patient underwentemergency surgery before the surgery the compartment pressure of her left leg was measured by simpleneedle manometry the pressures were as follows mmhg mmhg mmhg and mmhg in theanterior lateral superficial posterior and deep posterior compartments respectively some muscle tissuesin the anterior and deep posterior compartments werenecrotic for double incision fasciotomy a relaxationincision was made on her left knee and theaffected area was irrigated and debrided fig aftertheandtherefore wepolymyxin b directhemoperfusion pmxdhp to trap endotoxins andcontinuoususinghemofeel ch13 w toray medical co ltdurayasu japan as a slightly curved gnr that wasoxidase positive was detected in her blood we diagnosed her with necrotizing fasciitis and septic shockcaused by vibrio species we changed the antibioticsfrom meropenem to ceftriaxonelevofloxacin andminocycline we used the pmxdhp once again andtapered the dose of noradrenalin gradually wediscontinued noradrenalin on day postoperativelyon day postoperatively the anism was identifiedas novc theantibiotics wasconfirmed postoperatively on day and we discontinued levofloxacin table although the patient™sgeneral condition improved there was a discharge ofpus from the postoperative wound on day postoperatively a second debridement was performedseveral muscles in the patient™s left leg including theanterior tibial muscle had become necrotic and thenecrosis had spread to her knee on day postoperatively an aboveknee amputation was performedher vital signs and laboratory data obtained since admission are shown in fig her condition improvedand she was discharged weeks after admissionsusceptibility ofdiscussion and sixteen cases of necrotizing fasciitis caused by novchave been previously reported table [“] themajority of patients were exposed to seawater or hadan injury in rare cases vigorous massage is one ofthe risk factors of necrotizing fasciitis howeverthe patient in the present case had a risk of novcinfection because of colon cancer and immunosuppression due to chemotherapy as she received chemotherapy within a month thusthein this case 0ctsuruta bmc infectious diseases page of fig t2weighted magnetic resonance images of the patient™s lower legs a coronal image b axial image these images show that the soleusand posterior tibial muscles on the left lower leg indicated by red arrows are swollen and inflamedfig photographs of lesions in the patient™s leg the patient™s leg before surgery shows multiple large blisters 0ctsuruta bmc infectious diseases page of table susceptibility of antibioticsantibioticsampicillinminimal inhibitory concentrations piperacillinceftazidimeimipenemcilastatinamoxicillinclavulanategentamicinminocyclinechloramphenicolsulfamethoxazoletrimethoprimlevofloxacinfosfomycins s s ‰¦s s s ‰¦s ‰¦s ‰¦s ‰¦r the novc remains unknown assource ofthepatient did not report any exposure to sea water oreating seafood the only potential cause of injury toher left leg was the massage she received thereforewe speculate that the massage might have been thesource ofthe novc based on the circumstantialevidence we administered blood purification therapyusing pmxdhp and venovenous hemodiafiltrationfor septic shock although no previous studies havereported the use of pmxdhp for novc a studyreported the use of pmx for v vulnificus thirdgenerationtetracyclineandfluoroquinolone were used for severe vibrio infections tetracycline combined with the fluoroquinoloneorcephalosporinfollowed by oral fluoroquinolones or doxycycline wasrecommended for invasive novc infections [ ]an in vitro study revealed that cefotaxime and minocycline have a synergistic effect in the treatment forcephalosporinsaparenteralthirdgenerationfig change of vital signs and laboratory data during the hospital admission a changes in the patient™s vital signs during days “ ofhospitalization b changes in patient™s blood biochemistry during days “ of hospitalization atiii antithrombin iii crp creactive proteinfdp fibrinfibrinogen degradation products map mean arterial pressure nad noradrenaline 0ctsuruta bmc infectious diseases page of age sex underlying diseasestable clinical characteristics of patients with nono vibrio cholerae necrotizing fasciitisyear ofreportsourcesurgery amputation multiple debridementand antibiotics ticarcillinclavulanate imipenemgentamicin clindamycinrisk factors mdiabetes mellitussurvived usatreatmentoutcome country oantigen epidemiologicexposureexposure of achronic plantar ulcerto sand in abathhouse mcirrhosissurgery cefotaxime minocycline cefotaximesurvivedtaiwan f mcirrhosis congestiveheart failurecirrhosis diabetesmellitus mhepatitis csurgery ceftriaxonediedtaiwansurgical debridement ceftazidime doxycyclinediedtaiwan o56surgery antibiotics thirdgeneration cephalosporindoxycyclinediedtaiwanhandling seafood mhepatitis steroidssurgery antibiotics thirdgeneration cephalosporindoxycyclinesurvivedtaiwan mcirrhosissurgery clindamycin ceftazidime tetracyclinesurvivedtaiwansurgery antibioticssurgery antibioticsdiedtaiwandiedtaiwan m m mcirrhosis hepatitis cdiabetes mellituscirrhosis hepatitis bhepatitis c diabetesmellituscirrhosis diabetesmellitusexposure to seawaterprobable woundinfectionconsumption of rawseafoodseawater exposureinsect bite woundinfectionminor abrasionexposed to seawaterseawater exposuresurgery antibioticsdiedtaiwanseawater exposure mcirrhosissurgery antibioticssurvivedtaiwanseawater exposure mcopdsurgery antibioticssurvivedtaiwan mhiv hepatitis ccirrhosis mdiabetes mellitus michthyosis cellulitisnone mcopd chronic constructive pulmonary diseasesurgical debridement daptomycin levofloxacinsurviveditalyo137surgical debridement piperacillintazobactamfosfomycinsurgical debridement piperacillintazobactamtigecycline metronidazolesurgical debridementpenicillin gentamicin metronidazolesurvived austriadiedaustriaseawater exposuresurvived croatia o8seawater exposurev cholerae infections as patients with novcbacteremia require antibiotic treatment for at least month we administered ceftriaxone and minocycline for month necrotizing softtissue infectionscaused by novc are more lethal than those causedby v vulnificus to conclude we treated a woman with necrotizingfasciitis and septic shock caused by novc this caseillustrates that early fasciotomy and debridement arenecessary forsevere necrotizing fasciitis caused bynovc and prolonged intensive care may be requiredafter surgeryo139 vibrio cholerae ntprobnp nterminal probrain natriuretic peptidepmxdhp polymyxin b direct hemoperfusionacknowledgementsnoneauthors™ contributionskt tu tw kn and ku treated the patient kt tu hf reviewed the literatureand mainly wrote this report kn tw ku reviewed the literature andmodified this paper based on specialty orthopedics emergency departmentinfectious disease all authors have read and approved the manuscriptfundingnoneabbreviationsbun blood urea nitrogen fdp fibrinogen degradation productsgnr gramnegative rod mri magnetic resonance imaging novc nono1availability of data and materialsall data are included in this published 0ctsuruta bmc infectious diseases page of ethics approval and consent to participatethis case report was approved by the ethics review committee atminaminara general hospital and was conducted in accordance with thedeclaration of helsinki consent for participation is not applicableconsent for publicationwritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images a copy of the writtenconsent is available for review by the editor of this journalcompeting intereststhe authors declare that they have no competing interestsauthor details1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japan 2orthopedic departmentminaminara general hospital nara japan 3infectious diseases departmentminaminara general hospital nara japan 4department of emergency andcritical care medicine nara medical university nara japanreceived march accepted august referencesgardner ad venkatraman kv the antigens of the cholera group of vibriosj hyg lond “ hirk s huhulescu s allerberger f lepuschitz s rehak s weil s necrotizing fasciitis due to vibrio cholerae nono1nono139 after exposureto austrian bathing sites wien klin wochenschr “ dobrović k rudman f ottaviani d crnek sÅ¡ leoni f Å¡krlin j a rare case ofnecrotizing fasciitis caused by vibrio cholerae o8 in an immunocompetentpatient wien klin wochenschr “jain akc varma ak mangalanandan kh kumar h bal a surgical outcome ofnecrotizing fasciitis in diabetic lower limbs j diab foot comp “ikeda t kanehara s ohtani t furukawa f endotoxin shock due to vibriovulnificus infection eur j dermatol “su ba tang hj wang yy liu yc ko wc liu cy in vitro antimicrobialeffect of cefazolin and cefotaxime combined with minocycline against vibriocholerae nono1 nono139 j microbiol immunol infect “tsai yh huang tj hsu rww weng yj hsu wh huang kc necrotizing softtissue infections and primary sepsis caused by vibriovulnificus and vibrio cholerae nono1 j trauma “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations morris jg nono group vibrio cholerae a look at the epidemiology of anoccasional pathogen epidemiol rev “li m shimada t morris jg jr sulakvelidze a sozhamannan s evidence forthe emergence of nono1 and nono139 vibrio cholerae strains withpathogenic potential by exchange of oantigen biosynthesis regions infectimmun “dalsgaard a albert mj taylor dn shimada t meza r serichantalergs o characterization of vibrio cholerae nono1 serogroups obtained froman outbreak of diarrhea in lima peru j clin microbiol “isaacmárquez ap lezamadávila cm eslavacampos c navarroocaña acraviotoquintana a serotypes of vibrio cholerae nono1 isolated fromwater supplies for human consumption in campeche mexico and theirantibiotic susceptibility pattern mem inst oswaldo cruz “hughes jm hollis dg gangarosa ej weaver re noncholera vibrioinfections in the united states clinical epidemiologic and laboratoryfeatures ann intern med “deshayes s daurel c cattoir v parienti jj quilici ml de la blanchardière anono1 nono139 vibrio cholerae bacteraemia case report and literaturereview springerplus mubarak sj owen ca doubleincision fasciotomy of the leg fordecompression in compartment syndromes j bone joint surg am “ wagner pd evans sd dunlap j ballonlanda g necrotizing fasciitis andseptic shock caused by vibrio cholerae acquired in san diego californiawest j med “ko w chuang y huang g hsu sy infections due to nono1 vibrio choleraein southern taiwan predominance in cirrhotic patients clin infect dis “ cheng nc tsai jl kuo ys hsueh pr bacteremic necrotizing fasciitis causedby vibrio cholerae serogroup o56 in a patient with liver cirrhosis j formosmed assoc “tsai yh hsu rww huang kc chen ch cheng cc peng kt systemicvibrio infection presenting as necrotizing fasciitis and sepsis a series ofthirteen cases j bone joint surg am “ changchien ch bacteraemic necrotizing fasciitis with compartmentsyndrome caused by nono1 vibrio cholerae j plast reconstr aesthetic surg“ maraki s christidou a anastasaki m scoulica e nono1 nono139 vibriocholerae bacteremic skin and soft tissue infections infect dis lond “ ottaviani d leoni f rocchegiani e canonico c masini l pianetti a unusual case of necrotizing fasciitis caused by vibrio cholerae o137 j clinmicrobiol “ 0c"
Colon_Cancer
acute myeloid leukemia aml is the most common type ofacute leukemia in adults caused by the clonal expansion ofundiï¬erentiated myeloid progenitor cells although mostpatients with aml can achieve complete remission by inductionchemotherapy the recurrence rate remains high and thus is themain factor aï¬ecting the outcomes of aml patients relapseoften develops from minimal residual disease in the protectivebone marrow bm microenvironment a comprehensiveunderstanding of the bm microenvironment is conducive to thediagnosis and personalized treatment of aml leukemic cellscytogenetics and molecular aberrations are the main factorsfor risk stratification in patients with aml in additionthe bm microenvironment also plays a very important role thein the homing and survival ofbm microenvironment contains various componentssuchas immune cells stromal cells endothelial progenitor cellsextracellular matrix growth factors and cytokines theinteraction between leukemic cells and bm microenvironmentaï¬ects resistance to chemotherapy in aml the modulationof bm microenvironment in aml is currently undergoingpreclinical research and early clinical trials molecular inhibitorssuch as cxcr4 inhibitors vla4 inhibitors and eselectininhibitors are currently undergoing clinical trials immunecells and stromal cells are important components of the bmmicroenvironment and are also the main ‚uencing factorsof leukemia development the estimate program isa common method to explore the microenvironment of manytumors recently it has also been used to explore theprognostic genes in the microenvironment of aml patients“ most studies have focused on diï¬erentially expressedgenes degs however the interaction and relationship betweengenes are open to investigate moreover the coding genes wereextensively explored but regions that encoded lncrnas andmirnas were less wellstudiedweighted gene coexpression network analysis wgcnais an algorithm commonly used in systems biology toexplore the correlation between gene sets and the clinic functionalization is achieved by constructing a freescalecoexpression gene network wgcna can identify highlyrelated genes and aggregate them into the same genetic modulecurrently wgcna is used in multiple fields such as cancer andnervous system or to identify potential biomarker candidates ornew therapeutic targets from genetic data “the competition endogenous rna cerna hypothesis was anew regulatory mechanism between noncoding rna ncrnaand messenger rna mrna proposed by salmena in in this theory crosstalk between cernas is achieved byabbreviations aml acute myeloid leukemia auc area under curve bmbone marrow cam cell adhesion molecules cerna competing endogenousrnas deg diï¬erentially expressed gene david the database for annotationvisualization and integrated discovery go gene ontology icam1 intercellularadhesion molecule il10ra interleukin receptor subunit alpha keggkyoto encyclopedia of genes and genomes ppi proteinprotein interactiontcga the cancer genome atlas tlr6 toll like receptor tlr8 toll likereceptor tom topological overlap matrix wgcna weighted correlationnetwork analysisimmunerelated cerna network of amlcompetitively combining shared mirnas in recent yearsthe cernas hypothesis has attracted widespread attention in thestudy of molecular and biological mechanisms of tumorigenesisand development for example previous studies have foundthat lncrnarelated cernas were involved in the biologicalprocesses of glioblastoma and breast cancer theresearch on cernas of leukemia was generally based on thediï¬erential genes screened by leukemia and normal controls but no known module based on cernas network related tomicroenvironment in leukemia has been set upinformation ofthe aml cohortin this study mrnas mirnas and lncrnas data andclinicalfrom the cancergenome atlas tcga database were used to calculate theimmune and stromal scores of these aml cases using theestimate algorithm diï¬erentially expressed mrnas andlncrnas were applied to wgcna to identify the modulesmost relevant to the aml immune microenvironment thenthe immunerelated lncrnamirnamrna cerna networkwas established to screen genes with clinical significance thesefindings will help to better understand the role oftumormicroenvironment in aml and shed light on the developmentand progression of amlmaterials and methodsdata acquisitionall data sets of aml patients were downloaded from tcgadatabase1 the data used in this study met the following criteria excluding samples combined with other malignancies samples with lncrnas and mirnas and mrnas detection datafinally all lncrnas mrnas and mirnas expression profilesof aml specimens and the corresponding clinical followupdata were downloadedidentification of differentially expressedgenesthe estimate algorithm2 was used to calculate the immunescores and stromal scores of aml samples diï¬erentiallyexpressed genes degs such as lncrnas mirnas and mrnaswere identified between high and low score groups stratified bythe median value of immune scores and stromal scores usinglimma package all q values use fdr to correct the statisticalsignificance of the multiple test lncrnas and mrnas withlog fc and fdr were regarded as diï¬erentiallyexpressed while mirnas with log fc and fdr were regarded as diï¬erentially expressed then all the degs wereentered into r version auckland nz united states forcluster analysis based on the expression value of each sample inits respective data set the results were expressed in a clustergrameach column represents a sample and each row represents theexpression level of a given gene1portalgdccancergov2sourcefenetprojectsestimateprojectfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amldavid3 wasgo and pathway enrichment analysesthe database for annotation visualization and integrateddiscoveryenrichedbiological themes of degs functions particularly go geneontology terms and kegg kyoto encyclopedia of genes andgenomes pathway enrichment p was set as thecutoï¬ criterionapplied to analyzeweighted gene coexpression networkanalysiswgcna is an algorithm for identification of gene coexpressionnetworksthrough highthroughput expression profiles ofmrnas or lncrnas with diï¬erent characteristics pairwisepearson correlation analysis was used to evaluate the weightedcoexpression relationship between all datasettopics in theadjacency matrix in this study wgcna was used to analyzemrnas and lncrnas to obtain the mrnas or lncrnas mostrelevant to aml immune microenvironmentcerna network construction andanalysisaccording to the results of wgcna we selected all mrnasand lncrnas in the most relevant module turquoise anddiï¬erentially expressed mirnas to construct a cerna networkbriefly the associated cerna network in aml was constructedfollowing three stages prediction of lncrnamirna inorder to make lncrnas and mirnas map into the interactionssuccessfully we used the miranda4 and pita5 to get targetedlncrnas that mirnas may regulate prediction of mirnamrna three online databases miranda4 targetscan6 andmirwalk7 were used simultaneously for target mrna prediction construction of lncrnamirnamrna cerna networkthe cerna network was constructed based on the negativelyregulating target relationships of mirna“mrna and mirna“lncrna correlation pairsppi network constructionthe retrieval of interacting genes string database8 wasutilized to construct a protein“protein interaction ppi networkof the degs identified in the cerna network the interactingpairs with a confidence score greater than were considered assignificant and were retained the degree represents the numberof interaction partnerssurvival analysiskaplan“meier plots weretherelationship between the overall survival of aml patientsand the expression level of mrnas lncrnas and mirnasthe statistical significance of the correlation was tested by theconstructed to illuminatelogrank test and p was considered significant theanalysis was conducted using the r package of survivalstatistical analysisgraphpad prismtm san diego ca united states or rversion auckland nz united states software was usedfor all data analyses diï¬erences across groups were comparedusing kruskal“wallis test for continuous variables diï¬erenceswere considered significant when p resultsimmune and stromal scores areassociated with aml clinicalparameterswe obtained gene expression profiles and clinical informationof aml patients from tcga database supplementarymaterial among them were male and were female with a median age range “ years oldaccording to the fab classification there were cases of m0 m1 cases m2 cases m3 cases m4 cases m5 cases m6 cases and m7 case the estimate algorithm was used tocalculate the immune scores and stromal scores of aml patientsthe median immune score was range from to and the median stromal score wasˆ’ range fromˆ’ to we analyzed the relationship between immune scores andstromal scores and clinical parameters of aml patients caseswith m4 subtype aml had the highest immune scores while caseswith m3 subtype had the lowest immune scores p figure 1a similarly m4 cases had the highest stromal scoreswhereas m0 subtypes had the lowest p figure 1baccording to cytogenetics aml patients were divided intothree groups favorable intermediatenormal and poor therewas an obvious correlation between the cytogenetic risk andthe immune scores p figure 1cfavorable vsintermediate p favorable vs poor p intermediate vs poor p but no significant correlationbetween the cytogenetic risk and the stromal scores was observedp figure 1dscoreand high immunestromalusing the median immune or stromal score as a thresholdaml patients were divided into two groups with lowimmunestromalscoresurvival analysis showed that the survival rate of aml patientswith low immune scores was significantly higher than that ofpatients with high immune scores p figure 1ehowever there was no significant diï¬erence in survival betweenpatients with low stromal scores and those with high stromalscores p figure 1f3httpdavidncifcrfgov4httpwwwmicrorna5genieweizmannacilpubsmir07mir07_exehtml6httpwwwtargetscan7http12920671508tringdbidentification of differentially expressedgenes based on immune scoresand stromal scoressetting the cutoï¬ criteria as log fc and fdr we identified mrnas figure 2a and lncrnasfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure immune and stromal scores are associated with aml clinical parameters ab distribution of immune scores a and stromal scores b for aml fabsubtypes cd the correlation between immune scores c and stromal scores d and aml cytogenetic risk ef kaplan“meier survival curve based on immunese and stromal scores f aml acute myeloid leukemiafigure heatmap of differentially expressed genes in the high and low immunestromal score groups a mrnas b lncrnas and e mirnas based onimmune scores c mrnas d lncrnas and f mirnas based on stromal scoresfigure 2b based on immune scores and mrnasfigure 2c and lncrnasfigure 2d based onstromal scores setting the cutoï¬ criteria as log fc and fdr we identified and mirnas based onimmune scores figure 2e and stromal scores figure 2frespectively the degs oflow vs high immunethefrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top go terms in each of biological process were performed for functional enrichment clustering analysis a top25 significant go terms based onupregulated genes in immune scores b top25 significant go terms based on upregulated genes in stromal scores c top25 significant go terms based ondownregulated genes in immune scores d top25 significant go terms based on downregulated genes in stromal scores go gene ontologyscore or stromal score groups were illustrated in the heatmap figure aml and thus require further research to determine theirbiological contributionfunctional enrichment analysis of degsbased on the david the databasefor annotationvisualization and integrated discovery gene annotation tool weperformed go analyses of both upregulated and downregulateddegs the top go biological process indicated that theupregulated degs based on immune or stromal scores wereprimarily enriched in neutrophil degranulation regulationof immune response signal transduction and ‚ammatoryresponse figures 3ab while the downregulated degs basedon immunestromal scores were primarily enriched in rrnaprocessing regulation of translation regulation of transcriptionand cell diï¬erentiation figures 3cd subsequently weperformed kegg kyoto encyclopedia of genes and genomespathway enrichment and interrelation analysis kegg analysisrevealed that the upregulated degs were mainly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingpathway hematopoietic cell lineage and cell adhesion moleculescams pathways figures 4ab while the downregulateddegs were mainly enriched in ribosome metabolism pi3kaktsignaling pathway transcriptional dysregulation in cancer andmirnas in cancer figures 4cd above analyses revealedthatthese degs play a vital role in the development ofconstruction of weighted correlationnetwork analysis and identification ofkey modulesbased on the results of survival analysis degs based on immunescores were selected for subsequent analysis the best β valuein the lncrnamrna coexpression network was which wascalculated using the diï¬erential lncrnas diï¬erentialmrnas and their expression data in leukemia samples nextthe method of dynamic tree cutting was used to producecoexpression modules finally modules of lncrnamrnacoexpression networks were generated and the heat map plot oftopological overlap matrix tom was shown figure 5a eachmodule was calculated and plotted with its corresponding clinicalcharacteristics correlation analysis showed that turquoisemodule displayed the highest relationship with aml immunescores r which included mrnas and lncrnasfigure 5b these mrnas were further used to performthe gene enrichment analysis the genes were most relatedto neutrophil degranulation immune response ‚ammatoryresponse signal transduction and tolllike receptor signalingpathway figure 5c in addition genes were highly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top kegg pathway analysis were performed for functional enrichment clustering analysis a top25 significant kegg pathways based onupregulated genes in immune scores b top25 significant kegg pathways based on upregulated genes in stromal scores c top25 significant kegg pathwaysbased on downregulated genes in immune scores d top25 significant kegg pathways based on downregulated genes in stromal scores kegg kyotoencyclopedia of genes and genomespathway metabolic pathways and hematopoietic cell lineage bykegg analysis figure 5dcerna network constructionsince the turquoise module showed the highest relationshipwith aml immune scores we selected lncrnas and mrnas inthe turquoise module and diï¬erentially expressed mirnasbased on immune scores to construct a cerna network firstlybased on the pita and mircode online database that matchespotential mirnas with lncrnas a total of lncrnamirnapairs contained lncrnas and mirnas then we searchedfor the mrnas targeted by the diï¬erentially expressed mirnasusing three target gene prediction websites miranda mirwalkand targetscan using these websites we detected and target mrnas respectively based on the venn intersectionanalysis target mrnas were selected subsequently wematched the predicted target genes with the mrnas in theturquoise module then we constructed the cerna networkby integrating the mirnalncrnamrna interactions at lasta final lncrnamirnamrna cerna network was constructedwith lncrnas mirnas and mrnas figure 6aprotein“protein interaction ppinetwork analysisto further explore the interplay among the mrnas in cernawe constructed a ppi network based on the string theretrieval of interacting genes online database figure 6b inthe network tlr8 toll like receptor icam1 intercellularadhesion molecule tlr6 toll like receptor and il10rainterleukin receptor subunit alpha had higher degrees and respectively supplementary table the genes encoding these proteins have been confirmed tobe associated with immune microenvironment and leukemiaprogression “association between mrnas mirnasand lncrnas in cerna and overall amlsurvivalwe further analyzed the prognostic values of mrnas mirnasand lncrnas in the cerna network subjects were dividedinto highexpression and lowexpression cohorts accordingto the median value ofthese genes for overall survivalfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure wgcna was used to analyze genetic modules a cluster diagram of coexpression network modules based on topological overlap in mrna andlncrna b study the relationship between each module with their corresponding clinical characteristics c go analysis showed the gene symbols and geneinteractions in the turquoise module d kegg analysis was used to study the pathway enrichment in the turquoise module wgcna weighted correlation networkanalysis go gene ontology kegg kyoto encyclopedia of genes and genomesthrough the package survivalthe highexpression and lowexpression cohorts were splitfor the logrank testin rsoftware out of lncrnas ac0099485 cmahp cta331p31 fcgr2c grk6p1 linc00539 linc01272 mipepp3psmb8as1 rp11266l95 rp11320g101 rp11421f163rp11439e1910 rp11792a83 and stag3l2 out of mirnas hsamir125b5p and hsamir3383p and out of mrnas agpat3 ankrd27 cbx2 ccnd2cd300lb cyth1 erg gdf11 igf1r kiaa0513 kiaa0930larp1 lfng lpcat1 nrep nudt16 pou2f2 ppm1hptafr qsox2 rab3d ralgps2 siglec7 slc43a2 srsf6tnfaip2 tns3 trib1 zbtb5 znf70 and znrf1 wereassociated with overall survival according to the logrank testp representative genes are shown in figure age ‰¥ years vs years and risk group favorablevsintermediatenormal vs poor were also associated withoverall survival according to the logrank test p and respectively the above mentioned lncrnas mrnas and mirnas were brought into further multivariatecox proportional hazard regression analysis with age and riskgroup finally mrnas ccnd2 erg lpcat1 nudt16ralgps2 tnfaip2 and znf70 lncrnas cmahp fcgr2cpsmb8as1 rp11266l95 rp11320g101 rp11792a83 andstag3l2 and mirnas hsamir125b5p and hsamir3383p were independently associated with overall survival table discussionin recent years studies about the roles of gene mutationsand chromosomalin the occurrence anddevelopment of aml and their prognostic values have madesignificant progress however the bm microenvironmentwhich also plays an important role in the pathophysiologytranslocationsfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure cerna network construction and protein“protein interaction network a a lncrnamirnamrna cerna network was constructed by lncrnas mirnas and mrnas b a ppi network was constructed based on the string database the rectangle represents micrornas the circle represents mrnasand the triangle represents lncrnas the red represents upregulation in ishigh and the green represents downregulation in ishigh the size of the dot representsthe regulatory capacity of the mrna and larger points indicate stronger regulatory capability cerna competing endogenous rnas ppi protein“protein interactionprocess in aml are poorly understood therefore mosttreatments previously targeted only tumor cells butfewtargeted the tumor microenvironmentindepth researchon the microenvironment of leukemia will help to furtherunderstand the mechanism of leukemia development and mayfind new targets for microenvironment treatment this studyscreened microenvironmentrelated genes based on the tcgadatabase and further established microenvironmentrelatedlncrnamirnamrna cerna networks through wgcnafirst we calculated the immune scores and stromal scores ofaml patients based on the estimate algorithm and found thatthese scores were related to the fab typing of aml in additionimmune scores were significantly correlated with cytogenetic riskand overall survival estimate is a new algorithm to inferthe level of stromal and immune cells in tumor tissues andtumor purity using gene expression data high immunescores in the bm samples from patients with poor prognosisindicated that more immune cells were recruited in their bmfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure correlation between mrnas mirnas and lncrnas in cerna and overall aml survival in tcga kaplan“meier survival curves with the logrank testwere performed for the representative mrnas mirnas and lncrnas cerna competing endogenous rnas aml acute myeloid leukemia tcga the cancergenome atlasmicroenvironment this may be due to that aml cells activelyshape the bm environment and immune cells to promote diseaseprogression through cellular structural and functional changes however there was no significant correlation betweenstromal scores and cytogenetic risk or survival of aml patientssuggesting that the proportion of stromal cells were comparablein diï¬erent group possible explanation may be that stromal cellsplay an important role in solid tumors while its role inleukemia is not as strong as in solid tumors “then we identified diï¬erentially expressed mrnas mirnasstromaland lncrnas based on the immune scores orscores functional enrichment analysis indicated thatthesedegs were mainly involved in immune and ‚ammatoryresponses consistent with these results previous studies haveshown that the biology of the immune system is essentialfor the formation of a complex bm microenvironment in recent yearsthe immunologicalcharacteristics of aml has increased and the developmentof eï¬ective aml immunotherapy strategies has attractedwidespread attention the understanding ofin recent years important advances in cerna coexpressionnetwork research have developed rapidly the disruption offrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amltable multivariate cox proportional hazard regression analysis of lncrnas mrnas and mirnasgenesccnd2erglpcat1nudt16ralgps2tnfaip2znf70cmahpfcgr2cpsmb8as1rp11266l95rp11320g101rp11792a83stag3l2hsamir125b5phsamir3383phr 95ci “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “pthe cerna network balance is a major cause for tumorigenesis therefore understanding the complex interactions betweendiï¬erent cerna networks will lead to an indepth understandingof gene regulatory networks and has implications for cancertreatment in addition the lncrnamirnamrna cernanetwork can predict the prognosis of the disease for examplea lncrnamirnamrna cerna network was established basedon rnaseq data of breast cancer from tcga which consistsof mirnas lncrnas and mrnas multiplexcox regression analyses showed that four of these lncrnasadamts9as1 linc00536 al3914211 and linc00491 havesignificant prognostic value wang identified lncrnas mirnas and mrnas from a database toconstruct a lncrnamirnamrna cerna network inthe network a univariate and multivariate cox proportionalhazard regression analysis was used to establish a survivalmodel with target mrnas hoxa9 insr krit1 mybspry2 ube2v1 wee1 and znf711 where auc area undercurve is indicating the sensitivity and specificity ofprognostic prediction however the screening of diï¬erentialgenes in this study was based on leukemia patients andnormal people and did not focus on tumor microenvironmentso far there is no cerna research based on the leukemiamicroenvironment in our research the screening of diï¬erentialgenes was based on the immune score then wgcna wasused to identify the modules most relevantto the amlimmune microenvironment then using wgcna and mirnaprediction websites a lncrnamirnamrna cerna networkconsisting of lncrnas mirnas and mrnaswas constructedsubsequently we built a ppi network predicting theinteraction among the proteins encoded by the degs inthe cerna network tlr8 icam1 tlr6 and il10ra hadhigher degrees tlr8 and tlr6 are members of the tolllike receptor family which is upstream to the transcriptionittransportationthe innate immune system andfactor nfκb and part ofplays an importantin progression of aml roleicam1 is one of the cams a large class of transmembraneproteinsinvolved in the binding of cells to another cellor extracellular matrix and involved in cell proliferationdiï¬erentiation movementandtissue structure the protein encoded by il10ra is areceptor for interleukin which has been shown to mediatethe immunosuppressive signal ofinterleukin and thusinhibits the synthesis of pro‚ammatory cytokines and isreported to promote survival of progenitor myeloid cellsthrough the insulin receptor substrate2pi3kakt pathway these results indicated that this novel cerna networkwere closely associated with immune microenvironment andprogression of amlapoptosisfurthermore lncrnas mirnas and mrnas withprognostic significance were screened out which could be usedas biomarkers for prognosis among the genes with prognosticsignificance in our module of immunerelated cerna networkthere were aml related reports about cbx2 ccnd2 ergigf1r larp1 lfng nudt16 pou2f2 ptafr rab3dsiglec7 srsf6 tnfaip2 trib1 zbtb5 and znrf1 themost reported of which were erg ccnd2 and ifg1r ergtranslocation was involved in the occurrence and developmentof aml and high expression of erg was a poor prognosticfactor for patients with normal karyotype aml ccnd2mutations were more common in cbfaml and it was also afrequent mutation event in t aml ccnd2 leadedto increased phosphorylation of retinoblastoma proteins leadingto significant cell cycle changes and increased proliferation ofaml cell lines nicolas chapuis found that igf1spontaneous lesions played a key role in pi3kakt activation ofaml cells providing strong evidence for targeting igf1r as apotential new therapy for aml the functions of agpat3ankrd27 cd300lb cyth1 gdf11 kiaa0513 kiaa0930lpcat1 nrep ppm1h qsox2 ralgps2 slc43a2 tns3and znf70 in aml have not been reported we identified lncrnas with clinical significance among them only cmahpwas reported to be related to mllpositive aml andother lncrnas have not been reported in leukemia twomirnas including hsamir125b5p and hsamir3383p havebeen reported to be associated with a variety of cancers “ but no studies have been reported related to aml all theseunreported mrnas mirnas and lncrnas may be potentialnovel biomarkers or therapeutic targets for amlis importantto note that our current research haslimitations we selected the target data from the tcga publicdatabase only through the biological algorithm method weshould further verify the results of this in clinical patientsin further studyconclusionin summary a comprehensive bioinformatics analysis wasperformed on the aml dataset in tcga with an emphasison the immune microenvironment using wgcna andfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlmirna prediction programs an immunerelated lncrnamirnamrna cerna network was established and degs withprognostic value were further identified further studies of thesegenes are needed in the clinic and may provide new insights intothe pathogenesis of aml this study increases our understandingof the complex interactions between aml tumor cells and the bmmicroenvironment and may provide novel prognostic factors andtherapeutic targetsdata availability statementall data sets of aml patients were downloaded from the cancergenome atlas tcga database portalgdccancergovauthor contributionsyfl cw and zj conceptualization and design sw dataacquisition and writing “ original draft sw ly yx and dzmethodology sw and cw data analysis and interpretationyjl and yfl writing “ review and editing yfl cw and zjproject administration all authors contributed to the andapproved the submitted versionfundingthis work was supported by the national natural sciencefoundation of china grant numbers and u1804191and the henan medical science and technology research projectgrant number acknowledgmentswe thank the tcga database for the availability of the datasupplementary materialthe supplementary materialonline202001579fullsupplementarymaterialfor this can be foundwwwfrontiersins103389foncatreferences ferrara f schiï¬er ca acute myeloid leukaemia in adults lancet “ doi 101016s0140673612617279 zeng z shi yx samudio ij wang ry ling x frolova o targetingthe leukemia microenvironment by cxcr4 inhibition overcomes resistanceto kinase inhibitors and chemotherapy in aml blood “doi 101182blood200805158311 shafat ms gnaneswaran b bowles km rushworth sa the bone marrowmicroenvironment“home of the leukemic blasts blood rev “ doi 101016jblre201703004 bullinger l döhner k döhner h genomics of acute myeloid leukemiadiagnosis and pathways j clin oncol “ doi 101200jco ayala f dewar r kieran m kalluri r contribution of bonemicroenvironment to leukemogenesis and leukemia progression leukemia “ doi 101038leu2009175 uy gl rettig mp motabi ih mcfarland k trinkaus km hladnik lm a phase study of chemosensitization with the cxcr4 antagonist plerixaforin relapsed or refractory acute myeloid leukemia blood “doi 101182blood201110383406 rashidi a uy gl targeting the microenvironmentin acute myeloidleukemia curr hematol malig rep “ doi 101007s11899 austin r smyth mj lane sw harnessing the immune system in acutemyeloid leukaemia crit rev oncol hematol “ doi 101016jcritrevonc201604020 yehudairesheï¬ s attiasturgeman s sabbah r gabay t musallam rfridmandror a abnormal morphological and functional nature ofbone marrow stromal cells provides preferential support for survival of acutemyeloid leukemia cells int j cancer “ doi 101002ijc yoshihara k shahmoradgoli m martínez e vegesna r kim h torresgarciaw inferring tumour purity and stromal and immune cell admixture fromexpression data nat commun doi 101038ncomms3612 yan h qu j cao w liu y zheng g zhang e identification of prognosticgenes in the acute myeloid leukemia immune microenvironment based ontcga data analysis cancer immunol immunother “ doi101007s00262019024087 huang s zhang b fan w zhao q yang l xin w identification ofprognostic genes in the acute myeloid leukemia microenvironment aging “ doi 1018632aging102477 ni j wu y qi f li x yu s liu s screening the cancer genome atlasdatabase for genes of prognostic value in acute myeloid leukemia front oncol doi 103389fonc201901509 langfelder p horvath s wgcna an r package for weighted correlationnetwork analysis bmc bioinformatics doi yao y zhang t qi l zhou c wei j feng f integrated analysis of coexpression and cerna network identifies five lncrnas as prognostic markersfor breast cancer j cell mol med “ doi 101111jcmm14721 spiers h hannon e schalkwyk lc smith r wong ccy o™donovan mc methylomic trajectories across human fetal brain development genomeres “ doi 101101gr180273114 liu q jiang c xu j zhao mt van bortle k cheng x genomewide temporal profiling of transcriptome and open chromatin of earlycardiomyocyte diï¬erentiation derived from hipscs and hescs circulat res “ doi 101161circresaha116310456 salmena l poliseno l tay y kats l pandolfi pp a cerna hypothesisthe rosetta stone of a hidden rna language cell “ doi101016jcell201107014 karreth fa pandolfi pp cerna crosstalk in cancer when cebling rivalriesgo awry cancer discov “ doi 10115821598290cd13 zhang k li q kang x wang y wang s identification and functionalcharacterization of lncrnas acting as cerna involved in the malignantprogression of glioblastoma multiforme oncol rep “ doi103892or20165070 wang jd zhou hs tu xx he y liu qf liu q prediction ofcompeting endogenous rna coexpression network as prognostic markers inaml aging “ doi 1018632aging101985 ritchie me phipson b wu d hu y law cw shi w limma powersdiï¬erential expression analyses for rnasequencing and microarray studiesnucleic acids res 43e47 doi 101093nargkv007 kanehisa m goto s kegg kyoto encyclopedia of genes and genomes nucleicacids res “ brenner ak bruserud ø functional tolllike receptors tlrs are expressed
Colon_Cancer
" the widely recognized anticancer potential of aspirin has created a broad interest to explore theclinical benefits of aspirin in cancer therapy however the current understanding of the molecular mechanismsinvolved in the anticancer potential of aspirin remains limitedmethods cancer stemness assays which contained aldh side population chemoresistance sphere formationand tumorigenesis were performed to validate aspirin function in vitro and in vivo histone modification assay wasperformed to check the effect of aspirin on histone methylation as well as the activity of hdac and kdm6abinhibitor in vivo assay was performed to evaluate therapeutic effects of various inhibitor combination mannersresults in regards to in vitro studies aspirin diminishes cancer cell stemness properties which include reducing thealdh subpopulation side population chemoresistance and sphere formation in three cancer types in regards toin vivo studies aspirin decreases tumor growth and metastasis and prolongs survival in addition our resultsshowed that aspirin inhibits inflammationrelated stemness gene expression especially icam3 identified by a highthroughput sirna platform in regards to the underlying molecular mechanism of action aspirin reduces histonedemethylase kdm6ab expression that mediates histone methylation and suppresses gene expression via a coxindependent manner in regards to therapeutic strategies aspirin combined hdm inhibitors icam3 downstreamsignaling srcpi3k inhibitors or icam3 inhibitor lifitigrast prevents cancer progression in vivos the aforementioned findings suggest a molecular model that explains how aspirin diminishes cancercell stemness properties these findings may provide novel targets for therapeutic strategies involving aspirin in theprevention of cancer progressionkeywords aspirin histone methylation cancer stemness icam3 cox therapeutic strategies correspondence shenwenzhi2011126com1department of pathology and institute of precision medicine jining medicaluniversity hehua road jining chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang stem cell research therapy page of cancer stem cell csc was found as the chief culprit toinitiate tumor occurrence to enhance tumor malignancyand to cause tumor recurrence whereby the maintenanceof cancer cell stemness mainly depends on the tumormicroenvironment or also called the œniche [“] currently the specific properties of csc were identified likehigh aldh1 activity aldehyde dehydrogenase sidepopulation chemoresistance and other cd moleculescd44 cd133 or markers sox2 oct4 nanoglgr5 positive in cancer clinical therapy which targetedhighly tumorigenic cscs may provide new targets orinsight for cancer therapy however unfortunately cscshad demonstrated a relative resistance to conventionalchemotherapy and radiotherapy moreover the cancer cellstemness and the resultant tumor initiationmalignancycould be maintained by the tumorassociated inflammation factors within the tumor microenvironment niche[ ] our previous work presented a mediumthroughput sirna screen platform to identify inflammation genes that regulate cancer cell stemness and obtainedseveral novel candidates agents that target these genesmay inhibit both inflammation and cancer cell stemnessat the same timeaspirin a nonsteroidal antiinflammatory drugiscommonly used as an antipyretic analgesic antiinflammatory and antithrombotic agent [ ] recentobservational and epidemiological studies have shownthat regular prolonged use of aspirin reduces the riskfor several cancers eg colorectal esophageal breastlung prostate liver and skin cancers [“] althoughthe benefits of aspirin for cancer patients have beenwidely appreciated the mechanism remains unclear previous studies attribute the anticancer potential of aspirin to the inhibition of cyclooxygenase2 cox2which is upregulated in various cancer cells [ ] ofnote an increasing body of evidence suggests that aspirin exhibits anticancer effects in a coxindependentmanner histone modification is a reversible process mediated bythe epigenetic enzymes [ ] histone methylation andacetylation are two important chemical modifications thatact in transcriptional activation or inactivation chromosome packaging and dna damagerepair [ ] histone demethylases hdms and histone deacetylaseshdacs are the key enzymes that remove methyl andacetyl groups respectively to regulate gene transcriptionin this regard aspirin was reported to affect hdacs expression and suppress progression of some cancers like aspirin mediates h3k27 acetylation to prevent coloncarcinogenesis and aspirin cooperates with p300 to activate h3k9 acetylation further to promote colorectal cancer cell apoptosis [ ] however the specific rolesand mechanisms of aspirinmediated histone methylationin cancer stemness remains insufficient thus we studiedthe role of aspirin on histone methylation and the attendantand cancerprogressioneffects on cancercellstemnessthe aldh subpopulationour results indicated that aspirin diminishes various cancer cell stemness properties which include reducingside populationchemoresistance and sphere formation in three cancertypes in vitro aspirin inhibits tumor growth and metastasis as well as prolongs survival in vivo aspirininhibits inflammationrelated stemness genes especiallyicam3aspirin reduces histone demethylasekdm6abexpression which mediates histone methylation respectively with a coxindependent manner and aspirin hdm inhibitors aspirin icam3downstream signaling srcpi3k inhibitors and aspirin icam3 inhibitor lifitigrast all reduce cancer progressionin vivo the abovementioned findings demonstrate apromising mechanism of action and potential therapeutic strategy of aspirin in the prevention of cancerprogressionmethodscell culturemdamb231 breast cancer cell and a549 lung cancercell were purchased from atcc hepg2 was obtainedfrom the chinese academy of sciences mdamb231breast cancer cell and hepg2 liver cancer cell were cultured in dmem medium a549 lung cancer cell was cultured in medium allculture media weresupplemented with fbs gibco and were grown at °c in co2 incubators all cells were passaged forless than months before renewal from frozen earlypassage stocks and tested to ensure thatthey weremycoplasma negativecytotoxicity assayaspirin was purchased in sigma cat a2093 and dissolved in dmso mdamb231 a549 and hepg2 cellswere cultured in 96well plate and treated with variousconcentrations and mm for a549 cells and mm for mdamb231 cells and and mm forhepg2 cells of aspirin for h cell activity was testedby applying cck8 kit dojindo china following themanufacturer™s instructionsaldefluor assaythe aldeflour assay kit stemcell technologies vancouver canada was used to measure aldh enzymaticactivity in three cancer cell lines mdamb231 a549and hepg2 in brief cells were treated with aspirin for h and — cells were suspended in aldeflour 0czhang stem cell research therapy page of assay buffer containing aldh1 substrate and incubated for min at °c cells treated with the specific aldh inhibitor deab served as the negative control stained cellswere analyzed on bd facs calibur flow cytometer bdbiosciences san jose ca data analysis was performedusing flowjo software tree star inc ashland orside population assaymdamb231 a549 and hepg2 cells treated with aspirin for h were harvested and resuspended in prewarmed staining buffer pbs buffer added fbs at adensity of — cellsml hoechst dye wasadded at a final concentration of μgml μgmla549 and μgml hepg2 in the presence or absence of μm fumitremin c ftc the followingsteps were described previously [ ]cell apoptosis assaythe mdamb231 a549 and hepg2 cells were treatedwith aspirin and cisplatin ddp μgml for h simultaneously then harvested and resuspended in prewarmed staining buffer pbs buffer added fbs at adensity of — cellsml apoptotic cells were stainedwith propidiumiodide and annexinvfitc bd biosciences flow cytometry analysis was performed by facscalibur cytometer bd biosciences in which a minimum of events were recordedsphere formation assaycells were collected and rinsed to remove serum thendissociated to singlecellsuspension in serumfreedmemf12 medium supplemented with iumlpenicillin μgml streptomycin ngml human recombinant epidermal growth factor hregf ngmlhuman recombinant basic fibroblast growth factorbfgf and b27 supplement invitrogen cells weresubsequently cultured in ultralow attachment 24wellplates at a density of cells per well then treatedwith aspirin all the time until the spheres were formedanimal studyfemale balbc mice at “ weeks were separated randomly into several groups n ‰¥ — 4t1luci cellswere inoculated sc into each mouse at the right axillafor lung metastasis assay at days after injection micewere treated intraperitoneally with aspirin mgkg aspirin mgkg every days and dmso used as thecontrol for chemoresistance assay at days after injection mice were first treated intraperitoneally with cisplatin mgkg then treated with aspirin mgkgaspirin mgkg every days and dmso used as thecontrolnodscid mice at “ weeks were separated randomly into several groups n ‰¥ — a549luci cellswere inoculated sc into each mouse for inhibitor treatment assay days after tumor cells injection micewere first treated intraperitoneally with aspirin mgkgor kdm6ab inhibitor gsk j1 mgkg or src inhibitor a new specific highefficient src inhibitor [ ] mgkg or pi3k inhibitor ly294002 mgkg orlifitegrast mgkg dmso used as the controltumor volume mm3 was measured with calipers andcalculated by using the standard formulalength —width22 the individual measuring the mice was unaware of the identity of the group measured primarytumor tissues were harvested and separated into threeparts one was formalin fixed paraffin embedded andsectioned for ihc staining the other two were brokenby the tissue homogenizer then used for rna trizoland protein ripa lysis buffer extraction animal usecomplied with nankai university and jining medicaluniversity animal welfare guidelines western blottingthe western blot steps were described previously [ ]the special primary antibodies used in this assay arelisted in supplementary table s1 all western data wasthe representative image of three biologically independent repeats the results were quantified using image jsoftware national institutes of health baltimore mdand analyzed by graphpad prism5 software graphpadsoftware san diego ca usanuclear fractionation analysiscells were harvested and the cytoplasmic and nuclearfractions were separated and extracted with an nepernuclear and cytoplasmic extraction kit thermo fisherscientific inc ma usa h3k3me marker proteinswere detected by western blotimmunofluorescencecells grow on glass slides and tumor tissue slices werefixed in paraformaldehyde and labeled with primaryantibodies overnight at °c followed by incubation withspeciesappropriateroomtemperature for h nuclei were stained with dapi andimages were obtained using a leica dm4000 uprightmicroscope or confocal fluorescence microscopy nikontokyo japan [ ]antibodiessecondaryatchromatin immunoprecipitation assaythe assay was performed with an ezzyme chromatinprep kit millipore according to the manufacturer™sprotocol antihistone modification marker antibodieswere used to precipitate dna crosslinked with histone modification markers respectively and normal rabbitigg was used in parallel as a control enriched dna wasthen used as a template to assess the binding intensity of 0czhang stem cell research therapy page of histone modification markers to putative binding sitesin the icam3 promoter primers used in this assay arelisted in supplementary table s2immunohistochemistryimmunohistochemistry was performed on tumor tissuesections from the mice primary antibodies raise againstthe target proteins at a dilution overnight the expression levels of the proteins were evaluated accordingto the percentage of positive cells in each tumor tissuesections the images were recorded by olympus bx51epifluorescent microscopy under a — or — objective olympus co tokyo japan changes in apoptosis in the three cancer cell lines usingthe cell apoptosis assay after cisplatin ddp treatmentour results indicated that apoptosis increases in theaspirintreated groups versus controls and thereby reduces cisplatin chemo resistance fig 1e f in orderto determine the effects of aspirin on cancer cell stemness we next investigated the changes in cell sphere formation in the three cancer cell lines using the sphereformation assay our results showed that sphere formation decreases in the aspirintreated groups versus controls fig 1g h the abovementioned findings suggestthat aspirin diminishes cancer cell stemness propertiesin vitrostatistical analysisall data were analyzed using graphpad prism5 softwaregraphpad software san diego ca usa values wereexpressed as means ± sem p values were calculatedusing a twotailed student™s t test two groups or oneway anova more than groups unless otherwisenoted a value of p was used as the criterion forstatistical significance an asterisk indicates a significantdifference with p two asterisks indicate a significant difference with p and three asterisks indicatea significant difference with p [ ]resultsaspirin diminishes cancer cell stemness properties in vitroin order to establish the proper working concentrationsof aspirin in various cancer cells we determined theic50 of aspirin in a549 lung cancer cells mdamb231breast cancer cells and hepg2 liver cancer cells using acytotoxicity assay our results showed a 107mm ic50in a549 lung cancer cells a 43mm ic50 in mdamb breast cancer cells and a 97mm ic50 in hepg2liver cancer cells fig s1 based on the ic50 we choseworking concentrations of and mm aspirin fora549 lung cancer cells and mm aspirin formdamb231 breast cancer cells and and mmaspirin for hepg2 liver cancer cells in our studiesthe aldh subpopulation decreasesin order to determine the in vitro effects of aspirin oncancer cell stemness we investigated aldh subpopulation changes in a549 lung cancer cells mdamb231 breast cancer cells and hepg2 liver cancer cellsusing the aldh staining assay our results indicatedthatin theaspirintreated groups versus controls fig 1a b inorder to determine the effects of aspirin on cancer cellstemness we next investigated the changes in the sidepopulation in the three cancer cell lines using the sidepopulation assay our results indicated that the sidepopulation decreases in the aspirintreated groups versuscontrols fig 1c d in order to determine the effects ofaspirin on cancer cell stemness we next investigated theaspirin diminishes cancer cell metastasis and stemnessproperties in vivoin order to determine the effects of aspirin on cancercell metastasis and stemness in vivo we implanted 4t1luciferase cells into the fourth fat pad of female balbcmice seven days after implantation we ip injected themice with mgkg aspirin mgkg aspirin ordmso control group times per week fig 2a ourresults showed thattumor volume decreases in theaspirintreated groups versus the control fig 2b however we found that the body weight did not change inthe aspirintreated groups versus the control fig 2c inaddition we found that the survival time increases in theaspirintreated groups versus control fig 2d with respect to the effect of aspirin on cancer cell metastasiswe found thatlung metastasis decreases in aspirintreated groups versus the control fig 2e“g with respect to the effect of aspirin on cancer cell stemnessproperties we found thatthe immunocytochemicalstaining of sox2 and oct4 stemness markers decreasesin the aspirintreated groups versus dmso controlsfig 2h i the abovementioned findings suggest thataspirin diminishes cancer cell metastasis and stemnessproperties in vivoaspirin reduces cancer cell chemoresistance in vivoin order to determine the effects of aspirin on cancercell chemoresistance in vivo we implanted 4t1luciferase cells into the fourth fat pad of female balbcmice eight days after implantation we ip injected themice with mgkg cisplatin mgkg aspirin mgkgcisplatin mgkg aspirin or dmso control groupevery days fig 2j our results showed that tumor volume decreases in the cisplatinaspirintreated versus thedmso control fig 2k however we found that thebody weight did not change in the cisplatinaspirintreated versus the dmso control fig 2l in additionwe found that the survival time increases in the cisplatinaspirintreated groups versus the dmso controlfig 2m we also found that the rate of tumor growth 0czhang stem cell research therapy page of absllec evitisop hdladeabctrl2mm5mm 4mm10mm ctrleddp10ugml2mm5mm 4mm10mm agpehagpeha549ctrl 5mm 10mm ctrl 2mm 4mm sllec evitisop hdlasllec evitisop hdlahepg2fctrl 5mm 10mm slliec ssotpopaa549 slliec ssotpopactrl 5mm 10mm ctrl 2mm 4mm hepg2 slliec ssotpopactrl 5mm 10mm cblockctrl2mm5mm 4mm10mm agpeha549d noitlaupopeds ia549 noitlaupopeds ictrl 5mm 10mm ctrl 2mm 4mm fig see legend on next pagehepg2h noitlaupopeds ictrl 5mm 10mm rebmun erehpsgctrl2mm5mm 4mm10mm agpehhepg2ctrl 5mm 10mm ctrl 2mm 4mm rebmun erehpsrebmun erehpsctrl 5mm 10mm 0czhang stem cell research therapy page of see figure on previous pagefig aspirin restrains cancer cell stemness properties in vitro a aldh staining assay was performed to check aldh subpopulationpercentage in the three cancer cell lines with or without aspirin treatment b statistic results of aldh subpopulation percentage were shown cside population assay was performed to detect sp percentage in three cancer cell lines with or without aspirin treatment d statistic results of sppercentage were shown e facs was performed to detect cell resistance to cisplatin and the percentage of apoptotic cells was shown f statisticresults of apoptosis cells percentage were shown g sphere formation assay was performed to check the cell sphere formation ability in the threecancer cell lines with or without aspirin treatment scale bars μm h statistic results of sphere amounts were shownwas slower in the cisplatinaspirintreated versus thedmso control fig 2n with respect to the effect of aspirin on cancer cell stemness properties we found thatthe immunocytochemical staining of sox2 and oct4stemness markers decreases in the cisplatinaspirintreated groups versus dmso controls fig 2o p theabovementioned findings suggest that aspirin reducescancer cell chemoresistance in vivoaspirin inhibits the expression of inflammationrelatedstemness genes in vitro and in vivoour previously published report established a mediumthroughput sirna screening platform that identifies inflammation genes that regulate cancer cell stemness specifically we identified several novel candidate genesthat decrease oct4 expression and the aldh subpopulation both of which characterize stemness fig 3ain order to determine whether aspirin decreases theexpression of these novel candidate genes to further diminish cancer cell stemness we investigated the expression of novel candidate genes and stemness markerssox2 and oct4 in a549 lung cancer cells mdamb231 breast cancer cells and hepg2 liver cancer cellsusing western blot our results showed that icam3ccl16 pde3a prtn3 sox2 and oct4 protein expression decreases in the aspirintreated groups versuscontrols fig 3b fig s2a we also found that icam3ccl16 pde3a prtn3traf6 bcar1 il1a il1bnfkb1 ikbkb sox2 and oct4 mrna expression decreasesin the aspirintreated group versus controlfig 3c moreover in icam3 ccl16 pde3a prtn3traf6 bcar1 il1a il1b nfkb1 sox2 and oct4the protein expression decreasesindicated byimmunofluorescencestainingaspirintreatedmbamd231 fig 3d and a549 cells data not shownversus the controlasin thein order to confirm the above in vitro results we theninvestigated mrna and protein expression in tumorsfrom and 46day aspirintreated miceversus control using qpcr and western blot our resultsdemonstrated that icam3 pde3a prtn3 traf6bcar1 il1a il1b nfkb1 ikbkb sox2 and oct4mrna expression decreasesin the aspirintreatedgroups versus control fig 3e in addition we foundthat icam3 pde3a prtn3 traf6 bcar1 il1ail1b nfkb1 sox2 and oct4 protein expressionsimilarly decreases in the aspirintreated groups versuscontrol fig 3f fig s2b the abovementioned findingsexpression ofsuggesttheinflammationrelated stemness genesin vitro andin vivoaspirin decreasesthataspirin mediates histone methylation to regulate targetgenes expressionin order to determine the mechanism underlying the action of aspirin we explored the regulatory effect of aspirin on histone methylation markers in a549 lungcancer cells mdamb231 breast cancer cells andhepg2 liver cancer cells using western blot our resultsindicated thatthe expression of h3 trimethylationmarkersie h3k43me h3k93me h3k273meh3k363me and h3k793me increases in the aspirintreated higher concentration groups versus controlfig 4a s3a we also found that the expression of histone demethylases ie kdm6a and kdm6b decreasesin the aspirintreated higher concentration groups versuscontrol fig 4a in addition we studied the protein expression of h3k43me h3k93me h3k273meh3k363me h3k793me and h3 in a549 lung cancercells mdamb231 breast cancer cells and hepg2 livercancer cells using immunofluorescence ourresultsshowed that the protein expression of the h3 methylation markers within the nucleus increases in the aspirintreated groups versus control fig 4b to further support this we extracted the nuclear proteins of eachgroup and detected these h33me markers the resultsalso showed that the expression of h33me markers wasincreased within the nucleusin the aspirintreatedgroups versus control fig 4c s3bin order to identify the role of h3 methylation in regulating selected inflammationrelated stemness genes wemeasured the amount of icam3 dna fragments in h3modification marker pulldowned dnas in a549 lungcancer cells mdamb231 breast cancer cells andhepg2 liver cancer cells using the chipqpcr assaywe selected icam3 since our previous studies demonstrated that icam mediates cancer cellinflammationand stemness ourtheamount of icam3 dna fragments in the various h3methylation marker pulldowned dnas decreases in alllines fig 4c the abovementionedthree cancer cellfindingssuggesthistoneresults demonstrated thatreducesthataspirin 0cbmcemuovl romutdliavvrus noitcarfctrlasp 50mgkgasp 100mgkgdayscg thgew ydobictrlasp 50mgkgasp 100mgkg days of treatmentectrlasp50 asp100ctrlasp 50mgkgasp100mgkggsedoni ssatsat emgnulfhctrlasp asp ctrlasp asp ——xostcoi sllec xos sllec tcoctrl asp50asp100ctrl asp50 asp100ctrl asp50 asp100zhang stem cell research therapy page of a4t1luci injectionaspirin injectionsurvivalday timej4t1luci injectionaspirin cisplatin 2mgkg injectionsurvivalday kmcemuovl romutctrlcisplatinasp 25cisplatinasp50cisplatin dayslg tihgew ydobctrlcisplatinasp 25cisplatinasp50cisplatin days of treatmentmliavvrus noitcarfncisplatin treatedctrlasp asp50yadyadctrlcisplatinasp 25cisplatinasp50cisplatintimefig see legend on next pageop sllec xoscisplatin treatedctrlasp asp50xostcoctrl asp25 asp50cisplatin treated sllec tcoctrl asp25 asp50cisplatin treated 0czhang stem cell research therapy page of see figure on previous pagefig aspirin suppresses cancer cell metastasis and stemness in vivo a schema of the metastasis model established by subcutaneousimplantation of 4t1luci cells into the 4th pair of mammary fat pad of balbc mice b tumor growth curve of 4t1luci with or without aspirintreatment c the body weight of balbc mice in the course of aspirin treatment d the survival curve of balbc mice inoculated with 4t1luciwith or without aspirin treatment e the representative luciferase images showing the 4t1luci tumors at the primary site and lung metastasissites with or without aspirin treatment f representative he staining images of 4t1luci tumors metastasis to the lung with or without aspirintreatment scale bars lower panel μm g statistic results of metastasis loci of 4t1luci tumors metastasis to the lung with or without aspirintreatment h immunohistochemistry staining of sox2 and oct4 in 4t1luci primary tumors with or without aspirin treatment representativeimages with — magnification were shown scale bars μm i statistic results of sox2 or oct4 positive cells in 4t1luci primary tumors withor without aspirin treatment j schema of the chemoresistance model established by subcutaneous implantation of 4t1luci cells into the 4thpair of mammary fat pad of balbc mice k tumor growth curve of 4t1luci with or without aspirin treatment in the presence of cisplatin l thebody weight of balbc mice in the course of aspirin treatment in the presence of cisplatin m the survival curve of balbc mice inoculated with4t1luci with or without aspirin treatment in the presence of cisplatin n the representative luciferase images showing 4t1luci tumors at theprimary sites with or without aspirin treatment on day before cisplatin administration and day after cisplatin administrationo immunohistochemistry staining of sox2 and oct4 in 4t1luci primary tumors with or without aspirin treatment in the presence of cisplatinrepresentative images with — magnification were shown scale bars μm p statistic results of sox2 or oct4 positive cells in 4t1luciprimary tumors with or without aspirin treatment in the presence of cisplatindemethylase ie kdm6a and kdm6b expressionwhich mediates histone methylation and thereby inhibits gene expression in vitroaspirin mediates h3 methylation to regulate icam3expression in vivoin order to confirm the above in vitro results we nextexamined h3 methylation marker expression in tumorsfrom aspirintreated mice versus control using immunocytochemistry our results demonstrated that the h3methylation marker immunostaining within the nucleusincreases in the aspirintreated group versus controlfig 4d e we also found that the amount of icam3dna fragments in the various h3 methylation markerpulldowned dnas decreasesin the aspirintreatedgroup versus control indicating that icam3 expressionis blocked fig 4f these findings suggest that aspirinmediates h3 methylation and thereby regulates icam3expression in vivoside populationaspirin mediates h3 methylation to regulate icam3expression via a coxindependent mannerin order to determine the role of cox in aspirinmediated h3 methylation and targeted gene expressionwe knocked down cox1 and cox2 expression in a549cells respectively fig 5a s3c and examined thealdh populationand chemoresistance the results showed that the aldh population fig 5b e and side population fig 5c f were decreased in shcox1 or shcox2 cells treated with aspirincompared to shcox1 or shcox2 cells treated withdmso and also the aldh population and side population were decreased in shcox1 or shcox2 cellstreated with aspirin compared to shctrl treated with aspirin moreover the apoptosis was increased in shcox1or shcox2 cells treated with ddp and aspirin comparedto shcox1 or shcox2 cells treated with ddp anddmso ctrl and also the apoptosis was increased inshcox1 or shcox2 cells treated with ddp and aspirincompared to shctrltreated with ddp and aspirinfig 5d g in addition western blot results displayedthat the h3 trimethylation markers were increased andthe histone demethylases ie kdm6a and kdm6bwere decreased in shcox1 or shcox2 cells treated withaspirin compared to shctrl treated with aspirin fig 5hs3d accordingly as the new target genes icam3 expression was decreased in shcox1 or shcox2 cellstreated with aspirin versus shctrl treated with aspirinfig 5i s3e these findings suggest that aspirin mediates h3 methylation and thereby regulates icam3 expression via a coxindependent manneraspirin combined with hdm kdm6ab or icam3signaling inhibitors diminish cancer progression in vivoour previous work proved that icam3 could mediatesrcpi3k signaling to promote cancer cell stemness inorder to investigate the use of aspirin combined withhdm kdm6ab or icam3 signaling inhibitors as thetherapeutic strategies we implanted a549luciferasecells into the fourth fat pad of male nodscid micetwentythree days after implantation we injected ip themice with mgkg aspirin mgkg aspirin mgkg kdm6ab inhibitor gsk j1 mgkg aspirin mgkg src inhibitor mgkg aspirin mgkg pi3kinhibitor ly294002 mgkg aspirin mgkg lifitigrast icam3 inhibitor or dmso control group every days fig 6a our results showed that tumor size andtumor volume decreases in the aspirintreated groupand the aspirin inhibitortreated groups versus dmsocontrol fig 6b c however we found that the bodyweight did not change significantly in the aspirintreatedgroup and the aspirin inhibitortreated groups versusdmso control fig 6din addition we found that the survival time increasesin the aspirintreated group and the aspirin inhibitortreated groups versus dmso control fig 6e these 0czhang stem cell research therapy page of fig see legend on next page 0czhang stem cell research therapy page of see figure on previous pagefig aspirin inhibits the expression of inflammationrelated stemness genes in vitro and in vivo a schematic representation of the sirna screenleft summary of the results from the rnai screen right b western blot examining the expression of inflammatory candidates and stemnessproteins sox2 oct4 in a549 mdamb231 and hepg2 cells with or without aspirin treatment c quantitative pcr examining the mrnaexpression of inflammatory candidates and stemness genes sox2 oct4 in a549 mdamb231 and hepg2 cells with or without aspirintreatment d immunofluorescence staining of inflammatory candidates and stemness genes sox2 oct4 in a549 mdamb231 and hepg2 cellswith or without aspirin treatment scale bars μm e quantitative pcr examining the mrna expression of inflammatory candidates andstemness genes sox2 oct4 in 4t1luci tumors separated from balbc mice treated with aspirin for different survival days f western blotexamining the expression of inflammatory candidates and stemness genes sox2 oct4 in 4t1luci tumors separated from balbc mice treatedwith aspirin for different survival dayssuggestresultsthat aspirin combined with hdmkdm6ab or icam3 signaling inhibitors diminishcancer progression in vivo and may serve as the therapeutic strategiesproposed model of aspirin inhibits cancer cell stemnessand cancer progressionbased on our findings we propose the following modelfig aspirin inhibits histone demethylase hdm expression which then mediates histone methylationh3k43me h3k93me h3k273me h3k363meh3k793me respectively these h3 methylations theninhibit the expression of various inflammationrelatedstemness genes previously identified by highthroughputsirna screening il1a il1b icam3 ccl16 traf6pde3a prtn3 nfkb1 ikbkb bcar1 using theicam3 gene as a representative of the inflammationrelated stemness genes by the aspirinmediated h3modifications restrain icam3 promoter activity andcause icam3 expression is inhibited thus aspirin maydiminish cancer cell stemness properties and cancer progression in vitro and in vivo by inhibiting the expressionof various inflammationrelated stemness genes mostinterestingly the above process was not depending oncox expression as the therapeutic strategies aspirincombined various inhibitors suppressed tumor progression effectivelydiscussionthe widely recognized anticancer potential of aspirin aclassical nonsteroidal and antiinflammatory drug hascreated a broad interest to explore the clinical benefitsof aspirin in cancer therapy [“] previous findingsby many investigators have establishe
Colon_Cancer
authorsann c gregory olivier zablockiahmed a zayed allison howellbenjamin bolduc matthew b sullivancorrespondencesullivan948osueduin briefat least studies to date have looked atthe human gut virome but with limitedconsistency gregory and zablocki curate and aggregate these data toprovide a systematic virome databaseuse it to assess study biases globalecological patterns and show howviromes evolve throughout the humanlifespanhighlightsd assembly of gut metagenomes from studiesexposed viral populationsd interstudy analyses reveal strong study biases at the viralpopulationleveld viral population detection was higher in bulk versus vlpenriched metagenomesd gut viral diversity is agedependent across healthy westernpeoplegregory cell host microbe “november ª elsevier inc101016jchom202008003ll 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcethe gut virome database reveals agedependentpatterns of virome diversity in the human gutann c gregory145 olivier zablocki134 ahmed a zayed13 allison howell1 benjamin bolduc13and matthew b sullivan12361department of microbiology ohio state university columbus oh usa2department of civil environmental and geodetic engineering ohio state university columbus oh usa3center of microbiome science ohio state university columbus oh usa4these authors contributed equally5present address department of microbiology and immunology rega institute for medical research vibku leuven center formicrobiology leuven belgium6lead contactcorrespondence sullivan948osuedu101016jchom202008003summarythe gut microbiome profoundly affects human health and disease and their infecting viruses are likely asimportant but often missed because of reference database limitations here we built a human gut viromedatabase gvd from viral p or microbial metagenomes from individuals representing countries assess its effectiveness and report a metaanalysis that reveals agedependent patternsacross healthy westerners the gvd contains unique viral populations approximately speciesleveltaxa and improves average viral detection rates over viral refseq and imgvr nearly 182fold and 26foldrespectively gvd metaanalyses show highly personalized viromes reveal that interstudy variability fromtechnical artifacts is larger than any ˜˜disease™™ effect at the population level and document how viral diversitychanges from human infancy into senescence together this compact foundational resource these standardization guidelines and these metaanalysis findings provide a systematic toolkit to help maximize ourunderstanding of viral roles in health and diseaseintroductionthe human gut microbiome is now thought to play an integralrole in health and disease clemente gilbert lynch and pedersen schmidt persistent alterations in the structure diversity and functionof gut microbial communities”dysbiosis”are increasinglyrecognized as key contributors in the establishment and maintenance of a growing number of disease states frank human microbiome project consortium qin including obesity turnbaugh andcancer yoshimoto gut dysbiosis can developfrom complex interplays between host cognate microbiotaand external environmentalfactors mirzaei and maurice shreiner within the gut microbial consortium the bacteriome has been the most extensively studied where significant shifts in population dynamics havebeen observed between healthy and diseased individualszhang however emerging views mirzaei andmaurice ogilvie and jones tetz suggest that the gut virome plays an important role in homeostatic regulation and disease progression through multipleinteraction paths with the cooccurring bacteriome and evendirectly with human immune system components keen anddantas the first step in studying viruses in complex communities isbeing able to detect them problematicallyidentifying viralsequences in large mixedcommunity datasets is notoriouslychallenging because viruses lack a universal viral markerrohwer and edwards as opposed to bacterial 16srrna for example human gut microbiome studies have mostcommonly used sequence homology searches with blast orkraken wood and salzberg against ncbi viral referencesequence database refseqwwwncbinlmnihgovgenomeviruses aclame a mobile element genome database [leplae ] or custom hidden markov modelhmm databases eg prokaryotic virus orthologous groups[pvogs] [grazziotin ] although there is now a suiteof virus identification tools available including deepvirfinderren marvel amgarten vibrantkieft and virsorter roux only the latterhas been used in the human gut microbiome literature to dateand all are dependent upon reference genome databases tosome degree further once viruses are detected there is nostandard applied on how viral contigs translate into ˜˜species™™level sequences that are to be used as a ˜˜working™™ virus poolcell host microbe “ november ª elsevier inc 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcefigure overview of studies comprising the gut virome database gvdglobal heatmap of the world showing the number and distribution of studies per country each white box represents a different continent and contains informationabout the number of individuals sampled represented by the filled human pictograms and percentage of the total gvd sequencing effort for vlpenriched red piecharts and bulk metagenomes yellow pie charts of each country studied within that continentsee also table s1for downstream analysis the lack of viral analysis standardscould partly explain the estimated highly variable “mirzaei and maurice rates of virus detection betweenstudies in addition factors such as differences in sample processing shkoporov broad underrepresentationof viral genome space in reference databases wang lack of culturable host gut microbes wang and interindividual variation add further variability shkoporov further although viral reference datasets are beinggenerated at unprecedented rates roux thesenew data are rarely incorporated for crosscomparisons whichwould ‚ate virus novelty in new datasets andor leaves manyvirus sequences undetected in response to these challengesand to enable viromecentric research in health and diseasewe sought to establish a comprehensive easyaccess databasededicated to human gut viruses this effort would enable futuregut microbiome research by augmenting virus detection andhelping establish processing standards for human gut viruseshere we collected and curated human gut metagenomes previously studied for viruses and published as ofoctober to build the human gut virome database gvd evaluated its utility against the best available databases national center for biotechnology information [ncbi] viral refseqand integrated microbial genomevirus [imgvr] [paezespino ] and used it in metaanalyses to assess methodological effects and establish largescale patterns of gut viromediversity during the course of the human lifespan the gvd™s human gut metagenomic datasets derive from studiesand encompass individuals from countries that originated either from viruslike ps vlps or whole microbialcommunities bulk as well as several datasets that includedrna sequencing data derived from vlps all these datasetswere previously studied for viruses but by using highly variable cell host microbe “ november methods for the gvd we in silico reprocessed these data toidentify viral populations and rigorously remove contaminationthis gvd resource is now available on ivirus bolduc 2017a and will be regularly updatedresults and discussionthe gvd contains unique viral populationsdominated by phagesto build the gvd metagenomic samples from individuals were processed from datasets publicly available as ofdecember n see table s1 along with one unpublished dataset where access was granted prior to publicationthese studies represent tbp of sequence data derivedfrom a spectrum of gut virome study areas including thefollowing healthy gut viromes of infants lim reyes and adults ly manrique minot rampelli aswell as individuals experiencing fecal microbiota transplantfmt for autism and clostridium difficile infection broecker chehoud draper kang zuo ‚ammatory bowel disease ibd fernandes norman pe´ rezbrocal zuo hiv infection monaco type i and ii diabetes aiemjoy kramna´ ma zhao malnutrition reyes and chronic fatigue syndrome giloteaux and hypertension han see table s1 these datasets were globally distributed figure however most of the studies originated from the united states of gvd studies and the highest number of sampled individuals and base pairs bps sequenced came from chinese cohorts of individuals and bp sequenced in the gvd all 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003resourcereads from both bulk and vlp metagenomes and ofthe gvd respectively were processed consistently assembledinto contigs and virallike sequences were identified by usingthree independent methods figure s1 see method detailseach lowscoring prediction was validated by crosscomparisons between methods and subsequently evaluated for falsepositives by detecting whether candidate virus sequences contained benchmarking universal singlecopy orthologs buscorelated host singlecopy genes sima˜ o and for thepresence of viral family proteins vpfspaezespino confidence scores associated with each virus detectionmethod and potential false positives are reported in table s2 toavoid duplicate viral genomes andor partial virus genomesacross the datasets contigs were dereplicated by clusteringsequences according to percentage of average nucleotide identity ani and sequence length multiple reports brum 2015a duhaime and sullivan duhaime gregory 2019a roux have revealed that ani was a suitable threshold for defining a set of closelyrelated discrete ˜˜viral populations™™ followon studies suggestthat this cutoff establishes populations that are largely concordant with a biologically relevant viral species definition bobayand ochman gregory 2019a using this clustering strategy we identified highly variable numbers of uniqueviral populations per study range “ viral populationsmean figure s2a the gvd comprises viralcontigs and viral populations r5 kb or r15 kb and circular contigs n50 bp l50 bp and mostlybacteriophages of gvd for context ncbi™s viral refseq v98 released january database holds viruses of eukaryotes bacteria and archaea from all environments combined specifically for bacteriophages the gvdcontains 12fold more than the entire set of cultured phage isolates in viral refseq to date thus the gvd greatly augments therepertoire of known phages in the human gut importantly due toa lack of negative controls across out of the studies in thegvd there is a chance that some of the viral populationsincluded in the gvd might result from contamination thispaucity of negative controls is currently a limitation to gut viromestudiestaxonomically of gvd viral populations are bacterialviruses ie phages are eukaryotic viruses and are archaeal viruses figure 2a the eukaryotic viruseswere taxonomically diverse from families dominated by singlestranded dna ssdna families anelloviridae genomoviridae and circoviridae all of which havebeen previously reported in the datasets underlying the gvdmonaco with the exception of genomoviridaethree singlestranded positivesense rna virus families weredetected table s3 represented by viral populations of the gvd the human picornaviridae was the most represented parechoviruses coxsackievirus cosaviruses enterovirus and hepatovirus along with plant or fungal viruses ofthe alphaflexviridae and virgaviridae and one putative memberof cruliviridae detection of plant viruses has been reportedbefore zhang and is likely the result of transient passage through dietary habits human picornaviruses associatedwith gastrointestinal tract disorders were to be expected andmost derived from a cameroonian patient cohort selected forllgastroenteritis symptoms in which the study design includedrna sequencing yinda the low number of recovered rna viruses of the gvd see tables s2 and s3 inthe gvd might stem in part from having a few studies out of that included viral rna sequencing more importantly thelikely biggest factor contributing to low rna virus detection isthat de novo rna virus identification method development isan ongoing effort shi starr such thatrna virus diversity in gut viromes and generally in viral metagenomes is likely vastly undersampled and that our detection islimited to homology to wellcharacterized pathogens zhang among the phages did not have internationalcommittee on taxonomy of viruses ictv classification and theremaining fraction comprised of doublestranded dna dsdnatailed phage families siphoviridae myoviridae podoviridae andackermannviridae microviridae and inoviridae see table s2twentyfour unknown archaeal viral populations were detectedbut none with close genome andor gene homology to any of theclassified archaeal viruses notably our naive viral taxonomicassignments using ˜˜a majorityrules approach™™ see method details led to taxonomic assignments that recent literature hasshown are erroneous and due to methodological artefactssuch as phycodnaviridae and mimiviridae sutton so we manually removed such taxa thus given that most ofthe viral populations are represented by fragments of their genomes taxonomic assignments using the ˜˜a majorityrulesapproach™™ willimprove and be refined as more completegenome representatives are sequenced and assembled nonetheless the high number of unclassified phages likely resultsfrom the underrepresentation of human gut phages in referencedatabases and further highlights how much viral diversity remains to be characterized in the human gutto fill this phage and archaeal virus taxonomic classificationgap we used an extensively validated adriaenssens bolduc 2017b jang bin genomebased genesharing network strategy that de novo predictsgenuslevel groupings ˜˜viral clusters™™ [vcs] from viral population data a network figure 2b computed from gvdphage genomes only those kb in length of gvd and reference phage genomes from ncbi viral refseqversion revealed vcs of these vcs were exclusively composed of gvd genomes viral genomes or 18 of gvd genomes whereas vcs contained genomesfrom both refseq and the gvd viral genomes or 18 ofgvd genomes and vcs were exclusively composed of refseq taxa thus the gvd augments the current number of ictvrecognized phage genera approximately 35fold although notexplored here given that our goals focused on taxonomic classification the shared protein content within and between vcscalculated in our network analyses could be used to guideqpcr assays for nextgeneration sequencing validationmonaco and kwon andor tracking of viruses at eitherthe viral population or genera level under changing conditionskramna´ next we sought to link the gvd phage and archaeal viralpopulations to their hosts by using in silico strategies seemethod details in total we were able to identify the hostsdown to the microbial taxonomic family genome taxonomyparks of 18database [gtdb] taxonomycell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcefigure the gut virome database gvda pie charts showing the number of bacteriophages eukaryotic viruses and archaeal viruses in the gvd center and their familial taxonomic composition by thebacteriophages left and the eukaryotic viruses rightb genesharing taxonomic network of the gvd including viral refseq viruses v88 refseq viruses are highlighted in red every node represents a virus genomewhereas connecting edges identify significant genesharing between genomes which form the basis for their clustering in genuslevel taxonomyc concentric pie chart showing the number of annotated bacterial host phyla inner and family outer of the gvd viruses host taxonomy follows thegtdb database taxonomic classifications and putative host information per each viral population is listed in table s2 see also figures s1 and s2 and tables s2s3 and s6n of the viral populations see table s2 the mostcommon identifiable hosts figure 2c across gvd viral populations belonged to the bacterial phyla firmicutes gtdb firmicutes firmicutes_a and firmicutes_c combined andbacteroidetes gtdb bacteroidota consistent withour knowledge that firmicutes and bacteroides are the mostprominent bacterial phyla in the human gastrointestinal tracteckburg notably firmicutes typically outnumberbacteroidetes in unhealthy individuals with metabolic anddigestive disorders broecker chehoud ley nicholson norman ott zhao and gvd metagenomes are biased toward unhealthy individuals of themetagenomes comprising of the bps sequenced whichmight account for the increased firmicutes viral populationsin gvdthe gvd significantly improves virus detection overcurrent viral genome databasesto assess the value of the gvd we quantitatively evaluated virusidentification sensitivity between multiple databases bycomparing the number of viral populations identifiable by readrecruitment against gvd ncbi™s viral refseq v96 doe™s imgvr v4 paezespino and the individual virome databases from each study figure see method details to controlfor assembly improvements since the original metagenome andor virome datasets were published for the latter we individuallyassembled the original viromes into viral populations for read cell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003resourcellfigure gvd as a reference database increases viral population detectionboxplots showing median and quartiles of the number of viral populations detected per study using the individual virome viral refseq v96 jgi imgvr v4 vd databases all pairwise comparisons were performed by using mannwhitney u tests nonsignificant p values are denoted as ˜˜ns™™see also figure s3 and table s4recruitment ncbi viral refseq was the most commonly usedviral genome database across the studies surveyed here beingused in of studies where the specific database used wasdocumented information on the genome database used was unavailable for three studies see table s1 and hosted virusgenomes already dereplicated as of v96 november usedhere in comparison the imgvr database was not documented as being used by any of the studies gathered despitethe latest release v4 july used in this study containingnearly two orders of magnitude more virus genomes andgenome fragments virus contigs though not dereplicated for comparison purposes to the gvd see method details we dereplicated the imgvr contigs the same way aswe did the gvd to obtain viralpopulationlevel genomes thisyielded viral populations for the imgvr databasein out of the total studies tested figure the gvdenabled the detection of significantly more viruses than viral refseq v96 mannwhitney u tests p [average] ± [standard deviation]fold increase and individual viromesmannwhitney u tests p 6fold ± 40fold increasenotably the proportion of the metagenome mapping to thegvd was highly variable between studies figure s2b and asexpected a higher proportion of vlpenriched metagenomesmapped to the gvd than did bulk metagenomes figure s2cthere was a single study reyes in which no viruseswere detected see method details in all databases queried inthis analysis in comparison to imgvr we detected more viruses with the gvd in all studies from total of whichwere in a significant manner mannwhitney u tests p 26fold ± 21fold increase figure five of the remaining fourteen studies had too low of a sample size andor number of detected viruses to statistically compare the gvd and imgvradditionally we tested the ability of the gvd to increase thenumber of viral populations detected in a study not included inthe gvd clooney figure s3a we saw similar results and gvd significantly outperformed viral refseq v96 andthe individual virome while having a nonsignificant higher median number of viral populations detected than imgvrwhen we considered the number of reads that recruitedacross the different databases across all studies significantlymore reads mannwhitney u tests p were recruited tothe gvd than to any other database across out of the studies figure s3b after gvd imgvr was the next best performing database for viral detection in the human gut given thatour tests showed an average of 64fold ± 120fold increase overviral refseq mannwhitney u tests p imgvr was expected to surpass viral refseq because it aggregates both cultivated reference virus genomes from refseq prophages and uncultivated virus genomes andorfragments from many environments including multiple humanbody sites paezespino overall the significant increase in virus detection by the gvd over other databases highlights the low representation of gut viruses in refseq and thusdemonstrates the value of the gvd for sequencebased virusidentification in human gut microbiome datasets thus giventhat the gvd significantly improves viral detection over currentviral genome databases we used the gvd as the database forall remaining analyses in this studythe human gut virome is highly person specificin light of the current hypothesis of a ˜˜core™™ gut virome manrique we were first curious whether any gvd viralcell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcefigure individual viromes study databases and crossstudy comparisonsshown at the top left is a hierarchically clustered heatmap showing the number of viral populations shared within and between studies clustered into four groupsi“iv viral population cooccurrence network per individual within each study per group shown on the bottom right is a hierarchically clustered heatmapshowing the number of viral genera shared within and between studies clustered into three groups viral genus cluster cooccurrence networks per metagenomewithin each study per group colored dots and pictograms next to study names in heatmaps represent metagenome type and a common disease studies acrossall studies in gvd respectivelysee also figure s4population was found across a high percentage or all metagenomes in the gvd on average ± average ± sd range“ viral populations were detected per metagenome butnot a single viral population was found across all metagenomesin fact the most ubiquitous viral population in the gvd wasfound in only of the metagenomes viral populationsoccurred in more than of the metagenomes and most or of the viral populations were only sporadicallydetected at all of the metagenomes figures s4a ands4b table s4 further we specifically looked at the prevalenceof crassphages a wellrecognized multigenera family ofphages known to be widespread in gut viromes guerin figure s4b in total we identified crassphage populations see method details of which had genomes kb cell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003resourcellfigure vlpenriched vlp and bulk metagenomes comparisons for studying viruses in the human guta“c boxplots showing median and quartiles of the number of assembled contigs per base pair sequenced per study a of vlp and bulk metagenomes b ofvlp metagenomes with and without mda and c of the different vlpenrichment methodologies across the studies outlier dots were removed from plot c tobetter show the range of values the n value above each box plot represents the number of studies using each vlpenrichment methodd scatter plot with a linear regression line showing the number of assembled viral contigs per bp sequenced per study with vlp and bulk metagenome studiesidentified by different colors in the inset is a venn diagram showing the number of gvd viral populations that originated from vlp or bulk or both types ofmetagenomese boxplots showing median and quartiles of the number of viral populations detected per bp sequenced per individual of vlp and bulk metagenomesf boxplots showing median and quartiles of the number of assembled contigs per bp sequenced top left and the median contig length top right for vlp andbulk metagenomes processed for the same samples in the shkoporov bottom connected dot plot showing the number of viral populations detectedper bp sequencedby using vlp and bulk metagenomes for each individual in the shkoporov study all pairwise comparisons were performed by usingmannwhitney u tests nonsignificant p values are denoted as ˜˜ns™™see also figure s4that clustered into vcs ie genuslevel grouping bygenomebased genesharing networks jang bin although togetherthese crassphage populations areubiquitous across the gvd samples there was not one crassphage viral population found universally and the most widespread crassphage population occurred in only of samplesthese findings support the mounting evidence for highly personal gut viromes as recently highlighted in twins morenogallego and in ten healthy adults during a yearlongmonitoring period shkoporov although the latterstudy pointed to the potential of a core virome at higher taxonomic levels we failed to recover any universally shared viralvcs approximately genus level taxonomy figures s4c ands4d given that the most ubiquitous vc was only present in of the metagenomes that same study suggesting thepotential of a highertaxon core only looked at ten healthywestern adults and the metagenomes in the gvd representeda diversity of people from different geographical regions andages thus further studies are necessary to resolve whether acore virome does exists at higher taxonomic levelsvaried processing methodology prevents populationlevel bdiversity interstudy comparisonsnext given a systematically processed gvd and its demonstrably improved virus detection capability we sought to determine whether global clustering patterns would emerge via a gvdbdiversity betweensample changes in population composition metaanalysis to this end we performed population cooccurrence analyses at two levels of resolution per study andacross metagenomes within studies and then evaluated whatlevel of metadata best captured the resultant variation methodology disease state etc to assess populationoverlap between studies we counted the number of gvd viral populationsthat recruited reads within and between different gvd studiescell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llie the same viral population was detected in metagenomein both studies compared we expected studies exploringsimilar disease states would share the greatest number of viralpopulations however hierarchical clustering of the studies onthe basis of the number of shared viral populations revealedthat this was rare and mostly not the case and studies exploringthe viromes of diseased individuals eg ibd clostridium difficile infection and diabetes did not cluster together figure top left”heatmap instead we saw that the different studies hierarchically clustered into four groups i“iv and that this clustering was weakly driven around metagenome type many bulkmetagenomes clustered together into group iiibecause the gvd studies did cluster into four distinct groupsfigure top left”heatmap we next tested whether any metadata best captured the resultant variation across the metagenomes within each study within each group using an inversecovariance analysis ie combined partial correlations acrossshared viral population between metagenomes to subclusterthe different metagenomes across the studies in each groupwe found that the metagenomes within a study subclusteredtogether irrespective of geographical origin health status andor diet figure top left”networks notably the group iii metagenomes derived mostly from bulk metagenomes were moreclosely subclustered but they still subclustered strongly bystudy this perstudy subclustering implies that even withinthese grouped studies metagenomes from different studiesare not comparable because the interstudy variation is drivenby methodological impacts these results reveal that althoughmethodology does not affect the number of viral contigs recovered it does affect the recovered types of viruses see upcomingfindings comparing methodological effects interestingly whenwe looked at genuslevelie vclevel cooccurrence wesaw that there are still strong groupings a“c at the study levelbut within each group metagenomes across these studiesshare many vcs figure bottom right thus bdiversitymetaanalysis across all studies exploring the effect of ˜˜disease™™across gvd studies is not possible at the population level butwithin similarly processed studies it might be possible at thegenus levelto enrich or not to enrich viruses recovered from bulkversus viruspenriched metagenomesfrom a pragmatic point of view we next wondered whethergvd datasets could inform experimental design specifically cell host microbe “ november resourcefigure more gut viruses are temperatephages than in the soil and oceanspie charts showing the percentages of temperatephages found in the human gut gvd datasetsoils isogenie dataset and oceans globaloceans viromes datasetto study viruses is sequencing effort better put into metagenomes of bulk or purified vlps the gvd™s gut metagenomes are roughly evenly divided acrossthese two metagenome types with bulkand vlp metagenomes contributing tbp 18 of gvd and tbp 18 of gvd of data respectively although most samplesonly have one or the other data one study samples shkoporov provided both bulk and vlp metagenomesfor sampleswe first assessed whether there was a difference in de novoviral recovery between vlp and bulk metagenomes figure 5awe measured viral recovery by using the number of viral contigs kb or kb and circular in length ie not dereplicatedviral populations assembled per bp sequenced per study giventhat the viral contigs assembled from samples within the samestudy are often pooled these analyses revealed no significantdifference mannwhitney u test p in the number of viralcontigs assembled per bp sequenced between vlp and bulkmetagenomes which contrasts viralrecovery results frompermafrost soils where vlp metagenomes outperform bulkmetagenomes by 2fold trubl however viral recovery from the gvd™s vlp metagenomes was heterogeneous sowe evaluated how vlp methodology affected viral recoveryfirst although multiple displacement amplification mdaisknown to provide nonquantitative metagenomic datasets withboth systematic and stochastic biases solonenko yilmaz we found no significant difference mannwhitney u test p in viral recovery between nonmdaand mdatreated metagenomes figure 5b nonetheless itwas notable that mdatreated vlp studies were significantly enriched in eukaryotic ssdna viruses mannwhitney u tests p a known bias of mda figure s5a second we tested theeffect of vlp enrichment strategies which ranged fromremoving human and bacterial cells to enrich for vlps centrifugation filtration cscl gradients and nucleases to concentrating the vlps centricon concentration and peg precipitationagain we found no significant difference in the number of viralcontigs recovered kruskalwallis test p across
Colon_Cancer
nasopharyngeal carcinoma npc is an epithelial malignancy with high morbidity rates in the east and southeast asia the molecular mechanisms of npc remain largely unknown we explored the pathogenesis potential biomarkers and prognostic indicators of npcmethods we analyzed mrnas long noncoding rnas lncrnas and micrornas mirnas in the whole transcriptome sequencing dataset of our hospital five normal tissues vs five npc tissues and six microarray datasets normal tissues vs npc tissues downloaded from the gene expression omnibus gse12452 gse13597 gse95166 gse126683 and gse70970 gse43039 differential expression analyses gene ontology go enrichment kyoto encyclopedia of genes and genomes kegg analysis and gene set enrichment analysis gsea were conducted the lncrnamirnamrna competing endogenous rna cerna networks were constructed using the miranda and targetscan database and a protein“protein interaction ppi network of differentially expressed genes degs was built using search tool for the retrieval of interacting genes string software hub genes were identified using molecular complex detection mcode networkanalyzer and cytohubbaresults we identified mrnas 14mirnas and lncrnas as shared degs related to npc in seven datasets changes in npc were enriched in the chromosomal region sister chromatid segregation and nuclear chromosome segregation gsea indicated that the mitogenactivated protein kinase mapk pathway phosphatidylinositol3 oh kinaseprotein kinase b pi3kakt pathway apoptotic pathway and tumor necrosis factor tnf were involved in the initiation and development of npc finally hub genes were screened out via the ppi networks several degs and their biological processes pathways and interrelations were found in our current study by bioinformatics analyses our findings may offer insights into the biological mechanisms underlying npc and identify potential therapeutic targets for npccorrespondence chenchuanben2010126com yuanji xu and xinyi huang contributed equally to this work department of radiation oncology fujian medical university cancer hospital fujian cancer hospital no fuma road fuzhou fujian people™s republic of chinafull list of author information is available at the end of the the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cxu a0et a0al cancer cell int page of keywords nasopharyngeal carcinoma npc bioinformatics analysis gene expression omnibus geo differentially expressed genes degs gene ontology go competing endogenous rna cerna network nasopharyngeal carcinoma npc is an epithelial malignancy originating from the inner mucosal lining of the nasopharynx in there were an estimated new cases of npc worldwide accounting for of all cancer sites and deaths due to npc accounting for of all cancer sites the incidence of npc is geographically imbalanced with new cases mainly concentrated in east and southeast asia especially in south china [ ] the estimated agestandardized incidence rate of npc is per in china and only per in north america [ ] the oncogenesis and progression of npc are strongly associated with hereditary susceptibility environmental or random aspects and epsteinbarr virus ebv infection in the early stages of npc the main pathogenesis is related to ebv infection indeed the expression of ebvdna can be used for the monitoring and followup of npc patients and maybe a useful indicator for riskstratification strategies however despite the great advances in medical technology in recent years such as the application of intensitymodulated radiotherapy and optimized chemotherapy strategies the detection and treatment of npc remain challenging epidemiological investigations have shown that although the incidence and mortality rates of npc have been greatly reduced over the past decade even in endemic areas the survival rate of npc patients remains unsatisfactory due to local recurrence and distant metastasis especially in patients with advancedstage disease thus noninvasive cancerspecific biomarkers for early diagnosis and precision treatment are urgently requiredmicroarray and bioinformatics analyses have enabled researchers to screen the genetic alterations in npc and have proven to be convenient methods for identifying potential biomarkers in other diseases these analytic methods have uncovered several biomarkers with proven prognostic value and potential clinical applications in npc for example one study discovered a novel long noncoding rna lncrna named linc01385 involved in npc development and functional analysis demonstrated that linc01385 could serve as a therapeutic target in npc microarray and rnasequencing techniques have been used to identify differentially expressed genes degs and signaling pathways related to the oncogenesis and development of npc one study analyzed two microarray datasets to identify degs in normal tissue samples and npc tissue samples however the falsepositive rate for the two datasets was potentially high and the limited sample size may have led to unreliable results due to the substantial heterogeneity among the patients zhang et a0al investigated three microarray datasets to identify degs and hub genes that may serve as potential diagnostic biomarkers for npc both these studies analyzed only chip datasets and did not include sequencing data which would lead to offsets in the studies thus the precise molecular mechanisms and biological processes underlying npc remain largely unknown and must be urgently investigated to develop a precise curative treatment for npcin recent years competitive endogenous rna cerna has provided a new way to study the molecular mechanism of cancer he et a0al found that circgfra1 may serve as cerna to regulate gfra1 expression by sponging mi34a in triple negative breast cancer cerna is a transcript that can compete shared mirnas and regulate one another at the posttranscription level and the cerna networks link the function of mrnas to the function of micrornas mirnas lncrnas circular rnas and other rnas cerna can act as mirna sponges thereby affecting mirna expression cerna regulation network refers to the regulatory network with cerna participationtherefore in this study we aimed to explore the molecular pathogenesis potential biomarkers and prognostic indicators of npc by analyzing the full transcriptome sequencing data from fujian cancer hospital along with six microarray datasets acquired from the gene expression omnibus geo to identify degs between npc samples and normal tissue samples our findings may guide the precision treatment of npcmaterials and a0methodssample collection and a0preparationfresh nasopharyngeal tissues were collected from five npc patients who were treated in fujian cancer hospital between november and may normal nasopharyngeal tissues from five healthy donors were also collected all tissue samples were frozen using liquid nitrogen the five npc patients consisted of three men and two women with a median age of a0years the age and sex of five donors were matched to the npc patients two patients had stage iii npc while three patients had stage iva npc according to the 8th edition of the current international union against canceramerican joint committee on cancer guidelines for npc the 0cxu a0et a0al cancer cell int page of ethics committee of fujian cancer hospital approved the human tissue samples related to this work project ethics number sq201901801 the fresh tissue samples were removed from liquid nitrogen and subjected to total rna extraction using the trizol method the purity photometer® spectrophotometer implen ca usa of the extracted rna was determined using a nano the rna concentration was measured using the qubit® rna assay kit and a qubit® fluorometer life technologies ca usa and the rna integrity was evaluated using the rna nano assay kit of the bioanalyzer system agilent technologies ca usarna sequencinga total of a0μg rna from each tissue sample served as the somal rna was eliminated using epicentre ribozero„¢ input material for the rna sample arrangements riborrna removal kit epicentre usa the rrnafree residue was removed using ethanol precipitation next depleted rna by using the nebnext® ultra„¢ direcsequencing libraries were produced from the rrnational rna library prep kit for illumina® neb usa in brief fragmentations were implemented by bivalent cations below the high temperature in nebnext first strand synthesis reaction buffer5x the firststrand cdna thus obtained was compounded using a stochastic hexamer primer and mmulv reverse transcriptase rnase h then secondstrand cdna synthesis was carried out using dna polymerase i and rnase h in the reaction buffer dntps with dutp were substituted for dttp residual overhangs were turned into blunt ends through exonucleasepolymerase activities after the adenylation of the ² ends of the dna fragments the nebnext adapter carrying a hairpin loop structure was ligated to initiate hybridization to optimize the cdna fragments with a length of “ a0bp we purified the library fragments with the ampure xp system beckman coulter beverly usa next a0 μl user enzyme neb usa was applied with the sizeselected adaptorligated cdna at a0°c for a0min and then at a0°c for a0 min thereafter pcr was conducted using phusion highfidelity dna polymerase general pcr primers and index x primer finally the obtained products were refined ampure xp system and the library quality was evaluated on the agilent bioanalyzer system after the library was constructed qubit20 was used for preliminary quantification the library was diluted to a0ngμl then used the agilent system was used to determine the insert size of the library after the insert size was confirmed to be as expected qpcr was used to confirm the valid concentration a0 nm and accurate quantification of the library to ensure library quality when the library was deemed eligible varying libraries were pooled to meet the demands of valid concentration and enable offline data volume hiseq sequencing was conducted these wholetranscriptome sequencing data were termed the fjch datasetmicroarray datageo httpwwwncbinlmnihgovgeo is a public genomics dataset repository which collects highthroughput sequencing data chips and microarrays we downloaded the following six gene expression datasets from geo the mrna gene expression datasets gse12452 and gse13597 the mirna gene expression datasets gse43039 and gse70970 and the lncrna gene expression datasets gse95166 and gse126683 all six datasets were annotated using r software version via annotation documents all datasets were from the species homo sapiens and the dataset type was microarray expression profile details of every dataset study are provided in table a0identification of a0degsto identify degs between normal tissue samples and npc samples we analyzed the microarray data by using the limma package and a multivariate linear model of the adjusted tstatistic the cutoff criteria were as follows log fold change absolute value of log2 in table associated microarray datasets from a0the a0gene expression omnibus geo databasereferencepmidrecordtissue platformnormal cancerfjchdodd et al bose et al zheng et al unknownnpcnpc gse12452 gse13597npc gse126683 npcunknowm gse95166npcillumina hiseqtm2500miseqtm[hgu133_plus_2] affymetrix human genome u133 plus array[hgu133a] affymetrix human genome u133a arrayagilent045997 arraystar human lncrna microarray v3 probe name versionarraystar human lncrna microarray v20 agilent_033010 probe name versionlyu et al bruce et al gse43039 gse70970npcnpcccdtmmirna850version 4p14ncounter® human mirna assay v10 nanostring 0cxu a0et a0al cancer cell int page of the fold change of gene expression ‰¥ and adjusted p value ‰¤ enrichment analyses of a0npcgene ontology go is the main bioinformatics tool for gene annotation and analysis of the biological processes bps of genes and gene products which involves annotation of bps molecular functions mfs and cellular components ccs go analysis of degs was conducted using upsetr the kyoto encyclopedia of genes and genomes kegg is a bioinformatics database resource for determining the highlevel functions and uses of cells and anisms from their genomic information to investigate the functional and pathway enrichment in go and kegg we used upsetr to identify the modules involved in biological functions gene set enrichment analysis gsea is a knowledgebased method for the translation of genomewide expression profiles we analyzed pathways using gsea and identified each functional cluster using clusterprofiler the cutoff criteria were a false discovery rate and p value construction of a0the a0lncrna‘mirna‘mrna interaction networkthe lncrnamirna interactions were predicted using the mircode tool httpwwwmirco de which is described as œa map of putative mirna target sites in the long noncoding transcriptome three convenient online databases namely mirdb httpwwwmirdb mirtarbas httpmirta rbase mbcnctuedutw and targetscan httpwwwtarge tscan were used to predict the target mrnas of the mirnas data with five or more binding sites were retained we selected the mrnas at the intersection of the three databases as the predictive targets of mirnas for the construction of lncrna“mirna“mrna cerna networks two separate cerna networks were constructed using upregulated and downregulated rnas and these were visualized using cytoscape https cytos cape version a popular online bioinformatics database protein‘protein interaction network constructionthe protein“protein interaction ppi network was predicted using the search tool for the retrieval of interacting genes string httpstrin gdb version online database significant insights into the underlying mechanisms of npc can be provided by investigating the interactions between proteins all degs of mrnas were predicted using string and a comprehensive score over was regarded as statistically significant cytoscape version https cytos cape was used to visualize biological network and integrate the data the molecular complex detection mcode version algorithm of cytoscape was used for detecting densely connected regions in the ppi network which represented the most closely related gene sets among the degs networkanalyst https wwwnetwo rkana lystcafaces homexhtml a visual analysis platform for the networkbased metaanalysis of gene expression data was used to visualize the proportion of degs cytohubba a cytoscape plugin was used to filter out the top hub genes with the strongest connections to the other genes in the merged networkstatistical analysismost statistical analyses were conducted using the bioinformatics tools mentioned above and the version of r software is version differential expression levels of mrna mirna and lncrna were obtained using a twotailed student™s ttest for the identification of deg benjamini and hochberg false discovery rate method were performed to adjust pvalue functional and pathway enrichment analyses were analyzed by the hypergeometric test and bonferroni correction variables were expressed as mean ± standard deviation a p value was regarded as notably significantresultsdata collection and a0preprocessingto determine whether there was clustering or outliers in the sample set the differences between the clustering of the mrna fig a01a“c lncrna fig a01d“f and mirna fig a01g h expression matrixes of the npc and normal tissue samples in different datasets were examined using threedimensional principal component analysis pca the results showed that npc was well distinguished from the normal tissue samplesidentification of a0degs in a0npcto identify the degs in npc the mrna lncrna and mirna expression profiles were analyzed using the limma package the results showed that mrnas lncrnas and mirnas were dissimilarly expressed logfc ‰¥ adjusted p ‰¤ fig a0 between the npc and normal tissues of these mrnas lncrnas and mirnas were significantly upregulated while mrnas lncrnas and mirnas were significantly downregulated in total degs were shared among the three mrna datasets fig a02d differentially expressed lncrnas delncrnas were shared among the three lncrna datasets fig a02h and differentially expressed mirnas demirnas were shared among the two mirna datasets fig a02k those degs may provide new insight into the biological mechanisms of npc and serve as 0cxu a0et a0al cancer cell int page of fig principal component analysis pca showing the clustering of mrna long noncoding rna lncrna and microrna mirna expression matrices in different samples and different datasets a“c pca of mrna expression between the nasopharyngeal carcinoma npc cluster and normal tissue cluster in the fjch a gse12452 b and gse13597 c datasets the purple dots represent the npc samples and the blue dots represent the normal tissue control samples d“f pca of lncrna expression between the npc cluster and normal cluster in the fjch d gse95166 e and gse126683 f datasets the blue dots represent the npc samples and the red dots represent the normal tissue control samples g h pca of mirna expression between the npc cluster and normal cluster in the gse70970 g and gse43039 h datasets the green dots represent the npc samples and the blue dots represent the normal tissue control samplespotential therapeutic targets for npc functional roles of delncrnas shared among the three lncrna datasets are provided in additional file a0 table a0s1 and functional roles of demirnas shared among the two mirna datasets are provided in additional file a0 table a0s2construction of a0the a0cerna networkto explore the role of mirnas and corresponding target mrnas as well as corresponding lncrnas in npc we predicted the target mrnas of the demirnas and lncrnas that may have interrelations with mirnas the results may help to better explain the 0cxu a0et a0al cancer cell int page of fig volcano plots of the distributions of degs in different datasets a“c volcano plots of the distributions of demrnas in the fjch a gse12452 b and gse13597 c datasets e“g volcano plots of the distributions of delncrnas in the fjch e gse126683 f and gse95166 g datasets i j volcano plots of the distributions of demirnas in the gse43039 i and gse70970 j datasets differentially expressed genes degs were those with a fold change of and a pvalue of in the mrna expression matrix lncrna expression matrix and mirna expression matrix upregulated degs are mapped as red spots and downregulated degs are mapped as green spots genes without notable variation are labelled as black spots d venn diagram of the degs among the mrna expression datasets fjch gse12452 and gse13597 h venn diagram of the degs among the lncrna expression datasets fjch gse43039 and gse70970 k venn diagram of the degs among the mirna expression datasets gse43039 and gse70970critical regulatory functions of mirnas mrnas and lncrnas the interaction of upregulated and downregulated mirnas with delncrnas was predicted based on mircode the prediction of target mrnas of upregulated and downregulated mirnas was performed using three databases mirdb mirtarbas and targetscan lncrnas mirnas and mrnas were included in the upregulated and downregulated lncrnamirnamrna cerna networks respectively fig a0 3a b the blue red and green nodes represent mirnas lncrnas and mirnas respectively additional files tables s3 s4 shows the details of the interactions of the upregulated and downregulated mirnas and mrnas respectively additional file a0 tables s5 and s6 shows the details of the interactions of the upregulated and downregulated mirnas and lncrnas respectivelygo and a0kegg analyses of a0degsto further analyze the possible functions of the degs we conducted biological analyses by using clusterprofiler and upsetr the results suggested that the degs were significantly enriched in go and kegg terms the go analysis showed that the following bps were notably enriched among the degs chromosome segregation nuclear chromosome segregation sister chromatid segregation mitotic sister chromatid segregation negative regulation of chromosome anization fig a0 4a the following mfs were largely enriched in atpase activity protein serinethreonine kinase activity atpase activity coupled tubulin binding catalytic activity acting on dna dna dependent atpase activity dna helicase activity and singlestranded dna dependent atpase activity fig a04b finally the following ccs were found to be largely enriched in the chromosomal region condensed chromosome chromosome centromeric region 0cxu a0et a0al cancer cell int page of fig interaction networks of mrnamirnalncrna in nasopharyngeal carcinoma npc a a cerna network of upregulated genes b a cerna network of downregulated genes the blue red and green nodes represent predictive mirnas predictive long noncoding rnas lncrnas and predictive micrornas mirnas respectivelyfig upsetr plots showing the distributions of the gene ontology go annotations associated with the differentially expressed genes degs in nasopharyngeal carcinoma npc a biological processes b molecular functions c cellular components d upsetr plot showing the distribution of pathways associated with the degs in npc based on kyoto encyclopedia of genes and genomes kegg analysiscondensed chromosome centromeric region nuclear chromosome telomeric region and condensed chromosome kinetochore fig a04c the kegg pathway analysis suggested that degs in npc were largely enriched in the cell cycle dna replication and small cell lung cancer fig a0 4d the results suggested that chromosomal 0cxu a0et a0al cancer cell int page of dysfunction was closely related to the development of npcgsea of a0npc‘related genesto explore the biological functions of the degs involved in npc gsea was applied the mrna expression profile of the fjch dataset was subjected to gsea by means of clusterprofiler the analysis showed that the following biological pathways were overrepresented in the npc tissues as compared to the normal tissues the mitogenactivated protein kinase mapk signaling pathway the phosphatidylinositol3 oh kinaseprotein kinase b pi3kakt signaling pathway fig a0 5a the apoptotic pathway and the tumor necrosis factor tnf signaling pathway fig a0 5b the pathways found our study were involved with cancer progression metastasis and apoptosisppi network analysis of a0degsthe string database was used version to explore the ppi network based on the correlations among the degs in npc the obtained data were then examined using cytoscape software the ppi network of degs was constructed using mcode to obtain the vital gene module the networkanalyzer plugin was applied to further analyze the ppi network according to the scores the cytohubba plugin was used to analyze the hub genes associated with npc and the following genes with the top grades were deemed to be hub genes nusap1 racgap1 prc1 kif4a top2a pbk kif2c tpx2 cenpu oip5 ttk mad2l1 ndc80 birc5 melk cenpf foxm1 tyms cdk1 and cep55 fig a0 those genes may contribute to the investigation of biological mechanisms and uncover underlying therapeutic targets for npcfig gene set enrichment analysis gsea of the gene expression profiles of the fjch dataset a gsea shows that the mitogenactivated protein kinase mapk pathway and the phosphatidylinositol 3kinaseprotein kinase b pi3kakt pathway are concentrated in nasopharyngeal carcinoma npc b gsea reveals that the apoptosis pathway and the tumor necrosis factor tnf pathway are concentrated in npc 0cxu a0et a0al cancer cell int page of fig proteinprotein interaction ppi networks a a ppi network of differentially expressed genes degs constructed using string software b most relevant gene sets in the ppi network extracted using mcode c further analysis of degs using networkanalyzer d the top hub genes with the most correlations identified using cytohubbago and a0kegg analyses of a0hub genesto analyze the functions of the top hub genes we again conducted biological analyses by using clusterprofiler and upsetr the results suggested that the hub genes were significantly enriched in go and kegg terms go analysis showed that changes in the following bps of hub genes were notably enriched in chromosome segregation nuclear chromosome segregation sister chromatid segregation mitotic sister chromatid segregation microtubule cytoskeleton anization involved in mitosis and regulation of chromosome segregation fig a07a in addition the changes in the following mfs were mainly enriched in protein serinethreonine kinase activity tubulin binding microtubule binding and protein cterminus binding fig a0 7b finally changes in the following ccs of degs were enriched in the chromosomal region spindle condensed chromosome chromosome centromeric region kinetochore microtubule midbody condensed chromosome centromeric region condensed chromosome kinetochore and mitotic spindle fig a0 7c kegg pathway analysis indicated that the degs in npc were mainly enriched in the cell cycle cellular senescence oocyte meiosis progesteronemediated oocyte maturation and platinum drug resistance fig a07d enrichment analyses of the hub genes were similar to the results of the analyses of the degs hence the findings obviously 0cxu a0et a0al cancer cell int page of fig upsetr plots showing the distributions of the gene ontology go annotations associated with the hub genes of nasopharyngeal carcinoma npc in the case of a biological processes b molecular functions and c cellular components and d upsetr plot showing the distribution of pathways associated with the hub genes of npc based on kyoto encyclopedia of genes and genomes kegg analysissuggested that chromosomal dysfunction was a vital contributor to the tumorigenesis of npcdiscussionin this work we performed a comprehensive analysis of the full transcriptome sequencing dataset of fujian cancer hospital and six microarray datasets downloaded from the geo repository to uncover degs between npc tissues and normal nasopharyngeal tissues we identified differentially expressed mrnas demrnas demirnas and delncrnas among the seven datasets and constructed a lncrnamirnamrna network of npc go enrichment analysis kegg enrichment analysis and gsea proved that the enriched components and pathways among the degs associated with npc were inseparable from the chromosome dysfunction mapk signaling pathway and pi3kakt signaling pathway discovered in npc we also identified the top hub genes in the ppi network related to npc and the results of the enrichment analysis of the hub genes were similar to those of the degsstudies have shown that lncrnamirnamrna networks play significant roles in the development and progression of tumors by constructing visual networks we can see the interaction between degs of different molecular types the lncrnamirnamrna network constructed in our study indicated that in npc mrnas could be regulated by lncrnas via corresponding mirnas li et a0al identified mirnas including highly expressed mirnas and lowly expressed mirnas from the serum of npc patients with different radiosensitivity these mirnas were found to have remarkable differences between the patients fold change ‰¥ or ‰¤ and p the highly expressed mirna hsamir6088 and the lowly expressed mirna hsalet7f13p from the above study were also found in our cerna networks we also identified hsamir29a3p and hsamir103a3p as demirnas which were recently found to act as circulating biomarkers of npc with fairly good diagnostic accuracy for detecting npc as compared with controls area under the curve the radioresistant npc cne2ir cell 0cxu a0et a0al cancer cell int page of line has been shown to overexpress jun guo et a0al identified junrelated mirnas by using mirdip software including hsamir200b3p hsamir1395p hsamir200c3p hsamir95p and hsamir92b3p thus jun could promote tumorigenesis and tumor development qing et a0 al found that inhibiting cjun expression could enhance radiosensitivity and induce cell cycle arrest and apoptosis the above results show that cerna networks can offer insights into the complex regulation patterns of npc and potentially facilitate the individualized treatment of npcgo analyses of the bps of the degs associated with npc showed that the negative regulation of chromosome segregation nuclear chromosome segregation sister chromatid segregation mitotic sister chromatid segregation and negative regulation of chromosome anization were closely associated with the oncogenesis of npc among the cc annotations chromosomal region condensed chromosome chromosome centromeric region condensed chromosome centromeric region condensed chromosome kinetochore and nuclear chromosome telomeric region were notably related to npc several studies have reported on the chromosomal aberrations involved in the carcinogenesis of npc including chromosomal gains or losses [ ] loss of heterozygosity chromosomal rearrangements and chromosomal imbalances in one study loss of heterozygosity on 3p was observed in “ of primary npc specimens and almost of precancerous lesions tan et a0al hypothesized that apoptosis induced by oxidative stress may lead to cadmediated chromosomal breakage after incorrect dna repair cells that survive apoptosis may carry chromosomal rearrangements leading to the tumorigenesis of npc to investigate common genetic variations in npc natasya et a0al screened out cases of npc in the malaysian population by the comparative genomic hybridization cgh technology and the results showed chromosomal changes in all npc cases enrichment analyses of the hub genes identified in our study were greatly compliant with the results of the enrichment analyses of the degs thus the above findings clearly implicate chromosomal dysfunction as an important contributor to the carcinogenesis of npcgsea showed that the mapk signaling pathway pi3kakt signaling pathway apoptotic pathway and tnf signaling pathway were the top four pathways associated with npc the enriched pathways identified in our investigation are related to tumor progression metastasis and
Colon_Cancer
" in head and neck cancer hnc the relationship between a delay in starting radiotherapy rt andthe outcome is unclear the aim of the present study was to determine the impact of the amount of time beforetreatment intervention tti and the growth kinetics of individual tumors on treatment outcomes and survivalmethods two hundred sixtytwo hnc patients with primary tumors treated with definitive chemo rt wereretrospectively analyzed the tti was defined as the time interval between the date of histopathologic diagnosisand the first day of the rt course volumetric data on tumors were obtained from diagnostic and rt planningcomputer tomography ct scans in order to calculate the tumor growth kinetic parametersresults no significant association between locoregional control or causespecific hazards and tti was found thelog hazard for locoregional recurrence linearly increased during the first days of waiting for rt although this wasnot significant the median tumor volume relative increase rate and tumor volume doubling time was 32dayand days respectively and neither had any impact on locoregional control or causespecific hazards the association between a delay in starting rt and the outcome is complex and does not harm allpatients waiting for rt further research into imagingderived kinetic data on individual tumors is warranted inorder to identify patients at an increased risk of adverse outcomes due to a delay in starting rtkeywords head and neck cancer radiotherapy waiting time treatment delay outcome with new cases and deaths reported in head and neck cancer hnc is the eighth mostcommon and most lethal cancer in men worldwide in addition to surgery and systemic therapy radiotherapy rt is one of the cornerstones for treatment of thiscancer owing to the rising cancer incidence rate in ageing populations and the widening list of indications forirradiation the demands for rt have increased dramatically over the past decades [ ] the increasing complexity of pretreatment diagnostics and rt technology correspondence pstrojanonkoisi1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia4chair of oncology faculty of medicine university of ljubljana ljubljanasloveniafull list of author information is available at the end of the has led to delays in treatment decisionmaking and thereduction in linear accelerator throughputthat hasresulted in a significant imbalance between the demandfor rt and the availability of rt capacities in manypublically funded health systems this is also the case inslovenia [ ]due to obvious ethical reservations the only way tostudy the impact of delays in starting rt on treatmentoutcomes are retrospective observational analyses of cohorts from different institutions or countries intuitively one would expect that the prolongation of thetime taken before treatment intervention tti is harmful to patients both the likelihood of tumor growth andthe acquisition of a metastatic phenotype increases as afunction of time furthermore advanced tumors aremore difficult to treat than smaller tumors the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cžumer radiation oncology page of indeed a systematic review of pertinent literature fromthe period “ by chen showed an increasein the risk of local hnc recurrence of for everymonth of delay in definitive rt however certainstudies included in chen™s metaanalysis and also somemore recent reports negated the association betweenthe delay in definitive rt and the increased risk of treatment failure [“] several different biases inherent inretrospective analyses either related to the quality ofdiagnostic procedures and treatment or to the inhomogeneity of the studied population as well as a selectionbias ie patients with fasttumor progression or ahigher burden of symptoms receive priority in treatment and significant variability in the kinetics of individual hnc cases may abolish the effect of tti inoutcomes [“] however if patients with advancedor fastgrowing tumors have to wait longer the magnitude of this effect may be overestimated furthermore no compelling relationship between treatmentdelay and prognosis was found in some other cancertypes [“]in order to determine what would be an acceptabletti in hnc patients treated with definitive rt or concurrent chemoradiation we aimed to analyze the impactof tti and growth kinetics of individual tumors on theoccurrence of localregional failure distant metastasisand survival in the present study of a cohort of slovenepatients with hncmethodsin a retrospective study patients with oral cavity oropharyngeal hypopharyngeal or laryngeal squamous cellcarcinoma scc who were treated with curativeintentdefinitive rt with or without concurrent chemotherapybetween january and december at the institute of oncology ljubljana slovenia were includedpatients with t1n0 or t2n0 glottic cancer were left outof this cohort the “ period was chosen because of fluctuations in the waiting time for irradiationas a result of intensive renovations and expansion in thedepartment of radiotherapy that took place over thistime span from patients™ medical and rt charts wecollected information on clinical gender age onset ofsymptoms date and type of disease recurrence anddeath tumor histology site of origin tnm stage andtreatment characteristics rt technique regimen anddose duration of rt type of concurrent chemotherapy[cct] and the number of cycles administered thetnm stage was determined according to the 7th editionof the uicc classification systemfor analysis of the impact of tumor growth kinetics ontreatment outcomes the volumes ml of primary tumors and neck nodal metastases as marked on diagnostic and rt planning computer tomography ct scanswere compared patients with the same basic clinicaldisease and treatment characteristics as indicated abovebut with both sets of ct scans available were selectedfor this part of the study diagnostic ct scans were performed through the acquisition of mm thin ct sections whereas planning ct scans had a slice thicknessof mm both with intravenous iodine contrast enhancements sets with extensive artefacts were excluded forthe purposes of rt planning patients were positionedsupine on the flat tabletop and a fivefixation pointthermoplastic mask was used lymph nodes were considered positive if the smallest diameter was more than cm andor the necrotic center or extracapsular extension was seen if available segmentation was guided bymagnetic resonance imaging mri sets and the resulting contours around the primary tumors and metastaticneck nodes represented a consensus between two radiation oncologists and a radiologist volumes of primarytumors and metastatic neck nodes were separately calculated by a computer software program used for rt planning xio computerized medical systems inc stlouis usa eclipse varian medical systems inc paloalto usa the end points in this part of the study werechanges in the tumornodal volume and tnm stage thecalculation of the primary tumor volume doubling timeand their impact on the treatment outcomestatisticsthe study protocol was approved by the republic ofslovenia national medical ethics committee no “ for retrospective studies a written consentis deemed unnecessary according to national regulationsbasic descriptive statistics were reported with meansstandard deviations and ranges for numerical variablesand as percentages for categorical variables in patientswith two simultaneous hncs some characteristics werereported in regards to patients while others were reported in regards to tumorsthe survival curves were computed using the kaplanmeier estimator and the aalenjohansen estimator wasused to estimate the cumulative probabilities of competing risks the effect of covariates was analyzed using amultiple cox regression analysis with all the analysesthe data were censored at a fiveyear followupwhen focused on the survival of patients the analyseswere completed with patients as the units and the timewas measured from the first day of therapy until deaththe overall survival os regardless of the cause ofdeath and the absolute risk cumulative probability ofdying due to index cancer were reported in the cox regression only the index cancer deaths causespecifichazard csh were considered to be events of interestin the analyses where locoregional control lrc wasof primary interest the calculations were performed in 0cžumer radiation oncology page of regards to tumors excluding the nonresponders to rtie those with residual local or regional tumors at “ weeks after rt completion for the latter group weconcluded that it is the radioresistance of tumor cellsthat are responsible for the persistence of the diseaseafter therapy and not that patients had to wait for rtthe followup time was calculated from the last day oftherapy the estimated cumulative probability of localandor regional recurrence distant metastases lrcprobability of being still alive and without local andorregional recurrence and diseasefree survivaldfsprobability of being still alive and without events locoregional and distant failure and deaths were the events ofinterest were reported all the analyses were conductedin regards to the tumors as independent units this assumption was checked in the sensitivity analysis andallowed for gamma frailtythe assumptions of the cox regression were checkeda nonlinear effect a spline with degrees of freedomwas allowed for the numerical variables and the proportional hazards assumption was tested using schoenfeldresidualsthe tti was defined as the time interval between thedate of histopathologic diagnosis and the first day of thert course tumor growth kinetics was expressed as thetumor volume relative increase rate per day and as thetumor volume doubling time in daysthe tumor volume relative increase rate per day was 13 calculated as 12v t 2ðþþv t 1ðt2 ˆ’ t1where vt1 gross tumor volume at time t1 ieon diagnostic ct scans and vt2 gross tumor volume at time t2 ie on planning ct scans it was reported as the percentage increase was reported as per day the tumor volume doubling time was calculated asðv t 2ðv t 1ðln 2ð þ t ˆ’ t þþlnþsince a onetoone relationship existed between thetwo only the tumor volume relative increase rate whichrequires no extrapolation was considered for modellingall analyses were performed using r statistical software version and a pvalue below was considered statistically significantresultsimpact of time to treatment initiationbetween and patients with oral cavity oropharyngeal hypopharyngeal or laryngeal primarysccs were treated with definitive chemo radiotherapywith curative intent there were men and women aged “ years mean the majority oftumors originated in the oropharynx tumors in patients and were tnm stage iv tumors in patients the tti ranges from to days with mean days the distribution of the tti is only slightlyasymmetric with median and interquartile rangeiqr “ fig patients were irradiated with dimensionaltechnique or 3dimensionalconformal isocentric technique to the median rt dose gy iqr “ delivered in gy dailyfractions iqr “ concurrently to rt patientsreceived chemotherapy consisted of platinbased monochemotherapy patients or mitomycinecbleomycin combination patients the characteristics of patients and their tumors are shown in table treatment outcome and survivalclinical andor radiologic assessment at “ weeksposttherapy confirmed disease persistence locally andor regionally in cases ie in patients and thesepatients were excluded from further analyses of lrcand dfs thus patients with tumors were analyzed and during followup a local andor regional relapse was recorded in cases with a mediantime from treatment completion of months range “ months the two and fiveyear probability of localrecurrence after the end of treatment was and respectively whereas the regional relapse was and respectively after threeyears posttherapy we only observed a small increase ie inlocal recurrence probability whereas the probability ofregional relapse was stable after the third year followupthe probability of occurrence of distant metastases at years was and at years it was at the second and fifthyear followup the lrc was confidence interval [ci] and ci respectively and the dfs was ci and ci respectivelyin the fifth year posttreatment out of patients had died the index cancer was the reason in of the cases during the first years posttherapythe probability of cancerrelated death steeply increasedfrom zero to but later the changes were less pronounced at the second and fifth year posttreatment theos independent of the cause of death was ci and ci respectivelyeffect of covariatesin the univariate analysis the following parameters weretested for lrc and csh age gender stage of diseasetype of treatment rt vs rt cct and tti for rtas continuous variable results are presented in table 0cžumer radiation oncology page of fig distribution of ttitable clinical characteristics of patients and their tumorscharacteristicsgenderno femalemaleage yearsatumor locationboral cavityoropharynxhypopharynxlarynxtumor stagebstage istage iistage iiistage ivtreatmentbradiotherapyconcurrent chemoradiotherapyrt duration daysatime to treatment interventiona ± “ ± “ ± “the same parameters were also included in the multivariate model table the occurrence of locoregional recurrence was significantly related to the diseasestage p whereas the relationship with agewas only of marginal significance p higherage had a lower hazard no significant associationwith the type of treatment could be found p table the log hazard for locoregional recurrenceseemed to linearly increase during the first days oftable effect of covariates on locoregional control and indexcancerspecific hazard n patients with tumors anunivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherap cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt““““““““““““ a mean ± sd rangeb eleven patients had two simultaneous primary tumorsci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapy 0cžumer radiation oncology page of table impact of tti on locoregional control and indexcancerspecific hazard n patients with tumors amultivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt““““““““““““ ci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapywaiting for rt although the association between thehazard and tti wasregardless ofwhether we made an allowance for nonlinearity ornot fig 2ainsignificantthe hazard of dying due to the index cancer ie cshwas found to be favorably associated with a lower disease stage p and the addition of cct to rtp whereas age gender and tti did not reachthe level of statistical significance table fig 2bassumptions and sensitivity analysisproportional hazards for all included variables and thelinearity assumption for age were analyzed and presented graphically some indication of nonproportionalhazards could be found in gender but allowing for nonproportional hazards did not change the interpretationof the other covariates as a part of the sensitivity analysis the assumption of the dependence of the tumorsbelonging to the same individual was relaxed but nochanges in the interpretation of the results could be observed as an additional confounder the timedependentcovariate œduration of rt was considered but its effectdid not prove to be importantimpact of tumor growth kineticsdiagnostic and planning ct scans median interval days range “ days were available from patientsfive of these patients had two primary tumors the majority of patients were males with a meanage of years range “ and the patients hadprimary tumors located in the oropharynx table when two ct sets were compared the original tstage of the primary tumor was increased ie upgradedin two cases and the nodal stage was increased in six patients due to the limited number of npositive casesn only volumes of primary tumors werecompared between the two ct sets for the calculationof the tumor volume relative increase rate per dayand tumor doubling time the absolute increase intumor volume ranged from ˆ’ to cm3 per daymedian no increase in volume was observed infive patients ct scans in these five patients were takenfig trend of the hazard for locoregional recurrence a and for index cancerspecific death b 0cžumer radiation oncology page of table characteristics of patients with available diagnostic andplanning ct scanscharacteristicsgenderno femalemaleage yearsatumor locationboropharynxhypopharynxlarynxstagebstage istage iistage iiistage ivtherapybradiotherapyconcurrent chemoradiotherapytumor volume relative increase rate dayatumor volume doubling time daysaa mean ± sd rangeb five patients had two simultaneous primary tumors “ “ “from days to days apart median days the median tumor volume relative increase rate was perday median range “ and the mediantumor volume doubling time was days range “days no difference was observed when these two parameters were compared between different tumor stagestreatment outcome and survivalamong the tumors the time taken for local andorregional relapse and the occurrence of distant metastaseswere assessed for tumors in which treatment resultedin clinicalradiological eradication of the disease at “ weeks posttherapy local andor regional relapse wasrecorded in nine cases with a median time fromtreatment completion of months range “ monthsthe two and fiveyear probability of local recurrenceafter the end of the treatment was and respectively whereas for the regional relapse it was and respectively at years posttherapy only anincrease in the local recurrence probability of wasobserved whereas the probability of regional relapseremained stable the two and fiveyear probability ofdistant metastases was at two and years thelrc was ci and ci respectively and the dfs was ci and ci respectivelyin the yearperiod after the start of treatment outof patients died the index cancer was thereason in cases at two and years posttreatment the os rates were ci and ci respectivelyeventseffect of covariatesdue to the low number of events local andor regionalrecurrence ninecancerspecific deaths events only univariate regression models were fittedthe following covariates were tested in the modelsoverall disease stage initial tumor volume tumor doubling time and relative increase rate day of primarytumor volume measures of tumor kinetics did not showany impact on lrc or csh only an inverse relationshipbetween the initial primary tumor volume and csh wasobserved hr for index cancerspecific death perevery cm3 increase in the volume of primary tumorstable there was no difference in the value of dfsbetween patients with a primary tumor volume relativeincrease rate 1day and 1day p fig discussionwhile it is intuitively anticipated and confirmed by theresults of the metaanalysis that a delay in starting rtwould have a negative impact on the treatment of patients with hnc this association was not confirmed inour study we only found a statistically insignificantupward trend in the risk of locoregional recurrence forthe first days of rt delay in addition the differencesin the growth kinetics between individual tumors whichwere studied in a smaller group of patients were considerable but did not appear to be of significance for theprediction of treatment outcomesobviously the relationship between tti and diseaseprognosis in patients with hnc is more complex than itmight seem at first glance the first negative impact ofwaiting for treatment to begin is the risk that the tumorwill increase in size andor metastasize during this timemaking it harder to treat or resulting in it becoming untreatable [ ] however the time for a tumor to growis only one of the factors that determines prognosis andthe absence of a statistically significant association between tti and the treatment outcome in our study andis thus not surprising [“]many other reportsmoreover no obvious methodological differences couldbe found between these negative studies and the positive studies that confirmed the association between ttiand treatment outcomes [“] in both groups thereare individual studies that have similar sample sizestumor sitestage mix and periods covered speaking infavor of comparable quality of diagnostics therapy andstatistics across the studies 0cžumer radiation oncology page of table effect of stage and tumor kinetics on locoregional control and overall survivalparameterstnm stageiv vs iiiivtuper cm3vtu relative increase rateper dayvtu relative increase rate‰¤ vs 1dayvtu volume of primary tumorlocoregional controlhr ci““““pvaluecause specific hazardhr ci““““pvalueon the contrary in more recently reported analyses ofthe national cancer registries data an adverse effect ofwaiting for radiotherapy was clearly established [“]however the results from this type of analysis should betaken with caution not only due to the limitations inherent in the tumor registry data unmeasured confounding selection biasincomplete data and codingerrors but also because the effect of delaying rt oncancerspecific outcomes was not evaluated in additionpatients irradiated in postoperative and definitive settings were not analyzed separately overall the researchmethodology and interpretation used in these studieswere criticized and the magnitude of the effect that theysupposedly demonstrated was questioned in the present study a linear increase in the log hazardfor locoregional recurrence was found during the first days of waiting for rt although it was not statisticallysignificant it is possible that unknown confounders thatwere not accounted in our analysis eg tumor growth kinetics reduced the statistical power of the tti the resultsfrom fortin and naghavi who reported on theincreased risk of locoregional failure with tti daysand days respectively [ ] are the most comparable to our own however optimal ttithresholdsfig impact of primary tumor volume relative increase rate to diseasefree survival in patients with no residual disease at “ weeks posttherapy 0cžumer radiation oncology page of identified in different studies showed considerable variations pointing to the uncertainty of such calculations andtheir dependence on the characteristics of the analyzedpopulation [ “] the possible role of classical prognostic factors such as location of primary tumor diseasestage and addition of cct is not expected to be relevant inthis respect as in our and other similar studies the statistical significance of tti was verified by multivariateanalysisthe effect of tti on treatment outcomes however isnot only conditioned by the duration of waiting for rtbut also on the rate of tumor growth in hnc a vast heterogeneity in tumor cell kinetics has been observed conditioned by the local milieu from which it arises whichdiffers from patient to patient [ ] historicallydifferent methods were explored to evaluate tumor cellkinetics but did not succeed in providing clinically relevant kinetic parameters [ ] more recently a comparison of two sets of imaging data acquired at twodifferent time points was successfully employed for thispurpose usually volumetric data are extracted from diagnostic and rtplanning ct scans for the calculation ofdifferent parameters reflecting the rate of tumor growtheg tumor volume doubling time or absolutepercentagetumor volume increase per day despite some differencesin the calculation methodology across studies a large variation in the individual values of kinetic parameters wasseen in all of them including ours indicating that all ofthe studied populations represent a unique mix of slowand fast growing tumors [“] among our patientsfive had no measurable increase in primary tumor volumeeven when the interval between ct scans was up to days the inverse relationship between kinetic parametersdetermined by the comparison of two ct datasets andtreatment outcomes was implied or even confirmed inseveral smaller studies pointing to potential clinical utilityof imagingderived kinetic data [“] in our grouphowever no such association was found a small numberof patients and “ in contrast to other studies “ the inclusion of different tumor sites could contribute to the negative result as well as differences in the changes in kineticproperties triggered by rtccr in individual tumors tumor radiosensitivity may also influence the effect oftti on outcome while to some extent it may be evaluated before rt begins eg by molecular profiling identification of hypoxic cells and the determination of hpvstatus in oropharyngeal primary tumors in daily clinicalpractice it is usually not considered when planning treatment [ ] in order to diminish the impact of intrinsic tumor radiosensitivity the patients in our study withresidual disease at “ weeks postrt were excludedfrom the analysis of lrc we hypothesized that the inability to achieve a complete tumor response to chemoradiation was due to the radiobiological characteristicsof the disease and not the delay in starting rt howeverthe exclusion of these patients from the analysis did notaffect the end resultsour study has weaknesses which are mostly due to itsretrospective nature however for obvious ethical reasons this is an inevitable feature of studying delays inthe initiation of cancer therapy we are aware that thereis always some doubt as to the accuracy of the diagnosticprocedures the staging the quality of planning and theimplementation of rt in the case of retrospective research nonetheless the same restraint exists in the caseof other similar studies and even more so in the case ofanalyses of cancer registry datasets an important feature of our data set is that no patients were lost duringthe followup and in our opinion is free from manyhidden biases that larger studies inevitably bring withsince almost half of our patients had oropharyngeal carcinoma missing information regarding the p16 or human papillomavirus hpv tumor status could be ofimportance howeverin the cohort of oropharyngealcancer patients treated at our institution between and only had a hpvrelated tumor thus we reasonably assume that the impact of p16hpv tumor status on the study results was negligible inaddition in hpvpositive cases perni found nosignificant association between tumor growth velocitycalculated from serial pretreatment ct scanslocaland distant control or os and the same was reportedby chu who measured metabolic growth velocityusing pretreatment petct scans [ ]other drawbacks to consider include technical limitationsin ct scan acquisition the accuracy of presentation of actual tumor volume on ct images and the precision of thedelineation of gross tumor volume to minimize errors inthe estimation of comparative tumor volumes only highquality pairs of ct images were selected for the analysis oftumor kinetics in addition physical findings documentedin clinical records and when available diagnostic mriswere used for this purpose and labelled tumor volumes represented a consensus between two experienced radiationoncologists and a radiologist all dedicated to hnc management like many other studies [“ “] the comorbidity burden was not registered in our patientsalthough it should be taken into account when assessingsurvival outcomes finallylags in the prebiopsyperiod were not addressed in our study including patientdelay and delays in referral and diagnostics which may addsignificantly to the total tti these are also costly and potentially fatal but can be successfully reduced by effectivecoordination between providers [“]sthe relationship between tti and treatment outcomesis multifaceted so the controversy of published results is 0cžumer radiation oncology page of not surprising in this study we found that delays in theonset of rt do not harm all patients as tti is a problem in many public health systems further research iswarranted and should focus on two areas evaluatinglarge population surveys with highquality data andtreatmentrelated outcomes not just os and the prognostic relevance of imagingderived kinetic data of individualtumors which appeared promising in severalsmaller and statistically underpowered studies in orderto obtain a tool to identify patients at increased risk oftreatment failure due to delays in starting rt in a situation without clear knowledge to whom waiting for irradiation is harmful the only possible recommendationcould be that the waiting time for rt should be œasshort as reasonably achievable asara abbreviationshnc head and neck cancer rt radiotherapy tti time to treatmentintervention scc squamous cell carcinoma cct concurrent chemotherapyct computer tomography mri magnetic resonance imaging os overallsurvival csh causespecific hazard lrc locoregional control dfms distantmetastasisfree survival dfs diseasefree survival iqr interquartile rangeci confidence interval hpv human papillomavirusacknowledgementsthis study was financially supported by the slovenian research agencyprogram no p30307authors™ contributionsstudy concepts žumer b strojan p study design žumer b pohar perme mstrojan p data acquisition all authors quality control of data strojan p dataanalysis and interpretation pohar perme m strojan p statistical analysispohar perme m manuscript preparation all authors manuscript editingstrojan p manuscript review žumer b pohar perme m strojan p the authors read and approved the final manuscriptfundingthis study was financially supported by the slovenian research agencyprogram no p3“availability of data and materialsthe datasets analyzed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study protocol was approved by the republic of slovenia national medicalethics committee no “ all patients gave consent for using theirdata for study purposes at the start of their treatment for retrospective studies awritten consent is deemed unnecessary according to national regulationsconsent for publicationthe republic of slovenia national medical ethics committee approved thestudy which was conducted in accordance with the ethical standards laiddown in an appropriate version of the declaration of helsinki theneed for consent was waived by the republic of slovenia national medicalethics committeecompeting intereststhe authors declare that they have no competing interestsauthor details1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia 2institute of biostatistics and medicalinformatics faculty of medicine university of ljubljana ljubljana slovenia3department of radiology institute of oncology ljubljana ljubljanaslovenia 4chair of oncology faculty of medicine university of ljubljanaljubljana sloveniareceived may accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin “ world health anization who life expecta
Colon_Cancer
" laparoscopic tumorspecific mesorectal excision tsme preserving the left colic artery and superiorrectal artery is still a technically challenging procedure we conducted this study to demonstrate the feasibility ofthis procedure for upper rectal cancermethods a total of patients with upper rectal cancer were retrospectively analyzed in our cancer centerbetween april and april these patients were treated with either laparoscopic tsme n orlaparoscopic total mesorectal excision tme n in the tsme group the left colonic artery and superior rectalartery were preserved while they were not in the tme groupresults the operation time in the tsme group was longer than that in the tme group ± min vs ± min p furthermore the number of resected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p the blood loss between the tsme and tmegroups was not significant no mortality occurred in either the tsme or tme groups one patient in the tme groupunderwent conversion to laparotomy the total postoperative complication rates in the tsme and tme groups were and respectively there was no difference in severe complications between the two groupsanastomotic leakage and stenosiss laparoscopic tsme preserving the left colic artery and superior rectal artery can be safely conductedfor upper rectal cancerkeywords laparoscopic surgery rectal cancer tumorspecific mesorectal excision superior rectal artery leftcolonic artery tme correspondence 237721898qqcom 250537471qqcomhuxiang_zc1978sinacom chi zhang haotang wei wenqing hu and yueming sun contributedequally as joint first authors7department of general surgery yizhen people™s hospital clinical medicalcollege yangzhou university yangzhou jiangsu province china3department of gastrointestinal surgery changzhi people™s hospital theaffiliated hospital of changzhi medical college changzhi shanxi provincechina1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang world of surgical oncology page of introductiontotal mesorectal excision tme is an important surgical technique to prevent the local recurrence of rectal cancer on the other hand tme may not besuitable for every case of rectal cancer such as rectosigmoid junction and upper rectal cancers the resection range of tme reaches cm below the inferiorborder of the tumor and has acquired an adequatecure rate reported in previous studies for patientswith rectosigmoid junction and upper rectal cancers this tumorspecific resection according to thetumorsite or t staging is called tumorspecificmesorectal excision tsme itsudeck™s critical point at the rectosigmoid junction isdescribed as the point of origin of the last sigmoid arterial branch originating from the inferior mesenteric artery ima the anastomosis between the lastsigmoidal artery and superior rectal artery sra is absent in some people to avoid the risk of postoperativeischemic necrosis anastomotic leakage colitis and delayed stricturetosudeck™s point for cases where anastomosis may be absent or insufficiently present in addition the rate ofis desirable to ligate proximalabsence of the left colic artery lca is which maybe associated with a risk of anastomotic leakage due toinsufficient vascularization of the proximal colonic conduit this study introduces the procedure and technicalpoints of laparoscopic tsme with preservation of thelca and sra the operation is still a technically challenging procedure we conducted this study to demonstrate the feasibility of this procedure for upper rectalcancer and shortterm prognosismethodspatientslaparoscopic tsme preserving the lca and sra wasperformed on patients with upper rectal cancer fromapril to april in the same period patients with upper rectal cancer underwent standardtme surgery this study was conducted in accordancewith approved guidelines this study was approved bythe institutional review board of the first affiliatedhospital of dalian medical university written informedconsent was obtained from all patientsfig ima 3d cta 0czhang world of surgical oncology page of equipmentangled ° 10mm diameter 3d laparoscope insufflation equipment and bipolar electrosurgical deviceaesculap german harmonic vascular closure systemjohnson usa 10mm and 5mm port trocars teleflexmedical usa laparoscopic linear staplers mm inlength covidien usa hemolock polymer lockingsurgical clips teleflex medical usa and a circularstapler ethicon endosurgery usa were used in thisstudypreoperative preparationinferior mesenteric artery ima 3d cta examinationshould be performed before the operation to assess themesenteric vascular vessel types fig intestinal preparation was performed days before the operation andprophylactic intravenous antibiotics were used beforethe operation for min central venous catheterizationwas performed after general anesthesia the surgicalposture was the starboard lithotomy position with thehead lower and feet higherthe operating surgeon and camera assistant stood onthe patient™s right side and the first assistant stood atthe patient™s left foot side the laparoscopic monitor wasplaced on the patient™s right foot side the trocar for thelaparoscope was inserted from the right paraumbilicalside and four ports were used as working ports fig surgical techniquesthis surgical technique was characterized by thoroughlymph node dissection based on neurovascular preservation and dissection of the left colon and sigmoid andfig position of the trocarupper rectal vessels along the inferior mesenteric vesselsthe region of operation was the superficial layer of thenerve sheath on the vascular surface the left colonicand superior rectal vessels needed to be preserved andthe vascular branch from the sigmoid vessels and theblood vessels from the superior rectal vessels to the intestinal wall were selected and severed according to thetumor positionfirst we adopted a lateral approach by opening themonks™ white line along the descending and sigmoidcolon reaching the splenic flexure as the cephalad dissection point the correct plane of dissection wasachieved by toldt™s fascia we usually used bipolar electrosurgical devices and bipolar scissors to separate thiscorrect plane with gentle blunt and sharp dissectionthe ureter and other retroperitoneal structures weresafely protected by staying in this plane we continuedto dissect along the plane to the root of the ima thehypogastric nerves were visible the nerves were carefully protectedthen the dissection began at the position of the sacralpromontory the junction of the sigmoid mesentery andretroperitoneum from the previous dissection plane in thefirst step ideally we dissected the presacral space belowthe sra from the left side across the midline to the rightside attentively protecting the hypogastric nerves whileusing a bipolar electrosurgical device fig 3a the distaldissection endpoint was approximately “ cm below thetumor we needed to open the peritoneal reflection anddissect the lateral ligament of the rectum by protectingthe neurovascular bundle nvb using a harmonic vascular closure system in some patients we placed the dissected colon and mesocolon to the right celiac side andthoroughly revealed the left side of the mesocolon wecarefully employed dissection in the correct plane on thevessels to avoid tissue damage for the realization of enbloc resection the technique in this step is to identify therelationship between the left colic artery inferior mesenteric vein imv to the ima and sra and the branch ofthe arteriae sigmoideae fig 3b this vascular bundle canbe traced from the origin of the ima to the rectal segmentapproximately “ cm below the inferior border of thetumor fig 3cthe second step was performed using a medial approach this step involved thorough lymph node dissection based on neurovascular preservation the leftcolonic and superior rectal vessels need to be preservedand the sigmoid vessels and vessel branch from the superior rectal vessels to the intestinal wall were selectedand severed according to the tumor positiondissection at the correct presacral space and cephaladdissection to the ima could be employed our generalmedial approach was to begin at the presacral space andobtain a connection with the plane ofthe lateral 0czhang world of surgical oncology page of fig a dissection the presacral space below the superior rectal artery sra approached from the left side across the midline to the right sideattentively protected hypogastric nerves while using a bipolar electrosurgical device b identification of the relationship between left colic arteryimv to the ima and sra and the branch of the arteriae sigmoideae c tracing this vascular bundle from the origin of the ima to the rectumsegment approximately “ cm below the inferior border of the tumor d ligation of arteriae sigmoideae and vascular branch from sra eligation of arteriae sigmoideae and preserving left colonic vasculature f excision of the mesorectum just underneath the rectal wall about “cm and avoiding injury to the rectal wall and sra g tsme preserving left colic artery and superior rectal arteryapproach pelvic dissection was performed from the entrance of the pelvic cavity down to the pelvic floor wecould identify both the hypogastric nerve fibers and pelvicnerve by using highdefinition 3d laparoscopy and preservethem the imvleft colic artery bundle was then carefullytraced to the junction position from the ima and lymphnode no253 was dissected the pelvic nerves and ureterwere already carefully insulated and the circumference ofthe ima could be revealed the mesocolon could be freedfrom the retroperitoneal position by anterior dissection bygently applying a bipolar electrosurgical device we dissected the sra and blood vessels from the sra to the intestinal wall and dissected lymph nodes no252 andno251 at this point we had completed lymph node dissection and completely clarified the relationship betweenthe lca imv ima sra and arteriae sigmoideae finallywe ligated the arteriae sigmoideae and vascular branchfrom the sra into the intestinal wall fig 3d while preserving the left colonic vasculature fig 3e energy devicesand hemolocks were used widely in this step 0czhang world of surgical oncology page of after the above procedure was completed we separated the rectal wall from the mesorectum with an adequate distance from the tumor in accordance with thet stage and position of the tumor using a harmonic vascular closure system in order to provide enough spaceto insert an endoscopic linear stapler we excised themesorectum about “ cm just underneath the rectalwall fig 3f careful surgery was performed to avoid injury to the rectal wall and sra then the endoscopic linear stapler was fixed the rectum was transected andsatisfactory tsme preservation of the left colic and superior rectal arteries was shown fig 3glastly a small 5cm incision was made at the leftlower abdomen and the specimen was taken outside ofthe abdomen and transected intraabdominal presacralanastomosis was performed by double stapling techniques after inserting the anvil head of a 28mm circularstapler into the oral side of the sigmoid colon doubledrains were placed and no diverting stoma wasperformedin the tme group the inferior mesenteric artery wassevered at the root the colon was severed cm awayand digestive tract reconstruction methods were similarto the tsme groupstatisticsspss190 version was used for statistical analysis categorical variables were compared using a χ2 test continuous variables were presented as the mean standarddeviation or median range these variables were compared using a mannwhitney u test p values of were considered statistically significantresultsthe general characteristics of the included patients arelisted in table there were men and women in the tsme group and men and women in the tme group the meanage was ± years and ± years in thetsme and tme groups respectively there were no significant differences in preoperative comorbidity tumorsize depth of invasion and lymph node metastasis between groups the average distance between the tumorand anus of the tsme group was ± cm andthe distal margin was ± cm the pathologicalstages of the patients for the tsme group were as follows stage i stage iia stage iib stage iic stage iiia and stage iiib theproportion of patients with normal preoperative carcinoembryonic antigen cea was approximately of patients had cea levels between and ngml of patients had cea levels between and ngml and only patients had cea levels ngmltable clinicopathological features between the tsme andtme groupsfactorsage yearstme n ± tsme n ± p valuegendermalefemalebmi kgm2comorbidity ± ± cardiovascular disease respiratory diseasediabetes mellitushistological type differentiated typeundifferentiated type tumor size mm ± ± t categoryt1t2t3t4n categoryn0n1n2 conversion to open surgery operation time min ± ± blood loss ml ± ± lymph node dissection ± ± the operation time in the tsme group was longerthan that in the tme group ± vs ± p table furthermore the number ofresected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p table the blood loss between groupswas not significantly different table the averagehospital stay in the tsme group was a little shorter thanthat in the tme group ± days vs ± days table no mortality occurred in either group one patient inthe tme group underwent conversion to laparotomy dueto bowel ischemia in the distal colon table the totalpostoperative complication rates in the tsme and tmegroups were and respectively table forsevere complications between the two groups anastomotic leakage and stenosis the severity of complicationswas claviendindo classification grades “ and therewas no significant difference between groups 0czhang world of surgical oncology page of tsme n tme n p value ± table postoperative complicationsfactorspostoperative hospital stay days ± mortalitymorbidityabsentpresentanastomotic leakagebleedingabdominal abscessileuswound infectionanastomotic stenosisurinary tract infectionascitesurinary retentionpneumoniacardiacrelated complications discussionin the british surgeon heald proposed tme for rectalcancer and pointed out that the anatomical level of tmewas clear so that the operative quality can be assessed the main concerns were a higher anastomotic leakage ratelonger operative time and higher blood loss after tme lopezkostner pointed out that tme was the standard operation performed for lower rectal cancers tme isnot necessary for cancers of the upper rectum therefore the tsme technique was introduced to achieve satisfactory local control and low morbidity partial mesorectalexcision is applied in tsme according to willian™s report in only of patients had distal intraluminal diffusion cm pollett and nicholls observed that there were no differencesin the local recurrence rate of rectal cancer between distal margins cm “ cm and cm a randomized prospective study of nsabb the national surgicaladjustburst and bowel project showed that the localrecurrence rate was not significantly different betweendistal rectal margins cm “ cm and cm according to the practice parameters for the management of rectal cancer edition a 2cm distal margin is more acceptable than cm but a 5cm distalmargin is still recommended total mesorectal resectiontme should be used for tumors located in the middleand lowerregardless oftwothirds ofwhether itis performed with low anterior resectionlar or combined abdominal and perineal resectionapr for tumors in the upper onethird of the rectumresection of the mesentery can be carried out accordingto the tumor situation and the distance between thethe rectumdistal margin and tumor should be cm the recommended grade was 1a tme was performed according to the distance between the distal margin of the rectal tumor and anus cm while tsme was performed for patients with adistance between the distal end of the rectal tumor andanus of “ cm in the author™s medical departmentoncological outcomes after surgery can be divided intotwo aspects longterm survival and local recurrence ratelaw reviewed patients the 5year local recurrence rate for tme and partial mesorectal excisionpme for proximal cancer was and respectively the disease stage was associated with a higher riskof local recurrence there was no difference in the localrecurrence rates of tme and pme the 5year cancerspecific survival rates with and without tme were similarat and respectively kim reportedthat the 5year cancerspecific survival rate was andthe local recurrence rate was with cases of rectalcancer after tsme with pathologic stages i“iii the riskfactors affecting cancerspecific survival rate were the ptstage pn stage positive distal resection margin and positive circumferential resection margin the risk factors affecting local recurrence were the pn stage positive distalresection margin and positive circumferential resectionmargin another study from a korean reviewed experience in patients with rectal cancer showed that theoverall local recurrence rate was the 5year local recurrence rates were and in stages i iiand iii respectively the 5year cancerspecific survivalrates were and in stages i ii and iiirespectively the risk factors were the pn stage and circumferential resection margin zakir performed an analysis with years of experience in rectal cancer patients who underwent laparoscopic andopen tsme surgery the 5year local recurrence rate was the overall 5year and cancerspecific survival rateswere and respectively there was no difference in the local recurrence rate between laparoscopic oropen resection the overall and cancerspecific survivalrates were and in the laparoscopic surgerygroup and and in the open surgery group respectively the results showed that laparoscopic surgerywas better than open surgery in overall and cancerspecific survival there was no difference in survival in patients with stage i however the survival rates in patientswith stages ii and iii among the laparoscopic surgerygroup were better than those in the open surgery groupwhich shows the superiority of laparoscopic tsme surgery for the longterm prognosis of rectal cancerkorean scholars conducted a study on the safety andprognosis of tsme after neoadjuvant chemotherapy forrectal cancer patients received 5fu with leucovorinchemotherapy and radiotherapy cgy for cycles 0czhang world of surgical oncology page of leadership was tsme was performed “ weeks later the resultsshowed that the overall complication rate was empiricalinternal construction was the 5year survival rate was and the 5yeardiseasefree survival was at present chinasouth korea and the usa have formulated similarguidelines for preoperative radiotherapy and chemotherapy for middle and low rectal cancer but there is nospecific reference data for preoperative radiotherapy andchemotherapy for upper rectal cancer the purpose ofthis paper is to introduce a new method of tsme anddiscuss the safety of the operation longterm survivaland local recurrence have not been discussedtsme surgery based on tme is now accepted as astandard for rectal cancer surgery and laparoscopic rectal cancer resection is accepted widely in the world eventhough it is a challenging procedure for surgery bloodloss in the laparoscopic group is well shown with anaverage of to ml the average blood loss inour study was ml lower than that reported in the literature we can identify neurovascular lesions usinghighdefinition 3d laparoscopy to preserve them and weuse a bipolar electrosurgical device to reduce injurywhich is beneficial for accurate operationthe overall complication rate in laparoscopic tsmeoperation was lower than that in the open operationgroup the rate of anastomotic leak showed no statistical difference between the two operation methods theaverage leak rate for rectal cancers was zakir reported that the overall complicationrate was in tsme for rectal cancer patients therate of anastomotic leakage was in the open tsmegroup and in the laparoscopic tsme group therewas no statistical difference between groups in our studythe incidence rate of postoperative anastomotic leakagewas three patients had complications after surgeryand the overall complication rate was the threecomplications were wound infection fluid collection andurinary retention with a claviendindo grading of “yoo evaluated the optimal duration of urinarycatheterization after tsme for rectal cancer logistic regression analysis was performed to determine the risk factors for urinary retention the variables including age sexasa grade surgical procedure tnm stage tumor position preoperative radiotherapy duration of urinarycatheterization and time of surgery were not significantrisk factors for urinary retentionat present a 3d laparoscopic system aesculapgerman is used in laparoscopic surgery in our department single and reduced portlaparoscopic surgeryrobot operations and tatme operations are not usedfor tsme the surgeons who performed tsme had morethan years of experience in gastroenterostomy and hadexperience with open tsme the difficulty of the tsmeoperation is the management of the mesorectum seiji has reported on the management of the mesorectumin the narrow pelvis which our treatment method is basedon first the right part of the mesorectum is lifted fromthe right side of the sigmoid mesocolon to expose the inferior mesenteric artery and vein left colonic vessels sigmoid colonic vessels and superior rectum vessels theassistant lifts the left mesentery of the sigmoid colon exposes the above vessels expands the sigmoid mesocolonagain penetrates the mesentery from the right side andexposes the surrounding vessels expansion of the pelviccavity along the vessels is continued and the mesorectumis repaired from the left to the right side “ cm above thetumor according to the location ofthebranches of the severed vessels are determined and “cm of the intestinal wall is repaired the rectum is dissected using an endogia staplerthe tumorlaparoscopic tsme has been used for rectal cancer andcan obtain satisfactory functional results compared to openresection and tme we do not think that the reduction inthe hospital stay is due to the acceleration of the intervention as per enhanced recovery after surgery eras butis due to an increase in the doctors™ confidence in reducingthe risk of postoperative complications after vascular preservation threedimensional cta examination is importantfor the preoperative evaluation of sigmoid colonvascular classification and intraoperative management ofthe sigmoid and left colon vessels however preoperativeexamination could not obtain information on the trafficbranch the biggest advantage of this operation is themaintenance of the blood supply of the proximal and distalintestines and the sufficient length of the intestine so thereis no need for temporary defunctioning stoma temporarydefunctioning stoma only increases the complexity of theoperation and closure of the temporary stoma increasesthe risk of complications in addition the results of the statistical analysis showed that the number of lymph nodes inthe tsme group was greater than that in the tme groupit cannot be concluded that tsme was significantly betterthan tme for lymph node dissection suggesting thattsme was not inferior to tmeslaparoscopic tsme with preserved left colic and superior rectal arteries is a technically challenging procedureintact visceral pelvic fibro is protected with even greateraccuracy than other techniques by 3d laparoscopywhich offers an optimal vision tsme with preserved leftcolic and superior rectum arteries did not increase therisk of operation compared with tme but increased thesurgeon™ s confidence in patient outcomes thereforelaparoscopic tsme with preserved left colic and superior rectal arteries can be safely performed for rectal cancer patients as an alternative to tme 0czhang world of surgical oncology page of abbreviationstme total mesorectal excision tsme tumorspecific mesorectal excisionima inferior mesenteric artery imv inferior mesenteric vein sra superiorrectal artery nvb neurovascular bundle pme partial mesorectal excisionacknowledgementsnoneauthors™ contributionslz yx and xh designed the study cz yx hw qz zd and whcollected and analyzed the data ma lz ys and xh interpreted the datalz and cz drafted the manuscript lz ma yx and xh revised themanuscript the authors read and approved the final manuscriptfundingthis study was supported by the jiangsu natural science foundationbk20180274 this funding supported the collection analysis andinterpretation of the dataavailability of data and materialsall experimental data used to support these findings are included in theethics approval and consent to participatethis study was approved by the institutional review board of the firstaffiliated hospital of dalian medical university written informed consent forpublication was obtained from all patientsconsent for publicationwritten informed consent was obtained from the patients and legalguardian for the publication of these patientscompeting intereststhe authors declare that they have no conflicts of interestauthor details1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province china 2department ofgastrointestinal surgery the third affiliated hospital of guangxi medicaluniversity nanning guangxi province china 3department ofgastrointestinal surgery changzhi people™s hospital the affiliated hospital ofchangzhi medical college changzhi shanxi province china 4department ofcolorectal surgery the first affiliated hospital of nanjing medical universitynanjing china 5department of gastrointestinal surgery the first people™shospital of dali city dali yunnan province china 6department ofgastrointestinal surgery graduate school of medicine university of tokyotokyo japan 7department of general surgery yizhen people™s hospitalclinical medical college yangzhou university yangzhou jiangsu provincechinareceived february accepted august heald rj husband em ryall rd the mesorectum in rectal cancer surgerythe clue to pelvic recurrence br j surg “ macfarlane jk ryall rd heald rj mesorectal excision for rectal cancerlancet “lee ky factors influencing oncologic outcomes after tumorspecificmesorectal excision for rectal cancer j korean soc coloproctol “ williams ns dixon mf johnston d reapppraisal of the centimetre rule ofdistal excision for carcinoma of the rectum a study of distal intramuralspread and of patients™ survival br j durg “ pollett wg nicholls rj the relationship between the extent of distalclearance and survival and local recurrence rates after curative anteriorresection for carcinoma of the rectum ann surg “ wolmark n fisher b wieand hs the prognostic value of the modificationsof the dukes™ c class of colorectal cancer an analysis of the nsabp clinicaltrials ann surg “ monson jrt weiser mr buie wd chang gj rafferty jf prepared by thestandards practice task force of the american society of colon and rectalsurgeons practice parameters for the management of rectal cancerrevised dis colon rectum “law wl chu kw anterior resection for rectal cancer with mesorectalexcision a prospective evaluation of patients ann surg “kim sh bae kb kim jm oncologic outcomes and risk factors forrecurrence after tumorspecific mesorectal excision of rectal cancer cases j korean soc coloproctol “kim nk min bs kim js hur h lee ky sohn sk oncologic outcomesand safety after tumorspecific mesorectal excision for resectable rectalcancer a single institution™s experience with patients with rectalcancer j korean soc coloproctol “ zakir k mohamed wai lun law outcome of tumorspecific mesorectalexcision for rectal cancer the impact of laparoscopic resection world jsurg “kim nk baik sh seong js oncologic outcomes after neoadjuvantchemoradiation followed by curative resection with tumorspecificmesorectal excision for fixed locally advanced rectal cancer impact ofpostirradiated pathologic down staging on local recurrence and survivalann surg “ poon jt law wl laparoscopic resection for rectal cancer a review annsurg oncol “ yoo be kye bh kim hj early removal of the urinary catheter aftertotal or tumorspecific mesorectal excision for rectal cancer is safe discolon rectum “seiji o takashi t kazuki s a new laparoscopic surgical procedure toachieve sufficient mesorectal excision in upper rectal cancer int j surgoncol httpsdoi1011552011708439publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesenker w e thaler h t cranor m l polyak t total mesorectal excision inthe operative treatment of carcinoma of the rectum j am coll surg “lopezkostner i lavery c hool gr rybicki la fazio vw total mesorectalexcision is not necessary for cancer of the upper rectum surgery “zaheer s pemberton jh farouk r dozois rr wolff bg ilstrup d surgicaltreatment of adenocarcinoma of the rectum ann surg “sudeck p ueber die gefässversung des mastdarmes in hinsicht auf dieoperative gangrän muenchen med wschr “van tonder jj boon jm becker jhr anatomical considerations onsudeck™s critical point and its relevance to colorectal surgery clin anat“cirocchi r randolph j cheruiyot i systematic review and metaanalysis of the anatomical variants of the left colic artery color dis httpsdoi101111codi14891 0c"
Colon_Cancer
section all disclosure information for gie editors can befound online at httpwwwgie contentconflictofinterest cme editors and their disclosures are as followsprasad g iyer md associate editor for cmeconsultingadvisoryspeaking olympus research supporttakeda pharmaamit rastogi md associate editor for cmeconsultingadvisoryspeaking olympuskarthik ravi md cme editordisclosed no relevant financial relationshipswilliam ross md cme editorconsultingadvisoryspeaking boston scientific olympusara sahakian md cme editordisclosed no relevant financial relationshipsbrian weston md cme editordisclosed no relevant financial relationshipsall cme activities including their associated sare copyrighted by the asge gastrointestinal endoscopy volume no wwwgie 0ccme activitycontinuing medical education questions september question objectivedemonstrate triaging advanced gi endoscopy procedures during the covid19 pandemictriaging advanced gi endoscopy procedures during the covid19 pandemicquestion due to the resurgence of covid19 in your area healthofficials issue a mandate to cease elective proceduresbased on the findings of the current study a consensuswas reached that all of the following advanced gi procedures can be safely deferred for weeks exceptpossible answers aea ercp for asymptomatic choledocholithiasisb radiofrequency ablation for barrett™s esophagus withhighgrade dysplasiac endoscopic mucosal resection for barrett™s esophaguswith nodular highgrade dysplasiad endoscopic mucosal resection for a cm adenomatous colon polyp with highgrade dysplasiae eus for incident pancreatic and common bile duct dilation on ct or mri normal liver function testslookup sawhney ms bilal m pohl h triaging advanced gi endoscopy procedures during the covid19 pandemic consensus recommendationsusing the delphi method gastrointest endosc question objectivedescribe the learning curve for ablative therapy for dysplastic barrett™s esophagus and the effect of center volumes onoutcomeslearning curves and the ‚uence of procedural volume for the treatment of dysplastic barrett™sesophagusquestion a 53yearold man returns for surveillance endoscopy ofc3m5 nondysplastic barrett™s esophagus initially diagnosed years prior the patient has been compliant with twicedailyproton pump inhibitor therapy and denies any heartburnacid regurgitation or dysphagia he has no other significantmedical history you perform an egd which again reveals ac3m5 barrett™s segment without any nodularity biopsyspecimens are obtained and subsequently reveal multifocallowgrade dysplasia which is subsequently confirmed by anexpert gi pathologist after a subsequent discussionregarding ablative therapy with radiofrequency ablationrfa versus continued surveillance the patient wishes topursue ablation you discuss referral to a new colleague whojoined your practice several months earlier the colleague isthe first endoscopist to perform rfa at your institution andhas treated a total of patients the patient inquireswhether he would be more likely to have a successfuloutcome if he is referred to a moreexperienced endoscopistat a highvolume center which of the following is truepossible answers ada the patient is more likely to achieve complete remission of intestinal metaplasia crim if he undergoesablation at a center that has treated more than patientsb the patient is more likely to achieve complete remission of dysplasia crd if he undergoes ablation at acenter that has treated more than patientsc the patient is more likely to achieve crim if he undergoes ablation by an endoscopist who has treatedmore than patientsd the patient is more likely to achieve crd if he undergoes ablation by an endoscopist who has treatedmore than patientslookup lipman g markar s gupta a learning curves and the ‚uence of procedural volume for the treatment of dysplastic barrett™s esophagusgastrointest endosc wwwgie volume no gastrointestinal endoscopy 754e1 0ccme examquestion objectiveexplain the utility of a new gi bleeding prediction score for patients with upper gi bleedingthe horibe gi bleeding prediction score a simple score for triage decisionmaking in patients withsuspected upper gi bleedingquestion you are called to the emergency department to see apatient who has just arrived with the complaint of blacktarry stools the emergency department physician has notyet seen the patient but the nurse relays vital signs labresults and history sufficient to calculate a harbingerscore according to the study by horibe in thismonth™s issue this score is used to assess risk of which ofthe followingpossible answers ada deathb need for blood transfusionc need for endoscopic interventiond presence of highrisk stigmatalookup horibe m iwasaki e bazerbachi f horibe gi bleeding prediction score a simple score for triage decisionmaking in patients with suspectedupper gi bleeding gastrointest endosc question objectivereport the longterm efficacy and safety of eusguided hepaticogastrostomy for treatment of malignant biliary obstructionlongterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy inpatients with malignant biliary obstructionpossible answers ada success rate of eushgs is less than b adverse events occur in less than of casesc recurrent biliary obstruction occurs in about onethirdof patientsd prior biliary drainage has no impact on the likelihood ofrecurrent biliary obstructionquestion a 58yearold man is transferred to your hospitalformanagement of a pancreatic head mass causing obstructive jaundice and gastric outlet obstruction an endoscopic ultrasound with fineneedle aspiration of the massconfirmed pancreatic adenocarcinoma and a contrastenhanced computed tomography scan demonstratednumerous liver lesions compatible with metastatic disease a duodenal stent was placed which precluded anercp procedure for biliary drainage biliary drainage isnecessary before chemotherapy can be initiated variousoptions are discussed with the patientincluding eusguided hepaticogastrostomy eushgs according tothe current study by nakai which of the following isaccuratelookup nakai y sato t hakuta r longterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy in patients withmalignant biliary obstruction with video gastrointest endosc 754e2 gastrointestinal endoscopy volume no wwwgie 0ccme activitycontinuing medical education answers september question correct response erationale for correct responsethe covid19 pandemic has necessitated many elective procedures to be rescheduled in order to mitigate the spreadof infection and conserve vital resources choosing which advanced endoscopic procedures should be performed or safelydeferred during the covid19 pandemic may be a complex decision14 the aim of the current study was to provideguidance for triaging advanced endoscopic procedures using a modified delphi method the delphi method is a validatedand structured technique to obtain expert consensus which is useful in the present situation in which outcome data arelimited156 the following important patient outcomes were used avoidance of deathprolongation of life avoidance of cancercancer progression avoidance of major surgery andor hospitalization and improvement orpalliation of symptoms the following procedural timing categories were used timesensitive emergent schedulewithin week timesensitive urgent schedule within to weeks or nontime sensitive defer for weeks andthen reassess the timinga prespecified consensus threshold of was achieved in of advanced endoscopy indications reviewed by expert gastroenterologists one hundred percent consensus was achieved in of indications in of indications to consensus was achieved the only indication for which consensus could not be achieved was œincidentallyfound pancreatic duct dilation mm and common bile duct dilation mm on ct scan or mri with normal liverfunction teststhis study provides a decisionmaking framework for endoscopists to determine the timing for endoscopic proceduresduring and after the pandemic the decision to perform endoscopy during the covid19 pandemic needs to balance therisks associated with delaying the procedure in the individual patient with the risk of viral exposure to patients and healthcare providerstakehome message triaging advanced gi procedures during the covid19 pandemic may be facilitated by thecurrent consensus recommendations using the delphi method individual practice and patient factors must also beconsideredreferences sawhney ms bilal m pohl h triaging advanced gi endoscopy procedures during the covid19 pandemic consensus recommendations usingthe delphi method gastrointest endosc sultan s lim jk altayer o aga institute rapid recommendations for gastrointestinal procedures during the covid19 pandemic gastroenterology epub mar joint gi society message on covid19 available at httpsgi20200315jointgisocietymessageoncovid19 accessed march gastroenterology professional society guidance on endoscopic procedures during the covid19 pandemic available at httpswebfilesgilinksmediajoint_gi_society_guidance_on_endoscopic_procedure_during_covid19_final_impending_3312020pdf accessed april hsu cc sandford ba the delphi technique making sense of consensus pract assess res eval “ donohoe h stellefson m tennant b advantages and limitations of the edelphi technique implications for health education researchers am j healtheduc question correct response crationale for correct responseendoscopic eradication therapy for dysplastic barrett™s typically uses endoscopic mucosal resection of visible lesionswith subsequent ablation of the flat barrett™s segment with rfa this approach has demonstrated high clinical successwith estimates that crd is achieved in over and crim in over of treated patients1 however studies assessingthe learning curve associated with attaining proficiency with endoscopic therapy of dysplastic barrett™s and the effect ofcase volumes at centers on outcomes have yielded unclear results23 consequently current guidelines suggesting minimum numbers of cases to achieve competence for individual endoscopists or reflecting quality for centers are based onlimited evidence4wwwgie volume no gastrointestinal endoscopy 754e3 0ccme answersin this month™s issue of gie lipman and colleagues5 report results from a retrospective study examining patientstreated in the uk rfa registry to assess crd crim and dysplasia recurrence based on endoscopist experience andcenter case volumes5 a total of consecutive patients were identified from centers which were further divided into patients treated at lowvolume centers patients treated patients treated at mediumvolume centers patients treated and patients treated at highvolume centers patients treated rates of crd and crim at months did not differ based on center volumes however dysplasia recurrence washigher in patients treated at lowvolume centers further analysis using a riskadjustment cumulative risk sum curve wasdone to assess the effect of the learning curve on individual endoscopists a significant reduction of crd was seen at cases whereas a similar reduction of crim was seen at cases takehome message in conclusion this study suggests that the learning curve for rfa may be relatively short withfewer than cases required to achieve competency further center volume may have a very limited effect on outcomesfuture prospective studies are still required to assess the optimal number of training cases required to achieve endoscopicablative competencyreferences haidry rj dunn jm butt ma radiofrequency ablation and endoscopic mucosal resection for dysplastic barrett™s esophagus and early esophagealadenocarcinoma outcomes of the uk national rfa registry gastroenterology pasricha s cotton c hathorn ke effects of the learning curve on efficacy of radiofrequency ablation for barrett™s esophagus gastroenterology fudman di lightdale cj poneros jm positive correlation between endoscopist radiofrequency ablation volume and response rates in barrett™sesophagus gastrointest endosc fitzgerald rc di pietro m ragunath k british society of gastroenterology guidelines on the diagnosis and management of barrett™s oesophagusgut lipman g markar s gupta a learning curves and the ‚uence of procedural volume for the treatement of dysplastic barrett™s esophagus gastrointest endosc question correct response drationale for correct responsefor decades gastroenterologists have been searching for a straightforward method to determine whether a patientpresenting with an upper gastrointestinal bleed is at high risk of needing hospital admission therapeutic interventions ordeath multiple scoring systems have been proposed but systematic reviews have found them all flawed to various degrees12 however one of the betterknown scores proposed by blatchford has been recommended in recentguidelines to assess risk of requiring an intervention34 yet the quest for a more predictive model continues with somegroups turning to machine learning methods for an answer5in this month™s issue of gie horibe 6 report on a simple 3factor assessment to predict the likelihood of findinghighrisk stigmata hrs on endoscopy which they call harbinger because their median time to endoscopy was only hours the prevalence of hrs was quite high up to even in patients deemed to be low risk for stigmata predictablythe intervention rate was more than double that in a large international trial vs comparing harbinger toother scoring systems including blatchford™s the authors found their model to be superior this outperformance couldhave been predicted because the other models were designed to detect risk of other endpoints such as mortality or needfor intervention not hrsin addition the performance of the model was not consistently strong across the study institutions because auc valuesfor harbinger ranged from to between institutions with no overlap in confidence intervals between the topand low scores6 concerns about how harbinger would perform in other settings are reasonable as factors like access toppis varies across marketsin a resourceconstrained world the ability to predict need for interventions would seem more helpful than predictingendoscopic appearance although hrs are linked to the need for interventions the rapid time to endoscopy skews thelinkage by ballooning the prevalence of hrs many of these patients are likely to have far less ominous endoscopic appearances if the endoscopy was done hours after presentationreferences ramaekers r mukarram m smith cam the predictive value of preendoscopic risk scores to predict outcomes in emergency department patientswith upper gastrointestinal bleeding a systematic review acad emerg med de groot nl bosman jh siersema pd prediction scores in gastrointestinal bleeding a systematic review and quantitative analysis endoscopy blatchford o murray wr blatchford m a risk score to predict need for treatment for uppergastrointestinal hemorrhage lancet barkun an almadi m kuipers ej management of nonvariceal upper gastrointestinal bleeding guideline recommendations from the internationalconsensus group ann int med 754e4 gastrointestinal endoscopy volume no wwwgie 0ccme answers shung dl au b taylor ra validation of a machine learning model that outperforms clinical risk scoring systems for upper gastrointestinalbleeding gastroenterology horibe m iwasaki e bazerbachi f horibe gi bleeding prediction score a simple score for triage decisionmaking in patients with suspected uppergi bleeding gastrointest endosc stanley aj laine l dalton hr comparison or risk scoring systems for patients presenting with upper gastrointestinal bleeding international multicenter prospective study bmj 2017356i6432question correct response crationale for correct responsebiliary drainage options may be limited by gastric outlet obstruction or surgically altered anatomy eusguided hepaticogastrostomy eushgs provides an effective alternative to ercp and percutaneous drainage in these complex cases1additionally eushgs has been compared to ercp for primary biliary drainage and outcomes were found to be comparable2 however potential adverse events of eushgs such as stent migration and peritonitis can be severe and longterm data thus far have been lacking3in the current issue of gie nakai and colleagues4 present a retrospective study of patients who underwent eushgs for drainage of malignant biliary obstruction partially covered metallic stents ranging in length from to cm wereused to create the hepaticogastrostomy technical and functional success rates were and respectively adverseevents occurred in of patients with the most common being fever and abdominal pain peritonitis occurred in andcholangitis in of patients no cases of stent dislodgement were reported recurrent biliary obstruction rbo occurredin of patients in a median time of months short length of the intragastric portion of the stent and prior biliarydrainage were both associated with recurrent biliary obstruction of the patients who had rbo underwent successfulreintervention with eushgstakehome message eusguided hepaticogastrostomy is an effective alternative for longterm biliary drainage recurrences can be successfully managed with repeat hepaticogastrostomyreferences nakai y isayama h yamamoto n safety and effectiveness of a long partially covered metal stent for endoscopic ultrasoundguided hepaticogastrostomy in patients with malignant biliary obstruction endoscopy paik wh lee th park dh eusguided biliary drainage versus ercp for the primary palliation of malignant biliary obstruction a multicenter randomized clinical trial am j gastroenterol giovannini m eusguided hepaticogastrostomy endosc ultrasound 20198s35s9 nakai y sato t hakuta r longterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy in patients with malignant biliary obstruction with video gastrointest endosc wwwgie volume no gastrointestinal endoscopy 754e5 0c'
Colon_Cancer
" lung carcinoma is a prominent cause of mortality among patients with cancer previous studies have reported the vital role of long noncoding rnas lncrnas in the malignant progression of lung cancer lncrna rp11284f219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown therefore the present study aimed to elucidate the role of lncrna rp11284f219 in lung carcinoma progression the expression of rp11‘284f219 in lung cell lines and tissues was measured using reverse transcription‘quantitative pcr the endogenous expression of rp11284f219 was silenced using rna interference and cell viabilities were measured with a cell counting kit‘ assay the invasion and apoptosis of cells were determined via transwell assays and flow cytometry respectively the protein expression levels were measured by western blotting an increased expression of rp11‘284f219 was identified in both lung carcinoma tissues and cells knockdown of rp11‘284f219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis the present study identified the existence of a direct interaction between rp11‘284f219 and microrna mirnamir6273p mechanistically it was demonstrated that rp11284f219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of mir6273p furthermore cell division cycle and apoptosis regulator ccar1 was identified as a target gene of mir6273p the in vivo tumor growth assay also demonstrated that the knockdown of rp11‘284f219 suppressed tumor growth upregulated mir6273p and downregulated correspondence to dr yuan wang department of medical imaging the first affiliated hospital of xi'an jiaotong university west yanta road xi'an shaanxi pr chinaemail wangyuan8003126comabbreviations ccar1 cell division cycle and apoptosis regulator nsclc non‘small cell lung cancer sclc small cell lung cancerkey words rp11284f219 lung carcinoma proliferation invasion microrna6273p ccarccar1 in the xenograft model of nude mice thus the present findings indicated the tumor promoting functions of rp11284f219 in the progression of lung carcinoma and provided a novel lncrnamirna axis as a target for the management of lung cancerintroductionpulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women furthermore of lung cancer cases are categorized as nonsmall cell lung cancer nsclc while the remaining are classified as sclc although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentiallong noncoding rnas lncrnas are nucleotides in length and have little or no protein coding capacity the mechanisms via which lncrnas regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small rnas generating different splice variants via regulating mrna splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes in recent years previous studies have reported that various human cancer types exhibit lncrnas dysfunction and these lncrnas are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells in lung cancer lncrna metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncrna hox transcript antisense rna is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with nsclc furthermore studies have shown that the expression of lncrna urothelial carcinoma‘associated 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionis significantly upregulated in nsclc and may induce resistance to treatment of egfr‘tyrosine kinase inhibitors by activating the aktmtor pathway lncrna rp11284f219 was primarily discovered in a pancancer transcriptomic analysis lncrna rp11‘284f219 exists as a cluster of three annotated lncrnas rp11284f219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas however the specific function and the underlying mechanism of rp11284f219 in lung carcinoma remain unknownto the best of our knowledge the present study demonstrated for the first time that lncrna rp11284f219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer together with microrna mirnamir6273p and cell division cycle and apoptosis regulator ccar1 the regulatory axis of rp11‘284f219mir‘‘3pccar1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo the present study aimed to investigate rp11284f219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the rp11‘284f219mir‘‘3pccar1 axismaterials and methodstissue samples and cell lines between may and jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range ‘ years nine male patients four female patients who were diagnosed and treated in first affiliated hospital of xi'an jiaotong university the samples were dissected during the surgery and immediately flash‘frozen in liquid nitrogen and transferred to ‘Ëšc storage for further extraction of both rna and protein all the tissue samples were obtained with written informed consent from the patients the protocol was approved by the first affiliated hospital of xi'an jiaotong university approval no a normal lung epithelial cell line beas2b and lung carcinoma cell lines ncih460 ncih1299 and a549 were purchased from american type culture collection atcc and cultured according to the atcc guidelines 293t cells were purchased from procell life sciencetechnology co ltd and cultured in dmem supplemented with fbs cat no ‘ atcc and 1x penicillin‘streptomycin thermo fisher scientific inc beas2b cells were cultured in bronchial epithelial growth medium begm cat no cc‘ clonetics corporation according to the manufacturer's instructions nci‘h460 and nci‘h1299 cells were cultured in rpmi‘ medium cat no ‘ atcc and a549 cells in f12k medium cat no ‘ atcc supplemented with fbs cat no atcc and 1x penicillin‘streptomycin thermo fisher scientific inc all cells were culture at ˚c with co2rna extraction and reverse transcription‘quantitative pcr rt‘qpcr total rna from both tissue samples and cell lines were extracted using trizol® reagent invitrogen thermo fisher scientific inc for each sample ng total rna was reverse transcribed to synthesize the first‘strand cdna using the primescript rt reagent kit takara bio inccdna samples were diluted times to perform the rt‘qpcr using sybr premix ex taq takara bio inc on a cfx96 realtime pcr detection system biorad laboratories inc expression levels of mrnas lncrnas and mirnas were normalized to gapdh the primers used for rtqpcr analyses were as follows gapdh forward '‘aac gac ccc ttc att gac c‘' and reverse '‘tcc acg aca tac tca gca cc‘' rp11‘284f219 forward '‘agg att ggc act cac ttc gg‘' and reverse '‘tct ctc acc acg tct ggt ct‘' and ccar1 forward '‘ctg atg gct agc cct agt atg ga‘' and reverse '‘tgc ctt tca tgc cca cta aaa ‘' the temperature protocol used to perform rt was ˚c for h followed by ˚c for min thermal conditions of pcr reactions were initial denaturation at ˚c for min followed by cycles for sec at ˚c and sec at ˚c the mrna expression levels were determined using the 2δδcq method oligonucleotides and cell transfection the small interfering rna sirna synthetic negative control sinc rp11284f219 sirnas sirp11284f219 mirnc mir‘‘3p mimics and mir‘‘3p inhibitor were purchased from shanghai genepharma co ltdall primer sequence information is presented in table i at a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency all of the oligonucleotides were transfected at a final concentration of nm using lipofectamine® reagent invitrogen thermo fisher scientific inc according to the manufacturer's instruction cells were collected at h posttransfection for subsequent experimentscell counting kit cck‘ assay and edu labeling of prolif‘erating cells a cck‘ was used for cell proliferation assay the cells were seeded into ‘well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions dojindo molecular technologies inc the optical density was measured at nm using a spectrophotometer thermo fisher scientific incfor the edu assay cells were incubated with µm edu cat no ab219801 abcam for h at ˚c and fixed with formaldehyde at room temperature for min after a brief washing with pbs click reagent was added into each well and incubated in the dark for min at room temperature followed by pbs washing the cells were stained with µgml dapi at room temperature for min images were captured using a fluorescence microscope nikon corporation and measured using adobe photoshop software adobe systems inc the edu labeled cells were analyzed with moflo astrios beckman‘coulter inc magnification x200transwell assay and flow cytometry measurement of cell apoptosis transwell assays were performed with a coating of matrigel bd biosciences mixed with culture medium mixed at ratio at ˚c for h a total of 1x105 cells in µl serum‘free medium were added to the upper layer of the transwell chambers µm pore size corning inc and cultured for h the lower chamber contained the culture medium with fbs the migrated cells were fixed with 0concology reports table i sequence of sirnas and mirna mimics and inhibitorsoligonucleotides si‘nc si‘rp11‘284f219 mir‘nc mir‘‘3p mimics mir‘‘3p inhibitor mir microrna sirna small interfering rna nc negative controlsequence '†’'uucuccgaacgugucacguttuauuggcaccaaggauagcucguuaaucggcuauaauacgcucuuuucuuugagacucacuucuuuucuuugagacucacu paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope magnification x200cellular apoptosis was detected using the apoptosis detection kit cat no kgf001 nanjing keygen biotech co ltd according to the manufacturer's instructions cells were stained with fluorescein isothiocyanateconjugated annexin v and pi after incubated for min at ˚c in the dark µl 1x binding buffer was added to each tube and stained cells were analyzed using bd facs canto ii flow cytometry facs calibur bd biosciences data were analyzed using flowjo software version tree star incluciferase reporter assay the rp11284f219 wildtype wt or mutant mut '‘untranslated region '‘utr and ccar1 wt or mut '‘utr sequences were cloned into the pmirglo plasmid youbio httpwwwyoubiocn cat no vt1439 the vectors µgml were co‘transfected with mir‘nc or mir6273p mimic nm and renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using lipofectamine® reagent invitrogen thermo fisher scientific inc cell lysates were collected at h after transfection and the luciferase activities were detected with the dualluciferase reporter assay system promega corporation according to the manufacturer's instructionswestern blotting cell were lysed using ripa lysis buffer sigma‘aldrich merck kgaa and protein concentrations were assessed with the bca protein assay kit according to the manufacturer's instructions beyotime institute of biotechnology shanghai china equal amounts µg of cell protein lysates were loaded and separated by sds‘page transferred to a pvdf membrane and blocked with non‘fat milk at room temperature for h the membranes were then incubated with ccar1 primary antibody cat no ab70243 abcam overnight at ˚c followed by incubation with goat anti‘mouse or goat anti‘rabbit igg‘horseradish peroxidase conjugate secondary antibodies cat no ab205718 abcam at room temperature for h gapdh cat no ab181602 abcam was used as loading control the signals were detected using the ecl system protein simple according to the manufacturer's instructionsin vivo tumorigenicity analysis in mice male balbc nude mice age weeks weight ‘ g were obtained from beijing vital river laboratory animal technology co ltd and housed at a room temperature of ˚c with a h lightdark cycle the mice were maintained in an individually ventilated cage system under specific pathogen‘free conditions temperature ˚c humidity and fed with sterile food and water free access to evaluate the effect of rp11‘284f219 knockdown on the growth of lung carcinoma in vivo 5x106 sinc or sirp11284f219 treated nci‘h1299 cells in µl serum‘free medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin the animals were monitored daily and the following criteria for humane endpoint was used severe tumor burden mm in diameter difficulty breathing significant body‘weight loss and clinical signs such as prostration hypothermia and significant abdominal distension tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] then weeks after inoculation the mice were euthanized by co2 inhalation co2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat the xenografts were imaged and weighedthe total rna was then extracted from the xenografts as aforementioned animal care and study were approved by the institutional animal care and use committee of the first affiliated hospital of xi'an jiaotong university approval no target prediction potential target mirnas of rp11284f219 were predicted using lncbase v2 httpcarolinaimisathena‘ innovationgrdiana_toolswebindexphprlncbasev2index the target genes of mir‘‘3p were predicted using three bioinformatics algorithms targetscanv72 httpwwwtargetscanorgvert_72 and mirdb httpwwwmirdborgmininghtmlstatistics analysis data were analyzed using the graphpad prism software graphpad software inc and presented as the mean ± sd from ‰¥ independent experiments a two‘tailed unpaired student's t‘test or one‘way anova with tukey's post‘hoc analysis were performed to evaluate the statistical significance p005 was considered to indicate a statistically significant difference 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure rp11‘284f219 expression is upregulated in lc tissues and cell lines a expression of rp11‘284f219 in lc tissues in comparison with adjacent healthy tissues was analyzed using rtqpcr p0001 vs adjacent tissues n13 b expression of rp11‘284f219 in human lung carcinoma cell lines ncih460 ncih1299 and a549 compared with normal human lung epithelial cell line beas2b was analyzed using rtqpcr p005 p0001 vs beas‘2b n3 lc lung carcinoma rt‘qpcr reverse transcription‘quantitative pcrresultsexpression of rp11‘284f219 is upregulated in lung carci‘noma to investigate the potential role of rp11284f219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma the results demonstrated that the expression of rp11‘284f219 was significantly upregulated in tumor tissues compared with healthy tissues fig 1a the expression of rp11‘284f219 was also analyzed in human lung carcinoma cell lines ncih460 ncih1299 and a549 and normal human lung epithelial cell line beas2b consistent with the findings in the tissue samples the expression of rp11‘284f219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line fig 1b these results indicated that rp11284f219 may serve an oncogenic role in lung carcinomaknockdown of rp11‘284f219 exerts anti‘oncogenic effects in lung carcinoma cells to study the specific role of rp11284f219 in lung carcinoma cells rp11284f219 sirna was transfected into ncih1299 and ncih460 cells fig 2a after transfection the proliferation of these cells was measured using cck‘ and edu assays fig 2b‘d the results suggested that knocking down rp11‘284f219 significantly reduced the proliferation of lung carcinoma cells compared with the nc group fig 2bd the invasiveness of si‘rp11‘284f219 transfected cells also significantly decreased as indicated by the data from the transwell assay fig 2f to further validate the invasive capability a rt‘qpcr assay was performed to detect the expression levels of invasion‘related genes and the results identified that both mmp2 and mmp9 were significantly decreased when rp11‘284f219 was downregulated fig s1the results of flow cytometry measurement based apoptosis assay suggested that cells transfected with sirp11284f219 had a higher apoptotic rate compared with the sinc transfected group fig 2e these data demonstrated the antitumor effects of rp11‘284f219 knockdown in lung carcinoma cells indicating an oncogenic role of rp11284f219rp11‘284f219 directly interacts with mir‘‘3p based on the prediction of the online tool lncbase v2 from diana prediction module httpcarolinaimisathena‘innovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream mirnas of rp11284f219 the first five mirnas in the output list were tested among the predicted potential targets it was found that mir6273p had the most significant upregulation in ncih1299 cells transfected with sirp11284f219 fig s2using sequence alignment it was identified that mir‘‘3p was partially complementary with the '‘utr of rp11‘284f219 fig 3a subsequently 293t cells were transfected with the pmirglorp11284f219wt or mut vector containing the wt or mut sequence of rp11284f219 '‘utr with or without mir‘‘3p mimics results from the luciferase reporter assay suggested that mir6273p mimics significantly decrease the signal of rp11‘284f219‘wt transfected cells but not the rp11‘284f219‘mut transfected cells indicating a direct interaction between the two non‘coding rnas fig 3a furthermore transfection of sirp11284f219 into ncih1299 and ncih460 cells resulted in the suppression of endogenous rp11‘284f219 leading to a significant increase in mir‘‘3p expression fig 3b thus these findings suggested an inhibitory effect of rp11‘284f219 on the expression of mir‘‘3p in lung carcinoma cellsthe expression of mir‘‘3p was detected in both lung carcinoma tissues and cell lines it was demonstrated that mir‘‘3p was significantly downregulated in carcinoma tissues fig 3c and ncih460 ncih1299 and a549 cells fig 3d compared with healthy tissues and cells collectively these data suggested a direct interaction between rp11284f219 and mir6273p in which rp11284f219 suppresses the expression of mir‘‘3prp11‘284f219 regulates the proliferation and invasiveness of lung carcinoma cells via mir‘‘3p to rescue the antitumor effects of sirp11284f219 in lung carcinoma cells the mir‘‘3p inhibitor which specifically downregulates the expression of mir‘‘3p was transfected into nci‘h1299 and nci‘h460 cells fig 4a the results from the cck‘ and edu assays demonstrated that treatment with si‘rp11‘284f219 0concology reports figure rp11‘284f219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis a rp11‘284f219 knockdown was achieved via rp11‘284f219 sirna and the knockdown efficiency was verified using reverse transcription‘quantitative pcr n3 cell counting kit‘ assay was performed to measure the proliferation of b ncih1299 and c ncih460 cells after transfection with sirp11284f219 compared with the si‘nc group n5 d an edu assay was performed to measure the proliferation of nci‘h1299 and nci‘h460 cells after transfection with si‘nc and si‘rp11‘284f219 magnification x200 e flow cytometry analysis was performed to determine the effects of rp11‘284f219 knockdown on apoptotic rates in nci‘h1299 and nci‘h460 cells n3 f transwell assay was performed to determine the effects of rp11‘284f219 knockdown on nci‘h1299 and nci‘h460 cell invasion n3 magnification x200 p005 p001 vs control group nc negative control sirna small interfering rna od optical density and mir‘nc significantly decrease the proliferation of both ncih1299 and ncih460 cells fig 4bd however the administration of mir‘‘3p inhibitor partially reversed the antiproliferative effect of sirp11284f219 indicating that rp11284f219 regulates the proliferation of lung carcinoma cells partially via mir6273p fig 4bd in addition the 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure rp11‘284f219 directly interacts with mir‘‘3p a binding site between rp11‘284f219 and mir‘‘3p that was identified using the diana tools and a luciferase reporter assay was conducted in pmirglorp11284f219wt or mut treated cells in the presence of mir6273p mimics or mir‘nc n3 p005 vs mir‘nc b expression of mir‘‘3p in nci‘h1299 and nci‘h460 cells transfected with si‘rp11‘284f219 was analyzed using rtqpcr p001 vs si‘nc n3 mir‘‘3p expression in c lc tissues and d nci‘h460 nci‘h1299 and a549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using rt‘qpcr n3 p005 p001 vs adjacent tissue or beas‘2b cells nc negative control sirna small interfering rna wt wild‘type mut mutant mir microrna lc lung carcinoma mir‘‘3p inhibitor restored the reduction in the number of ncih1299 and ncih460 cells that migrated through the transwell membrane induced by si‘rp11‘284f219 treatment fig 4f these data indicated the participation of mir6273p in the rp11284f219mediated invasive effectthe qpcr assay results identified that both mmp2 and mmp9 expression levels were restored in rp11284f219downregulated cells when mir6273p was inhibited compared with the mir‘nc group fig s3 in addition transfection with mir‘‘3p inhibitor also diminished the pro‘apoptosis effect of si‘rp11‘284f219 in both ncih1299 and ncih460 cells fig 4e therefore it was suggested that rp11284f219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of mir‘‘3prp11‘284f219 regulates ccar1 via targeting mir‘‘3p to further evaluate how rp11‘284f219 exerts an oncogenic role via mir‘‘3p the publicly available algorithms of targetscan httpwwwtargetscanorg and mirdb were used which identified ccar1 as a potential target for mir6273p fig 5a in order to validate this prediction mir6273p mimic was transfected into cells and the transfection efficiency was assessed the results demonstrated that transfection of mir6273p mimic increased the expression of mir‘‘3p by times compared with cells transfected with mirnc fig s4after validating the upregulation of mir6273p mimic a ccar1wt vector was constructed which contained the wt binding site between mir‘‘3p and the ccar1 '‘utr and ccar1mut vector containing the mut sequence fig 5a the results from luciferase reporter assays indicated that compared with the mirnc group the mir6273p mimic significantly decreased the luciferase activity of ccar1‘wt treated cells but not the ccar1‘mut treated cells suggesting a direct binding of mir‘‘3p to the '‘utr of ccar1 fig 5b increased expression levels of ccar1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues fig 5c moreover a significant decrease in both mrna and protein expression levels of ccar1 was detected upon transfecting ncih1299 and ncih460 cells with mir6273p mimics fig 5d and e thus ccar1 may be a direct target of mir‘‘3p in lung carcinoma cells and tissuesrp11‘284f219 knockdown inhibits tumor growth and the expression of ccar1 in vivo in order to investigate the effect of rp11284f219 on in vivo tumorigenicity ncih1299 cells were transfected with sinc or sirp11284f219 and injected into the nude mice after weeks a significantly 0concology reports figure rp11‘284f219 regulates proliferation and invasiveness in lung carcinoma cells via mir‘‘3p a expression of mir‘‘3p in nci‘h1299 and nci‘h460 cells transfected with mir‘nc or mir‘‘3p inhibitor was detected using rt‘qpcr analysis n3 p005 vs mir‘nc cell counting kit‘ assay was performed in b nci‘h1299 and c nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor n5 d edu assay was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor magnification x200 e flow cytometry analysis was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor n3 f transwell assay was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor magnification x200 n3 p005 vs si‘nc nc negative control sirna small interfering rna od optical density mir microrna 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure mir‘‘3p directly targets ccar1 a bioinformatic analysis of the predicted binding sites between the ccar1 '‘untranslated region and mir‘‘3p b luciferase reporter assay in ccar1‘wt or ccar1‘mut treated cells in the presence of mir‘nc or mir‘‘3p mimic n3 p005 vs mir‘nc c ccar1 expression in lc tissues compared with adjacent healthy tissues was analyzed using rt‘qpcr n13 p001 vs adjacent tissue expression of ccar1 in nci‘h1299 and nci‘h460 cells transfected with mir‘nc or mir‘‘3p mimics was detected using d rt‘qpcr and e western blotting n3 p005 vs mir‘nc mir microrna nc negative control wt wild‘type mut mutant rt‘qpcr reverse transcription‘quantitative pcr ccar1 cell division cycle and apoptosis regulator lc lung carcinoma slower proliferative rate of the tumors was observed in the sirp11284f219 group compared with the sinc group fig 6a and b furthermore the tumor volume and weight were significantly decreased in the si‘rp11‘284f219 group compared with the control group fig 6a and b rtqpcr analysis also demonstrated that compared with the sinc group the tumors in the si‘rp11‘284f219 group expressed higher levels of mir6273p fig 6c and lower levels of ccar1 fig 6d providing further evidence to the existence of the rp11‘284f219mir‘‘3pccar1 regulatory axis in lung carcinoma tumor tissuesdiscussionthe present study investigated the function of rp11284f219 in lung carcinoma it was initially found that rp11284f219 was significantly upregulated in both lung cancer tissues and cell lines following the deduction of a potential oncogenic role of this lncrna sirp11284f219 was transfected into ncih460 and ncih1299 cells and it was demonstrated that knockdown of rp11‘284f219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells in the mechanistic studies using online prediction tools and in vitro assays the results indicated that mir‘‘3p directly interacts with rp11‘284f219 by binding to its '‘utrthe function of mir627 was initially reported in colorectal cancer crc padi found that when upregulated by calcitriol mir627 targets the histone demethylase jumonji domain containing 1a to increase methylation of histone h3k9 and suppresses the proliferative factors of crc cells thus inhibiting the proliferation of crc both in vitro and in vivo moreover in crc sun discovered the role of mir‘ in vitamin d‘enhanced efficacy of irinotecan via inhibition of the cytochrome p450 enzyme‘mediated intratumoral drug metabolism mir‘ is also reported to be a potential non‘invasive diagnostic marker in gastric and breast cancer types in pulmonary diseases mir627 is downregulated in patients with chronic obstructive pulmonary disease and targets the high‘mobility group box protein to inhibit its expression thus improving transforming growth factorβ1‘induced pulmonary fibrosis the present results demonstrated the inhibitory effect of rp11‘284f219 on the expression of mir‘‘3p in addition it was identified that the mir‘‘3p inhibitor can neutralize the anti‘tumor effects of rp11‘284f219 knockdown indicating that rp11‘284f219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating mir‘‘3p this anti‘tumor role of mir6273p under the regulation of rp11284f219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human crc gastric and breast cancer types 0concology reports figure rp11‘284f219 knockdown inhibits tumor growth in vivo a macroscopic image of xenografted tumors b tumor volume in nude mice injected with nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 measured over weeks n5 c weight of tumors in nude mice at weeks after injection of nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 n5 expression levels of d mir‘‘3p and e ccar1 in the tumor tissues from nude mice injected with nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 for weeks were detected using reverse transcription‘quantitative pcr n5 p005 p001 p0001 vs si‘nc mir microrna nc negative control sh short hairpin rna ccar1 cell division cycle and apoptosis regulator using the publicly available rna interaction prediction algorithms the current study identified that ccar1 which was initially shown as the target gene of mir6273p is also regulated by rp11‘284f219 furthermore the regulatory axis of rp11‘284f219mir‘‘3pccar1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model the interaction between rp11‘284f219 and mir‘‘3p and the interaction between mir‘‘3p and ccar1 were demonstrated by the dual‘luciferase assay although this method has been used to validate rna‘rna interactions in previous studies other assays such as rna pulldown and rna binding protein immunoprecipitation that would provide more direct evidence for the rnarna and rna‘protein interactions should be performedccar1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as adriamycin and etoposide subsequently kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors in human breast cancer cells as ccar1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells in crc cells ou reported that ccar1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor ccar1 is essential for the expression of wnt target genes as well as the neoplastic transformation of crc cells in gastric cancer cells researchers have revealed the cooperation between ccar1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells in lung cancer ccar1 was reported to be an effector of doxorubicin‘induced apoptosis moreover muthu demonstrated that certain chemical compounds that bind with ccar1 can increase the expression of ccar1 and induce apoptosis however a contradictory conclusion was reported in a recent study which observed that ccar1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate corroborating this finding in the current study via the targeting of mir‘‘3p the expr
Colon_Cancer
ulcerative colitis is a type of ‚ammatory bowel disease thatcan potentially lead to cancer e age of onset of colitis istypically “ years old and it can seriously threaten thequality of life of patients e immunopathogenesis andimmunosuppressive treatment of colitis are currently theresearch topics of significant interest e research goals areto diagnose and treat colitis in order to prevent exacerbationof the disease e drugs used to treat colitis in the clinicoften have adverse eï¬ects after their longterm applicationanother crucial area of colitis research is focused on thediscovery of functional foods that can prevent colitis withoutside eï¬ects natural plants including aegle marmeloslinn also have the intervention eï¬ect on colitis a recentstudy has shown that the intestinal flora is closely related tocolitis and that the intestinal flora participates in the mucosalimmune response bacteria are an important promoter of‚ammatory bowel disease e symptoms of colitis can bealleviated by regulating the intestinal flora preventing floralimbalance and increasing the number of probiotics yak yoghurt is a natural fermented food that is rich innutrients and is common in the minority areas of theqinghai tibet plateau previous research has suggested thatyak yoghurt exerts various physiological activities such asantioxidationimmunitycholesterolreductionand 0cevidencebased complementary and alternative medicineenhancement e qinghai tibet plateau has a specificclimate and unique environment for the fermentation of yakyoghurt additionally the availability of yak milk and specialtibetan fermentation utensils eg certain fermentationmicroanisms can make the flavor and quality of yakyoghurt diï¬er greatly from ordinary fermented milk aprior study on the intestinal physiological activity of lacticacid bacterial species in yak yoghurt showed that the lacticacidproducing bacteria isolated from yak yoghurt hadantioxidant and constipation preventing eï¬ects in this studythe potential eï¬ects of lactobacillusplantarum ys4 lpys4 on oxazolidoneinduced colitiswere investigated for the first time e findings provide apossible foundation for further development of lpys4especially its application in functional food or medicineratio � solution massnormal group were daub treated with ml of ethanolwith those in the remaining four groups being daub treated with ml of oxazolidonesolvent � after treatment for days the mice wereanesthetized en the blunt head of a silicone tube wasinserted into the intestinal tract from the anus of the mouse at adepth of cm e mice in normal group were administeredwith ml of ethanol solution while those in theremaining four groups were administered with ml of oxazolidone solution mass ratio � solvent � ethanoltwenty seconds later the catheters were removed and the micewere lifted up by their tails for half a minute on the last dayof treatment day all the mice were sacrificed by decapitation and their plasma samples and colon tissues were collected e length and weight of the colon were documentedexperiment animal materials materials and methodsand reagentsexperimental strain the strain was isolated from yak yoghurt in the yushu area of qinghai province china by ourteam it was named lpys4 and stored in china center fortype culture collection cctcc wuhan china nom2016750 e negative control strain lb was purchasedfrom the cctcc no ab fifty male balbc mice weeks old were purchasedfrom the experimental animal center of chongqingmedical university certificate no syxk yu “oxazolone was purchased from sigmaaldrich co llcusa il2 il10 et1 sp ss and vip serum cytokine kitswere purchased from biolegend inc usa gsh sodmpo and mda kits were purchased from nanjing jiancheng bioengineering institute nanjing china trizol reagent oligodt18 rnase dntp mlv primer bca proteinquantitative kit aps temed sdspage pvdf membrane first antibody and second antibody were purchasedfrom ermo fisher scientific inc usa instruments and equipment imark microplate readerwas purchased from biorad usa steponeplus pcrinstrument was purchased from ermo fisher scientificinc usa tanon chemiluminescence imager waspurchased from tanon science and technology co ltdchina sas v91 statistical software package was purchasedfrom sas institute inc usalb animal grouping and intervention a total of balbcmale mice were assigned to five groups model groupnormal group lactobacillus bulgaricustreatmentgroup and highdose lpys4h and lowdoses lpys4l treatment groups and there were mice in each groupe mice in lb lpys4h and lpys4l groups were fedwith — — and — cfukg ml livingbacteria physiological saline of each corresponding straindaily by oral gavage for consecutive days and normal andmodel groups were fed with ml physiological salineafter days of treatment the abdomens of all mice wereshaved with an area of cm — cm e mouse abdomens in detection of endothelin1 et1 substance p spsomatostatin ss and vasoactive intestinal peptide vipconcentrations in serum samples e whole blood samplesof mice were allowed to clot at room temperature for h andthen centrifuged at rpmmin for min after collecting the serum samples the concentrations of et1 sssp and vip were detected using commercial kits determination of interleukin2 il2 and interleukin10il10 levels in serum samples e mouse serum sampleswere prepared according to section en the serumlevels of il2 and il10 cytokines were assessed usingcommercial kits determination of glutathione gsh malondialdehydemda myeloperoxidase mpo and superoxide dismutasesod activities in colon tissues a mixture of g colontissue and ml normal saline was prepared at weightratio after homogenizing the mixture the activities of gshmda mpo and sod in colon tissues were evaluated usingcommercial kits pathological observation of he staining e lesionsite — — cm of colon was cut with a scalpel etrimmed tissue and corresponding label were placed in neutral formalin solution for h e colon tissue wasdehydrated embedded sliced dewaxed stained and thendehydrated transparent and sealed finally the pathologicalstate of colon tissue was observed under a microscopebx43 olympus tokyo japan qpcr assay total rna was isolated using rnazol andthen diluted to the final concentration of μgμl for cnasynthesis μl of the diluted rna extract was taken andcdna was prepared using a reverse transcriptase kit enthe cdna template μl was added into μl of sybrgreen pcr master mix and μl of forward primer andreverse primer each table qpcr amplification wascarried out for cycles under the reaction conditions of95oc s °c s °c s and °c s followed by 0cevidencebased complementary and alternative medicineckitinosenosnnossequencegene nametable sequences of primers used in this studyforward ²agagagatcgggttcaca3²reverse ²cacagaactgagggtaca3²forward ²tcgtccaacttctgggctctt3²reverse ²ccttctcttcctcccctctcttc3²forward ²tcagccatcacagtgttccc3²reverse ²atagcccgcatagcgtatcag3²forward ²catagcccaggtaaagcacaat3²reverse ²gaacactccagaatcgtcaactcforward ²tcagggactacgctgcgaaag3²reverse ²aagagctggcagaccgactca3²forward ²tgcaccaccaactgcttag3²reverse ²gatgcagggatgatgttc3²Î´ctdetection gene δctgapdh measured according to the following equation ˆ’δct �cycle under the reaction conditions of °c s and°c s gapdh was used as internal control for thisdetermination and the relative mrna expression was²gapdhscf western blotting mg of tissue samples was mixedwith μl of pmsf and ml of ripa and then homogenized at rmin 4oc for min protein quantificationwas conducted using the bca protein quantitative kit andthe protein samples were diluted to μgml en thediluted protein and sample buï¬er were mixed at heatedat 100oc for min and icebathed for min subsequentlyacrylamide starting buï¬er resolving buï¬er temed aps and diï¬erentiated water were mixed in specific proportions in order to prepare sdspage separation glue andconcentration glue e prestained samples and proteinladder were placed into the sample hole of the rubber sheetrespectively and then the proteincontaining sdspageglue was subjected to vertical gel electrophoresis for minafter activation with methanol for min the pvdf wereblocked with skimmed milk in — tbst solution for hen the blocked pvdf membranes were rinsed with — tbst followed by incubation with the primary antibodyat °c for h after washing with — tbst for times thesecondary antibody was incubated at °c for h lastly theprotein bands were visualized by supersignal west picoplus chemiluminescent substrate and the images werecaptured using a chemiluminescence imager multiple comparisons were conducted using onewayanova followed by tukey™s test statistical analysis e average value of three experimental results was determined and the statistical softwaresas was used to analyze whether there was a significantdiï¬erence between each group at the level of p longest in normal group ± cm while being the results eï¬ect of lpys4 on colon parameters e experimentalresults demonstrated that the length of mouse colon was thepared to those in the remaining four groups in contrast thethe ratio of colon weightlength was the highest in normalpgml and the lowest concentrations were found for sslpys4h group and these eï¬ects were better than those of eï¬ect of lpys4 on the serum contents of et1 sp ss andvip in mice it can be seen from figure that the serumml was decreased in normal mice compared to that of theremaining four groups e colitis model mice exhibited theopposite results in which the highest concentrations wereserum concentrations of ss and vip in colitis mice andmarkedly decrease those of et1 and sp more importantlythe eï¬ects were better after treatment with lpys4hshortest in the colitis model group ± cm similarlygroup ± while being the lowest in the colitis modelgroup ± figure it was found that lpys4 couldsignificantly p attenuate the decline in colon lengthand weightlength ratio induced by colitis ± cm and± for lpys4l group ± cm and ± forlb ± cm and ± concentrations of ss ± pgml and vip± pgml in normal mice were increased comlevel of et1 ± pgml and sp ± pgobserved for et1 ± pgml and sp ± ± pgml and vip ± pgml interestingly lpys4 could significantly p improve the± ± ± and ± pglb ± ± ± and ±± pgml in colitis model mice was signifigroups p following the treatment with lpys4 anhigher in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml and the serum levels of il10 werelower in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml p gsh ± μmolmg and sod ± μmolhighest while those of mpo ± mumg and mda± nmolmg were the lowest among the five± μmolmg and sod ± μmolgprotwere significantly reduced p while those of mpo± mumg and mda ± nmolmg wereremarkably increased p in the model group mice eï¬ect of lpys4 on the serum concentrations of il2 andil10 in mice it can be seen from figure that the serumcontentand il10± pgml eï¬ect of lpys4 on the activities of mpo sod gsh andmda in mouse colon as shown in figure the activities ofincrease in il2 and a decrease in il10 cytokine level wereobserved notably the serum levels of il2 were markedlyml and the eï¬ects of lpys4 were better than those ofgroups after induced by oxazolidone the levels of gshcantly reduced and raised compared to the remaining fourgprot in the colon tissue of the normal group were the pgmlofil2 0cevidencebased complementary and alternative medicinebacmice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgfigure ac colon length and colon weightcolon length of each group of mice data are presented as the mean± standard deviationa“d diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4land mda levels p such eï¬ect was stronger comparedto lbtreated mice ± μmolmg ± μmolgprot ± mumg and ± nmolmg more± μmolgprot in colon tissue and markedly decreased those of mpo ± mumg and mda± nmolmg when compared to colitis model groupand the goblet cells were increased compared with the modelgroup among them lpys4h had the most obvious eï¬ecton improving colon tissue which indicated that lpys4 couldreduce the colon injury caused by dss and the high efficiencyeï¬ect was also enhanced with the increase of concentrationlpys4 could markedly attenuate the decline in gsh andsod levels and prevented colitisinduced increase in mpo± μmolmgspecifically treatment with lpys4h significantly increasedthesodlevelsandof pathological observation as shown in figure in thenormal group the epithelial cells of colon mucosa were intactthe ‚ammatory cells were normal without ltration andthe goblet cells were arranged orderly without congestionand edema in the model group the epithelial cells of colontissue were obviously damagedthe intestinal wall wasthickened and edema ‚ammatory cell ltration andgoblet cells were reduced after treatment with lb and lpys4 congestion edema and cell ltration were alleviated eï¬ect of lpys4 on mrna and protein expression inmouse colon as shown in figures and colitis inductioncould lead to the upregulated mrna and protein expressionof inos in mouse colon but downregulated the relativeexpression of ckit enos nnos and scf treatment withlpys4h could increase the relative expression of nnosenos ckit and scf and decrease that of inos in the colontissues of colitis mice such eï¬ects were stronger than thoseof lpys4l or lb treatment group discussione ratio of colon weightlength is employed as a vitalstandard for assessing colitis in vivo e colon length ofnormalmodellblpys4llpys4hadccb0020406080100normalmodellblpys4llpys4hcolon length cmcolon lengthadccb00100200300400500normalmodellblpys4llpys4hcolon weightcolon lengthcolon weightcolon length 0cevidencebased complementary and alternative medicineabfigure a et1 b ss c sp and d vip serum levels of each group of mice data are presented as the mean± standard deviationa“d diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4lmice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgdcfigure a il2 and b il10 serum levels of each group of mice data are presented as the mean± standard deviation a“e diï¬erentletters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4l mice treatedwith a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentration oflactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgbadabbc0050100150200250normalmodellblpys4llpys4het1 level pgmlet1aedcb00100200300400500600700normalmodellblpys4llpys4hss level pgmlssvipdabbc00100200300400500600700normalmodellblpys4llpys4hsp level pgmlspaedcb00100200300400500600700normalmodellblpys4llpys4hvip level pgmlvipadccb050100150200250300normalmodellblpys4llpys4hil2 level pgmlil2eabcd02004006008001000normalmodellblpys4llpys4hil10 level pgmlil10 0cevidencebased complementary and alternative medicineabfigure a mpo b no c gsh and d mda colon tissue levels of each group of mice data are presented as the mean± standarddeviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent testlpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a highconcentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgcdcolitis mice was shorter on average than that of control miceand the ratio of colon weightlength was lower in colitis micethan in control mice howeverit appeared thattreatment with lpys4 could attenuate the decline in colonlength and weightlength ratio induced by colitisa prior study has shown that vasoconstriction of theendothelin can lead to colonic mucosa erosion and ulceration which in turn can exacerbate the progression of colitis ss however can reduce gastrointestinal ‚ammationby suppressing the production of gastric acid and othergastrointestinal fluids us a decrease in ss level can inducethe secretion of gastrointestinal fluids thus aggravatingcolitis excessive accumulation of sp can induce colitisbut after antagonizing it has been shown to relieve colitis invivo vip inhibits no production by regulating thetranscriptional activity of inos in the colon tissue thusprotecting the intestinal mucosa besides vip can also ‚uence certain immune aspects of colitis in this studylpys4 inhibited colitis by downregulating the levels of etand sp and upregulating those of ss and vipil2 is an eï¬ector cytokine produced by cells whichhas been closely associated with colitis cells regulate the‚ammatory response that causes colitis and il2 is involved in the suppression of ‚ammatory process andseverity reduction of colitis by ‚uencing cells il is another cytokine produced by treg cells with immunoinhibitory eï¬ects which plays a significant role in thedevelopment of colitis ‚ammatory bowel diseaseibd is a chronic refractory intestinal ‚ammatory diseasemainly including ulcerative colitis uc and crohn™s diseasecd although the etiology of ibd is still unclear theimbalance between and has been recognized as themain cause of mucosal damage in ibd under the action ofdiï¬erentiation factor il10 regulatory t cells derived fromintestinal mucosa associated lymphoid tissue can correct deviation by secreting high level of il10 andmedium level of tgfβ so as to achieve the purpose oftreating ‚ammatory bowel disease to a certain extent it was observed that lpys4 could increase the level of il2thus regulating immunity and alleviating colitis and lpeabcd00100200300400normalmodellblpys4llpys4hmpo level mumgmpoadccb00100200300400normalmodellblpys4llpys4hsod level μmolgprotsodaedcb0020406080100120normalmodellblpys4llpys4hsgh level μmolmggshdabbc000510152025normalmodellblpys4llpys4hmda level nmolmgmda 0cevidencebased complementary and alternative medicinefigure observation of colon pathology in mice by he stainingys4 could inhibit the colitis and reduce the secretion of ile aggregation of neutrophils began to decline afterintestinal ‚ammation and a large amount of them enteredinto the circulation and migrated to tissues at the sametime free radicals such as reactive oxygen species and reactive nitrogen species gathered in large quantities which inturn led to damage and toxicity of colon tissue and furtheraggravated colitis after colitis the levels of sod andgsh were reduced in colon tissue while those of mda andmpo were elevated our findings also indicated that colitiscould lead to decrease in gsh and sod levels as well asincrease in mda and mpo levels [ ] in addition lpys4 attenuated colitis by inhibiting the transcriptional responses to oxidative stressnos can be divided into nnos enos and inos it hasbeen reported that no produced by enos plays a key rolein response to colonic tissue damage and excessive nogenerated by inos promotes colitis damage enoscontrols the production of no to keep the colonic tissue ina normal state which plays an important role in reducingcolitisinduced colonic injury e presence of excessiveno aggravates colon damage nnos can also controlthe level of no in tissue and protect the tissue from beingdamaged by excessive no in this study lpys4upregulated the expression of enos and nnos in the colonand downregulated that ofthereby attenuatingcolitisinosulcerative colitis not only shows hematochezia anddiarrhea but also presents colonic motility disorders it hasbeen proved that interstitial cells of cajal icc are related tocolonic motility dysfunction and directly participate in theprogression of colitis as a specific marker of gastrointestinalicc ckit is a transmembrane glycoprotein specificallyexpressed on icc cell membrane ckit gene located onchromosome 4q1213 belongs to protooncogene and itsproduct is tyrosine kinase type iii as a receptor of scfckit can regulate the proliferation and diï¬erentiation ofhematopoietic stem cells through a series of signalingpathways [ ] scf exerts a direct eï¬ect on ‚ammatorybowel disease by regulating the function and number oficc scf can interact with its ligand ckit and the dysregulation of scfkit signaling pathway may decrease theproliferation and diï¬erentiation of icc thus exacerbatingcolitis [ ] e abnormal expression of scfckitsignaling pathway can also change the physiologicalfunction of icc weaken gastrointestinal motility andaggravate intestinal dysfunction in the present studylpys4 could inhibit colitis by regulating the expressionlevels of scf and ckiticc autophagy regulation has become a new target forthe treatment of intestinal motility disorder in ulcerativecolitis because the drug treatment of colitis often hasside eï¬ects and once stopped it is easy to relapse ereforethe use of natural harmless substances through the regulation of icc prevention and treatment of colitis canmaintain longterm health a study has shown thataurantii fructusimmaturus and atractylodis macrocephalae rhizoma can inhibit the autophagy of cajal stromalcells induced by glutamate which may play an inhibitoryrole in colitis meanwhile there is also a study showingthat lactic acid bacteria can regulate icc thus regulatingnormallblpys4llpys4hmodel 0cevidencebased complementary and alternative medicineacbdthe mean± standard deviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestlyfigure eï¬ect of lpys4 on a nnos b enos c inos d ckit and e scf mrna expression in mouse colon data are presented asesignificantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillusbulgaricus — cfukgintestinal function and protecting the intestine isstudy also confirmed that the lpys4 can regulate the scfckitsignaling pathway and the scfckit signalingpathway is an important icc regulatory pathway erefore the lpys4 may also inhibit the colon by regulating iccaedcb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupnnosadccb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupscfaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineacbdmean± standard deviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly sigfigure af eï¬ect of lpys4 on nnos enos inos ckit and scf protein expression in mouse colon data are presented as thenificantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillusbulgaricus — cfukgef conclusionin this study oxazolone was used to induce colitis in balbcmice and the inhibitory eï¬ects of lpys4 on colitis weredetected rough the observation of colon tissues and serum samples of mice it was found that lpys4 treatmentcould alleviate colitis by restoring the levels of ‚ammatoryindicators closer to those measured in healthy control miceis work suggests that lpys4 is superiorquality lactic acidbacteria with a potential role in colitis treatment and provides a foundation for further research and developmentabbreviationslpys4 lactobacillus plantarum ys4lbqpcr quantitative polymerase chain reactionhelactobacillus bulgaricushematoxylineosinnormalmodellblpys4llpys4hckitenosinosnnosscfβactinadcbcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupnnosaedcb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosadccb0010203040506070normalmodellblpys4llpys4hrelative to multiple of model groupscfadccb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineendothelin1substance psomatostatinvasoactive intestinal peptideinterleukin2interleukin10et1spssvipil2il10mpo myeloperoxidasesodgshmda malondialdehydeenosnnos neuronal nitric oxide synthaseinducible nitric oxide synthaseinosscfstem cell factorsuperoxide dismutaseglutathioneendothelial nitric oxide synthasedata availabilityno data were used to support this studyconflicts of intereste authors of this manuscript state that they do not haveconflicts of interest to declareauthors™ contributionsruokun yi and fang tan contributed equally to this workruokun yi and fang tan performed the majority of theexperiments and wrote the manuscript huayi suo wenfengli xianrong zhou and jianfei mu contributed to the dataanalysis xin zhao and peng xie designed and supervised thestudy and read the final manuscriptacknowledgmentsis research was funded by national key rd program ofchina 2018yfd0502300 children™s research institute ofnational center for schooling development programmeand chongqing university of education csdp19fs01103theand technology research program ofchongqing municipal education commission kjzdk201901601 and research project of chongqing universityof education ky2015tbzc chinasciencereferences r k yi f tan w liao et al œisolation and identification oflactobacillus plantarum hfy05 from natural fermented yakyogurt and its eï¬ect on alcoholic liver injury in mice microanisms vol no p j liu f tan x h liu et al œexploring the antioxidanteï¬ects and periodic regulation of cancer cells by polyphenolsproduced by the fermentation of grape skin by lactobacillusplantarum kfy02 biomolecules vol no p t s olson b k reuter k g e scott et al œe primarydefectfrom a nonhematopoietic source journal of experimental medicinevol no pp “ ileitis originatesin experimental b manandhar k r paudel b sharma and r karkiœphytochemical profile and pharmacological activity of aeglemarmelos linn journal of integrative medicine vol no pp “ x zhou h liu j zhang et al œprotective eï¬ect of lactobacillus fermentum cqpc04 on dextran sulfate sodiuminduced colitis in mice is associated with modulation of thenuclear factorκb signaling pathway journal of dairy science vol no pp “ m c arrieta k madsen j doyle and j meddings œreducing small intestinal permeability attenuates colitis in theil10 genedeficient mouse gut vol no pp “ h suo x zhao y qian et al œerapeutic eï¬ect of activatedcarboninduced constipation mice with lactobacillus fermentum suo on treatment international journal of molecular sciences vol no pp “ y qian h suo m du et al œpreventive eï¬ect of lactobacillus fermentum lee on activated carboninduced constipation in mice experimental and eerapeutic medicinevol no pp “ x zhao y qian h suo et al œpreventive eï¬ect of lactobacillus fermentum zhao on activated carboninduced constipation in mice journal of nutritional science andvitaminology vol no pp “ x long y pan and x zhao œprophylactic eï¬ect ofkudingcha polyphenols on oxazolone induced colitis throughits antioxidant capacities food science and human wellnessvol no pp “ k zhu g huang j xie x zhou j mu and x zhaoœpreventive eï¬ect of flavonoids from wushan shencha malus doumeri leaves on ccl induced liver injury foodscience nutrition vol no pp “ x zhao j zhang s yi et al œlactobacillus plantarumcqpc02 prevents obesity in mice through the pparα signaling pathway biomolecules vol p w strober i j fuss and r s blumberg œe immunologyofmucosalmodels ofinflammation annual review of immunology vol no pp “ jl song y qian gj li and x zhao œanti‚ammatoryeï¬ects of kudingcha methanol extract ilex kudingcha cjtseng in dextran sulfate sodiuminduced ulcerative colitismolecular medicine reports vol no pp “ y qian x zhao jl song et al œinhibitory eï¬ects of resistant starch rs3 as a carrier for stachyose on dextransulfate sodiuminduced ulcerative colitis in c57bl6 miceexperimental and eerapeutic medicine vol no pp “ x y chen x zhao h w wang et al œprevent eï¬ects oflactobacillus fermentum hy01 on dextran sulfate sodiuminduced colitis in mice nutrients vol no p y qian a l lei x j liu et al œinhibitory eï¬ects oflactobacillus plantarum ys2 in dextran sulfate sodiuminduced c57bl6j mice colitis science and technology of foodindustry vol no pp “ s chen x zhao p sun j qian y shi and r wangœpreventive eï¬ect of gardenia jasminoides on hclethanolinduced gastric injury in mice journal of pharmacologicalsciences vol no pp “ l xie z h xing r x jiang et al œeï¬ect of sanpi decotionon the expression of il2 and il10 of mice with ulcerativecolitis chinese journal of experimental traditional medicalformulae vol no pp “ l hang s kumar a m blum j f urban m c fantini andj v weinstock œheligmosomoides polygyrus bakeri infection decreases smad7 expression in intestinal cd4 t cells 0cevidencebased complementary and alternative medicinewhich allows tgfβ to induce il10producing regulatorytcells that block colitis ee journal of immunology vol no pp “ j zhang q li y wei et al œprocess design of the antioxidant shuidouchi and its eï¬ect on preventing dextransulfate sodium dssinduced colitis in mice via antioxidantactivity applied sciences vol no p c fiocchi œ‚ammatory bowel disease new insights intomechanisms of ‚ammation and increasingly customizedapproaches to diagnosis and therapy current opinion ingastroenterology vol no pp j zhang r yi y qian p sun x zhao and z yangœlactobacillus plantarum cqpc06 activity prevents dextransulfate sodiuminduced colitis by regulating the il8 pathway journal of food science vol no pp “ j zhang x chen jl song et al œpreventive eï¬ects oflactobacillus plantarum cqpc07 on colitis induced bydextran sodium sulfate in mice food science and technologyresearch vol no pp “ lh chen jl song y qian x zhao hy suo and j liœincreased preventive eï¬ect on colon carcinogenesis by use ofresistant starch rs3 as the carrier for polysaccharide ofinternationallarimichthysjournal of molecular sciences vol no pp “ x feng j zhang y qian et al œpreventative eï¬ects oflactobacillus plantarum ys3 on oxazoloneinduced balbccolitis in mice applied biological chemistry vol no pp “ swimming bladdercrocea j m wang m li r tang et al œeï¬ect of yiqi jianpitongbian recipe on icc and scfckit signaling pathway incolon tissue of slow transport type constipation rats chinesearchives of traditional chinese medicine vol no pp “ j feng j gao s zhou et al œrole of stem cell factor in theregulation of icc proliferation and detrusor contraction inrats with an underactive bladder molecular medicine reports vol no pp “ c lu h lu x huang et al œcolonic transit disordermediated by downregulation of interstitial cells of cajalanoctamin1 in dextran sodium sulfateinduced colitis micejournal of neurogastroenterology and motility vol no pp “ y c dai l zheng y l zhang et al œjianpi qingchangdecoction regulates intestinal motility of dextran sulfate sodiuminduced colitis through reducing autophagy of interstitial cells of cajal world journal of gastroenterologyvol no pp “ s yan y z yue m m sun et al œsuppressive eï¬ect ofaurantii fructus immaturus and atractylodis macrocephalaerhizoma on glutamic acidinduced autophagy of interstitialcells of cajal journal of integrative medicine vol no pp “ c li sp nie kx zhu et al œeï¬ect oflactobacillusplantarumncu116 on loperamideinduced constipation inmice international journal of food sciences and nutritionvol no pp “ r yadak m breur a
Colon_Cancer
agar oligosaccharides aos produced by agarase has a good application prospect agar amarine polysaccharide producing from red algae is widely used in food industry pharmaceuticalindustry and biological engineering wonjae as the main component of agaragarose is composed of 36anhydrolgalactose lahg and dgalactose dgal bondededited bybaolei jiachungang university south koreareviewed bychangro leemyongji university south koreago furusawauniversiti sains malaysia usmmalaysiacorrespondencechunsheng lilichunshengscsfriaccnxianqing yangyangxqscsfriaccnyxqgd163comspecialty sectionthis was submitted toevolutionary and genomicmicrobiologya section of the frontiers in microbiologyreceived may accepted july published august citationli c li c li l yang x chen sqi b and zhao y comparativegenomic and secretomic analysisprovide insights into unique agardegradation function of marinebacterium vibrio fluvialis a8 throughhorizontal gene transferfront microbiol 103389fmicb202001934frontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio fluvialisalternately by α13 and 14 linkages araki agaroseoligosaccharidesincluding aos and neoagaroligosacchridesnaos are agarose degradation products which exhibit variousphysiological functions such as antioxidant kang anti‚ammatory activity enoki whitening eï¬ectkim immune modulation mussatto and mancilha and antitumor activity bhattarai and kashyap there are two methods to produce agaroseoligosaccharidesincluding acid hydrolysis and agarase biodegradation comparedwith acid hydrolysis agarase biodegradation can be easy tocontrol agaroseoligosaccharide types and produce the productswith fixed polymerization degrees in an environmentally friendlyway without damage of structure xu therefore it isvery important to develop agarases in the industrial productionof agaroseoligosaccharidesmost agarases are produced by marine gramnegative bacteriaincluding agarivorans zhang pseudoalteromonaswang li microbulbifer ma zhu alteromonas seo etc vibrio isalso an important genus of marine gramnegative bacteria toproduce agarases and various strains have been reported abouttheir agarase production characteristics such as vibrio sp jt0107jam vibrio sp cn41 liao vibrio spzc1 su vibrio sp ejy3 yu vibriosp po303 dong and vibrio sp v134 zhang andsun the agarases produced by these strains have been wellcharacterized and classified however the genomic informationand specific taxonomic status of these strains in vibrio remainpoorly understood here we isolated and characterized a marinebacterium vibrio sp a8 with high agar degradation ability froma red algae in the south china sea this strain was identifiedas vibrio fluvialis and showed similarity with v fluvialisnbrc103150 by 16s rrna gene sequencing to date only threegenome sequencing projects of v fluvialis have been completedaccording to the ncbi genome database however interestinglyno agarase genes have been found in these genomes based on thegene sequences and annotation data therefore it is significantlyuseful and interesting to study whether vibrio sp a8 belongs tov fluvialis whether this strain can produce agarase and why thisstrain possesses unique agar degradation abilityin this study the complete genome of novel agardegradingbacterium vibrio sp a8 was sequenced using a combinationof nextgeneration sequencing and thirdgeneration sequencingtechnology the microbialfunctions were annotated andanalyzed against the gene databases to investigate the geneticelements related to agar degradation comparative genomicanalysis between vibrio sp a8 and similar strains in vibriowas performed to ascertain the taxonomy position ofthisstrain and to explain whether these bacteria had a commonability of agar degradation secretomic analysis using nanolcmsms was performed to study the molecular propertiesof agarase in the secretome of this strain followed by theanalysis of the biochemical properties of purified agarase thisstudy provides information about novel agarase in vibrio andfills in the gap on the genomic information and taxonomyposition of the agardegrading strain in this genus which isvaluable for better understanding the agar degradation pathwayin the biofilm of red algae surface and providing insightsinto biotechnological applications of agarase for production ofagaroseoligosaccharidesmaterials and methodsisolation and biochemical identificationof strain a8marine bacterium strain vibrio sp a8 with efficient agardegradation ability was isolated from the rotten red algaegracilaria lemaneiformis in the south china sea firstly grotten algae was added into ml sterilized saline water aftersufficient vortex oscillation the solution was spread on lb agerplates peptone yeast extract powder nacl and agar ph using gradient dilution after cultivation at —¦cthe strains with concave around their colonies were selected andinoculated on fresh lb ager plates for h at —¦c the plateswere then stained with lugol™s iodine solution i2 and ki colonies with a clear zone formed by agar degradation weredetermined as agardegrading strains the strain with the highestagar degradation ability was maintained on lb agar slant peptone yeast extract powder nacl and agar ph and was used for further studybiochemical identification of vibrio sp a8 was carried outusing the vitek gn test kit biomrieux according to themanufacturer™s instructionsagaraseproducing conditionoptimizationvibrio sp a8 was first precultured by transferring a loopfulof slant culture to liquid lb medium peptone yeastextract powder and nacl ph and incubated at —¦cand rmin for h thereafter the strain was transferredto ml liquid agaraseproducing medium yeast extractpowder and agar in artificial seawater ph in mlerlenmeyer flaskunless otherwise stated in the agaraseproducing experimentsthe inoculation size of the strain after preculture was theph value of the medium was the culture temperature was—¦c and the culture time was h diï¬erent carbon sources galactose glucose sucrose agar agar galactose agar glucose or sucrose agar were first added in the agaraseproducingmedium and then the eï¬ects of diï¬erent concentrations of theoptimum carbon source on the agarase activity in the mediumwas studied the optimum nitrogen source for the agaraseproduction was selected by changing the yeast extract powderin the medium into other nitrogen sources peptone nh4no3 nh42so4 nh4cl or nh4cl yeast extract powder the eï¬ects of nacl concentrations “ ph value “ temperature “—¦c and culture time“ h on the agarase activity in the medium were studied todetermine the optimum culture conditions of vibrio sp a8the agarase activity was determined by measuring the releaseof the reducing sugars using the 35dinitrosalicylic acid dnsfrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio fluvialisfigure effect of a carbon source with yeast extract powder b agar concentration with yeast extract powder c galactose concentrationwith yeast extract powder and agar d nitrogen source with agar and galactose e yeast extract powder concentration with agarand galactose f nacl concentration with agar galactose and yeast extract powder g ph with agar galactose yeast extract powder and nacl h temperature with agar galactose yeast extract powder and nacl at ph and i culture timewith agar galactose yeast extract powder and nacl at —¦c and ph on the agarase activity in artificial seawater cultivation with straina8 bars labeled with the same letter are not statistically different p tested by oneway anova and multiple comparison tukey test pearson correlationanalysis was tested between agarase activity and od600 at different culture timemethod miller briefly the medium µl aftercultivation was incubated with melted agar ml at —¦cfor min the solution was then placed in boiling water for min to stop the enzymatic reaction subsequently the dnsreagent ml was added to the reaction solution which washeated in a boiling water bath for min when dropped toroom temperature the mixture was diluted with distilled waterto ml and the released reducing sugars were quantifiedspectrophotometrically at nm one unit of agarase activitywas defined as the amount of enzymes that released µg ofreducing sugars per minute under these experimental conditionswhole genome sequencing assemblyand annotationthe genomic dna from vibrio sp a8 was extracted and purifiedusing a wizard genomic dna purification kit promega thewhole genome was sequenced using a combination of pacbiors ii smrt and illumina sequencing platforms for illuminasequencing the genomic dna sample over µg was shearedinto “ bp fragments using a covaris m220 focusedacoustic shearer covaris illumina sequencing libraries weregot from these fragments using the nextflextm rapid dnaseqkit bioo scientific and were then used for pairedend illuminasequencing — bp on an illumina hiseq — ten machineillumina for pacific biosciences sequencing an aliquot of µg dna was spun in a covaris gtube covaris at rmin for s dna fragments were then purified endrepaired and ligated with smrtbell sequencing adapters pacificbiosciences the resulting sequencing library was purified threetimes using agencourt ampure xp beads beckman coultergenomics and sequenced on one smrt cell pacific biosciencesusing standard methodsthe complete genome was assembled using both the pacbioand illumina reads feng briefly the pacbio readswere assembled into a contig using hgap and canu thecircular step was checked and finished manually generating acomplete genome with seamless chromosomes and plasmids theillumina reads was used for the error correction of the pacbioassembly results using pilon the cds trna and rrna wasfrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio fluvialistable genomic features of strain a8featuregenome size bpdna gcchromosomeplasmidproteincoding genestrnasrrnastandem repeatsgenes assigned to gogenes assigned to coggenes assigned to kegggenes with pfam domainsgenes coding for cazymesgenes related to secondary metabolismgenes related to virulent factenes related to antibiotic resistancegenes coding for secretory proteinsgenes coding for transport proteinsgenes coding for transmembrane proteinsgene islandsvaluerespectively predicated by glimmer version trnascanse version and barrnap version the predicted cdsswere annotated from nr swissprot pfam go cog keggand cazyme database using sequence alignment tools such asblast blast2go eggnog diamond and hmmer each set ofquery proteins were aligned with the databases and annotationsof the bestmatched subject evalue ˆ’ were used fene annotation the gene islands were predicted using theisland viewer bertelli the circular genomic plot ofpmtb21 was plotted by circos song h the agarase and naos hydrolase gene sequences werecompared with the gene sequences available from the ncbidatabase phylogenetic tree ofthese gene sequences wereperformed using mega based on the maximumlikelihoodmethod after multiple alignments of the sequences by clustalx larkin numbers represented the frequencieswith which the tree topology wasreplicated after bootstrap iterationscomparative genomic analysisgenomic data of the similar vibrio species analyzed in this studywere obtained from the ncbi the homologous genes wereanalyzed using orthomcl li with the followingparameters evalue 1eˆ’ percent identity cutoï¬ markov‚ation index phylogenetic analysis was performed byra — ml stamatakis after multiple alignments of singlecopy of homologous genes using clustal x larkin to compare the genetic relationship among vibrio species theaverage nucleotide identity ani value was calculated usingjspecies richter and rossellomora saturationsecretomic analysislabelfree quantitative lcmsms was performed to study thesoluble extracellular proteins of vibrio sp a8 the extracellularproteins were isolated by ammonium sulfate fractionationˆ¼ ammonium sulfateredissolved inphosphate buï¬er solution moll ph and thendesalted by dialysis bag the bradford method bradford was used to determine the protein concentration ofsample the protein sample µg was digested using trypsinpromega at —¦c overnight equal volume of formic acidwas mixed with digested sample and centrifuged at — gfor min at room temperature the supernatant was slowlyloaded to the c18 desalting column washed by ml of washingsolution formic acid and acetonitrile three times andthen eluted twice using ml of elution buï¬er formicacid and acetonitrile the eluents were lyophilized andreconstituted in µl of formic acid peptide sampleswere injected and analyzed using a q exactive hfx massspectrometer thermo coupled with nanoeasynlc uhplc thermo peptides were first trapped onto a homemade c18 nanotrap column cm — µm µm and theneluted onto a homemade analytical column cm — µm µm via a min gradient of “ acetonitrile with formic acid at a flow rate of nlmin “ min “ min “ “ min “ “ min ““ min “ “ min “ ms full scan rangedfrom mz to with a target value of at aresolution of at mz the top precursors of thehighest abundant in the full scan were fragmented using higherenergy collisional dissociation and analyzed in msms with anautomatic gain control target value of and normalizedcollision energy of raw msms data were searched againstthe genome of vibrio sp a8 obtained in this study usingproteome discoverer thermo for abundance calculationmass spectrometric signal intensities of peptide precursor ionsbelonging to each protein were divided by the total abundanceof all detected proteins the mass spectrometry proteomics datahave been deposited to the proteomexchange consortium viathe pride perezriverol partner repository withthe dataset identifier pxd019302phylogenetic tree of agarases based on their amino acidsequences was performed using mega based on the neighborjoining method after multiple alignments of the sequences byclustal x larkin the sequences of agarases thatwere similar to that in vibrio sp a8 were obtained from uniprotdatabase numbers represented the frequencies with which thetree topology was replicated after bootstrap iterationsagarase purification and propertiesthe extracellular proteins were isolated using ammoniumsulfate fractionation ˆ¼ ammonium sulfate saturationredissolved in phosphate buï¬er solution moll ph and then desalted by dialysis bag the agarase purification wasperformed by deae sephadex anionexchange chromatographythe proteins were eluted by nacl solution at moll theeluent collected from the protein peak was tested for the agarasefrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio fluvialisfigure circular maps of a chr1 and b chr2 of strain a8 the circles represent from inner to outer gc skew gc content rrna and trna cog assignments for cdss on the reverse strand cog assignments for cdss on the forward strand scale in mbfrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio fluvialisfigure phylogeny of vibrio species based on singlecopy core orthologs a core homologous genome genes was computed by the genome of straina8 along with complete vibrio genomes obtained from public database b phylogenetic analysis was performed by ra — ml after multiple alignments of singlecopy of homologous genes using clustal x c venn diagram showing homologous and unique genes among v fluvialis strainsfrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio fluvialistable heatmap of the ani value between the conserved regions in the genomes of strains in vibriostrains in vibrio v fluvialis a8 v fluvialis v fluvialis ak 1296a21 v fluvialis atcc v furnissii fdaargos_777 v furnissii nctc v tubiashii atcc v anguillarum v coralliilyticus snuty1species on the first column and first row of the table are represented in the same numeric order the numbers show the percentage similarity between the conservedregions of the genomes where the colors vary from yellow low similarity to blue high similarityactivity the eluent with the agarase activity was used for thestudy of agarase propertiesunless otherwise stated in the study of agarase propertiesthe diluted enzyme solution µl was incubated with melted agar ml at —¦c and ph for min in whichcondition the agarase activity was set as the agarasesolution was incubated with ml of gl carrageenan alginchitosan cellulose or amylum to study the substrate specificity ofagarase one unit of enzyme activity was defined as the amount ofenzymes that released µg of reducing sugars per minute underthese experimental conditions using dns method as mentionedpreviously to study the optimal reaction temperature theagarase activity was measured at diï¬erent temperatures and —¦c the optimal ph of the agarase wasassayed in a ph range of “ in mm na2hpo4citratebuï¬er ph “ mm phosphate buï¬er ph “ and mm glycine“naoh buï¬er ph the eï¬ects of diï¬erentmetal ions nacl fecl2 fecl3 cacl2 cuso4 kcl or mgcl2at mmoll on the agarase activity were also studied the sameamount of agarase solution was placed in the water bath at and —¦c respectively for “ min to study the agarasethermostability the data were fitted to first order plots andplots of lnra residual activity versus time were constructedto calculate kd the halflife of the enzyme t12 was calculatedusing the following equationt12 ln kdwhere t12 is the time required for the residual activity reducingto half and kd is the deactivation rate constantresults and discussionagaraseproducing conditionoptimization of strain a8the eï¬ects of medium components and culture conditions onthe agarase production of vibrio sp a8 are shown in figure diï¬erent carbon sources showed obviously diï¬erent eï¬ects onthe agarase activity in the medium figure 1a when glucosegalactose sucrose or agar was added to the medium alonethe agarase activity was the highest in the presence of agarhowever the agarase activity decreased after glucose and sucrosewere added to the medium possibly because the coexistenceof glucose or sucrose reduced the utilization of agar by thisstrain thus inhibiting the secretion of agarase the additionof galactose enhanced the agarase activity with the agar as theonly carbon source probably resulting from the improved straingrowth by galactose therefore the agar and galactose werechosen as the carbon sources of the agarproducing mediumthe eï¬ects of agar and galactose concentrations on the agaraseactivity in the medium were respectively studied as shown infigures 1bc the agarase activity in the medium first increasedand then decreased with the increasing agar or galactoseconcentrations the optimum agar and galactose concentrationwas respectively and diï¬erent agarase activities was found in the medium withthe addition of diï¬erent nitrogen source figure 1d anicnitrogen source was more suitable for the agarase production ofvibrio sp a8 than inanic nitrogen source the agarase activitywas highest when yeast extract powder was the only nitrogensource in the medium the agarase activity was not significantlydiï¬erent at various yeast extract powder concentrations “ figure 1e the highest agarase activity was observed at yeast extract powderbased on artificial seawater the diï¬erent concentrations ofnacl were added to determine the optimum osmotic pressurefor the agarase production of vibrio sp a8 figure 1f theagarase activity was improved by nacl but was significantlyinhibited by “ nacl this might be because the excessiveosmotic pressure inhibited the strain growthleading to thedecrease of its agarase production diï¬erent ph values had great‚uence on the agarase activity in the medium figure 1gthe optimum ph was at which the agarase activity was uml similar to ph the culture temperature showedsignificant ‚uence on the agarase production of strain a8figure 1h the optimum culture temperature was —¦c atwhich the agarase activity was as high as umlthe eï¬ect of culture time on the agarase production of vibriosp a8 is shown in figure 1i the agarase activity increasedslightly at “ h markedly rised at “ h and then reachedequilibrium after h the growth curve was in accordancefrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio fluvialisfigure genes related to agar degradation in the genome of v fluvialis a8 a number of genes in each cazyme class b number of genes related topolysaccharide degradation in ghs genomic islands gi contained genes related to agar degradation c gene3149 coding for α13lna2 hydrolase andgene3151 gene3152 and gene3153 coding for agarase were in gi d gene3109 coding for agarase was in gi e the gc content in the gi gi chr2 and genome of v fluvialis a8 as well as in the genomes of v vulnificus strainswith the agaraseproducing curve pearson correlation analysisshowed that the growth of strain a8 was significantly correlatedwith agarase activity p indicating that the positive‚uence of strain growth on the agarase productionthe optimum medium for agarase production of vibrio spa8 was determined as agar galactose yeastextract powder and nacl in artificial seawater and theoptimum culture conditions were determined as temperatureat —¦c ph at and incubation time for h underthese conditionsthe fermentationbroth was uml which was higher than thatbefore optimizationthe agarase activity offrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio fluvialistable genes related to degradation of agar and relative compounds in the genome of v fluvialis a8gene idlocationstrandstartendgene lengthprotein lengthannotationgh familygene3109gene3151gene3152gene3153gene3149gene0378gene1513gene3148gene4670gene1669gene3155chr2chr2chr2chr2chr2chr1chr1chr2chr2chr1chr2ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’agaraseagaraseagaraseagaraseα13lna2 hydrolasegalactosidasedgalactosidasegalactosidasegalactosidaseαgalactosidaseαgalactosidasegh50gh50gh50gh50gh117gh2gh2gh2gh2gh36gh36genome features of strain a8the genomic features of vibrio sp a8 are presented in table the complete genome of vibrio sp a8 was bp gc in length composed oftwo circular chromosomeschr1 bp gc figure 2a and chr2 bp gc figure 2b and no plasmidwas detected the genome contained proteincodinggenes trna genes and rrna genes and all rrnatable genes involved in degradation of agar and relative compounds in thegenomes of strains in vibrio based on homologous gene analysisstrains in vibrioagar degradation gene numbersagarase neoagarobiosegalacto sidasehydrolasev fluvialis a8v fluvialis v fluvialis ak 1296a21v fluvialis atcc v furnissii fdaargos_777v furnissii nctc v tubiashii atcc v anguillarum v coralliilyticus snuty1 v coralliilyticus re98 v antiquaries ex25 v parahaemolyticusforc_014 v alginolyticus k10k4 v natriegens atcc v diabolicusfdaargos_105 v campbellii atccbaa1116 v owensii v180403 v breoganii ff50 v vulnificus cect v casei dsm v nigripulchritudo sfn1 present ˆ’ absent ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ genes were located in chr1 there were genes assigned to the go terms including genes in biologicalprocess genes in molecular function and genesin cellular component the most abundant go annotationincluded the metabolic process genes membrane genes and catalytic activity genes in respectivecategory supplementary figure s1 there were genes assigned to the cog terms and high percentageof genes was classified into the amino acid transport andmetabolism genes transcription genes inanicion transport and metabolism genes energy productionand conversion genes and carbohydrate transport andmetabolism genes as well as the function unknown genes supplementary figure s1 in addition genes were allocated to the kegg pathways in whichthe carbohydrate metabolism genes global and overviewmaps genes amino acid metabolism genes andmembrane transport genes were the predominant pathwayssupplementary figure s1identification of strain a8strain a8 was initially identified as vibrio sp a8 based on 16srrna gene sequence to determine the exact species of thisstrain the comparative genomic analysis between a8 and similar strains in vibrio was studied among the vibrio strains homologous genes were found and constructed the coregenome figure 3a the genome phylogenetic tree was thenconstructed based on the core homologous genome figure 3bthe phylogenetic tree was capable of grouping various strainsof a single species into a single cluster strain a8 clustered withv fluvialis atcc v fluvialis and v fluvialisak 1296a21 suggesting that these four strains might harborsimilar biological properties the biochemical identification andcharacterization observations for strain a8 were made on vitek gn system supplementary table s1 strain a8 was identifiedas v fluvialis with of probability on the basis of aboveresults the strain a8 could be determined as v fluvialis andwas renamed as v fluvialis a8 the ani similarity analysis alsoproved that strain a8 belonged to v fluvialis it was reported thatthe strains could be considered as the same prokaryotic specieswhen the ani value was over richter and rossellomorafrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio fluvialis as shown in table intraspecies similarity of v fluvialiswas higher than the highest similarity with v fluvialisa8 was v fluvialis atcc reaching which wasconsistent with the result of phylogenetic tree although homologous genes were found among these v fluvialis strainsthere were still unique genes in v fluvialis a8 figure 3camylumcelluloserecognitiongenes related to agar degradation invibrio fluvialis a8carbohydrateactive enzymes cazymes that are responsiblefor the complex carbohydrate metabolism mainly includethecarbohydratebinding module cbmssynthesis glycosyltransferases gts and degradation glycosidehydrolases ghs polysaccharide lyases pls carbohydrateesterases ces and enzymes of auxiliary activities aasof the carbohydrates huang there were genes coding for cazymes in the genome of v fluvialis a8table this strain had a high fraction of carbohydratedegrading enzymes especially ghs genes which couldincluding agar agarohydrolyze various polysaccharidesoligosaccharidesandchitin figure hemicelluloseagarases are classified into αagarase ec andagarase ec according to their cleavage position onagarose αagarase catalyzes the α13 linkage to produce aoswhile agarase produces naos by split of 14 linkage fuand kim agarases are grouped into ghs based on thesequence similarity eg agarase grouping into the gh16gh50 gh86 and gh118 families and αagarase only groupinginto the gh96 family su in this study a total offour genes coding for agarase which all belonged to gh50family were found in the genome of v fluvialis a8 amongthe agarase genes the gene3151 gene3152 and gene3153arranged in order from to on the forward strandof chr2 while the gene3109 was located on the reverse strandof chr2 from to table naos can be furthercatalyzed by αnaos hydrolase ec which has beenclassified into gh family and can recognize neoagarobiosena2 neoagarotetraose na4 and neoagarohexaose na6 assubstrate ariga watanabe in thegenome of v fluvialis a8 the gene3149 coding for α13lna2 hydrolase was classified into gh family this enzymecan hydrolyze na2 and produce lahg and dgal watanabe it was reported that galactosidase in vibrio spejy3 could act on aos to release dgal lee there were four genes of galactosidase and two genes ofαgalactosidase in the genome of v fluvialis a8 which mightcontribute to its ability of agar degradation the abundant genesof agarase naos hydrolase and galactosidase in v fluvialis a8oï¬er an enormous advantage in the saccharification of agar inred algae for its use as a biomass feedstock for the productionof fermentable sugarto study whether the strains closely related to v fluvialisa8 shared the common ability of agar degradation the agardegrading genes of these strains were analyzed by comparativegenomic analysis table interestingly although the vibriostrains exhibited the genes coding for galactosidase the homologsfor agarase and na2 hydrolase were not observed in all othertested strains even in the same species v fluvialis v fluvialis ak 1296a21 and v fluvialis atcc thissuggested that v fluvialis a8 was probably diï¬erent fromother similar strains and possessed the unique agar degradationability although the agardegrading genes were not found inthe tested genomes another vibrio sp strain ejy3 exhibitedsimilar agarolytic system with v fluvialis a8 and also couldproduce four agarases gh50 and one αnaos hydrolasegh117 yu horizontal gene transfer also knownas lateral gene transfer refers to nonsexual transmission ofgenetic material between unrelated genomes oliveira as the most important form of horizontal gene transfergenomic island gi contains numerous genes associated witha variety of biological functions godde it hasbeen reported that horizontal gene transfer in the bacteriaaï¬ected the phenotypes such as thermotolerance and multidrugresistance of closely related strains hegstad matsutani horizontal gene transfer also givesbacteria unique polysaccharide degradation ability alteromonasmacleodii harbored an alginolytic system within a kbgi which comprised five putative alginate lyases and othercazymes neumann the marine bacteriumpseudoalteromonas haloplanktis ant505 acquired a gi with afunctional pathway for pectin catabolism including two multimodular pectate lyases pela and pelb hehemann a novel agarase gene aga1 in an inland soil agardegradingbacterium paenibacillussp ssg1 might be horizontallytransferred from marine bacteria via human microbiota song t in the genus vibrio horizontal gene transfer mainlycontributes to the virulence of strains le roux and blokesch deng however few studies of horizontal genetransfer related to agar degradation were reported in the genusvibrio in the genome of v fluvialis a8 there were gisin which genes located accounting for over of totalproteincoding genes interestingly all the gis were located inthe chr2 of this strain the genes coding for agarase and α13lna2 hydrolase were all contained in the gis the gene3151gene3152 gene3153 and gene3149 were in the gi figure 4cwhile the gene3109 were in the gi figure 4d theseresults proved that these genes related to agar degradation wereobtained from other species through horizontal gene transferthe gene sequence similarity was further studied to determine theprobable source using the phylogenetic tree analysis figure 4dthe gene3109 gene3151 gene3152 gene3153 and gene3149in vibrio vulnificus vawere found most similar to thatidentity with v vulnificuswgs18058 percentvawgs18047 percent identity with v vulnificusvawgs18038 percent identity with v vulnificus vawgs18058 percent identity with and v vulnificusvawgs18058 percent identity with respectivelyfurthermore the gc content in the gi and gi was and respectively which was obviously lower thanthat in the genome gc and chr2 gcof v fluvialis a8 but much closer to that in the genome ofv vulnificus strains “ gc figure 4e thesefrontiers in microbiology wwwfrontiersinaugust volume 0cli agarase production by vibrio fluvialistable proteins with the highest abundance and enzymes belonging to the ghs in the secretome of v fluvialis a8protein idgene idannotationmw [kda]calculated pirelative abundance proteins with thehighest abundanceenzymes belongingto the ghsgene3819gene2266gene2186gene3152gene2187gene4060gene4061gene2267gene3797gene2482gene4295gene3152gene4670gene4149gene3263gene0718gene1292gene0815gene3730zinc proteaseflagellin protein fladflagellin protein flaaagaraseflagellin protein fladhemolysin hlyathermolabile hemolysin precursorflagellin protein flacmetalloproteasemembrane proteinaminopeptidaseagarasegalactosidasechitodextrinasechitodextrinasechitodextrinasepullulanaseglycosidaseglycosyl hydrolaseresults indicated thatv vulnificus strains through horizontal gene transferth
Colon_Cancer
" to improve the postoperative prognosis of patients with lung cancer predicting the recurrence highrisk patients is needed for the efficient application of adjuvant chemotherapy however predicting lung cancerrecurrence after a radical surgery is difficult even with conventional histopathological prognostic factors thereby anovel predictor should be identified as lipid metabolism alterations are known to contribute to cancer progressionwe hypothesized that lung adenocarcinomas with high recurrence risk contain candidate lipid predictors this studyaimed to identify candidate lipid predictors for the recurrence of lung adenocarcinoma after a radical surgerymethods frozen tissue samples of primary lung adenocarcinoma obtained from patients who underwent a radicalsurgery were retrospectively reviewed recurrent and nonrecurrent cases were assigned to recurrent n andnonrecurrent n groups respectively extracted lipids from frozen tissue samples were subjected to liquidchromatographytandem mass spectrometry analysis the average total lipid levels of the nonrecurrent andrecurrent groups were compared candidate predictors were screened by comparing the folding change and pvalue ofttest in each lipid species between the recurrent and nonrecurrent groupsresults the average total lipid level of the recurrent group was times higher than that of the nonrecurrent groupp a total of lipid species were increased folding change ‰¥ p and lipid species were decreasedfolding change ‰ p in the recurrent group among these candidates increased sphingomyelin smd351 inthe recurrent group was the most prominent candidate predictor showing high performance of recurrence predictionauc sensitivity specificity accuracy we propose smd351 as a novel candidate predictor for lung adenocarcinoma recurrence our finding cancontribute to precise recurrence prediction and qualified postoperative therapeutic strategy for lung adenocarcinomastrial registration this retrospective study was registered at the umin clinical trial registry umin000039202 on 21stjanuary keywords lung adenocarcinoma prognostic factor recurrence prediction lipid mass spectrometry correspondence kahyohamamedacjp1department of cellular and molecular anatomy hamamatsu universityschool of medicine handayama higashi ward hamamatsu shizuoka japan5international mass imaging center hamamatsu university school ofmedicine handayama higashi ward hamamatsu shizuoka japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctakanashi bmc cancer page of lung cancer is one of the leading causes of cancerrelatedmortality worldwide radical resection is the standardtreatment for stage i“ii nonsmall cell lung cancer nsclc however the postoperative survival rate remainsunsatisfactory despite complete resection among patientswith nsclc who received complete resection experience local or distant disease recurrence thereforeadjuvant chemotherapy should be administered to improve survival after a radical surgery adjuvant chemotherapy has been shown to reduce therisk of death due to lung cancer recurrence [“] nonetheless not all patients who underwent radical surgerybenefit from adjuvant chemotherapy because some ofthem are already successfully healed without adjuvantchemotherapy therefore patients highly at risk for recurrence who are likely to benefit from adjuvant chemotherapy should be identified for the efficient applicationof adjuvant chemotherapyadenocarcinoma is the most common histologicaltype of nsclc accounting approximately of allnsclc cases in lung adenocarcinomas severalprognostic factors obtained by histopathological evaluations of surgical specimens have been reported to datesuch as lymph node metastasis pleural invasion lymphatic vessel invasion [ ] blood vessel invasion[ ] adenocarcinoma subtype of micropapillary pattern and spread through air space stas [ ]however predicting lung cancer recurrence after radicalsurgery is still difficult because data on the direct relationship between conventional prognostic factors and recurrence are limited furthermore subjective judgmentsof conventional prognostic factors are considered to hinder accurate recurrence prediction and its retrospectivevalidation accordingly novel recurrence predictors withhigh objectivity are strongly expectedexampleprevious studies demonstrated that lipid metabolismalterations in cancer contribute to cancer cell proliferation and invasion [ ] and some lipids have beensuggested as prognostic factors in several cancer typesforthe number of phosphatidylcholinepc321 in recurrent cases of primary triplenegativebreast cancer tnbc is higher than in that of nonrecurrent cases and thereby pc is suggested as acandidate predictor for tnbc recurrence oleicacid attenuation is correlated with shorter progressionfree period in clear cell renal carcinoma with regard to lung cancer although nsclc is reportedly characterized by drastic changes in phospholipid profiles ascompared to the normal lung tissue and contains different lipid profiles according to the histologic subtypes no lipidomic approach to investigate the prognosticfactor for nsclc has been used based on these previous studies we hypothesized that lung adenocarcinomaswith high recurrence risk have different lipidomes fromthat of lung adenocarcinomas with low recurrence riskand specific lipids that can be considered as candidatesas novel predictive factors for recurrencein this study lipid species that can be considered as potential predictors for lung adenocarcinoma recurrence aftera radical surgery were identified by comparing lipidomes ofprimary lung adenocarcinomas between recurrent andnonrecurrent cases using liquid chromatography“tandemmass spectrometry lc“msmsmethodspatients and tissue samplesretrospective frozen tissue samples of primary lungadenocarcinoma obtained from patients who underwentradical surgery from january to december athamamatsu university hospital were examined radicalsurgery was defined as complete resection performedwith lobectomy or pneumonectomy accompanied by systematic lymph node dissection at stage i or ii and ascomplete resection achieved by segmentectomy orwedge resection with or without lymph node samplingat stage i tissue samples of primary tumors were collected immediately after the resection and stored at ˆ’ °c after a rapid freezing in liquid nitrogen histopathological diagnosis was performed by experienced pathologists according to the world health anizationcriteria pathological staging was identified based on the8th edition ofthe tnm classification for lung andpleuraltumors patients were followedup withcomputed tomography ct of the body trunk and biochemicalantigencea every months during the first years thenevery months until more than years after the surgerywhen cea was elevated ‰¥ ngml without any ctfindings of recurrence head magnetic resonance imaging and systemic positron emission tomography wereperformed for the detection of brain metastasis or bonemetastasiscarcinoembryonicexamination ofin patient selection clinical records of these tissuesamples were retrospectively reviewed patients withpathological stage i or ii indicated for radical surgeryand with major histological subtypes of invasive adenocarcinoma lepidic papillary acinar or solid predominant were analyzed patients who received inductionchemotherapy or radiotherapy and those with other subtypes of adenocarcinoma were excludedthencases withoutand with recurrence wereassigned to nonrecurrent and recurrent groups respectively recurrence was defined as radiological imagingbased findings of distant or locoregional recurrencewithin years whereas no recurrence was defined as nofindings of distant or locoregional recurrence in ‰¥ yearsafter the radical surgery in the nonrecurrent group 0ctakanashi bmc cancer page of cases with followup period of years were excludedin the recurrent group cases with recurrence in theform of pleural dissemination were excluded assumingthe possible attribution with insufficient surgical marginfinally cases for recurrent and for nonrecurrentgroups were subjected for analysishistological evaluationparaffinembedded tissue blocks were sectioned at μmthick sections stained by hematoxylin“eosin he wereexamined for adenocarcinoma subtypesizelymph node metastasis and stas d2“ stain wasused to evaluate lymphatic vessel invasion and elasticavan gienson stain to evaluate blood vessel invasion allhistologicalsections were reviewed by experiencedpathologiststumorchemicalsmethanol chloroform glacial acetate and ultrapurewater were purchased from wako pure chemical industries osaka japan the 12dilauroylsnglycero3pcavanti polar lipids alabaster al pc 120_120 wasused to calibrate standard lipid levelslipid extraction from the cancer tissueeach weight of the frozen tissue samples was measuredusing sartorius analytical lab balance cpa224s sartorius ag göttingen germany additional file supplemental table after the weight measurement modifiedblighdyer methods were performed for lipid extractiontissue samples were transferred into glass tubes and ml of methanol ml of chloroform and mlof m glacial acetate were subsequently addedthen mmol of pc 120_120 per mg of sampletissue was added and subsequently followed by 10minextraction at room temperature after the extraction ml of chloroform was added and vortexed sequentially ml of m glacial acetate was added andvortexed extracted samples were subjected to centrifugation at rpm for min extracted anic layerswere transferred into new glass tubes and were evaporated until completely dried using mivac duo lv genevacextracted lipid wasdissolved with μl of methanol and μl of the dissolved lipids were diluted again with methanol proportional to the weight of the original tissue samples so thatthe concentration of pc 120_120internal controlwill be as similar as possible among casesipswich england thelipid analysis by liquid chromatography“tandem massspectrometry lc“msmsextracted lipids from collected frozen tissue sampleswere analyzed using q exactive„¢ hybrid quadrupoleorbitrap„¢ massanequipped withspectrometerelectrospray ionization source and connected to an ultimate system thermo scientific μl of the extracted lipid samples were injected and separated onacculaim c18 column mm × mm μmthermo scientific components of mobile phase awere as follows wateracetonitrilemethanol vvv mm ammonium formate and formic acidthe components of mobile phase b were as followsacetonitrileisopropanol vv mm ammonium formate and formic acid for elution the flow ratewas set at μlmin a set of linear gradient startingat solvent b was used and linearly increased to b in min maintained at b until minthen decreased linearly to b from min to min and finished with b for the last min theoverall run time was min ms instrument conditionswere as follows sheath gas flow rate auxiliary flowrate sweep gas flow rate capillary temperature °c slens rf level probe heater temperature °c and spray voltage of kv in positive mode and kv in negative mode fullms mode conditions forquantification were as follows ms scan range “ resolution agc target × and maximum injection time was ms for identification top datadependent ms2 method with a resolution of was used the agc target was × and themaximum injection time was ms stepped normalizedcollision energies of and for the positivemode and and for the negative mode wereapplied spectral data were acquired in the mz range of“ mz using an xcalibur v30 software thermoscientifictolerance ppmlipid identification and quantificationlipidsearch„¢ software version mitsui knowledgeindustry tokyo japan was used to identify and quantify lipid species parameter settings for identificationwere followings database hcd retention time min search type product_qex precursor tolerance ppm and productidentificationquality filters of a b and c were used quantificationwas performed at mz tolerance of ± with retentiontime range from ˆ’ min to min alignment of theidentified lipid species among cases was performedwith retention time tolerance of molecules that areannotated as redundant lipid names with different calculated mz and retention times were regarded as independentisomersinadditional file œduplicationannotatedasdata processingtrend analysis between the nonrecurrent and recurrentgroups was performed by comparing the average totallipid level between the two groups and principal 0ctakanashi bmc cancer page of component analysis pca intensities of lipids recordedin the xcalibur v30 software and monoisotopic peakarea values of lipid species identified by lipidsearch„¢software were normalized by dividing with the areavalues of internal control pc 120_120 the total lipidlevel of each case was defined as an accumulation ofnormalized intensities of lipids normalized area valueswere subjected for pcafor respective lipid species pvalues were calculatedusing the student ttest to compare area values betweenthe two groups to screen candidate lipids for recurrence prediction lipidomes were compared between thenonrecurrent and recurrent groups by describing volcano plots with log10 pvalue for vertical axis andlog2 folding change for horizontal axis the foldingchange for a lipid was defined as an average area valueof the recurrent group divided by that of the nonrecurrent group significance was determined at pvaluesof folding change of ‰¥ or ‰ statistical analysisdemographic information and associations with clinicalcharacteristics were evaluated using the fisher exact testcategorical variables or the mann“whitney utest forcontinuous variables the student ttest was used tocompare the average totallipid amounts of the nonrecurrent and recurrent groups and to describe volcanoplots recurrentfree survival rfs was determined asthe time from operation until the first disease recurrenceor death survival curve was described using thekaplan“meier method the optimal cutoff values todiscriminate the two groups were determined using thereceiver operating characteristic roc curve analysisthe area under the roc curves aucs were calculatedto validate the discrimination abilities of candidate lipidsspearman™s rank correlation analysis was used to validatethe correlation among candidate lipid predictors allstatistical analyses except for the ttest were performedusing r the r foundation for statistical computingvienna austria version the student ttest wasperformed with œttest of excel„¢ microsoft redmond usa pvalues of were considered assignificantresultsclinicopathological characteristics of patient cohortclinicopathological characteristics of patients are shownin table in this study cohort tissue samples from nonrecurrent and recurrent cases were analyzedamong the characteristics of these two groups differences in pathological stage p lymph node metastasis p and blood vessel invasion p were statistically significant the and 2year rfs rateof the recurrent group was and with median rfstime of range “ monthsrespectivelyfile supplemental fig the medianadditionalfollowup time ofthe nonrecurrent and recurrentgroups was range “ and range “months respectivelytrend analysis between the nonrecurrent and recurrentgroupsthe frozen tissue samples were subjected to lc“msms and the total lipid level of cases was calculated byaccumulating normalized intensities of lipids notablythe average total lipid level of the recurrent group was times higher than that of the nonrecurrent groupp fig a total of lipid species wereidentified and quantified by analyzing the mass spectraldata using a lipidsearch„¢ software the full list of identified lipid species is presented as additional file which were also subjected to pca the pca plot didnot show clear separation between the recurrent andnonrecurrent groups however the recurrent group exhibited partial separations between the first three principal components additional file supplemental fig these results suggested differences of lipidome betweenthe recurrent and nonrecurrent groups which urged usto screen lipids to distinguish the two groupsscreening of candidate lipids for recurrence predictionto screen lipids with different levels between the twogroups volcano plots of the identified lipids were described first and lipidomes between the nonrecurrentand recurrent groups were compared fig the volcano plotidentified lipid species with relativeamounts significantly different between the two groupsfolding change ‰¥ or ‰ pvalues thenumber of lipids that increased and decreased in the recurrent group was and respectively these increased or decreased lipid species consisted of varioushead groups additional file increased lipid speciesshown in red decreased lipid species shown in greenthen based on prominent distributions of the volcanoplot we narrowed the candidate lipids increased inthe recurrent group to the following molecules fig biotinylbluephosphoethanolamineceramidecerd420 sphingomyelin smd351 cerd180_240pc monoether phosphatidylcholine mepc346echolesterol ester che241 mepc 408e and che20 as for the lipids that decreased in the recurrent groupthe following four molecules were annotated fig bluearrows pointing to green plots monohexosylceramidehex1cert421 otriglyceride tg150_140_140pc 182_182 and lysophosphatidylcholine lpc120biotinylpe303arrowsplotspointingtoredthe relative amounts of these lipid species were evaluated with their distributions by comparing the two 0ctakanashi bmc cancer page of table clinicopathological characteristics of the nonrecurrent and recurrent groupscharacteristicsmedian age rangenonrecurrent n “gender malefemalesmoking history ˆ’pathological stage iiimedian tumor size mm rangeadenocarcinoma subtypelepidicpapillaryacinarsolidlymph node metastasis ˆ’pleural invasion ˆ’lymphatic vessel invasion ˆ’blood vessel invasion ˆ’micropapillary component ˆ’spread through air space ˆ’driver gene mutationegfr ˆ’alk ˆ’surgical procedurelobectomywedge resectionadjuvant chemotherapyindication stage ia3iibreceivedrecurrent stylelocoregionaldistant “““recurrent n “ “pvalue““abbreviations alk anaplastic lymphoma kinase egfr epithelial growth factor receptroups fig 3a and b in all tested lipids distributionsbetween the two groups were well separated enough toestablish the cutoff values whereas only few markedoutliers were foundwe next calculated the cutoff values and auc of these lipids to evaluate their discrimination ability for diseaserecurrence and the following final candidates with topthree auc were selected smd351 cerd420 and tg 150_140_140 table respective lipidspecies can be found in additional file with the followingidentical numbers smd351 cerd420 andtg 150_140_140 these three final lipid candidates were annotated as the following ions [smd351 h] [cerd420 hcoo] and [tg 150_140_140 nh4] in the lipidsearch„¢ software additional file msms for [smd351 h] [cerd420 hcoo]and [tg 150_140_140 nh4] demonstrated production peaks corresponding to phosphocholine severalfragments compatible with fragmentation of cerd42 with concomitant oxidation reaction two fragmentsproduced by neutralfatty acid fa140 orfa respectivelyadditional file supplemental fig consequentlythe annotations ofthe final candidates by lipidsearch„¢ software were consistent with the results ofmsmsfrom tg 150_140_140loss ofamong these three candidate predictors smd351was found to be positively correlated with cerd420spearman™s rank correlation coefficient[rs] p tg 150_140_140 was inversely correlated with smd351 rs ˆ’ p and tg150_140_140 was weakly inversely correlated withcerd420 rs ˆ’ p additional file supplemental fig 0ctakanashi bmc cancer page of conventionalpathologicalvalidation of recurrence prediction ability among thefinal lipid candidatestable shows the sensitivity specificity and accuracyof the final candidate lipid predictors compared withfactorstheprognosticlymph node metastasis and blood vesselinvasionwhich were identified as significant recurrent factorsin this cohort sensitivity of all three candidate lipidpredictors is superior to that of lymph node metastasis patients with lymph node metastasis all of themwere hilar or lobar lymph node metastasis corresponded to those in stage ii among the recurrentgroup in this study cohort half of the study population had stage i whereas the other half had stage iias lymph node metastasis can be detected amongstage ii cases the sensitivity of lymph node metastasiswas consequently lower than those of three candidatelipid predictors which detected both stage i and stageii hencethese three predictors were superior tolymph node metastasis for patient screening whencomparing the candidate lipid predictors and bloodvesselshowed predictionto those of blood vesselabilities higher or equalinvasion only smd351fig comparison of total lipid levels between the recurrent andnonrecurrent groups the average total lipid level of the recurrentgroup was times higher than that of the nonrecurrentgroup p fig volcano plots of identified lipid species each plot represents a lipid species to be identified the relative amount of lipid speciesred plots were increased fc ‰¥ right side of in the horizontal axis pvalue in vertical axis and that of lipid species greenplots were decreased fc ‰ left side of ˆ’ in the horizontal axis pvalue in vertical axis in the recurrent group nineincreased lipids showing prominent distributions and all decreased lipid species were annotated for candidate predictors blue arrowsabbreviations cer ceramide che cholesterol ester fc folding change hex1cer monohexosylceramide lpc lysophosphatidylcholine mepcmonoether phosphatidylcholine pc phosphatidylcholine pe phosphoethanolamine sm sphingomyelin tg triglyceride 0ctakanashi bmc cancer page of fig comparisons of relative amount distributions between the nonrecurrent and recurrent groups are shown for increased a and decreasedb lipid species in the recurrent group boxplots show the upper percentile upper quartile median lower quartile and lower percentilemaximum and minimum values are shown in dots pvalues for significance and fcs are presented for each lipid species abbreviations cerceramide che cholesterol ester fc folding change hex1cer monohexosylceramide lpc lysophosphatidylcholine mepc monoetherphosphatidylcholine pc phosphatidylcholine pe phosphoethanolamine tg triglycerideinvasion in allvalidation points therefore wepropose smd351 as the most hopeful candidate forrecurrence predictiondiscussionin this study candidate lipid predictors for lung adenocarcinoma recurrence after a radical surgery were retrospectively screened and smd351 was found as themost prominent predictor showing that the predictionability was superior to that of conventional pathologicalprognostic factors in this small cohortthe average total lipid level was significantly high inthe recurrent group in this study furthermore thenumber ofincreased lipid species was considerablyhigher than that of decreased lipid species in the recurrent group these results were consistent with that of 0ctakanashi bmc cancer page of table auc rank of candidate lipid predictors determined byroc curverankcutoff valuespeciessmd351cerd420tg150_140_140cerd180_240pc182_182che241pc412biotinylpe303lpc120hex1cert421 omepc408eche201mepc346eauc ci “ “ “ “ “ “ “ “ “ “ “ “ “lipids with top three auc were selected as final candidate predictorsboldfaced notationsabbreviations auc area under the roc curve ci confidential interval rocreceiver operating characteristicprevious studies that showed an accelerated lipid synthesis in cancer cells contributing to tumor phenotypes such as cellular membrane building stimulationof signaling pathways for growth and proliferation orsurvival under hypoxic conditions by supporting glycolysis [ ] increased total lipid level in the recurrent group may be biologically plausible because theaggressiveness may be supported by accelerated lipidsynthesisthe number of smd351 and cerd420 two of finalcandidate predictors were increased in the recurrentgroup sm and cer are major bioactive components oflipid rafts on the cellular membrane sm is synthesized from cer by sm synthase sms which transfersthe phosphocholine head group from phosphatidylcholine to cer and results in concomitantly producingtable comparison of sensitivity specificity and accuracyamong the three final candidate predictors and conventionalhistopathological prognostic factorspredictors for recurrencecandidate lipid predictorsspecificitysensitivityaccuracysmd351cerd420tg150_140_140pathological prognostic factorslymph node metastasisblood vessel invasionsmd351 showed the most excellent prediction abilityabbreviations cer ceramide sm sphingomyelin tg triglyceridediacylglycerol dag sm reconversion to cer is catalyzed by sphingomyelinase smase increased smabundance and sms activity have been reported to playa critical role in cell proliferation and survival in severalcancer types [“] with regard to lung cancer metabolic changes in sphingolipids are suggested to correlatewith chemoresistance phenotype and the total smlevel in cancer tissues is reportedly lower than that ofthe normallung tissue in patients with nsclc this is speculated in the report that decreased sm abundance in lung cancer tissues may be attributable to highconsumption of serine precursor by highly proliferatingcancer cells cer accumulation in the lungs has beensuggested to participate in both cell apoptosis andtumorigenesis under cigarette smokeinduced oxidativestress taking together these knowledge and significant positive correlation between smd351 h andcerd420 in this study increased synthesis flow of certoward sm in the recurrent group was suggested actually significant increase on the total sm p leveland increased tendency on total cer p anddag p levels in the recurrent group were observed in this study cohort additional file supplemental fig this result supports the suggestion ofstrong synthesis flow of cer toward sm the sm andcer levels were not compared between the tumor tissuesand normal lung tissues in this study because normallung tissue samples were lacking nonetheless increasedsmd351 and cerd420 in the recurrent group in thisstudy is consistent with previous studies [“ ]based on the following explanation among lung adenocarcinomas with high sm and cer consumption casesthat can maintain increased sm and cer synthesis havehighly aggressive phenotypes resulting in recurrencedecreased tg 150_140_140 in the recurrent groupwas also included in the final candidate predictors although tg abundance in the lung cancer tissue has not yetbeen explored to date tg level in colon cancer is reportedto be lower as the disease progresses suggesting that energysupply for colon cancer with higher degree of malignancymay depend on tg hydrolysis inconsistent with theprevious study the total tg level in this study revealedno significant difference between the nonrecurrent and recurrent groups p possible explanation for decreased tg 150_140_140 in the recurrent group is thataggressive recurrent lung adenocarcinoma that may preferably consume specific tg species for energy supplythe difficulty of predicting lung cancer recurrenceusing histopathological prognostic factors may be partlyattributed to subjective judgement in addition althoughthe degree of histopathological prognostic factors widelyvaries their judgements have been performed qualitatively [ “] thereby these methods may hinder accurateretrospectiverecurrence prediction and its 0ctakanashi bmc cancer page of between representativerecurrentimages of papillarytype adenocarcinomavalidation conversely excellent prediction ability ofsmd351 that is superior to histopathological factorswas considered for its high objectivity and quantitativevalues actuallyit was difficult to predict recurrentprognosis objectively from the conventional histopathologicalthemost popular tissue subtype with no significant differenceand nonrecurrent cases whereas the mass spectrum intensitiesof [smd351 h] were markedly higher in the recurrent case to help recurrence prediction additional file supplemental fig furthermore as high smd351level was detected in all recurrent cases including stagei and stage ii cases with high specificity and accuracysmd351 was considered to be widely applicable for recurrence prediction in postoperative patients whounderwent radical surgeryseveral limitations in this study should be acknowledged first this retrospective study is performed on asmall sample size due to difficulty of obtaining frozensurgical specimens with clinical information that meetour inclusion criteria thereby verifying the reproducibility of using other validation cohorts was difficult thusidentified lipid predictors did not exceed above the œcandidate levels and further large cohort studies should beconducted to validate candidate predictors identified inthis study as rigid predictors for lung adenocarcinomarecurrencebecause a large number of candidate lipid species species relative to the small number of samplesize cases were screened for candidate predictorsone candidate that shows nearperfect discriminationability can be bound to be identified third adjacentnormal lung tissue samples were lacking hence the difference between the abundance of the identified candidate lipid predictors in the normal lung tissue of therecurrent group and that of the nonrecurrent groupwas not able to be compared fourth because the nonrecurrent group in this study included five cases that received adjuvant chemotherapy the nonrecurrent groupmay possibly include the recurrence highrisk casesamong them recurrence might be prevented by adjuvantchemotherapy moreover the nonrecurrent group inthis study included two cases with recurrence predictionpositive for smd351 additional file supplementalfig among the two cases one patient received adjuvant chemotherapy and the other did not the formercase may be considered as highly at risk for recurrencewhich was prevented by adjuvant chemotherapy thelatter may be an exceptional case that cannot be ruledout by smd351 fifth because lc“msms is not auniversal examination in the clinical field examining alarge number of surgical specimens for recurrence prediction using lc“msms is difficult to utilize thefindings of this study in a clinical field lipid predictorsshould be replaced with other molecules that can be examined by universal methods such as immunohistochemistry of sms or smase involved in the smmetabolism additionallyin thisstudy included histopathological type of adenocarcinomaonly as a topic for future study squamous cell carcinoma a major histological subtype behind adenocarcinomarecurrent predictorsshould be explored forthrough the lipidomic approachthe sample cohortswe propose that smd351 is a hopeful candidate predictor for lung adenocarcinoma recurrence after a radical surgery our findings provide novel insights on themechanisms oflung adenocarcinoma recurrence andcan contribute to the development of precise recurrenceprediction and qualified postoperative therapeutic strategy for lung adenocarcinomasupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020073061additional file supplemental table weights of the frozen tissuesamples each weight of the frozen t
Colon_Cancer
" when we encounter patients who present with both a neck mass and nephrotic syndrome bothmalignancy and kimura™s disease need to be evaluated as the therapeutic strategies differ vastly between themcase presentation we present the case of a 27yearold male patient with neck mass and nephrotic syndromethe presence of both eosinophilia and elevated immunoglobulin e levels were concerning for kimura™s diseasewhich is an allergic syndrome defined by eosinophilic granulomas of neck soft tissue along with peripheraleosinophilia the eventual final diagnosis however was sclerosing mucoepidermoid carcinoma of parotid glandwith both eosinophilia and membranous nephropathy following the surgical resection of the mass the nephroticsyndrome completely resolved detailed histopathological assessments of both the parotid gland and renal tissue were key aspects ofthe diagnosis and management to exclude kimura™s diseasekeywords membranous nephropathy nephrotic syndrome sclerosing mucoepidermoid carcinoma with eosinophilia membranous nephropathy can occur in the setting ofmalignant tumors and often recovers following definitivetreatment of malignancy sclerosing mucoepidermoidcarcinoma with eosinophilia is a rare variant of mucoepidermoid carcinoma for which surgical resection isgenerally recommended as a primary treatment thereare currently no published reports of nephrotic syndromeassociated with mucoepidermoid carcinoma kimura™s correspondence teimamumedutoyamaacjp1the second department of internal medicine university of toyama sugitani toyama toyama japanfull list of author information is available at the end of the disease which is a benign syndrome accompanied byeosinophilic granulomas of neck soft tissue often found inyoung men of eastasian descent sometimes accompaniesrenal disease and can be treated by steroid therapy [ ]we present here a young male patient who sufferedmucoepidermoid carcinoma of right parotid grand withlocalized spread to lymph nodes and secondary membranous nephropathy both of which had significant eosinophilic infiltration the presence of peripheral eosinophiliaand elevated immunoglobulin e level has a broad differential diagnosis with vastly different treatment pathways the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cfujioka bmc nephrology page of case presentationa 27yearold japanese male patient without a history ofany allergic syndromes was admitted to our institutewith bilateral peripheral edema proteinuria and swellingof the right parotid gland cytology of the parotid glandand lymph node biopsy showed no malignancy thougheosinophilic infiltration in the lymph node was observedhe was diagnosed with nephrotic syndrome with gg of creatinine of proteinuria gdl of serum albuminand mgdl of lowdensity lipoprotein mild peripheral eosinophilia μl and elevated immunoglobuline iuml were also present immunoglobulin gwas mgdl soluble interleukin receptor was uml c3 was mgdl c4 was mgdl andtotal complement activity was umlkidney sizes were mm right and mm leftwe performed a kidney biopsy to further investigate themechanism of the observed nephrotic syndrome onlight microscopy the glomerular basement membraneexhibited mild diffuse thickening with spike formationfig 1a eosinophilic interstitialinfiltration was alsoseen fig 1b immunofluorescence staining showed diffusegranular deposits of immunoglobulin g and c3 along theglomerular capillary walls fig 1c immunoglobulin g4 wasnot predominant for immunoglobulin g subclass and kappaand lambda light chains had equal intensity in the immunofluorescence staining immunofluorescence staining for thephospholipase a2 receptor pla2r and the thrombospondin type domaincontaining 7a thsd7a were negativethe electron microscopy showed globalsubepithelialelectrondense deposits and spike formation of the glomerular baseline membrane fig 1d he was diagnosed withstage ii secondary membranous nephropathycomputed tomography showed a mm of tumor inthe right parotid grand accompanied by a mmlymphoid focus fig both of which showed uptake byfluorodeoxyglucoseposition emission tomography weat this point suspected malignant disease instead of analternative benign presentation such as kimura™s diseaserepeat biopsies eventually demonstrated carcinoma andlymph node metastasis t3n2m0 stage ivabefore surgical excision steroid pulse therapy methylprednisolone mg — days was performed leading tothe partial reduction of proteinuria and eosinophilia atone month following pulse therapy the patient underwentsuccessful right parotidectomy with neck dissection theresected specimen was consistent with the diagnosis ofsclerosing mucoepidermoid carcinoma with eosinophiliaa variant of mucoepidermoid carcinoma pt3n2bfig the proteinuria achieved complete remissionand eosinophilia normalized without steroid therapyduring the 12month followup with concomitantpostoperative radiation therapy gy 30fr he hashad no recurrence of disease over twoyear followupfig histopathological findings in kidney biopsy specimen a diffuse thickness of the glomerular basement membrane with spike formationarrowhead periodic acidmethenaminsilver stain b infiltration of eosinophils in renal interstitium hematoxylineosin stain c diffuse granulardeposits of immunoglobulin g along the glomerular capillary walls immunofluorescence stain for igg d global subepithelial electrondensedeposits and spike formation of the glomerular baseline membrane electron microscopy 0cfujioka bmc nephrology page of fig head computed tomography shows tumor at right parotid gland a and lymphadenopathy of right neck bdiscussion and we diagnosed mucoepidermoid carcinoma with eosinophilia and excluded kimura™s disease with successivebiopsies and detailed histopathological assessment thepatient did clinically well following careful exclusion ofkimura™s disease for which the correct therapeutic pathway was surgical resection instead of steroid therapyboth kimura™s disease and mucoepidermoid carcinomawith eosinophila have several common characteristicsincluding neck mass with lymph node enlargementeosinophilia and high serum immunoglobulin e levelmucoepidermoid carcinoma is the most commonmalignancy among salivary glandorigin tumors andalthough rareit can occur in younger patients fig a tumor of right parotid gland hematoxylineosin stain showing invasion of tumor cells surrounded by sclerotic stroma and numerouseosinophils b normal tissue of right parotid gland hematoxylineosin stain also showing infiltration of eosinophil 0cfujioka bmc nephrology page of mucoepidermoid carcinoma has several variants sclerosing mucoepidermoid carcinoma with eosinophilia is oneof more rarely encountered variants the exact mechanism of why this carcinoma is associated with peripheraleosinophilia and infiltration is uncertain prior studieshave suggested that tumorderived cytokine release mightbe an important pathobiological mechanism [“]kimura™s disease is a rare syndrome accompanied byeosinophilic granulomas of neck soft tissue peripheraleosinophilia and high immunoglobulin e level it is mostoften observed in young eastasian males [ ] this is abenign chronic granulomatous disease characterized byfollicular lymphomas with eosinophilic infiltration thedisease can be treated by steroids or other forms ofsystemic immunosuppression prior reports suggestthat renal disease with nephrotic syndrome may occur inup to of patients with kimura™s disease the pathogenesis of kimura™s disease remains unknowntrauma infection an immunoglobulin emediated hypersensitivity reaction or an autoimmune process mightstimulate cytokine release that stimulates eosinophil production of note there are no published reports ofkimura™s disease in the presence of a confounding malignancy in this case atypical change with eosinophilic interstitial infiltration and negative expression of pla2r andthsd7a on renal biopsy suggested secondary membranous nephropathy instead of a de novo origin to our knowledge this is the first published report ofmucoepidermoid carcinoma associated with membranousnephropathy it is well known that patients with malignancies may have secondary membranous nephropathy “ cases have been reported in lung prostate andcolon cancers and even with hematologic malignancies the mechanism of why malignancyrelated secondarymembranous nephropathies can occur remains unknownbut several hypotheses are proposed antibodystimulation from tumor antigens immunologically similarto an endogenous podocyte antigen leading to in situ immune complex formations shed tumor antigens mightform circulating immune complexes that are subsequentlytrapped in the glomerular capillary wall circulating antibodies might also react to the tumor antigens that havealready been planted in the subepithelial location and an extrinsic process including viral infection or underlyingabnormal immune response might be responsible for bothdiseases in this case a tumorassociated immunoreactionmight have had a considerable impact on the developmentof a secondary membranous nephropathy the detailedmechanism of renal interstitial infiltration of eosinophilscontinues to remain uncertainthough tumorsecretedcytokine release might be the causative pathwayabbreviationspla2r phospholipase a2 receptor thsd7a thrombospondin type domaincontaining 7aacknowledgementsnot applicableauthors™ contributionshf contributed to analyzing and interpretation of data and writing themanuscript tk contributed to interpretation of data and writing themanuscript ti contributed to interpretation of data and writing themanuscript kka contributed to analyzing and interpretation of data andwriting the manuscript hy contributed to managing the patient andassessing the data ha contributed to managing the patient and operationtn contributed to analyzing and interpretation of pathological findings kkicontributed to analyzing and interpretation of data and writing themanuscript the authors read and approved the final manuscriptfundingthere was no fundingavailability of data and materialsall the date supporting our findings is contained within the manuscriptethics approval and consent to participatethe case report was approved by the medical ethics committee of theuniversity of toyama the patient provided written informed consent forparticipation in the study at university of toyamaconsent for publicationthe patient received all information regarding this case report writteninformed consent for publication in bmc nephrology was obtained from thepatientcompeting intereststhe authors declare that they have no competing interestsauthor details1the second department of internal medicine university of toyama sugitani toyama toyama japan 2department ofotorhinolaryngology head neck surgery university of toyama toyamajapan 3department of diagnostic pathology university of toyama toyamajapanreceived december accepted august referencesrow pg cameron js turner dr evans dj white rh ogg cs membranous nephropathy longterm followup and association withneoplasia q j med “kimura t on the unusual granulation combined with hyperplastic changesof lymphatic tissue trans soc pathol jpn kuo tt shih ly chan hl kimura's disease involvement of regional lymphnodes and distinction from angiolymphoid hyperplasia with eosinophiliaam j surg pathol “guevaracanales jo moralesvadillo r guzm¡narias g cavavergiº ceguerramiller h montesgil je mucoepidermoid carcinoma of the salivaryglands a retrospective study of cases and review of the literature actaodontol latinoam “urano m abe m horibe y kuroda m mizoguchi y sakurai k sclerosing mucoepidermoid carcinoma with eosinophilia of the salivaryglands pathol res pract “hu g wang s zhong k tumorassociated tissue eosinophilia predictsfavorable clinical outcome in solid tumors a metaanalysis bmc cancergatault s legrand f delbeke m loiseau s capron m involvement ofeosinophils in the antitumor response cancer immunol immunother “anagnostopoulos gk sakorafas gh kostopoulos p disseminatedcolon cancer with severe peripheral blood eosinophilia and elevated serumlevels of interleukine2 interleukine3 interleukine5 and gmcsf j surgoncol “ 0cfujioka bmc nephrology page of natov sn strom ja ucci a relapsing nephrotic syndrome in a patient withkimura's disease and iga glomerulonephritis nephrol dial transplant “ yamada a mitsuhashi k miyakawa y membranous glomerulonephritisassociated with eosinophilic lymphfolliculosis of the skin report of a caseand review of literature clin nephrol “terada n konno a shirotori k fujisawa t atsuta j ichimi r mechanism of eosinophil infiltration in the patient with subcutaneousangioblastic lymphoid hyperplasia with eosinophilia kimura's diseasemechanism of eosinophil chemotaxis mediated by candida antigen and il int arch allergy immunol suppl “ couser wg primary membranous nephropathy clin j am soc nephrol“leeaphorn n kue app thamcharoen n ungprasert p stokes mb knight elprevalence of cancer in membranous nephropathy a systematic review andmetaanalysis of observational studies am j nephrol “ beck lh jr membranous nephropathy and malignancy semin nephrol“publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
Colon_Cancer
" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearman™s correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days ˆ’ to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturer™s instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearman™s correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients™ cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time × power time × power–¼–¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci “ pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci “ pgml and pgml ci“ pgml respectively the median level of il p40 before the mwa treatment was pgml ci “ pgml there was a slight increase to pgml ci “ pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci “ pgml and increasedto pgml ci “ pgml days afterthe mwa treatment and to pgml ci “ pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci “ pgml there was a significant increase at h postmwa with a median level of pgml ci “ pgml the median level ofil1 before the mwa treatment was pgml ci “ pgml and a significantincrease wasnoted days after the mwa treatment pgml ci “ pgml the median level of il6before the mwa treatment was pgml ci“ pgml and significantly increased daysafter the mwa treatment pgml ci “ pgml the median level ofil8 before themwa treatment was pgml ci “ pgml and increased significantly to pgml ci“ pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci “ pgml and increasedsignificantly days after the mwa treatment pgml ci “ pgml the median level oftnfα before the mwa treatment was pgml ci “ pgml and increased significantlyto pgml ci “ pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of œenergy time × power time × power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional “10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aœheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ h postmwa pgml ci “ ci “ –¼ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa –¼ 2fold vs premwa days postmwa pgml ci “ ci “ ci “ ci “ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ days postmwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 it™s mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of œin situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaˆ’energyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearman™s rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the œenergyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wasœcancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a œcancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled œabscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as œdifferentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmed™s work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors™ contributionsjz conceptualization data curation writing“original draft and writing“review and editing ql conceptualization and writing“review and editingmm conceptualization and writing“review and editing brconceptualization and writing“review and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writing“review and editing rl conceptualization data curation formalanalysis visualization writing“original draft and writing“review and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the œsix one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett “bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144“rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol “yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res “lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218“jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine “ gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol “ ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology “ chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc “ huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres “ alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res “kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a “ onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother “ yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget “ yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490“erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol “ den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res “ zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology “ zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim
Colon_Cancer
" laparoscopic tumorspecific mesorectal excision tsme preserving the left colic artery and superiorrectal artery is still a technically challenging procedure we conducted this study to demonstrate the feasibility ofthis procedure for upper rectal cancermethods a total of patients with upper rectal cancer were retrospectively analyzed in our cancer centerbetween april and april these patients were treated with either laparoscopic tsme n orlaparoscopic total mesorectal excision tme n in the tsme group the left colonic artery and superior rectalartery were preserved while they were not in the tme groupresults the operation time in the tsme group was longer than that in the tme group ± min vs ± min p furthermore the number of resected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p the blood loss between the tsme and tmegroups was not significant no mortality occurred in either the tsme or tme groups one patient in the tme groupunderwent conversion to laparotomy the total postoperative complication rates in the tsme and tme groups were and respectively there was no difference in severe complications between the two groupsanastomotic leakage and stenosiss laparoscopic tsme preserving the left colic artery and superior rectal artery can be safely conductedfor upper rectal cancerkeywords laparoscopic surgery rectal cancer tumorspecific mesorectal excision superior rectal artery leftcolonic artery tme correspondence 237721898qqcom 250537471qqcomhuxiang_zc1978sinacom chi zhang haotang wei wenqing hu and yueming sun contributedequally as joint first authors7department of general surgery yizhen people™s hospital clinical medicalcollege yangzhou university yangzhou jiangsu province china3department of gastrointestinal surgery changzhi people™s hospital theaffiliated hospital of changzhi medical college changzhi shanxi provincechina1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang world of surgical oncology page of introductiontotal mesorectal excision tme is an important surgical technique to prevent the local recurrence of rectal cancer on the other hand tme may not besuitable for every case of rectal cancer such as rectosigmoid junction and upper rectal cancers the resection range of tme reaches cm below the inferiorborder of the tumor and has acquired an adequatecure rate reported in previous studies for patientswith rectosigmoid junction and upper rectal cancers this tumorspecific resection according to thetumorsite or t staging is called tumorspecificmesorectal excision tsme itsudeck™s critical point at the rectosigmoid junction isdescribed as the point of origin of the last sigmoid arterial branch originating from the inferior mesenteric artery ima the anastomosis between the lastsigmoidal artery and superior rectal artery sra is absent in some people to avoid the risk of postoperativeischemic necrosis anastomotic leakage colitis and delayed stricturetosudeck™s point for cases where anastomosis may be absent or insufficiently present in addition the rate ofis desirable to ligate proximalabsence of the left colic artery lca is which maybe associated with a risk of anastomotic leakage due toinsufficient vascularization of the proximal colonic conduit this study introduces the procedure and technicalpoints of laparoscopic tsme with preservation of thelca and sra the operation is still a technically challenging procedure we conducted this study to demonstrate the feasibility of this procedure for upper rectalcancer and shortterm prognosismethodspatientslaparoscopic tsme preserving the lca and sra wasperformed on patients with upper rectal cancer fromapril to april in the same period patients with upper rectal cancer underwent standardtme surgery this study was conducted in accordancewith approved guidelines this study was approved bythe institutional review board of the first affiliatedhospital of dalian medical university written informedconsent was obtained from all patientsfig ima 3d cta 0czhang world of surgical oncology page of equipmentangled ° 10mm diameter 3d laparoscope insufflation equipment and bipolar electrosurgical deviceaesculap german harmonic vascular closure systemjohnson usa 10mm and 5mm port trocars teleflexmedical usa laparoscopic linear staplers mm inlength covidien usa hemolock polymer lockingsurgical clips teleflex medical usa and a circularstapler ethicon endosurgery usa were used in thisstudypreoperative preparationinferior mesenteric artery ima 3d cta examinationshould be performed before the operation to assess themesenteric vascular vessel types fig intestinal preparation was performed days before the operation andprophylactic intravenous antibiotics were used beforethe operation for min central venous catheterizationwas performed after general anesthesia the surgicalposture was the starboard lithotomy position with thehead lower and feet higherthe operating surgeon and camera assistant stood onthe patient™s right side and the first assistant stood atthe patient™s left foot side the laparoscopic monitor wasplaced on the patient™s right foot side the trocar for thelaparoscope was inserted from the right paraumbilicalside and four ports were used as working ports fig surgical techniquesthis surgical technique was characterized by thoroughlymph node dissection based on neurovascular preservation and dissection of the left colon and sigmoid andfig position of the trocarupper rectal vessels along the inferior mesenteric vesselsthe region of operation was the superficial layer of thenerve sheath on the vascular surface the left colonicand superior rectal vessels needed to be preserved andthe vascular branch from the sigmoid vessels and theblood vessels from the superior rectal vessels to the intestinal wall were selected and severed according to thetumor positionfirst we adopted a lateral approach by opening themonks™ white line along the descending and sigmoidcolon reaching the splenic flexure as the cephalad dissection point the correct plane of dissection wasachieved by toldt™s fascia we usually used bipolar electrosurgical devices and bipolar scissors to separate thiscorrect plane with gentle blunt and sharp dissectionthe ureter and other retroperitoneal structures weresafely protected by staying in this plane we continuedto dissect along the plane to the root of the ima thehypogastric nerves were visible the nerves were carefully protectedthen the dissection began at the position of the sacralpromontory the junction of the sigmoid mesentery andretroperitoneum from the previous dissection plane in thefirst step ideally we dissected the presacral space belowthe sra from the left side across the midline to the rightside attentively protecting the hypogastric nerves whileusing a bipolar electrosurgical device fig 3a the distaldissection endpoint was approximately “ cm below thetumor we needed to open the peritoneal reflection anddissect the lateral ligament of the rectum by protectingthe neurovascular bundle nvb using a harmonic vascular closure system in some patients we placed the dissected colon and mesocolon to the right celiac side andthoroughly revealed the left side of the mesocolon wecarefully employed dissection in the correct plane on thevessels to avoid tissue damage for the realization of enbloc resection the technique in this step is to identify therelationship between the left colic artery inferior mesenteric vein imv to the ima and sra and the branch ofthe arteriae sigmoideae fig 3b this vascular bundle canbe traced from the origin of the ima to the rectal segmentapproximately “ cm below the inferior border of thetumor fig 3cthe second step was performed using a medial approach this step involved thorough lymph node dissection based on neurovascular preservation the leftcolonic and superior rectal vessels need to be preservedand the sigmoid vessels and vessel branch from the superior rectal vessels to the intestinal wall were selectedand severed according to the tumor positiondissection at the correct presacral space and cephaladdissection to the ima could be employed our generalmedial approach was to begin at the presacral space andobtain a connection with the plane ofthe lateral 0czhang world of surgical oncology page of fig a dissection the presacral space below the superior rectal artery sra approached from the left side across the midline to the right sideattentively protected hypogastric nerves while using a bipolar electrosurgical device b identification of the relationship between left colic arteryimv to the ima and sra and the branch of the arteriae sigmoideae c tracing this vascular bundle from the origin of the ima to the rectumsegment approximately “ cm below the inferior border of the tumor d ligation of arteriae sigmoideae and vascular branch from sra eligation of arteriae sigmoideae and preserving left colonic vasculature f excision of the mesorectum just underneath the rectal wall about “cm and avoiding injury to the rectal wall and sra g tsme preserving left colic artery and superior rectal arteryapproach pelvic dissection was performed from the entrance of the pelvic cavity down to the pelvic floor wecould identify both the hypogastric nerve fibers and pelvicnerve by using highdefinition 3d laparoscopy and preservethem the imvleft colic artery bundle was then carefullytraced to the junction position from the ima and lymphnode no253 was dissected the pelvic nerves and ureterwere already carefully insulated and the circumference ofthe ima could be revealed the mesocolon could be freedfrom the retroperitoneal position by anterior dissection bygently applying a bipolar electrosurgical device we dissected the sra and blood vessels from the sra to the intestinal wall and dissected lymph nodes no252 andno251 at this point we had completed lymph node dissection and completely clarified the relationship betweenthe lca imv ima sra and arteriae sigmoideae finallywe ligated the arteriae sigmoideae and vascular branchfrom the sra into the intestinal wall fig 3d while preserving the left colonic vasculature fig 3e energy devicesand hemolocks were used widely in this step 0czhang world of surgical oncology page of after the above procedure was completed we separated the rectal wall from the mesorectum with an adequate distance from the tumor in accordance with thet stage and position of the tumor using a harmonic vascular closure system in order to provide enough spaceto insert an endoscopic linear stapler we excised themesorectum about “ cm just underneath the rectalwall fig 3f careful surgery was performed to avoid injury to the rectal wall and sra then the endoscopic linear stapler was fixed the rectum was transected andsatisfactory tsme preservation of the left colic and superior rectal arteries was shown fig 3glastly a small 5cm incision was made at the leftlower abdomen and the specimen was taken outside ofthe abdomen and transected intraabdominal presacralanastomosis was performed by double stapling techniques after inserting the anvil head of a 28mm circularstapler into the oral side of the sigmoid colon doubledrains were placed and no diverting stoma wasperformedin the tme group the inferior mesenteric artery wassevered at the root the colon was severed cm awayand digestive tract reconstruction methods were similarto the tsme groupstatisticsspss190 version was used for statistical analysis categorical variables were compared using a χ2 test continuous variables were presented as the mean standarddeviation or median range these variables were compared using a mannwhitney u test p values of were considered statistically significantresultsthe general characteristics of the included patients arelisted in table there were men and women in the tsme group and men and women in the tme group the meanage was ± years and ± years in thetsme and tme groups respectively there were no significant differences in preoperative comorbidity tumorsize depth of invasion and lymph node metastasis between groups the average distance between the tumorand anus of the tsme group was ± cm andthe distal margin was ± cm the pathologicalstages of the patients for the tsme group were as follows stage i stage iia stage iib stage iic stage iiia and stage iiib theproportion of patients with normal preoperative carcinoembryonic antigen cea was approximately of patients had cea levels between and ngml of patients had cea levels between and ngml and only patients had cea levels ngmltable clinicopathological features between the tsme andtme groupsfactorsage yearstme n ± tsme n ± p valuegendermalefemalebmi kgm2comorbidity ± ± cardiovascular disease respiratory diseasediabetes mellitushistological type differentiated typeundifferentiated type tumor size mm ± ± t categoryt1t2t3t4n categoryn0n1n2 conversion to open surgery operation time min ± ± blood loss ml ± ± lymph node dissection ± ± the operation time in the tsme group was longerthan that in the tme group ± vs ± p table furthermore the number ofresected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p table the blood loss between groupswas not significantly different table the averagehospital stay in the tsme group was a little shorter thanthat in the tme group ± days vs ± days table no mortality occurred in either group one patient inthe tme group underwent conversion to laparotomy dueto bowel ischemia in the distal colon table the totalpostoperative complication rates in the tsme and tmegroups were and respectively table forsevere complications between the two groups anastomotic leakage and stenosis the severity of complicationswas claviendindo classification grades “ and therewas no significant difference between groups 0czhang world of surgical oncology page of tsme n tme n p value ± table postoperative complicationsfactorspostoperative hospital stay days ± mortalitymorbidityabsentpresentanastomotic leakagebleedingabdominal abscessileuswound infectionanastomotic stenosisurinary tract infectionascitesurinary retentionpneumoniacardiacrelated complications discussionin the british surgeon heald proposed tme for rectalcancer and pointed out that the anatomical level of tmewas clear so that the operative quality can be assessed the main concerns were a higher anastomotic leakage ratelonger operative time and higher blood loss after tme lopezkostner pointed out that tme was the standard operation performed for lower rectal cancers tme isnot necessary for cancers of the upper rectum therefore the tsme technique was introduced to achieve satisfactory local control and low morbidity partial mesorectalexcision is applied in tsme according to willian™s report in only of patients had distal intraluminal diffusion cm pollett and nicholls observed that there were no differencesin the local recurrence rate of rectal cancer between distal margins cm “ cm and cm a randomized prospective study of nsabb the national surgicaladjustburst and bowel project showed that the localrecurrence rate was not significantly different betweendistal rectal margins cm “ cm and cm according to the practice parameters for the management of rectal cancer edition a 2cm distal margin is more acceptable than cm but a 5cm distalmargin is still recommended total mesorectal resectiontme should be used for tumors located in the middleand lowerregardless oftwothirds ofwhether itis performed with low anterior resectionlar or combined abdominal and perineal resectionapr for tumors in the upper onethird of the rectumresection of the mesentery can be carried out accordingto the tumor situation and the distance between thethe rectumdistal margin and tumor should be cm the recommended grade was 1a tme was performed according to the distance between the distal margin of the rectal tumor and anus cm while tsme was performed for patients with adistance between the distal end of the rectal tumor andanus of “ cm in the author™s medical departmentoncological outcomes after surgery can be divided intotwo aspects longterm survival and local recurrence ratelaw reviewed patients the 5year local recurrence rate for tme and partial mesorectal excisionpme for proximal cancer was and respectively the disease stage was associated with a higher riskof local recurrence there was no difference in the localrecurrence rates of tme and pme the 5year cancerspecific survival rates with and without tme were similarat and respectively kim reportedthat the 5year cancerspecific survival rate was andthe local recurrence rate was with cases of rectalcancer after tsme with pathologic stages i“iii the riskfactors affecting cancerspecific survival rate were the ptstage pn stage positive distal resection margin and positive circumferential resection margin the risk factors affecting local recurrence were the pn stage positive distalresection margin and positive circumferential resectionmargin another study from a korean reviewed experience in patients with rectal cancer showed that theoverall local recurrence rate was the 5year local recurrence rates were and in stages i iiand iii respectively the 5year cancerspecific survivalrates were and in stages i ii and iiirespectively the risk factors were the pn stage and circumferential resection margin zakir performed an analysis with years of experience in rectal cancer patients who underwent laparoscopic andopen tsme surgery the 5year local recurrence rate was the overall 5year and cancerspecific survival rateswere and respectively there was no difference in the local recurrence rate between laparoscopic oropen resection the overall and cancerspecific survivalrates were and in the laparoscopic surgerygroup and and in the open surgery group respectively the results showed that laparoscopic surgerywas better than open surgery in overall and cancerspecific survival there was no difference in survival in patients with stage i however the survival rates in patientswith stages ii and iii among the laparoscopic surgerygroup were better than those in the open surgery groupwhich shows the superiority of laparoscopic tsme surgery for the longterm prognosis of rectal cancerkorean scholars conducted a study on the safety andprognosis of tsme after neoadjuvant chemotherapy forrectal cancer patients received 5fu with leucovorinchemotherapy and radiotherapy cgy for cycles 0czhang world of surgical oncology page of leadership was tsme was performed “ weeks later the resultsshowed that the overall complication rate was empiricalinternal construction was the 5year survival rate was and the 5yeardiseasefree survival was at present chinasouth korea and the usa have formulated similarguidelines for preoperative radiotherapy and chemotherapy for middle and low rectal cancer but there is nospecific reference data for preoperative radiotherapy andchemotherapy for upper rectal cancer the purpose ofthis paper is to introduce a new method of tsme anddiscuss the safety of the operation longterm survivaland local recurrence have not been discussedtsme surgery based on tme is now accepted as astandard for rectal cancer surgery and laparoscopic rectal cancer resection is accepted widely in the world eventhough it is a challenging procedure for surgery bloodloss in the laparoscopic group is well shown with anaverage of to ml the average blood loss inour study was ml lower than that reported in the literature we can identify neurovascular lesions usinghighdefinition 3d laparoscopy to preserve them and weuse a bipolar electrosurgical device to reduce injurywhich is beneficial for accurate operationthe overall complication rate in laparoscopic tsmeoperation was lower than that in the open operationgroup the rate of anastomotic leak showed no statistical difference between the two operation methods theaverage leak rate for rectal cancers was zakir reported that the overall complicationrate was in tsme for rectal cancer patients therate of anastomotic leakage was in the open tsmegroup and in the laparoscopic tsme group therewas no statistical difference between groups in our studythe incidence rate of postoperative anastomotic leakagewas three patients had complications after surgeryand the overall complication rate was the threecomplications were wound infection fluid collection andurinary retention with a claviendindo grading of “yoo evaluated the optimal duration of urinarycatheterization after tsme for rectal cancer logistic regression analysis was performed to determine the risk factors for urinary retention the variables including age sexasa grade surgical procedure tnm stage tumor position preoperative radiotherapy duration of urinarycatheterization and time of surgery were not significantrisk factors for urinary retentionat present a 3d laparoscopic system aesculapgerman is used in laparoscopic surgery in our department single and reduced portlaparoscopic surgeryrobot operations and tatme operations are not usedfor tsme the surgeons who performed tsme had morethan years of experience in gastroenterostomy and hadexperience with open tsme the difficulty of the tsmeoperation is the management of the mesorectum seiji has reported on the management of the mesorectumin the narrow pelvis which our treatment method is basedon first the right part of the mesorectum is lifted fromthe right side of the sigmoid mesocolon to expose the inferior mesenteric artery and vein left colonic vessels sigmoid colonic vessels and superior rectum vessels theassistant lifts the left mesentery of the sigmoid colon exposes the above vessels expands the sigmoid mesocolonagain penetrates the mesentery from the right side andexposes the surrounding vessels expansion of the pelviccavity along the vessels is continued and the mesorectumis repaired from the left to the right side “ cm above thetumor according to the location ofthebranches of the severed vessels are determined and “cm of the intestinal wall is repaired the rectum is dissected using an endogia staplerthe tumorlaparoscopic tsme has been used for rectal cancer andcan obtain satisfactory functional results compared to openresection and tme we do not think that the reduction inthe hospital stay is due to the acceleration of the intervention as per enhanced recovery after surgery eras butis due to an increase in the doctors™ confidence in reducingthe risk of postoperative complications after vascular preservation threedimensional cta examination is importantfor the preoperative evaluation of sigmoid colonvascular classification and intraoperative management ofthe sigmoid and left colon vessels however preoperativeexamination could not obtain information on the trafficbranch the biggest advantage of this operation is themaintenance of the blood supply of the proximal and distalintestines and the sufficient length of the intestine so thereis no need for temporary defunctioning stoma temporarydefunctioning stoma only increases the complexity of theoperation and closure of the temporary stoma increasesthe risk of complications in addition the results of the statistical analysis showed that the number of lymph nodes inthe tsme group was greater than that in the tme groupit cannot be concluded that tsme was significantly betterthan tme for lymph node dissection suggesting thattsme was not inferior to tmeslaparoscopic tsme with preserved left colic and superior rectal arteries is a technically challenging procedureintact visceral pelvic fibro is protected with even greateraccuracy than other techniques by 3d laparoscopywhich offers an optimal vision tsme with preserved leftcolic and superior rectum arteries did not increase therisk of operation compared with tme but increased thesurgeon™ s confidence in patient outcomes thereforelaparoscopic tsme with preserved left colic and superior rectal arteries can be safely performed for rectal cancer patients as an alternative to tme 0czhang world of surgical oncology page of abbreviationstme total mesorectal excision tsme tumorspecific mesorectal excisionima inferior mesenteric artery imv inferior mesenteric vein sra superiorrectal artery nvb neurovascular bundle pme partial mesorectal excisionacknowledgementsnoneauthors™ contributionslz yx and xh designed the study cz yx hw qz zd and whcollected and analyzed the data ma lz ys and xh interpreted the datalz and cz drafted the manuscript lz ma yx and xh revised themanuscript the authors read and approved the final manuscriptfundingthis study was supported by the jiangsu natural science foundationbk20180274 this funding supported the collection analysis andinterpretation of the dataavailability of data and materialsall experimental data used to support these findings are included in theethics approval and consent to participatethis study was approved by the institutional review board of the firstaffiliated hospital of dalian medical university written informed consent forpublication was obtained from all patientsconsent for publicationwritten informed consent was obtained from the patients and legalguardian for the publication of these patientscompeting intereststhe authors declare that they have no conflicts of interestauthor details1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province china 2department ofgastrointestinal surgery the third affiliated hospital of guangxi medicaluniversity nanning guangxi province china 3department ofgastrointestinal surgery changzhi people™s hospital the affiliated hospital ofchangzhi medical college changzhi shanxi province china 4department ofcolorectal surgery the first affiliated hospital of nanjing medical universitynanjing china 5department of gastrointestinal surgery the first people™shospital of dali city dali yunnan province china 6department ofgastrointestinal surgery graduate school of medicine university of tokyotokyo japan 7department of general surgery yizhen people™s hospitalclinical medical college yangzhou university yangzhou jiangsu provincechinareceived february accepted august heald rj husband em ryall rd the mesorectum in rectal cancer surgerythe clue to pelvic recurrence br j surg “ macfarlane jk ryall rd heald rj mesorectal excision for rectal cancerlancet “lee ky factors influencing oncologic outcomes after tumorspecificmesorectal excision for rectal cancer j korean soc coloproctol “ williams ns dixon mf johnston d reapppraisal of the centimetre rule ofdistal excision for carcinoma of the rectum a study of distal intramuralspread and of patients™ survival br j durg “ pollett wg nicholls rj the relationship between the extent of distalclearance and survival and local recurrence rates after curative anteriorresection for carcinoma of the rectum ann surg “ wolmark n fisher b wieand hs the prognostic value of the modificationsof the dukes™ c class of colorectal cancer an analysis of the nsabp clinicaltrials ann surg “ monson jrt weiser mr buie wd chang gj rafferty jf prepared by thestandards practice task force of the american society of colon and rectalsurgeons practice parameters for the management of rectal cancerrevised dis colon rectum “law wl chu kw anterior resection for rectal cancer with mesorectalexcision a prospective evaluation of patients ann surg “kim sh bae kb kim jm oncologic outcomes and risk factors forrecurrence after tumorspecific mesorectal excision of rectal cancer cases j korean soc coloproctol “kim nk min bs kim js hur h lee ky sohn sk oncologic outcomesand safety after tumorspecific mesorectal excision for resectable rectalcancer a single institution™s experience with patients with rectalcancer j korean soc coloproctol “ zakir k mohamed wai lun law outcome of tumorspecific mesorectalexcision for rectal cancer the impact of laparoscopic resection world jsurg “kim nk baik sh seong js oncologic outcomes after neoadjuvantchemoradiation followed by curative resection with tumorspecificmesorectal excision for fixed locally advanced rectal cancer impact ofpostirradiated pathologic down staging on local recurrence and survivalann surg “ poon jt law wl laparoscopic resection for rectal cancer a review annsurg oncol “ yoo be kye bh kim hj early removal of the urinary catheter aftertotal or tumorspecific mesorectal excision for rectal cancer is safe discolon rectum “seiji o takashi t kazuki s a new laparoscopic surgical procedure toachieve sufficient mesorectal excision in upper rectal cancer int j surgoncol httpsdoi1011552011708439publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesenker w e thaler h t cranor m l polyak t total mesorectal excision inthe operative treatment of carcinoma of the rectum j am coll surg “lopezkostner i lavery c hool gr rybicki la fazio vw total mesorectalexcision is not necessary for cancer of the upper rectum surgery “zaheer s pemberton jh farouk r dozois rr wolff bg ilstrup d surgicaltreatment of adenocarcinoma of the rectum ann surg “sudeck p ueber die gefässversung des mastdarmes in hinsicht auf dieoperative gangrän muenchen med wschr “van tonder jj boon jm becker jhr anatomical considerations onsudeck™s critical point and its relevance to colorectal surgery clin anat“cirocchi r randolph j cheruiyot i systematic review and metaanalysis of the anatomical variants of the left colic artery color dis httpsdoi101111codi14891 0c"
Colon_Cancer
" preproofsevere acute respiratory syndrome coronavirus sarscov2 is a highly contagious zoonotic pathogen that has exacted heavy public health social and economic tolls in february the world health anization acronymed the disease caused by sarscov2 as covid19 for coronavirus disease the number of confirmed covid19 infections which has been detected in at least countries has reached worldwide as of april with deaths according to the us centers for disease control and prevention cdc1 many cases of covid19 resolve quickly however the disease which like other respiratory pathogens that cause common cold symptoms is believed to be transmitted through respiratory droplets infection with covid19 can also lead to significant morbidity and death this is particularly the case for cancer patients moreover because the signs and symptoms of covid are easily misattributed to the sequelae of cancer itself such as pulmonary embolism or its treatment such as nausea and diarrhea diagnosis may be delayed or missed potential covid rule out criteria based on the wells˜ criteria for pulmonary embolism another protean disease entity are provided as a decisionmaking aid this review summarizes the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis rationale to treat the cancer or not treatment and prevention of covid19 with an emphasis on implications in cancer keywords covid19 sarscov2 cancer introduction sarscov2 the rna virus responsible for the illness which has been named covid19 for coronavirus disease2019 the year it was diagnosed has sent shockwaves and dominated the news cycle due to its pandemic spread from the point of origin in wuhan china to the rest of the world declared a public health emergency of international concern pheic by the world health anization who2 sarscov2 is the third highly pathogenic novel zoonotic bat coronavirus sonamed because of the crownlike spikes on its surface to have emerged the first was sars coronavirus now named sarscov1 in with a fatality rate of and 0c preproofthe second was middle east respiratory syndrome mers in with a fatality rate of where the camel was the intermediate host3 sarscov1 merscov and sarscov2 belong to the betacoronavirus genus which are enveloped positivestranded rna viruses whose approximately nucleotide genome serves as an mrna template for the translation of viral proteins4 the virion contains four proteins spike envelope membrane and nucleocapsid and the host receptor with which the spike surface glycoprotein of sarscov2 engages is angiotensin converting enzyme ace25 the biology of sarscov2 is described in more detail in figure a zinc metallopeptidase enzyme ace2 which is abundantly present in lung and gastrointestinal epithelial cells6 not only mediates viral entry through receptormediated endocytosis7 but also the efficiency of viral replication8 its expression is upregulated with older age smoking the antihypertensives angiotensin converting enzyme ace inhibitors and angiotensin receptor blockers arbs thiazolidinediones tzds a class of oral antidiabetic drugs and ibuprofen risk factors which may increase susceptibility to the covid19 virus infection and which are common in generally elderly multimorbid cancer patients9 preproofthe clinical spectrum of sarscov2 ranges from mild upper respiratory tract infection with fever sore throat headache cough and potentially nausea and diarrhea the majority of cases recover without serious complications to severe pneumonia with sequelae that include acute respiratory distress syndrome ards cytokine storm and death10 because sars is an acronym for severe acute respiratory syndrome digestive manifestations including inappetence nausea abdominal pain and diarrhea of covid19 resulting from binding of the virus to the ace2 receptor in the gi tract may precede respiratory symptoms11 gastrointestinal manifestations are potentially underappreciated and overlooked as sentinel symptoms that herald the onset or persistence of disease especially in cancer patients and may contribute to delayed or missed opportunities for testing diagnosis and containment unlike sarscov1 which seems to have disappeareddied out12 and mers which reappears only sporadically sarscov2 is less lethal with a fatality rate between although this number is highly uncertain and debated13 but much more infective consequently public panic and economic disruption have ensued resulting in wartimelike mobilization efforts to mitigate its spread old age smoking and comorbidities such as diabetes morbid obesity immunosuppression frailty and cardiovascular disease appear to predispose to worse outcomes possibly secondary to impaired t and b cell responses notably covid19 infection is associated with lymphopenia and delayed development of the adaptive immune response which appears to correlate with prolonged virus clearance and more severe disease progression15 the first pillar of defense against infection is hand washing avoidance of face touching and minimization of close contact ie social distancing with selfquarantine and selfisolation16 in case of exposure or evidence of covid19 symptoms respectively the second prophylactic pillar is vaccination with specific viral antigens or mrnas which are not yet publicly available although the company moderna has reportedly started testing an mrna vaccine in healthy volunteers and multiple other vaccination strategiesplatforms appear to be in progressunder development17 0c transmission and prevention according to the world health anization who21 sarscov2 is spread persontoperson mainly via aerosol inhalation from sneezing coughing or exhalation 22and via fomitetoface contact since depending on the surface material the virus may remain viable and infectious for hours to days figure fecaloral transmission has also been hypothesized because diarrhea was a common feature with sars and mers and diarrhea and other digestive issues have also been reported in patients with covid1925 26notably transmission of sarscov2 is not limited to symptomatic individuals ie those with fever cough sore throat myalgias or dyspnea but also to asymptomatic or subclinically infected carriers of the virus which is problematic from the perspective of disease control 27and highlights the importance of containment measures including isolation and quarantine the basic reproduction number r0 of sarscov2 is which means that on average for every patient an additional individuals are infected because coronaviruses may persist on inanimate surfaces like metal glass or plastic for up to days careful disinfection with or greater ethanol for small surfaces or sodium hypochlorite for larger surfaces is recommended preproofdiagnosis and clinical features preproofthe available evidence is limited but clinical courses and outcomes of covid19 are likely to be worse in patients with cancer especially given the clear association between severity of disease and older age and higher levels of comorbidity the overall case fatality rate cfr for the general covid19infected population is around but in cancer it rises to overall and to in italy19 this cfr in cancer patients compares to for no comorbid conditions for cardiovascular disease for diabetes for hypertension and for chronic respiratory disease the aim of this review is to summarize and condense the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis treatment and prevention of covid19 with a special focus on cancer in the united states the test of choice for sarscov2 is a nasopharyngeal swab specimen or sputum if a productive cough is present on which a reversetranscriptase pcr rtpcr assay or an enzymelinked immunoassay eia directed particularly at the envelope e rnadependent rna polymerase rdrp spike protein s and nucleocapsid n genes is performed the fda has also approved an antibody test29 a positive test for sarscov2 in a symptomatic patient generally confirms the diagnosis of covid19 with the caveat that false positive and false negative tests have been documented if initial testing is negative but a high index of suspicion and pretest probability for covid19 remains on the basis of patient signs and symptoms then retesting is indicated in patients with high indexes of clinical suspicion and equivocal or negative test results the who recommends that lower respiratory tract specimens which contain the highest viral loads should be obtained since nasopharyngeal swabs may miss some infections30 according to cdc guidelines disease is excluded on the basis of two 0c preproofconsecutive negative tests respiratory tests separated by ‰¥ hours however in the presence of suggestive symptoms rectal swabs may also be indicated since the ace2 enzyme to which the virus binds is abundantly present in rectal epithelia cells31 the differential diagnosis for sarscov2 in cancer is extremely broad and includes conditions such as foreign body aspiration toxicities from chemotherapy and radiation tumor progression postobstructive pneumonia malignant obstruction atelectasis pulmonary embolism pneumonitis pulmonary edemafluid overload immunotherapyrelated pneumonitis copd exacerbation q fever adenovirus bocavirus coronavirus 229e hcov 229e coronavirus hku1 hcov hku1 coronavirus nl63 hcov nl63 coronavirus oc43 hcov oc43 human metapneumovirus hmpv influenza a influenza a subtype h1n1pdm09 influenza a subtypes h1 and h3 influenza b parainfluenza virus ““ piv “ respiratory syncytial virus ab rsv ab rhinovirusenterovirus hrvev bordetella pertussis legionella pneumophila and mycoplasma pneumoniae32 the diagnosis of sarscov2 is complicated by the possibility of simultaneous coinfection with other respiratory viruses33 which is especially true for immunosuppressed cancer patients whose susceptibility to microanisms is increased the heightened infectious risk for cancer patients underscores the importance of screening them at presentation with extended viral respiratory panel testing given that coinfection may impact management decisions since conceptually at least the morbidity of covid19 and the risk of severe illness should increase in the presence of a second or third virus preproofunlike infection with influenza for example covid19 signs and symptoms may vary considerably depending on the dose of viral inoculum route of inoculation concomitant medications and underlying health status34 to include fever dry cough fatigue sputum production shortness of breath sore throat headache myalgia or arthralgia chills nausea or vomiting nasal congestion diarrhea hemoptysis and conjunctival congestion with an incubation period of to days after exposure36 presymptomatic or minimally symptomatic infection may majorly drive transmission especially since detected viral loads are similar in both symptomatic and asymptomatic patients3738 populations of concern include the elderly smokers vapers and dual users those of any age with preexisting chronic medical conditions those receiving particular medications or therapies which upregulate the ace2 receptor or suppress the immune system and those from lower socioeconomic classes a conglomeration of factors which are often present in cancer patients as depicted in figure while the surveillance focus for covid19 is on the respiratory tract enteric symptoms are a potentially underappreciated overlooked and misattributed manifestation of disease as stated earlier and this is especially the case for cancer patients where gastrointestinal toxicity occurs routinely from chemotherapy ie cisplatincarboplatinoxaliplatin irinotecan 5fluorouracil ifosfamide from targeted agents ie erlotinib imatinib bortezomib temsirolimus sunitinib regorafenibsorafenib and bevacizumab39 and from locally advanced or metastatic disease therefore abdominal complaints in cancer patients which are potentially but not automatically attributable to underlying disease justify further investigation especially if persistent worsening or new particularly because sarscov2 transmission may occur via the fecal“oral route40 0c preproofabnormal laboratory findings in covid19 include lymphopenia percent prolonged prothrombin time percent elevated lactate dehydrogenase percent elevated ast and alt “ percent elevated highly sensitive hs crp and elevated procalcitonin however because these parameters routinely fall well outside of the normal reference range in cancer patients it is difficult to confirm or refute the presence of disease on this basis alone chest radiographs and chest ct abnormalities are similarly nonspecific since the most common features multifocal groundglass opacities and consolidation mimic other pneumonias41 significant antibody production is observed after infection but it is unknown whether this helps or harms since antibodydependent enhancement ade may potentiate viral entry and the induction of a severe inflammatory response42 universal screening of cancer patients for covid19 is desirable but logistically impossible for the foreseeable future since diagnostic tests are in short supply and simply not always readily available43 hence covid19 rule out criteria are proposed in table as a potential decisionmaking aide mémoire which separates patients into low and highrisk groups by analogy to the wells˜ criteria for pulmonary embolism4445 preventive measures focus on selfisolation social distancing with a 6foot 2m separation46 frequent hand washing with soap and water andor use of hand sanitizers patient isolation during clinical care use of masks to help prevent aerosol transmission and flushing with the lid closed to control socalled •toilet plume– in an asco guidance immunocompromised cancer patients are advised to minimize exposure to sick contacts and large crowds48 for healthcare personnel the use of personal protective equipment such as n95 masks ffp3 masks gowns eye protection gloves and gowns is mandated49 preproof vaccination and immunity vaccination efforts and the related topic of whether those who have recovered from covid19 develop protective immunity have drawn great attention the latter has implications on whether people who test positive for sarscov2 antibodies can be safely assumed to be immune and at negligible risk of contracting or transmitting the disease there have been case reports of patients who have recovered from covid19 and had recurrence of rtpcr positivity approximately one month after initial diagnosis with only one patient exhibiting significant clinical symptoms and another having a mild intermittent cough50 but while not zero the risk of transmissibility or recurrence of symptomatic disease in recovered patients has yet to be quantified and the paucity of currently available reports of recurrence in the setting of a pandemic suggests that it is low a separate practical question will be whether antibodybased tests prove to have sufficient sensitivity and specificity to identify people who had asymptomatic infections developed immunity and can return to normal activities without jeopardizing disease containment efforts immunity may be due to antibodies cell mediated immunity or a combination of the two previous experience with using plasma from convalescent patients to treat severe cases of the first sars and mers as well as limited experience with covid19 suggests that antibody mediated immunity alone is clinically beneficial even during acute infection51 safety concerns about antibodies have been raised based on preclinical studies of sarscov vaccination in 0c preproofferrets showing hepatotoxicity52 and of vaccination against feline infectious peritonitis virus another coronavirus leading to more severe disease when kittens were subsequently challenged with the virus53 although animal models may not be representative of human hostpathogen interactions the nature of sarscov and sarscov2 antibodies are likely different as crossneutralization was not observed invitro54 and experience with convalescent plasma has not borne evidence of antibody mediated enhancement of infection in acutely infected patients the potential risk deserves attention if vaccination is proposed for the entire population t cell responses are also readily observable in patients who recover from coronavirus infections55 and memory t cell responses alone were protective in mice56 with the potential advantage of longer persistence of memory t cell responses compared to humoral immunity when clinical data on vaccine candidates becomes available cancer patients may face different considerations surrounding vaccination than the general population particularly patients with hematologic malignancies being treated with agents targeting b cells who would derive greater benefit from vaccines eliciting cell mediated than antibody responses preproofpathogenesis and pathology relating to ace2 and ras signaling the ace2 enzyme a key regulator of the reninangiotensin system ras57 to which the virus binds through its surface spike proteins is particularly abundant in the digestive tract lungs kidney heart and blood vessels where pathology from sarscov2 occurs58 a peptidase that catalyzes the conversion of angiotensin ii angii referred to as •the quintessential perpetrator of inflammation– to angiotensin ang ace2 mediates antiproliferative and vasodilatory functions that oppose the vasoconstrictive and inflammatory functions of angiotensin converting enzyme ace60 the binding of sarscov2 to ace2 leads to downregulation of ace2 expression potentially through increased internalization and shedding from the cell surface with decreased ang1“ generation and increased ang ii levels as a consequence61 this unfavorably shifts the balance of the renin angiotensin system ras from the vasoprotective ace2ang17 axis to the aceang iiangiotensin at1 receptor axis and drives a proinflammatory profibrotic and proliferative response62 as shown in figure fang et al63 contend that because thiazolidinediones ibuprofen and angiotensin converting enzyme ace inhibitors and angiotensin ii typei receptor blockers arbs substantially increase the expression of ace2 they facilitate sarscov2 infection and therefore the risk of severe and fatal covid19 in contrast alghatrif et al64 present a diametrically opposed hypothesis that downregulated ace2 signaling is responsible for sarscov2induced acute lung injury ali acute respiratory distress syndrome ards and cytokine storm and that aceis and arbs are beneficial precisely because they increase ace2 expression and activity furthermore according to alghatrif et al lower ace2 levels and hence higher baseline oxidative stress and inflammation6566 are present in older comorbid individuals such as cancer patients which renders them more susceptible to severe covid19 than younger noncomorbid individuals with increased ace2 levels and lower baseline inflammation as shown in figure furthermore low ace2 may promote tumor progression and conversely ace2 overexpression is associated with antiangiogenesis and tumor regression67 in summary then 0c preproofdespite the concerns and controversy68 surrounding the use and continuation of aceisarbs during the sarscov2 epidemic it is likely that the pros outweigh the cons especially in cancer patients due to their potential antitumor and anticovid19 effects69 in line with ras involvement emerging data suggest that sarscov2 infection may induce serious cardiovascular injury or exacerbate existing cardiovascular disease cardiovascular sequela includes heart failure arrythmias disseminated intravascular dissemination dic and troponin elevation which may closely correlate with disease severity and the likelihood of inhospital death70 liu et al71 propose a mechanism whereby the virus which lowers hemoglobin hb levels72 binds to the porphyrin of heme and displaces iron thereby compromising the oxygencarrying capacity of red blood cells and exacerbating the hypoxemia since chloroquine and the experimental anticancer agent rrx001 also bind to porphyrins they may competitively interfere with binding by the virus rationale for continuation or discontinuation of cancer therapy preproofthe benefitrisk calculus that informs the decision whether and how to treat with anticancer therapy falls into a •gray zone– about which no consensus exists leading to a therapeutic dilemma on the one hand zhang et al73 in annals of oncology reported a strong association in patients of them with lung cancer between antineoplastic therapy in the past days and severe effects of covid19 hr4079 ci p0037 on this basis the authors recommend treatment interruption dose reduction or substitution of cytotoxic chemotherapy with nonimmunosuppressive options eg checkpoint inhibitors if available especially in the case of lung cancer patients that are already prone to develop respiratory infections and complications74 similarly heavily immunosuppressed patients such as those who have undergone hematopoietic stem cell transplantation are also particularly susceptible to viral respiratory infections these findings are supported by a nationwide analysis of data75 in china from covid19 patients of which were diagnosed with cancer this patient cohort experienced a higher incidence of severe events vs p and the administration of chemotherapy or surgery was found to have increased the risk of death andor intensive care unit admission even after adjusting for age sex and comorbidities odds ratio or ci “ p while these studies are limited by small sample sizes the data suggests that cancer predisposes to more severe disease therefore since inperson contact increases the risk of transmission several institutions have mandated realtime video or telephone interactions alternatively referred to as telehealth77 postponed surgeries biopsies endoscopies scans and routine investigations when possible and in line with esmo guidelines78 encouraged conversion from the intravenous to the oral route eg 5fluorouracil to capecitabine etoposide and vinorelbine on the other hand the immediate existential threat of progressive disease for which death is an impending imminent certainty rather than a remote possibility in the absence of treatment likely outweighs the theoretical risk of sarscov2 infection even in lower risk disease for example in situ or localized prostate breast and head and neck cancer delayed treatment is 0c preproofpotentially conducive to tumor development and progression and thus may unfavorably impact prognosis79 hanna et al have proposed a triage strategy80 which prioritizes treatment for those patients with imminent risk of early mortality from acute leukemias aggressive lymphomas metastatic germ cell tumors oncologic emergencies such as spinal cord compression chemoradiotherapeuticresponsive cancers such as head and neck cervical and anal cancers and neoadjuvant or adjuvant therapyresponsive tumor types such as stage iii colon cancer and deprioritizes visits for surveillance and survivorship however in the absence of a •one size fits all– consensus recommendation which is unlikely since cancer is so genetically diverse and heterogeneous the decisionmaking process and the subsequent treatment plan are individualized and to be determined tbd on casebycase basis taking into account multiple factors including the risk of cancer recurrence if therapy is delayed modified or interrupted the type of therapy eg surgery radiation chemotherapy checkpoint inhibitors and stem cell transplantation extent of comorbidities concomitant medications patient preferences physicianpatient relationship race age the number of cycles of therapy completed and treatment tolerance in terms of specific cancerrelated conditions asco makes the following heavily qualified recommendations81 \uf0b7 growth factor prophylaxis for neutropenia and neutropenic fever even at lower levels of risk as well as empiric antibiotics for acute care \uf0b7 erythropoietinstimulating agents for anemia prophylaxis and transfusion when necessary depending on the patient context and underlying comorbidities preproof treatment based on the high transmissibility of the virus82 the main nonpharmacologic countermeasures to mitigate or delay the impact of covid19 include rigorous hand hygiene use of facemasks respiratory etiquette ie coughing or sneeze into the upper sleeve or elbow not the hands flushing with the lid down to prevent bioaerosolization as well as quarantine stay at home policies and workplace and school closures which have upended the social cultural political and economic status quo no specific treatment or vaccine is currently available although promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir the mainstay of medical therapy includes symptomatic care such as supplemental oxygen antibiotics and hemodynamic and mechanical ventilatory support if indicated for septic shockmultiple an failure and respiratory failure respectively83 over active clinical treatment trials are underway84 these include vaccines as well as a number of different agents some with promising preliminary data as mentioned above and also those with potential anticancer activity which will hopefully serve a double purpose first to treat covid19 and second as an adjunct to bridge the time gap until the patient is recovered and the primary antineoplastic is startedrestarted as shown in table 0c preproof conclusions the alarming spread of the covid19 pandemic has disproportionately affected cancer patients an atrisk population both from the standpoint of increased disease severity and disruption to care which includes widespread suspension of clinical trials in the united states that are already fraught with barriers to enrollment and participation85 86because the symptoms of covid19 are nonspecific underlying symptoms from the cancer eg dyspnea cough fever fatigue diarrhea etc which overlap with those from the viral infection may obscure and delay the diagnosis hence if the covid19specific rapid reverse transcriptase polymerase chain reaction rtpcr test is not readily available andor in short supply which is currently the case diagnosis will depend on the maintenance of a high index of clinical suspicion especially in advanced cancer patients who check all the boxes for risk factors such as older age frailty disability immunosuppression generalized systemic inflammation and multiple comorbidities eg hypertension diabetes and cardiorenovascular diseases that predispose to severe disease and death preproofthese comorbidities are commonly treated with renin angiotensin system blockers such as angiotensinconverting enzyme inhibitors aceis or angiotensinreceptor blockers arbs which increase levels of ace2 the continued use of aceisarbs is the centerpiece of an intense debate because on the one hand sarscov2 coopts ace2 for target cell entry but on the other ace2 overexpression may counterbalance vasoconstriction and profibrotic processes and thereby reduce the incidence or mortality associated with covid19“associated ali or acute respiratory distress syndrome another controversy involves whether or not to continue cancer treatment given the high transmissibility potential of the virus however since no expert consensus recommendations have been issued to date and prognosis stage and responses to therapy are highly heterogeneous the riskbenefit tradeoff and subsequent treatment plan are highly individualized and context dependent currently the focus of treatment is infection control appropriate symptomatic care and oxygen therapy no approved medication or vaccine has been developed but promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir and several repurposed agents with antitumor properties are under investigation including thalidomide and rrx001 which may hopefully bridge the gap from the time covid is first diagnosed until the primary anticancer therapy is restarted finally multiple comparisons have been made between the allout mobilization efforts to combat covid19 with the massive scaleup of human and material resources that occurred during world war ii8788 in the words of winston churchill prime minister of great britain from whose intrepid fighting spirit iron will and intransigent defiance of tyranny galvanized the resolve of an entire nation to fight on in the face of seemingly impossible odds oncologists on the frontlines that have answered the call should •never worry about action only inaction– 0c preproof declaration of interests the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper references sitammagari k skandhan a dahlin awhat hospitalists need to know about covid19 medscape mar httpswwwmedscapecomviewarticle924596 meo sa alhowikan am alkhlaiwi t meo im halepoto dm iqbal m usmani am hajjar w ahmed n novel coronavirus 2019ncov prevalence biological and clinical characteristics comparison with sarscov and merscov eur rev med pharmacol sci feb24420122019 chan jf kok kh zhu z et al genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan emerg microbes infect kuba k imai y rao s et al a crucial role of angiotensin converting enzyme ace2 in sars coronavirusinduced lung injury nat med zhang h kang z gong h et al the digestive system is a potential route of 2019ncov infection a bioinformatics analysis based on singlecell transcriptomes [epub ahead of print] biorxiv de wit e van doremalen n falzarano d munster vj sars and mers recent insights into emerging coronaviruses nat rev microbiol “ w li et al angiotensinconverting enzyme is a functional receptor for the sars coronavirus nature hughes s covid19 and angiotensin drugs help or harm medscape march httpswwwmedscapecomviewarticle927542 yang x yu y xu j et al clinical course and outcomes of critically ill patients with sarscov2 pneumonia in wuhan china a singlecentered retrospective observational study [published online ahead of print feb ] [published correction appears in lancet respir med feb ] lancet respir med 2020s2213 gu j han b wang j covid19 gastrointestinal manifestations and potential fecaloral transmission [published online march ] gastroenterology ruiheng x chance missed but still there memoirs at the 10th anniversary of sars outbreak j thorac dis aug 5suppl s90“s93 jiang s don't rush to deploy covid19 vaccines and drugs without sufficient safety guarantees nature mar5797799321 doi 101038d41586020007519 peiris js guan y yuen ky severe acute respiratory syndrome nat med dec suppls8897 zheng h zhang m yang c et al elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid19 patients cell mol immunol2020 tognotti e lessons from the history of quarantine from plague to influenza a emerg infect dis “ httpswwwnihgovnewseventsnewsreleasesnihclinicaltrialinvestigationalvaccinecovid19begins httpswwwascoascocoronavirusinformationcareindividualscancerduringcovid19 onder g rezza g brusaferro s casefatality rate and characteristics of patients dying in relation to covid in italy published online march doi101001jama20204683 hanna tp evans ga booth cm cancer covid19 and the precautionary principle prioritizing treatment during a global pandemic nat rev clin oncol who advice on the use of masks in the community during home care and in healthcare settings in the context of the novel coronavirus 2019ncov outbreak jan rodriguezmorales aj macgregor k kanagarajah s patel d schlagenhauf p going global travel and the novel coronavirus travel med infect dis httpsdoi101016jtmaid2020101578 httpswwwnejmdoifull101056nejmc2004973 lu cw liu xf jia zf 2019ncov transmission through the ocular surface must not be ignored the lancet preproof 0c preproof holshue ml et al first case of novel coronavirus in the united states n engl j med yeo c kaushal s yeo d enteric involvement o
Colon_Cancer
" heat shock transcription factor1 hsf1 was overexpressed to promote glutaminolysis and activatemtor in colorectal cancer crc here we investigated the mechanism for cancerspecific overexpression of hsf1methods hsf1 expression was analyzed by chromatin immunoprecipitation qrtpcr immunohistochemistrystaining and immunoblotting hsf1 translation was explored by polysome profiling and nascent protein analysisbiotin pulldown and m6a rna immunoprecipitation were applied to investigate rnarna interaction and m6amodification the relevance of hsf1 to crc was analyzed in apcmin and apcmin hsf1ˆ’miceresults hsf1 expression and activity were reduced after the inhibition of wntcatenin signaling by pyrvinium orcatenin knockdown but elevated upon its activation by lithium chloride licl or catenin overexpression thereare much less upregulated genes in hsf1ko mef treated with licl when compared with licltreated wt mefhsf1 protein expression was positively correlated with catenin expression in cell lines and primary tissues aftercatenin depletion hsf1 mrna translation was impaired accompanied by the reduction of its m6a modificationand the upregulation of mir4553p which can interact with ²utr of hsf1 mrna to repress its translationinterestingly inhibition of mir4553p rescued catenin depletioninduced reduction of hsf1 m6a modificationand mettl3 interaction both the size and number of tumors were significantly reduced in apcmin mice whenhsf1 was genetically knockedout or chemically inhibiteds catenin suppresses mir4553p generation to stimulate m6a modification and subsequenttranslation of hsf1 mrna hsf1 is important for catenin to promote crc development targeting hsf1 could bea potential strategy for the intervention of catenindriven cancerskeywords colorectal cancer catenin hsf1 translation mir4553p m6a rna modification correspondence wangx118zjueducn jinhczjueducn1department of medical oncology cancer institute of zhejiang university sirrun run shaw hospital school of medicine zhejiang university hangzhouchina2labortary of cancer biology key lab of biotherapy in zhejiang sir run runshaw hospital school of medicine zhejiang university hangzhou chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csong molecular cancer page of introductioncolorectal cancer crc is the third most common cancer with high mortality rate globally the accumulation of various genetic and epigenetic changes activatesmultiple oncogenic signaling critical for the pathogenesisof crc such as wntcatenin signaling pathway its activation will eventually initiate a transcriptiondependent oncogenic process to promote cell cycle progression and apoptosis resistance while the mechanismfor activated wntcatenin signaling to promote crcdevelopment has been wellexplored no therapeuticstargeting this pathway has been successfully developedin addition to proliferation activation and apoptosisresistance metabolism reprogramming is one of important hallmarks of cancer cells for example cancercells favor glycolysis instead of oxidative phosphorylationfor glucose metabolism even in aerobic conditionswhich was wellknown as warburg effect [ ] pyruvate the last product of glycolysis is converted into lactate rather than acetylcoa acetyl coenzyme a fortca tricarboxylic acid cycle or the krebs cycletherefore targeting enhanced glycolysis has been proposed as novel options in the prevention and treatmentof human cancers including crc however as a metabolism hub tca cycle is important in both energyproduction and biosynthesis therefore it needs to bereplenished by anaplerotic reactions such as glutaminolysis previously we reported that heat shock transcription factor hsf1 stimulated glutaminolysis toactivate mtor and promote crc development by upregulating the expression of glutaminase gls1 thecritical enzyme in glutaminolysis hsf1 expressionwas increased in crc and had a positive correlationwith shorter diseasefree survival dfs however theupstream mechanism for hsf1 overexpression in crcwas still uncleargene expression can be controlled by multiple processesincluding transcription mrna degradation translationand protein degradation while gene translation and protein degradation have been extensively investigated moreand more studies focused on mrna translation by exploring the effect of noncoding rnas such as micrornasmirnas and new modifications of mrna including n6methyladenine m6a modification [ ] mirnas canform a mirnainduced silencing complex mirisc toposttranscriptionally regulate gene expression by inhibiting capdependent initiation and stimulating mrna deadenylation [ ] on the other hand as one of the mostabundant modifications in mrna m6a modification ofmrnas usually promotes translation by recruiting initiation factors such as eif3 to the ² end of the mrna while mirnas and mrna m6a modifications play a distinct role in mrna translation the interplays betweenthem were not clarifiedin this study we found that activated wntcateninsignaling stimulated hsf1 translation to promote crcdevelopment byrepressing hsf1 mrnatargetingmir4553p to increase m6a modification of hsf1mrna therefore targeting hsf1 translation could be anew strategy for the intervention of crc and other cancers driven by activated wntcatenin signalingmaterials and methodscell antibodies and chemicalshuman crc celllines sw480 sw620 dld1 rkowere obtained from the american type culture collection atcc all cells were routinely cultured in rpmi invitrogen “ or dmem invitrogen“ supplemented with fetal bovine serumall cells were incubated at °c with co2 and humidity the following antibodies were used for western blotting and ihc hsf1 12972s cell signalingtechnology cst ab52757 abcam catenin 8480scst actin l cst flag f1804“ sigmacyclind1 ab134175 abcam cleaved parp1 9541scst mettl3 a8370 abclonal gls1 ap8809babgent pyrvinium p0027 licl cycloheximide r750107 chloroquine c6628 mg132 and pd150606 d5946 were purchased from sigmaaldrichinterfering rna sirnasirna mirna mimicsinhibitors transfectiontargeting cateninsmallmettl3 and micrornas were synthesized by genepharma shanghai china and ribobio guangzhouchina the sequence of these sirnas and mirnaswere listed in additional file table s1 sirnas andmirna mimicsinhibitors were transfected into cellsseeded overnight by lipo2000 invitrogen usa or lipofectamine rnaimax transfection reagentinvitrogenusa according to the manufacturer™s instructionsluciferase activity assaythe plasmid of catenin reporter was gifted from profximei wu zhejiang university for hsf1 activity assaya fragment containing x hse were synthesized andinserted into the pgl3basic vectors promega corporation usa the plasmid was cotransfected with prlrenilla and catenin sirna by using lipo2000 invitrogen usa or treatment with pyrvinium by xtremegene hp dna transfection reagent roche usa ²utr segment of the hsf1 was cloned by pcr andinserted into the vector pmirreporter promegathe mutation of mir455 binding sites in hsf1 ²utrwas generated by quick sitedirected mutagenesis“ stratagene usa the resultant plasmidswere cotransfected with prl renilla and mir455 mimicsby usinglipo2000 invitrogen usa h post 0csong molecular cancer page of transfection the luciferase activity was measured by thedualglo luciferase assay system promega corporation usachromatin immunoprecipitation chipchip analysis was conducted with the simplechip„¢ enzymatic chromatin ip kit cst usa antibodies usedwere antihsf1 12972s cst tcf7l2c48h11 cstand flag f1804“ sigma the primers used for thepcr analysis of precipitated dna were shown inadditional file table s2 for flagchip assay theflagcatenin vector was transiently overexpressed bytransfection after h the enrichment of flagcateninon col27a1 promoter was measured by the chip kitimmunoblotting and immunohistochemistryimmunoblotting and immunohistochemistry ihc assays were performed as previously reported for immunoblotting total proteins were extracted with ripabuffer supplemented with protease inhibitors rocheusa after heating the protein sample to “ °c for min celllysates were transferred to polyvinylidenefluoride pvdf membranes after the membranes wereblocked in milk primary antibody with gentle agitation overnight at °cfor ihc assay was performed in a tissue array containing cases of colonic tissues primary antibodies usedwere listed above the degree of immunostaining wasassessed by independent pathologists and evaluated byassigning a score of “ scores were defined as follows no staining faint staining moderate staining and strong staining final scores of and were regardedas low expression whereas scores of and consideredas high expressionapoptosis detectioncell apoptosis was measured by flow cytometry analysisand western blotting for flow cytometry analysis ofapoptosis cells were harvested and resuspended in μl x binding buffer μl fluorescein isothiocyanatefitc annexin v and propidium iodide pi bd biosciences usa were added to the cell suspensionand then incubated for min at room temperatureafter that the samples were attenuated with 400ul xbinding buffer and analyzed by acs caliburflowcytometerpuromycinlabellingto detect the change of nascent hsf1 synthesis x cells were plated in cm dish afterthe giventreatment cells were incubated with biotindcpuromycin nu925bios jena bioscience for hcells were lysed with np40 buffer mm trishclph mm nacl np40 glycerolcontaining 1x protease inhibitor cocktail after adequatecentrifugation the supernatant was incubated with 80ulstreptavidin sepharose beads ge17“ sigma byrotating at °c for h to overnight the mixture waswashed by np40 buffer for times and subjected towestern blotting using hsf1 antibodyseparatestranslatingpolysome profilingpolysome profilingor nontranslating mrnas on a sucrose gradient according tothe number of bound ribosomes as previously described in brief cells were grown to confluence before collection cells were incubated with μgml ofcycloheximide for min then cells are lysed by polysome buffer [ mmoll kcl mmoll mgcl2 triton x100 μgml cycloheximide mmoll heparin and uml rnase inhibitor takara 1x cocktail] for min on ice lysates were centrifuged rpm for min and the supernatant was layered onto a to sucrose gradient gradients were then centrifuged at rpm for min at °c and polysomebound fractions were collected using an isco densitygradient fractionation system isco lincoln ne withcontinuous monitoring based on a260nm wavelengththe rna in each fraction was extracted using trizol reagent invitrogen and analyzed by realtime rtpcrbiotin pull down assaybiotin pull down assay was performed as described previously cells were transfected with biotinylatedmir4553p probes for h and resuspended using lysisbuffer mm tris ph mm nacl mmmgcl2 uml superasein mm dtt igepal protease inhibitors lysates were incubated withprepared streptavidin beadsge healthcare yeasttrna sigma was used for blocking lysates at °c for h then washed times with binding and wash buffer mm trishcl ph mm edta m naclfinally the bound rnas were extracted and purified forqpcrrna immunoprecipitation rip assayrip assay was performed by magna riptm rnabinding protein immunoprecipitation kitmilliporeno17“ briefly × cells were lysed in μlrip lysis buffer and immunoprecipitated with antibodiesofinterest and protein g magnetic beads at °covernight followed by six times of washes in washingbuffer and protein digestion at °c total rna wasisolated and subjected to rtpcr analysis followingantibodies were used for rip n6methyladenosine synaptic mettl3 a8370 abclonal igg millipore 0csong molecular cancer page of rnasequencing2x106 wt mef and hsf1 ko mef were plated andcultured overnight following day cells were treatedwith mm licl for h cells were collected with trizol reagent the total rna was processed by nebnext®polya mrna magnetic isolation module to enrichmrna and the product rna was used for constructionlibrary via kapa stranded rnaseq library prep kitillumina sequencing libraries denatured by mnaoh to generate singlestranded dna as amplified insitu illumina cbot truseq sr cluster kit v3cboths gd4013001 illumina the ends of the generatedfragments were used to run cycles by the illuminahiseq sequencer all the experimental steps afterthe rna extraction were conducted in kangcheng biotechnology co ltd aksomics shanghai china rnasequencing was performed three timesanimal experimentsanimal care and experiments were conducted in compliance with institutional animal care and use committeeand nih guidelines the c57bl6 j mice and apcminmice were purchased from model animal research center of nanjing university marc nanjing chinahsf1 ko mice reported previously were used to generate apcmin mice hsf1ˆ’ subsequently groupsof mice wild type apcmin apcmin hsf1ˆ’ and apcmin treated with knk437 as previously reported were fed with highfat diet kcal fat for monthsthe intestine was dissected flushed with pbs and cutopen longitudinally along the main axis the number oftumors was counted and the sizes oftumors weremeasuredstatisticsall data were expressed as mean ± sd unless specifiedthe student™s ttest was performed for statistical significance analysis p value was considered as statistically significantresultscatenin activates hsf1 in crcin an effort to explore potential regulations of hsf1 wescreened chemicals generating a gene expression patternsimilar to hsf1 depletion by connective map httpportalsbroadinstitutecmapinterestingly a recently reported inhibitor of wntcateninsignaling pyrvinium had a similar effect on genomewide gene expression as hsf1 depletion fig 1aand additional file figs1ab moreover the expression signature related to wntcatenin signaling was positively correlated with the hsf1 signature fig 1b indicating a potential connection of hsf1 withwntcatenin signaling indeed pyrvinium attenuated[ ]the activity of a luciferase reporter driven by hsf1 binding sites hse heat shock response elements [ ]fig 1c and reduced the expression of wellknown transcriptional targets of hsf1 such as hsp90aa1 hspa4hspb1 and hsph1 fig 1d and additionalfile figs1c chromatin immunoprecipitation chip assayfurther confirmed the reduced interaction of hsf1 withits transcriptional targets fig 1e and additional file figs1d in consistence with pyrvinium knockdown ofcatenin by sirna also decreased hsf1 activity fig1f reduced the expression of hsf1 targets fig 1g andadditional file figs1e and attenuated the interactionof hsf1 with its targets fig 1i and additional file figs1f furthermore hsf1 targets were upregulated bythe potent gsk3 inhibitor licl in colorectal cancer cellline rko which had a low level of catenin expressionfig 1hto explore the biological relevance of hsf1 activationto catenin signaling we profiled gene expression ofwild type mouse embryonic fibroblasts wt mef andhsf1 knockout mef hsf1 ko mef before and afterlicl treatment ncbi geo gse151119 while only genes were upregulated in licl treatedhsf1 komef there were genes significantly upregulated inwt mef after licl treatment fig 1j among them genes displayed a dependence on hsf1 since theirexpression levels failed to be upregulated by licl treatment once hsf1 was depleted fig 1k in fact their expression levels had a high correlation with theexpression of a previously reported hsf1 signature fig1l furthermore genes had a hse heatshock response element hse within their promoter regions fig 1m and additional file meaning that theyare most likely bona fide targets of hsf1 indeed someof them such as tma16 dedd2 hspa9 and kif21a havebeen confirmed as the target of hsf1 by chipseqncbi geo gse57398 fig 1n and additional file figs1g taken together these results indicated that catenin can positively regulate hsf1catenin stimulates hsf1 protein translationto delineate how catenin regulates hsf1 we quantitated protein levels of hsf1 before and after inhibitingcatenin both pyrvinium and catenin depletion reduced the protein level of hsf1 fig 2a and b in contrast overexpression of exogenous catenin increasedhsf1 protein level fig 2c furthermore hsf1 proteinlevel was increased after activating wntcatenin signaling by licl treatment in both rko and mef cellsfig 2d in addition hsf1 expression correlates wellwith catenin expression in primary tissues fig 2e andf p chisquare test all of these data indicatedthat catenin upregulates hsf1 protein expression 0csong molecular cancer page of fig wntcatenin signaling activates hsf1 a chemicals influencing gene expression in a similar manner to hsf1 inhibition were screened byconnective map analysis b the correlation of wntcatenin signaling signature and hsf1 signature was detected by gepia c the effect ofpyrvinium on hsedriven promoter activity was explored by luciferase reporter assay d the effects of pyrvinium on the targets of hsf1 wereanalyzed by rtpcr e binding of hsf1 to the promoters of hsf1 targets in crc cells treated with or without pyrvinium was determined by chipf the luciferase assays of hse before and after catenin knockdown were shown as in c g and h the mrna levels of hsf1 targets with catenin knockdown or licl treatment were analyzed by rtpcr i binding of hsf1 to its targets promoter in crc cells before and after cateninknockdown was analyzed by chip j volcano plot displays differentially regulated genes in dhsf1 compared to wt parental cells with licl reddots indicate significantly regulated genes based on adjusted pvalue and logfold change logfc p log2fc k differential geneexpression analysis in wt and hsf1ˆ’ mef treated with licl were performed by rnaseq numbers of upregulated genes in two cells wereshown in venn graph l the correlation of putative hsf1dependent genes from k with reported hsf1 signature was detected by gepia mnumbers of putative hsf1dependent genes with or without hse in their promoters were summarized n representative hsf1 chipseqtracks ncbi geo gse57398 for hsecontaining genes are shown asterisks indicate p 0csong molecular cancer page of fig catenin stimulates hsf1 protein translation a the effect of pyrvinium on the protein expression of hsf1 was explored by westernblotting b the effect of catenin knockdown on hsf1 protein level was analyzed by western blotting c the protein level of hsf1 before andafter catenin overexpression was analyzed by western blotting d the effect of licl on hsf1 protein level in rko and mef was analyzed bywestern blotting e the expression of catenin and hsf1 in colorectal tissue was analyzed by immunohistochemistry staining f the correlationbetween catenin expression and hsf1 expression in colorectal tissue was analyzed by chisquare test p g the effect of catenindepletion on hsf1 with puromycin labeling was determined by western blotting h amount of hsf1 mrna in various polysome fractions wasanalyzed by rtpcrp however there were no apparent alterations in hsf1mrna level after catenin knockdown or pyrviniumtreatment additional file figs2a and s2b meanwhile the halflife of hsf1 protein was also not changedbefore and after catenin knockdown additional file figs2c inhibitors of proteasome autophagy and calpains all failed to reverse hsf1 protein downregulationinduced by catenin knockdown additionalfile 0csong molecular cancer page of figs2d all of these results implied that catenin affects hsf1 protein expression mostlikely via translationregulation therefore puromycin labeling assay wasemployed to monitor the synthesis of nascent hsf1 protein as expected the puromycin labeling of hsf1was reduced by catenin depletion fig 2g to furtherconfirm it monopolysome fractions from cytoplasmicextracts of crc cells before and after catenin depletion were collected by sucrose gradient centrifugationthe subsequent rtpcr analysis revealed that catenindepletion considerably reduced the presence of hsf1mrna in the polysome fraction but increased in nontranslating ribosome fractions fig 2h in summary catenin upregulates hsf1 expression by stimulatinghsf1 protein translationhsf1 protein translation is regulated by mir4553pas micrornas mirnas play an important role inregulating the efficiency of protein translation we wondered whether hsf1 protein translation was regulatedby micrornas based on bioinformatics screening bytargetscan mirdb and starbase some micrornas including mir4553p mir2145p mir4315p mir184mir4903p and mir375 were proposed to target ²utr of hsf1 mrna fig 3a after functional validation by western blotting mir4553p and mir2145pbut not other micrornas were capable to suppress theexpression of hsf1 protein in crc cells fig 3b andadditional file figs3a however mir2145p but notmir4553p also reduced hsf1 mrna level additionalfile figs3b what™s more an inhibitor of mir4553prather than mir2145p rescued the downregulation ofhsf1 protein by catenin knockdown fig 3c and additional file figs3c indicating that mir4553p mightbe relevant to catenininvolved regulation of hsf1protein translation indeed mir4553p inhibited the activity of luciferase driven by wild type hsf1 mrna ²utr but not its mutant unable to bind mir4553p fig3d the interaction of mir4553p with hsf1 mrnawas further confirmed by biotin pull down assay fig3e based on the analysis of tcga data httpmirtvibmssinicaedutwthe expression of mir4553p islower in colon adenocarcinoma than in normal tissuesadditional file figs3d similarly qpcr analysis revealed lower levels of mir4553p in crc tissues than inadjacent nontumor tissues fig 3f additionally wehad confirmed high expression of hsf1 in the same cohort of human crc tissues previously indeedmir4553p expression was negatively correlated with theexpression of hsf1 fig 3g on the other handmir4553p similar to hsf1 inhibition as we reportedrecently reduced the expression of hsf1 targets induced the viability inhibition and apoptosis activation ofcolorectal cancer cells fig 3hj and additional file figs3eg the seed sequence of micrornas was important for targeting mrna by basepairing indeed the seed sequence mutant of mir4553p could notdownregulate the protein level of hsf1 additional file figs4a confirming the importance of mir4553p totarget hsf1 protein expression in a word mir4553ptargets hsf1 mrna ²utr to inhibit its translationm6a modification of hsf1 mrna stimulates its proteintranslationin addition to microrna mrna modifications such asn6methyladenosine m6a play important roles in theregulation of hsf1 translation interestingly we noticedthat the matching sites of mir4553p seed sequence inhsf1 mrna ²utr contains a typical motif of m6amodification fig 4a which was supported by bioinformatic analysis httpwwwcuilabcnsramp fig 4b andadditional file figs4b moreover we have done themerip sequencing in sw620 and found that the ²utr region of hsf1 has one m6a modification site intriguingly this sequence is completely complementary tothe seed sequence of mir4553p additionalfile figs4c pcr analysis after merip m6a rna immunoprecipitation further confirmed m6a modification ofhsf1 mrna fig 4c what™s more the activity of luciferase driven by the mutant hsf1 mrna ²utr whichwas unable to bind mir4553p but retains the m6amodification site sequence drach d a g or u r a or g h a u or c [“] was higher than theactivity of luciferase driven by wild type hsf1 mrna²utr fig 3dindicating the importance of m6amodification to hsf1 expression as the main component of the methyltransferase œwriter complex [ ]mettl3 was also bound to hsf1 mrna fig 4donce its expression was depleted m6a modification ofhsf1 mrna was decreased fig 4e in consistencewith its potential roles in promoting protein translationsuch a reduction of hsf1 mrna m6a modification reduced hsf1 protein expression fig 4f and nascenthsf1 protein synthesis fig 4g furthermore mettl3depletion considerably reduced the presence of hsf1mrna in the polysome fraction but increased in nontranslating ribosome fractions fig 4h while hsf1mrna or the stability of hsf1 protein were not changed additionalfile figs4d and s4e moreoverhsf1 protein was decreased with the knockdown ofythdf1 which was the reader protein of hsf1 m6amodification fig 4i to sum up m6a modification ofhsf1 mrna was relevant to stimulate its translationcatenin suppresses mir4553p to increase hsf1 mrnam6a modificationnext we explored the interplay between mir4553p andm6a modification of hsf1 mrna both hsf1 mrna 0csong molecular cancer page of fig hsf1 protein translation is regulated by mir4553p a overlap of hsf1targeting micrornas predicted by targetscan mirdb and starbaseb the effect of mir4553p on hsf1 protein was analyzed by western blotting c the effect of mir4553p inhibitor on catenin knockdowninduced hsf1 downregulation was determined by western blotting d luciferase activity assay was used to analyze the effect of mir4553p onthe activity of ²utr with or without mir4553p binding sites p e the binding between biotinmir4553p and hsf1 mrna wasdetermined by biotin pull down assay p f expression of mir4553p in pairs of fresh crc tissues and adjacent nontumor tissues wasanalyzed by qpcr g the correlation of hsf1 protein and mir4553p in pairs of fresh crc tissues and adjacent nontumor tissues wasanalyzed h the effect of mir4553p on viability of crc cells was explored by mts assay i the effect of mir4553p on apoptosis of crc cells wasanalyzed using flow cytometry after pi and annexin vfitc double staining j apoptosis of crc cells treated with or without mir4553p wasdetermined by western blottingm6a modification and its binding to mettl3 were decreased by the overexpression of wild type mir4553pbut not its mutant unable to bind to hsf1 mrna ²utr figfigs5aand additional5afileinterestingly mettl3 depletion not only reduced m6amodification of hsf1 mrna fig 4e but also enhanced the interaction of mir4553p with hsf1 mrnafig 5b and additionalfile figs5b while the 0csong molecular cancer page of fig m6a modification of hsf1 mrna stimulates its protein translation a the sites of hsf1 ²utr binding to the seed sequence of mir4553pwas consistent with m6a rna modification elements œdrach b bioinformatic prediction of m6a modification in ²utr of hsf1 mrna c m6amodification of hsf1 mrna was analyzed by merip p d binding of mettl3 to hsf1 mrna was detected by rip p e m6amodification of hsf1 mrna with or without mettl3 depletion was analyzed by merip p f the protein level of hsf1 before and aftermettl3 depletion was detected by western blotting g the effect of mettl3 knockdown on hsf1 synthesis was determined by western blottingafter puromycin labeling h amount of hsf1 mrna in various polysome fractions was analyzed by rtpcrp i the effect of ythdf1knockdown on hsf1 protein level was analyzed by western blottingexpression of mature and primary mir4553p was notupregulated additional file figs5cd these resultsindicated that mir4553p may compete with mettl3for the m6a modification of hsf1 mrna thus inhibitinghsf1 protein translation furthermore the binding ofmir4553p to hsf1 mrna was not changed by ythdf1deletion additional file figs5e indicating that thetranslation repression of hsf1 mrna was more likely tobe mediated directly by the reduced m6a modification ofhsf1 0csong molecular cancer page of fig catenin suppresses mir4553p to increase hsf1 mrna m6a modification a m6a modification and mettl3 interaction of hsf1 mrnawith wt or mutant of mir4553p were analyzed by rip b the interaction between biotinmir4553p and hsf1 mrna with or without mettl3depletion was analyzed by biotin pull down c the effect of mir4553p inhibitor andor catenin knockdown on m6a modification of hsf1mrna was analyzed by merip d the effect of mir4553p inhibitor andor catenin knockdown on the interaction of mettl3 with hsf1 mrnawas analyzed by rip e the effect of catenin knockdown on interaction of mir4553p and hsf1 mrna was analyzed by biotin pull down f andg the levels of mature f and primary g mir4553p with catenin knockdown or licl treatment were determined by rtpcr h the correlationof col27a1 and mir455 was analyzed in linkedomics httplinkedomics i the effect of catenin depletion or licl on mrna level ofcol27a1 was analyzed by rtpcr j the interaction of catenintcf7l2 and hsf1 promoter was determined by chip k the correlation of catenin protein expression with the rna level of col27a1 was detected by linkedomics httplinkedomicsindeed mir4553pcatenindepletioninduced decrease of hsf1 mrna m6a modification fig 5c and additional file figs5f meanwhilethe interaction of mettl3 with hsf1 mrna wasinhibitorrescuedabrogated by depleting catenin fig 5d and additionalfile figs5g accompanied by the increased interactionof mir4553p to hsf1 mrna fig 5e and upregulationof mature fig 5f and additionalfile figs5h 0csong molecular cancer page of precursor additional file figs5i and primary mir4553p fig 5g and additional file figs5j in contrastwhen catenin was upregulated by overexpression or licltreatment both mature mir4553p fig 5f and additionalfile figs5h and primary mir4553p fig 5g and additional file figs5j were downregulatedprimary mir4553p was derived from a premirna hairpin encoded in intron of the collagen gene col27a1 additional file figs5k actually col27a1 expression was significantly correlated with the expression ofmir455 httplinkedomics fig 5h consistent withthis we observed col27a1 mrna levels were increasedupon catenin depletion while decreased after licl treatment fig 5i and additional file figs5l moreover catenintcf7l2 complex could interact with the promoterof col27a1 while the pair of primers negativechipprimer at a position far away from the promoter region couldnot enrich col27a1 fig 5j and additionalfile figs5m meanwhile the protein expression of cateninwas negatively correlated with rna level of col27a1httplinkedomics fig 5k these results indicatedthat the transcription of col27a1 was inhibited by wntcatenin signalingleading to decreased biogenesis ofmir4553p therefore catenin facilitates the shift frommir4553p binding to m6a modification in hsf1 mrnaby suppressing mir4553p expression eventually promoting hsf1 protein translationboth genetic and chemical inhibition of hsf1 attenuatecolorectal carcinogenesis in micein light of these in vitro findings we further exploredthe relevance of hsf1 to colorectal carcinogenesis inapcmin and apcmin hsf1ˆ’ mice since the interaction of mouse mir4553p and mouse hsf1 mrnaseems to be well conserved mouse mir4553p seed sequence cagucca the binding site in mouse hsf1mrna ²utr tggactg the expression of hsf1 andits downstream target gls1 were increased whilemir4553p expression was reduced in intestine tissuesfrom apcmin mice compared with normal c57bl6mice fig 6a and b after fed with highfat diet for months these apcmin mice developed multiple tumorsin the intestine fig 6c however both the size andnumber of tumors were significantly reduced in apcminmice treated with a chemical inhibitor of hsf1 knk437and apcmin hsf1ˆ’ mice fig 6d accompanied bythe downregulation of hsf1 targets fig 6e all of theseresults confirmed that hsf1 is a novel downstream target of wntcatenin signaling important to promotecrc developmentdiscussiongenetic changes in components of wntcatenin signaling such as deletion of the apc gene and ctnnb1mutations have been frequently detected in many typesof human cancers all of these muta
Colon_Cancer
" ethnopharmacological relevance herba patriniae has been used for thousands of years in china as a traditional chinese medicine with heatclearing and detoxicating effects it is applied widly for the treatment of rheumatoid arthritis diarrhea acute hepatitis pelvic inflammatory disease and ulcerative colitis in clinic two species namely patrinia scabiosaefolia fisch ps and patrinia villosa juss pv from the caprifoliaceae family are considered as herba patriniae in the pharmaceutical industry aim of the review this paper aims to comprehensively outline the traditional uses botanical description phytochemistry pharmacology toxicology quality control pharmacokinetics and patents of herba patriniae and elaborate the samedifferent characteristics between ps and pv materials and methods detailed information of herba patriniae was collected from various online databases pubmed web of science google schola china national preproof 0c knowledge infrastructure database national intellectual property administration prc national medical products administration and those published resources msc thesis and books results a total of compounds have been identified in herba patriniae including triterpenoid saponins flavonoids anic acids irids and volatiles a very distinct difference was observed that ps is rich in triterpenoid saponins and volatiles while pv contains more flavonoids two source species of herba patriniae gave similar pharmacological effects on anticancer antiinflammatory antioxidant antimicrobial sedative and hypnotic effects but there were no reports were on antipruritic proangiogenic and antidiarrheal effects for ps and no studies on antidiabetic effects for pv generally herba patriniae showed nontoxic in the clinical dose but mild side effects such as temporary leukopenia dizziness and nausea could be found when large and excessive dosage is used a variety of compounds have been quantified for the quality control of ps and pv the variety growth environment growth time and harvest time not only affected the contents but also the pharmacological activities of the bioactive compounds in the past year patents for compositions containing pv and ps have been filed mainly involving human health hygiene agriculture and animal husbandry unfortunately the research on pharmacokinetics is insufficient only the prototype components and metabolites were repored after intragastric administration of total flavonoids extract from pv in rats herba patriniae has displayed a significant medicinal value in clinic but the differences in phytochemistry pharmacological effects and the content of compounds have been found between two official recorded species about side effects and pharmacokinetic characteristics the differeces between two species have not been well studied for a better clinical use of herba patriniae it is urgent to establish systematic pharmacology quality control pharmacokinetics and clinical researches on the samedifferent characteristics between ps and pv keywords herba patriniae traditional uses phytochemistry pharmacology quality control preproof 0c cells a549 human lung cells cancer aspartate polysaccharide mixture ast list of abbreviations 3t3l1 preadipocytes 5fuhct8 human ileocecal adenocarcinoma cells a2780 human ovarian cancer cells a375s2 human melanoma cells a498 human renal abts carcinoma 'azinobis3ethylbenzothiazoline6sulphonic acid ags human gastric cancer cells akt protein kinase b alt alanine aminotransferase ap acute pancreatitis ap3 aminotransferase bax bcl2associated x protein bcl2 bcell lymphoma2 bclxl b cell lymphoma factor xl bel7402 human hepatoma cells bv2 mouse microglia cells caco2 human colon cancer cells cox2 cyclooxygenase2 crc colorectal cancer dai disease activity index dpph 22diphenyl1picrylhydrazyl ec50 half maximal effective concentration emt epithelialmesenchymal transition fak focal adhesion kinase gcms gas chromatographymass spectrometer glut4 glucose transporter gsh glutathione h2o2 hydrogen peroxide hela human cervical cancer cells hepg2 human hepatoma cells hl60 human promyelocytic leukemia cells ho1 heme oxygenase1 hplc high performance liquid chromatography hsp heat shock proteins hsp heat shock proteins ht1080 human fibrosarcoma cells ht29 human colon carcinoma cells huvecs human umbilical vein endothelial cells ic50 inhibitory concentration icam1 intercellular adhesion molecule icr institute of cancer research il1 interleukin1 beta il6 interleukin il8 interleukin inos inducible nitric oxide synthase irs insulin receptor substrate k562 human malignant myeloid cells ldh lactate dehydrogenase lps lipopolysaccharides mcf7 human breast cancer cells mda malondialdehyde mdamb231 human breast cancer cells mpo myeloperoxidase mrna messenger ribonucleic acid nfκb nuclear factor κb ngf nerve growth factor no nitric oxide nqo1 quinine oxidoreductase nrf2 nuclear factor erythroid 2related factor o2 superoxide anion oh hydroxyl radical pcna proliferating cell nuclear antigen pid pelvic inflammatory disease ps patrinia scabiosaefolia fisch pv patrinia villosa juss raw2647 mouse leukemic monocyte macrophage ros reactive oxygen species rsv respiratory syncytial virus sars severe acute respiratory syndrome sd sprague dawley sgc7901 human gastric cancer cells smmc7721 hepatocellular carcinoma cells stat3 signal transducer and activator of transcription sw480 human colon carcinoma cells tc50 half toxic concentration tcm traditional chinese medicine tgf transforming growth factor beta ti drug treatment index tnfα tumor necrosis factor alpha taoc total antioxidant capacity tsod total superoxide dismutase u14 mice cervical cancer cells u266 human multiple myeloma cancer cells u937 human lymphoma cells uc ulcerative colitis uv ultraviolet preproof 0c table of contents introduction traditional uses botany phytochemistry triterpenoid aglycones and triterpenoid saponins flavonoids anic acids irids volatiles other compounds pharmacology anticancer effect antiinflammatory effect antioxidant effect antimicrobial antiviral and antifungi effects sedative and hypnotic effects others toxicity quality control pharmacokinetics patented formulations and perspectives acknowledgements conflict of interest author contribution references preproof 0c introduction herba patriniae as known as œbai jiang cao in chinese is a traditional chinese medicine tcm originally recorded in œshen nong™s herbal classic as a middle grade medicinal material which has been used for thousands years besides korean ancient pharmacopaea œdonguibogam also record its medical value and it has been used for more than years in korea jeon et al it possesses the tcm properties of pungent and bitter in flavor and slightly cold in nature and has been classified to the stomach large intestine and liver meridians xiao two official species of patrinia scabiosaefolia fisch ps and patrinia villosa juss pv figure were considered as herba patriniae in chinese pharmacopoeia edition and chinese provincial pharmacopoeias these two plants have been widely used for more than years with good biological activities of clearing heat and detoxification eliminating carbuncle and expelling pus dispelling blood stasis and relieving pain through an analysis of ancient and modern literatures herba patriniae was mostly used in intestinal carbuncle lung carbuncle gynecological epigastric pain postpartum blood stasis and eczema in ancient times chen and han modern pharmacological studies have found that it has effects of anticancer antiinflammation antipathogenic microanisms antioxidation sedation and hypnosis wang et al 2019a nowadays herba patriniae is widely used in the respiratory system digestive system genitourinary system gynecology dermatology and other multidisciplinary diseases in clinical practice zhu and jiang and the number of applied patents increases every year httppsssystemcnipagovcn in view of its high content of amino acids vitamins minerals and other nutrients herba patriniae is not only regarded as a potherb with healthy value but also processed into tea products su et al zeng et al zhong et al in the past decades an increasing number of scholars have studied the chemical constituents and pharmacological effects of herba patriniae interestingly based on these studies we found that there are many differentsame characteristics between ps and pv both of them are official species for herba patriniae but differentiated clinical uses of them in different diseases may be better for the clinical outcome unfortunately we cannot found a comprehensive and updated review on the samedifferent characteristics of the two sources of herba patriniae and actually these two species also have not been differentiated in clinical uses therefore this review aims to systematically summarize the similarities and differences from the preproof 0c aspects of the traditional uses botanical description phytochemistry pharmacology and quality control of these two species of herba patriniae as well as being evidences for their clinical application and further research figure two species of herba patriniae a patrinia villosa juss b patrinia scabiosaefolia fisch a httpwwwcvhaccnspmcshcsh0005548 b httpwwwcvhaccnspmsyaufsyauf010108 traditional uses herba patriniae has a wide geographical distribution mainly in east asia and north america he et al some plants such as sonchus arvensis l sonchus asper vill sonchus oleraceus l etc may be confused as herba patriniae lu and hence these adulterants of herba patriniae should be exclude when clinical use traditionally according to records of œshen nong™s herbal classic 神农本草经 œcompendium of materia medica 本 草 纲 目 and œsynopsis of the golden chamber 金匮要略 œtai ping sheng hui fang 太平圣惠方 œpu ji fang 普 济方 œsheng ji zong lu 圣济æ»å½• œqian jin yi fang 千金翼方 and œqian jin fang 千金方 ancient doctors have used the whole herbs and roots of herba patriniae for disease treatment such as the stomach intestine liver gallbladder and gynecological diseases tian and tian zhu and jiang herba patriniae was recorded in chinese pharmacopoeia edition for the treatments of appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain swollen wellingabscess and clove sores pharmacopoeia committee of the ministry of health of p r china in addition herba patriniae is also preproof 0c recorded in the standards of traditional chinese medicine in many provinces of china table in miao nationality herba patriniae is also called œjia jiang le and used to treat rheumatoid arthritis colds and diarrheal qiu wang in dong medicine lu yi medicine drug control institute of yunnan chuxiong health bureau and dai medicine shi ps is called œnyangt ngeec liongc bail jangl œshe wei long and œpa hong respectively its whole herb is used to treat infantile diarrhea schizophrenia and infantile tinea capitis respectively ps is also called ba gai bao in zhuang medicine and its root is used to treat icteric hepatitis furuncles and snakebites shi pv is called œbitter vegetable by she nationality biological products identification institute of the ministry of health and œpao zi tong by tujia nationality peng and guan its whole herb can be used to treat appendicitis intestinal febrile symptoms constipation mammary abscess blister carbuncle and qi stagnation pv is also called œba gai lan and œhong pa in zhuang medicine biological products identification institute of the ministry of health and dai medicine shi respectively and its root is used to treat jaundice hepatitis furuncle local ulceration caused by snake injury and infantile convulsion moreover in korea people usually use the roots or whole plants of ps and pv as a traditional herbal medicine to treat appendicitis inflammation wound healing edema abscesses endometritis and abdominal pain after childbirth kang et al yang et al in recent years herba patriniae has been extensively applied in clinical practice in china especially in gynecology such as postpartum pain mastitis dysmenorrhea and tubal obstructive infertility liu 2019a it is noteworthy that herba patriniae is one of the most important ingredients in many prescriptions of tcm which is effective in diarrhea he acute hepatitis song pelvic inflammatory disease zhang 1997a typhoid fever paratyphoid fever sun ulcerative colitis liu anal cryptitis shi pelvic endometriosis yan and qiu acute pancreatitis he et al 2019b itching wang and wang gastroesophageal reflux disease benign prostatic hyperplasia rhinosinusitis mumps and phlebitis kong and zhao zhu and jiang a powder composed of coicis semen radix aconiti lateralis preparata and herba patriniae is a classic prescription for treating intestinal carbuncle in the œsynopsis of the golden chamber which is clinically used to treat chronic appendicitis chronic pelvic inflammatory disease and chronic prostatitis ji in addition a powder containing herba preproof 0c patriniae in the prescription is also used to treat sinusitis acute purulent tonsillitis and recurrent upper respiratory tract infection qin and diao zhu and jiang moreover it showed significant efficacy in the treatment of psoriasis vulgaris yan et al keshan disease scientific research cooperation group of herba patriniae in yan'an city for the prevention and control of keshan disease and chronic pelvic inflammation jia in the form of tablets in the chinese pharmacopoeia edition there are chinese herbal medicine prescriptions containing herba patriniae among which kangfu xiaoyan shuan and yifei qinghua gao are used to treat gynecological diseases and respiratory diseases respectively while longqing pian nankang pian niaosaitong pian and qianliexin jiaonang are used to treat genitourinary diseases state pharmacopoeia commission of p r china a summary of the traditional and traditional and clinical preparation of herba patriniae in china is given in table the tender stems and leaves of herba patriniae are rich in nutrients fresh in taste and grow in the mountains without environmental pollution it is a highquality vegetable that urban and rural residents like to eat pv tea is also abundant in hubei province and fujian province jiang 2019a xu et al herba patriniae is not only used in human health but also in agriculture fishery and animal husbandry interplanting herba patriniae in the newly reclaimed tea garden can increase the natural vegetation and reduce soil erosion and surface runoff caused by rainstorm erosion in the rainy season chen the combination of herba patriniae and other medicinal plants can be used to treat poisoned wound of cattle by agkistrodon acutus bitting crawling bee disease liver and skin diseases of turtle and fish and postpartum abdominal pain in cattle chen li shi zhao preproof 0c table the information of herba patriniae in national and local standards in china standards standard of traditional chinese medicine in hunan province standard of traditional chinese medicine in shandong province standard of traditional chinese medicine in heilongjiang province standard of traditional chinese medicine in liaoning province standard of traditional chinese medicine in sichuan province standard of traditional chinese medicine in guizhou province chinese pharmacopoeia edition application acute appendicitis diarrhea enteritis hemorrhagic leucorrhea red eye pterygium postpartum abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles acute appendicitis diarrhea hemorrhagic leucorrhea postpartum blood stasis abdominal pain swelling and pain of eye hepatitis boils and carbuncles acute appendicitis diarrhea dysentery postpartum blood stasis abdominal pain conjunctivitis boils and carbuncles acute appendicitis and its abdominal pain postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles dosage g standardsetting department hunan food and drug administration g g g g g g shandong medical products administration heilongjiang medical products administration liaoning food and drug administration sichuan food and drug administration guizhou medical products administration pharmacopoeia committee of the ministry of health of p r china preparation name yiyi fuzi baijiang san 薏苡附子败酱散 machixian heji 马齿苋合剂 aiye san 艾叶散 baijiang san 败酱散 baijiang san 败酱散 baijiang tang 败酱汤 baijiang tang 败酱汤 table traditional and clinical preparation of herba patriniae in china formulation main compositions powder coicis semen aconiti lateralis radix praeparata herba patriniae decoction portulacae herba isatidis folium arnebiae radix herba patriniae persicae semen carthami flos paeoniae radix rubar powder powder artemisiae argyi folium angelicae sinensis radix paeoniae radix alba dipsaci radix achyranthis bidentatae radix herba patriniae herba patriniae angelicae sinensis radix chuanxiong rhizoma paeoniae radix alba cinnamomi cortex powder herba patriniae moutan cortex cinnamomi cortex siphonostegiae herba aucklandiae radix decoction herba patriniae notopterygii rhizoma et radix dianthi herba aurantii fructus cinnamomi cortex persicae semen decoction herba patriniae reference jin gui yao lüe ãŠé‡‘匮要略㋠zhang 1997b surgery of chinese medicine ãŠä¸­åŒ»å¤–科学㋠beijing traditional chinese medicine hospital tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang pu ji fang ãŠæ™®æµŽæ–¹ã‹ volume zhu tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao qian jin yi fang ãŠåƒé‡‘翼方㋠volume sun preproof 0cpreparation name baijiang tang 败酱汤 baijiang tang 败酱汤 baijiang yin 败酱饮 changyong tang 肠痈汤 decoction chenzhou sheyao pian 郴州蛇药片 chure jili wan 除热蒺藜丸 tablets pills danggui xi tang 当归洗汤 danggui yin 当归饮 ganyan chongji 肝炎冲剂 decoction decoction electuary formulation main compositions decoction herba patriniae rhei radix et rhizoma persicae semen decoction herba patriniae cinnamomi cortex siphonostegiae herba moutan cortex aucklandiae radix decoction herba patriniae angelicae sinensis radix bambusae caulis in taenias rehmanniae radix moutan cortex glycyrrhizae radix et rhizoma herba patriniae zingiberis rhizoma recens poria coicis semen platycodonis radix liriopes radix salviae miltiorrhizae radix et rhizoma paeoniae radix alba rehmanniae radix pv tribuli fructus rhei radix et rhizoma herba patriniae cinnamomi cortex ginseng radix et rhizoma aconiti lateralis radix praeparata coicis semen coptidis rhizoma astragali radix abri herba angelicae sinensis radix aurantii fructus immaturus paeoniae radix alba tetrapanacis medulla angelicae sinensis radix angelicae pubescentis radix angelicae dahuricae radix sanguisorbae radix herba patriniae angelicae sinensis radix herba patriniae dipsaci radix paeoniae radix alba rehmanniae radix bambusae caulis in taenias bupleuri radix angelicae sinensis radix paeoniae radix alba paeoniae radix rubra citri reticulatae pericarpium aurantii fructus curcumae radix cyperi rhizoma salviae miltiorrhizae radix et rhizoma scrophulariae radix artemisiae scopariae herba isatidis radix herba patriniae huangdan tang 黄疸汤 decoction jiedu dihuang wan 解毒地黄丸 pills gualou san powder artemisiae scopariae herba gardeniae fructus lonicerae japonicae flos forsythiae fructus herba patriniae isatidis radix paeoniae radix rubra paeoniae radix alba bupleuri radix perillae caulis platycodonis radix sojae semen germinatum rehmanniae radix astragali radix trichosanthis radix scutellariae radix liriopes radix mantidis ootheca rhei radix et rhizoma ginseng radix et rhizoma gardeniae fructus cistanches herba peucedani radix cimicifugae rhizoma paeoniae radix alba anemarrhenae rhizoma vaccariae semen polygalae radix herba patriniae jujubae fructus trichosanthis semen herba patriniae asari radix et rhizoma zingiberis rhizoma magnoliae officinalis cortex platycodonis radix ginseng radix et rhizoma saposhnikoviae radix reference sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao qian jin fang ãŠåƒé‡‘方㋠volume （sun ） gu jin ming fang ãŠå¤ä»Šåæ–¹ã‹ yan and liu qian jin fang ãŠåƒé‡‘方㋠volume （sun ） qian jin fang ãŠåƒé‡‘方㋠volume （sun ） sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao study on the treatment of common diseases with traditional chinese medicine ãŠå¸¸è§ç—…的中 医 æ²» 疗 研 究 ㋠teaching and research group of traditional chinese medicine the first affiliated hospital of xi'an medical college lin zheng yi an yi fang ãŠä¸´è¯åŒ»æ¡ˆåŒ»æ–¹ã‹sun sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume preproof 0c preparation name 栝蒌散 lanwei xiaoyan wan 阑尾消炎丸 lanweiyan heji 阑尾炎合剂 formulation main compositions pills lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba spatholobi caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix decoction lonicerae japonicae flos taraxaci herba herba patriniae forsythiae fructus rhei radix et rhizoma paeoniae radix rubra toosendan fructus aucklandiae radix persicae semen lanweiyan tang 阑尾炎汤 lanwei yihao xiaoyan wan 阑尾ä¸å·æ¶ˆç‚Žç‰‡ lenge xiaoji tang 棱莪消积汤 lishi zhiyang pu yao 理湿止痒扑药 lidan tuihuang tang 利胆é黄汤 neibu wuxiang wan 内补五香丸 qianliexian tang 前列腺汤 qumai wan 瞿麦丸 decoction pills decoction powder decoction pills decoction pills yinqiao hongjiang jiedu tang decoction rhei radix et rhizoma moutan cortex persicae semen paeoniae radix alba salviae miltiorrhizae radix et rhizoma bupleuri radix lonicerae japonicae flos forsythiae fructus herba patriniae coicis semen lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix talci pulvis sargentodoxae caulis sparganii rhizoma curcumae rhizoma salviae miltiorrhizae radix et rhizoma paeoniae radix rubra corydalis rhizoma moutan cortex persicae semen coicis semen sargentodoxae caulis herba patriniae kochiae fructus bombyx batryticatus dictamni cortex angelicae dahuricae radix schizonepetae spica artemisiae scopariae herba herba patriniae alumen glycyrrhizae radix et rhizoma talcum cinnabaris artemisiae scopariae herba herba patriniae isatidis radix curcumae radix gardeniae fructus aquilariae lignum resinatum olibanum aucklandiae radix caryophylli flos dipsaci radix rehmanniae radix praeparata paeoniae radix alba magnoliae officinalis cortex herba patriniae ginseng radix et rhizoma poria cervi cornu salviae miltiorrhizae radix et rhizoma lycopi herba paeoniae radix rubra persicae semen carthami flos olibanum myrrha vaccariae semen citri reticulatae pericarpium toosendan fructus foeniculi fructus angelicae dahuricae radix herba patriniae taraxaci herba dianthi herba realgar vaccariae semen rehmanniae radix ephedrae herba imperatae rhizoma herba patriniae saposhnikoviae radix achyranthis bidentatae radix rhei radix et rhizoma lonicerae japonicae flos forsythiae fructus sargentodoxae caulis herba patriniae moutan cortex gardeniae fructus paeoniae radix rubra persicae semen coicis semen corydalis rhizoma toosendan fructus olibanum myrrha reference zhao beijing chinese traditional patent medicine specification ãŠåŒ—京市中成药规范㋠volume beijing municipal bureau of health selected data of acute abdomen treated by integrated traditional chinese and western medicine ãŠä¸­è¥¿åŒ»ç»“合治疗æ¥è…¹ç—‡èµ„æ–™é‰ç¼–ã‹ affiliated hospital of guangzhou college of traditional chinese medicine lin zheng yi an yi fang ãŠä¸´è¯åŒ»æ¡ˆåŒ»æ–¹ã‹ sun compilation of traditional chinese medicine preparations ãŠä¸­è¯åˆ¶å‰‚汇编㋠cao obstetrics and gynecology ㊠妇 产 科 å­¦ ã‹shanghai college of traditional chinese medicine selection of medical prescriptions of cixi and guangxu ãŠæ…ˆç¦§å…‰ç»ªåŒ»æ–¹é‰è®®ã‹ chen gu jin ming fang ãŠå¤ä»Šåæ–¹ã‹ yan and liu tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang surgery of chinese medicine ãŠä¸­åŒ»å¤–科学㋠beijing traditional chinese medicine hospital qian jin yi fang ãŠåƒé‡‘翼方㋠volume sun obstetrics and gynecology ㊠妇 产 科 å­¦ ã‹shanghai college of traditional chinese preproof 0c formulation main compositions preparation name 银翘红酱解毒汤 danhuang quyu jiaonang 丹黄祛ç˜èƒ¶å›Š qianlieping jiaonang 前列平胶囊 fuping jiaonang 妇平胶囊 fuyan kangfu jujue pian 妇炎康复å’嚼片 fuyan kangfu pian 妇炎康复片 fuyan kangfu jiaonang 妇炎康复胶囊 fuyan kangfu keli 妇炎康复颗粒 fuyanxiao jiaonang 妇炎消胶囊 fuyanqing xiji 妇炎清洗剂 capsules capsules capsules tablets tablets capsules granules capsules lotion xiaoer reke koufuye 小儿热咳口服液 oral liquid kangfu xiaoyan shuan 康复消炎栓 kangfu xiaoyan jiaonang suppository capsules astragali radix salviae miltiorrhizae radix et rhizoma dioscoreae rhizoma smilacis glabrae rhizoma angelicae sinensis radix spatholobi caulis euryales semen houttuyniae herba sparganii rhizoma curcumae rhizoma scorpio herba patriniae cinnamomi cortex atractylodis macrocephalae rhizoma zingiberis rhizoma praepatum eupolyphaga steleophaga corydalis rhizoma toosendan fructus sophorae flavescentis radix herba patriniae salviae miltiorrhizae radix et rhizoma paeoniae radix rubra persicae semen carthami flos lycopi herba pyrrosiae folium olibanum myrrha reference medicine httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa fagopyri dibotryis rhizoma violae herba curcumae rhizoma herba patriniae polygoni perfoliati herba solidaginis herba httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae trichosanthis radix rhei radix et rhizoma moutan cortex atractylodis rhizoma linderae radix httpswwwyaozhcom nmpa taraxaci herba herba patriniae coicis semen paeoniae radix rubra atractylodis rhizoma angelicae sinensis radix chuanxiong rhizoma cyperi rhizoma corydalis rhizoma alismatis rhizoma ephedrae herba armeniacae semen amarum forsythiae fructus rhei radix et rhizoma trichosanthis fructus mori cortex herba patriniae carthami flos glycyrrhizae radix et rhizoma sophorae flavescentis radix violae herba herba patriniae andrographis herba suis fellis pulvis taraxaci herba arnebiae radix aloe httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa state pharmacopoeia commission of p r china httpswwwyaozhcom taraxaci herba herba patriniae paeoniae radix rubra coicis semen angelicae sinensis radix atractylodis rhizoma chuanxiong rhizoma cyperi rhizoma alismatis rhizoma corydalis rhizoma httpswwwyaozhcom nmpa preproof 0c formulation main compositions reference preparation name 康妇炎胶囊 manshenning heji 慢肾宁合剂 nankang pian 男康片 decoction tablets astragali radix cinnamomi ramulus epimedii folium rehmanniae radix asini corii colla poria alismatis rhizoma scutellariae radix herba patriniae moutan cortex leonuri herba httpswwwyaozhcom nmpa paeoniae radix rubra rehmanniae radix praeparata cistanches herba glycyrrhizae radix et rhizoma taraxaci herba pyrolae herba phellodendri chinensis cortex carthami flos houttuyniae herba epimedii folium fubi fructus atractylodis macrocephalae rhizoma astragali radix cuscutae semen violae herba herba patriniae chrysanthemi indici flos angelicae sinensis radix sophorae fructus notoginseng radix et rhizoma sophorae flavescentis radix bletillae rhizoma cnidii fructus artemisiae argyi folium herba patriniae lonicerae japonicae flos portulacae herba saposhnikoviae radix alumen borneolum syntheticum glycyrrhizae radix et rhizoma pv sucrose dextrin astragali radix codonopsis radix glehniae radix liriopes radix agrimoniae herba bistortae rhizoma fritillariae cirrhosae bulbus asteris radix et rhizoma platycodonis radix armeniacae semen amarum herba patriniae glycyrrhizae radix et rhizoma codonopsis radix trionycis carapax paridis rhizoma atractylodis macrocephalae rhizoma astragali radix citri reticulatae pericarpium eupolyphaga steleophaga rhei radix et rhizoma persicae semen scutellariae barbatae herba herba patriniae poria coicis semen curcumae radix sappan lignum ostreae concha artemisiae scopariae herba cyperi rhizoma polygoni cuspidati rhizoma et radix ardisiae japonicae herba sedi herba scutellariae barbatae herba magnoliae officinalis cortex herba patriniae arnebiae radix wenyujin rhizoma concisum lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba spatholobi caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba sargentodoxae caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix lonicerae japonicae flos herba patriniae taraxaci herba moutan cortex toosendan fructus paeoniae radix rubra rhei radix et rhizoma persicae semen aucklandiae radix state pharmacopoeia commission of p r china httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa state pharmacopoeia commission of p r china httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa atractylodis rhizoma poria acori tatarinowii rhizoma plantaginis semen indigo naturalis phellodendri amurensis cortex talcum herba patriniae salviae miltiorrhizae radix et rhizoma ht
Colon_Cancer
" many suggest that shared decisionmaking sdm is the most effective approach to clinical counseling itis unclear if this applies to surgical decisionmakingespecially regarding urgent highlymorbid operations in thisscoping review we identify s that address patient and surgeon preferences toward sdm in surgerymethods we used the preferred reporting items for systematic reviews and metaanalyses extension for scopingreviews prismascr to develop our protocol medline embase and cochrane databases were searched frominception through title review identified peerreviewed empirical s that addressed patientsurgeon preferences toward sdm in surgery identified s underwent full review by two independentinvestigators we addressed the following questions what is known from existing empirical evidence aboutpatients™ andor surgeons™ surgical decisionmaking preferences why might patients andor surgeons prefer sdm does acuity of intervention impact surgical decisionmaking preferences outcome measures included studymethods surgical specialty diagnosis study locationsetting typenumber of subjects acuity of intervention surgeonpatient decisionmaking preferences and factors associated with favoring sdm data was analyzed in microsoft excelresults s were identified with duplicates yielding s for title review swere included in final analysis of s discussed oncologic decisionmaking of these focused on breastcancer of s included patients included surgeons of s found surgeons favored sdm demonstrated surgeons favored surgeon guidance of s demonstrated patients favored sdm showedpatients favored surgeon guidance showed patients preferred independent decisionmaking the most commonfactors for patients favoring sdm included female gender higher education and younger age for surgeons the mostcommon factors for favoring sdm included limited evidence for a given treatment plan multiple treatment optionsand impact on patient lifestyle no s evaluated decisionmaking preferences in an emergent settings there has been limited evaluation of patient and surgeon preferences toward sdm in surgical decisionmaking generally patients and surgeons expressed preference toward sdm none of the s evaluated decisionmaking preferences in an emergent setting so assessment of the impact of acuity on decisionmaking preferences islimited extension of research to complex emergent clinical settings is neededkeywords surgery shared decision making ethics correspondence ericacarlisleuiowaedu1program in bioethics and humanities university of iowa carver college ofmedicine iowa city usa3department of surgery university of iowa hospitals and clinics iowa cityusafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cshinkunas bmc medical informatics and decision making page of over the past several decades physician paternalism hasbeen systematically rejected and respect for patient autonomy has emerged as a leading ethical priority in clinical counseling shared decisionmaking sdm aprocess by which physicians and patients actively worktogether to integrate care plans that are responsive topatient goals and values has been advocated as a clinicalcounseling approach that promotes patient autonomy byencouraging patients to participate in clinical decisionmaking [“] along with its presumed promotion ofpatient autonomy data suggesting that sdm reduceshealth care costs and improves quality of care have ledto relatively widespread incorporation of sdm intohealth policy despite this implicit acceptance ofsdm relatively limited data exist regarding patient orphysician preferences toward sdm such data seem tobe especially lacking in surgical decisionmakingby supporting patient autonomy sdm places somelimits on the extent to which a physician™s influenceguides a patient™s decisions some ethicists have arguedthat such prioritization of patient autonomy is criticallyimportant and that even subtle attempts by a physicianto sway a patient toward a particular decision violates respect for patient autonomy however others have argued that if attempts to promote patient autonomy aretoo strong or rigid the emphasis on selfdeterminationmay be inconsistent with patients™ wishes for more professional guidance [ ] in fact there is an emergingbody of literature that suggests that patients may prefermore physician guidance during medical decision making [ “] these findings prompt one to questionwhether autonomyheavy approaches to sdm in clinicalcounseling are always consistent with patient preferencesor whether patients would at least sometimes prefer aless autonomous and more guided approach to clinicalcounselingwith respect to the physician™s perspective it is important to note that studies have shown physicians to besomewhat reluctant to incorporate sdm into clinicalpractice one reason for this may be a sense thatwhen a physician overly prioritizes patient autonomythere is lessening of the physician™s role such that the fiduciary nature of the patientphysician relationship isundermined prioritization of patient autonomy and integration of sdm into clinical counseling has left somephysicians feeling that their role has become one ofmerely offering patients the information necessary tomake their own œinformed decisions rather than trulyengaging in a fiduciary relationship with the patient this is illustrated in a recent narrative that describes anencounter in which a physician reviewed all options fortreatment of nonischemic cardiomyopathy with her patient but was stopped by the patient before she couldmake a recommendation with the request that the patient be allowed time to independently reflect and makea decision that was best for him in the physician™s reflection on the encounter she notes œsince the decisionwas his it was no longer mine i had informed him buthad i been his doctor perhaps such efforts to assure patient autonomy and sdm limit the role of thephysician in patient counseling these types of reportscall for further investigation so we can better understandphysician preferences toward shared decision makingconcerns about the appropriateness of sdm may beparticularly pronounced in surgical decision making giventhe often dramatic and irreversible outcomes associatedwith surgery these concerns may further escalate whenconsidering emergent highly complex operations that areassociated with a high risk of mortality or morbidity in aninitial effortto better understand preferences towardsdm in surgical decision making we reviewed the literature regarding parent and surgeon preferences towardsdm in pediatric surgery we found that there wasmarkedly limited data available of the existing sthe predominant focus was on parent preferences towarddecision making in elective nonurgent procedures therewas limited data regarding surgeon preferences and virtually no discussion of preferences for decision making inmore urgent settings the purpose of this review is to gain a more thorough understanding of patient and surgeon preferences toward sdmin adult surgery we chose to conduct a scoping review because there is limited published data on patient and surgeondecision making preferences particularly when surgery isconsidered urgent or emergent scoping reviews are a valuable methodology because they allow for the mapping of important concepts and research gaps in a defined area ofstudy by comprehensively identifying reviewing and summarizing the existing information from the literature specific research questions addressed in our scoping reviewincluded what is known from existing empirical evidence about patients™ andor surgeons™ surgical decisionmaking preferences why might patients andor surgeonsprefer sdm does acuity of intervention impact surgicaldecisionmaking preferencesmethodsprotocol designour scoping review protocol follows arksey and o™malley™s methodological framework as well as the preferred reporting items for systematic reviews andmetaanalyses extension for scoping reviews prismascr this protocol has not been registeredidentifying relevant studiesafter ascertaining our research questions we worked inconjunction with an experienced medicallibrarian to 0cshinkunas bmc medical informatics and decision making page of identify relevant studies we followed the preferredreporting items for systematic reviews and metaanalyses prisma guidelines for reporting the identified screened eligible and included studies fig after drafting refining and finalizing our search strategies we searched three bibliographic databases from inception through november medline embaseand cochrane databases the final search strategies forall three databases are outlined in additional file thefinal search results were imported into endnote versionx91 and yielded sstudy designeligibility criteriainclusion and exclusion criteria were defined a priorireview was limited to english language no translatorsavailable peerreviewed published literature only empirical studies were included review was limited todecision making preferences of surgeons andor adultpatients decision making preferences were loosely defined and included œpreferred role œperceived role œexpectationsœdesires and œsatisfaction with actualdecisionmaking role s in the following categories were excluded reviews letters to the editor editorialssuggested models of care patient educationhandouts decision making tools animal studies and s related to pediatric surgery in addition we excluded s without accessible full textliterature reviewafter duplicates were removed by the primary authorlas we were left with s to screen twoof the authors las and emc independently reviewedall titles and s and jointly decided to exclude s based on the eligibility criteria theremaining s were selected for full text reviewfig flow diagram of study selection 0cshinkunas bmc medical informatics and decision making page of following full text review additional s were excluded because they either did not pertain to an adultsurgical population or to decisionmaking preferences inthe surgery setting disagreements were resolved by discussion between the two authorssubjects gender acuity ofsurgeryothercharting the datafor each of the included s two of the authorslas and cjk independently ed the followingoutcome measures study methods quantitativequalitativemixed methods surgical specialty cancer diagnosisyesnounclear study location usnonus study setting inpatientoutpatient type of subject patientsurgeon number oftheintervention electiveurgentemergentunclear surgicaldecision to be made surgery v nonoperative managementchoice among different surgical proceduresdecision on timing ofsurgeonpatientdecision making preferences shared decision makingsurgeon guided decision making independent decisionmaking and surgeonpatient factors associated with favoring sdmacuity ofthe intervention was defined as followsemergent immediate need for surgery to preserve lifeurgentsurgery is required within the next days orweeks and elective surgery is not required notablycancer resections were considered urgent however subsequentreconstruction was considered elective iebreast cancer resection with subsequent reconstructionthe control preferences scale which is a fivepointmeasure used to gauge preferred involvement in medicaldecision making was adapted to define surgeonpatient preferences as follows shared decision makingsdm the patient and surgeon prefer to make the decision regarding surgery together surgeon guided decisionmaking sg the preference is for the surgeon to guidedecision making either entirely or in part while the patient takes a more œpassive role independent decisionmaking idm the preference is for the patient to take amore œactive role in decision making either partly orentirely independent from the surgeonthe data ion form was a modified version ofthe one we used for a literature review we conducted ondecision making preferences in the pediatric surgical setting discordant opinions were discussed at weeklymeetings the third author emc was available to mediate if consensus could not be reached data was analyzed in microsoft excel resultssummarizing collating and reporting the resultssummarizing the results s were identified medline n embase n cochrane n duplicateswere removed and s underwent title review seventyfour s were included in thefinal analysis because they specifically addressed existingempirical evidence about patient andor surgeon decision making preferences toward sdm in adult surgerysetting table provides a summary of allincludedscollating and reporting the resultstable provides frequencies for the characteristics of allincluded s over half of the s were quantitative n and performed outside of the us n sixtyseven included outpatient surgeries fourteen surgical subspecialties were representedwith the most s originating from surgical oncology n general surgery n orthopedic surgery n and urology n fifty s discussed decision making for patients with cancer and of these focused onbreast cancer most s assessed a choice betweenoperative and nonoperative management n or an option among different surgical procedures n sixtyeight of the s included patients ofthese demonstrated that patients preferredsdm showed that patients favored a surgeonguided approach and revealed a patient preference for independent decision making the most common factors for patients favoring sdm included femalegender higher education and younger ageonly of the s assessed surgeons™ preferences of these found that surgeons preferredsdm while demonstrated that surgeons favoreda more surgeonguided decisionmaking approach thefactors most commonly listed for surgeons favoringsdm included limited evidence for a given treatmentplan multiple treatment options and impact on patientlifestylenone of the s evaluated patient decisionmakingpreferences in an emergent setting out of the sthat assessed patient decisionmaking preferences in theelective surgery setting preferred sdm threeout of four of the s assessing surgeon decision making preferences in the elective surgery settingreported that surgeons preferred sdm in six out of nine of the s surgeons also preferred sdm in theurgent surgery setting in s patients were fairlysplit on their decisionmaking preference when it cameto urgent surgeries with desiring sdm and favoring a more surgeonguided approachonly s looked at both patient and surgeon decision making preferences in a little over half ofthese s n there was discordance between patient and surgeon decision making preferences 0cshinkunas bmc medical informatics and decision making page of table characteristics of included sacuity of andthedate ofpublicationinterventionstudy populationmajor findings related to decisionmaking dm preferencesalmyroudi ananian andersen asghari ashraf avis ballinger breast cancerpatients breast cancerpatientsurgentelective breast cancersurvivorsurgent hospitalized patients on surgical wardsunclear patients undergoingeither immediate ordelayed breastreconstructionelective hernia repair patientselective breast cancerpatientsurgentbeaver colorectal cancerpatientsurgentbeaver belue health professionalscaring for colorectalcancer patients weresurgeons cardiologists making adecision about surgery patients with coronaryartery diseaseblumenthalbarby left ventricular assistdevice patients andcandidatesburton older breast cancerpatientsbutow patient advocates breast cancersurgeonsurgenturgenturgenturgenturgent preferred a passive role acollaborative role46 an active role of women choosing breastreconstruction œdecided with surgeon of these patients were satisfied withthe information receivedon average reported being œveryinvolved i made all the decisions myself were content with dm roleœstrongly desire to receive informationand participate in decisionmaking were in the œinformedconsumeristgroup when it came to actual dm of these patients were satisfied with theinformation receivedœexpectations of participation can besummarized as ˜being told™ and ˜going into get it fixed™ œfelt their healthcare professionalshad surgical preferences for thembelieved that clinical issues determinedthese preferences but still knew thechoice was theirsœwanted to be well informed andinvolved in the consultation process butdid not necessarily want to use theinformation they received to makedecisionsœshared decision making was favored byhealth professionalsphysicians œprefer patients whoactively participate in the decisionpatients œprefer the physician tomake the decision sdm œprefer to make the decision on theirownœdeferred heavily to clinicians preferred œpatientcentred œdoctorcentred sdm of surgeons and of patientadvocates preferred sdm breast cancer survivors urgentcampesino cohen patients with localizedprostate cancerurgentcorriere patients undergoingelectivespanishspeaking latinas preferredœphysician treatment recommendationsenglishspeaking latinas and africanamericans preferred sdmmost viewed the surgeonguided approach as œappropriate and welcome preferred œchoosing together withdm themerelated tomajorfindingsaptsgsurg“factors associated with favoringsdmyounger age higher educationsdm “type of procedureidm“younger age level of educationincomesdm “female level of educationidmsg““sdm “sg““sdmyounger patient agesgsdm““sgsgidmsdm sdmsdmsgsg““englishspeakingsdm “multiple treatment options type 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferencesfactors associated with favoringsdmdm themerelated tomajorfindingsaptsurg elective vascularprocedures prostate cancersurvivors hand surgeons patients with triggerfinger breast cancerpatientsurgentelectiveurgentcuypers doring durifbruckert gainer the provider preferred œhaving theprovider choose for them preferred a collaborative role an active role a passive roleof proceduresdm “higher education younger agehigher sespatients œpreferred to decide forthemselves surgeons preferred sdmidmsdmwanted to participate in decisions butœperceived sdm as an obligation becauseit did not seem to fit with their idea of aproper doctorpatient relationshipsg“trust in surgeon support fromfamily written information fromsurgeon frail and olderpatients care teammembers includessurgeonsunclearboth patients and care team membersœsupported a formal approach to sdmsdm sdmghane general surgerypatientsgolden clinicians weresurgeonsgong patients with carpaltunnel syndromeelectiveurgentœpreferred relatively high levels ofdecisional control on averagem out of sd most felt that they practiced sdm eventhough they did not tend to distinctlyprompt patient dm preferencesidm“male good health high healthliteracy“sdmelective preferred sdmsdm “hack breast cancerpatientshageman hand surgeons patients with carpaltunnel syndromehawley breast cancerpatientsheggland hausken health professionalsfrom surgical wards patients who underwentsurgical treatmenturgentelectiveurgentelectiveheggland hausken surgical patients surgeonselectiveurgentheggland [ ]henderson shum physicians workingin surgical wardsunclear surgical and medicalpatientselectiveurgent preferred a collaborative role an active role a passive rolesurgeons preferred œpatient andprovider make a shared decisionpatients preferred that œthe patientdecidesactual dm role sdm œpatientbased œsurgeonbased preferreddm role content with level of dminvolvementhealth professionals majority preferred aœshared or œinformed model patientsabout half preferred a œshared orœinformed model and the other halfpreferred a œpaternalistic modelsurgeons the majority preferred anœinformed model ¦ patient is giveninformation and left to make thedecision patients preferred aœpaternalistic model and preferredsharedphysicians on average rated decisionmaking control a which means thatœphysicians were not reluctant to involvepatients in decisionmaking processeswhere active role shared and passive “ the mean dm value for thesevere scenario was moderatescenario was mild scenario was history of surgical procedureimportance of family memberopinions having privateinsuranceage nonwidowed longerduration postopsdm “idmsdm“sdmidmsdmfemalesdmsgsgidm“sdmsdm “younger age noncriticalcondition 0cdm themerelated tomajorfindingsaptsdm “surgfactors associated with favoringsdmfemale higher educationsgsg“““female no stomasdmmore years in practice morecomfort in pulmonary nodulemanagementcollege degree higher selfefficacysdm “sdm “sg“sdm “œfemales indicated that they would like tohave more input in the decisionmakingprocess than the males v onthe controlled preferences scaleœguidance by the clinician was identifiedas most important œactive role of patientin treatment decision making regardedas less important preferred a passive role sdm an active roleperceived role œshare decisionsequally with the patient œallowthe patient to decide œdecide forthemselves after considering the patient™sopinionœmost patients wanted to decide on theirtreatment options together with theirphysician preferred sdm preferred tomake the decision œwith physician inputœ wanted their surgeons to make arecommendation and when given followed the recommended treatmentplan preferred a collaborative role inpatients in twosurgical unitsunclear lung cancer patientsurgenthenderson hopmans hou colorectal cancerpatientsiaccarino clinician members ofthe american thoracicsocietyurgenturgentihrig prostate cancerpatientsjanz breast cancerpatients newly diagnosedbreast cancer patientsurgenturgenturgent breast cancerpatientsurgentjohnson keating keating lally shinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferences lung or colorectalcancer patientsurgent œpatient controlled sdm œphysician controlledsdm “married better prediagnosishealth status caucasian strongevidence for procedure breast cancer patients urgentœwomen™s lack of sharing theirpreferences with their surgeons and thesurgeons™ lack of making treatmentrecommendations resulted in what wasmore likely informed than shared decisionmaking preferred sdm preferred œthechoice to be their own preferred œtodelegate the decisionactual role sdm madedecision with œsurgeon input weresatisfied with dm levelactual dm œjoint patientdoctor decision œdoctor advocated œpatient asked preferred dm contentwith level of dm involvementthe surgical group showed a morepassive role in both their preferred andactual dm roleœsurgeon™s recommendationand fear of dying from cancer played themost important role in dmin surgery preferred a œdirectivecommunication style a œnondirective communication styleidm“sdm “younger agesdm “sdm “femalesgsg““sdmsglam breast cancerpatientslantz breast cancerpatientsurgenturgentlarsson patients scheduledfor invasive surgeryelectiveurgenturgentlee patients with earlygastric cancermarkovic martinez newly diagnosedgynecologic cancerpatients newly diagnosedbreast cancer patientsurgent 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferences surgeonsunclearmcguire mendick breast cancer patients surgeonsurgentmeredith surgical patients surgeonsunclear older breast cancerpatientsurgent patients withirritable bowel diseaseelective breast cancer patients urgentman morishige moumjid nam patients with carpaltunnel syndromeelectiveomar op dendries consecutive patientsbeing seen in a multidisciplinary stone clinicelective liver transplantcandidates and recipientsurgentorsino end stage renaldisease patientselectiveœmany physicians saw their role as anexpert who educates the patient butretains control over the decisionmakingprocessothers took a more collaborativeapproach encouraging patients toassume decisional prioritysurgeons œmade most decisions forpatients patients œgenerally lacked trustin their own decisions and usually soughtsurgeons™ guidancepatients œmajority agreed that thesurgeon should supply them with the˜pros™ and ˜cons™ of all measures toaddress the problem and it was for themultimately to decide what was right forthem surgeons œnot enthusiastic at theprospect of devoting more time todiscussing surgical alternatives risks andcomplications and outlook indicators fortheir patients benefitin surgery preferred sdm aœdoctorcentered approach œ aœpatientcentered approach œthought having a physician involvethem in the decisions concerning theirtreatment was very importantœmost were satisfied with the informationgiven and the possibility of participatingto the treatment decisionmakingprocessœi prefer that my doctor and i shareresponsibility œœi prefer that mydoctor makes the final decision aboutwhich treatment will be used butseriously considers my opinion œwould rely on the physician™srecommendationœ wished to be involved in makingthe decision to accept or not accept aliver for transplantation preferred œequal responsibility an œautonomous role adecision driven by the health care teamdm themerelated tomajorfindingsapt“surgsdmsgfactors associated with favoringsdmmultiple treatment optionsincreased risk impact ofprocedure on patient lifestylemoral contentsgsgsdm sgpatients strong evidence forprocedure surgeons multipletreatment options impact ofprocedure on patient lifestylesdm “older agecomorbidities surgical historyuse of biologics treated at anacademic hospital being marriedsdm “sdm “sdm “sg“sdm “sdm “younger agepieterse ramfelt rectal cancer patients surgical oncologistsurgentthe majority of patients and clinicianspreferred sdmsdm sdmpatients female highereducation rectal or colon cancerpatientsurgent of rectal cancer patients ofcolon cancer patients preferred acollaborative rolesdm “younger ageratsep patients with lumbardisc herniationelective preferred sdmsalkeld rectal or coloncancer patientsurgent preferred a surgeonguided approach sdm a more independent dm rolesdm “sg“desire for more disease specificinformationfemale younger age history ofradiation 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferencessantema patients with eitherabdominal aorticaneurysm or peripheralarterial occlusive diseaseelective preferred sdmseror young breast cancerpatientsurgentsidana young prostatecancer patientsurgentsnijders stiggelbout kiebert gi surgeonsurgent cancer patients surgical patientsunclearpreferred a more passive approach preferred œfully passive and preferred fairly passive preferred sdm an œinformeddecision made by myself based oninformation a passive roleœmost patients were offered only onetreatment option and little sdm wasseenœthe physician should make the decisionsbut strongly consider my opinion wasselected most frequentlyfactors associated with favoringsdmtrust in doctor doctor has aclear communication styledoctor listens enough time forconsultationhigher education type ofproceduredm themerelated tomajorfindingsaptsdm “surgsg“sdm ““sgsg“younger age femalesung patients with pelvicfloor disordertyler ellis newly diagnosedrectal cancer patientsuldry patients undergoingelective gi surgeryvogel breast cancerpatientswang breast cancerpatientsurgenturgent spine clinic patientselectiveweiner essis wilson patients undergoingmajor thoracicabdominal operationsurgentelectivewoltz patients withdisplaced midshaftclavicular fractureelectiveurgent preferred a collaborative role an active role a passive rolesdm “ of total mesorectal excision patientsand of local excision patientspreferred sdmsdm “higher education younger ageelective preferred an active roleidmyounger age male level ofeducationhigher anxiety scores multipletreatment options preferred a passive role anactive role sdm preferred a collaborative role a passive role an active roleœthe majority of patients felt that thephysician rather than the patient shouldmake the basic treatment decision preferred a œpatientdriven role sdm a œsurgeondriven rolesg“sdm “sgidm““ preferred sdm œautonomousrole a passive rolesdm “ ovarian cancerpatientsziebland adecision making preference dm decision making sg surgeonguided sdm shared decision making idm independent decision makingdx diagnosis pt patient surg surgeonœpreferred their medical team to decideon their behalf or œ˜going along with™their doctor™s recommendationurgentsg“out of these s three focused on elective surgeriesin orthopedics one on urgent surgeries in cardiac surgery one on both elective and urgent surgeries in general surgery and one was unclear on the acuity of theinvention but occurred in general surgerydiscussionshared decision making has been highlighted as a desirable approach to clinical counseling however it isunclear if this applies to surgical decision making particularly when considering surgical counseling in settingsof emergent complex highlymorbid operations inour scoping review of the adult surgical literature wefound relatively few studies that address patient and surgeon preferences toward sdm in surgery we found thata large proportion of existing s on preferences toward sdm address elective outpatient procedureswhile patients did seem to prefer sdm in these controlled settings it is possible that patients and surgeonsmay prefer more surgeon guidance when discussingemergent complex operations that have a high risk ofmorbidity or mortality further studies that specifically 0cshinkunas bmc medical informatics and decision making page of table frequencies for characteristics of all included sn variablesurgical specialtyastudies noncologygeneral surgeryorthopedicsurologygynecologycolorectalthoraciccardiacplastic surgerytransplantationvascularneurosurgeryentotolaryngologyophthalmologycancer diagnosisyesnounclearstudy methodsqualitativequantitativemixed methodsstudy locationusnonusstudy settinginpatientoutpatientbothtype of subjectspatients onlysurgeons onlybo
Colon_Cancer
the incidence and death rate of nonsmall cell lung cancer nsclc in china ranks the first among the malignant tumors circular rna circrna was reported to be involved in the progression of nsclc our study aimed to investigate the underlying mechanism of circ_0020123 in nsclc progressionmethods quantitative realtime polymerase chain reaction qrtpcr was used to detect the expression of circ_0020123 mir5905p and thrombospondin thbs2 in nsclc tissues and cells cell proliferation and migration were examined by cell counting kit8 cck8 assay and transwell assay respectively flow cytometry assay was used to detect the apoptosis of nsclc cells the protein levels of ki67 matrix metalloprotein9 mmp9 cleavedcaspase9 cleavedcasp9 and thbs2 were detected by western blot the targets of circ_0020123 and mir5905p were predicted by starbase and targetscan and then confirmed by dualluciferase reporter assay and rna immunoprecipitation rip assay the animal experiment showed the effect of circ_0020123 on tumor growth in vivoresults the expression of circ_0020123 was upregulated in nsclc tissues and cells functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of nsclc cells interestingly circ_0020123 directly targeted mir5905p and inhibition of mir5905p reversed the knockdown effects of circ_0020123 on nsclc cells more importantly thbs2 was a target of mir5905p and thbs2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in nsclc cells finally suppression of circ_0020123 inhibited tumor growth in vivo through mir5905pthbs2 axis circular rna circ_0020123 regulated thbs2 by sponging mir5905p to promote cell proliferation and migration and inhibit cell apoptosis in nsclc cellskeywords nsclc circ_0020123 mir5905p thbs2highlights circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in nsclc cellscorrespondence bskrju163comdepartment of thoracic surgery lianyungang second people™s hospital no hailian east road haizhou district lianyungang jiangsu china circ_0020123 directly targeted mir5905p and mir5905p downregulation reversed the knockdown effects of circ_0020123 on nsclc progression thbs2 acted as a target of mir5905p and overthe effects of expression of thbs2 reversed circ_0020123 knockdown on nsclc progression downregulation of circ_0020123 suppressed tumor growth in vivo through mir5905pthbs2 axis the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cwang a0et a0al cancer cell int page of lung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis small cell lung cancer sclc accounts for about of all lung cancer cases however nonsmall cell lung cancer nsclc accounts for of lung cancer and the a0years overall survival rate os is only about therefore it is important to find the effective treatment and potential molecular targets of nsclc progressioncircular rna circrna is a single stranded rna molecule with a closed circular structure recently amounts of circular dna have been discovered and most of which were thought to be the byproducts of typical splicing [ ] previous reports indicated that the expression of circrna was tissuespecific and the change of its expression intensity was associated with some diseases [“] furthermore circrna was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] for example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging mir34a5p to regulate cc chemokine ligand ccl22 meanwhile hsa_circ_0043256 participated in the progression of nsclc cells by mediating the cinnamaldehyde treatment a previous report suggested that circ_0020123 acted as an oncogene in nsclc and circ_0020123 regulated zincfingerenhancer binding protein zeb1 and enhancer of zeste homolog ezh2 by competitively binding with mir144 to induce cell progression and migration these reports suggested that circ_0020123 was a vital factor in the pathogenesis of nsclc and its function and molecular mechanism need to be further studiedas a small endogenous rna microrna mirna is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers recently some aggregated mirnas have been found in prostate cancer such as mir221222 mir143145 mir23b27b241 and mir1133a which were downregulated and had tumor inhibiting functions a previous study found that circulating mir5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy besides overexpression of mir5905p reduced the development of nsclc cells and regulated the expression of epithelialmesenchymal transformation emtrelated proteins by targeting the signal transducers and activators of transcription stat3 however the precise mechanism by which mir5905p affects nsclc needs further investigationthrombospondin thbs2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer colorectal cancer and nsclc a previous report suggested that thbs2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by mir20a tian et a0al found the expression and clinicopathological features of thbs2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis however the molecular function of thbs2 in nsclc remains poorly definedin this study the targeting relationship between circ_0020123 and mir5905p was firstly detected the effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesmaterials and a0methodspatients and a0specimensnsclc tissues and the adjacent healthy lung tissues were taken from nsclc patients in the lianyungang second people™s hospital all volunteers signed written informed consents nsclc tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at ˆ’ a0 °c for further experiments this research was approved by the ethics committee of lianyungang second people™s hospitalcell culture and a0cell transfectiontwo nsclc cell lines a549 and h1299 and one normal lung cell line imr90 were obtained from the beijing concorde cell library beijing china a549 h1299 and imr90 cells were cultivated in dulbecco™s modified eagle medium dmem hyclone logan ut usa supplementing with fetal bovine serum fbs hyclone and cultured in an incubator at a0„ƒ with co2small interfering rna sirna targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin rna shrna targeting circ_0020123 shcirc_0020123 mir5905pinhibitors sirna negative control sinc shnc and ncinhibitors were all obtained from biomics biotech jiangsu china full length of thbs2 cdna sangon biotech shanghai china was subcloned into pcdna31 plasmid ekbioscience shanghai china then cell transfection was performed by lipofectamine thermo fisher scientific waltham ma usa 0cwang a0et a0al cancer cell int page of rna isolation and a0quantitative real‘time polymerase chain reaction qrt‘pcrthe trizol reagent invitrogen carlsbad ca usa was used for extracting the total rnas next the reversed transcription was carried out by rtpcr kit invitrogen the qrtpcr was performed using the abi sybr green master mix invitrogen the primers in our study were as follows f5²ttc gga cga ccg tca aac at3² and r5²agg atc cct gca cca caa tg3² for circ_0020123 f5²tga aag acg tga tgg cac ac3² and r5²ctt cca ttt tgg for mir5905p f5²aga agg ggt ttt tgg3 ² ctg ggg ctc att tg3² r5²agg ggc cat cca cag tct tc3² for glyceraldehyde3phosphate dehydrogenase gapdh f5²gcg gct ggg tct att tgt c3² and r5²gca gga ggt gaa gaa cca tc3² for thbs2 f5²att gga acg ata cag aga agatt3² and r5²gga acg ctt cac gaa ttt g3² for u6 gapdh and u6 were the internal parameterscell counting kit‘ cck‘ assaythe proliferation viability of a549 and h1299 cells were detected by the cellcounting kit8 msk wuhan china a549 and h1299 cells were cultivated into a 96well plate with a density of × a0cellswell and incubated in a0°c for or a0h then a0μl fresh medium and cck8 solution was added after incubation at a0°c for a0h the od values were detected by the multiskan ascent microplate reader abcam cambridge ma usatranswell assaytranswell chamber corning life sciences corning ny usa was used to detect cell migration firstly the serumfree dmem thermo fisher scientific was fixed with cell suspension cells and seeded into the upper chamber and the dmem containing serum was put into the lower chamber after incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed finally the migrated cells were stained with crystal violet corning life sciences and observed by using a microscopeflow cytometryfirstly a549 and h1299 cells were cultured and pbs was used for washing cells then the binding buffer was used to resuspend cells and the annexin vfluorescein isothiocyanate vfitcpropidium iodide pi apoptosis detection kit thermo fisher scientific was used to stain cells finally cell apoptosis was detected by flow cytometry thermo fisher scientificwestern blot analysisthe total proteins of nsclc tumors or cells were collected by ripa lysis buffer sangon biotech then the proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis sdspage and transferred to polyvinylidene fluoride pvdf membranes thermo fisher scientific the skimmed milk was added and incubated with primary antigapdh antibody invitrogen carlsbad ca usa antiβactin antibody invitrogen antiki67 antibody invitrogen antimatrix metalloprotein9 mmp9 antibody invitrogen anticleavedcaspase9 cleavedcasp9 antibody invitrogen or antithbs2 antibody invitrogen at a0°c overnight finally the membranes were incubated with the secondary antibody for a0 h at room temperature the results were viewed using kodak film developer fujifilm japandual‘luciferase reporter assaysthe wild type circ_0020123 sequences circ_0020123wt mutant circ_0020123 sequences circ_0020123mut wild type thbs2 ²utr sequences thbs2wt mutant thbs2 ²utr sequences thbs2mut were cloned into pgl3 luciferase reporter plasmid promega madison wi usa then the plasmid and mir5905p or mirnc were cotransfected into a549 and h1299 cells by lipofectamine thermo fisher scientific after transfection for a0h the dualluciferase reporter assay system promega was performed to detect the luciferase activityrna immunoprecipitation ripfirstly the magna rip rnabinding protein immunoprecipitation kit gzscbio guangzhou china was performed to verify the relationship between circ_0020123 and mir5905p in brief the magnetic beads and antiago2 antibody abcam were added into cells and incubated for a0h then the proteinase k and the phenol“chloroformisoamyl alcohol reagent were added for purifying rnas finally qrtpcr was used to measure circ_0020123 enrichmentanimal experimentsthe 4weekold balbc male nude mice vitalriver beijing china were raised in a sterile environment for 0cwang a0et a0al cancer cell int page of experiments then pbs was used to suspend a549 cells × transfected with shcirc_0020123 or shnc next the nude mice were divided into two groups n a549 cells transfected with shcirc_0020123 or shnc were shcirc_0020123 or shnc inoculated into the nude mice the tumor volume was detected every a0 days after a0days the nude mice were euthanatized and the tumor weight was detected besides the tumor tissues from each group were collected to detect the expression of circ_0020123 mir5905p and thbs2 the animal experiment was approved by the animal experimentation ethics committee of lianyungang second people™s hospitalstatistical analysisthe software graphpad prism was performed for statistical analysis the data was displayed as mean ± standard deviation sd the significant difference was calculated by student™s t test and oneway analysis of variance anova p was considered as statistically significantresultscirc_0020123 was a0upregulated in a0nsclc tissues and a0cellsto begin with qrtpcr was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in nsclc tissues compared with the adjacent healthy tissues fig a0 1a similarly the expression of circ_0020123 in nsclc cells a549 and h1299 was markedly higher than that in normal cells imr90 fig a01b from these data it is speculated that circ_0020123 might be acted as an oncogene in nsclcfig circ_0020123 was upregulated in nsclc tissues and cells a qrtpcr was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b the expression of circ_0020123 in normal cell line imr90 and nsclc cell lines a549 and h1299 was detected by qrtpcr p downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0nsclc cellsto investigate the functional effects of circ_0020123 on nsclc cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into a549 and h1299 cells firstly the transfection efficiency was detected by qrtpcr fig a02a next cck8 was used to detect the proliferation and the results showed that the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced fig a0 2b moreover the migration of a549 and h1299 cells was significantly downregulated by circ_0020123 knockdown fig a02c in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with sinc fig a02d finally the protein levels of cell proliferationrelated protein ki67 and cell migrationrelated protein mmp9 were inhibited while cell apoptosisrelated protein cleavedcasp9 was upregulated in nsclc cells transfected with sicirc_00201231 or sicirc_00201232 fig a02e these data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in nsclc cellscirc_0020123 directly targeted mir‘‘5pby searching in the online software starbase the potential binding sites between circ_0020123 and mir5905p were detected fig a0 3a to confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123wt reporter plasmid was reduced by mir5905p mimic while the circ_0020123mut reporter plasmid activity was not changed in a549 and h1299 cells fig a03b furthermore the expression of mir5905p was lower in a549 and h1299 cells compared with that in imr90 cells fig a0 3c in contrast mir5905p expression was elevated in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a0 3d finally the rip assay was also used to confirm the targeting relationship between circ_0020123 and mir5905p and the results showed that circ_0020123 and mir5905p were enriched in antiago2 group fig a03emir‘‘5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0nsclc cellsto further explore the functional effects between circ_0020123 and mir5905p mir5905pinhibitor was established qrtpcr was used to detect the transfection efficiency fig a0 4a interestingly mir5905p was upregulated in a549 and h1299 cells transfected with sicirc_00201231 while the expression of mir5905p was recovered in cells transfected with 0cwang a0et a0al cancer cell int page of fig downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of nsclc cells a the transfection efficiency of sicirc_00201231 and sicirc_00201232 in a549 and h1299 cells was detected by qrtpcr b cck8 assay was used to detect the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 c the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by transwell assay d flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 e the protein levels of cell proliferation related protein ki67 cell migration related protein mmp9 and cell apoptosis related protein cleavedcasp9 were detected by western blot p fig a0sicirc_00201231 mir5905pinhibitors 4b moreover circ_00201231 knockdown inhibited cell proliferation and migration while the mir5905p inhibitor reversed these effects fig a0 4c d in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 was increased which was abolished by mir5905pinhibitor fig a0 4e similarly mir5905p inhibitors reversed the effects on the protein levels of ki67 mmp9 and cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 fig a0 4f these results that mir5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of nsclc cellsindicated mir‘‘5p targeted thbs2 in a0nsclc cellsthe thbs2 ²utr was predicted to contain the binding sites of mir5905p through the online software targetscan fig a05a then the dualluciferase reporter assay was used to confirm the targeting relationship the results showed that cotransfection of mir5905p and thbs2wt significantly limited the luciferase activity in both a549 and h1299 cells the luciferase activity was not altered in cells cotransfected with mir5905p and thbs2mut fig a05b importantly the mrna and protein level of thbs2 was enahnced in nsclc cells fig a05c d we further explored whether circ_0020123 affected the functions of thbs2 in nsclc cells the mrna and protein expression of thbs2 were repressed in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a05e f 0cwang a0et a0al cancer cell int page of fig circ_0020123 directly targeted mir5905p a the binding site between circ_0020123 and mir5905p was detected by the online software starbase b the luciferase activity of circ_0020123wt or circ_0020123mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p was detected by dualluciferase reporter assay c qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells d the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr e rip assay was used to confirm the relationship between circ_0020123 and mir5905p p thbs2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0nsclc cellsbased on the work ahead of us the pcdna31thbs2 was constructed then the qrtpcr and western blot were used to detect the transfection efficiency and the thbs2 expression was increased in a549 and h1299 cells transfected with pcdna31thbs2 fig a0 6a b in addition the proliferation and migration rates of a549 and h1299 cells transfected with sicirc_00201231 pcdna31thbs2 were higher than that transfected with sicirc_00201231 fig a0 6c d meanwhile a similarly phenomenon was also observed in cell apoptosis the pcdna31thbs2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis fig a0 6e furthermore the effects of circ_0020123 deletion on ki67 mmp9 and cleavedcasp9 protein levels were also reversed by thbs2 overexpression fig a0 6f these data suggested that overexpression of thbs2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisreduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123mir‘‘5pthbs2 axisto further explore the function of circ_0020123 in nsclc cells the shcirc_0020123 was constructed and the xenograft tumor was established then a549 cells transfected with shcirc_0020123 or shnc were injected into the nude mice the xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shnc group fig a07a moreover tumor weight was inhibited by circ_0020123 knockdown fig a0 7b furthermore the expression circ_0020123 and thbs2 was decreased while the mir5905p was increased in xenograft tumor transfected with shcirc_0020123 fig a0 7c western blot assay also revealed that the protein level of thbs2 was repressed by circ_0020123 knockdown fig a07d finally the digital tomosynthesis dts was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group fig a07e the results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivodiscussionclinically only a few nsclc patients were diagnosed at an early stage and treated by surgical resection more than of nsclc patients were diagnosed with the advanced stage or metastatic tumors thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of nsclcrecently circrna was no longer considered as a random product in the rna shearing process and its biological significance and function in malignant tumors 0cwang a0et a0al cancer cell int page of fig mir5905p downregulation reversed circ_0020123 knockdown effects in nsclc cells a qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells transfected with mir5905pinhibitors b the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was detected by qrtpcr c the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was tested by cck8 assay d transwell assay was used to measure the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors e flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors were detected by western blot p had received more and more attention previous reports revealed that circ_0020123 was involved in the development of nsclc moreover the level of circ_0020123 was elevated in nsclc cells consistently we found that the expression of circ_0020123 was markedly higher in nsclc tissues and cells moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of nsclc cells in a0 vitro besides circ_0020123 promoted tumor growth in a0vivoendogenous circrnas could act as microrna sponges to inhibit their function and some studies linked mirna sponges to human diseases including cancer a previous study indicated that circrna ctransferrin receptor ctfrc regulated tfrc by sponging mir107 to facilitate bladder carcinoma development mir5905p was studied in different cells such as airway smooth muscle cells colon epithelial cells and nsclc cells however the potential relationship between mir5905p and circrna has not been researched in this study circ_0020123 directly targeted mir5905p and mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc progression these data provided a clue to the therapeutic strategy for nsclc 0cwang a0et a0al cancer cell int page of fig mir5905p targeted thbs2 in nsclc cells a the potential binding site between thbs2 ²utr and mir5905p was predicted by the online software targetscan b dualluciferase reporter assay was used to measure the luciferase activity of thbs2wt or thbs2mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p c qrtpcr was used to detect the mrna expression of thbs2 in nsclc cells d the protein level of thbs2 in nsclc cells was tested by western blot e the mrna expression of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr f western blot was used to measure the protein level of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 p our study also confirmed that mir5905p could target thbs2 directly in nsclc cells thbs2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase mmp genes involved in tissue formation and repair [ ] a previous document suggested that thbs2 acted as a target of mir2213p and participated in lymph node metastasis in cervical cancer the data in this research showed that the expression of thbs2 in nsclc cells was markedly higher than normal healthy cells furthermore overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells suggesting that circ_0020123 promoted the progression of nsclc cells through mir5905pthbs2 axisin our research showed that the expression of circ_0020123 was higher in nsclc tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of nsclc cells and also suppressed tumor growth in a0 vivo moreover circ_0020123 directly targeted mir5905p while mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc cells more importantly circ_0020123 regulated the expression of thbs2 by sponging mir5905p and upregulation of thbs2 reversed the effects of circ_0020123 knockdown on nsclc cells therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cwang a0et a0al cancer cell int page of fig overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells a b the mrna and protein expression of thbs2 in a549 and h1299 cells transfected with pcdna31thbs2 was detected by qrtpcr and western blot c cck8 assay indicated the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 d the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was measured by transwell assay e the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was detected by flow cytolysis assay f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 were detected by western blot p apoptosis of nsclc cells by sponging mir5905p to regulate thbs2results and develop the manuscript all authors read and approved the final manuscriptabbreviationsnsclc nonsmall cell lung cancer circrna circular rna qrtpcr quantitative realtime polymerase chain reaction cck8 cell counting kit8 mmp9 matrix metalloprotein9 cleavedcasp9 cleavedcaspase9 cleavedcasp9 cleavedcaspase9 rip rna immunoprecipitation zeb1 zincfingerenhancer binding protein ezh2 zeste homolog stat3 signal transducers and activators of transcription thbs2 thrombospondin acknowledgementsnot applicableauthors™ contributionslw collaborated to design the study lz were responsible for experiments analyzed the data yw wrote the paper all authors collaborated to interpret fundingnoneavailability of data and materialsplease contact corresponding author for data requestsethics approval and consent to participatethis research was approved by the ethics committee of lianyungang second people™s hospital the animal experiment was approved by the animal experimentation ethics committee of lianyungang second people™s hospitalconsent for publicationall listed authors have actively participated in the study and have read and approved the submitted manuscript 0cwang a0et a0al cancer cell int page of fig reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123mir5905pthbs2 axis a a total of × a549 cells transfected with shcirc_0020123 or shnc were injected into nude mice to establish the xenograft tumor tumor volume was measured every d after injection b tumor weight was measured on d c the expression of circ_0020123 mir5905p and thbs2 in xenograft tumor was measured by qrtpcr d the protein level of thbs2 in xenograft tumor was evaluated by western blot e the number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis dts p competing intereststhe authors declare that they have no competing interestsreceived april accepted july references bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin “ abe h takase y sadashima e fukumitsu c murata k ito t kawahara a naito y akiba j insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value cancer cytopathol “li c zhang l meng g wang q lv x zhang j li j circular rnas pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer j cancer res clin oncol “ belousova ea filipenko ml kushlinskii ne circular rna new regulatory molecules bull exp biol med “ zhang z xie q he d ling y li y li j zhang h circular rna new star new hope in cancer bmc cancer li l chen y nie l ding x zhang x zhao w xu x kyei b dai d zhan s guo j zhong t wang l zhang h myodinduced circular rna cdr1as promotes myogenic differentiation of skeletal muscle satellite cells biochim biophys acta gene regul mech “ greco s cardinali b falcone g martelli f circular rnas in muscle function and disease int j mol sci weng xd yan t liu cl circular rna_larp4 inhibits cell migration and invasion of prostate cancer by targeting foxo3a eur rev med pharmacol sci “ deng n lei d huang j yang z fan c wang s circular rna expression profiling identifies hsa_circ_0011460 as a novel molecule in severe preeclampsi
Colon_Cancer
" gastric neoplasms containing neuroendocrine carcinoma nec components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure nec and mixed tumors containing neccomponents we aimed to investigate whether there is a distinct difference in overall survival os between gastricneoplasms containing nec components and gastric adenocarcinomamethods surgically resected gastric neoplasms containing nec components n and gastricadenocarcinomas n from january to december at peking university cancer hospital wereretrospectively analysed patients were categorized into a surgical group and a neoadjuvant group and adjustedusing propensity score matching in the two groups gastric neoplasms containing nec components were dividedinto pure nec and mixed tumors with less than ghminen between and ghminen andmore than ghminen neuroendocrine carcinoma components os was compared between thesegroups and the gastric adenocarcinoma groupresults the os of gastric neoplasms containing neuroendocrine nec components was poorer than that of gastricadenocarcinomas in the surgical group regardless of whether the percentage of neuroendocrine cancercomponents was less than between and more than or cox multivariable regressionanalysis suggested that tumor category neoplasms containing nec components or gastric adenocarcinoma wasan independent risk factor for prognosis interestingly among patients receiving neoadjuvant therapy thedifference was not significantcontinued on next page correspondence buzhaodecjcrcn jijiafuhscpkueducn jiahui chen anqiang wang and ke ji contributed equally to this workdepartment of gastrointestinal surgery key laboratory of carcinogenesisand translational research ministry of education peking university cancerhospital institute no fucheng road haidian district beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cchen bmc cancer page of continued from previous pages gastric neoplasms containing any proportion of nec components had poorer overall survival thangastric adenocarcinoma in patients treated with surgery directly indicating that these neoplasms are moremalignant than gastric adenocarcinoma among the patients receiving neoadjuvant therapy the difference inoverall survival was not significant which was in sharp contrast with the results of the surgery group suggestingthat neoadjuvant therapy may have a good effect in the treatment of these neoplasmskeywords neuroendocrine carcinoma gastric adenocarcinoma overall survival gastric neoplasms containing neuroendocrine carcinomanec components are a heterogeneous subgroup ofgastric cancer with highly aggressive behavior and poorprognosis and include pure necs and mixed tumorscontaining nec components every yearthere areapproximately million new cases of gastric cancerworldwide and gastric neoplasms containing nec components account for approximately “ of thesecases [ ] given the low incidence there is little comprehensive basic and clinical research to systematicallyguide the treatment of these gastric neoplasms makingthe prognosis of these tumors unsatisfactory [“]according to the world health anizationwho digestive neuroendocrine tumor classificationneuroendocrine neoplasm nen can be divided intothree categories based on ki67 levels and mitotic counts— hpf grade g1 ki67 ‰ mitoses grade g2 ki67 ‰ ‰ mitoses‰ grade g3ki67 mitoses meanwhile the americanjoint committee on cancer ajcc defines highly differentiated nen as a neuroendocrine tumor net and thepoorly differentiated nen as a neuroendocrine carcinoma nec based on the degree of tumor cell differentiation generally g1 g2 and rare welldifferentiated g3nens belong to the nets while poorly differentiatedg3 nens belong to necs[ ] gastric mixedneuroendocrinenonneuroendocrineneoplasm gminen is a special type of gastric nen that is definedas containing more than of both neuroendocrineand nonneuroendocrine components accountingfor approximately of all gnens and of gastricneuroendocrine carcinomas gnecs [“] for thosemixed tumors with less than or more than neuroendocrine carcinoma components there is no uniform definition consideringthe heterogeneity ofminen and the malignancy degree of the different components in the tumor la rosa [ ] proposeddividing minen into three categories highgradeintermediategrade and lowgrade highgrade minenconsists of nec and carcinomaadenoma intermediategrade mimen consists of net and carcinoma and lowgrade minen consists of net and adenoma thereforein this study gastric highgrade mixed neuroendocrinenonneuroendocrine neoplasm ghminen was defined as gastric cancer containing more than of bothneuroendocrineadenocarcinomacomponentscarcinomaandgenerally the prognosis of mixed tumors is largely determined by the most malignant component kim found that gnec has shorter progressionfree survival pfs than gastric adenocarcinoma huang found that the prognosis of patients with more than of neuroendocrine cancer components is significantly poorer than that of patients with less than components all of these studies provide evidence thattumors containing neuroendocrine cancer componentsmay contribute to a worse prognosis therefore wehypothesized that a mixed tumor containing neuroendocrine carcinoma components would have a worse prognosis than pure adenocarcinoma alone we sought tofind studies on the overall survival os comparison between ghminen and gastric adenocarcinoma butfailed thus we think that a study of the comparison ofthe os of ghminen and gastric adenocarcinoma willprovide a valuable supplement to current research on ghminen to overcome the bias caused by the differences between the covariates in the comparison we usedpropensity score matching psm to match importantfactors such as age gender tumor location tumor sizepathological staging and adjuvant chemotherapy between the two groups making the research results morereliablemethodspatient selectionwe retrospectively collected patients diagnosed withgastric nens and underwent radical resection at pekinguniversity cancer hospital beijing from january to december the inclusion criteria were as follows pathologically confirmed pure nec or tumorcontaining nec components no other tumors werediagnosed before the operation complete clinicopathological information and survival information thatcould be obtained through followup patients diagnosedwith cm1 or ct4b before surgery or died from perioperative complications were excluded from the study 0cchen bmc cancer page of patients with gastric adenocarcinomas undergoing radical surgery were randomly selected for psm analysesperformed the chisquared test and mannwhitney utest were used to further verify the matching resultsfollowupwe followed the patients at least twice a year serumtumor markers test gastroscope and computed tomography ct scans were used to reexamine patients aftersurgery depending on the patients™ status magneticresonance imaging mri and positron emission tomography computed tomography petct were alsoconsidered for patients who cannot regularly visit ourcenter for postoperative examination we use telephonefollowup to obtain survival informationdiagnosis and classificationwe reevaluated the diagnosis and classification of ghminen mixed tumors with less than or morethan neuroendocrine carcinoma components werealso included in this study which were defined as ghminen and ghminenrespectively atumor consisting of nec is defined as pure necall neuroendocrine tumors were identified diagnosedand classified by two independent pathologists in accordance with the who classification of tumors neuroendocrine components were identified byhistological features and immunohistochemical specificity marks such as synaptophysin syn chromogranina cga and neuro cell adhesion molecule cd56 orncam the tumor staging described in the study wasbased on the ajcc 8th edition tnm staging guidelines all possible disagreements were discussed in ourstudy groupdefinition of variables and groupsin this study patients were divided into a surgical groupand a neoadjuvant group based on whether they had received neoadjuvant therapy before surgery patients inthe surgery group were assessed by the ptnm stagingsystem while patients in the neoadjuvanttreatmentgroup were assessed by the yptnm staging system osrefers to the time from surgery to the last followup thetime of death or the end ofloss offollowup or other cause of deathfollowup egpropensity score matchingto accurately compare the prognosis of ghminenand gastric adenocarcinoma we employed psm to balance the differences between the two groups psm wasperformed through the pamatching plugin in spss software logistic regression models were used toestimate propensity scores based on gender age tumorlocation tumor size and pathological staging given a caliper width nearest neighbor matching wasstatistical analysisall statistical analyses were performed using spss statisticalsoftware ibm united states the chisquared test and mannwhitney u test were used forstatistical analysis of categorical variables and continuous variables respectively kaplanmeier method wasused for the comparison of os the logrank test wasused to compare survival rates multivariable cox proportional hazards models were used to identify predictors of survival outcome p was regarded as thethreshold of significanceresultspatient selection and psm resultsbetween and among the patients treated atthe gastrointestinal cancer center of peking universitycancer hospital a total of patients with gastric neoplasms containing nec components met the inclusioncriteria for the study including cases of pure necand cases of mixedtype of these patients a total of patients received neoadjuvant therapy nec ghminen ghminen ghminen while the remaining patients receivedsurgery directly nec ghminen ghminen ghminen there were aninsufficient number of patients in group ghminen group to conduct effective statistical analysisso we combined the ghminen group with thenec group for further analysis we also randomly selected patients with gastric adenocarcinoma whounderwent radical surgery among them patientsreceived neoadjuvant therapy and the remaining patients were treated with surgery directly fig immunohistochemical specificity markers were utilizedto identify the neuroendocrine components fig 2asyn was expressed in almost all neoplasms containingnec components while the positive rates ofcga and cd56 were much lower and respectively no significant difference in the positiverate of syn and cga was observed between pure nec ghminen ghminen and ghminenfig 2b c only the positive rate of cd56 was found tobe higher in the pure nec group than that in the ghminen group fig 2dtherefore priorto os comparison psm wasperformed to ensure that there were no significant differences in patient gender age tumor location tumorsize pathological staging and adjuvant chemotherapybetween the two groups 0cchen bmc cancer page of fig flow chart of patient enrolmentcomparison of os between all patients with neccomponents and patients with gastric adenocarcinoma inthe surgical group and neoadjuvant groupbefore psm we compared the survival curves between all patients with nec components and patientswith gastric adenocarcinoma by the kaplanmeiermethod fig apparently patients with nec components had a poorer os than those with gastricadenocarcinoma fig 3a p in the surgicalgroup in contrast no significant difference was observed between the patientsreceiving neoadjuvanttherapy fig 3b p according to the proportion of nec components patients were classifiedinto pure nec ghminen ghminenand ghminen the os was also comparedbetween patients with adenocarcinomaand thesegroups and the results were similar to the overallcomparison fig 3c dfig illustrations of immunohistochemical staining patterns in gastric neoplasms containing nec components a an overview of the expressionof syn cga and cd56 in tumors containing nec components b syn expression in different nec component groups c cga expression indifferent nec component groups d cd56 expression in different nec component groups cd56 neuro cell adhesion molecule cgachromogranin a nec neuroendocrine carcinoma syn synaptophysin pvalue 0cchen bmc cancer page of fig see legend on next page 0cchen bmc cancer page of see figure on previous pagefig comparison of os between gastric neoplasms containing nec components and gastric adenocarcinoma a os comparison betweengastric neoplasms containing nec components and gastric adenocarcinoma before psm in the surgical group b os comparison between gastricneoplasms containing nec components and gastric adenocarcinoma before psm in the neoadjuvant group c os comparison between differentnec content groups pure nec ghminen ghminen and ghminen and gastric adenocarcinoma before psm in the surgicalgroup d os comparison between the different nec content groups and gastric adenocarcinoma before psm in the neoadjuvant group e oscomparison for patients in the surgical group after psm f os comparison for patients in the neoadjuvant group after psm nec neuroendocrinecarcinoma os overall survival psm propensity score matchingbefore psm significant differences between the baseline characteristics were observed in the surgical groupand the neoadjuvant group table table to balance the clinicopathological differences between the twogroups psm was performed to ensure that there wereno significant differences in patient gender age tumorlocation tumor size pathological staging and adjuvantchemotherapy between the two groups the detailedclinicopathological characteristics before and after psmare shown in table and table as a result patients with nec components and patients with gastric adenocarcinoma were matchedin the surgical group table patients with nec components also had a poorer os than those with gastricadenocarcinoma fig 3e p multivariable analysis showed that adjuvant therapy tumor category andtnm stage werefactorstable independent prognosticto investigate whether neoadjuvant therapy had an effect on os patients with nec components and patients with gastric adenocarcinoma were matched inthe neoadjuvant group table interestingly kaplanmeier analysis showed that among patients receivingneoadjuvant therapy there was still no significant difference in os between the two groups fig 3f p comparison of os between patients with differentproportions of nec components and patients with gastricadenocarcinomato investigate whether the level of nec componentshad an effect on os in the surgical group ghminen ghminen pure nec and pure nec plus ghminen were compared with gastric adenocarcinoma after psm the results showed that even thegroup with the lowest proportion of nec componentsthe ghminen group had a poorer os thanadenocarcinoma fig 4a p as expected theghminen pure nec and pure nec plus ghminen groups each with relatively high proportionsof nec components had worse os than the gastricadenocarcinoma group fig 4bd p detailed clinical information after matching isshown in additional file tables s1s4psm was also performed in the neoadjuvant group incontrast to the results of the surgery group in the purenec group containing the highest proportion ofnec componentstill no significantdifference in os from gastric adenocarcinoma fig5d the other three groups with lower nec contentwere also notfrom gastricadenocarcinoma in terms of os fig 5ac detailedclinicopathologicaland afterpsm are shown in additional file tables s5s8characteristics beforethere wassignificantly differentdiscussionamong gastric neuroendocrine neoplasms the tumorcontaining nec components is a special type includingpure nec and mixed tumor containing nec components the incidence of these tumors is extremely lowbut they are more invasive and have a poorer prognosisthan welldifferentiated gnens [ ]received neoadjuvantin previous study kim found that in patientschemotherapywho had notprogressionfree survivalpfs of pure gnec waspoorer than that of gastric adenocarcinoma while thepfs of mixedtype tumors was not significantly differentin kim™sfrom that of gastric adenocarcinoma study the mixed type was defined as net mixed withgastric cancer rather than nec net is much less malignant than nec [ ] this may be the reason whythere was no significant difference in os between mixedtype and gastric adenocarcinomas in addition mixed tumors with less than or more than of nec components were not included in that study which webelieve was a deficit of the study pfs is an important indicator for evaluating prognosis in many cases it can reflect the trend of os based on kim™s research resultswe regarded tumors containing nec components as awhole and found that the os of these tumors was poorerthan that of adenocarcinoma in the surgical group inthe comparison of os between mixed tumors with different proportions of nec components and gastricadenocarcinoma the results for pure nec cases wassimilar to kim™s while the os of mixed tumors was alsopoorer than that of gastric adenocarcinoma whether theproportion of neuroendocrine cancer components wasless than between and or more than which was not mentioned in kim™s study cox multivariable regression analysis showed thattumor categoryneoplasm with nec component or adenocarcinoma 0cchen bmc cancer page of table comparison of clinicopathological characteristics before and after psm in surgical grouppatient characteristicsunmatched comparisonpatients with neccomponents n p valuematched comparisonpatients with neccomponents n age year mean ± sdgender malefemalebmi mean ± sdadjuvant therapyyesnotumor locationupper thirdmiddle thirdlower thirdentiretumor size cm‰¥ cmtype of gastrectomytotal gastrectomydistal gastrectomy ± ± proximal gastrectomy surgical procedureopenlaparoscopict staget1t2t3t4n stagen0n1n2n3m stagem0m1ptnm stageiiiiiiiv gastricadenocarcinoman ± ± ± ± p value gastricadenocarcinoman ± ± bmi body mass index minen mixed neuroendocrinenonneuroendocrine neoplasm nec neuroendocrine carcinoma psm propensity score matchingpatients with nec components nec high grade minen high grade minen and high grade minen 0cchen bmc cancer table comparison of clinicopathological characteristics before and after psm in neoadjuvant groupmatched comparisonpatient characteristicsunmatched comparisonpatients with neccomponents n age year mean ± sdgender malefemalebmi mean ± sdadjuvant therapyyesnotumor locationupper thirdmiddle thirdlower thirdentiretumor size cm‰¥ cmtype of gastrectomytotal gastrectomydistal gastrectomyproximal gastrectomysurgical procedureopenlaparoscopict staget0t1t2t3t4n stagen0n1n2n3m stagem0m1yptnm stageiiiiiiiv ± ± gastricadenocarcinoman ± ± p valuepatients with neccomponents n ± ± page of p valuegastricadenocarcinoman ± ± bmi body mass index minen mixed neuroendocrinenonneuroendocrine neoplasm nec neuroendocrine carcinoma psm propensity score matchingpatients with nec components nec high grade minen high grade minen and high grade minen 0cchen bmc cancer page of table univariate and multivariate analyses of survival after psm in surgical grouppatient characteristicsunivariate analysishr ci“multivariate analysishr cip valueage yeargendermale vs femalebmiadjuvant therapyyes vs notumor size‰¥ cm vs cmtumor categorycarcinoma with nec component vsgastric adenocarcinoma vstype of gastrectomytotal gastrectomydistal gastrectomyproximal gastrectomysurgical procedurelaparoscopic vs opentnm stageiiiiiiivp value“““ ““““““““““ “ ““““““““tumor size and tnm staging were independent risk factors for prognosis this suggests that the prognosis ofgastric neoplasms with nec components is substantiallydifferent from that of gastric adenocarcinoma and evena small percentage of nec components can alsoimpair prognosis which challenges the current cutoffvalue of the proportion of each component that must theoretically be greater than was set in andsince who has also adopted this standard to define minen this largely avoids the overdiagnosisof minen in tumors with only focal neuroendocrinemarker expression and no corresponding morphologicalchanges in additionit also prevents clinicians fromdealing with these rare neoplasms too often withoutguidelines nevertheless it is now being questionedby an increasing number of scholars the componentsin mixed tumors are not evenly distributed for large tumorsthe randomness of biopsy and postoperativepathological sampling causes the proportion of eachcomponent to fluctuate greatly making it difficult to describe the proportion of each component precisely park compared the os between tumors with morethan nec components and gastric adenocarcinomawith or without less than nec and they found thattumors with an nec composition of more than hada worse prognosis this suggests that even a small proportion of malignant components can affect prognosis while in park™s study for unknown reasons the authors did not compare the prognosis of mixed tumorswith nec components less than with gastricadenocarcinomas directly nor did they compare allneccontaining tumors as a whole with gastric adenocarcinoma which we believe was a deficit of the studyin our study we regarded tumors containing neccomponents as a whole and found that the os of thesetumors was poorer than that of adenocarcinoma in thesurgical group in addition we also found that the os ofmixed tumors with less than between and more than nec components or pure nec wasworse than that of gastric adenocarcinoma analysis ofimmunohistochemical markers show that there was nosignificant difference in the positive rate of syn and cgabetween different nec content groups only the positiverate of cd56 was found to be higher in the pure necgroup than that in the ghminen group therole of cd56 in the diagnosis of nec is still controversial however syn and cga are two wellrecognized 0cchen bmc cancer page of fig comparison of os between gastric neoplasm with different proportions of nec and gastric adenocarcinoma in the surgical group aoverall survival comparison between ghminen and gastric adenocarcinoma b overall survival comparison between ghminen andgastric adenocarcinoma c overall survival comparison between ghminen plus pure nec and gastric adenocarcinoma d overall survivalcomparison between pure nec alone and gastric adenocarcinomamarkers therefore from the results of immunohistochemistry we believed that there was no significantlydifference in tumors containing nec componentsstudies on the molecular mechanism of pathogenesisshow that nec components and adenocarcinoma components have similar genomic abnormalities similarlosses of heterozygosity loh and mutations at multiple loci and key oncogenes such as tp53 apc and rbgenes all these results imply that the two componentsin the mixed tumor may have a common origin and acquire biphenotypic differentiation during carcinogenesis[“] moreoverin the who definition of mixedneuroendocrine and nonneuroendocrine neoplasms ofother ans ie lung and thyroid no minimumpercentage for either ingredient is established thereforewe believe that mixed tumors containing nec components are actually of the same origin have similar biological characteristics and are differentfrom gastricadenocarcinoma we propose considering mixed tumorscontaining nec components as a whole rather than defining them based on the definition for both tumorcomponents which has not been raised by other studiespreviously many studies have confirmed the efficacyof neoadjuvant chemotherapy in gastric adenocarcinoma[ ] in a retrospective study involving patientsma found that neoadjuvant chemotherapy improves the survival of patients with nec and hminenof the stomach van der veen reported that 0cchen bmc cancer page of fig comparison of os between gastric neoplasm with different proportions of nec components and gastric adenocarcinoma in theneoadjuvant group a overall survival comparison between ghminen and gastric adenocarcinoma b overall survival comparisonbetween ghminen and gastric adenocarcinoma c overall survival comparison between ghminen plus pure nec and gastricadenocarcinoma d overall survival comparison between pure nec and gastric adenocarcinomaneoadjuvant chemotherapy could not benefit the survivalof patients with mixed tumors containing nec components however because only eight patients wereincluded in the neoadjuvant group van™s results arequestionable in our study among patients receivingneoadjuvanttherapy no significant difference in osbetween mixed tumor and gastric adenocarcinoma wasobserved even for the pure nec group with the highestnec contentthere was no significant differencesuggesting that neoadjuvant therapy may have a positiveeffect on these neoplasmsalthough this is only a singlecenter retrospectivestudy the sample we reported is considerable for thisrare disease which can provide new ideas for clinicaland basic research in addition we proposed treatingall gastric neoplasms containing nec components asa whole and found that neoadjuvanttherapy mayhave a good effect on these neoplasms in the futurewe will conduct more genomics studies to confirmour ideas this study also has its limitations due tothe lack of recurrence and detailed chemotherapy information we were unable to compare progressionfree survival and analyse the effects of differentchemotherapy regimens as a retrospective study despite our performing psm in advance selection biascannot be completely avoided in addition since theexact proportion of each componentin the mixedtumor could not be obtained we could not determine 0cchen bmc cancer page of whether there is a cutoff value for the diagnosis ofthe mixed tumor with nec componentless than so we could only treat all mixed tumors withnec component as a wholesour study demonstrated that gastric neoplasms withnec components regardless of the proportion of components have poorer overall survival than gastric adenocarcinomaindicating a higher degree of malignancythan gastric adenocarcinoma among the patients receiving neoadjuvant therapy the difference in overallsurvival was not significant which was in sharp contrastwith the results of the surgery group suggesting thatneoadjuvant therapy may have a good effect on theprognosis of these malignancies therefore for this typeof malignancy we should adopt more aggressive andpowerful treatments than those used for gastric adenocarcinoma to improve the prognosis of patients neoadjuvant chemotherapy may be a good way to improve theefficacy offor these tumors at advancedstagestreatmentsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072817additional file table s1 comparison of clinicopathologicalcharacteristics before and after psm of 30ghminen patients insurgical group table s2 comparison of clinicopathologicalcharacteristics before and after psm of ghminen patients in surgicalgroup table s3 comparison of clinicopathological characteristics beforeand after psm of 70ghminen plus pure nec patients in surgicalgroup table s4 comparison of clinicopathological characteristics beforeand after psm of pure nec patients in surgical group table s5 comparison of clinicopathological characteristics before and after psm of 30ghminen patients in neoadjuvant group table s6 comparison ofclinicopathological characteristics before and after psm of ghminen patients in neoadjuvant group table s7 comparison of clinicopathologicalcharacteristics before and after psm of 70ghminen plus pure necpatients in neoadjuvant group table s8 comparison of clinicopathological characteristics before and after psm of pure nec patients in neoadjuvant groupabbreviationsajcc american joint committee on cancer ct computed tomography ghminen gastric highgrade mixed neuroendocrinenonneuroendocrineneoplasm gnec gastric neuroendocrine carcinoma hpf high power fieldminen mixed neuroendocrinenonneuroendocrine neoplasmnec neuroendocrine carcinoma nen neuroendocrine neoplasmnet neuroendocrine tumor mri magnetic resonance imaging os overallsurvival petct positron emission tomography computed tomographypfs progressionfree survival psm propensity score matching who worldhealth anizationacknowledgmentsthanks to dr zhongwu li of the department of pathology peking universitycancer hospital and his colleagues for their assistance in pathologicaldiagnosis and review thanks to all colleagues in the department ofgastrointestinal surgery of peking university cancer hospital and dr jianghong from the statistics department for their assistance in this studyauthors™ contributionsall authors contributed to the study conception and design jc performeddata collection and wrote the manuscript aw wrote and t revised hemanuscript kj helped with statistical analysis and prepared the illustrationszb edited the manuscript jj conceived the study and reviewed themanuscript all authors read and approved the final manuscriptfundingthis work was supported by the national science foundation for youngscientists of china beijing youth talent plan qml20191101 andscience foundation of peking university cancer hospital “ thefunders had no role in study design data collection and analysis decision topublish or preparation of the manuscriptavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe study was approved by the ethics committee of peking universitycancer hospital and the patients™ written consent was also obtained writteninformed consent for publication was obtained and stored in pekinguniversity cancer hospitalconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived may accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin “ matsubayashi h takagaki s otsubo t iiri t kobayashi y yokota t advanced gastric glandularendocrine cell carcinoma with 1year survivalafter gastrectomy gastric cancer “park jy ryu mh park ys park hj ryoo by kim mg prognosticsignificance of neuroendocrine components in gastric carcinomas eur jcancer “la rosa s inzani f vanoli a klersy c dainese l rindi g histologiccharacterization and improved prognostic evaluation of gastricneuroendocrine neoplasms hum pathol “ishida m sekine s fukagawa t ohashi m morita s taniguchi h neuroendocrine carcinoma of the stomach morphologic andimmunohistochemical characteristics and prognosis am j surg pathol“rayhan n sano t qian zr obari ak hirokawa m histological andimmunohistochemical study of composite neuroendocrineexocrinecarcinomas of the stomach j med investig ““jiang sx mikami t umezawa a saegusa m kameya t okayasu i gastriclarge cell neuroendocrine carcinomas a distinct clinicopathologic entityam j surg pathol “ohike nan la rosa s who classification of tumours of endocrineans 4th ed lyon iarc press amin mb edge sb ajcc cancer s
Colon_Cancer
during the covid19 pandemic emergency departments have noted a significant decrease in strokepatients we performed a timely analysis of the bavarian telestroke tempis œworking diagnosis databasemethods twelve hospitals from the tempis network were selected data collected for january through april in years through were extracted and analyzed for presumed and definite ischemic stroke is amongst otherdisorders in addition recommendations for intravenous thrombolysis rtpa and endovascular thrombectomy evtwere noted and mobility data of the region analyzed if statistically valid groupcomparison was tested with fisher™sexact test considering unpaired observations and apvalue was considered significantresults upon lockdown in midmarch we observed a significant reduction in recommendations for rtpa compared to the preceding three years [“] vs p¼ recommendations for evt werep¼ reflecting its increasing importance following the covid19 lockdown midmarch the number ofevt decreased back to levels in “ [“] vs p¼ absolute numbers of issignificantly higher in january to midmarch compared to “ [“] vs decreased in parallel to mobility datas the reduced stroke incidence during the covid19 pandemic may in part be explained by patientavoidance to seek emergency stroke care and may have an association to population mobility increasing mobilitymay induce a rebound effect and may conflict with a potential second covid19 wave telemedical networks maybe ideal databases to study such effects in nearreal timekeywordstelestroke covid19 lockdown stroke thrombolysisdate received may date accepted june introductionimplementation of social distancing to combat theimpact of corona virus pandemic sequelae has emergedas the major strategy to contain the spread of infectiongiven the lack of specific treatments for covid19 andlimited intensive care resources1 major concerns forstroke neurologistsin this extraordinary scenarioinclude the following a rapid specific managementof cases of acute stroke with possible covid19from initiation of the stroke call in the preclinical setting through the ambulance system emergency department and hospital stroke department and in the1department of neurology university of regensburg bezirksklinikumregensburg tempis telemedical stroke center regensburg germany2cts herdecke germany3department of neurology tempis telemedical stroke centeracademic teaching hospital of the university of munich mu¨nchen klinikharlaching munich germanycorresponding authorfelix schlachetzki md department of neurology university ofregensburg center for vascular neurology and intensive care tempistelemedical stroke center bezirksklinikum regensburguniversit‚¬atsstr84 regensburg germanyemails felixschlachetzkiklinikuniregensburgde 0c of telemedicine and telecare bthe factneuroradiological department when needed to aid instroke diagnosis and treatmentthatpatients with mild stroke symptoms or transient ischemic attacks tias may be reluctant to request hospitaladmission for acute stroke23 and c that covid19itself is associated with severe stroke syndromes this issuggested in a recent case series of covid19 patientsfrom wuhan china focusing on neurological symptoms that described cerebrovascular events in of cases especially in elderly patients and in thosewith more severe infections also authors of a secondcase series reported unusual cases of young covid19patients yrs with large vessel stroke and otherauthors reported three stroke patients with coagulopathy and antiphospholipid antibodies in the context ofsevere covid infections4“the number ofin contrast several stroke departments in germanyincluding our own the usa and china have noted asignificant drop in the number of stroke patient admissions during the corona pandemic7 data on this phenomenon are still scarce howeverin a descriptivereport by morelli from piacenza lombardyitaly covering the period february appearance ofthe first sarscov2 patient recorded in italy to march stroke admissionsdecreased from an average of with largevessel occlusions lvos to two tias one lvoand three lacunar strokes8 using a commercial neuroimaging database with the rapid software platform kasangra and hamilton observed a decrease in stroke imaging procedures with the nadirfollowing the first statewide stayathome order in theusa9 the decrease was observed in all age sex andstroke severity subgroups within all participatinghospitals which processed overall patientsbetween july and april cardiologistsin france observed a similar significant drop in admissions to nine intensive cardiac care units after initiationof social distancing and selfquarantine in midmarch overall there are scarce data available on theimpact of the covid19 infection itself on cardiovascular morbidity including cerebral stroke11aims and hypothesisthe primary aim of this study was to evaluate the effectof the covid19 pandemic lockdown on stroke consultations and treatment recommendations using theacute consultant database of the telestroke networktempis12 we focused on data collected during thefirst four months of which included the emergence of the corona virus pandemic in southeasternbavaria through the first two months of social distancingregion shutdown we compared these data withcomparable data collected during the same months inthe years “methodsdata from daily consultations at clinics withoutneurology departmentsin the telestroke networktempis form the basis of this study the consultationstook place between january and april in the years“ all data were pseudonymized weextracted the actual working diagnoses based on telemedical consultation and neuroimaging results mainlycerebral computed tomography two major databaseswere used to calculate the population within these districts wwwdestatisde and experiencearcgiscomexperience478220a4c454480e823b17327b2bf1d4pagepage_1 this retrospective study was approvedby the local ethics committee of the university ofregensburg and performed in accordance with guidelines of the declaration of helsinkimobility data available at wwwapplecomcovid19mobility were extracted these data were generated from the relative request volume for directionsin munich germany compared with a base volumeon january to observe the relationship ofmobility and the reported stroke decline in piacenzawe also extracted mobility data from milan close topiacenza italy8the major ˜working diagnostic groups™ were asfollows a ischemic stroke b tia c intracranialhaemorrhage d epileptic seizure e migraine andf other disorder including facial palsy headacheand brain tumour also included were cases in whichthere were recommendations for iv thrombolysis ivrtpa or endovascular therapy evt thrombectomyfor lvoexploratory descriptive summary statistics withmean values and standard deviations were appliedin an analysis of data covering january through aprilin years “ in comparison with data coveringthe same period in counts are presented as agraphic display showing incidences standardized to15day periods if statistically valid especially percentage of recommendations for iv thrombolysis andthrombectomy groupcomparison was tested withfisher™s exact test considering unpaired observationsa pvalue was considered significantresultsthere were telemedical consultations during thespecific time frames investigated and the population inthe geographical areas covered by these rural hospitals is most hospitals reside in areas with ahigh number of covid19 cases figure 1a the 0cschlachetzki number of covid19“positive cases in the whole ofbavaria rose from five at the end of february to cases on april the public lockdownwas initiated on march however the recommendation of personal quarantine for people who hadtravelled to northern italy was broadcast earlier on march in munich applevr mobility trends demonstrated a decrease in walking activity in midmarch to “ “ to “ of the baseline levelin milan lombardy italy on march walking activity began to decrease soon reaching “ of baselineactivity and remaining fairly constantthereafterfigure 1boverall consultations were analysed and excluded being nonacute consultations within thenetwork ie followup examinations statistically significant changes in the number of recommendations foriv thrombolysis were observed in figure 1cwhile in “ iv thrombolysis was recommendedin of consultations with suspected ischemicstroke of the frequency of this recommendation decreased to of in p¼ no differences in the number of ivthrombolysis recommendations were observed duringthe time period covering january to march in “ vs in notfigure a incidence of new covid19 infections in bavaria on april red dots indicate network hospitals and green andyellow squares depict the two academic stroke centres that alternate weekly for the tempis consult service modified with permission from the bavarian state office for health and food safety httpwwwlglbayerndegesundheitinfektionsschutzinfektionskrankheiten_a_zcoronaviruskarte_coronavirus b mobility data according to covid19 mobility trends reports apple thedata reflect requests for routing in apple maps for munich which resides in the centre of the tempis network and for milan nearpiacenza where the first decline in the number of strokes was reported morelli 8 horizontal dotted line indicates reportedreduced stroke activity in piacenza c recommendations absolute numbers for application of iv thrombolysis and thrombectomyvertical dashed line indicates the official beginning of lockdown in bavaria time and patient numbers on yaxis are standardized to15day periods xaxis in each month to compensate for shorter february and longer january and march months j1¼ january first half j2 “ january second half f¼ february m¼ march a¼ april d working diagnoses of the telestrokeconsultations vertical dashed line indicates the official beginning of lockdown in bavaria time and patient numbers on yaxis arestandardized to 15day periods xaxis in each month to compensate for shorter february and longer january and march months j1¼ january first half j2 “ january second half f¼ february m¼ march a¼ april 0c of telemedicine and telecare significant no trend in fewer recommendations forevt was observed between march and april in compared with the same time periods in “ of vs “ of however in the preceding time frame january to march significantly more recommendations for thrombectomy were made comparedwith “ of vs of in “ p¼ the data reflect the development of consultationsand treatment recommendations for lvo in the network from onward the number of recommendations for evt steadily rose with increasing evidencefor recanalization even in later time windows andincreasing employment of computed tomography angiography in the tempis network table shows thedevelopment of consultations in up to the end ofthe study including the lockdown period it shows adrop in the number of consultations and more importantly fewer recommendations for iv thrombolysisand evt which suggests fewer incidences of ischemicstroke severities table figure 1dalthough bavaria is the state with the highestnumber of covid19 cases in germany especially inour region we only performed five telestroke consultations for the network hospitals in which possiblecovid19 infection was discussed including a singlepatient with stroke symptoms and feverdiscussionthe tempis telestroke working data confirm thecurrent observation of a low stroke incidence insoutheastern bavaria with relative proportions of theworking diagnosis remaining similar the number ofcases of disabling stroke from intracranial haemorrhage and ischemic stroke requiring iv rtpa or evtalso diminished challenging the theory that onlypatient avoidance to call for emergency treatment isresponsible for this phenomenon this study also demonstrates the potential and importance of telestrokenetworks in the current covid19 pandemic313the observation of fewer stroke cases during thecovid19 pandemic seems to contradict two essentialassumptions with regard to stroke risk a sarcov is a strong risk factor for stroke and b physicalinactivity in a lockdown setting may increase the riskof stroke especially among elderly persons firstsarcov2 may induce hypercoagulability and highlevels of creactive protein ddimer and interleukin6placing patients at risk to develop thrombotic complications14 in a series of intensive care unit patientsin the netherlands reported by klok only threestrokes complicated the course of covid19 whereasthe majority of complications included pulmonarythrombosiscatheterassociatedembolism n¼ and peripheral venous thrombosisn¼ andobservations in case series that concurrent covid infection complicates or triggers unusual ischemicstroke may well prevail but case control studies focusing on this phenomenon are urgently needed to affirmor deny the assertion5 second physical inactivity has aprofound effect on atrial fibrillation obesity diabetesmellitus management and hypertension among othersand contradicts current recommendations on mid andlongterm stroke prevention16 a recent study in consecutive patients with nonstsegment elevationacute coronary syndromes acss and optical coherence tomography of the culprit lesion reported bykato found that the combination of greaterphysical activity outdoor acs onset and high bodymass index had a significant effect on the incidence ofcoronary plaque erosion17 interestingly mobility datasuch as those provided by the apple mobility databasevr demonstrated a parallel reduction in incidencesof stroke and acs in three published papers8“ inaddition to oursour data confirm the observation from morelli who termed the phrase ˜baffling case of ischemicstroke disappearance™ these authors also discuss thatthis effect cannot be totally explained merely by thereluctance of patients to call for help in a stroke emergency because the number of cases presenting withsevere stroke requiring evt and the number of generalconsultations in tempis also decreased an analysisbased on a large database associated with the application of rapid software in acute stroke by kansagra is in line with our observation that also severestroke patients diminished during the early lockdownphase9 the number of ischemic core volumes “ml and greater than ml were observed to decreaseby and respectively core volumes “ ml decreased by and and very smallcore infarct volumes measuring “ ml decreased the decrease in the number of very small infarctvolumes may well be explained by the generally proposed hesitation to seek emergency care while thereduction in large ischemic core volumes is morelikely due to fewer lvos as observed in our studywith a sharp decline in iv thrombolysis and thrombectomy recommendationsanother explanation may be a concurrent low infection rate with other viruses that can trigger atherosclerosis and plaque rupture resulting in neuro andcardiovascular morbidity18 the lockdown not onlyreduces physical activity strict social distancing anduse of facial masks should also lead to low rates ofexposure to and transmission of other common virusesand allergens that by themselves appear to triggerstroke19 additional studies with detailed analyses of 0cschlachetzki stekcarberauqsniatadhtnomehtfoshtgneltnereffidrofstnemtsudajtuohtiwtubsdoirepkeewotnidedvdiilirpa“yraunajrofsnoitatlusnocforebmunlatotlebatrpa“rpa“ram“ram“bef“bef“naj“naj“sopdvocilatotairavabnisesacnoitaiveddradnatsdnaseulavnaemni“morfatadwohssipmetkrowtenekortsnoitatlusnoceetl] 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06[snp p¡ 8a 06[¡snp§p§§§§ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[  8a 06[§ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[lsisyobmorhtvi 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06[ 8a 06 06[ 8a 06[ 8a 06 06[ytilibadaerrettebrofldoblynoerasrebmunldobskcattaicmehcsitneisnartatiamotamehlarudbushdsegahrromehidonhcarabushasegahrromeahlainarcartnihciigndeebllainarcartnsnsn¼¼¼¼bp§pcpp§§ ¡iids 06“naemsnoitatlusnocekortsicmehcsids 06“naemds 06“naemymotcebmorhtds 06“naemds 06“naema tids 06“naemh cids 06“naemhashdsbcieniargimeruziessrehto 0c of telemedicine and telecare symptom onsettodoor times stroke severity neuroimaging and inflammatory markers are needed tounderstand the reason for the reduced number of revascularization therapies requested during the covid19pandemiclimitations of the studyanalysis of daily working diagnoses in the tempistelestroke network has the advantage of being highlytimely yet it lacks specificity because the final diagnosismay differ from the initial one this may be compensated by the creation of a large common database fortelestroke networks that incorporates corrections forthe actual population covered analyses of otherstrokerelated databases such as the one associatedwith rapid software healthcare provider databasesand common stroke registries for quality control thedecrease in the number of thrombectomy recommendations in our cohort midmarch did not reach statistical significance when compared with the sameperiod in years through because rates forthis procedure increased according with levels of evidence2021 in agreement with this development thrombectomy recommendations by tempis neurologists in prior to the covid19 pandemic occurred morefrequently than in previous yearssour study using the tempis telestroke database confirms lower incidences of ischemic stroke and otheracute neurological disorders requiring consultationsuch as intracerebral haemorrhage seizure disorderand migraine next to a reluctance within the population to seek immediate medical assistance for acutestroke the covid19 lockdown which resulted inless physical activity and fewer other common infections may also be responsible for the fewer numberof patients with severe stroke especially those withintracranial haemorrhage and those eligible for recanalization therapies if lockdownassociated factors areindeed responsible for a lower stroke incidence we mayexpect a rebound effect following the lockdown periodwith an increased incidence of stroke as well as ofmyocardial infarcts and traumatic brain injuries aspatients™ frailty may have increased during the lockdown analyses of large stroke databases may revealfurther insights into this phenomenon however telestroke networks such as tempis may be ideal tools tomonitor stroke occurrence in real timeacknowledgmentsthe authors acknowledge all consulting neurologists intempis and colleaguesin badin partner hospitalsebersbergburglengenfeldreichenhalleggenfeldenerding freising kelheim mu¨ hldorf rotthalmu¨ nstervilsbiburg dingolfing and zwiesel the authors like tothank jo ann elison ma elsdfor editing thispaper for english grammar and languagedeclaration of conflicting intereststhe authors declared no potential conflicts of interest withrespect to the research authorship andor publication of thisarticleanonymized data are available on requestfundingthe authors received no financial support for the researchauthorship andor publication of this articleorcid idfelix schlachetzkiorcidorg0000000161672597references jawaid a protecting older adults during social distancing science khosravani h rajendram p notario l protectedcode stroke hyperacute stroke management during thecoronavirus disease covid19 pandemic stroke “ markus hs and brainin m covid19 and stroke a global world stroke organization perspective int jstroke “ mao l jin h wang m neurologic manifestationsof hospitalized patients with coronavirus disease inwuhan china jama neurol “ oxley tj mocco j majidi s largevessel stroke asa presenting feature of covid19 in the young n engl jmed e60 zhang y xiao m zhang s coagulopathy andantiphospholipid antibodies in patients with covid19n engl j med e38 zhao j rudd a and liu r challenges and potentialsolutions of stroke care during the coronavirus disease covid19 outbreak stroke “ morelli n rota e terracciano c the baffling caseofischemic stroke disappearance from the casualtydepartment in the covid19 era eur neurol “ kansagra ap goyal ms hamilton s collateraleffect of covid19 on stroke evaluation in the unitedstates n englnejmc2014816 online ahead of printj meddoi huet f prieur c schurtz g one train may hideanother acute cardiovascular diseases could be neglectedbecause of the covid19 pandemic arch cardiovasc dis “ bansal m cardiovascular disease and covid19diabetes metab syndr “ audebert hj schenkel j heuschmann pu effectsof the implementation of a telemedical stroke networkthe telemedic pilot project for integrative stroke care 0cschlachetzki tempis in bavaria germany lancet neurol “ patel uk malik p demasi m multidisciplinaryapproach and outcomes of teleneurology a reviewcureus e4410 terpos e ntanasisstathopoulos i elalamy i hematological findings and complications of covid am j hematol “ klok fa kruip m van der meer njm incidenceof thrombotic complications in critically ill icu patientswith covid19 thromb res “ kyu hh bachman vf alexander lt physicalactivity and risk of breast cancer colon cancer diabetesischemic heart disease and ischemic stroke eventsaystematic review and doseresponse metaanalysis forthe global burden of disease study bmj i3857 kato a minami y katsura a physical exertion asa trigger of acute coronary syndrome caused by plaqueerosion j thromb thrombolysis “ grau aj urbanek c and palm f common infectionsand the risk of stroke nat rev neurol “ pagliano p spera am ascione t infections causing stroke or strokelike syndromes infection “ campbell bcv donnan ga lees kr endovascular stent thrombectomy the new standardof care for large vessel ischaemic stroke lancet neurol “ vinny pw vishnu vy and padma srivastava mvthrombectomy to hours after stroke n engl jmed 0c'
Colon_Cancer
coutard b valle c de lamballerie x canard b seidah ng and decroly e the spike glycoprotein of the new coronavirus 2019ncovcontains a furinlike cleavage site absent in cov of the same clade antiviral res 101016jantiviral2020104742 cao z wu y faucon e and sabatier jm sarscov2 covid19 keyroles of the ˜reninangiotensin™ systemvitamin d impacting drugand vaccine developments infect disord drug targets “ 1021741871526520999200505174704 zhang z chen l zhong j gao p and oudit gy ace2ang17 signaling and vascular remodeling sci china life sci “s1142701446933 paz ocaranza m riquelme ja garcia l jalil je chiong m santos ras counterregulatory reninangiotensin system incardiovascular disease nat rev cardiol “ 101038s4156901902448 ziegler cgk allon sj nyquist sk mbano im miao vn tzouanas cn sarscov2 receptor ace2 is an interferonstimulatedgene in human airway epithelial cells and is detected in specific cell subsets across tissues cell “101016jcell202004035 marrero mb schieffer b paxton wg heerdtt l berkt bc delafontaine p direct stimulation of jakstat pathway by theangiotensin ii at1 receptor j med chem “ perrotta f matera mg cazzola m and bianco a severe respiratory sarscov2 infection does ace2 receptor matter respir med 101016jrmed2020105996 cheng h wang y and wang g anprotective effect of angiotensinconverting enzyme and its effect on the prognosis of covid19 jmed virol “ 101002jmv25785 de lang a osterhaus adme and haagmans bl interferon Î and interleukin4 downregulate expression of the sars coronavirusreceptor ace2 in vero e6 cells virology “ the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 mcgonagle d sharif k o™regan a and bridgewood c the role of cytokines including interleukin6 in covid19 induced pneumonia andmacrophage activation syndromelike disease autoimmun rev linkinghubelseviercomretrievepiis1568997220300926101016jautrev2020102537 ye q wang b and mao j the pathogenesis and treatment of the ˜cytokine storm™ in covid19 j infect “linkinghubelseviercomretrievepiis0163445320301651 101016jjinf202003037 behrens em and koretzky ga review cytokine storm syndrome looking toward the precision medicine era arthritis rheumatol “ 101002art40071 guo xj and thomas pg new fronts emerge in the ‚uenza cytokine storm semin immunopathol “s002810170636y ronco c and reis t kidney involvement in covid19 and rationale for extracorporeal therapies nat rev nephrol “101038s4158102002847 wu c chen x cai y xia j zhou x xu s risk factors associated with acute respiratory distress syndrome and death in patientswith coronavirus disease pneumonia in wuhan china jama intern med “ 101001jamainternmed20200994 jose rj and manuel a covid19 cytokine storm the interplay between ‚ammation and coagulation lancet respir med “ murakami m kamimura d and hirano t pleiotropy and specificity insights from the interleukin family of cytokines immunity “ 101016jimmuni201903027 o™shea jj schwartz dm villarino av gadina m mcinnes ib and laurence a the jakstat pathway impact on human disease andtherapeutic intervention annu rev med “ 101146annurevmed051113024537 lawrence t the nuclear factor nf k b pathway in ‚ammation cold spring harb perspect biol “ catanzaro m fagiani f racchi m corsini e govoni s and lanni c immune response in covid19 addressing a pharmacologicalchallenge by targeting pathways triggered by sarscov2 signal transduct target ther 101038s4139202001911 li q and verma im nfκb regulation in the immune system nat rev immunol “ 101038nri910 tergaonkar v correa rg ikawa m and verma im distinct roles of iκb proteins in regulating constitutive nfκb activity nat cell biol “ 101038ncb1296 tergaonkar v nfκb pathway a good signaling paradigm and therapeutic target int j biochem cell biol “linkinghubelseviercomretrievepiis1357272506001245 101016jbiocel200603023 correa rg matsui t tergaonkar v rodriguezesteban c izpisuabelmonte jc and verma im zebrafish ikappab kinase negativelyregulates nfkappab activity curr biol “ 101016jcub200506023 wong et and tergaonkar v roles of nfκb in health and disease mechanisms and therapeutic potential clin sci “portlandpresscomclinsci116645168639rolesofnfκbinhealthanddiseasemechanismsand101042cs20080502 morrison dk map kinase pathways cold spring harb perspect biol a011254 101101cshperspecta011254 mizutani t fukushi s saijo m kurane i and morikawa s phosphorylation of p38 mapk and its downstream targets in sarscoronavirusinfected cells biochem biophys res commun “ 101016jbbrc200405107 grimes jm and grimes kv p38 mapk inhibition a promising therapeutic approach for covid19 j mol cell cardiol “101016jyjmcc202005007 cuadrado a and nebreda ar mechanisms and functions of p38 mapk signalling biochem j “101042bj20100323 han j lee jd tobias ps and ulevitch rj endotoxin induces rapid protein tyrosine phosphorylation in 70z3 cells expressing cd14 jbiol chem “ jiang y chen c li z guo w gegner ja lin s characterization of the structure and function of a new mitogenactivated proteinkinase p38 j biol chem “ 101074jbc2713017920 li z jiang y ulevitch rj and han j the primary structure of p38Î a new member of p38 group of map kinases biochem biophys rescommun “ linkinghubelseviercomretrievepiis0006291x96916629 101006bbrc19961662 jiang y gram h zhao m new l gu j feng l characterization of the structure and function of the fourth member of p38 groupmitogenactivated protein kinases p38δ j biol chem “ 101074jbc2724830122 zarubin t and han j activation and signaling of the p38 map kinase pathway cell res “ 101038sjcr7290257 cuevas bd abell an and johnson gl role of mitogenactivated protein kinase kinase kinases in signal integration oncogene “ httpwwwnaturecoms1210409 101038sjonc1210409 alonso g ambrosino c jones m and nebreda ar differential activation of p38 mitogenactivated protein kinase isoforms depending onsignal strength j biol chem “ 101074jbcm007835200 brancho d mechanism of p38 map kinase activation in vivo genes dev “ 101101gad1107303 parker cg hunt j diener k mcginley m soriano b keesler ga identification of stathmin as a novel substrate for p38 deltabiochem biophys res commun “ linkinghubelseviercomretrievepiis0006291x98992506101006bbrc19989250 rouse j cohen p trigon s morange m alonsollamazares a zamanillo d a novel kinase cascade triggered by stress and heatshock that stimulates mapkap kinase2 and phosphorylation of the small heat shock proteins cell “linkinghubelseviercomretrievepii0092867494902771 1010160092867494902771 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 tan y rouse j zhang a cariati s cohen p and comb mj fgf and stress regulate creb and atf1 via a pathway involving p38 mapkinase and mapkap kinase2 embo j “ 101002j146020751996tb00840x xu w wang x tocker am huang p reith mea liuchen ly functional characterization of a novel series of biased signalingdopamine d3 receptor agonists acs chem neurosci “ 101021acschemneuro6b00221 kotlyarov a neininger a schubert c eckert r birchmeier c volk hd mapkap kinase is essential for lpsinduced tnfαbiosynthesis nat cell biol “ httpwwwnaturecomsncb0699 10103810061 kontoyiannis d pasparakis m pizarro tt cominelli f and kollias g impaired onoff regulation of tnf biosynthesis in mice lacking tnfaurich elements immunity “ linkinghubelseviercomretrievepiis1074761300800382101016s1074761300800382 saccani s pantano s and natoli g p38dependent marking of ‚ammatory genes for increased nfκb recruitment nat immunol “ 101038ni748 dienz o and rincon m the effects of il6 on cd4 t cell responses clin immunol “ 101016jclim200808018 velazquezsalinas l verdugorodriguez a and rodriguez ll the role of interleukin during viral infections front microbiol “ zhang c wu z li jw zhao h and wang gq the cytokine release syndrome crs of severe covid19 and interleukin6 receptoril6r antagonist tocilizumab may be the key to reduce the mortality int j antimicrob agents 101016jijantimicag2020105954 matthay ma ware lb and zimmerman ga the acute respiratory distress syndrome j clin invest “101172jci60331 zhang h rostami mr leopold pl mezey jg o™beirne sl strulovicibarel y expression of the sarscov2 ace2 receptor inthe human airway epithelium am j respir crit care med “ 101164rccm2020030541oc tian s xiong y liu h niu l guo j liao m pathological study of the novel coronavirus disease covid19 throughpostmortem core biopsies mod pathol “ 101038s413790200536x sungnak w huang n b ´ecavin c berg m queen r litvinukova m sarscov2 entry factors are highly expressed in nasalepithelial cells together with innate immune genes nat med “ 101038s4159102008686 lukassen s chua rl trefzer t kahn nc schneider ma muley t sars cov2 receptor ace and tmprss are primarilyexpressed in bronchial transient secretory cells embo j “ 1015252embj20105114 leung jm yang cx tam a shaipanich t hackett tl singhera gk ace2 expression in the small airway epithelia of smokersand copd patients implications for covid19 eur respir j 10118313993003006882020 dhawale vs amara vr karpe pa malek v patel d and tikoo k activation of angiotensinconverting enzyme ace2 attenuatesallergic airway ‚ammation in rat asthma model toxicol appl pharmacol “ 101016jtaap201606026 bradding p richardson m hinks tsc howarth ph choy df arron jr ace2 tmprss2 and furin gene expression in the airwaysof people with asthma implications for covid19 j allergy clin immunol “ 101016jjaci202005013 vareille m kieninger e edwards mr and regamey n the airway epithelium soldier in the fight against respiratory viruses clinmicrobiol rev “ 101128cmr0001410 sabroe i parker lc dower sk and whyte mkb the role of tlr activation in ‚ammation j pathol “101002path2264 frieman m and baric r mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation microbiol mol biolrev “ 101128mmbr0001508 lafferty ei qureshi st and schnare m the role of tolllike receptors in acute and chronic lung ‚ammation j ‚amm httpwwwjournal‚ammationcomcontent7157 10118614769255757 kawai t and akira s signaling to nfκb by tolllike receptors trends mol med “101016jmolmed200709002 darby wj mcnutt kw and todhunter en niacin nutr rev “ 101111j175348871975tb05075x yoshikawa t hill te yoshikawa n popov vl galindo cl garner hr dynamic innate immune responses of human bronchialepithelial cells to severe acute respiratory syndromeassociated coronavirus infection plos one e8729101371journalpone0008729 dediego ml nietotorres jl reglanava ja jimenezguardeno jm fernandezdelgado r fett c inhibition ofnfkappabmediated ‚ammation in severe acute respiratory syndrome coronavirusinfected mice increases survival j virol “101128jvi0257613 imai y kuba k neely gg yaghubianmalhami r perkmann t van loo g identification of oxidative stress and tolllike receptor signaling as a key pathway of acute lung injury cell “ 101016jcell200802043 ding y feng q chen j and song j tlr4nfκb signaling pathway gene single nucleotide polymorphisms alter gene expression levelsand affect ards occurrence and prognosis outcomes medicine baltimore e16029 101097md0000000000016029 totura al whitmore a agnihothram s sch ¨afer a katze mg heise mt tolllike receptor signaling via trif contributes to aprotective innate immune response to severe acute respiratory syndrome coronavirus infection mbio “101128mbio0063815 olejnik j hume aj and m ¨uhlberger e tolllike receptor in acute viral infection too much of a good thing plos pathog “101371journalppat1007390 okumura a rasmussen al halfmann p feldmann f yoshimura a feldmann h suppressor of cytokine signaling is aninducible host factor that regulates virus egress during ebola virus infection j virol “ 101128jvi0173615 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 o™brien tr thomas dl jackson ss prokuninaolsson l donnelly rp and hartmann r weak induction of interferon expression bysarscov2 supports clinical clin infect dis 101093cidciaa453 chang yj liu cyy chiang bl chao yc and chen cc induction of il8 release in lung cells via activator protein1 byrecombinant baculovirus displaying severe acute respiratory syndromecoronavirus spike proteins identification of two functional regions j immunol “ 104049jimmunol173127602 wang w ye l ye l li b gao b zeng y upregulation of il6 and tnfα induced by sarscoronavirus spike protein in murinemacrophages via nfκb pathway virus res “ 101016jvirusres200702007 zhang x wu k wang d yue x song d zhu y nucleocapsid protein of sarscov activates interleukin6 expression throughcellular transcription factor nfκb virology “ 101016jvirol200704009 toews gb cytokines and the lung eur respir j suppl “ 101183090319360100266001 russell b moss c rigg a and van hemelrijck m covid19 and treatment with nsaids and corticosteroids should we be limiting theiruse in the clinical setting ecancermedicalscience “ 103332ecancer20201023 severgnini m takahashi s rozo lm homer rj kuhn c jhung jw activation of the stat pathway in acute lung injury am jphysiol lung cell mol physiol “ 101152ajplung003492003 zhao j yu h liu y gibson sa yan z xu x protective effect of suppressing stat3 activity in lpsinduced acute lung injury amj physiol lung cell mol physiol l868“l880 101152ajplung002812016 pedroza m schneider dj karmoutyquintana h coote j shaw s corrigan r interleukin6 contributes to ‚ammation andremodeling in a model of adenosine mediated lung injury plos one e22667 101371journalpone0022667 goldman jl sammani s kempf c saadat l letsiou e wang t pleiotropic effects of interleukin6 in a œtwohit murine modelof acute respiratory distress syndrome pulm circ “ 101086675991 rincon m and irvin cg role of il6 in asthma and other ‚ammatory pulmonary diseases int j biol sci “107150ijbs4874 swanson kv deng m and ting jpy the nlrp3 ‚ammasome molecular activation and regulation to therapeutics nat rev immunol “ 101038s4157701901650 zhao c and zhao w nlrp3 ‚ammasome ” a key player in antiviral responses front immunol “ orzalli mh smith a jurado ka iwasaki a garlick ja kagan jc an antiviral branch of the il1 signaling pathway restrictsimmuneevasive virus replication an antiviral branch of the il1 signaling pathway restricts immuneevasive virus replication mol cell 825e5“840e5 101016jmolcel201807009 shi c and nabar nr sarscoronavirus open reading frame8b triggers intracellular stress pathways and activates nlrp3 ‚ammasomescell death discov 101038s4142001901817 siu k yuen k castanorodriguez c ye z yeung m fung s severe acute respiratory syndrome coronavirus orf3a proteinactivates the nlrp3 ‚ammasome by promoting traf3dependent ubiquitination of asc faseb j “101096fj201802418r kusov y tan j alvarez e enjuanes l and hilgenfeld r a gquadruplexbinding macrodomain within the œsarsunique domain isessential for the activity of the sarscoronavirus replicationtranscription complex virology “101016jvirol20150601 chang ys ko bh ju jc chang hh huang sh and lin cw sars unique domain sud of severe acute respiratory syndromecoronavirus induces nlrp3 ‚ammasomedependent cxcl10mediated pulmonary ‚ammation int j mol sci “103390ijms21093179 sun dz song cq xu ym and dong xs role of the mapk pathway in human lung epitheliallike a549 cells apoptosis induced byparaquat genet mol biol e20190137 10159016784685gmb20190137 arcaroli j yum hk kupfner j park js yang ky and abraham e role of p38 map kinase in the development of acute lung injury clinimmunol “ 101006clim20015108 li sw wang cy jou yj yang tc huang sh wan l sars coronavirus papainlike protease induces egr1dependentupregulation of tgf1 via rosp38 mapkstat3 pathway sci rep “ yoshida k kuwano k hagimoto n watanabe k matsuba t fujita m map kinase activation and apoptosis in lung tissues frompatients with idiopathic pulmonary fibrosis j pathol “ 101002path1208 qian f deng j wang g ye rd and christman jw pivotal role of mitogenactivated protein kinaseactivated protein kinase in‚ammatory pulmonary diseases curr protein pept sci “ 1021741389203716666150629121324 renda t baraldo s pelaia g bazzan e turato g papi a increased activation of p38 mapk in copd eur respir j “1011830903193600036707 mercer ba and d™armiento jm emerging role of map kinase pathways as therapeutic targets in copd int j chron obstruct pulmon dis “ feng y fang z liu b and zheng x p38mapk plays a pivotal role in the development of acute respiratory distress syndrome clinics “ 106061clinics2019e509 mizutani t signaling pathways of sarscov in vitro and in vivo mol biol “ 9783642036835˙ mizutani t signal transduction in sarscovinfected cells ann ny acad sci “ 101196annals1408006 cheng vcc hung ifn tang bsf chu cm wong mml chan kh viral replication in the nasopharynx is associated withdiarrhea in patients with severe acute respiratory syndrome clin infect dis “ 101086382681 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 arabi ym alomari a mandourah y alhameed f sindi aa alraddadi b critically ill patients with the middle east respiratorysyndrome a multicenter retrospective cohort study crit care med “ 101097ccm0000000000002621 wong sh lui rns and sung jjy covid19 and the digestive system j gastroenterol hepatol “101111jgh15047 luo s zhang x and xu h don™t overlook digestive symptoms in patients with novel coronavirus disease covid19 clingastroenterol hepatol “ 101016jcgh202003043 xiao f tang m zheng x liu y li x and shan h evidence for gastrointestinal infection of sarscov2 gastroenterology 1831e3“1833e3 101053jgastro202002055 young be ong swx kalimuddin s low jg tan sy loh j epidemiologic features and clinical course of patients infected withsarscov2 in singapore jama jamanetworkcomjournalsjamafull2762688 101001jama20203204 xu y li x zhu b liang h fang c gong y characteristics of pediatric sarscov2 infection and potential evidence for persistentfecal viral shedding nat med “ 101038s4159102008174 tang a tong z wang h dai y li k liu j detection of novel coronavirus by rtpcr in stool specimen from asymptomatic childchina emerg infect dis “ httpwwwnccdcgoveid266200301 htm 103201eid2606200301 zhang w du rh li b zheng xs yang xl hu b molecular and serological investigation of 2019ncov infected patientsimplication of multiple shedding routes emerg microbes infect “ 1010802222175120201729071 yeo c kaushal s and yeo d enteric involvement of coronaviruses is faecaloral transmission of sarscov2 possible lancetgastroenterol hepatol “ 101016s2468125320300480 van doremalen n bushmaker t morris dh holbrook mg gamble a williamson bn aerosol and surface stability ofsarscov2 as compared with sarscov1 n engl j med “ 101056nejmc2004973 setti l passarini f de gennaro g barbieri p perrone mg borelli m airborne transmission route of covid19 why meters6feet of interpersonal distance could not be enough int j environ res public health 103390ijerph17082932 perlman s and netland j coronaviruses postsars update on replication and pathogenesis nat rev microbiol “101038nrmicro2147 m ¨onkem ¨uller k fry l and rickes s covid19 coronavirus sarscov2 and the small bowel rev esp enferm dig “ wang d hu b hu c zhu f liu x zhang j clinical characteristics of hospitalized patients with novelcoronavirusinfected pneumonia in wuhan china jama jamanetworkcomjournalsjamafull2761044101001jama20201585 pan l mu m yang p sun y wang r yan j clinical characteristics of covid19 patients with digestive symptoms in hubei chinaam j gastroenterol “ 1014309ajg0000000000000620 budden kf gellatly sl wood dla cooper ma morrison m hugenholtz p emerging pathogenic links between microbiota andthe gutlung axis nat rev microbiol “ 101038nrmicro2016142 camargo smr singer d makrides v huggel k pos km wagner ca tissuespecific amino acid transporter partners ace2 andcollectrin differentially interact with hartnup mutations gastroenterology 872e3“882e3linkinghubelseviercomretrievepiis0016508508018933 101053jgastro200810055 hashimoto t perlot t rehman a trichereau j ishiguro h paolino m ace2 links amino acid malnutrition to microbial ecology andintestinal ‚ammation nature “ 101038nature11228 br ¨oer a juelich t vanslambrouck jm tietze n solomon ps holst j impaired nutrient signaling and body weight control in naneutral amino acid cotransporter slc6a19deficient mouse j biol chem “ 101074jbcm111241323 ma xm and blenis j molecular mechanisms of mtormediated translational control nat rev mol cell biol “101038nrm2672 bostancklo ˘glu m temporal correlation between neurological and gastrointestinal symptoms of sarscov2 ‚amm bowel dis “101093ibdizaa131 louveau a smirnov i keyes tj eccles jd rouhani sj peske jd structural and functional features of central nervous systemlymphatic vessels nature “ 101038nature14432 jessen na munk asf lundgaard i and nedergaard m the glymphatic system a beginner™s guide neurochem res “s1106401515816 wu y xu x chen z duan j hashimoto k yang l nervous system involvement after infection with covid19 and othercoronaviruses brain behav immun “ 101016jbbi202003031 liu k pan m xiao z and xu x neurological manifestations of the coronavirus sarscov2 pandemic j neurol neurosurgpsychiatry “ 101136jnnp2020323177 xydakis ms dehganimobaraki p holbrook eh geisthoff uw bauer c hautefort c smell and taste dysfunction in patients withcovid19 lancet infect dis s14733099 “ 101016s1473309920302930 moriguchi t harii n goto j harada d sugawara h takamino j a first case of meningitisencephalitis associated withsarscoronavirus2 int j infect dis “ 101016jijid202003062 baig am khaleeq a ali u and syeda h evidence of the covid19 virus targeting the cns tissue distribution hostvirus interaction andproposed neurotropic mechanisms acs chem neurosci “ 101021acschemneuro0c00122 helms j kremer s merdji h clerejehl r schenck m kummerlen c neurologic features in severe sarscov2 infection nengl j med “ 101056nejmc2008597 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 panizmondolfi a bryce c grimes z gordon re reidy j lednicky j central nervous system involvement by severe acuterespiratory syndrome coronavirus2 sarscov2 j med virol “ 101002jmv25915 zhou l zhang m wang j and gao j sarscov2 underestimated damage to nervous system travel med infect dis linkinghubelseviercomretrievepiis1477893920301101 101016jtmaid2020101642 panciani pp saraceno g zanin l renisi g signorini l battaglia l sarscov2œthreesteps infection model and csfdiagnostic implication brain behav immun “ 101016jbbi202005002 desfes m le coupanec a dubeau p bourgouin a lajoie l dub ´e m human coronaviruses and other respiratory virusesunderestimated opportunistic pathogens of the central nervous system viruses 103390v12010014 fazakerley jk and walker r virus demyelination j neurovirol “ 10108013550280390194046 murray rs cai gy hoel k zhang jy soike kf and cabirac gf coronavirus infects and causes demyelination in primate centralnervous system virology “ 101016004268229290757g lane te and buchmeier mj murine coronavirus infection a paradigm for virusinduced demyelinating disease trends microbiol “101016s0966842x97817684 stohlman sa and hinton dr viral induced demyelination brain pathol “ 101111j175036392001tb00384x li z liu t yang n han d mi x li y neurological manifestations of patients with covid19 potential routes of sarscov2neuroinvasion from the periphery to the brain front med “ s1168402007865 netland j meyerholz dk moore s cassell m and perlman s severe acute respiratory syndrome coronavirus infection causes neuronaldeath in the absence of encephalitis in mice transgenic for human ace2 j virol “ 101128jvi0073708 bohmwald k galvez n r´Ä±os m and kalergis am neurologic alterations due to respiratory virus infections front cell neurosci 103389fncel201800386 montalvan v lee j bueso t de toledo j and rivas k neurological manifestations of covid19 and other coronavirus infections asystematic review clin neurol neurosurg 101016jclineuro2020105921 zhou f yu t du r fan g liu y liu z clinical course and risk factors for mortality of adult inpatients with covid19 in wuhanchina a retrospective cohort study lancet “ linkinghubelseviercomretrievepiis0140673620305663101016s0140673620305663 varga z flammer aj steiger p haberecker m andermatt r zinkernagel as endothelial cell infection and endotheliitis incovid19 lancet “ 101016s0140673620309375 bostanciklio ˘glu m sarscov2 entry and spread in the lymphatic drainage system of the brain brain behav immun “101016jbbi202004080 li yc bai wz and hashikawa t the neuroinvasive potential of sarscov2 may play a role in the respiratory failure of covid19 patientsj med virol “ 101002jmv25728 li y bai w hirano n hayashida t taniguchi t sugita y neurotropic virus tracing suggests a membranouscoatingmediatedmechanism for transsynaptic communication j comp neurol “ 101002cne23171 reichard rr kashani kb boire na constantopoulos e guo y and lucchinetti cf neuropathology of covid19 a spectrum ofvascular and acute disseminated encephalomyelitis ademlike pathology acta neuropathol “s00401020021662 kanberg n ashton nj andersson lm yilmaz a lindh m nilsson s neurochemical evidence of astrocytic and neuronal injurycommonly found in covid19 neurology nneurologycontentneurologyearly20200616wnl0000000000010111fullpdf101212wnl0000000000010111 xiao l haack kkv and zucker ih angiotensin ii regulates ace and ace2 in neurons through p38 mitogenactivated protein kinase andextracellular signalregulated kinase signaling am j physiol cell physiol c1073“c1079 101152ajpcell003642012 tang n li d wang x and sun z abnormal coagulation parameters are associated with poor prognosis in patients with novel coronaviruspneumonia j thromb haemost “ 101111jth14768 mehta p mcauley df brown m sanchez e tattersall rs manson jj covid19 consider cytokine storm syndromes andimmunosuppression lancet 101016s0140673620306280 koralnik ij and tyler kl covid19 a global threat to the nervous system ann neurol “ 101002ana25807 toscano g palmerini f ravaglia s ruiz l invernizzi p cuzzoni mg guillainbarr ´e syndrome associated with sarscov2 nengl j med nejmc2009191 101056nejmc2009191 madjid m safavinaeini p solomon sd and vardeny o potential effects of coronaviruses on the cardiovascular system jama cardiol “ jamanetworkcomjournalsjamacardiologyfull2763846 epelman s shrestha k troughton rw francis gs sen s klein al soluble angiotensinconverting enzyme in human heartfailure relation with myocardial function and clinical outcomes j cardiac fail “ 101016jcardfail200901014 wild ps dimmeler s and eschenhagen t an epidemiological study exploring a possible impact of treatment with ace inhibitors orangiotensin receptor blockers on ace2 plasma concentrations j mol cell cardiol “ 101016jyjmcc202003018 wu z and mcgoogan jm characteristics of and important lessons from the coronavirus disease covid19 outbreak in china jama jamanetworkcomjournalsjamafull2762130 101001jama20202648 zheng yy ma yt zhang jy and xie x covid19 and the cardiovascular system nat rev cardiol “101038s4156902003605 ruan q yang k wang w jiang l and song j clinical predictors of mortality due to covid19 based on an analysis of data of patients from wuhan china intensive care med “ s0013402005991x the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 cheng r and leedy d covid19 and acute myocardial injury the heart of the matter or an innocent bystander heart “101136heartjnl2020317025 zhu l carretero oa xu j harding p ramadurai n gu x activation of angiotensin ii type receptor suppresses tnfαinducedicam1 via nfκb possible role of ace2 am j physiol heart circ physiol h827“h834 101152ajpheart008142014 dzimiri n receptor crosstalk implications for cardiovascular function disease and therapy eur j biochem “101046j14321033200203181x wheelerjones cpd cell signalling in the cardiovascular system an overview heart “101136hrt2005072280 houliston ra pearson jd and wheelerjones cpd agonistspecific cross talk between erks and p38 mapk regulates pgi synthesis inendothelium am j physiol physiol c1266“c1276 101152ajpcell20012814c1266 adam lp franklin mt raff gj and hathaway dr activation of mitogenactivated protein kinase in porcine carotid arteries circ res “ 10116101res762183 nebreda ar and porras a p38 map kinases beyond the stress response trends biochem sci “linkinghubelseviercomretrievepiis0968000400015954 101016s0968000400015954 wang y huang s sah vp ross j brown jh han j cardiac muscle cell hypertrophy and apoptosis induced by distinct membersof the p38 mitogenactivated protein kinase family j biol chem “ 101074jbc27342161 saurin at martin jl heads rj foley c mockridge jw wright mj the role of differential activation of p38mitogenactivatedprotein kinase in preconditioned ventricular myocytes faseb j “ 101096fj990671com podewski ek hilfikerkleiner d hilfiker a morawietz h lichtenberg a wollert kc alterations in janus kinase jaksignaltransducers and activators of transcription stat signaling in patients with endstage dilated cardiomyopathy circulation “10116101cir000005754582749ff patel ab and verma a covid19 and angiotensinconverting enzyme inhibitors and angiotensin receptor blockers what is the evidencejama “ jamanet
Colon_Cancer
" breast cancer bc is the most commonly diagnosed malignant cancer in women bc is the main cause of cancerrelated death in women and seriously threatens the life and health of women worldwide micrornas mirnasmirs have been reported to regulate the development and progression of different types of cancer however the regulatory functions of mir1885p in bc have not been thoroughly demonstrated in this present research we identified that mir‘‘5p was downregulated in bc tissues and several bc cell lines downregulation of mir‘‘5p was significantly associated with advanced tnm stage moreover we identified that mir‘‘5p mimics significantly inhibited proliferation using cck8 assay colony formation and xenograft animal model suppressed invasion and migration detected by transwell invasion assay and increased the cellular apoptosis of bc cells as determined by cell apoptosis assay moreover mir1885p mimics also reduced the expression of nfκb p65rel to further investigate its regulatory mechanism transcription factor zinc finger protein zfp91 was predicted as the targeted protein of mir1885p by bioinformatic method we confirmed their specific binding by dual luciferase dlr assay we demonstrated that the overexpression of mir1885p significantly inhibited the expression of zfp91 in bc cell lines and reduced the expression of nfκb p65rel an inverse correlation was found between the expression of mir1885p and zfp91 in bc tissues importantly we demonstrated that the restoration of zfp91 was able to block the effect of mir1885p on the progression of mdamb231 cells therefore our study showed that mir1885p may be one of the important indicators and could inhibit the progression of human bc via targeting the zfp91nf‘κb p65rel signaling pathway suggesting that mir1885p may be a promising future target for bc treatmentcorrespondence to dr zhaoyu liu department of radiology shengjing hospital of china medical university sanhao street shenyang liaoning pr chinaemail liuzy1226126comkey words breast cancer mir1885p zinc finger protein zfp91 proliferation apoptosisintroductionbreast cancer bc is one of the most commonly diagnosed malignancy in the world the mortality rate for bc ranks first among all female malignant tumors globally the number of newly diagnosed bc cases reached approximately million in accounting for almost of cancer cases among women bc exhibits a complex pathogenesis and is a clinically heterogeneous disease with a wide range of clinical behaviors and treatment responses although many dysregulated molecular pathways have been discovered in bc the development of effective therapeutic methods has been limited it is urgent to discover novel molecules to suppress bc proliferation induce apoptosis and inhibit invasion and provide potential therapeutic strategies to improve the survival and quality of life of bc patients micrornas mirnasmirs are a class of endogenous noncoding rnas of approximately nucleotides in length which are generally located in unstable regions of the human genome and are usually dysregulated in malignant tumors to regulate gene functions mirnas regulate target genes through binding to the 'untranslated regions 'utrs of the target mrnas subsequently inhibiting gene expression through regulation of the targeted proteins mirnas play an important role in many tumor cellular processes such as proliferation cell cycle apoptosis invasion and metastasis and participate in almost all signaling pathways mir1885p has been reported to be an inhibitor of tumor growth and metastasis in prostate cancer and hepatocellular carcinoma however to the best of our knowledge the functions of mir1885p in bc remain elusivein the present study we detected the expression of mir1885p in tumor tissues of bc patient tissues and several bc cell lines furthermore we investigated its regulatory role in bc proliferation apoptosis and invasion we also predicted and confirmed the targeted protein of mir‘‘5p transcription factor zinc finger protein zfp91 elucidating the regulatory mechanisms of mir1885p in bcmaterials and methodspatients and tissues one hundred paired bc tissue specimens including malignant and normal tissues used in this study were obtained from shengjing hospital of china medical university shenyang liaoning china during the period 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91from january to december with the informed consent of patients the age range of the patients was from to years with a mean age of years all experiments were approved by the ethics committee of shengjing hospital of china medical university no 2016ps18j the samples were snap‘frozen in liquid nitrogen and stored at ‘Ëšc tnm staging system was performed for tumor grading of bc and for evaluating and staging of patients respectively which was carried out according to the 7th edition of the american joint committee on cancer ajcc tnm classification system cell cultivation the bc cell lines mdamb231 atcc crmhtb26 bt549 atcc htb122 and mcf7 atcc crl3435 were cultured in rpmi1640 medium sigma‘aldrich merck kgaa containing heat‘inactivated fetal bovine serum fbs mp biomedicals penicillin‘streptomycin invitrogen thermo fisher scientific inc no the nonmalignant mammary epithelial cell line mcf 10a atcc crl10317 was cultivated in dmemf12 ham's mixture supplemented with equine serum hyclone ge healthcare egf ngml insulin µgml hydrocortisone mgml and cholera toxin ngml all from sigma‘aldrich merck kgaa all cells were incubated at ˚c in a humidified co2 atmosphererna isolation and quantitative qpcr total rnas from tissues or cells were isolated using rnx„¢plus reagent cinnagen and cdna was synthesized using the primescript„¢ rt reagent kit takara according to the manufacturer's instructions qpcr was performed using a sybr premix extaq„¢ kit takara with the following primer sets on the abi qpcr system applied biosystems actin was used to normalize the relative expression of the target genes mirnas were detected through a miscript ii rt kit qiagen in a fluorescence thermal cycler bio‘rad laboratories inc the primers for mir1885p and the reference gene u6 were purchased from novland biopharm the thermocycling condition were ˚c for min followed by cycles of ˚c for sec and ˚c for 1min followed by a hold at ˚c the relative expression ratio of mir‘‘5p was quantified using the 2δδcq method the relative expression of mir1885p was normalized to u6 the primer sequences are listed in table iplasmid preparation the coding region of human zfp91 was amplified from human breast cancer cell mda‘mb‘ cdna library by pcr then we cloned the prepared zfp91 fragment into pcmv‘tag2b stratagene to obtain pcmv‘tag2b‘zfp91 the primer sequences are listed in table icell transfection the mir1885p mimics and mirnc were purchased from thermo fisher scientific inc firstly lipofectamine transfection reagent thermo fisher scientific inc was used to transfect mir1885p mimics mirnc into mdamb231 cells in accordance with the manufacturer's instructionsafter the whole detection the pcmvtag2b vector pc was transfected into mdamb231 cells with mirnc ncpc or mir1885p mimics pcmir1885p pcmv‘tag2b‘zfp91 was transfected into mda‘mb‘ cells with mir‘‘5p mimics mir‘‘5pzfp91 as the cells were grown to mir mimics or vector was transfected into cells according to the manufacturer's instructions then cells were cultivated for up to h finally the total rna and protein were extracted and properly stored for further researchcell proliferation assay cell counting kit8 cck8 kit dojindo was performed to detect cell proliferation based on the reduction of wst‘ to wst‘ formazan briefly the mdamb231 cells were seeded in a 96well plate at a density of 5x103 cellswell on the day of the experiment the cells were transfected with empty vector and mir1885p cck8 reagent was added into the culture medium at the indicated time and incubated for min the absorbance at nm was measured by a microplate readercolony formation assay mdamb231 cells from the different treated groups were seeded in a 60mm dish containing cells followed by a 14day cultivation at ˚c with co2 the supernatant was discarded and cells were washed twice with pbs the colonies were fixed in paraformaldehyde for min and then stained with giemsa staining solution solarbio science technology co ltd beijing china for min colonies were counted and images were captured under an inverted microscope nikon tokyo japan this assay was repeated timescell apoptosis assay mdamb231 cells were stained by annexin valexa fluor488propidium iodide pi staining to identify the apoptotic mdamb231 cells after transfection with mir1885p for h mdamb231 cells were stained with annexin valexa fluor488 for min on ice followed by the addition of pi solution for the secondary staining process all experimental procedures were strictly protected from lights the data were calculated by flowjo software v87 tree star after facs calibur bd biosciences analysiscell migration and invasion assays after the counting mdamb231 cells in the different groups were inoculated equally at a density of 5x105 cellsml in the upper compartments of polycarbonate membrane filters cell migration and invasion assays were performed uncoated for the migration assay and coated with matrigel bd biosciences for the invasion assay after h the migrated and invaded cells in the membrane were fixed with methanol and then stained with crystal violet for min at room temperature cells were observed under a light microscope with magnification x100western blotting protein samples extracted from tissues or cultivated cells were lysed in ripa buffer containing protease and phosphatase inhibitor cocktail and incubated at ˚c followed by the quantified measurement of protein using bca kit fujifilm wako pure chemical corp after protein samples µgeach sample were loaded and separated on sdspage gels for electrophoresis the proteins were then transferred onto a polyvinylidene difluoride pvdf membranes millipore usa the membranes were blocked in wv skim milk for h at room temperature and incubated at ˚c overnight with primary antibodies anti‘zfp91 dilution 0concology reports bidirectional primer sequencetable i primer informationgene name f 'ccctctctcacatcccttgcat3'mir1885p r 'atcctgcaaaccctgcatgtg3' f '‘tgagacctacaaaccccactt‘'zfp91 r 'ccttttgggtaaacgtggacttt3'homoactin f 'ttcctccgcaaggatgacacgc3'r 'ccttttgggtaaacgtggacttt3' f 'cgggtttgttttgcatttct3'u6 snrna f 'agtcccag catgaacagctt3'zfp91 zinc finger protein homolog f forward r reverse abcam ab30970 and antivimentin dilution cell signaling technology inc antiecadherin dilution cell signaling technology inc ncadherin dilution cell signaling technology inc matrix metalloproteinase mmp2 dilution cell signaling technology inc mmp9 dilution cell signaling technology inc nfκb p65 dilution cell signaling technology inc relb dilution cell signaling technology inc and gapdh dilution cell signaling technology inc as internal control on the following day all membranes were incubated with antirabbit igg hrplabeled secondary antibodies dilution cell signaling technology inc finally the signals were detected and analyzed with the application of luminata forte western hrp substrate millipore in the biorad chemidox xrs imaging system biorad laboratoriesluciferase reporter assay to further investigate the specific correlation between mir‘‘5p and zfp91 targetscan wwwtargetscanorgmamm_31 and miranda wwwmicrornaorgmicrornahomedo were performed the zfp91 was selected to be the predicted targeting of mir1885p the fragments of the 'utr of zfp91 containing mir‘‘5p binding sites and its mutants were amplified by pcr and then the pcr products were inserted into pmirglo dualluciferase mirna target expression vector promega corp the reporter and control vector were transfected into 293t cells using lipofectamine thermo fisher scientific inc after cultivation for h the relative luciferase activity was examined by the dualluciferase reporter assay kit thermo fisher scientific inc according to the manufacturer's instructionspreparation of tumor xenograft animal model and treat‘ment with mir‘‘5p mimics thirtysix nude mice female weighing ± g were purchased from huafukang biotech beijing china the experiments were performed in the animal facility at the department of laboratory animal science of china medical university and approved by the animal ethics committee of shengjing hospital approval no nude mice were randomly divided into a control group n12 mir1885p group n12 and nc group n12 a density of 5x106 cells in logarithmic phase were transfected with 1x pbs control group nc or mir1885p then the different groups of cells were resuspended in 1x pbs and injected into the nude mice respectively then tumor size was measured every days using a slide caliper and the tumor volume v was calculated using the formula vlength x width22 after days the mice were euthanasia by cervical dislocation and the tumors were excised imaged weighed and stored properly for further investigationsstatistical analysis graphpad prism graphpad software inc was used to perform statistical analysis the results are represented as mean ± sd of at least independent experiments the comparisons between groups were evaluated by student' t‘test one‘way anova followed by tukey test was used to evaluate the differences for multiple comparisons the statistical significance of correlations between mir‘‘5p and zfp91 expression in bc tissue were analyzed by pearson's correlation coefficient p005 was considered to indicate a statistically significant differenceresultsmir‘‘5p is signif\ufeff\ufefficantly decreased in bc tissue and cell lines firstly we analyzed the expression level of mir1885p in cases of bc tissues and adjacent counterparts by rtqpcr the results showed that the level of mir1885p in bc tissues was significantly lower than that in the normal adjacent counterparts fig 1a p005 we also found that mir1885p was correlated with bc tnm stage fig 1b p005 the expression level of mir‘‘5p in advanced bc tumors was lower than that in early stage tumors suggesting that mir1885p is inversely correlated with the malignancy of bcwe also compared the expression level of mir1885p in the nonmalignant mammary epithelial cell line mcf10a and bc cell lines mdamb231 bt549 and mcf7 our data showed that the levels of mir1885p in the mdamb231 bt549 and mcf7 cells were lower than that in the mcf‘10a cells fig 1c p005 meanwhile the lowest mir1885p expression was detected in mdamb231 therefore mdamb231 cells were selected for further experimentsmir‘‘5p inhibits proliferation induces cell apoptosis and suppresses migration and invasion of bc cells as the expression of mir1885p in both bc cell lines and tumor tissues of bc patients were clearly downregulated we sought to investigate the effects of mir1885p on bc development by using both in vitro bc cell line cultivation and in vivo mouse tumor xenografts as shown in fig 2a transfection of mda‘mb‘ cells with mir‘‘5p mimics significantly elevated the expression level of mir1885p when compared to the control and mir‘nc groups p005 importantly the increased level of mir‘‘5p in mda‘mb‘ cells significantly inhibited the cell proliferation when compared to the control and mir‘nc groups fig 2b and c p005 it was also observed that the apoptotic mdamb231 cell numbers were significantly increased by the upregulation of mir‘‘5p when compared to the control and mirnc groups fig 2d 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure mir1885p is downregulated in bc tissue and bc cell lines a rtqpcr results showed that the level of mir1885p in bc tissues was downregulated compared with counterpart bc tissues the comparisons between groups were evaluated by student's ttest b relative expression level of mir1885p in patients at different clinical stages c relative expression level of mir1885p in bc cell lines mdamb231 bt549 and mcf7 relative to the normal human breast epithelial cell line mcf‘10a one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons in b and c all data are presented as means ± sd n3 bc breast cancerp005 importantly mir‘‘5p mimics significantly inhibited the invasion and migration abilities of the mdamb23 cells under transwell assay detection when compared to the control and mir‘nc groups fig 2e p005 moreover mir‘‘5p mimics significantly enhanced the expression of vimentin and ncadherin and reduced the level of ecadherin when compared to the control and mirnc groups fig 2f p005 the matrix metalloproteinases mmp2 and mmp9 mmp29 possess the ability to hydrolyze components of the basement membrane and stimulate tumor growth metastasis and epithelialmesenchymal transition emt mir‘‘5p mimics were demonstrated to significantly inhibit the expression of mmp2 and mmp9 fig 2f p005 these data provide robust evidence that mir1885p inhibits the tumor proliferation induces apoptosis reduces tumor invasion and migration and inhibits emt of bc which may be through the regulation of mmp29 expressionzfp91 is the downstream target of mir‘‘5p to further investigate the specific correlation between mir1885p and zfp91 targetscan wwwtargetscanorgmamm_31 and miranda wwwmicrornaorgmicrornahomedo were performed the results predicted that mir1885p possesses the binding sites of zfp91 fig 3a hence we sought to discover the regulatory mechanisms of mir1885p on bc development through targeting on zfp91 as hypothesized the upregulation of mir‘‘5p in mda‘mb‘ cells decreased zfp91 mrna and protein levels when compared to the mirnc and control groups fig 3b p005 moreover the luciferase assay confirmed that mir1885p specifically binds to the 'utr of zfp91 fig 3c p005 it was also discovered that the injection of mdamb231 transfected with mir1885p mimics in tumor xenograft mice inhibited zfp91 expression fig 6b p005 these results suggested that zfp91 is the downstream target gene of mir1885p in bcmir‘‘5p regulates bc cell progression through targeting zfp91 to further investigate the biological functions of mir1885p in bc development we established a zfp91‘overexpressing mda‘mb‘ cell line the expression of zfp91 was confirmed by rt‘qpcr fig s1 p005 with this system inhibition of zfp91 by mir‘‘5p mimics was reversed fig 4a p005 then it was found that the co‘transfection of mda‘mb‘ cells mir‘‘5pzfp91 group significantly enhanced the cell proliferation compared to that in pcmir‘‘5p group fig 4b and c p005 significantly suppressed cell apoptosis fig 4d p005 and significantly promoted invasion migration fig 4e p005 and emt fig 4f p005 in contrast with the mono‘transfection of mir1885p mimics in mdamb231 cells moreover the regulatory role of mir1885p on mmp2 and mmp9 was also reversed by overexpression of zfp91mir‘‘5p and zfp91 are correlated in tumor tissues of bc patients furthermore we examined the levels of zfp91 in the tumor tissues and adjacent normal tissues of bc patients the aberrantly high level of zfp91 was observed in the tumor tissues of the bc patients fig 5a p005 spearman's correlation analysis showed a significantly inverse correlation between mir‘‘5p and zfp91 in the bc patient tissues fig 5b p005 taken together these results further confirmed that the proliferation and apoptosis of bc is regulated by mir‘‘5pzfp91mir‘‘5p inhibits the proliferation of mda‘mb‘ cells and reduces the expression of zpf91 in a bc xenograft mouse model moreover to evaluate the regulatory role of mir1885p in a bc xenograft mouse model we injected the mdamb231 cells transfected with mir1885p mimics or mirnc into nude mice the results showed that mir1885p mimics inhibited the tumor volume and weight compared to the mir‘nc group fig 6a p005 protein expression and the mrna level of zpf91 were also suppressed by mir1885p mimics in the tumor tissues of the xenograft mouse model when compared with the mirnc and control groups fig 6b p005mir‘‘5pzfp91 axis regulates nf‘kbp65 and relb expression numerous studies have reported that zinc finger protein zfp91 promotes proliferation and tumorigenesis 0concology reports figure effect of mir1885p on bc cell line mdamb231 mdamb231 cells were transfected with mir1885p mimics and mirnc a the mrna levels of mir1885p in mdamb231 cells were determined by rtqpcr p005 the proliferation of mda‘mb‘ cells was examined by b cck‘ kit post transfection and c colony formation p005 compared to control group p005 compared to mir‘nc group d the apoptotic mda‘mb‘ cells were analyzed using annexin vpi staining and facs p005 e the invasion and migration capability of mda‘mb‘ cells were detected by transwell assay p005 f expression of e‘cadherin n‘cadherin vimentin mmp2 mmp9 and nf‘κb p65rel were detected by western blotting the data are represented as the mean ± sd n3 p005 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer mmp matrix metalloproteinase nc negative controlof different cancer types via regulation of the nfκb p65 pathway therefore to further investigate the regulatory mechanism of the mir‘‘5pzfp91 axis we detected the expression of nfκbp65 and relb in bc cells the results showed that mir‘‘5p mimics significantly reduced the expression of nfκbp65 and relb together fig s2a p005 moreover the co‘transfection of mir‘‘5p mimics and zfp91 also upregulated the expression levels of 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure zfp91 is a target of mir‘‘5p a zfp91 provides binding sites with mir‘‘5p b rt‘qpcr and western blotting were used for analysis of transcription and translation of zfp91 in vitro and in vivo p005 c the binding between mir‘‘5p and zfp91 was confirmed by luciferase assay p005 relative gene expression was normalized by gapdh expression the data are represented as the mean ± sd n3 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons in b the comparisons between two groups were evaluated by t‘test in c zfp91 zinc finger protein nfκbp65 and relb compared to the monotransfection of mir1885p mimics fig s2b p005 in summary these results illustrated that the mir‘‘5pzfp91 axis regulates the progression of bc via the noncanonical nfκb signaling pathwaydiscussionbreast cancer bc is one of the most common types of tumors diagnosed in women worldwide bc is the second leading cause of cancerrelated mortality worldwide in more than women were diagnosed with bc in china and almost percent of all newly diagnosed cancer cases were in women however the molecular mechanisms of bc still await elucidation and effective molecular targets for the diagnosis and treatment of bc are urgently required recently research has reported that mirnas are small noncoding rna molecules which regulate target protein expression to play critical roles as tumorpromotors or suppressors several studies have demonstrated that mir1885p promotes cell proliferation migration and metastasis in gastric cancer and hepatocellular carcinoma moreover iwakawa detected higher expression of mir1885p in stage iii breast cancer and tnbc wang reported that circulating mir1885p was upregulated in bc patients and associated with tnm of bc interestingly mir1885p was downregulated in bc mdamb231 and mcf7 cells moreover using gainof and lossoffunction analyses of mir1885p in breast cancer cells the authors demonstrated that mir1885p inhibited the proliferation and invasion of bc mdamb231 cells via targeting il6st however we demonstrated that the expression of mir1885p was drastically downregulated in bc tissue specimens which was also decreased in bc cell lines mdamb231 bt549 and mcf7 compared to normal breast epithelial cell line mcf10a moreover the downregulation of mir‘‘5p was significantly 0concology reports figure mda‘mb‘ cell proliferation and apoptosis are regulated by mir‘‘5pzfp91 pcmv‘tag2b vector pc was transfected into bc mda‘mb‘ cells with mir‘nc ncpc or mir‘‘5p mimics pcmir‘‘5p pcmv‘tag2b‘zfp91 was transfected into mda‘mb‘ cells with mir‘‘5p mimics mir‘‘5pzfp91 a the mrna levels of mir‘‘5p in the co‘transfected overexpressing zfp91mda‘mb‘ cells were quantified by rtqpcr p005 cell proliferation was measured by b the cck‘ kit and c colony formation assay p005 compared to control group p005 compared to mirnc group d cell apoptosis was detected by annexin vpi staining and facs p005 e the invasion and migration capability of mdamb231 cells were detected by transwell assay p005 f expression of e‘cadherin n‘cadherin vimentin mmp2 mmp9 and nf‘κb p65rel were detected by western blotting relative genes expression was normalized by gapdh expression the data are represented as the mean ± sd n3 p005 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer zfp91 zinc finger protein mmp matrix metalloproteinase nc negative control 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure mrna and protein levels of zpf91 in bc tissues a the mrna and protein levels of zpf91 in bc tissue and corresponding non‘tumor normal tissue were quantified using rt‘qpcr and western blot analysis the comparisons between two groups were evaluated by t‘test n non‘cancer tissue c cancer tissue p005 b correlation between zpf91 mrna and mir‘‘5p was analyzed using pearson's correlation coefficient the data are represented as the mean ± sd n100 bc breast cancer zfp91 zinc finger protein figure the regulatory role of mir1885p in a bc xenograft mouse model bc mdamb231 cells were transfected with mir1885p mimics and mirnc and then injected into nude mice a the tumor volume and weight in the tumor xenograft mouse were compared among the mir1885p mimics group mirnc group and control group p005 compared to control group p005 compared to mir‘nc group b the protein expression and mrna level of zpf91 were detected by c western blotting and rt‘qpcr the data are represented as the mean ± sd n3 p005 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer zfp91 zinc finger protein nc negative controlassociated with advanced tnm stage however to investigate the relationship of mir1885p and bc patient prognosis we found that kaplanmeier analysis of mir1885p was limited due to the small sample size in tcga these results illustrated that the downregulation of mir1885p may be related with bc progression in the clinic suggesting that mir1885p may be a valuable bc diagnostic indicator moreover we confirmed that mir1885p mimics considerably inhibited the proliferation induced the apoptosis and inhibited the invasion of bc cells suggesting that mir1885p plays an inhibitory role in bc cellstranscription factor zinc finger protein zfp91 was firstly identified in the mouse in which was found to be overexpressed in colon liver prostate stomach and breast cancer zfp91 has a molecular mass of kda with amino acids containing five zinc‘finger motives a leucine zipper a coiledcoil structure and nuclear localization sequences zfp91 was confirmed to be a transcription factor located in the cellular nucleus ma reported that zfp91 functions as an oncogene in cancer development by activating hif1α transcription the overexpression of zfp91 0concology reports was also found to result in the promotion of nkκb signaling pathway activation through increasing nkκb inducing kinase nik whose activity and overexpression are related to cancer progression in melanoma pancreatic breast and lung cancer the inhibition of zfp91 was demonstrated to promote apoptosis in bc stomach cancer cells colon cancer and endometrial cancer in addition the overexpression of zfp91 was found to increase the cancer cell growth rate and metastatic capability zfp91 was also reported to interact with cyclin‘dependent kinase inhibitor 2a cdkn2a which is an alternative reading frame arf tumor suppressor inhibiting the induction of p53dependent cell death to illuminate the molecular mechanisms of mir188 we predicted that il6st foxn2 zfp91 may be the targets of mir‘‘5p using targetscan and miranda furthermore peng reported that zfp91 is the target protein of mir‘‘5p in gastric cancer in addition overexpression of mir1885p was confirmed to inhibit the progression of breast cancer thus we chose the reported oncogene zfp91 for further investigation in the present study we confirmed that the 'untranslated region 'utr of zfp91 was bound by mir‘‘5p through dual luciferase assay moreover transfection of mir1885p mimics in mda‘mb‘ cells reduced the zfp91 mrna and protein levels together mirnas usually bind to the 'utrs of target mrnas and do not reduce the level of mrnas however mirnas also were reported to decay the target mrnas and decease mrna level restoration of zfp91 largely reversed the decreased proliferation and induced apoptosis which were both regulated by mir1885p overexpression moreover in the tumor xenograft mouse model we observed that the expression of zfp91 was downregulated by an increased level of mir1885p furthermore the expression of mir‘‘5p and zfp91 were negatively correlated in bc patient tissues therefore our studies confirmed that mir‘‘5p can inhibit the progression of human bc via targeting zfp91zfp91 has been reported to promote proliferation in colon cancer prostate cancer and gastric cancer ma reported that zfp91 activates nf‘kappabp65 to promote proliferation and tumorigenesis of colon cancer paschke identified that zfp91 is a noncanonical nf‘κb signaling pathway regulator with oncogenic properties in prostate cancer in the present study we also confirmed that a decrease in zfp91 could significantly inhibit nf‘κbp65 and relb expression in bc cells therefore mir1885p overexpression reduced zfp91 via the noncanonical nf‘κb signaling pathway to inhibit the progression of bcin conclusion our data showed that mir1885p is downregulated in bc cell lines and tissues and the downregulated expression of mir1885p is associated with the poor prognosis of patients with bc we further investigated that overexpression of mir1885p could inhibit proliferation and induce the apoptosis of mda‘mb‘ cells furthermore zfp91 was predicted and confirmed as a target gene of mirna‘‘5p and the effects of mir1885p on bc cells were dependent on the inhibition of zfp91 additionally a decrease in zfp91 significantly inhibited the nfκbp65 and relb expression in bc cells moreover the expression levels of mir‘‘5p and zfp91 were highly correlated with bc progression therefore we suggest that mir1885p can inhibit breast cancer progression via the zfp91nf‘κbp65 axis and may be a potential diagnostic indicator for bcacknowledgementsnot applicablefundingthis study was supported by the national natural science foundation of china grant nos and and talent projectavailability of data and materialsthe datasets used and analyzed during the current study are available from the corresponding author upon reasonable requestauthors' contributionszy and zl conceived and designed the study zy zc gy performed the experiments zy wrote the paper zy zl zc and gy reviewed the results and data and edited the manuscript all authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolvedethics approval and consent to participatetissues used in this study were obtained from shengjing hospital of china medical university shenyang liaoning china with the informed consent of patients and all experiments were approved by the ethics committee of shengjing hospital of china medical university no 2016ps18jpatient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences davidson ne armstrong sa coussens lm cruzcorrea mr deberardinis rj doroshow jh foti m hwu p k
Colon_Cancer
"tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh flavor and health benefits and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves™ drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneficial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneficial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health benefits and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2‹…egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg finally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals ‹…egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe field of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientific community and the general public indeedegcg has shown both prophylactic and therapeutic efficacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table [“]apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoh“o2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site specificity of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpsppar𝛾 cebp𝛼aco ppar𝛼 cpt1acad pgc1𝛼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis [“]accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent “ μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by flow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form biofilms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibiofilm efficacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results verified the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the efficacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microflorawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ “] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are final products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for five days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these findings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o
Colon_Cancer
" in the current research we have developed silver and iron nanops of isolated proanthocynidinpac from grape seed by green synthesis and evaluated for antimicrobial antioxidant activity and in vitrocytotoxicity against colon cancer cell linesresults one percent solution of isolated proanthocynidin in water was vigorously mixed with silver nitrate and ferric chloride solution and kept for h to yield pacagnp and pacfenp the synthesized nanops werecharacterized by uv ftir xrd and sem analysis and evaluated for antimicrobial potential against selectedmicrobes moreover the synthesized nanops were studied for dpph assay and in vitro cytotoxicity usingcolon cancer cell lines colo320dm and ht29 mtt srb and trypan blue assay uv spectroscopy confirmed thedevelopment of nanops sem analysis showed that the ps were aggregated in the size range of to nm antimicrobial potential was found to be less against staphylococcus aureus pseudomonas aeruginosa andescherichia coli whereas cytotoxicity of pacagnp and pacfenp against colo320dm and ht29 exhibited promisingresults as compared to the pure pac pacagnp and pacfenp exhibited ± and ± inhibition respectively against dpph radical whereas pure pac showed ± inhibition and standardascorbic acid exhibited ± inhibition of dpph radical the silver and iron nanops were successfully developed by green synthesis method usingisolated proanthocynidin which is economical and ecofriendly the use of metal nanops may open up a newopportunity for anticancer therapies to minimize the toxic effects of available anticancer drugs specifically intargeting specific sitekeywords proanthocynidin silver and iron nanops antioxidant activity cytotoxicity colorectal cancercolo320dm ht29 correspondence randivedheerajgmailcom1department of pharmaceutics rajarambapu college of pharmacy kasegaonwalwa sangli maharashtra indiafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 0cshejawal of genetic engineering and biotechnology page of highlights 0f the present research work is focused on greensynthesis of silver and iron nanops of isolatedproanthocynidin from grape seed extract 0f this is the first attempt to assess scientifically theanticancer efficacy of the developed metalnanops on in vitro colon cell lines ht29 andcolo320dm 0f the results of the study revealed that developednanops are having significant cytotoxic activityagainst colon cancer as compared with pureproanthocynidin phytoconstituents 0f this is an ecofriendly economical easy and rapidmethod of development of metal nanops withminimum usage of hazardous chemicals 0f use of isolated phytoconstituents which are devoidof toxic effects of all other anticancer drugs andbetter absorption as well as cellular uptake due tonanosize for targeting specific site will be a newerresearch area a foremost reason of cancer death in men and womenis colorectal cancer crc and it affects nearly millionpeople throughout the world every year it is thethird most frequently diagnosed cancer in both men andwomen about new cases and around deaths have been estimated to have occurred in inthe usa the occurrence of crc in china is lower thanthat of western countries but it has increased in thecurrent years and has become a substantial cancer burden in china mainly in the more developed areas epidemiologicalthe dailyconsumption of fresh fruits and vegetables is connectedwith a reduced risk of cancer the special effects may bemoderately attributed to the presence of several polyphenolic compounds which are known to exhibit antioxidant and free radicalscavenging properties literature hasshown thatit is very essential to deliver anticancer drug at the siteof action and the dosage form must exhibit high cellpenetration or permeability and drug solubility nanops may enter the human body via several routesthe probability of penetration depends on the size andsurface properties of ps and on the anatomicalstructure of the specific sites of the exposure routes nanotechnology can be termed as the exploitation ofmatter through specific chemical andor physical processes to produce materials with specific propertieswhich can be utilized in particular applications [ ] ananop can be a microscopic p or materialthat has at least one dimension less than nm in size[“] different bulk materials exhibit sole optical electrical thermal physical and chemical properties andhence they find a range of applications in the areas ofenvironment medicine chemistry energy agricultureinformationandheavy industry [ ] recently there has been a keeninterest in the green synthesis of nanops consumer goodscommunicationowing to the characteristic catalytic and optical properties of the metal nanops as compared to the bulkmaterial they have fetched greater attention to date inmultidisciplinaryin thepharmaceutical and cosmetic which position onwardgrowth in commercial interest and calling for effectualsynthesis procedures to match the growing demand ofsilver iron and gold nanops especiallyscientificareasayurvedic and herbalformulations available in themarket diverge in quality and therapeutic efficiencyowing to the differences in composition of the plantphytoconstituents [“] advancement in the ayurvedic herbal medicine has been revolutionized from theshowing of phytochemicals and pharmacological activities to elucidating their mechanisms of action and sitesof action also currently there is an increased interest in the herbal drugs and remedies for the treatmentof chronic diseases proanthocyanidins are the naturally available polyphenolic compounds which varied in chemical structure pharmacological action and characteristics andextensively available in fruits seeds vegetables nutsflowers and bark grape seed is an abundant source of proanthocyanidinpacs which is known to be a powerful inhibitor ofaromatase activity it is an enzyme expressed in higherlevels in cancerous than in usual breast tissues pacs belong to a bigger class of abundant plantderivedcompounds flavonoids which offers numerous valuablehealth effects largely because of its antioxidant properties [ ] it has been shown to defend against oxidative stress and tobaccoinduced dna damage andexhibited selective prominent cytotoxicity against somehuman cancers including colon lung breast prostateand gastric carcinomas [“]they are secondary plant metabolites available inmany diverse kinds of fruits vegetables and plantbasedbeverages and also available in cocoa apple grapes teaand red wine pac classes of condensed tannins areoligomers and polymers of catechin and ˆ’epicatechin and other related flavonoids chiefly linked by eitherbtype c4 †’ c6 or c8 or atype linkages c2 †’ o7grape seed extract gse falls into the btype categoryand grape seed extractrich diets have been connectedwith a reduced risk of chronic cardiac diseases andalso a variety of common cancers including colorectalcancer [“]proanthocyanins are strong free radical scavengers andare supposed to be contributors to the health benefits of 0cshejawal of genetic engineering and biotechnology page of fruits and vegetables [ ] proanthocyanidins obtained from grape seeds have been proven to protectagainst uv lightinduced carcinogenesis stop immunesuppression enhance interleukin il12 and decreaseil10 apple pacs have established synergistic effects with lysosomotropic compounds in increasing theanticancer properties targeting human colon cancerderived metastatic cells [ ]an environmentally friendly option is to prepare nanops dependent on three important parametersnamely solvent medium reducing and stabilizing or capping agent for nps therefore the present study isimportant with respect to the development of metalnanops of phytoconstituent specifically for targeting the cancer it has many advantages like being ecofriendly and costeffective and mainly the isolated phytoconstituents have prominent activity as compared withextract of plants and their parts moreover the side effects of the synthetic drugs can be avoided furtherin vivo animal study of the metal nanopbased formulation is the new area of researchmethodschemicalsproanthocynidin pac was obtained as a gift samplefrom influx healthcare mumbai maharashtra all thechemicals used in the study were of analytical grade silver nitrate agno3 and ferric chloride fecl3 were purchased from loba chem kolhapur ciprofloxacin wasobtained from okasa pharmaceutical satara celllinecolo320dm and ht29 were procured from nccspune maharashtramicroanism usedthe test anisms used in this study were staphylococcus aureusatcc pseudomonas aeruginosaatcc and escherichia coli atcc theculture was obtained from yashawantrao chavan college of science saidapur karad ms india”synthesis of metallic nanopssynthesis of proanthocyanidin silver nanopspacagnpspacagnps were synthesized using proanthocyanidinsolution and agno3 solution in accordance with theprocedure mentioned by phull with minor modification equal volumes of proanthocyanidinaqueous solution and silver nitrate solution were incubated at ambient temperature for “ h to obtainpacagnps synthesis of pacagnps was detected bynaked eye with a change of color from dark red to faintbrown which was confirmed by uv spectroscopy thecollected pacagnps were centrifuged for min at rpm and dried in vacuum chamber at °c shownin fig synthesis of proanthocyanidin iron nanopspacfenpsfor the synthesis of profenps proanthocyanidin aqueous solution and ferric chloride solution were used inaccordance with the procedure mentioned by raju with minor modification ferric chloride andproanthocyanidin solution were vigorously combined in a ratio of the mixture was then placed onan orbital shaker for h at an ambient temperature toobtain pacfenps the synthesis of pacfenps was observed with a color change from dark black to darkbrown which was also confirmed by uv spectroscopythe collected pacfenps were centrifuged for min at rpm and dried in vacuum chamber at °cshown in fig characterization of nanopsuvvis absorbance of pac pacagnp and pacfenpthe development of metal nanops of pacagnpand pacfenp by the proanthrocyanidin was recordedperiodically using a uv spectrophotometer shimadzuthe samples were diluted with ml of deionized waterand measured for uvvis spectrum after the formationof nanops that change in color deionized waterwas used as a blank for correction all samples were scanned from to nmsem and histogram analysis of pacagnp and pacfenpscanning electron microscopy sem is a normally usedmethod of evaluation and morphological analysis ofnanops at the nanometer to micrometer scale developed pacagnp and pacfenp were characterizedusing a highresolution scanning electron microscopeschottky field emission scanning microscope su5000the samples were prepared by a simple drop coating ofsuspended gold solution on to an electric clean glass andallowed the solvent to evaporate and the samples weredried atand analyzed in amicroscoperoom temperatureftir spectroscopy analysis of pac pacagnp and pacfenpto recognize the different biomolecules present in theproanthocynidin and the phytocompounds in ag and fenanops after synthesis were analyzed by ftirbruker alpha echo atr spectrum was recorded inthe range of “ cmˆ’xrd analysis of pacagnp and pacfenpthe green synthesized silver and iron nanops ofpac were evaluated by xrd analysis using an xrd xray diffractometer bruker d8 discover operated 0cshejawal of genetic engineering and biotechnology page of fig uv and synthesis npat a voltage of kv and ma with cu kα radiation inθ“2θ configurations the crystalline size was determinedfrom the width of the xrd peaks by assuming that theywere free from nonuniform strainsantimicrobial activity of pac pacagnp and pacfenpin vitro antimicrobial activity was performed using theagar well diffusion technique the sterile agar wasinoculated with the bacterial culture s aureus p aeruginosa and e coli for h at °c antimicrobial activities were tested on nutrient medium against s aureusp aeruginosa and e coli which are representative typesof grampositive and gramnegative anisms wellswere bored by using a sterile borer standard solutionciprofloxacin and test samples pac pacagnp andpacfenp mgml was prepared by dissolving the testsample in dmso were placed into the wells μlplates were then kept for h in the refrigerator to enableprediffusion of the extracts into the agar finally theplates were incubated overnight h at °c theantimicrobial activity was determined by measuring thediameter of zone of inhibition [ ]in vitro cytotoxicity studies of pac pacagnp andpacfenp by using mtt assayhuman ht29 cell and colo320dm were obtainedfrom the national center for cell sciences pune msindia the cell cultures were maintained indmem supplemented with fetal bovine serum thecells were plated at a density of × cells per well in a96well plate and cultured for h at °c the cellswere subsequently exposed the plates were incubatedfor h and cell proliferation was measured by adding μl of mtt thiazolyl blue tetrazolium bromide dye mgml in phosphatebuffered saline per well theplates were incubated for a further h at °c in a humidified chamber containing co2 formazan crystalsformed due to reduction of dye by viable cells in eachwell were dissolved in μl dmso and absorbancewas read at nmfinally the percent cytotoxicity of the compounds wascalculated by using the following formulapercent cytotoxicity ¼ reading of controlreading of treated cellsreading of control 02 since the absorbance was directly associated with thenumber of viable cells the percent viability was determined from the absorbancein vitro cytotoxicity studies of pac pacagnp andpacfenp by using srb assayhuman ht29 cells and colo320dm were maintainedin dmem supplemented with fetal bovine serumthe cells were plated at a density of × cells perwell in a 96well plate and cultured for h at °cthe cells were subsequently exposed to μgml compound after drug incubation μl tca waskept for h at °c then the plate was washed with 0cshejawal of genetic engineering and biotechnology page of tdw triple distilled water and air dried thereafter μl srb dye was added in each well and kept for min at room temperature again the plate was washedthree times with acetic acid and air dried finally μl tris buffer was added and the absorbance wasread at nmthe percent cytotoxicity of the compounds was calculated by using following formulapercent cytotoxicity ¼ reading of controlreading of treated cellsreading of control 02 in vitro cytotoxicity studies of pac pacagnp andpacfenp by using trypan blue assaythe dye exclusion test is used to find out the number ofviable cells present in a cell suspension it is based onthe principle thatlive cells possess undamaged cellmembranes that exclude certain dyes such as trypanblue eosin or propidium whereas dead cells do not exclude in this test a cell suspension is simply mixed withtrypan blue dye and then visually examined to determine whether cells take up or exclude dye in the studypresented here a viable cell will have undamaged a clearcytoplasm whereas a nonviable cell will have a bluecytoplasmfifty microliters of celllines of human ht29 cellsand colo d was taken in microcentrifuge tubethey were incubated for min and then added μlof all samples of nanops in concentration of μg mlˆ’ which were prepared by dissolving inphosphate buffer ph and dmso they were incubated in co2 incubator for min and thereafter trypan blue μl was added in each tube theywere further incubated for min in co2 incubatorand analyzed for total viable cells and nonviable cellsby using nubars slide antioxidant activity of pac pacagnp and pacfenp bydpph 22diphenyl2picryl hydrazyl hydrate assaythe scavenging ability of pac pacagnp and pacfenp on the stable free radical was calculated with themethod expressed by mensor twenty microliters of pac pacagnp and pacfenp solutions wasseparately added in three labeled testtubes subsequently ml of methanolic solution of dpph and ml of methanol were added to each test tube afterwhich all tubes were allowed to react at an ambienttemperature for min the control was prepared as described above without any extract and nanopsmethanol was used to correct the baseline after minof incubation the discoloration of the purple color wasmeasured under a uvvisible spectrophotometer theradicalscavenging activity was determined by the following formula scavenging activity ððþ ¼ aabs controlðþaabs sampleðaabs controlþþ 02 where a control is the absorbance of control samplepacagnp and pacfenp measured at nm and nm respectively and a control is the absorbance ofpac measured at nmstatistical analysisstatistical data ofthe cytotoxicity were assessed ongraphpad prism for windows bit with version results were analyzed by oneway anova withdunnett™s posttest analysis of variance the meanstandard error mean sem of all calculated values wasshown in each group a value of p or was considered statistically significantresultsuvvis spectroscopy of pac pacagnp and pacfenpwhen the aqueous pac was mixed with aqueousagno3 and fecl3 solution the color of the solutionchanged which indicated the formation of silver and ironnanops this change in color was due to the collective coherent oscillation of conduction electrons atthe surface of the nanops that interact with the oscillatingaphenomenon called surface plasmon resonance sprthis change in color indicated the reduction of ag andfe ions which was traced with uvvis spectroscopythe pac showed max at nm and silver nanop pacagnp possesses specific wavelength that canabsorb atmax nm whereas iron nanopspacfenp exhibit max at nm as shown in fig incidentelectriclightfieldoftheftir spectrum of pac pacagnp and pacfenpin the ftir of proanthocynidin fairly sharp peaks at and cmˆ’ were observed which indicate thepresence of the functional group present in the compound however the aromatic at ccvalencecmˆ’ the oh phenolic at ohvalence and chvalence arene alkane och3 the methoxylic at cmˆ’ and co stretching at appeared in the ir spectrum of complex as shown infig 2apacagnpthese characteristic vibrations after reduction of agions were shifted to new peaks at and cmˆ’which indicated the presence of the functional grouppresent in the compound however the aromatic at ccvalencethe oh phenolic atohvalencechvalencecmˆ’and 0cshejawal of genetic engineering and biotechnology page of fig a ftir b sem histogram c xrdarene alkane och3 the methoxylic at cmˆ’and co stretching at appeared in the irspectrum of complex as shown in fig 2ain addition bioreduction showed that the and cmˆ’ bands were suppressed in theagnp proanthocynidin and nps showed similar absorption bands indicating that nps might be stabilized byproanthocynidin on the basis of the orange yellow andbrownish green color of the biomass and the groups suggested by ftir analysisit was hypothesized thatproanthocynidin may be involved in silver nanopsynthesispacfenpthese characteristic vibrations after reduction of fe3ions were shifted to new peaks at and cmˆ’which indicated the presence of the functional grouppresent in the compound however the aromatic at ccvalencethe oh phenolic atohvalencechvalencecmˆ’and arene alkane och3 the methoxylic at cmˆ’and co stretching at appearin the irspectrum of complex as shown in fig 2a in additionbioreduction showed that the and cmˆ’ bands were suppressed in the fenpproanthocynidin and nps showed similar absorptionbandsindicating that nps might be stabilized byproanthocynidin on the basis of the orange yellow andblackish color of the biomass and the groups suggestedby ftir analysis it was hypothesized that proanthocynidin may be involved in iron nanop synthesissem and histogram analysis of pacagnp and pacfenpa scanning electron microscope was used to analyze thestructure of pacagnp and pacfenp nanops thatare developed and represented in fig 2b the nanops formed were aggregated having a size range of to nm this aggregation of the nanops can beminimized or prohibited by increasing the concentrationof the proanthocynidin extract histograms of both thenanops have been represented in fig 2b exhibiting 0cshejawal of genetic engineering and biotechnology page of average p size pacfenp μmand pacagnp μmxrd spectrum of pacagnp and pacfenpthe xrd pattern of the synthesized silver and ironnanops formed using proanthocynidin is shown infig 2c the diffraction peak at 2θ ° and subsequenthigher order reflections can be indexed to the ag andother facets of silver nanops by comparing jcpdsfile no in case of pacfenp the peak shown at2θ ° corresponds to the iron compared with standard xrd for iron jcpds data pdf no the xrd spectrum also revealed a weak peak around2θ ° which can be attributed to the phytochemicalcomponents it thus confirmed that the nanopsformed on the membrane consisted of crystalline xrdindicated possible multicomponent product formation athigher energyantibacterial activity of pac pacagnp and pacfenpthe zones of inhibition revealed that there is very little antimicrobial potential of pac pacagnp andpacfenp against pseudomonas aeruginosa staphylococcus aureus and e coli the results are highlightedin table and the zone of inhibition is depicted infig results of cytotoxicity of pac pacagnp and pacfenpthe results of cytotoxicity assay by mtt and srb assayhave been presented in table with respect to two different colon cancer cell lines namely colo320dm andht29 a variation in the results of different assay wasobserved howeverthe silver nanops exhibitedbetter activity than pure pac in both the methods theresults of mtt assay against ht29 demonstrated thatpacagnp showed maximum ± inhibitionrepresented in fig 4x a in case of colo320dm silver nanops exhibited ± inhibition asrepresented in fig 4x b whereas the srb assay ofpac pacagnp and pacfenp against ht29 revealed ± inhibition by pacagnp as shown in fig4x c in case of colo320dm srb assay a maximumof ± inhibition was exhibited by silver nanops as depicted in fig 4x dthe results of trypan blue assay revealed that silvernanopaticles pacagnp showed ± nonviability of colo320dm cell line when observed on amotic microscope whereas iron nanops pacfenpexhibited ± inhibition of ht29 cell lines asrepresented in table results of dpph 22diphenyl2picryl hydrazyl hydrateassay of pac pacaunp and pacfenpthe dpph activity results demonstrated effective freeradical percent scavenging potential of pac pacaunpand pacfenp as depicted in fig 4y as compared to ascorbic acid standardthe concentration responsecurves of dpph radicalscavenging activity of pacpacaunp and pacfenp are shown in table it wasobserved that pacagnps were more effective than pacextract and pacfenps at a concentration of μgmlˆ’ the scavenging activity of pacagnps was observed to be ± while at similar concentration for the pacfenps it was reported to be ± the outcome of antioxidants on dpph is thoughtto be due to their hydrogen donating abilitydiscussionengineered nanomaterials showed imperative benefitsdue to their unique nanostructure along with their significant properties for the designed applications metallic nps have been synthesized using several different methods such as chemical reduction electrochemical microbiological reduction ultrasonication methodand microwave radiation the present researchwork is focused on green synthesis of silver and ironnanops ofisolated proanthocynidin from grapeseed extract it is an ecofriendly economical easy andrapid method of development of metal nanopswith minimum usage of hazardous chemicals the developed silver and iron nanops were characterized byuv spectroscopy which showed the change in absorbance after development of nanops ftir spectrumprovides the information about the chemical change ofthe functional groups involved in bioreduction [ ]the ftir spectra of developed nanops confirmedthe formation of silver and iron nanops as characteristic vibrations after reduction of ag ions weretable results of antibacterial activity of pac pacagnp and pacfenp against selected microbial strainssrnosample namepac purepacagnppacfenpciprofloxacin stdvalues are expressed in triplicate mean sdzone of inhibition diameter mm against the selected microanismspseudomonas aeruginosa ± staphylococcus aureus ± ± ± ± ± ± e coli ± ± 0cshejawal of genetic engineering and biotechnology page of fig antimicrobial activityshifted to new peaks at and cmˆ’ and characteristic vibrations after reduction of fe3 ions wereshifted to new peaks at and cmˆ’ alsocharacteristic color change can be attributed to the surface plasmon resonance of deposited agnps and itclearly indicated the development of nanops the size of nanops and its morphology wereclearly observed in the sem images the average size fortable results of cytotoxicity of pac pacagnps and pacfenp by mtt and srb assay using colo320dm and ht29 cell linescompoundmtt assay against ht29 control”percent inhibitionmean odpercent viabilityproanthocynidin plainproanthocynidin agnpproanthocynidin fenpmtt assay against colo320dm control”proanthocynidin plainproanthocynidin agnpproanthocynidin fenpsrb assay against ht29 control”proanthocynidin plainproanthocynidin agnpproanthocynidin fenpproanthocynidin plainsrb assay against colo320dm control”proanthocynidin plainproanthocynidin agnpproanthocynidin fenpvalues are expressed in triplicate mean sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0cshejawal of genetic engineering and biotechnology page of fig graph of x cytotoxicity and y antioxidant dpphpacfenp was observed to be μm and μm for pacagnp the xrd resultsrevealed weak peak at 2θ ° for phytoconstituentwhereas diffraction peak at 2θ ° was observed for silver nanops and iron nanops showed thepeak at 2θ °in case of antimicrobial activity the developed silver andiron nanops exhibited little antimicrobial potential ascompared to pure proanthocynidin metal nps increase theantibacterial potential due to the formation of reactive oxygen species ros developed from different types of ironoxide nanops like feo fe2o3 and fe3o4 the freeradical produced in the reaction causes intracellular stressesthat can damage the dna of the cell the in vitro cytotoxicity results against ht29 andcolo320dm showed that pacagnp exhibited ± inhibition mtt assay and ± inhibitionofcolo320dm pacagnp demonstrated ± inhibition mtt assay and ± inhibition srbassayagainst ht29assaysrbincasein dpph assay pacfenp and pacagnp exhibited ± and ± inhibition respectivelythe obtained parameters of the characterization andevaluation of the nanops clearly revealed that ascompared to proanthocynidin the silver and iron nanops possess better antioxidant and anticancer potential against colorectal cancer thusthe use ofisolated phytoconstituents which are devoid of side effects and promoting better absorption and cellular uptake owing to their nanosize will certainly achieveeffective targeting and has to be provided greater attention as a newer research area also several review papershave been published about the synthesis of silver nanops using natural polymers like kcarrageenan andsynthetic polymers like poly vinyl pyrrolidone and polyethylene glycol to improve the strength and stability ofnanops as per the study of moustafa the use ofnatural and synthetic polymer for the development ofsilver nanops opened up new research area in medicinal and biological field along with food industry we have successfully synthesized pacagnps and pacfenps using proanthocynidin isolated from grape seedextract by employing a green synthesis method which istable results of cytotoxicity of pac pacagnps and pacfenp by using trypan blue assaysrnodrugcolo320dmpercent viability ± percent nonviable ± proanthocynidin plainproanthocynidin agnpproanthocynidin fenpvalues are expressed in triplicate mean sd ± ± ± ± ht29percent viability ± ± ± percent nonviable ± ± ± 0cshejawal of genetic engineering and biotechnology page of table antioxidant activity of dpph radicalscavenging activityconcentrations μg mlˆ’standard percent inhibition ± pac percent inhibition ± pacagnps percent inhibition ± pacfenps percent inhibition ± ic50 ± ± ± ± “ ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± values are expressed in triplicate mean sdconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of pharmaceutics rajarambapu college of pharmacy kasegaonwalwa sangli maharashtra india 2department of pharmaceuticalchemistry rajarambapu college of pharmacy kasegaon walwa sanglimaharashtra india 3department of pharmaceutics bharatividyapeeth college of pharmacy kolhapur maharashtra indiareceived may accepted august availability of data and materialsall the data required for the processing of the s are presented inthe œresults section supporting data was included separatelyethics approval and consent to participatenot applicable biao l tan s zhang x gao j liu z fu y synthesis andcharacterization of proanthocyanidinsfunctionalized ag nanopscolloids surf b biointerfaces “ gurushankar k gohulkumar m prasad nr rishnakumar n synthesischaracterization and in vitro anticancer evaluation of hesperetinloadednanops in human oral carcinoma kb cells adv nat sci nanoscinanotechnol “nanops j nanomater1“andsimpleenvironmentallybenignrelativelypacagnps and pacfenps were obtained with ps between and nm in size it is easy and costeffective and does not involve any harmful and poisonous chemicals all the other characterization like uvftir xrd and sem confirmed the development ofnanops we have observed significant antioxidantactivitycapacitiespacagnps and pacfenps exhibited a little antibacterial activity against the selected strains of microbes in anutshell the study showed that the developed nps fromthe isolated pac exhibit beneficial antioxidant and anticancer potential when assessed by three different in vitroassay methods with specifically colon cancer cell linesthus it may open up a new opportunity for anticancertherapies that need further researchradicalscavengingfreeandabbreviationspac proanthocynidin pacagnp proanthocynidin silver nanoppacfenp proanthocynidin iron nanop uv ultraviolet visiblespectroscopy ftir fourier transform infrared spectroscopy xrd xraydiffraction sem scanning electron microscope dmem dulbecco™s modifiedeagle™s medium dpph 22diphenyl2picryl hydrazyl hydrate mtt dimethylthiazol2yl25diphenyltetrazolium bromide srb sulforhodaminebacknowledgementsthe authors are thankful to the secretary of kes society kasegaon andprincipal of rajarambapu college of pharmacy kasegaon sanglimaharashtra for providing facilities we are also thankful to the managingdirector influx healthcare mumbai for providing pure phytoconstituentmarkers dr sandip patil md biocyte institute of research and developmentbird sangli mhauthors™ contributionsthe work was c
Colon_Cancer
"although the clinical development of immune checkpoint inhibitors icis therapy has ushered in a new era of antitumor therapy with sustained responses and significant survival advantages observed in multiple tumors mostpatients do not benefit therefore more and more attention has been paid to the identification and developmentof predictive biomarkers for the response of icis and more indepth and comprehensive understanding has beencontinuously explored in recent years predictive markers of icis efficacy have been gradually explored from theexpression of intermolecular interactions within tumor cells to the expression of various molecules and cells intumor microenvironment and been extended to the exploration of circulating and host systemic markers with thedevelopment of highthroughput sequencing and microarray technology a variety of biomarker strategies havebeen deeply explored and gradually achieved the process from the identification of single marker to thedevelopment of multifactorial synergistic predictive markers comprehensive predictivemodels developed byintegrating different types of data based on different components of tumorhost interactions is the direction offuture research and will have a profound impact in the field of precision immunooncology in this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of icis and discuss their future directions in achievingprecision immunooncologykeywords neoplasm immune checkpoint inhibitor predictive biomarker tumor mutation burden programmeddeath ligand1 immune checkpoint inhibitors icis therapy has usheredin a new era of antitumor therapy with sustained responses and significant survival advantages observed inmultiple tumors antiprogrammed cell death1programmed cell deathligand pd1pdl1 antibody hasbeen approved for secondline or firstline treatment in avariety of malignant neoplasms including melanoma lungcancer renal cell carcinoma rcc head and neck squamous cell carcinoma hnscc and gastroesophageal correspondence cuijwjlueducncancer center the first hospital of jilin university xinmin streetchangchun jilin chinacancer [ ] however despite the breakthrough in clinical treatment with icis most patients do not benefitpembrolizumab or nivolumab has an objective responserate orr of “ in firstline melanoma and insecondline nonsmall cell lung cancer nsclc [“]therefore in recent years more and more attentions havebeen paid to the identification and development of predictive biomarkers for the efficacy of icis and more indepth and comprehensive understanding has also beenobtained in recent yearsincluding new data on biomarkers of tumor genome and neoantigen tumor immune microenvironmentbiopsybiomarkers hostrelated factors and all of which havephenotypeliquid the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cbai biomarker research page of technologyimmunohistochemicalmade many new advances in the corresponding fieldswith the development and continuous improvement ofmultiplexhighthroughput sequencing and microarray technology a variety of biomarker strategies have emerged and graduallyrealized the process from the identification of singlemarker to the development of multifactorial synergisticpredictive markers the development of predictive biomarkers contributes to revealing the therapeutic mechanisms of icis and the interaction mechanisms betweentumor and host immunity achieving decisionmaking ofindividualized antitumorimmunotherapy monitoringefficacy and disease development guiding clinical trial design as well as for further understanding of drug resistance mechanisms and tumor prognosis in this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of icis it should be pointed out here that when reading and collating we try to read and include all therelevant s in the process of selecting s we include the authoritative s published in highlevel s or the latest research results and objectively describe and analyze their roles in this field as well as discuss the reasons that different research results may beinvolvedadvances of multiple predictive biomarkers toicis efficacyi tumor genome and neoantigen biomarkerstumor mutation burdensignificant correlations between high tumor mutationburden tmb and response to icis have been reported inseveral cancer types including urothelial carcinoma small cell lung cancer sclc nsclc [“]melanoma and human papilloma virus hpvnegative hnscc a metaanalysis of cancer typesshowed that the mean response rate was positively correlated with log tmb the national comprehensivecancer network nccn guidelines have adopted tmbas the recommended test for patients with nsclc receiving immunotherapy although the results in some clinicalstudies of rcc hpvpositive hnscc and melanoma receiving antipd1 after recurrence showedthat tmb alone also did not clearly distinguish respondersand predict os it is still exciting that multiple studies inthe american society of clinical oncology ascomeeting have confirmed the predictive value of tmb inimmunization or combination therapy keynote061study [ ] condor study eagle study epoc1704 study etc consolidating its status oftmb as an independent predictor and in april theus food and drug administration fda prioritized theapproval of tmb as a companion diagnostic biomarkerfor pembrolizumabnonetheless the cutoff values of tmb were defineddifferently across studies and assay platforms such asatezolizumab mtmb in urothelial cancer pembrolizumab mtmb in nsclc and atezolizumab‰¥ ‰¥ or ‰¥ mtmb in nsclc [“] andnivolumab plus ipilimumab ‰¥ mtmb in nsclc which needs further study to confirm the optimalcutoff value in different tumors moreover the ngspanels have approved by the fda that can be used to estimate tmb include the mskimpact and foundationone cdx panel the detection results of which arehighly consistent with whole exome sequencing wes[ ] and other solutions are under development astudy detecting tmb cutoff value at mtmb in nsclc patients with the foundationone platformcontaining a gene panel found that compared withtmbl patients overall survival os and dcr was significantly improved in tmbh patients treated withantipd1l1 drug both wes and targeted ngs a422cancergene panel performed in patients withnsclc treated with antipd1l1 demonstrated thattmbh population has a significantly better durableclinical benefitdcb and progressionfree survivalpfs these findings demonstrate the feasibility ofcomprehensive genomic profiling cgp but the designof optimal next generation sequencing ngs panel thatis more accurate comprehensive and costeffective isstill not clear in addition given that btmb was identified as a predictor of pfs but failed to differentiate patients with os benefits researchers consider the need toexplore other more precise factors eg allele frequencyaf a study that developed a new btmb algorithm intwo independent cohorts poplar and oak showedthat modified btmb low af btmb lafbtmb mutation counts with an af was significantly associated with favorable hr 95ci “ p pfs hr 95ci “ p andorr p after immunotherapy but required tobe prospectively validated finally static biomarkersare insufficient to accurately predict response due to thecomplexity of tumorimmune interactions a recent analysis of tumor genomewide dynamic detection in pretreatment and ontreatment melanomasfound thatpretreatment tmb was only associated with os in untreated patients while early 4week ontreatment changein tmb δtmb was strongly associated with antipd1response and os in the entire cohort the detectionof δtmb is helpful for early evaluating the response totherapy of patient but its clinical usability limited by thedifficulty in obtaining tissue samples and high price whileliquid biopsy discussed below might better 0cbai biomarker research page of in addition epigenetic changes are associated withtmb the latest study investigated the association between tmb and dna methylation dnam to explorepotential complimentary biomarkers for nsclc immunotherapies the results showed that high tmbnsclcs had more dnam aberrance and copy numbervariations cnvs showing certain value in predictingefficacy such as hox gene methylation status and tmb thus the correlated exploration of epigenetics hasattracted more attention in recent years and liquidbiopsybased epigenetic studies may become a future research direction exploration in chinese nsclc patientsshowed that nsclcs with high tmb had dnam aberrance and cnvs some insertion and deletion indelmutations can lead to frameshifts and more immunogenic neoantigens in the pancancer analysis of cancer types evaluated in the cancer genome atlastcga rcc had the highest indel mutation load andframeshift indel mutations were found to produce threetimes more candidate neoantigens per mutation thannonsynonymousnssnvs somatic copy number alterations scnas are another feature of the genomic landscape of tumors andpancancer tcga analysis revealed an inverse correlation between scnas atthe singlearm or wholechromosomelevel and immune infiltration in tumortypes tested and this result was subsequently replicated in a larger study of tcga single nucleotide variantsdna damage response pathwaysgenetic variation involved in dna mismatch repairmmr pathway can lead to microsatellite instabilitymsi a specific type of high tmb tumors and increased numbers of cd8 tumor infiltrating lymphocytestils pd1tils and indoleamine 23dioxygenaseido tumor cells have been shown in mmr deficiencydmmr colorectal cancer recently five clinical trials keynote016 including multipletumor types have shown that patients with dmmrmsih can achieve durable responses to pembrolizmabbased on this pembrolizumab is approved by the usfda for the treatment of any advanced solid tumor withdmmrmsih and nivolumab in combination with ipilimumab has also shown promising response in dmmrmsih colorectal cancer in addition dmmr canalso cause mutations in the dna polymerase gene epsilondelta polepold1increasing the mutationload and neoantigen load analysis of polepold1mutations in patients with different cancer typesshowed that patients with these mutations had significantly higher tmb and os therefore it may be an infordependentinidentifying patients who benefitaddition pathways of base excision repairberand prognostic markerfrom icis risk factorhomologous recombination repair hrr mmr in thedna damage response ddr signaling network contribute more significantly to tmb or neoantigens whichhave the highest levels when comutated it hadbeen identified that comutations in the ddr pathwaysof hrr and mmr or hrr and ber defined as comutare associated with increased levels of tmb neoantigenload and immune gene expression signatures comutpatients showed a higher orr and longer pfs or os indicating that comut can be used as predictors of response to icis and provide a potentially convenientmethod for future clinical practice specific mutated gene pathways in tumor cellsit is worth noting that alterations of signaling pathwaysin tumor cells affect the responsiveness to immunotherapy patients with mutations in the interferon ifnγpathway genes ifngr12 jak12 and irf1 are poorlyresponsive to icis treatment and confer resistance a study found that in patients receiving immunotherapytumor cells can downregulate or alter ifnγ signalingpathways such as lossoffunction alleles of genes encoding for jak12 and changes in stat1 to escape the influence of ifnγ resulting in poor efficacy andresistance recent studies suggest that inactivating mutations in a mammalian analog of the chromatin remodeling swisnf complex and unique genes of the pbafcomplex pbrm1 arid2 and brd7 lead to sensitivitiesto icis [ ] loss of function of the pbaf complexincreased chromatin accessibility to transcription regulator elements of ifnγ“inducible genes within tumorcells and subsequently increased production of cxcl9cxcl10 chemokines leading to more efficient recruitment of effector t cells into tumors in human cancers expression of arid2 and pbrm1 are related toexpression of t cell cytotoxicity genes which confirmedin pbrm1deficient murine melanomas with strongly infiltrated by cytotoxic t cells and responsive to immunotherapy [ ]in addition doublestranded rnadsrna editing enzyme adenosine deaminase acting onrna adar1 protein can block the ifnγ signalingpathway and lead to poor icis efficacy and resistanceloss of function of adar1 in tumor cells can reduce atoi editing of interferoninducible rna species and leadto dsrna ligand sensing by pkr and melanomadifferentiationassociated protein mda5 this resultsin growth inhibition and tumor inflammation respectively and profoundly sensitizes tumors to immunotherapy finally demethylation positively regulates thetranscriptional activity of some immunerelated genesincluding pdl1 and ifn signaling pathway genes sensitizingto anticytotoxic tlymphocyteassociatedprotein4 ctla4 therapy it 0cbai biomarker research page of in addition to the ifnγrelated signaling pathway alterations in other tumor genome such as tumor oncogenes and suppressor genes pathways and pathwaysrelated to tumor cell proliferation and infiltration canalso affect immunotherapy efficacy epidermal growthfactor receptor egfr and anaplastic lymphoma kinasealk mutations have been shown to be associated withreduced response rates to icis and low tmb and therefore the fda does not recommend firstline icistreatment in patients with egrf or alk positive tumors[ ] certain types of mutations in mdm2mdm4and arid1a can predict nonresponse to icis in hightmb tumors nsclc with kras and stk11 comutated was associated with reduced response andshorter survival in three independent cohorts of patientstreated with antipd1 therapy and stk11 deficiency was an independent indicator of poor antipd1response in nsclc with kras mutant however at the american association for cancer research aacrmeeting of patients in the keynote042 studynct02220894 update data were tested for stk11 andkeap1 and the results showed that patients could benefit from pembrolizumab regardless of stk11 and keap1status but patients with stk11 mutations did not respond well to chemotherapy but given that only ofall patients had mutation detection the results may beaffected in initial data from studies using targeted ngspanels suggested that duration of icistreatment was associated with certain braf and m terations butnot tmb status notch signaling pathway is associated with the occurrence development and prognosisof tumors especially with the biological function of cancer stem cells recent breakthrough findings have distinguished deleterious notch mutation showing that itcan be used as a potential predictor of favorable ici response in nsclc potentially via greater transcription ofgenes related to dna damage response and immune activation another tumorspecific inheritance thatmay influence icis efficacy is the aberrant expression ofendogenous retroviruses ervs pancancer analysisidentified a positive correlation of transcript expressionof ervs with tcell activity in various tumors andpatient prognosis furthermore with the improvement of precision detection technology the accurateanalysis of negative mutation sites helps to identify thepossibly effective ones for example the analysis of studydata of secondline pd1l1 inhibitor therapy found thatthe mpfs of patients with kras g12c or g12v was significantly better than that of patients with kras mutations at other sites in addition several pancancer biomarkers are recentlyapproved by the fda for example given the effectiveorr of and a disease control rate dcr of in secondline cholangiocarcinoma patients treated withanalysispemigatinib a new targeted therapy the recent fda approval of pemigatinib for the treatment of previouslytreated patients with locally advanced or metastatic cholangiocarcinoma with fibroblast growth factor receptor fgfr2 fusion or rearrangement and the comprehensive genomicassay foundationone cdxdeveloped by foundation medicine as a companiondiagnostic also exciting is the recent fda approval ofthe targeted anticancer drug capmatinib for the treatment of metastatic nsclc with met exon skippingmetex14 mutations including firstline patients andpreviously treated patients also using foundationonecdx as a companion diagnostic to help detect specificmutations present in tumor tissueimmunogenicity ofneoantigen loadneoantigen load the number of mutations actually targeted by t cells may be directly related to the responseto icis [“] a retrospective study showed thatclonal neoantigen burden was associated with the longeros in primary lung adenocarcinomas p traditionallycomputational neoantigen predictionshave focused on major histocompatibility complexmhc binding of peptides based on anchor residueidentities however neoantigen loads identified by thismethod are generally not superior to overall tmb inpredicting icis efficacy or survival in recent practice this neoantigen can be assessed by the difference inpredicted mhci binding affinity between the wildtypepeptide and the corresponding mutant peptide knownas the differential agretopicity index dai reflectingclinically relevanttumor peptide a high dai value indicates that the mutant peptidesignificantly increases binding affinity to mhc compared to the wildtype sequence and can generate moreimmune responses studies on previously published cohorts treated with three icis have shown that dai outperforms tmb and the traditionally defined neoantigenload in predicting survival [ ] in additionlowneoantigen intratumour heterogeneity might also be important for icis response analysis of the lung adenocarcinoma tcga database found that combining highmutational load and low intratumoral neoantigen heterogeneity was significantly associated with osand longer lasting clinical benefit than either variablealone anotherreported method for assessingneoantigen foreignness is based on sequence homologyof experimentally validated immunogenic microbial epitopes in the immune epitope database iedb butit does not account for all possible human leukocyteantigen hla contexts in addition the detection forneoantigen can be reflected from different levels such aspeptides or genomes a study developed the neopepseealgorithm using a machine learning approach incorporating 0cbai biomarker research page of integration of nine immunogenicity features and gene mutation expression levels and its application to melanoma and leukemia patients could improve the sensitivityand specificity of neoantigen prediction recently it has alsobeen shown that promoter hypermethylation of neoantigengenes may be an important mechanism for immune editingand tumor immune evasion indicating that combineddetection of tumor genome and epigenetics may providemore information for immunotherapy efficacyii tumor immune microenvironment phenotypebiomarkerscells is also considered separately as one of the biomarkersto distinguish the benefit population called immune positive score ips herbst showed that response toatezolizumab treatment was significantly associated withhigh levels of pdl1 expression on the surface of tils before treatment but not with pdl1 expression on tumorcells p finally other inhibitory immune pathways may affect the response to icis therapy including tcelllymphocyte activationgene3 lag3 and vdomain ig suppressor of tcell activation vista which can be used as potential biomarkers for icis responseimmunoglobulin3 tim3pdl1 expressiongiven that multiple studies in a variety of tumors havedemonstrated a positive correlation between pdl1 expression and response to icis or os even in firstlinecombination therapy [“] pembrolizumab is currently approved by the fda for use in patients with pdl1 pdl1 ‰¥ of tumor cells in firstline treatmentand ‰¥ in secondline treatment nsclc and pdl1immunohistochemistry ihc as a companion diagnosticfor antipd1 therapy in nsclc patients [ ] however some studies have not detected a significant correlation between pdl1 expression and response to icis[ ] and pdl1 negative patients can still benefitclinically with treatment with ici or combination treatment with icis with orrs ranging from to therefore pdl1 cannot yet be a comprehensive and independent biomarker in clinical practice in assessing efficacy with following challenges still existing firstlypdl1 assay and antibody are not standardized secondly pdl1 expression is temporally and spatiallyheterogeneous a study of metastatic nsclctreated with icis showed that pdl1 varies substantiallyacross different anatomic sites and during clinicalcourse being highest in adrenal liver and lymph nodemetastases and lower in bone and brain metastases andthe predictive value of pdl1 at different biopsy sites forthe benefit of icis in nsclc may vary higher pdl1 inlung or distant metastasis specimens was significantly associated with higher response rate pfs and os whilepdl1 in lymph node metastasis biopsy was not associated with either response or survival thirdly positive score and cutoff value of pdl1 expression is notstandardized at present pdl1 positive scoremainly focuses on the pdl1 expression level of tumorcells that is tumor proportion score tps but pdl1is also expressed on immune cells such as lymphocytesand macrophages and stromal cells thus the investigators introduce the concept of combined positive scorecps which is the proportion score of the sum of pdl1 expressed by tumor cells and tumorassociated immune cells in addition pdl1 expression on immuneresponseto icisimmunetreatmentbiomarkers of tumorinfiltrating immune cellsoverall immune status of tumor microenvironmentthe pattern of tumor immune infiltration can be broadlyclassified into immuneinflamed immuneexcluded andimmunedesert immuneinflamed is characterizedby the presence of cd8 and cd4 t cells in the tumorparenchyma accompanied by the expression of immunecheckpoint molecules indicating a potential antitumor immuneexcluded is characterized by the presence ofdifferent immune cell types in the aggressive margin orstroma of tumor but cannot infiltration into tumor parenchyma [ ] analysis of pretreatment samples forantipd1pdl1 revealed a relatively high abundance ofcd8t cells at the invasive margin in responders andserial sampling during treatment showed an increasedinfiltration of cd8t cells into tumor parenchyma while immunedesert phenotype is characterized by theabsence of abundant t cells in the parenchyma orstroma of tumors and poor response to icitreatment recentlyimmunoscore has been proposed as avalid marker for characterizing the immune status oftumor microenvironment tme classifying tumors aswell as predicting treatment response and prognosis which involves the density of two lymphocyte populations cd8 and memory [cd45ro] t cells in thecenter and invading margin of tumor mlecnik evaluated immunoscore in specimens of stagei“iv colorectal tumor and confirmed that it was significantly associated with pfs dfs and os and multivariate analysis also showed the superiority of immunoscorein predicting disease recurrence and survival the valueof immunoscore to predicting icis efficacy is being validated internationally in clinical trials of melanoma andnsclc a wider assessment of active immune responses withintme by immune gene expression profiling might effectively predict clinical benefit to icis strategies analysisof total rna and genes that were substantially differentbetween the patient groups in pretreatment tumor biopsies revealed atleast a 25fold increase in the 0cbai biomarker research page of expression of immunerelated genes in clinically active patientsincluding cytotoxic t cell markers egcd8a perforin granzyme b th1 cytokines or chemokines mhcii and other immunerelated genes egnkg7 ido1 ascierto screened morethan immunerelated genes in patients with recurrent breast cancer “ years after treatment and thosewithout recurrence more than years later and foundthat five genes igk gbp1 stat1 igll5 and oclnwere highly overexpressed in patients with recurrencefree survival in addition ifnγinduced immune genesignatures may be effective biomarkers for predicting theclinical benefit of treatment with icis the study developed ifnγ scores combining multiple immune variablesbased on gene signatures which were then extendedto gene signatures in a validation set of melanomapatients including genes encoding ifnγ granzymes ab perforin ido1 and other immunerelated genesboth gene scores showed significant associations withbest overall response rate and pfs optimized cutoffvalues for ifnγ scores based on receiver operatingcharacteristic curve roc curve can achieve a positivepredictive value of for responders and a negativepredictive value of for nonresponders immune cells with specific phenotypes in tmethe phenotype of tils also influences the efficacy oficis the study used singlecell mrna sequencingscrnaseq data analysis to identify two major cd8tcell phenotypes within melanoma memorylike andexhausted the proportion of which is strongly correlated with response to icis the research furtherfound that the transcription factor tcf7 is selectivelyexpressed in memorylike t cells so the ratio ofcd8tcf7 to cd8tcf7tils is strongly correlatedwith improved response and survival in melanoma patients treated with antipd1 balatoni found that of immune cells in tme were positivelyassociated with os after treatment including cd4 andcd8 t cells foxp3 t cells cd20 b cells cd134and cd137 cells and nkp46 cells and different immune cells at different sites were differently associatedwith clinical outcomes researchers found that only asmall proportion of cd8 tilsin tumors couldrecognize tumor mutationassociated antigens while another population bystander cells was insensitive anddifferential cd39 expression was the key molecule thatdistinguished the two populations analysis of peripheral blood from a patient with colorectal cancer whoresponded rapidly to pembrolizumab treatment showedhigh expression of cd39 on cd8 tils indicating thatcd39cd8til may be a promising predictive biomarker the fact of very low level of cd39 expression on cd8tils in of egfrmutant nsclc isconsistent with their low response rate to antipd1immunotherapyin addition a study showed that fc domain glycan ofthe drug and fcγ receptor fcγr expressed by the hostbone marrow cells could determine the ability of pd1tumorassociated macrophages tams to capture antipd1 drugs from the surface of t cells which leads topd1 inhibitor resistance and the association oftams and poor antipd1 response was reported inmelanoma cohorts antipd1 response was associated with an increase in cd8t cells and natural killercells nk cells and a decrease in macrophages andhigh intratumoral myeloid markers were associated witha nearly 6fold decrease in mpfs after antipdl1 therapy in rcc emphasizing the inhibitory role of myeloidcells in response to icis in conclusion immunecells in tme show a great promise in the developmentof predictive biomarkers for icisimmunerepertoirediversity of immune repertoires in tmeeffective t cell responses involve the activation and expansion of specific antigenreactive t cell clones so diversity ofin intratumoral orperipheral may correlate with icis responses and can bequantified as richness and clonality however theresults seem to be complex with some studies finding apositive correlation between til clonality and the response to icis before or after treatment whileothers showing that only an increase in til clonalityduring treatment is associated with the response to antipd1 [ ] others show that intratumoral t cellclonality is not associated with survival while peripheralt cell clonality is inversely associated with pfs and os tumeh further investigated whetherbaseline tils have a narrow t cell receptor tcr repertoire focusing on tumorspecific immune responsesand whether this narrow tcr repertoire correlates withpembrolizumab responses they found that respondingpatient had more restricted usage of the tcr beta chainie a more clonal less diverse population than patientswith progressive disease and showed a 10times increasein these clones after treatmentimplying a tumorspecific response to treatment in these patients notablybaseline tcr clonality was not highly correlated withtil density suggesting that some patients with restricted tcr clonality specific for tumor antigens maystill benefit from antipd1 therapy even though tildensity is low recently researchers have proposed theimmune repertoire irindex the average frequency ofshared tcr clones in t clones in tils and peripheralpd1cd8 t cells they found that neoantigenstimulated tcr agreed with irindex and patients withhigh irindex had better immune activation and highergene expression profiles geps score subsequently they 0cbai biomarker research page of confirmed the predictive value of irindex to icis efficacy dcrpfs but considering that it is difficult tosort out pd1cd8 t cells in tumor tissue based ontwo separate patient cohorts a research confirmed thattcr repertoire diversity and clonality of peripheral pd1cd8t cells may serve as noninvasive predictors ofclinical outcomes after icis in patients with nsclc the viewpoints of t cell diversity and tcr clonality as markers of icis efficacy need to be further validated in a large patient populationiiiliquid biopsy biomarkersperipheral blood cell biomarkersperipheral blood is a noninvasive source to explore potential biomarkers for icis and although associationswith clinical benefit and survival have been observed itseffectiveness has not been validated in prospective studies analysis of melanoma treated with ipilimumabshowed that improved os and pfs were associated withbaseline values of peripheral blood components including low absolute neutrophil countlow neutrophiltolymphocyte ratio nlr low absolute monocyte countlow frequency of myelogenous suppressor cells high frequency of foxp3 treg cells high lymphocyte frequencyhigh eosinophil count and clinical benefit also associated with the dynamic changes of blood markers duringincluding decreased foxp3treg concentratreatmenttions and increased lymphocyte and eosinophil counts reports in patients with melanoma treated withpembrolizumab and in patients with nsclc treatedwith nivolumab have shown that nlr is associated withworse tumor response [ ] multivariate analysis inmelanoma patients treated with antipd1 antibodiesshowed that nlr was the only factor associated withworse orr and shorter pfs indicating that nlr is astrong predictor of worse outcome in patients treatedwith ici low baseline lactate dehydrogenase ldhlevels high relativeabsolute eosinophil counts and relative lymphocyte counts were associated with prolongedos in antipd1 and ctla4 treated melanoma given that previous studies have proposed the importance of baseline derived nlr dnlr and ldhlevels as prognostic markers a recent study proposed acomposite prognostic index that comprehensively takesthe two factors into account lung immune prognosticindex lipi which characterized risk groups goodintermediate and poor the analysis of patients with advanced nsclc in randomized trialss
Colon_Cancer
mgat5 knockout ko in hek293 cells induces metabolic changes resulting in increased intracellular udpglcnac increasedglycolysis enhanced spare respiratory capacity and higher citrate flux from the mitochondria mgat5 ko cells express constitutively high mica mainly regulated onthe transcriptional level through opening of the chromatin at the mica promoter mica expression in mgat5 ko cells is dependent on citrate turnover and histoneacetylation blocking citrate flux inhibits mica expression in numerous cancer cell lines and we propose that this is a central metabolic regulation of mica andimmune surveillanceintroductionnatural killer nk and cd8 t cells monitor autologouscells for markers of tumorigenesis and stress these immunecells express the nkg2d receptor that recognizes nkg2dligands nkg2dls upregulated on the surface of transformedcells nkg2dl expression is in many ways a doubleedged sword upregulation of nkg2dls on cancer cellsenhance nk cell ltration and promote cancer cytotoxicity conversely numerous cancer cells maintain chronicnkg2dl expression and evade immune elimination bydownmodulating and impairing nkg2d receptor signaling“cancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdaci™sheatshock or shortchain fatty acids scfas that upregulatenkg2dls to date studies have primarily focused ondelineating transient nkg2dl induction whereas not much isknown about regulation of their constitutive expressionmetabolic reprogramming is a central hallmark of cancercancer cells use aerobic glycolysis that was initially believedto be a result of dysfunctional mitochondria howeverlater advances have shown that cancer cells often use aerobicglycolysis alongside mitochondrial oxidative phosphorylationoxphos mitochondria are not merely the powerhouseof the cell but also provide metabolites for anabolic pathwaysnecessary for cell growth citrate can be exported from thetricarboxylic acid tca cycle for biosynthetic purposes inthe cytosol citrate is cleaved by atp citrate lyase acly togenerate acetylcoa and oxaloacetate oaa citrateis an inhibitor of glycolysis thus to maintain high aerobicglycolysis cancer cells require low cytoplasmic citrate moreover conversion of citrate by acly is a critical regulatorof gene transcription by producing acetylcoa for histoneacetylation several of these cancerassociated metabolicfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionproperties are shared with other highly proliferating cells suchas activated t cellsexpression of nkg2dls is associated with hyperproliferation and thus with highly active metabolism two studies havelinked nkg2dl expression to active glycolysis whereasone study reports that inhibition of glycolysis increased basalnkg2dl expression in breast cancer cell lines thesestudies emphasize a link to proliferative cell metabolism andsuggest that the role of glycolysis in nkg2dl regulation iscontextspecificnkg2dls fall into two groups the ul16 binding protein ulbp16 and the mhc class i chainrelated proteins aand b mica and micb surface expression of each nkg2dlis regulated individually and at all levels of protein biogenesis we have previously shown that surface expression ofspecific mica alleles depends on nglycosylation nacetylglucosaminyltransferase v mgat5 is an oncoproteincatalyzing the formation of 16branched nglycans thatpromote surface retention of glycoproteins but it is notknown if mgat5 regulates surface expression of mica growthfactor receptors are examples of mgat5 substrates and mgat5overexpression is associated with growth adhesion invasionand metastasis of cancer “ inhibition of mgat5 reducestumor growth enhances the antitumor responses by cd4 tcells and macrophages and promotes th1 diï¬erentiation in this study we examine the metabolic regulation of thenkg2dl mica we discover that mica was increased aftermgat5 knockout ko in a metabolically dependent way anduse this as a model to investigate the regulatory mechanismsof constitutive mica expression we find that glycolysis andmitochondrial export of citrate promotes constitutive micatranscription in mgat5 ko cells a regulation that was alsoshown in several micaexpressing cancer cells in particularincreased mica transcription was associated with alteredchromatin accessibility of the mica promoter our findingssuggest that citrate drives a metabolic stress that modulateschromatin accessibility to facilitate basal mica transcription andthereby regulate immune surveillancematerials and methodsanimalsfemale nmri mice to 10weeks old taconic lille skensveddenmark were used and all studies were performed inaccordance with the danish act on animal experimentationwhich implements directive 201063eu on the protection ofanimals in scientific research the studies were approved by theanimal experimentation inspectorate ministry of environmentand food denmark license no healthmonitoring was carried out in accordance with federation forlaboratory animal science associations guidelinesreagents pharmacological inhibitorsand dna constructspharmacologicalfrom sigmaaldrich werecompoundsnacetyldglucosamine glcnac a3286 pugnac a7229carbonylcyanide2dg d61345aminoimidazole4carboxamide2deoxydglucosetrifluoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulfide bptes sml0601 potassium hydroxycitrate tribasicmonohydrate hc sodium dihydrogencitrate sodium acetate s5636 oxaloacetic acid oaa o41266mercaptopurine monohydrate 6mp azaserinea4142ribonucleotideaicar a9978 nacetylcysteine nac a9165 sodiumpropionate p1880 sodium butyrate b5887 dmso d2438pbs d8537 etomoxir sodium salt was purchased fromcayman chemicals ann arbor mi united states bristol bms303141 wasunited kingdom the gfpmycmicaˆ— and micaˆ— vectors containingthe coding sequences of micaˆ— or micaˆ— alleledownstream of a generic leader a gfp cassette and a myctag were provided by dr m wills university of cambridgecambridge united kingdom pgl3basic pgl3bluciferase vector was purchased from promega promegamadison wi united states e1751 micafirefly luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c o™callaghan university of oxfordoxford united kingdom from tocris biosciencepurification of peripheral bloodlymphocyteshuman peripheral blood mononuclear cells pbmcs wereisolated by histopaque1077 sigmaaldrich st louis mounited states separation from buï¬y coats obtainedfrom healthy blood donors the capital region blood bankcopenhagen university hospital copenhagen denmark toobtain peripheral blood lymphocytes pbls pbmcs weredepleted from monocytes by incubation with dynabeadsinvitrogen carlsbad ca united states as previouslydescribed pbls were activated in rpmi1640 withoutglucose gibco gaithersburg md united states supplemented with dialyzed fetal bovine serumfbs f9665 mm penicillinstreptomycin p4333 mmlglutamine g7513 mm sodium pyruvate s8636 and either mm dglucose g8769 or mm dgalactose g6404all purchased from sigmaaldrich pbls were activated withcd3cd28 beads invitrogen 11132d and 20uml hil2peprotech rocky hill nj united states for dayson day pbls were treated with ngml fr901228 nationalcancer institute bethesda md united states for hline pc3 and the keratinocytederived cellcell line cultivation and proliferationhuman embryonic kidneyderived hek293 cells the prostatecancer celllinehacat were purchased from american type culture collectionatcc manassas va united states nkg2d reporter cellct312 and the 2b4 parental cellline were kindly providedby chiwen chang trowsdale lab cambridge universitylines mdamb231 and mcf7 werethe breast cancer cellprovided by dr jos moreira departmentfor veterinaryfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressiondisease university of copenhagen denmark and henrikleï¬ers the state hospital copenhagen denmark respectivelythe cervical cancer cellline hela was provided by jesperjurlander the state hospital copenhagen denmark themelanoma cells skmel28 fm55m1 fm78 and fm86 andthe human colon adenocarcinoma cell lines ht29 and sw480were provided by dr per thor straten herlev universityhospital denmark hek293 mdamb231 and mcf7 cellswere cultured in dmem with glutamax gibco hela hacat pc3 fm55m1 fm78 fm86 skmel28 andsw480 were cultured in rpmi1640 sigmaaldrich r5886and ht29 were cultured in mccoy™s 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoy™s 5a for longterm cell culture inglucosegalactose cells were cultured in dmem medium withoutglucose gibco supplemented with dialyzedfbs mm penicillinstreptomycin mm sodium pyruvate and mm glucosegalactose all cells were kept at culture conditions—¦c and co2 and were passaged every “ daysfor proliferation assay wt and mgat5 ko cells wereseeded in — or — cellswell for each experimentcells were counted in triplicate wells after and h usingthe biorad tc20 automated cell counter biorad herculesca united statesgene editingmgat5 ko cells were generated by zinc finger nucleasetargeting in hek293 cells and subsequent cloning and selectionwas performed as described previously hek293cells were transfected with mrna sigmaaldrich or µgof endotoxin free plasmid dna using nucleofection on anamaxa nucleofector lonza copenhagen denmark mgat5ko clones were selected by loss of reactivity with lphaand clones were confirmed to have mgat5 mutations usingpcr and sequencinglentiviralmediated gene transfer was performed with anmgat5 encoding vector constructed by inserting the mgat5sequence generated as a bluntend pcr product from a vectorfrom hw university of copenhagen copenhagen denmarkinto an entry vector system using the pentr directionaltopo cloning kit invitrogen k243520k350020 followingmanufacturer™s protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were confirmed by sequencing at eurofins mwgoperons luxembourg colonies were amplified and plasmidswere purified with nucleobond xtra midi kit machereynagelduren germany mgat5 sequences were insertedinto plx302 lentiviral destination vector with lr clonase iienzyme mix invitrogen after proteinase k treatmentconstructs were transformed into dh5α using heatshock andsoc medium selected clones were amplified and dna waspurified using nucleobond xtra midi kit destination vectorswere checked for insertion using bsrgi digestion at —¦cmgat5coding lentiviral ps were packaged in hek293tcells transfected with a mix of µg pspax2 vector packagingvector µg pcmvvsvg envelope vector µg plx302vector carrying mgat5 and µl cacl2 to a final volume of µl the dna mixture was complexed with µl 2x hbsunder constant air flow and the transfection mix was addeddropwise to — hek293t cells in antibioticfree mediumcell culture medium was harvested days after transfection andviral p preparations were prepared by centrifugation at — g for min lentiviral ps were added to cells andincubated for h cells were cultivated in puromycin µgmlselection medium for weeks functional mgat5 expressionwas validated by lpha bindingtransient transfectiontransient transfections were performed as described previouslyusing amaxa nucleofector device lonza dna wasintroduced to — cells in µl nucleofector solution vlonza vca1003 and pulsed using the nucleofector programq001 for gfpmyctagged micaˆ— and micaˆ—constructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbriefly dnarna were prepared in µl cacl2 25mand adjusted to a final volume of µl dna mixture wascomplexed with µl 2x hbs hepes nacl na2hpo4and added dropwise to — cells scrambled sirnacontrol siidh1 and siidh2 ontarget plus smart poolswere purchased from ge healthcare dharmacon lafayetteco united statesfunctional assaysfor nkg2d downmodulation pbls were isolated as describedabove followed by depletion of cd4 cells using cd4 antibodyebioscience san diego ca united states anddynabeads mouse panigg invitrogen cd4depletedpbls were cultured in rpmi1640 sigmaaldrich r5886supplemented with human serum sigmaaldrich h3667 mm penicillinstreptomycin mm lglutamine and ngmlhil15 peprotech for days to enrich for nkcd8t cells nkg2d downmodulation assay was performed aspreviously described nkg2d ligands on eï¬ector cellshek293 wt or mgat5 ko cells were incubated with blockingnkg2dfc rd systems minneapolis mn united states1299nk or control igg1fc rd systems 110hg µgmlfor min at —¦c eï¬ector cells and target cells nkcd8t cells were mixed at indicated eï¬ectortarget ratios and spundown min — g to allow conjugate formation after h cocultivation nkcd8 t cells were analyzed for nkg2d surfaceexpression by flow cytometry using accuri c6 flow cytometerbd bioscience franklin lakes nj united statesfor the reporter cell assay the nkg2dreporter cell line2b4ct312 and the parental control 2b4 cell line target cells were mixed with eï¬ector cells wt or mgat5 ko cellsthat were either blocked with nkg2dfc or control igg1fc asdescribed above eï¬ector and target cells were cocultivated atdiï¬erent et ratios for “ h gfp expression of target cellswas assessed with accuri c6 flow cytometer for in vivo assaytarget cells were labeled with vybrant did celllabeling solutionfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioninvitrogen v22887 according to manufacturer™s protocol andinjected intraperitoneally together with wt or mgat5 kocells in a ratio — of each “ mice were usedper group target cells were harvested after approximately hwith peritoneal lavage and nkg2d activation of didpositivereporter cells were assessed as gfp expression with accuric6 flow cytometerwas assessed by accurate mass and retention time amrt plusfragment identification at two collision energies and evdetailed acquisition methodology has been described previously udpglcnacudpgalnac detected peak screened byexpected calculated mass could be of either compound as thesetwo sugars could not be separated chromatographically hencehas been reported as a putative metabolite pending confirmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturer™s protocolrandox crumlin united kingdom lc2389 reaction andanalysis was performed on an advia chemistry systemsiemens munich germanydntp levels were determined in — cells harvested withtrypsinization and pelleted by centrifugation for — g for min followed by resuspension of cell pellets in methanolfrozen in liquid nitrogen and boiled at —¦c for min sampleswere evaporated until dryness in a speedvac and whole cell levelsof dttp datp dctp and dgtp were determined using the dnapolymerase assay previously described lchrms metabolite profilingto determine intracellular metabolite levels cell pellets from — cells were resuspended in µl of cold methanolafter min sonication samples were prepared by svortex followed by min equilibration at room temperatureafter centrifugation at — g for min at —¦c µl supernatants were collected transferred to ultrafreemccentrifugal filter devices merck millipore ltd cork irelandand centrifuged at — g for min at —¦c from this µlwas transferred to lc vials and µl of each sample was pooledto a mixed qc samplelchrms was performed on a nity ii ultrahigh performance liquid chromatography uhplc systemcoupled to a ifunnel quadrupoletime of flight qtofmass spectrometer equipped with a dual ajs electrosprayionization source agilent technologies santa clara caunited states polar metabolites were separated on a sequantzichilic merck darmstadt germany column mm — mm µm p size coupled to a guardcolumn mm — mm µm p size and an inlinefilter mobile phases consisted of formic acid in water withsolvent a and formic acid in acetonitrile with solvent bthe elution gradient used was as follows isocratic step at bfor min b to b in min and maintained at bfor min then decreasing to b at min and maintainedfor min then returned to initial conditions over min and thecolumn was equilibrated at initial conditions for min the flowrate was mlmin injection volume was µl and the columnoven was maintained at —¦c the acquisition was obtainedwith a mass range of “ mz for where full scan highresolution data is acquired at three alternating collision energies ev ev and ev positive and negative raw lchrmsfiles were independently processed with an inhouse developedpcdl library for polar metabolites using profinder version b06agilent technologies identification of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular flux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density — cellswell ˆ¼ hbefore the experiment one hour prior to assay run cells wererinsed and switched to xf media agilent technologies with mm sodium pyruvate and mm glucose or galactose andincubated at —¦c co2free incubator for the mitochondrialstress tests ocr was measured under basal conditions andduring sequential injection of µm oligomycin sigmaaldrich µm fccp sigmaaldrich c2920 and µmrotenone rot sigmaaldrich r8875 µm antimycina aa sigmaaldrich a8674 reported basal respiration iscalculated from the third measuring point with ocr after rotand aa subtracted atpcoupled respiration display ocr afteroligomycin subtracted from the third measuring point andmaximal respiration is ocr after fccp with ocr after rotand aa subtractedfor measuring the eï¬ect of hc ocr was assessed h after aninjection of mm hc13c6glucose tracing experiment — cells were incubated for h in dmem medium withoutglucose supplemented with fbs mm sodium pyruvateand mm uniformly labeled [u13c]glucose cambridgeisotope laboratories tewksbury ma united states clm incubation medium samples were collected and cleared bycentrifugation — g for min cells were washed and detachedsterically intracellular metabolites were extracted in ethanoland centrifuged at — g for min —¦c to separatethe soluble extract supernatant from the insoluble componentspellet cell extracts and medium samples were lyophilizedand reconstituted in water for subsequent biochemical analysesextract samples were adjusted to ph with hcl and evaporatedto dryness under nitrogen flow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modified procedure from standards containing unlabeled metabolites of interest andcell extracts were separated and analyzed in a gas chromatographagilent technologies 7820a chromatograph jw gc columnhp5ms parts no 19091s433 coupled to a mass spectrometeragilent technologies 5977e the isotopic enrichment of themetabolites of interest was corrected for natural abundance of 13cusing the unlabeled standards and calculated according to data are presented as labeling of m x where m is the massof the unlabeled molecule and x is the number of labeled catomsin a given metabolite frontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionwestern blottingproteins were extracted using ripa buï¬er thermo scientificwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientific for minon ice lysates were sonicated times for s and clearedby centrifugation at rpm for min at —¦c proteinextracts were denatured at —¦c for min in nupage samplebuï¬er and dtt sigmaaldrich proteins were resolvedusing “ sdspage gels invitrogen and transferred tonitrocellulose membranes invitrogen ib301001 using the iblotdevice invitrogen for total protein stain membranes werewashed in ddh2o and stained with revert protein stainsolution licor biosciences lincoln ne united states according to manufacturer™s protocol membranes wereblocked in tbst blocking buï¬er licor biosciences “ probed with primary antibodies in tbs w tween and bsa overnight on a shaker at —¦c and washed intbs tween secondary antibody was from licorlicor biosciences “ and signals were visualizedby the odyssey fc imaging system licor biosciencesoglcnacylation was detected with rl2 oglcnacylationantibody abcam cambridge united kingdom ab2739 atpcitrate lyase acly was detected with rabbit acly antibodycell signaling and acly phosphorylation with rabbitphosphoacly ser455 antibody cell signaling flow cytometryadherent cells were detached in pbs w mm edta invitrogen or by pipetting cell surface staining was done aspreviously described and cells were analyzed on accuric6 flow cytometer bd bioscience antibodies used for thisstudy were mica rd systems fab1300a ulbp256 rdsystems fab1298p nkg2d rd systems fab139a ulbp1rd systems fab1380p ulpb3 rd systems fab1517aulbp4 rd systems fab6285a micab bd bioscience icam1 leinco technologies c170 mouse igg1antimyctag merck millipore micb rd systemsmab1599 or igg2b isotype control rd systems mab004detected with secondary antimouse igg biolegend san diegoca united states binding of fluorescently labeledaf647lpha invitrogen l32457 and fitcepha vectorlaboratories burlingame ca united states fl1121 was usedto measure surface levels of complex nglycans all isotypecontrols were purchased from bd biosciencefor staining with mitochondrial probes neutral lipid stainsor 2nbdg uptake — cells seeded the day prior toexperiment were washed once in pbs and incubated for minat —¦c and co2 in warm growth medium containing nmtetramethylrhodamine methyl ester perchlorate tmrm sigmaaldrich t5428 nm mitotracker green fm invitrogenm7514 or for h in growth medium with µm 2nbdginvitrogen n13195 bodipy invitrogen d3922 wasdiluted in warm serumfree medium in a dilution andshaken vigorously to solubilize the lipids immediately beforeloading into the cells for min cells were washed twice inpbs fbs and detached sterically prior to analysisthe soluble nkg2d“fc receptor 1299nk rd systemsand igg1“fc 110hg rd systems were labeled with zenonalexa fluor against human igg1 z25408 invitrogen priorto staining of melanoma cellsin forwardsidescatter plotsdata were acquired with an accuri c6 instrument usingaccuri c6 software and analyzed in flowlogic v721 inivaitechnologies mentone vic australia by gating on viablecellsfollowed bysingle cell gating by areaheightscatter plots fscafsch geometric mean fluorescent intensity mfi values aredisplayed in figures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and purified on directzol spincolumns zymoresearch irvine ca united states according to manufacturer™sprotocol cdna was generated using superscript cdnasynthesis kit invitrogen under standard pcr conditionsfollowing primersequences were used for quantitativertpcr with brilliant sybr green qpcr master mixkit mica mica_f tggcagacattccatgtttctgmica_r ctcgtcccaactgggtgttg ulbp2 ulbp2_f cagagcaactgcgtgacatt ulbp2_r ggccacaaccttgtcattctidh1 idh1_f ctatgatggtgacgtgcagtcg idh1_r cctctgcttctactgtcttgccidh2 idh2_f agatggcagtggtgtcaaggagidh2_r ctggatggcatactggaagcag glut1 glut1_fctgctcatcaaccgcaac glut1_r cttcttctcccgcatcatct glut2 glut2_f tacattgcggacttctgtgg glut2_r agactttcctttggtttctgg glut3glut3_f cagcgagacccagagatg glut3_r ttggaaagagccgattgtag glut4 glut4_f tgggcttcttcatcttcacc glut4_r gtgctgggtttcacctcctrplp0_fcctcgtggaagtgacatcgt rplp0_r cattcccccggatatgaggc realtime qpcr was performed on biorad cfx96 realtime thermal cycler c1000 touch and alltranscripts were normalized to housekeeping rplp0 transcripthousekeepingand rplp0asgeneluciferase reporter assaycells were transiently transfected using calciumphosphatetransfection as described above with firefly luciferase promotervectors µg and an sv40promoter driven renilla luciferasevector µg cells were harvested and snap frozen hpost transfection pellets were lysed in dualglo luciferasereagent promega e2920 and firefly luciferase activity wasanalyzed by luminometer microbeta ii perkinelmer walthamma united states renilla luciferase activity was recorded bythe instrument after subsequent addition of volume dualglo stop glo promega e2920 to correct for transfectionefficiency firefly luciferase signals were normalized to sv40renilla luciferase signals of corresponding sampleatacseqatacseq was performed as described previously foreach cellline cells were harvested from separatefrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioncultures and used to prepare tagmented chromatin replicatesof wt and replicates of mgat5 ko cell lines samplestotal quality of pcramplified sequencing libraries was assessedusing a tapestation instrument with high sensitivity dnascreentapes agilent libraries were sequenced as paired endreads on a single lane of an illumina hiseq4000 flow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were filtered toremove low quality duplicate and mitochondrial reads peakswere called using macs2 on merged reads from allsamples and diï¬erential peak accessibility between cell lines wasdetermined using edger with a threshold false discoveryrate of transcription factor binding motifs enriched indiï¬erentially accessible peaks were identified using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantification and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical significance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in figure legends using unpaired ttest in 1a 1c 1e 3ef3h 5c 7a 7ef paired ttest in 4fg 7d multiple ttest in 1b1d 3d 4ab one sample ttest in 2ac 3c 4c 4e 7g twowayanova in 3a 5df 5hi 6a 6e 7hi or oneway anova in5g level of statistical significance was determined by ˆ—p ˆ—ˆ—p and ˆ—ˆ—ˆ—p ˆ—ˆ—ˆ—ˆ—p resultsmgat5 knockout increases nkg2dlexpression and activates nkg2d in vitroand in vivoregulation of constitutive mica expression remains largelyunknown surface expression of certain mica alleles dependson nlinked glycosylation we questioned whetherthe cancerassociated glycosyltransferase mgat5 is required formica expression to assess the role of mgat5 in regulationof nkg2dl surface expression mgat5 ko clones weregenerated in hek293 cells remarkably mgat5 ko resultedin a permanently increased surface expression of the nkg2dlsmica micb and ulbp256 compared with parental wildtypewt cells figure 1a to confirm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspecifically to mgat5modified nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modifiednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modification of mgat5 expressiontherefore associated with substantial changes in constitutiveexpression of several nkg2dlsto verify the functionality of mgat5 koinduced nkg2dlswe tested nkg2d activation in a reporter cell line expressing1httpswwwencodeprojecthuman nkg2d coupled to dap10cd3ζ signaling and nuclearfactor of activated t cells nfatcontrolled gfp ultimatelyexpressing gfp in response to nkg2d activation nkg2dgfp activation was higher after cocultivation with mgat5ko cells than with wt cells figure 1b corresponding to theincreased nkg2dl expression in mgat5 ko cells figure 1athe reporter cells without nkg2d supplementary figure s1aremained inactivated indicating that the activation was nkg2dmediated figure 1b moreover blocking nkg2dls withsoluble nkg2dfc receptor impaired the activation furthervalidating nkg2d specificity supplementary figure s1bto test if mgat5 ko cells could activate nkg2d in vivowe adoptively injected nkg2d reporter cells together with wtor mgat5 ko cells into the peritoneum of nmri mice andmeasured gfp expression in reporter cells in line with ourin vitro data we observed a significant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspecific since the control reporter cellswere unaï¬ected figure 1c these data verify that mgat5 koinduced nkg2dls maintain their functional integrity in vivonkg2d is downmodulated upon activation to furtherexamine the functionality of nkg2dl expression causedby mgat5 ko we assessed nkg2d downregulation afterreceptor activation nkg2d was further downregulated oncd4depleted peripheral blood lymphocytes pbls after cocultivation with mgat5 ko cells than with wt cells and thisdownregulation was abolished by blocking nkg2dls with asoluble nkg2dfc receptor figure 1d combined these dataindicate that ko of mgat5 upregulates mica and ulbp256resulting in nkg2d activation in vitro and in vivoto ensure that the mica upregulation was a result of mgat5ko we stably transfected mgat5 into wt and mgat5 kocells lpha binding was restored within days after transfectionconfirming expression of functional mgat5 interestingly ittook multiple passages for mica expression to decrease to wtlevels figure 1e and supplementary figure s1c suggestingthat mica is regulated in response to a longterm adaptation toaltered mgat5 expressionudpglcnac upregulates micaexpressionlongterm mgat5 deficiency willlikely result in aberrantnglycosylation and an accumulation of the mgat5 donorsubstrate udpnacetylglucosamine udpglcnac to addressif mica was regulated by a change in nglycosylation inmgat5 ko cells we assessed the posttranslational regulationof mica by measuring surface expression of transgenicallyexpressed gfpmyctagged mica under a cytomegaloviruscmv promoter the mica alleles micaˆ— and micaˆ—are distinctly regulated posttranslationally and althoughmicaˆ— was upregulated in mgat5 ko cells the regulationwas minor and unlikely to account for the profound changein endogenously expressed mica figures 1a 2a micatranscripts on the other hand were highly increased in mgat5ko cells figure 2b as well as ulbp2 mrna supplementaryfigure s2a suggesting that nkg2dls are transcriptionallyfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionfigure mgat5 knockout increases nkg2dl expression and activates nkg2d in vitro and in vivo a surface expression of nkg2d ligands and binding offluorescently labeled lpha mgat5 modifications or epha mgat3 modifications on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by flow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean fluorescence intensity mfi b in vitro nkg2d activation measured as gfp expression in nkg2d negative reporter cells control and nkg2dexpressing nkg2d reporter cells target cells cocultivated with wt or ko cells effector cells for “ h at indicated effectortarget et ratios c nkg2dactivation in vivo measured on reporter cells as in b after activation by wt or ko at a ratio in peritoneum of nmri mice for approximately h gfp expressionin didlabeled reporter cells signifies nkg2d activation and is shown as gfp mfi values of cells from foursix mice per group d nkg2d downmodulation wasassessed on nkcd8 t cells target cells after cocultivation for h with wt or ko cells effector cells at indicated effectortarget ratios et nkg2dls on targetcells were blocked with nkg2dfc bl or unblocked with igg1fc un the graph depicts surface expression of nkg2d presented relative to surface nkg2dexpression on target cells alone e mica surface expression left and lphaepha surface binding right after lentiviral introduction of mgat5 into wt or kocells mfi values from antibody staining were corrected for isotype background staining 01mfi statistical analysis was performed by unpaired ttests in ace andmultiple ttest with fdr comparing wt and ko in bd p p p and p regulated in mgat5 ko cells notably we found that themgat5 substrate udpglcnac although indistinguishablefrom udpnacetylgalactosamine udpgalnac tended tobe higher in mgat5
Colon_Cancer
n6methyladenosine m6a regulators are involved in the progression of various cancers via regulating m6amodification however the potential role and mechanism of the m6a modification in osteosarcoma remains obscurein this study wtap was found to be highly expressed in osteosarcoma tissue and it was an independent prognosticfactor for overall survival in osteosarcoma functionally wtap as an oncogene was involved in the proliferation andmetastasis of osteosarcoma in vitro and vivo mechanistically m6a dot blot rnaseq and meripseq meripqrtpcrand luciferase reporter assays showed that hmbox1 was identified as the target gene of wtap which regulatedhmbox1 stability depending on m6a modification at the ²utr of hmbox1 mrna in addition hmbox1 expressionwas downregulated in osteosarcoma and was an independent prognostic factor for overall survival in osteosarcomapatients silenced hmbox1 evidently attenuated shwtapmediated suppression on osteosarcoma growth andmetastasis in vivo and vitro finally wtaphmbox1 regulated osteosarcoma growth and metastasis via pi3kaktpathway in this study demonstrated the critical role of the wtapmediated m6a modification in theprogression of osteosarcoma which could provide novel insights into osteosarcoma treatmentintroductionosteosarcoma is a primary malignant bone tumor thatis common among childhood and adolescents worldwide1despite the improvements including multiagent chemotherapy with surgery in recent years the 5year survival rate is for localized osteosarcoma and is for recurrent and metastatic osteosarcoma23 thereforea better understanding of molecular mechanism isurgent for developing novel therapeutic strategies forosteosarcomacorrespondence jinglei miao miaojinglei126com orjinsong li jinsongli_csu163com1department of orthopaedics the third xiangya hospital of central southuniversity tongzipo rd changsha hunan china2shanghai key laboratory of regulatory biology institute of biomedicalsciences and school of life sciences east china normal university shanghai chinafull list of author information is available at the end of the edited by a stephanouitn6methyladenosine m6a is the prominent dynamicmrna modification which is involved in various biological process by regulating mrna translocation translation and stability4is catalyzed by m6a writermethyltransferase removed by erasers rna demethylases and recognized by m6a readers involving in variousbiological progression5“ the formation of m6a is catalyzed by a methyltransferase mettl3 and mettl14form a core catalytic complex of methyltransferases that isstabilized by wtap8 recently mettl16910 mettl5zcchc411 and zc3h1312 were showed to play a criticalrole in compositing methyltransferase complex andfacilitating m6a methylation13 the eraser alkbh5 andfto has m6a demethylation activity to remove the m6amodification the reader proteins are from yth familyheterogeneous nuclear ribonucleoprotein hnrp familyand insulinlike growth factor mrnabinding protein the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cchen cell death and disease page of isfamily they recognize the m6a modification to adjustrna metabolisms14dynamic and reversible m6a regulation was reported tobe involved in various physiological and pathologicalprocesses including stem cell differentiation cardiachomeostasis adipogenesis neuronal disorders and spermatogenesis15“ recent years compelling evidence hasrevealed that m6a modification plays an important role inthe tumorigenesis of various cancers20“ for examplemettl3 was reported to play key role in the progressionof colorectal carcinoma25 bladder cancer2627 gastriccancer28 and pancreatic cancer29 ythdf2 was involvedthe progression of acute myeloid leukemia aml viaregulating hematopoietic stem cells hscs activity4 ftowas also reported to play a crucial role in the progressionof melanoma30 breast cancer31 gastric cancer32 andintrahepatic cholangiocarcinoma nevertheless the role ofm6a modification in osteosarcomastill poorlycharacterizedwtap a wilms™ tumor wt1 associated protein33has been reported to be critical in the biological processesincluding g2m transition and premrna splicing34“in addition accumulated studies identified the importantrole of wtap in the progression of various cancers37 forexample wtap function as oncogene in cholangiocarcinoma38 acute myeloid leukemia39 colon cancer40 ovariancancer41 and diffuse large bcelllymphoma42 recentstudies reported that wtap is strictly connected to afunctional m6a methylation complex43 here we revealedthe increased expression of wtap in osteosarcoma tissuewhich was associated with clinicopathological features andpoor prognosis in osteosarcoma patients wtap functionas an oncogenic gene and it promotes the m6a modification and progression of osteosarcoma mechanistically wtap induced the growth and metastasis ofosteosarcoma via downregulating hmbox1 expression inm6adependent manner further investigations demonstrated that wtaphmbox1 regulated osteosarcomagrowth and metastasis via pi3kakt pathway overallthese results imply that wtaphmbox1 may be animportant mechanism of osteosarcoma progression andserve as a novel therapeutically target of osteosarcomamaterials and methodsclinical samples and ethicsonehundredandfour paired fresh osteosarcoma metastatic os sample and nonmetastatic os sampleand normal tissues were obtained from patients withoutradiotherapy andor chemotherapy at the third xiangyahospital of central south university and then immediately frozen in ˆ’ °c for rna and protein extraction orformalxed and paraffinembedded for further usedinformed consent was obtained from each patient or theirguardians and the study were approved by the ethicsofficial of the cell death differentiation associationcommittee of the third xiangya hospital of centralsouth universitycell culture transfection and infectionthe human hfob119 cells and osteosarcoma cell linessjsa1 mg63 hos u2os and 143b were obtainedfrom the cell bank of the chinese academy of sciencesshanghai china the osteosarcoma cells were culturedin dmem with fbs gibco grand island ny usathe hfob119 cells were cultured in dmemf12 df with fbsthe sequences of shrna cloned into plko1 vectorand then shwtapplko1 or shhmbox1plko1 werecotransfected with packing and pax2 plasmids into293tf cells fortyeight hours after transfectionthelentivirus was collected and infected hos and u2os cellsfor h the μgml puromycin was used for screeninginfected cells the primers were listed in table s1western blotproteintechantiythdf2antipi3k ab191606 abcamthe proteins were obtained from cells and tissues beinglysed with ripa buffer and then separated by sdspage the antiwtap ab195380 abcam antihmbox1161231apab220163abcamantippi3kab182651 abcam akt 9272s cell signaling paktser473and antigapdhab181602 abcam were used for pvdf membrane incubating overnight at °c the protein expression was detected by incubating with antirabbit secondary antibodies4051s cellsignalingrnaseq analysis and qrtpcrtotal rnas were extracted from osteosarcoma tissueand cells using trizol thermofisher usa for rnaseqanalysis the library construction and illumina sequencingusing the illumina truseq rna sample prep kit sandiego ca usa for qpcr validation the cdna wasobtained using first strand cdna synthesis kittoyobo finally the mrna expression was detectedusing sybr green biorad california usa all primers were in table s1rna m6a dot blot assay and rna m6a quantificationtotal rnas were extracted from osteosarcoma cells bytrizol thermofisher usa and then nanodrop3000was used for detecting rna quality the m6a content wasdetected by epiquik m6a rna methylation quantification kit colorimetric epigentek usathe polya rnas and ng were spottedonto a nylon membrane ge healthcare for rna m6adot blot assay and then incubated with m6a antibody noabe572 merck millipore usa at °c overnight them6a dots was analyzed using an imaging system biorad usa 0cchen cell death and disease page of meripseq and meripqrtpcrtotal rnas were extracted from osteosarcoma cells bytrizol the dnafree fragmented rnas were incubatedwith magnetic dynabeads bounded antim6a antibodyabcam usa to enrichment the mrna with m6a andthen beads were treated with proteinase k and rna wasextracted for meripseq or validation by qrtpcr theprimers are in table s1cell proliferation assaysthe cck8 cell counting kit8 c0038 beyotimebiotechnology china was used for cell proliferationability44colony formation assaysthe cells were seeded into 6well plates with a density × cells per wells and cultured in dmem medium for weeks and then paraformaldehyde pfa was usedto fix the colonies and crystal violet was used to stain asprevious described45woundhealing assayosteosarcoma cells were cultured for h to reach confluency and then a straight artificial wound wasscraped with a μl pipette tip the cell migration abilitywas measured by photographing the distance at and h45transwell invasion assaysthe transwell chamber bd science bedford mausa was used for invasion assays as previously described1 in brief × osteosarcoma cells were seeded intothe upper chambers and incubated for h the invasivecells were counted and quantified for cellinvasion asprevious described45luciferase reporter assaysthe ²utr of hmbox1 was cloned into pmiglovector promega usa the putative m6a sites bases awere mutated into bases c in ²utr using sitedirectedmutagenesis kit thermo usa the wt and mutplasmids were transfected in osteosarcoma cells and thenthe dualluciferase assay kit promega was used fordetecting the luciferase activity44animal experimentsnude mice weeks male were used for tumor modelall animal care and handling procedures were approvedby the institutional animal care and use committee ofthe third xiangya hospital of central south universitychangsha china for the subcutaneous tumor model ×shwtapshhmbox1 u2os cells seeded into mice via subcutaneous injection tumor volume and tumor weightshhmbox1 orshncshwtap orofficial of the cell death differentiation associationwere measured to analyze tumor growth as previousdescribed46 for orthotopic xenografttumor modelshnc shwtap shhmbox1 or shwtapshhmbox1u2os cells were labeled with a luminescent dye and gfpand injected into the cavity of the tibia of mice thirtydays after injection tumor growth was detected for themetastasis model the cells were injected into the tail veinand the lung metastasis were detected days afterinjection using in vivo bioluminescence imaging systemimmunohistochemistryimmunohistochemistry ihc analysis was performedusing antiwtap ab195380 abcam antihmbox1161231ap proteintech as pervious described26bioinformatics analysisthe geo dataset gse87624 and gse46705 weredownloaded and analyzed by r version httpswwwrproject the different expressed gene wascalculated with edger package and identified by thethreshold criteria of log2 foldchange fc ‰¥ and adjp go and kegg analysis was performed toinvestigate the potential role of genes protein“proteininteraction ppi network was analyzed by the stringdatabasestatistical analysisthe spss spss inc chicago il usa was usedfor the statistical analyses using anova or student™s ttest in this study kaplan“meier analysis was used forsurvival the correlation between wtap and hmbox1were analyzed using pearson analysis statistical significance was defined p resultselevated wtap is associated with poor prognosis ofosteosarcoma patientsto reveal the important role of m6a modification inosteosarcoma we explored the expression levels of m6arelative genes in osteosarcoma tissue and normalbone tissue gse87624 normal and osteosarcomaas shown in figs 1a and s1a we examined the expressionof m6arelated genes in geo database and found thatwtap rbm15 ythdf1 and ythdf2 were differentlyexpressed in tumor tissue compared with control tissuethe m6a writers were reported to play key role on theprogression of various cancers so wtap was selected forfurther analysis next we detected the expression ofwtap in pairs of osteosarcoma tissue and the corresponding paratumor tissues from the third xiangyahospital of central south university txhcsu consistent with the geo results the significant higher mrnaand protein levels of wtap in osteosarcoma was detectedusing the qpcr and wb analysis fig 1b c moreover 0cchen cell death and disease page of fig the expression levels of wtap in osteosarcoma tissue and cell lines a wtap expression in osteosarcoma tissue compared with normalbone tissue from gse87624 b qpcr assay and c western blot assay revealed the wtap expression in osteosarcoma tissue and the correspondingparatumor tissues from txhcsu d the kaplan“meier survival analysis e univariate and multivariate survival analysis f establishment of the overallsurvival nomogram for osteosarcoma patients g timedependent roc analysis h the mrna expression levels of wtap in osteosarcoma cell linesand hfob119 cell line using qpcrthe osteosarcoma patients with high wtap showed pooroverall survival in txhcsu fig 1d and the univariateand multivariate cox analysis showed that wtap andmetastasis were the independent prognostic factor foroverall survival in osteosarcoma patients fig 1e theprognostic nomogram wasthepredictusedtoprobabilities of overall survival rates of osteosarcomapatients and the calibration curves showed has a goodconsistency between the prediction and the actual observation fig 1f in addition the years roc curve aucof wtap in osteosarcoma patients from txhcsu was fig 1g besides the overexpression of wtap wasofficial of the cell death differentiation association 0cchen cell death and disease page of table the association of wtap expression andclinicopathologic characteristics of osteosarcoma patientswtap acts as an oncogene that promoted cell proliferation migration and invasion in osteosarcomacharacteristics case number wtap expressionp valuehmbox1 is potential target of wtap in osteosarcomagendermalefemaleage‰¤tumor size‰¤ cm cmmetastasisyesnotnmiiiiiiivhighn lown p p p p p associated with tumor size metastasis and tnm stagetable and fig s1b finally the higher expression ofwtap was also detected in osteosarcoma cell lines sjsa mg63 hos u2os and 143b compared with that inthe human osteoblast hfob119 cell fig 1h collectively these results demonstrated that wtap is highlyexpressed in osteosarcoma and is correlated with its poorprognosissilenced wtap significantly represses osteosarcomaprogression in vitroto generateto further clarify the role of wtap in osteosarcomashwtap1 andwe next used shrna lentivirusshwtap2stable wtapknockdownosteosarcoma cells and analyzed the role of wtap oncells migrationinvasion and proliferation of osteosarcoma the shwtap1 and shwtap2 significantlyknockdown the expression levels of wtap in osteosarcoma cells fig 2a the cck8 assay and colonyformation assay showed that wtap deficiency significantly reduced proliferative capacity of osteosarcomacells fig 2b c the transwellinvasion and woundhealing assays showed that the invasion and migrationability of the osteosarcoma were significant reduced bysilenced wtap fig 2d e these results determine thatofficial of the cell death differentiation associationto reveal the potential mechanism by which wtapregulates the progression of osteosarcoma we performedrnaseq m6aseq in shwtapshnc u2os cell andclipseq to revealthe potential genes regulated bywtapmediated m6a modification the rnaseq resultsrevealed downregulated degs and upregulateddegs with logfc and adjp in shwtapgroup compared with shnc group fig s2a geneontology go showed that degs were enriched in neutrophil activation cell cycle and so on fig s2b kegganalysis showed that degs were enriched in metabolismrelated signal pathway fig s2c the m6aseq analysisidentified differentially m6a modification genes inwtapsilenced u2os cell compared with normal u2oscells table s2 the clipseq from gse46705 revealed wtaptargeted rna in wtap overexpressed cellusing parclip technology and then we obtained theoverlapped genes in rnaseq m6aseq and clipseq asshown in fig 3a genes were overlapped in three groupsincluding two upregulated gene slc3a2 casp7 andfour downregulated gene abr hs6st1 cd59 andhmbox1 consistent with these results slc3a2 andcasp7 were significantly upregulated and abr hs6st1cd59 and hmbox1 were significantly downregulated inosteosarcoma tissues from geo gse87624 databasefig s3a these results were also confirmed in theosteosarcoma tissues from our txhcsu data fig s3band then we evaluated the regulation of wtap on thesix candidates in osteosarcoma cells using qpcr amongwhich hmbox1 was the most significantly upregulatedgene in shwtap osteosarcoma cells fig 3b and wasselected for further analysis consistent with the mrnaexpressionthe protein level of hmbox1 was alsoremarkably increased by silenced wtap fig 3c wenext evaluated the relationship between wtap andhmbox1 expression in gse87624 and txhcsu as ourspeculation hmbox1 expression was negatively correlated with wtap expression r ˆ’ in gse87624and r ˆ’ in txhcsu fig 3d moreover weanalyzed the relationship between hmbox1 expressionclinicopathological features in osteosarcoma patientsfrom txhcsu hmbox1 expression was significantlyreduced with tumor size and metastasis table and figs3c and the survival analysis results showed that thepatients with low level hmbox1 had poor overall survival in osteosarcoma fig 3e moreover the univariateand multivariate analysis demonstrated hmbox1 as anindependent prognosticsurvivalp in osteosarcoma patients fig 3f we alsorevealedinof hmbox1expressionfactorfor overallthelower 0cchen cell death and disease page of fig silenced wtap significantly repressed osteosarcoma progression in vitro a the shwtap1 and shwtap2 downregulated wtapexpression b cck8 assay revealed that silenced wtap reduced the cell proliferation ability in osteosarcoma c colony formation assay showed thatsilenced wtap decreased the cloning number of osteosarcoma cells d transwell invasion and e woundhealing assay revealed the inhibition ofsilenced wtap on osteosarcoma cell invasion and migrationosteosarcoma cell lines sjsa1 mg63 hos u2os and143b than that in the human osteoblast hfob119 cellline fig 3g in these results indicated thathmbox1 is a potential target of wtap and is related topoor prognosis of osteosarcoma patientswtap regulates hmbox1 expression via m6a modificationin osteosarcomaas meripseq data revealed different m6a modificationof hmbox1 in nc and wtapsilenced cells we nextanalyzed whether wtap regulated the hmbox1official of the cell death differentiation association 0cchen cell death and disease page of fig hmbox1 is a potential target of wtap a the venn diagram was generated from differentially expressed genes in rnaseq m6aseq andclipseq in gse46705 the expression of the overlapped genes in hos and u2os cells with silenced wtap using b qpcr assay and c western blotassay d correlation analysis of wtap and hmbox1 in osteosarcoma from gse87624 and txhcsu e the kaplan“meier survival analysis f univariateand multivariate survival analyses g wtap hmbox1 expression in osteosarcoma cell lines and human osteoblast hfob119 cell lineexpression in an m6adependent manner using m6a dotblot and rna methylation quantification assay asexpected m6a levels were substantially decreased inwtapknockdown osteosarcoma cells compared controlosteosarcoma cells fig 4a moreover meripqpcrassay showed that hmbox1 was effectively enriched bym6aspecific antibody and enriched hmbox1 wasremarkably decreased by in wtapknockdown cells fig4b therefore we supposed that wtap could regulatem6a levels of hmbox1 according to the m6a rnaseqthe ²utr ofresulthmbox1 and the sramp httpwwwcuilabcnsramppredicted three very high confidence m6a sites at the ²utr of hmbox1 fig s4 to further prove the directedthe m6a modification was attarget role of wtap on hmbox1 with m6a modification we cloned the hmbox1 ²utr containing these m6a sites into pmirglo vectors and then we mutant thebases a into bases c in the predicted m6a sites fig3f in fig 3g the luciferase activity of hmbox1 wassignificantly increased by silenced wtap however theluciferase activity of mut hmbox1 did not affected bysilenced wtap in both hos and u2os cells fig 4cythdf2 is a well know m6a reader and plays animportant role in the progression of several cancers viaregulating mrna degradation figure 1s showed thatythdf2 was upregulated in osteosarcoma tissues ingse87624 in fig 4e ythdf2 evidently upregulated inosteosarcoma tissue and cells we next shed light on theofficial of the cell death differentiation association 0cchen cell death and disease page of table the association of hmbox1 expression andclinicopathologic characteristics of osteosarcoma patientscharacteristics case number hmbox1 expressionp valuehighn lown gendermalefemaleage‰¤tumor size‰¤ cm cmmetastasisyesnotnmiiiiiiivp p p p p expression of ythdf2 in osteosarcoma and the role ofythdf2 on hmbox1 expression in osteosarcoma disappointedly silenced ythdf2 showed no effects onhmbox1 expression in osteosarcoma cells fig 4f suggesting that wtap regulated m6amediated hmbox1expression in ythdf2independent mannertogether these data suggested that wtaprepressedhmbox1 expression via regulating m6a modification ofhmbox1 at its ²utrhmbox1 is involved in wtapmediated osteosarcomaproliferation and metastasis in vitrowe next explored whether wtap promoted osteosarcoma progression by regulating hmbox1 expressionas shown in fig 5a hmbox1 expression was evidentlyincreased by wtap knockdown and was decreased byhmbox1 knockdown in osteosarcoma cells the cck8results showed that the proliferation levels were increasedby shhmbox1 in wtapsilenced hos and u2os cellsfig 5b consistent with the cck8 results silencedhmbox1 also alleviated shwtapmediated repressionof cell proliferation in colony formation assay fig 5cthe similar effects of hmbox1 were also observed inwtapsilenced osteosarcoma cell using woundhealingand transwell invasion assays fig 5d and e in additionofficial of the cell death differentiation associationwestern blot results showed that silenced wtap evidently repressed the expression of mesenchymal markerscadherin and vimentin and induced the expression ofepithelial marker ecadherin which was attenuated byshhmbox1 in osteosarcoma cells fig 5f these datasuggested wtap promotes osteosarcoma cells proliferation and metastasis via repressing hmbox1 expressionhmbox1 inhibits osteosarcoma growth and metastasisin vivowe next verified the role of hmbox1 in vivo byinjecting shnc shwtap shhmbox1 and shwtapshhmbox1 u2os cells to induce subcutaneous osteosarcoma mice model orthotopic xenograft tumor modeland tail vein metastasis model the expression levels ofhmbox1 was significantly upregulated by silencedwtap in subcutaneous osteosarcoma tissue figs 6a ands5 and s6 moreover silenced wtap repressed thetumor size and tumor weight in subcutaneous osteosarcoma mice which was rescued by silenced hmbox1fig 6b we next used a luminescent dye and gfplabeled u2os cells to build an orthotopic xenografttumor model the bioluminescence imaging showed thatthe wtap knockdown reduced the proliferation ofu2os cells in situ while silenced hmbox1 alleviatedthis repression fig 6c to further detect the role ofwtap and hmbox1 on the metastatic ability ofosteosarcoma in vivo u2os cells were injected into nudemice via the tail vein the bioluminescence imagingshowed that silenced hmbox1 alleviated the repressionof silenced wtap on osteosarcoma metastasis fig 6dtaken together these results suggest that hmbox1 isinvolved in wtapmediated tumor growth and metastasis of osteosarcomawtaphmbox1 regulates the proliferation and metastasisof osteosarcoma partly by pi3kakt pathwaythe kegg results identified that pi3kakt pathwayscould be regulated by wtap fig s2b pi3kakt signaling pathway promotes the growth migration andinvasion of various cancers including osteosarcoma4748we hypothesized that wtaphmbox1 was involved inthe progression of osteosarcoma via regulating pi3kaktsignaling pathway as shown in fig 7a phosphopi3k andphosphoakt were evidently repressed by shwtap andinduced by shhmbox1 and shhmbox1 significantlyattenuatedandphosphoakt in both hos and u2os cells ly294002 api3kakt pathwaysreducedshhmbox1induced cell proliferation migration andinvasion in both hos and u2os cells fig 7b cthereforethese results imply that wtaphmbox1regulates the proliferation and metastasis of osteosarcomapartly via pi3kakt pathway fig shwtaprepressedphosphopi3kinhibitorremarkably 0cchen cell death and disease page of fig hmbox1 is negative correlation wtap expression and is associated to the poor prognosis of osteosarcoma a the m6a level in hosand u2os cells with silenced wtap b meripqpcr assay followed by qrtpcr revealed the hmbox1 m6a modification c wildtype or m6a sitemutant hmbox1 were cloned in pmirglo d luciferase reporter assays revealed the target role of wtap on the ²utr of hmbox1 e ythdf2expression in osteosarcoma tissue and cells f the effects of silenced ythdf2 on hmbox1 expression in osteosarcoma cellsdiscussionin the past several years m6a modification is considered as a pervasive internal modification of mrna andplays critical roles in the progression of a variety of humandiseases including cancers20 however the underlyinginvolvement of m6a regulators in osteosarcoma progression is still unclear in the present study we focused onthe role and underlying mechanism of wtap and itmediated m6a modification in the progression andmetastasis of osteosarcoma in this study wtap wasfirstly identified to upregulated which was associated withpoor prognosis of osteosarcoma functionally wtappromoted the growth and metastasis of osteosarcomain vitro and vivo mechanistically hmbox1 was identified as the target gene of wtap and it was regulated bywtap with m6a modification at the ²utr finallywtaphmbox1 regulated osteosarcoma growth andmetastasis in a pi3kaktdependent patternactually wtap was reported as an oncogene in various cancers37“ recent studies reported that wtapis strictly connected to a functional m6a methylationcomplex43 howeverfew study demonstrated theimportant role of wtap as a m6a regulator here weconcluded that wtap is not only upregulated but alsoplays key role on the m6a modification in osteosarcomanotably the aberrant of m6a modification is related toofficial of the cell death differentiation association 0cchen cell death and disease page of fig hmbox1 as a tumor suppressor was involved in wtapmediated progression a hmbox1 expression in osteosarcoma cell with shwtapand shhmbox1 knockdown of hmbox1 effectively alleviated wtapdependent b cell proliferation c colony formation d transwell invasion ande woundhealing assay f the protein expression level of emt transition related protein p various biological processes via regulating mrna stability splicing and translation next we shed light on thedownstream mrna that modified by wtapmediatedm6a modification by combining the data from rnaseqm6aseqshowed thathmbox1 is a potential target gene of wtap subsequently meripqpcr western blotand luciferasereporter assay results revealed that wtap repressedand clipseq theresultshmbox1 expressed with wtapdependent m6a modthe ²utr of hmbox1 thus wtapification atinvolved in tumorigenesis depending on its role of m6amodification although ythdf2 is a wellknown m6areader in several cancers we found that ythdf2 showedno effects on hmbox1 expression suggesting thatwtap regulated m6amediated hmbox1 expression inythdf2independent manner and the m6a readerofficial of the cell death differentiation association 0cchen cell death and disease page of fig silenced hmbox1 attenuated shwtaprepressed osteosarcoma growth and metastasis in vivo a the expression levels of hmbox1 inosteosarcoma mice models b knockdown of hmbox1 effectively alleviated shwtaprepressed osteosarcoma growth in mice c the orthotopicxenograft tumor and d lung metastasis were detected using a vivo bioluminescence imaging system representative images and a histogram areshown n each groupwhich was involved in the m6a modification of hmbox1need be further investigatedhmbox1 a homeobox containing protein49 wasreported to be a transcriptional repressor involving in thebiological processes in bone marrowderived stromacells50 nk cells5152 and vascular endothelial cells53recent studies demonstrated the dysregulated hmbox1in various cancers for example high expression ofhmbox1 was observed in gastric cancer and it wasassociated with the poor prognosis of gastric cancer54moreover hmbox1 also observed as tumor suppressorin ovarian cancer55 and cervical cancer56 hmbox1repressed the progression of ovarian cancer via regulatingcell proliferation and apoptosis55 hmbox1 repressedliver cancer progression via regulating autophagy as wellas and immune escape57 consistently hmbox1 isofficial of the cell death differentiation association 0cchen cell death and disease page of fig wtap hmbox1 was involved the progression of osteosarcoma via pi3kakt pathway a western blot analysis for the expression ofppi3k pi3k pakt and akt in osteosarcoma cells b cell proliferation c colony formation transwell invasion and woundhealing assay ofosteosarcoma cell treated with μm akt inhibitor ly294002 abcam ab120243 for hunclear how hmbox1 as a transcriptional repressorregulates pi3kakt pathway nonethelesssilencedhmbox1 only partly alleviated wtapmediated osprogressionthere are other potentialmolecular mechanisms regulated by wtapmediatedepigenetic modulation in osit means thatin summary we identified the elevated wtap inosteosarcoma and which is associated with poor clinicaloutcome and serves as an independent prognostic factorfor osteosarcoma patients wtap dramatically promotedosteosarcoma proliferation and metastasis via regulatinghmbox1 mrna stability in a m6adependent mannerwtaphmbox1 regulated osteosarcoma growth andmetastasis via pi3kakt pathway altogether our resultsdetermined wtap hmbox1 as a potential therapeutictarget for osteosarcomaacknowledgementsthis study was supported by national natural science foundation of chinagrant nos national natural science foundation of china grantnos natural science foundation of hunan province grant nos2018jj2617 natural science foundation of hunan province grant nos2016jj3176 national key research and development program of china no2016yfc1201800 natural science foundation of hunan province no2018sk2090author details1department of orthopaedics the third xiangya hospital of central southuniversity tongzipo rd changsha hunan china 2shanghai keylaboratory of regulatory biology institute of biomedical sciences and schoolof life sciences east china normal university shanghai china 3fourgynecological wards ningbo women and children™s hospital ningbozhejiang china 4the second xiangya hospital of central southuniversity changsha china 5school of basic medical science central southuniversity changsha china 6department of anatomy histology andembryology changsha medical university changsha chinafig a schematic model illustrating wtapmediated m6a regulationin osteosarcomaevidentlydownregulated and closely related to the poor prognosisof osteosarcoma in the present study in addition silencedhmbox1alleviated wtapknockdownmediated repression of osteosarcoma progression whichimplied the import roles of hmbox1 in wtapdrivenosteosarcoma development finally we analyzed thedownstream pathway of wtaphmbox1 in osteosarcoma pi3kakt pathway was reported to an important role in the progression of various cancers includingosteosarcoma58 we found that pi3kakt pathway we
Colon_Cancer
since leung reported a single case of lowdose aspirin lda induced multiple smallintestinal ulcers in many investigators have described ldainducedsmall intestinal mucosal injuries watanabe reportedthat all of their patients who used lda had small intestinallesions that were detected using capsule endoscopy 0cgastroenterology research and practicece iwamoto investigated patients whounderwent ce for occult bleeding and erosions wereobserved in cases of whom were taking lda ornonlda nonsteroidal anti‚ammatory drugs endo described small intestinal lesions in of subjects who took aspirin mg for weeks shiotani performed ce on young healthy individuals beforeand after medium doses of enteric aspirin were administered for days and rabeprazole mg was administered for week and found large erosions that included small intestinal ulcers in of the subjects recently direct oral anticoagulants doacs have beenadministered as alternatives to warfarin dabigatran is adirect thrombin inhibitor and rivaroxaban apixaban andedoxaban are factor xa inhibitors doacs have significantlyfewer side eï¬ects than warfarin including intracranial hemorrhage hence the number of patients taking doacs isgradually increasing [“] howeverthe findings frommetaanalyses have shown that compared with warfarinthe incidence of gastrointestinal gi bleeding is higher inassociation with doacs [ ] the causes of gi bleedingin association with doacs were bleeding from colon astric cancers and diverticular hemorrhages comparedwith warfarin dabigatran and rivaroxaban are associatedwith higher risks of gi bleeding depending on their dosesand the risk of gi bleeding associated with apixaban iscomparable edoxaban is associated with significantlyless gi bleeding at low doses mg once daily comparedwith warfarin but the risk is significantly greater at highdoses mg once daily but unlike western countries the rate of gi bleeding for both dabigatran and rivaroxaban is equivalent to warfarin in asian countries furthermore edoxaban tends to cause less digestive tractbleeding than warfarin esophageal ulcers caused bydabigatran have been described by toya kasai and okada and okada the tartaric acidcoating on dabigatran causes esophageal mucosal disorders because dabigatran persists in the midesophagus ifit is consumed without water however there havebeen no reports of smalllesions in patientswho receive doacs here we aimed to evaluate smallintestinal mucosal injuries in patients taking doacs usingvideo capsule endoscopy vceintestinal methodsthis study was a prospective openlabel nonblinded multicenter and observational study from september tomarch pat5ents taking doacs namely dabigatranrivaroxaban and apixaban for atrial fibrillation at saitamamedical university hospital keio university hospital saitama medical center and yokohama municipal citizen™shospital were enrolled patients with severe comorbiditiesincluding severe anemia and exacerbations of heart failurethat required blood transfusion crohn™s disease and ileuswere excludedthe hemoglobin hb and serum ferritin levels the esophagogastroduodenoscopy egd findings and colonoscopicfindings were examined vce pillcam sb2 given imagingfigure rednessfigure erosionltd yoqneam israel was performed to examine small intestinal lesions according to the doac used redness erosionulcer and angioectasia were checked redness was a red spotfigure and erosions were defined as small and superficialmucosal disruptions denuded of villi figure ulcers weredefined as large submucosal disruptions with a central areacovered with exudate and angioectasia was a patchy erythematous lesion figure the images were analyzed using theproprietary rapid software by an expert n h whohad performed more than vce examinations blindlythe type and location of smallbowel lesions were registeredalso the proportion of lesions detected between types ofdoac was evaluated and the hemoglobin hb and serumferritin levels were compared between patients with and without smallbowel lesionsthe study protocol accorded with the tenets of therevised declaration of helsinkiand it wasapproved by the institutional review boards at our institutions written informed consent was obtained from all ofthe patients this study was registered with the universityhospital medicalinformation network clinical trialsregistry umin000011527 october 0cgastroenterology research and practicetable patients™ characteristicsparametermean age years rangesex n malefemalecomorbid disease natrial fibrillationparoxysmal atrial fibrillationhypertensionhyperlipidemiacerebral infarctiondoac n years “dabigatranrivaroxabanapixabanbayaspirin ncelecoxib nppi nh2 blocker n “mean hb gdl rangedoac direct oral anticoagulant ppi proton pump inhibitor hbhemoglobinlower portion in patients table erosions wereobserved in patients and they were present in theupper portion in in the middle portion in and in the lower portion in patientstable erosions tended to occur less frequently in themiddle portion however the diï¬erence was not significantp compared with the upper portion p compared with the lower portion angioectasia was observedin patients and was present in the upper portionin patients and in the middle portion in patient and was absent from the lower portion table there were no ulcers in any patients erosions tended to bemore frequent in patients taking dabigatran or apixaban thanin patients taking rivaroxaban this diï¬erence was not significant p table no significant diï¬erences wereobserved regarding angioectasia among the patients takingthe diï¬erent doacs none of these patients had activebleeding from small intestinal lesionsthe mean hb concentrations in the patients with andlesions were gdl and gdlwithout smallbowelrespectively a diï¬erence which was not significant p the mean ferritin levels in the patients with and withoutsmallbowel lesions were mgdl and mgdl respectively a diï¬erence which was not significant p discussionthis study™s findings showed that of the patients who tookdoacs had redness had erosionsor small ulcers had angioectasia and had no abnormalities in their small bowel smallbowellesions were observed in of patients thereforethere was a high incidence of smallbowel lesions in patientsfigure angioectasiaibm®spss® statistical software version ibm corporation armonk ny usa was used for the statisticalanalyses the data were analyzed using ttests and fisher™sexact test resultsthirtythree patients were enrolled to participate in thisstudy but patients withdrew their consent and vce wasperformed on patients the patients™ mean age was years range “ years and males and females participated in this study twenty patients had atrial fibrillation had paroxysmal atrial fibrillation had hypertension hadhyperlipidemia and had cerebral infarction eight patientstook dabigatran took rivaroxaban and took apixabanthe mean duration of doac use was months months additionally patient took bayaspirin patienttook celecoxib patients took proton pump inhibitorsppis and patients took h2 receptor antagonists theaverage hb concentration was gdl range “ gdl table twentytwo patients underwent egdand atrophic gastritis was present in patients hiatal hernias in patients gastric polyps in patients erosive gastritisin patients gastric ulcer or ulcer scar in patients refluxesophagitis in patients endoscopic submucosal dissectionscar for early gastric cancer in patients and an esophagealulcer in patient nineteen patients underwent colonoscopyand colonic polyps were present in patients colonic diverticulum were present in patients and a rectal ulcer waspresent in patient none of these lesions detected by egdand colonoscopy had active bleedingthe patients evaluated with vce was redness in thelower esophagus was present in patient gastric erosionswere present in patients and gastric redness was presentin patient table the patient who had redness in thelower esophagus was taking apixaban smallbowel transitwas complete in of patients smallbowellesions were observed in of patients redness was observed throughout the small intestine in patients and it was present in the upper portionin in the middle portion in and in the 0cgastroenterology research and practicetable capsule endoscopy findingsentire small intestine observation rate n detection rate n esophagusstomachupper small intestinemiddle small intestinelower small intestineentire small intestineredness lower n erosions n redness n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions nangioectasia nno abnormalities n table findings at each small intestinal site according to the directoral anticoagulant useddabigatranrivaroxabanapixabansitefindingupper small intestineredness nerosions nangioectasia nmiddle small intestineredness nerosions nangioectasialower small intestineredness nerosions nangioectasia nentire small intestineredness nerosions nangioectasia ntaking doacs however none of these patients had activebleeding and most of the lesions were mild patients withsevere anemia or active bleeding were excluded from thisstudy hence only patients with mild symptoms wereincluded ldainduced lesions cause redness erosions andulcers [“] previous studies™ findings that describe the characteristics of smallbowel injuries associated with chroniclda use suggest that ulcers are observed mainly in the distalpart of the small bowel [“] in this study erosionstended to be observed less frequently in the middle portionof the small bowel in the patients taking doacs howeverthere were no significant diï¬erences regarding the distributions of the lesions there were no ulcers in any patientstherefore the intake of doac might not be related withsevere ulcers in the small intestinein this study angioectasia was observed in the upper andmiddle portions but not in the lower portion of the smallbowel kaufman used a transit timebased quartilemethod to evaluate patients with angioectasia whounderwent ce and found that most lesions were inthe first quartile igawa reported that while therewere no diï¬erences regarding the location of type 1a angioectasia among patients with occult gastrointestinal bleedingtype 1b angioectasia was relatively less frequent in the lowerportion compared with that in the upper and middle portionsof the small bowel the data reported before thereforeangioectasia might not be aï¬ected by the intake of doacscomparison of vce findings before and after the administration of d719acs is neededno significant relationships were determined in relationto the presence of the hb level or the serum ferritin levelbetween the patients with and without smallbowel lesionsin this study despite detecting abnormal findings in thesmall bowel no active bleeding was seen by vce and therewas no severe anemia in any patients in this study furthermore compared with warfarin the incidence of gi bleedingis higher in association with doacs the causes of gi bleeding in association with doacs are bleeding from colon astric cancers and diverticular hemorrhages however noneof these lesions detected by egd and colonoscopy had activebleeding doacs did not aï¬ect bleeding from the upper gitract and the colon in this studyno significant diï¬erences were observed among thedoacs in relation to smallbowel lesions the findings fromthe randomized evaluation of longterm anticoagulationtherapy trial of dabigatran showed that in the warfaringroup patients with gi tract bleeding had gastric canceror colonic cancer and that in the dabigatran group patients with gi tract bleeding had colonic cancer and patient had gastric cancer hence dabigatran mightinduce gi tract bleeding from colon cancer rivaroxaban apixaban and edoxaban compete directly with the s1pocket of factor xa and inhibit factor xa activity whereasdabigatran is a prodrug that is activated in the presence ofesterase in the gi tract plasma and liver the causes of themucosal damage by dabigatran were thought to be due todirect acting at the local area where it is absorbed in addition tartaric acid coats dabigatran tablets and the tabletscan cause mucosal damage if they are retained within theesophagus while we expected an increase in the frequencyof intestinal mucosal injury among the patients who tookdabigatran as a consequence of the tartaric acid coating this 0cgastroenterology research and practicestudy™s findings did not demonstrate a higher rate of smallbowel lesions associated with the use of this doac themechanisms underlying mucosal injuries caused by doacsother than dabigatran remain unclear smallbowel lesionsincluding redness erosions and angioectasia might be moreeasily detected by performing ce on patients who takedoacs because the doacs might cause bleeding thatcould facilitate the detection of the lesionsthis study has several limitations while this was a multicenter study the sample size was small hence more patientsshould be accrued and investigated in the near future moreover this study only included data that described the patients™ï¬ndings after the administration of the doacs and datadescribing the findings before their administration wereabsent therefore it remains unclear whether small intestinallesions are directly induced by doacs studies of patients™ï¬ndings before and after the administration of doacs areneeded in the near future furthermore patients with severeanemia and overt bleeding were excluded from this studyand most of the enrolled patients did not have bleeding orhad minor bleeding patients taking edoxaban were notincluded in this study because edoxaban was not available injapan when this study began conclusionlesionssmallbowelincluding redness erosions smallulcers and angioectasia were detected in of patientswho took doacs more patients using doacs should beinvestigated using ce in the near futureabbreviationsdoac direct oral anticoagulantvideo capsule endoscopyvceldalowdose aspirincapsule endoscopycegastrointestinalgihemoglobinhbegdesophagogastroduodenoscopyproton pump inhibitorppidata availabilitythis manuscript describes a study that was aimed at evaluating direct oral anticoagulant doac related to smallbowellesions using video capsule endoscopy we believe that ourstudy makes a significant contribution to the literaturebecause its findings showed that many patients takingdoacs had smallbowel lesions however most lesions wererelatively mild and they did not cause bleedingconflicts of interestthe authors have no conflicts of interest to disclose that arerelevant to this studyauthors™ contributionshiroyuki imaeda was responsible for the conception anddesign and final approval of the article minoru yamaokahiroyuki imaeda naoki hosoe kazuaki yoneno ryukanno hisashi mitsufuji takahiro sasaki and keijiyamamoto were responsible for enrollment of patientshiroyuki imaeda and naoki hosoe were responsible foranalysis and interpretation of the data minoru yamaokaand hiroyuki imaeda were responsible for drafting of thearticle naoki hosoe was responsible for the critical revision of the article for important intellectual content takanori kanai toshimasa yamamoto toshihide mimuraharuhiko ogata nobuo araki and hidetomo nakamotowere the supervisorsreferences w k leung i bjarnason v w s wong j j y sung andf k l chan œsmall bowel enteropathy associated withchronic lowdose aspirin therapy lancet vol no t watanabe s sugimori n kameda œsmall bowelinjury by lowdose entericcoated aspirin and treatment withmisoprostol a pilot study clinical gastroenterology andhepatology vol no pp “ j iwamoto y mizokami y saito œsmallbowel mucosalinjuries in lowdose aspirin users with obscure gastrointestinalbleeding world journal of gastroenterology vol no pp “ h endo k hosono m inamori œincidence of smallbowel injury induced by lowdose aspirin a crossover studyusing capsule endoscopy in healthy volunteers digestionvol no pp “ a shiotani k haruma r nishi œrandomized doubleblind pilot study of geranylgeranylacetone versus placebo inpatients taking lowdose entericcoated aspirin lowdoseaspirininduced small bowel damage scandinavian journalof gastroenterology vol no pp “ s j connolly m d ezekowitz s yusuf œdabigatranversus warfarin in patients with atrial fibrillation the newengland journal of medicine vol no pp “ m r patel k w mahaï¬ey j garg œrivaroxaban versuswarfarin in nonvalvular atrial fibrillation new england journal of medicine vol pp “ c b granger j h alexander j j v mcmurray œapixaban versus warfarin in patients with atrial fibrillation thenew england journal of medicine vol no pp “ c t ruï¬ r p giugliano e braunwald œcomparison ofthe efficacy and safety of new oral anticoagulants with warfarinin patients with atrial fibrillation a metaanalysis of randomised trials lancet vol no pp “ c s miller a dorreen m martel t huynh and a n barkun œrisk of gastrointestinal bleeding in patients taking nonvitamin k antagonist oral anticoagulants a systematic reviewand metaanalysis clinical gastroenterology and hepatologyvol no pp “1683e3 d caldeira m barra a ferreira œsystematic reviewwith metaanalysis the risk of major gastrointestinal bleeding 0cgastroenterology research and practicewith nonvitamin k antagonist oral anticoagulants alimentary pharmacology therapeutics vol no pp “ t yamashita y koretsune y yang œedoxaban vs warfarin in east asian patients with atrial fibrillationan engageaftimi subanalysis circulation journal vol no pp “ m hori m matsumoto n tanahashi œrivaroxaban vswarfarin in japanese patients with atrial fibrillation circulation journal vol no pp “ y toya s nakamura k tomita œdabigatraninducedesophagitis the prevalence and endoscopic characteristicsjournal of gastroenterology and hepatology vol no pp “ k kasai e ishida and y kobayashi œtwo cases of esophagealulcer caused by dabigatran gastroenterological endoscopyvol pp “ m okada and k okada œexfoliative esophagitis and esophageal ulcer induced by dabigatran endoscopy vol supplement pp e23“e24 t vanassche j hirsh j ginsberg and j eikelboom œanspecific bleeding patterns of anticoagulant therapy lessonsfrom clinical trials thrombosis and haemostasis vol pp “ h endo k hosono m inamori œcharacteristics ofsmall bowel injury in symptomatic chronic lowdose aspirinusers the experience of two medical centers in capsule endoscopy journal of gastroenterology vol no pp “ i watari s oka s tanaka œcomparison of smallbowelmucosalinjury between lowdose aspirin and nonaspirinnonsteroidal anti‚ammatory drugs a capsule endoscopystudy digestion vol no pp “ h endo k hosono t higurashi œquantitative analysisof lowdose aspirinassociated small bowel injury using a capsule endoscopy scoring index digestive endoscopy vol no pp “ d kaufman g leslie n marya œsmallintestinalangioectasia characterization risk factors and rebleedingjournal of clinical gastroenterology vol no pp “ a igawa s oka s tanaka œmajor predictors and management of smallbowel angioectasia bmc gastroenterologyvol no 0c'
Colon_Cancer
" exercise has been extensively utilised as an effective therapy for overweight and obesityassociatedchanges that are linked to health complications several preclinical rodent studies have shown that treadmillexercise alongside an unhealthy diet improves metabolic health and microbiome composition furthermorechronic exercise has been shown to alter hypothalamic and adipose tissue gene expression in dietinduced obesityhowever limited work has investigated whether treadmill exercise commenced following exposure to anobesogenic diet is sufficient to alter microbiome composition and metabolic healthmethods to address this gap in the literature we fed rats a highfathighsugar westernstyle cafeteria diet andassessed the effects of weeks oftreadmill exercise on adiposity dietinduced gut dysbiosis as well ashypothalamic and retroperitoneal white adipose tissue gene expression fortyeight male spraguedawley rats wereallocated to either regular chow or cafeteria diet and after weeks half the rats on each diet were exposed tomoderate treadmill exercise for weeks while the remainder were exposed to a stationary treadmillresults microbial species diversity was uniquely reduced in exercising chowfed rats while microbiome compositionwas only changed by cafeteria diet despite limited effects of exercise on overall microbiome composition exerciseincreased inferred microbialreduced fat mass and altered adipose andhypothalamic gene expression after controlling for diet and exercise adipose il6 expression and liver triglycerideconcentrations were significantly associated with global microbiome compositions moderate treadmill exercise induced subtle microbiome composition changes in chowfed rats but didnot overcome the microbiome changes induced by prolonged exposure to cafeteria diet predicted metabolic functionof the gut microbiome was increased by exercise the effects of exercise on the microbiome may be modulated byobesity severity future work should investigate whether exercise in combination with microbiomemodifyinginterventions can synergistically reduce diet and obesityassociated comorbiditieskeywords obesity microbiome exercise hypothalamus white adipose tissue cafeteria dietfunctions involved in metabolism correspondence mmorrisunsweduau1department of pharmacology school of medical sciences unsw sydneynsw australiafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cleigh nutrition metabolism page of introductionoverweight and obesity leads to reductions in physicaland mental health and quality of life resulting in increased direct and indirect costs to the global economy along with gross metabolic changes obesity is associated with altered fecal microbial species diversity and composition separate studies involving transferof obese human fecal microbiome samples induced fatgain in na¯ve mice and supplementation withakkermansia muciniphila improved insulin sensitivityand reduced body weightin overweight and obesepeople providing some evidence for a potential roleof diet and obesityassociated gut microbiota changes inadiposity and metabolic dysfunctionweight loss through lifestyle intervention is an effective strategy for reducing obesityrelated comorbidities one such intervention is moderate exercise a practical and sustainable approach for people with overweight and obesity while exercising at this intensityis unlikely to cause weight loss independent of caloricrestriction it confers cardiovascular and metabolic benefits and assists with weight maintenance [ ] furthermore regular exercise is known to improve glucoseregulation and insulin sensitivity as well as reducingcardiovascular disease and cancer risk andthere is increasing interest in the effects of exercise onthe gut microbiotathe first study to indicate an effect of exercise on fecalmicrobiome showed that elite professional athletes exhibited a distinct microbiome composition with increased microbial species diversity however sinceathletes consume a distinct diet from healthy people inthe community further work has been undertaken in rodents to identify the specific effects of exercise on fecalmicrobiome a recent systematic review of primarily rodent studies concluded that while there was no consistent effect of exercise on microbial species richnessexercise increases the relative abundance of firmicutes the different types of exercise used forced versusvoluntary have been shown to exert different effects onfecal microbiome composition in mice [ ] whichmay in part explain the inconsistent findingsfurthermore there were considerable differences inexercise duration used “ weeks which may contribute to the range of responses observed microbiomecompositional changes were observed in mice maintained on a healthy diet following weeks of moderatetreadmill exercise and after weeks in mice fed ahighfat diet in contrast eight weeks of lowtomoderateconfer microbiomecompositional changes in mice fed a highfat diet furthermore most studies examining the effects oftreadmill exercise on fecal microbiome in dietinducedobesity used a design wherecoexercise wasexercisedidnotadministered with highfat diet making it difficult totranslate the findings to people in terms of implementing exercise after a history of unhealthy eating andobesityhere we sought to examine whether moderate treadmill exercise in rats could exert benefits to gut microbiome composition following exposure to either ahealthy or a highfat highsugar westernstyle cafeteriadiet we aimed to investigate whether any changes ingut microbiome were associated with altered gene expression in white adipose tissue wat and the hypothalamus which are known to be affected by bothobesogenic diets and exercisematerials and methodssubjects and diet manipulationthis protocol was approved by the animal care andethics committee of unsw sydney in accordance withthe australian guidelines for the use and care of animalsfor scientific purposes national health and medicalresearch councilfortyeight male spraguedawley rats“ weeks g animal resource centre australia werehoused 3box °c h lightdark and handleddaily for one week while maintained on standard chow kjg premium rat and mouse maintenance dietgordon™s specialty stock feeds australia and water adlibitumfollowingacclimatization weightmatched groupswere randomly allocated to chow plus water or cafeteria diet n rats n cages per group ad libitumwhich comprised sucrosesolution alongsidecommerciallyproduced cakes cookies and savoury foods in addition to chow and water body weight and h food intake were measured twice weekly and food intake was calculated assuming equalintake per rat ineach cage body composition was measured during week by echomri900 echomri llc usacafsedcex cafeteria sedentarytreadmill exercisefollowing weeks half the rats in each dietary conditionwere allocated to treadmill exercise generating weightmatched groups chow sedentary csed chowexerciseandcafeteria exercise cafex for weeks until the day before sacrifice moderate exercise consisted of mmin for min five days a week at zero incline the firstweek comprised two training sessions “ mminafter which time spent at mmin was gradually increased sedentary rats were placed in stationary treadmills during the exercise protocol exercising rats wereclosely monitored three cafex rats showed signs of fatigue distress or vocalisation and were removed fromthe treadmill and thereafter exercised at a lower 0cleigh nutrition metabolism page of intensity mmin for min these rats were analysed together with moderately exercised cafex ratssample collectionat weeks of diet rats were deeply anaesthetized ketaminexylazine mgkg intraperitoneally bodyweight nasoanal length girth and blood glucose weremeasured following induction of anesthesia rats weredecapitated for trunk bloodthe hypothalamus within coronal block defined byrostrocaudal limits of circle of willis was rapidly dissected and collected retroperitoneal and gonadal watand liver were dissected and weighed one fecal pelletwas removed from the distal colon tissue and feceswere snap frozen in liquid nitrogen and stored at °cprotein and triglyceride measurementsplasma leptin and insulin concentrations were measuredusing commercial kits cat90040 and cat90060crystalchem inc usa plasma and liver triglyceridecontent were measured spectrophotometrically usingtriglyceride reagentroche diagnostics australia ptyltd australia at °c alongside a standard curvegenerated from glycerol standard g77935ml sigmaaldrich pty ltd australia livers were extracted byhomogenization in chloroformmethanol and incubated overnight nacl was added and samples werevortexed and centrifuged g for min the lowerphase was then evaporated at °c under nitrogen gasdried extract was redissolved in absolute ethanol andmeasured spectrophotometrically retroperitoneal watil6 content was determined using a duoset il6 ratelisa dy506 rd systems inc following manufacturers™ recommendationsstatistical analysesdata were analyzed using twoway betweensubjectsanova while measures over time were analysedusing 3way mixed anova posthoc pairwise comparisons were performed using a tukey adjustmentwhere appropriate thsd and presented in the associated figures and tables only when p pearson™scorrelations were used to identify associations allanalyses were completed using ibm spss statistics australiafecal dna extraction microbiome community sequencingand statistical analysesdna extraction was performed using the powerfecaldna isolation kit mobio laboratories usa microbial community composition was assessed by illuminaamplicon sequencing — bp miseq chemistry v4region 515f806r primer pair using a standard protocol sequence data were analyzed using mothur using modified commands from miseq sop including alignment with the silva database singletonremoval chimera checking with uchime and classification against the latest rdp training set sequence depthwas normalized by subsampling to total clean readsper samplecompletedusing calypso withotuoperationaltaxonomic unitcorrelationswerethebenjaminihochberg false discovery rate fdr procedure used to control for multiple tests fdrcorrected deseq2 was performed using the phyloseq r package for the negative binomial walk test indeseq2 otu abundances were analyzed usingspss with kruskalwallisfollowed by nonparametric bonferronidunn posthoc testing whereappropriate otus ofinterest were identified usingsina aligner testsrna extraction and gene expression assaysrna was extracted from hypothalamus and retroperitoneal wat using tri reagent sigmaaldrich pty ltdaustralia following dnase i treatment catalogue merck australia or μg of rna wat andhypothalamus respectively were reverse transcribed toproduce cdna high capacity reverse transcriptasekit thermofisher scientific usa gene expression wasassessed using taqman inventoried gene expression assays life technologies australia pty ltd australia seesupplementary table genes of interest were normalized against the geometric mean of the two most stablehousekeeping genes gapdh and hprt1 for wat hprt1and b2m for hypothalamus identified by the normfinder package analysis of relative gene expressionwas performed using the δδct method normalized toan independent calibrator made from all samplesdblmalpha diversity metrics distancedbased linear modellingpermutational multivariate anovapermanova nonmetric multidimensional scalingnmds and canonical analysis of principal coordinateswere completed using primer v6 primere ltdplymouth united kingdom all primer analysesutilized a braycurtis similarity matrix constructed atthe otu levelphylogenetic investigation of communities by reconstruction of unobserved states picrust was performed using galaxy web to predict putative functionsthrough metagenomic prediction from the 16s otudata using greengenes for taxonomic classification pathway counts were compared across groupsusing fdrcorrected kruskalwallis tests followed bynonparametric bonferronidunn posthoc testing whereappropriate 0cleigh nutrition metabolism page of resultsenergy intake body weight and composition and watgene expressionover the 7week study cafeteriafed rats ate more thantwice the energy consumed by chowfed groups fig 1acsed kjrat cex kjrat cafsed kjrat cafex kjrat when energy intake wasstratified into pre and duringexercise intervention asignificantinteraction between time and diet wasobserved fig 1b f p which appeared to be due to increased cafeteria diet intake whilerats were exercising although this comparison did notreach statistical significance significant interactions between time and diet were also identified for fat andfig shortterm treadmill exercise reduces body weight gain and fat mass and alters wat expression without affecting energy intake a energyintake over the study and b average weekly energy intake before and during treadmill exercise intervention c body weight of the study and dbody weight gain during treadmill exercise intervention e fat mass as a percentage of body weight and f absolute lean mass data expressedas mean ± sem n for cage data n “ for individual data data were analyzed by twoway anova followed by tukeyadjusted posthoctesting ap relative to csed bp relative to cex cp relative to cafsed 0cleigh nutrition metabolism page of carbohydrate intakes supplementary figure 1a f p and supplementary figure 1c f p respectively where both macronutrient intakes increased in the cafeteriafed rats during exercise protein intake while unaffected by time orexercise was elevated by cafeteria diet supplementaryfigure 1b f p sucrose intake increased over time supplementary figure 1d f p but was not affected by exerciseall rats gained body weight over time fig 1c andcafeteriafed rats gained significantly more than chowfed controls prior to f p and during fig 1d f p exercise exercisesignificantly reduced weight gain overall f p thsd posthoc comparisons showed thatcompared to csed and cex cafsed p and p and cafex p and p gained significantly more weight over the exercise intervention ofnote cafsed rats gained significantly more weight thancafex rats over the exercise intervention p body composition data following weeks of treadmillexercise showed that relative fat mass was significantlyincreased by cafeteria diet fig 1e f p absolute fat mass presented in table and reduced by exercise f p thsdposthoc comparisons revealed that relative fat mass inboth cafeteriafed groups were significantly greater thanboth chowfed groups lean mass however showed onlyan overall diet effect fig 1f f p with significantly more lean mass in cafeteriafed ratsthan chowfed ratscafeteria di so increased nasoanallength girthplasma insulin and triglycerides with no effect of exercise table plasma leptin levels were significantly increased by diet f p and reducedby exercise f p unfasted bloodglucose did not differ between groups table in line with plasma leptin retroperitoneal and epidydimal fat pad weights were significantly greater in cafeteria than chowfed rats f p andf p for retroperitoneal and epidydimal fat pads respectively and were significantly reduced by exercise overall f p and f p for retroperitoneal and epidydimalfat pads respectively see table microbial species diversity and microbiome compositionmicrobial species diversity was assessed using shannon™sdiversity index microbial species richness and microbialspecies evenness shannon™s diversity and evenness weresignificantly reduced by cafeteria diet overall f p fig 2a and f p fig 2c respectively and thsd posthoc comparisonsshowed that cafex rats exhibited reduced evenness relative to cex p and reduced shannon™s diversityrelative to csed p no significant differenceswere observed for bacterial species richness fig 2bmicrobiome composition at the otu level was signifipseudof p cantly affected by diet and cage pseudof p butnot by exercise when assessed using 4way permanova permutations and confirmed with nontable anthropometric measures at tissue collection and plasma measuresmeasurecafsedcsedcexterminal body weight gnasoanal length cmgirth cmliver scoreheart weight g ± ± ± 262ab ± ± ± ± ± ± ± ± cafex ± 124ab ± 01b ± 04ab ± 022ab ± 003ab ± 681ab ± 047ab ± 021ab ± ± 043ab ± 121ab ± 025ab ± 241abmain effects pvalueexercisediet interaction ± ± 06ab ± 017ab ± 007ab ± 1627ab ± 043ab ± 054ab ± ± 032ab ± 143ab ± 032ab ± 252ababsolute fat mass g echomri ± ± rp fat pad weight bw ± ± epidydimal fat pad weight bw ± ± blood glucose mmoll ± ± plasma insulin ngmlplasma leptin ngml ± ± ± ± plasma triglycerides mmoll ± ± liver triglycerides mgg tissueblood and plasma measures performed unfasted data expressed as mean ± sem n “ data were analyzed using twoway anova followed by posthocmultiple comparisons with a tukey hsd correction liver score was analysed using a kruskalwallis test followed by nonparametric bonferronidunnposthoc testingap relative to csed bp relative to cex ± ± 0cleigh nutrition metabolism page of fig see legend on next page 0cleigh nutrition metabolism page of see figure on previous pagefig impact of cafeteria diet and treadmill exercise intervention on fecal microbiota and inferred microbiome function at weeks a shannon™sdiversity b microbial species richness and c evenness data expressed as mean ± sem n “ data were analyzed by twoway anovafollowed by tukeyadjusted posthoc testing d nonmetric multidimensional scaling braycurtis permutations showing similarity betweenfecal microbiota samples at weeks e muribaculum_otu72 identified by deseq2 adjusted p as differentially abundant with exercise inchowfed rats f relative abundance of otu72 at weeks data expressed as boxandwhisker plots min iqr max n “ data were analyzedusing kruskalwallis test followed by nonparametric dunnbonferroni posthoc testing g amino acid metabolism h overall energy metabolism idglutamine and dglutamate metabolism and j one carbon pool by folate predicted using picrust from fecal microbiome data at weeks dataare expressed as boxandwhisker plots min iqr max n “ were analyzed by kruskalwallis tests fdradjusted overall pvalue to accountfor multiple relevant pathways included in analysis followed by nonparametric bonferronidunn posthoc comparisons posthoc symbols ap relative to csed bp relative to cex cp relative to cafsedmetric multidimensional scaling fig 2d supplementary figure shows groups differences in microbiomecomposition at the phylum leveldeseq2 analyses were used to identify otus differentially enriched with exercise exposure amongst the top otus while exercise was not associated with differentially expressed otus in cafeteriafed rats muribaculum_otu72 was significantly depleted in cex ratsrelative to csed fig 2e relative abundance in fig 2fthis otu was originally classified as akkermansia whenaligned with the rdp reference library muribaculum_otu72 was putatively identified as an unknown bacterium from the genus muribaculum using sina aligner alignment identitypredicted microbiome functionto determine whether the subtle microbiome composition changes observed with exercise affected microbiome function we inferred microbiome function usingpicrust following an fdr correction amino acidmetabolism fig 2g h p overallenergy metabolism fig 2h h p dglutamine and dglutamate metabolism fig 2i h p and one carbon pool by folate fig 2jh p exhibited overall group differences amino acid metabolism was significantly elevatedin cafex relative to csed p and cafsed p rats while the other processes were significantlyelevated in both exercised groups relative to csedwat and hypothalamic gene expressionexamination of wat inflammatory signaling andbrowning genes fig 3a revealed a significant interaction effect for ucp1 f p whilethsd posthoc comparisons showed that cafex ratsexhibited elevated ucp1 expression relative to cafsedp lep expression was significantly elevatedby cafeteria diet consumption f p while lepr expression was increased with exercise overall f p no significantdifferences wereproinflammatorymarkers or adipoq gene expressionobservedinhypothalamic gene expression was analyzed to determine if weeks of moderate exercise was sufficient toreverse dietinduced changes in expression of genes involved in feeding fig 3b blood brain barrier integrityand proinflammatory signaling fig 3c a significantinteraction effect was observed for npy gene expressionin the hypothalamus f p andthsd showed that cafsed rats exhibited downregulatednpy relative to csed p cex p andcafex rats p no significant differences wereobserved for pomc agrp npy1r lepr or insr crh geneexpression was significantly increased in cafeteriafedrats overall f p but was unaffected by exercise f p posthocthsd analysis revealed that cafex rats exhibited significantly upregulated crh relative to csed p andcex p both cln5 and glut1 were significantly increased incafeteriafed rats overall f p andf p respectively while no groupdifferences were apparent for cln5 using thsd comparisons glut1 expression was significantly elevated incafsed rats relative to csed controls p no significant differences were observed in ocln tjp1 or anyof the proinflammatory genes assessedassociations between variables of interest andmicrobiomewhen variables were assessed for their unique contribution to the variance in overall microbiome compositionseveral adiposity measures as well as retroperitonealwat il6 gene expression and hypothalamic crh andnpy expression were identified as significant predictorsof global microbiomecomposition supplementarytable when the contribution of variables of interestto overall microbiome composition was assessed whilecontrolling for diet and treadmill exercise both livertriglyceride concentration r2 p andretroperitoneal wat il6 gene expression r2 p were significant predictors of microbiomecomposition attable completemodel predicts of the variance in microbiomecompositionthe otu level 0cleigh nutrition metabolism page of fig gene expression in retroperitoneal wat hypothalamus and associations with gut microbiome changes a adipokine metabolic andinflammatory gene expression in retroperitoneal wat b feeding and stressrelated gene expression in the hypothalamus c bloodbrain barrierand proinflammatory gene expression in the hypothalamus adipoq adiponectin agrp agoutirelated protein cln5 claudin5 crh corticotrophinreleasing hormone glut1 glucose transporter il6 interleukin il10 interleukin10 il1b interleukin beta insr insulin receptor lep leptin leprleptin receptor npy neuropeptide y npy1r neuropeptide y receptor ocln occludin pomc proopiomelanocortin tjp1 tight junction protein tnf tumour necrosis factor ucp1 uncoupling protein data expressed as mean ± sem n “ data were analyzed by twoway anovafollowed by tukeyadjusted posthoc testing ap relative to csed bp relative to cex cp relative to cafsedporphyromonadaceae unclassified_otu106 wasincreased with cafeteria diet fig 4a h p and significantly associated with wat il6 geneexpression fig 4b interestingly this otu was alsocorrelated with hypothalamic npy genenegativelyexpression fig 4c il6 protein in wat exhibited asignificant interaction effect f p fig 4d and was positively associated with porphyromonadaceae unclassified_otu106 fig 4efollowing asimilar although nonsignificant trend to that observedwith il6 gene expression like il6 gene expression watil6 content was a significant independent predictor ofoverall microbiome composition supplementary table but was not a significant predictor in the final model 0cleigh nutrition metabolism page of table the shared contributions of diet exercise and variables of biological relevance on the variance observed in microbiomecomposition using distancebased linear modellingvariabledietpseudofpvaluessr2cumulative r2exerciseliver triglyceride contentwat il6 expressionsequential multiple regression captured by the braycurtis similarity matrix at the otu level max permutations involves interrogating the conditionalcontribution of each variable in order of entry into the model to determine whether variables contribute significantly to the variance explained in the presence ofother variables here diet and exercise conditions were added before any metabolic predictors were considered and the final model containing only statisticallysignificant covariates is shown metabolic predictors included in the sequential regression were selected based on their predictive value while trying to eliminatevariables with high covariance n “after controlling for diet and exercise sina alignerputatively identified porphyromonadaceae unclassified_otu106 as a strain of bacteroides eggerthii a gramnegative bacterium known to hydrolyze carbohydratesincluding simple sugars discussionwe found that weeks of moderate treadmill exercisereduced fat mass and plasma leptin concentrations andaltered wat expression ofsome adipokine andmetabolismassociated genes overall microbiome composition and microbial species diversity was changed bycafeteria diet but not by exercise however predictedmicrobial functions associated with metabolism were increased by exercise cafeteria dietinduced changes inhypothalamic npy and glut1 gene expression werereturned to control levels by exerciseexercise induced modest changes in gut microbiomecomposition that were statistically significant in chowfed rats only and the relative abundance of otu72 wassignificantly reduced in chowfed rats that exercisedcex rats relative to sedentary controls csed whilecafex rats exhibited reduced microbial species diversitythis reduction appeared dependent on cafeteria dietexposure rather than exercise to date work examiningthe effects of exercise on microbial species diversity hasproduced inconsistent results some rodent studieshave shown that exercise is associated with reductions infecal microbial species richness while others havereported no such effect after weeks of treadmill exercise we did not observechanges in overall microbiome composition our dataare in line with findings in humans showing thatchanges in microbiome composition are dependent onobesity status such that more severe obesity is associated with smaller effects of exercise since thecafeteria diet used here tends to produce a more severe metabolic phenotype than purified highfat diets[“] which to our knowledge have been used in allstudies investigating the interrelationship between exercise and diet on microbiome compositionthe dietinduced effects on microbiome composition here may bemore resistant to the effects of exercise than previouslyreported additionally a number of rodent studies report no differences in overall microbiome compositionwith exercise [ ] and there is evidence that this effect may be moderated by age which may have contributed to the inconsistent findings in the literaturepredicted microbial functions associated with metabolism specifically overall energy metabolism amino acidmetabolism one carbon pool by folate and dglutamineand dglutamate metabolism were increased in exercised rats this is in line with metagenomic resultswhere fecal microbiome from male elite athletes exhibited increased amino acid biosynthesis and overall energy metabolism relative to sedentary normalweightcontrols while picrust analysis produces predicted functional data unlike metagenomic analysis thisis an interesting finding that warrants followup to determine if and how exercise shifts the metabolic profileof the gut microbiome and whether any such shift is affected by exercise intensity and duration furthermoreconfirming these results across a range of diets would beuseful to determine whether the shift in microbial function with exercise is modulated by the macro andmicronutrients availablehere a moderate exercise intervention reduced fatmass and plasma leptin concentrations and increasedwat lepr gene expression in both exercised groupsand ucp1 gene expression in exercised cafeteriafedcafex rats uniquely increased ucp1 in wat depots isa marker of adipocyte beiging known to be promoted by exercise and is most likely related toexerciseinduced fat loss while there were no significanteffects of exercise on wat proinflammatory gene expression after controlling for diet and exercise wat il6expression wassignificantly associated with globalmicrobiome compositionwat is one of the major sources of il6 in obesehumans and mice [ ] which is a key component of the lowgrade systemic inflammation observedin overweight and obesity and is associated with 0cleigh nutrition metabolism page of fig relationship between otu106 and hypothalamic npy and wat il6 expression a relative abundance of otu106 data expressed as boxandwhisker plots min iqr max n “ data were analyzed using kruskalwallis test followed by nonparametric dunnbonferroni posthoctesting scatterplots for otu106 abundance and b il6 gene expression in wat and c npy gene expression in the hypothalamus showingoverall lines of best fit n d wat il6 protein content data expressed as mean ± sem n “ data were analyzed by twoway anovafollowed by tukeyadjusted posthoc testing e scatterplot for otu106 abundance and il6 protein content in wat showing overall lines of bestfit n ap relative to csed bp relative to cex cp relative to cafsedinsulin resistance wat il6 expression was stronglyassociated with the relative abundance of a strain ofbacteroides eggerthii otu106 a gramnegative sugarscavenging bacterium which is enriched in obesechildren relative to normalweight controls theassociations between wat il6 expression global microbiome composition and otu106 abundance are therefore likely to be due to the effects of cafeteria diet onboth adipose inflammatory processes and the gut microbiome however probiotic treatment with a strain of 0cleigh nutrition metabolism page of bifidobacterium in mice fed a highfat diet reducedwat macrophage infiltration and plasma il6 concentration indicating that changes to the gut microbiome may contribute to wat inflammatory signalingand il6 production furtherstudies determiningwhether specific bacterial species can modulate watil6 production are warranted as interventions thatcould reduce wat il6 expression in obesity may provide an avenue for preventing insulin resistance and type diabetesin contrast to the overall effect of exercise on watgene expression hypothalamic genes disrupted by cafeteria diet were typically normalized with exercise withno differences observed in chowfed rats npy wasdownregulated in cafsed rats as shown previously but normalized to control levels with exercise which isin line with other rodent work showing increased hypothalamic npy mrna and protein in response to bothacute and chronic exercise [“] hypothalamic glut1gene expression was increased in cafsed rats and reduced to control levels with exercise this is in contrastto studies showing that acute exercise increased glut1protein expression across the rat brain and prolonged exercise increases whole brain resting glucoseuptake in people further work investigating acuteand chronic exerciseinduced changes in glut1 expression in the hypothalamus and other brain regions isrequiredactivatescafeteria dietinduced crh upregulation in the hypothalam
Colon_Cancer
" rectus sheath block rsb is known to attenuate postoperative pain and reduce perioperative opioidconsumption thus a retrospective study was performed to examine the effects of bilateral rectus sheath blockbrsb in cytoreductive surgery crs combined with hyperthermic intraperitoneal chemotherapy hipecmethods a total of patients undergoing crshipec at our hospital were included patient information andanaesthesiarelated indicators were collected from the electronic medical record emr system all subjects weredivided into the following two groups the g group general anaesthesia and the gr group rsb combined withgeneral anaesthesia patients in the gr group received ropivacaine for brsb before surgery the primaryoutcomes included the total amount of remifentanil and rocuronium the total consumption of dezocine aftersurgery the visual analogue scale vas score and the patientcontrolled intravenous analgesia pcia input dose at h t6 h t7 h t8 h t9 and h t10 after surgery other outcomes were also recorded such aspatient demographic data the intraoperative heart rate hr and mean arterial pressure map and postoperativecomplicationsresults compared with the g group the gr group showed a shorter time to tracheal extubation p adecreased total amount of remifentanil and rocuronium p and a reduced vas score pcia input dose andnumber of pcia boluses at h h and h after surgery p however at h and h after surgery therewere no differences in the vas score of pain at rest or during motion between the two groups p moreoverthe incidence of hypertension emergence agitation delayed recovery hypercapnia and nausea and vomiting waslower in the gr group than in the g group p there were no differences in the changes in map and hrduring the surgery between the two groups p no complications associated with nerve block occurred brsb could provide shortterm postoperative analgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complications it is an effective and safe procedure in crshipeckeywords cytoreductive surgery hyperthermic intraperitoneal chemotherapy rectus sheath block generalanaesthesia analgesia correspondence trmzltz126comdepartment of anesthesiology beijing shijitan hospital capital medicaluniversity beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc anesthesiology page of radical cytoreductive surgery crs combined withhyperthermic intraperitoneal chemotherapy hipec isconsidered a standard for the treatment of peritonealcancer such as rectal cancer ovarian cancer peritonealpseudomyxoma and peritoneal mesothelioma thistechnique could prolong the longterm survival of patients with a decreased recurrence rate although thepositive results of this treatment have been proven inprevious studies [“] because of the large peritonealsurface area involved in this kind of surgery crshipecis time consuming and complex which presents agreat challenge for the anaesthesiologist in terms of perioperative managementdue to the stable respiratory and circulatory supportgeneral anaesthesia is the preferred choice in this surgery however long periods of general anaesthesia leadto drug accumulation in the body followed by increasedanaesthesiarelated complications including delayed recovery respiratory inhibition and cognitive dysfunction consequently exploring better anaesthesia methodsfor this surgery is still a major concerna new approach called ultrasoundguided bilateral rectus sheath block brsb has been proven to amelioratepostoperative pain and reduce the consumption of morphine [“] nonetheless there have been no reportson the application of general anaesthesia combined withbrsb in patients undergoing crshipec based on theinformation presented above this retrospective observational study was conducted to examine the efficacy andsafety of brsb in patients treated with crs and hipecmethodssubjectsall patients who underwent crs and hipec at beijingshijitan hospital between august and december were retrieved from the institutional database inthis study the exclusion criteria were as follows laparoscopic surgery with crshipec intraoperativeblood loss volume greater than ml mechanicventilation required after surgery and use of analgesictechniques apart from brsb and general anaesthesiaaccording to this standard a total of patients wereincluded and divided into the following groups generalanaesthesia g group n and general anaesthesiacombined with posterior rsb gr group n anaesthesia methodgeneral anaesthesia was consistently induced in all patients with intravenous propofol mgkg sufentanil μgkg and rocuronium mgkg invasive arterialpressure and central venous pressure were monitored byradial artery puncture flotracvigileo® edwards lifesciences irvine ca usa and internaljugular veinsitetargeteffectpuncture respectively after anaesthesia induction anaesthesia was maintained with sevoflurane and remifenconcentration “ ngmltanilkeeping the bispectral index bis between and rocuronium mgkg wasintermittently used tomaintain muscle relaxation in the gr group before anaesthesia induction patients received brsb under ultrasound guidance the puncture site was placed at theouter edge of the bilateral rectus abdominis at the levelof the umbilicus fig a a total of ropivacaine ml was injected into each side the spindleshapedspread of ropivacaine was observed between the posterior sheath of the rectus abdominis and the rectus abdominis itself implying success of the procedure fig b c patientcontrolled intravenous analgesia pciawas applied in both groups after the surgery sufentanil μgkg palonosetron hydrochloride mg was diluted to ml the dose was mlh and asingle dose was mlh with a 15min lockout intervalafter the surgery all patients were sent to the surgicalintensive care unit sicu if the visual analogue scalevas score at rest after surgery was ‰¥ dezocine mgwas used as a rescue analgesicdata collectionall the indicators we needed were obtained from theemr system the records included patient demographicdata patient medical history american society of anesthesiologists asa grade and new york heart association nyha grade the hr and map were recordedat the time before brsb t1 the time of anaesthesiat2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgeryt5 in addition the duration of the surgery time totracheal extubation the time after skin closure totalamount of remifentanil and muscle relaxants total fluidvolume urine volume and the total volume of allogeneicerythrocytes and plasma infused during the surgery wereall recorded moreover after surgery the occurrence ofhypertension the systolic blood pressure dropped bymore than of baseline blood pressure beforeanesthesia or the sbp mmhg during surgery nausea and vomiting hypoxemia spo2 or pao2 mmhg hypercapnia paco2 mmhg and emergence agitation during the recovery period were recorded the recovery period was considered as the timefrom switching off inhalation anaesthetics remifentaniland muscle relaxant to recovery of the patients™ abilitiesto command movement orientation as well as conscious state when the recovery period of patients is beyond min it was considered as delayed recovery thevas score for pain at rest and during motion the pciainput dose and the number of boluses at h t6 ht7 h t8 h t9 and h t10 after surgery 0cwang bmc anesthesiology page of the incomplete datachemotherapy crshipec during the year from to in our hospital one hundred and six patientsreceived brsb seventeen patients were excluded because ofincluding patientsundergoing intraoperative haemorrhage blood ml and other patients received mechanic ventilationbecause of acute respiratory distress syndrome ardsallergic shock and cardiac insufficiency thus patients with brsb were eventually obtained finally patients without brsb were randomly selected to analysis in this study fig statistical analysisspss software was used for statistical analysisnormal distribution data were recorded as the mean ±standard deviation sd and analysed by independentsamples t test for comparison between the two groupsnonnormally distributed data are presented as themedian range and were analysed by kruskalwallistest chisquared test or fisher™s exact test was used forcategorical data a p value of was considered statistically significantresultscharacteristics of study populationin total patients were included in the study thebaseline demographic and surgical variables of patientsare presented in table there were no significant differences in age sex body mass index bmi basic diseases asa grade nyha grade total surgery time totalfluid volume urine volume total volume of allogeneicerythrocyte infusion or total volume of plasma p however the time to tracheal extubation was shorter inthe gr group than in the g group p the totalamount of both remifentanil and rocuronium used wasless in the gr group than in the g group p thus posterior rsb could reduce the use of remifentaniland rocuronium during surgerychanges in haemodynamic parametersthe changes in hr and map are presented in table there were no significant differences in hr or map atany point in time t1 to t5 between the two groupsp the results suggest that brsb did not affectthe haemodynamics ofthe patient undergoing crshipecpainrelated indicatorstable shows the postoperative vas score the pca input dose and the number of pca boluses at h t6 ht7 h t8 h t9 and h t10 after surgeryas well as the dose of dezocine used as a rescue analgesic from t6 to t8 compared with the g group thegr group showed significantly decreased vas scores offig ultrasoundguided brsbas well as the dose of dezocine used as a rescue analgesic were also recordedin addition brsbrelatedcomplications such as peritoneal punctureinternalan injury and systemic toxicity were all recordeda total of patients underwent cytoreductive surintraperitonealcombined withgeryhyperthermic 0cwang bmc anesthesiology page of fig flow chart showing patient consecutive enrolment and analysis abbreviations crshipec cytoreductive surgery and hyperthermicintraperitoneal chemotherapy ga general anesthesia brsb bilateral rectus sheath block ards acute respiratory distress syndrome vas visualanalogue scalepain at rest and during motion p however at h and h after surgery there were no significant differences in the vas scores of pain at rest and duringmotion between the two groups p from t6 tot10 the pcia input dose and the number of pca boluses were also obviously reduced in the gr group compared with the g group p in addition as arescue analgesic the dose of dezocine after surgery inthe gr group was significantly lower than that in the ggroup p postoperative adverse eventsadverse events that occurred in the sicu are presentedin table after surgery there were cases withhypertension cases of emergence agitation cases ofdelayed recovery cases of hypercapnia and cases ofnausea and vomiting in the g group fewer cases of allof these events occurred in the gr group p there were no differences in the incidence of hypoxemiabetween the two groups p there were no complications associated with nerve block in either group 0cwang bmc anesthesiology page of table demographic and surgical variables mean ± sdage ysex malefemalebmi kgm2medical historydiabetes mellitus n yesnohypertension n yesnocoronary heart disease n yesnoasa grade iiiiiinyha grade iiitotal surgery time mintime to tracheal extubation minremifentanil mgrocuronium mgtotal fluid volume mlurine volume mltotal volume of allogeneic erythrocyte infusion mltotal volume of plasma mlg group n ± ± ± ± ± ± ± ± ± ± gr group n ± ± ± ± ± ± ± ± ± ± p value asa american society of anesthesiologists bmi body mass index calculated as weight in kilograms divided by height in metres squared nyha new york heartassociation g general anaesthesia gr bilateral rectus sheath block combined with general anaesthesia before bilateral rectus sheath block t1 the time ofanaesthesia t2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgery t5discussionin this retrospective study we examined the efficacy andsafety of brsb combined with general anaesthesia in patients undergoing crshipec regarding efficacy theresults show that ultrasoundguided brsb significantlyreduced the total dose of remifentanil used during thesurgery and shortened the time to tracheal catheter extraction which is consistent with the findings of previous studies of other surgeries [ ] in addition rsbreduced the total dose of rocuronium in this studytable haemodynamic parameters in both groups mean ± sdindextimepointgn ± grn ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± pvaluemmhghrbpmt1t2t3t4t5t1t2t3t4t5map mean arterial pressure hr heart rate g general anaesthesia gr bilateralrectus sheath block combined with general anaesthesiawhich may be associated with the high concentration ofropivacaine used in the studyrsb also effectively relieved postoperative pain in thisstudy we found that the vas scores of pain at rest andduring motion were all lower in the gr group than inthe g group at h after surgery however at h and h after surgery there were no differences in the vasscores of pain at rest and during motion between thetwo groups suggesting that the analgesic effects of asingle brsb remained within h after surgery thisresult may be different from the findings of others cho reported that at h after surgerythere were no differences in the vas scores of painat rest and during motion between the rsb and nonrsb groups the discrepant results may be related todifferences in the concentration of ropivacaine andthe physical constitution of patients a high concentration can prolong the duration of action of a localanaesthetic in this study we selected not ropivacaine additionallythese patientsundergoing crshipec may have been adaptive topain furthermore compared with the control groupthe rsb group showed a reduced totalinfused doseof sufentanil as pcia number of pca boluses within h after surgery and total dose of dezocine used asa rescue analgesic after surgery these results furtherprove the role of rsb in providing shortterm postoperative analgesia 0cwang bmc anesthesiology page of table painrelated indicators in both groups median [range]vas score of pain at rest [median range]t6t7t8t9t10vas score of pain during motion [median range]t6t7t8t9t10total infused dose of pcia [ml median range]t6t7t8t9t10cumulative number of pcia boluses [median range]t6t7t8t9t10total dose of dezocine as a rescue analgesic mggn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± grn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± p value vas visual analogue scale pcia patientcontrolled intravenous analgesia g general anaesthesia gr bilateral rectus sheath block combined with generalanaesthesia the time at h after surgery t6 h after surgery t7 h after surgery t8 h after surgery t9 and h after surgery t10table postoperative adverse events in both groupsadverse eventsn gn grn hypertensionemergence agitation delayed recoveryhypoxemiahypercapnia nausea and vomiting peritoneal punctureinternal an injurysystemic toxicity g general anaesthesia gr bilateral rectus sheath block combined withgeneral anaesthesiawe also examined the safety of rsb during the surgery ultrasoundguided brsb had no significant effectson the haemodynamics of patients during surgery compared with general anaesthesia alone in terms of postoperative adverse events the results show that comparedwith the control group the rsb group showed a reducedincidence of hypertension emergence agitation delayedrecovery hypercapnia and nausea and vomiting whichmight be correlated with the decreased analgesic andmuscle relaxant doses no rsbrelated complications occurred in any patient these data indicate that rsbcould reduce the risk of complications associated withgeneral anaesthesia and is safe for patientsrsb an established technique has regained popularityin clinical applications [“] previous studies havedemonstrated that this technique could achieve relaxation of the anterior abdominal wall [ ] bashandyreported that anterior branches of the t7t12 thoracicnerve and the l1 lumbar nerve travelled through thepvalue“““ 0cwang bmc anesthesiology page of plane of the transverse abdominis muscle entered the rectus abdominis sheath and distributed on the surface of theskin the main process of rsb is to inject local anaesthetics between the rectus abdominis and the posteriorsheath of the rectus abdominis therefore rsb exerteda good effect in terms of perioperative analgesia for medianabdominal incisions a midline incision is required inthis kind of surgery thus based on these results rsbcould meet the need for analgesia in these patientsin addition for a long time epidural analgesia eawas thought to be an effective method for abdominalsurgery [“] studies have proved that epidural analgesia could maintain a good analgesic effect and reduceperioperative opioid consumption including in this typeof surgery [ ] however the safety of ea in crshipec remains controversial especially regarding effectson coagulation and circulatory function coagulationdysfunction and profound fluid loss are the main characteristics of patients with peritoneal cancer [ ]which might limit the administration of eaalthough epidural catheter is standard of care insolanki™s guideline in our hospital epidural catheter in not the standard of care we performed generalanesthesia combined with epidural anesthesia in somepatients to reduce the consumption of intravenous drugsand provide perfect analgesia but coagulation dysfunction and profound fluid loss are the main characteristicsof patients with peritoneal cancer in our previous studywe found that the mean arterial pressure of patientsundergoing epidural anesthesia was difficult to be maintained in the surgery besides there were many patientswith coagulation dysfunction before surgery who werenot suitable for the epidural anesthesia these results wefound in clinical were similar with others™ researcheskajdi and colleagues reported a case of epidural haematoma in their study godden found that the incidence of hypotension in the ea group was obviouslyhigher than that in the rsb group consequentlyrsb could be a better choice than ea in crshipecadditionally there are some limitations to this studyfirst all the data of this study were collected from theemr system as this was a retrospective study the findings are not as persuasive as those of a randomized controlled study we plan to conduct prospective studies toexplore the comprehensive influence of rsb in this surgery second we only examined the application of brsbestablished with a single injection which provides only ashortterm analgesic effect the efficacy of continuousanalgesia with brs catheters in crshipec remains unclear and needs further exploration third in our the results are initially presented according to the different aspects the primary outcome of this study is thetotal consumption of remifentanil during the surgeryother indicators were belonged to second outcomessin brsb could provide good postoperativeanalgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complicationsthis is an easily applicable and safe procedure in crshipecabbreviationsrsb rectus sheath block brsb bilateral rectus sheath blockcrs cytoreductive surgery hipec hyperthermic intraperitonealchemotherapy emr electronic medical record vas visual analogue scalepcia patientcontrolled intravenous analgesia hr heart rate map meanarterial pressure bis bispectral index sicu surgical intensive care unitasa american society of anesthesiologists nyha new york heartassociation spo2 pulse oximetry paco2 partial pressure of carbon dioxidepao2 oxygen partial pressure bmi body mass indexacknowledgementsi would like to express my heartfelt thanks to the staff of the informationdata center of beijing shijitan hospital affiliated to capital medical universityauthors™ contributionswsh study design data collection writing paper lpf gt data collectionand data analysis gl coordinated the study and manuscript revision ltzstudy design and manuscript revision all authors read and approved thefinal manuscript all authors ensure the accuracy of the manuscript andagree to take personal responsibility for their contributionsfundingno fundingavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethis study was approved by the ethics committee of beijing shijitan hospitalaffiliated to capital medical university approval code research ethicsno69 this study is retrospective only anonymous data sources were usedand informed consent was not requiredconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesklaver ce musters gd bemelman wa punt cj verwaal vj dijkgraaf mgaalbers ag van der bilt jd boerma d bremers aj adjuvanthyperthermic intraperitoneal chemotherapy hipec in patients with coloncancer at high risk of peritoneal carcinomatosis the colopec randomizedmulticentre trial bmc cancer 201515undefined428van oudheusden tr braam hj nienhuijs sw wiezer mj van ramshorst bluyer md lemmens ve de hingh ih cytoreduction and hyperthermicintraperitoneal chemotherapy a feasible and effective option for colorectalcancer patients after emergency surgery in the presence of peritonealcarcinomatosis ann surg oncol “passot g vaudoyer d villeneuve l kepenekian v beaujard ac bakrin ncotte e gilly fn glehen o what made hyperthermic intraperitonealchemotherapy an effective curative treatment for peritoneal surfacemalignancy a 25year experience with procedures j surg oncol“arjonasánchez a barrios p boldoroda e camps b carrascocampos jmartín vc garcíafadrique a gutiérrezcalvo a morales r ortegapérez g hipect4 multicentre randomized clinical trial to evaluate safety andefficacy of hyperthermic intraperitoneal chemotherapy hipec with 0cwang bmc anesthesiology page of huepenbecker sp cusworth se kuroki lm lu p samen cd woolfolk cdeterding r wan l helsten dl bottros m continuous epiduralinfusion in gynecologic oncology patients undergoing exploratorylaparotomy the new standard for decreased postoperative pain and opioiduse gynecol oncol “teoh da hutton mj else s walker a lee a mack la epidural analgesia aprospective analysis of perioperative coagulation in cytoreductive surgeryand hyperthermic intraperitoneal chemotherapy am j surg “schmidt c creutzenberg m piso p hobbhahn j bucher m perioperativeanaesthetic management of cytoreductive surgery with hyperthermicintraperitoneal chemotherapy anaesthesia “kajdi me beckschimmer b held u kofmehl r lehmann k ganter mtanaesthesia in patients undergoing cytoreductive surgery withhyperthermic intraperitoneal chemotherapy retrospective analysis of asingle centre threeyear experience world j surg oncol solanki sl mukherjee s agarwal v thota rs balakrishnan k shah sb desain garg r ambulkar rp bhorkar nm society of oncoanaesthesia andperioperative care consensus guidelines for perioperative management ofpatients for cytoreductive surgery and hyperthermic intraperitonealchemotherapy crshipec indian j anaesth “ godden ar marshall mj grice as daniels ir ultrasonography guidedrectus sheath catheters versus epidural analgesia for open colorectal cancersurgery in a single centre ann r coll surg engl “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsmitomycin c used during surgery for treatment of locally advancedcolorectal carcinoma bmc cancer baratti d kusamura s iusco d gimondi s pietrantonio f milione mguaglio m bonomi s grassi a virzì s hyperthermic intraperitonealchemotherapy hipec at the time of primary curative surgery in patientswith colorectal cancer at high risk for metachronous peritoneal metastasesann surg oncol “chua tc robertson g liauw w farrell r yan td morris dl intraoperativehyperthermic intraperitoneal chemotherapy after cytoreductive surgery inovarian cancer peritoneal carcinomatosis systematic review of currentresults j cancer res clin oncol “li y zhou yf liang h wang hq hao jh zhu zg wan ds qin lx cui szji jf chinese expert consensus on cytoreductive surgery andhyperthermic intraperitoneal chemotherapy for peritoneal malignanciesworld j gastroenterol “ willschke h bosenberg a marhofer p johnston s kettner sc wanzel o kaprals ultrasonographyguided rectus sheath block in paediatric anaesthesiaanew approach to an old technique br j anaesth “azemati s khosravi mb an assessment of the value of rectus sheath blockfor postlaparoscopic pain in gynecologic surgery j minim invasive gynecol“ dingeman rs barus lm chung hk clendenin dj lee cs tracy s johnsonvm dennett kv zurakowski d chen c ultrasonographyguided bilateralrectus sheath block vs local anesthetic infiltration after pediatric umbilicalhernia repair a prospective randomized clinical trial jama surg “ relland lm tobias jd martin d veneziano g beltran rj mckee c bhalla tultrasoundguided rectus sheath block caudal analgesia or surgical siteinfiltration for pediatric umbilical herniorrhaphy a prospective doubleblinded randomized comparison of three regional anesthetic techniques jpain res 201710undefined2629“ xu l hu z shen j pm mq efficacy of usguided transversus abdominisplane block and rectus sheath block with ropivacaine anddexmedetomidine in elderly highrisk patients minerva anestesiol “ cho s kim yj jeong k moon hs ultrasoundguided bilateral rectus sheathblock reduces early postoperative pain after laparoscopic gynecologicsurgery a randomized study j anesth “li t ye q wu d li j yu j doseresponse studies of ropivacaine in bloodflow of upper extremity after supraclavicular block a doubleblindrandomized controlled study bmc anesthesiol bell jc rylah bg chambers rw peet h mohamed f moran bjperioperative management of patients undergoing cytoreductive surgerycombined with heated intraperitoneal chemotherapy for peritoneal surfacemalignancy a multiinstitutional experience ann surg oncol “landmann a visoiu m malek mm development of a novel technique forbilateral rectus sheath nerve blocks under laparoscopicguidance j pediatrsurg “kumar a wilson ga engelhardt te ultrasound guided rectus sheathblockade compared to perioperative local anesthetic infiltration in infantsundergoing supraumbilical pyloromyotomy saudi journal of anaesthesia“ bashandy gm elkholy ah reducing postoperative opioid consumption byadding an ultrasoundguided rectus sheath block to multimodal analgesiafor abdominal cancer surgery with midline incision anesthesiol pain med201443e18263 dowidar aerm ezz haa shama aae eloraby ma postoperative analgesiaof ultrasound guided rectus sheath catheters versus continuous woundcatheters for colorectal surgery a randomized clinical trial ˜† egypt journalof anaesth “karaarslan e topal a avci o tuncer uzun s research on the efficacy of therectus sheath block method agri “ piccioni f casiraghi c fumagalli l kusamura s baratti d deraco m arientif langer m epidural analgesia for cytoreductive surgery withperitonectomy and heated intraperitoneal chemotherapy int j surg 16pt a99“ vesterandersen m lundstrøm lh møller mh the association betweenepidural analgesia and mortality in emergency abdominal surgery apopulationbased cohort study acta anaesthesiol scand httpsdoi101111aas13461 0c"
Colon_Cancer
despite great advances in recent decades in screening diagnosisand curative surgery hepatocellular carcinoma hcc remainsthe second leading cause of cancerrelated mortality worldwidegrandhi siegel epidemiologicalevidence has confirmed that the longterm outcomes of patientswith hcc have notimproved significantly with the rapiddevelopment of surgical techniques madduru moreimportantly because of the limitations of systematic statustumor position and the need to preserve liver function morethan of patients are not eligible for surgical treatment evenafter curative resection prognosis remains unsatisfactory becauseof a high incidence of postoperative recurrence colecchia the initiation and maintenance of hcc is a complexand regulated process involving the accumulation of numerousgenetic changes over decades niu erkekoglu these sequential alterations not only endow normal livercells with neoplastic ability enabling uncontrolled growth butalso provide potential therapeutic targets and biomarkers thusfurther understanding of the initiation and maintenance of hccat the molecular level is crucial to prolonging survival and makingindividual treatment decisionsthe exive development of highthroughput technologyhas provided powerful tools for the molecular study of cancerschmidt and hildebrandt rna sequencing rnaseq and microarraysthe most representative methods ofthis technology are mature enough for use in commercialapplications mantione zhang duringthe past decades the genomewide transcriptional analysis ofgene expression has become critically important to gain betterinsight into the biological processes of hcc and other typesof cancer jin xiong in additionto the aberrant expression of transcripts studies have focusedon diï¬erent molecular levels multilevel omicsincludingcopy number variation epigenetic modifications nucleotidepolymorphisms and dna methylation especially in hcclee lin evidence obtained from thesestudies clearly demonstrates that hcc is a disease causedby cumulative aberrations at diï¬erentlevels of molecularregulation thus only a highthroughput multiomics analysiscan decipher the complex biology of hcc many previousstudies despite promising results focused only on the aberrantregulation of expression and its biological eï¬ects howeverstructural transcript variation in hcc which is heavily shaped byalternative splicing as has until recently been less well studiedaccording to the manual genome annotation project harrow pruitt there are only about proteincoding genes this number is obviously inconsistent withthe overall cellular complexity which includes at least distinct proteincoding sequences harrow thisdiscrepancy between the numbers of transcripts and proteincoding genes in human cells indicates the existence of anadditional mechanism between the transcriptional and the posttranslational levels that increases the coding capacity of thegenome through the as process a single rna precursorcan be spliced via distinct arrangements to generate rnaslandscape of as in hccwith diï¬erent structures and functions biamonti song this may be one ofthe main causesof cellular complexity and proteome diversity experimentalstudies on the eï¬ects of individual as events suggest that asmay change the biological function of a protein by regulatingits stability controlling its location modifying the mutualinteractions of proteins and even adding or deleting activedomains brett yang these findingssuggest that as well as expression abundance the balance ofdiï¬erent as events that result from the same rna precursormust be considered howeverthe latter consideration hasoften been neglected in previous studies in fact emergingdata from genomewide analyses feng kahles indicate that as occurs in more than ofmultiexon genes suggesting thatthe widespread existenceof as is the product of physiological processes rather thantranscription errorsin recent years the diagnostic and the therapeutic role ofas in many human diseases has attracted increasing attentionlargescale screening of as events has been performed usingexpressed sequence tag libraries although this approach isprone to a high rate of false positives venables exonjunction probes provide a higher experimental validation ratelapuk however this method has the disadvantageof being limited to known splice junctions owing to thelimited available techniques complicated mechanisms and hugenumbers involved transcriptomewide as dysregulation and itspotential associations with biological behavior in hcc haveremained uncharacterizedrnaseq not only supports the quantitative measurementof novel as events but also provides deeper coverage higheraccuracy and better resolution li y thus it maybe the most suitable of the currently available approaches foras study in recent years the cancer genome atlas tcgatomczak wang has accumulated a richand publicly available source of rnaseq data and correspondingclinical information this enables the analysis of as dysregulationin hcc at a genomewide level tcga includes rnaseqdata samples obtained from hcc patients together withtheir corresponding clinical information thereby facilitating theclinical analysis of hccrelated as events in a large cohorthowever without reliable and efficient bioinformatical methodsthe advantages of rnaseq in as analysis cannot be fullyexploited spliceseq a recently developed bioinformatics toolcan exactly match rna reads with gene splice graphs and ishelpful for accurately calculating complex or lowfrequency asevents ryan there has been a lack of studies combining largescalernaseq data with the corresponding clinical information tocomprehensively analyze as at singleexon resolution howeverthis is very necessary especially in hcc thereforein thecurrent study we comprehensively analyzed wholegenome asin the tcga hcc cohort to screen out hccrelated asevents and further studied the relationships of these eventswith clinical outcomes our findings suggest that certain hccrelated as events including nek2at and troptat havecritical roles in the progression and maintenance of hcc morefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccimportantly these hccrelated as events represent potentialnew therapeutic targetsmaterials and methodstumorlocationinvasioninformation ofdata curationclinicopathologicalthe hcc cohort andcorresponding rnaseq data were retrieved and downloadedfrom tcga1 to ensure appropriate protection of patientprivacy the tcga data were stratified according to data typeand level conforming to the publishing guidelines formulatedby tcga wang then the rnaseq data andcorresponding clinicopathological information were mutuallypaired using the unique tcga barcodes only patients whomet the criteria listed below were included grandhi patients with corresponding rnaseq data siegel patients with complete clinicopathological informationincluding localsex age distalmetastasis pathological stage diï¬erentiation grade lymph nodemetastasis and survival information madduru histological diagnosis of hcc and colecchia survival for at least month after the primary pathologicaldiagnosis spliceseq was used to determine rna splicing patternsand produce as profiles for each hcc patient as previouslydescribed li y zhu each as eventwas quantified using the percent spliced in psi value rangingfrom to a commonly used method to reflect the abundanceof as events in order to remove the eï¬ects of splicing noiseand generate as reliable a set of as events as possible a seriesof strict filters average psi ‰¥ percentage of samples withpsi ‰¥ was applied to the detected as events the interactivesets between the seven types of as were quantitatively analyzedand the results were visualized in upset plots using upsetrversion conway circlize version wasused to generate circos plots to depict the parent genes and theiras events in chromosomes gu the details of thedesign of the present study are illustrated in supplementaryfigure s1 all the methods used in this study were in line withthe relevant guidelines and regulationsidentification of deas and enrichmentanalysisto screen the diï¬erentially expressed alternative splicing deasevents between hcc and corresponding normal tissues the psivalue of each as event was determined in the tcga hcccohort hcc tissue samples and paired adjacent normaltissues a generalized linear model was applied to remove thebatch eï¬ects the deas were determined based on adjustedp adj p and associated log2 fold change fc values withadj p ‰¤ and log2fc ‰¥ representing as events thatwere downregulated and upregulated respectively biologicalfunction enrichment analysis was performed based on the deasparent genes gene ontology go and kyoto encyclopediaof genes and genomes kegg terms with false discoveryrate less than were considered to be significantly enrichedand were selected for further analysis enrichment analysis wasperformed using the clusterprofiler package version yu the parent genes of deas events were importedinto the string database and used to determine protein“protein interactions ppis a relationship network was thengenerated using cytoscape version su clusteranalysis was conducted using the average linkage agglomerationalgorithm and correlation distance metricsestablishment of hccrelated splicingcorrelation networka total of splicing factors sfs supplementary table s1were identified by comprehensive and handcurated screening ofthe literature all the sfs included in the current study had beenexperimentally validated in previous research giulietti and included heterogeneous nuclear ribonucleoproteinsproteins serineargininerich proteins and other proteinsbelonging to the celf fox khdrbs nova and elav familiesthe expression of each sf was obtained from the broadinstitute2 correlations between the psi values of deas and theexpression of sfs were analyzed by weighted gene coexpressionnetwork analysis version langfelder and horvath benjamini and hochberg correlation was used to adjust thepvalues the adjusted pvalues less than were consideredto indicate statistically significant diï¬erences cytoscape version was used to generate the correlation plotssurvival analysisall the included hcc patients were divided into two groupsbased on the psi value of each deas median cut and thetwo artificial categories were modeled as continuous variables toderive more easily interpretable hazard ratios based on overallsurvival os and diseasefree survival dfs cox regression wasperformed to evaluate the prognostic value of each deas eventlogrank test and kaplan“meier analysis were used to comparepatient survival between subgroups p was consideredas statistically significant the overall survivalrelated deaswere further analyzed in lasso regression to identity the mostpowerful prognostic markers finally a prognostic model wasconstructed for predicting the os in order to quantify the risk ofos a standard form of risk score rs for each colorectal cancercrc patient was calculated combine the levels of the psiι1 psii × lito divide the patients into the high or lowrisk group kaplan“meier curves were used to estimate the survival for patients in thetraining the testing and the validation sets between the highriskand the lowrisk groupspsii and lasso coefficients li risk score pnfunctional experiment of cxcl12splicing variants in hccthe human hcc cell line hepg2 was obtained from the chineseacademy of sciences committee on type culture collectioncell bank shanghai china the cell line was cultured in 1httpsportalgdccancergov2httpgdacbroadinstitutefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccprimers was used asgibco carlsbad ca united states supplemented with fetal bovine serum fbs bi beit haemek israel at —¦c with co2 total cdna from tissues was obtained as described abovethe pcr reaction was carried out using the forward primer5cid48tgcccttcagattgttgcac3cid48 common for allisoforms and theisoformspecific reverse primers 5cid48gctaactggttagggtaatac3cid48 and5cid48gctagcttacaaagcgccagagcagagcgcactgcg3cid48for np_9546371and np_0010290581 respectively bactin amplified usingthe forward 5cid48acactgtgcccatctagcagggg3cid48and reverse 5cid48atgatggagttgaaggtagtttcgtggat3cid48aloading control quantitative realtime pcr was performedin mx3005tm qpcr system with a mxpro qpcr software stratagene la jolla ca united states and sybr greendetection system the adherent hepg2 cells were transfectedwith the corresponding hiscxcl12 construct by the calciumphosphate method and cultured for h at h before collectingthem the cell supernatants were removed and when indicatedbrefeldin a was added to the fresh medium the collectedcells were left untreated or permeabilized with saponin andimmunolabeled with the his mab and a pegoat antiigsecondary antibody and analyzed by flow cytometry the cellinvasion assay was conducted using matrigelcoated chambers µm pore size corning costar corporation cambridgema united states in brief × cells were plated in theupper chamber coated with matrigel and supplemented withserumfree medium the lower chamber was filled with a culturemedium containing fbs incubation was carried out for h at —¦c following which the noninvasive cells were scrapedoï¬ with cotton swabs the cells that had successfully translocatedwere fixed with paraformaldehyde stained with crystalviolet and finally counted using an inverted microscope mttassay colony formation assay and soft agar growth assay wereperformed according to our previously described methods zhou protein structure homology modeling analysis wasperformed as previously described by using the online serverswissmodel waterhouse evaluation of the relationship of asclusters with clinicopathologicalfeaturesbased on the identified deas n the tcga hcc cohortin the current study was stratified by an unsupervised consensusapproach consensus cluster plus version wilkerson andhayes the optimal number of clusters was determinedby integrating the results of the elbow method and gap statisticthe relationship between clinical outcomes and as clusterswas evaluated using logrank test and kaplan“meier curves asdescribed by xiong 2018aresultslandscape of as event profiles in hccto systematically characterize the as events and their clinicalsignificance in hcc we collected rnaseq libraries andcorresponding clinicalinformation from hcc patientsthe tumor tissues and paired adjacent normal tissues from patients were sequenced simultaneously the includedpatients comprised males and femalesamong which patients developed recurrence and died of hcc the median followup period was months range “ months the general characteristicsof these hcc patients are fully detailed in supplementarytable s2 rnaseq data were associated with the clinicalthe corresponding patient via the tcgainformation ofbarcodes there were patients with rnaseq data bothfrom tumor tissue and adjacent normal tissue according tothe recommended analysis approach described in a previouslypublished study ryan we identified asevents from genes based on their splicing patterns theseas events could be roughly classified into seven types alternatepromoter ap mutually exclusive exons me retained intronri exon skipping es alternate acceptor site aa alternateterminator at and alternate donor site ad figure 1ato quantify the detected as events psi values were calculatedthese values measure the proportion of each detected splicingvariation in all of the expressed isoforms the expression ofcertain isoforms was fairly low psi and most of the asevents could not be stably detected in all of the given samplesafter screening average psi ‰¥ percentage of sampleswith psi ‰¥ a total of as events from geneswere obtained we further compared the variance in quantityof as events and the genes involved between tumor adjacentpaired normal and unpaired tumor tissues for diï¬erent splicingpatterns there were no significant diï¬erences in quantityvariations however on average one gene might have nearlythree as events figure 1b left panel moreover only annotated genes in this study stably underwent as figure 1bright panel notably diï¬erent as patterns may occur for a singlegene thus upset plots were used to depict the intersectionsbetween as types as demonstrated in figure 1c most of theparent genes only occurred in one type of as event whereascertain parent genes contained up to four types of as eventabout of the parent genes contained two or more asevents the arrangements of diï¬erent as types and as eventsbetween diï¬erent exonsintrons may be the major reason fortranscriptome diversity in order to comprehensively depict asevent profiles in hcc circos plots were used to visualize therelationships among as events and the corresponding parentgenes in chromosomes figure 1didentification of hccrelated deascomparing the variations in molecular components amongdiï¬erent pathological states using highthroughput techniquesis an eï¬ective way to screen key molecules this approachhas been widely used to identify diseaserelated molecules inprevious research xiong 2018ab it is reasonableto consider that significant diï¬erences in as events betweenprimary hcc tissues and adjacent normal tissues may be relevantto the initiation and maintenance of hcc in this study thetcga barcodes corresponding to tissue samples rnaseq data were analyzed from which as profiles of pairednormal and tumor tissues were finally extracted these pairedfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure landscape of alternative splicing as events in hepatocellular carcinoma hcc a diagrammatic sketch of the seven types of as events in the presentstudy alternate acceptor site aa alternate terminator at alternate promoter ap exon skipping es mutually exclusive exons me alternate donor site adand retained intron ri b number of as events and the corresponding parent genes illustrated according to as type left panel the color bar represents asevents filtered by criteria the black bar represents the corresponding genes involved in as each as type was divided into four groups based on the tissue sourcen normal tissue t tumor tissue pt paired tumor tissue npt unpaired tumor tissue number of detected as events asrelated genes filtered as events and thecorresponding genes right panel c intersection of parent genes between different as types n in hcc one gene may incur up to four types ofalternative splicing d circos plots depicting the distribution and the detailed alteration of as events and their parent genes in chromosomesas profiles were used to identify deas eventually deaswere identified from genes using a threshold of log2fc and adj p including aps ess ats risnine ads aas and one me figure 2a and supplementarytable s3 the top deas are listed in table notablythe proportion of as types between the filtered as and deaswas inconsistent the es events accounted for of filteredas but only of deas however the proportion of apevents rose from of filtered as to of deasfigure 2b these statistical findings suggest that as is notthe result of transcription errors but a tightly regulated processmoreover based on the identified deas the samples could beclearly separated into normal and tumor groups by unsupervisedhierarchical clustering figure 2c indicating that the deashad been reliably identified the psi values of deas eventsin diï¬erent hcc patients are illustrated in figure 2c as amatrix heat map the changes in color gradient intuitively revealthe heterogeneity of hcc a splice graph which representssplice junctions as edges and exons as rectangular nodes wasused to visualize some of the identified deas figure 2dfurthermore the diï¬erences in expression of these as eventsbetween primary hcc tissue and corresponding adjacent normaltissues are intuitively depicted in figure 2e taken together theseresults show that a significant variation of as occurred duringhcc initiation and maintenance indicating that the potentialrole of hccrelated as events requires further researchenrichment and interaction analysis ofdeasemerging evidence indicates that as could change a transcribedsequence directly with eï¬ects on expression abundance orprotein function thus the potential biological eï¬ects of deascould be determined by analyzing the corresponding proteinsas shown in supplementary figures s2a“c specific go termsclosely related to liver metabolism including negative regulationof hydrolase activity sterol homeostasis anic acid catabolicprocess and acidic amino acid transport were significantlyenriched by the parent genes of deas in addition certain keggpathways known to be involved in hcc were enriched includingthe cgmppkg signaling pathway the nfκb signaling pathwaythe mrna surveillance pathway and the phosphatidylinositolfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure identification of hepatocellular carcinoma hccrelated aberrant alternative splicing as a differences in as events between paired hcc tissue andparacancerous tissue volcano plot of the differentially expressed alternative splicing deas identified in hcc the blue and the red points represent the deas withstatistical significance logfc ‰¥ adj p b proportions of different as types among filtered as and deas c heat map of the deas the horizontal axisshows the clustering information of samples divided into two major clusters adjacent normal tissue n and paired tumor tissue n the left longitudinalaxis shows the clustering information of deas the gradual change of color from green to red represents the alteration of expression of deas from low to highd splice graph of some representative deas the thin exon sections represent untranslated regions and the thick exon sections represent coding regions theexons are drawn to scale and the connecting arcs represent splice paths e differences in percent spliced in values of as events between hcc and pairedadjacent normal tissuessignaling system supplementary figure s2d these resultssuggest that the parent genes of deas are critical in the biologicalregulation of hcc thus aberrant splicing of the transcribedsequences could influence their translation and change thecharacteristics of the resulting proteins therefore it is essentialto study as events from the perspective of ppi networks basedon the deasrelated genes a ppi network was establishedrepresenting not only normal interactions but also the potentialimpact of as events figure correlation network of sfs andhccrelated asas events are primarily regulated by sfs which attach to themrna precursor and aï¬ect the selection of exons and thechoice of splicing site aberrant as events in tumor tissuemay be orchestrated by a limited number of sfs for thisreason we conjecture that a few key sfs potentially regulatea large proportion of hccrelated as events to validate thisconjecture we first identified sfs supplementary table s1by comprehensive and handcurated screening of the literatureall of which had been previously experimentally validatedgiulietti then the copy number variation somaticmutations and expression abundance of these sfs in eachhcc patient were investigated using cbioportal figure 4avisualization using oncoprint revealed that each of the sfs harbored at least three molecular alterations figure 4aleft panel the most frequently aï¬ected sf was khdrbs3in which molecular alterations were detected in cases partly owing to the above changes the expressionabundance of the sfs showed a significant heterogeneity atan individual level figure 4a right panel the expressionprofiles of the sfs in diï¬erent cancer types also showedheterogeneous characteristics figure 4b more importantlythe expression of sfs also diï¬ered between paired normal andcancer tissues of the same hcc patient figure 4c nextcorrelation analyses were performed between the psi value ofeach deas event and the sfs according to the correlationcoefficient ttest p r a splicing regulatorynetwork was established as shown in figure 5a sfs weresignificantly correlated with deas events among which were downregulated and were upregulated several diï¬erentas events in the network were regulated by a single sf insome cases an sf had the opposite regulatory eï¬ect on diï¬erentas events figure 5a we also found that the same bindingsite as event could be competitively bound by diï¬erent sfsthese observations explain at least in part why one gene cangenerate several diï¬erent isoforms representative correlationsfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hcctable the top most different alternative splicing as eventssymbolas typeexonsfrom exonto exonmean nmean tlog fcadjusted pvaluedownregulatedmthfd2ligf2kif22gstz1serpinb5ppargfam3afip1l1tmem59tpm4cdh23mid1gnesamd12kif4afbxw7nek2padi4rnf115vti1aupregulatedrdm1nr1i3psphtmem145nr1i3gpr116piddnr1i3ano1cldn7sardhscp2znf331eno3pemtfn1tnfrsf10carhgef39igf2neil3apapadapatapaaatapapatapapatatapatatesatatatapatriesapriesapapesapatapesatapapatˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’1393eˆ’1593eˆ’4948eˆ’9941eˆ’2008eˆ’4387eˆ’8636eˆ’1393eˆ’4574eˆ’2519eˆ’1528eˆ’2202eˆ’6860eˆ’1522eˆ’2379eˆ’7699eˆ’1780eˆ’1843eˆ’4211eˆ’2609eˆ’1994eˆ’8164eˆ’1036eˆ’7765eˆ’1089eˆ’3569eˆ’1065eˆ’2135eˆ’5986eˆ’1038eˆ’1611eˆ’1110eˆ’2586eˆ’7736eˆ’2117eˆ’1072eˆ’4524eˆ’1151eˆ’1829eˆ’8136eˆ’between sfs and specific as events were illustrated using dotplots figures 5b“g for example the expression of esrp2 wassignificantly correlated with both es of ceacam1 figure 5cand at of epb41l5 figure 5fassociation of deas with prognosis ofhcc patientsa crossvalidation method was used to evaluate the accuracyof the survival data and the clinical information as shown insupplementary figure s3 stratifying patients according to thetnm stage resulted in separate kaplan“meier curves for bothos and dfs these results confirmed that the survival dataset forthe tcga hcc cohort although it contained censored data wasappropriate and informative for use in further survival analysistheeï¬ect of each deas on survival was determined by coxregression analysis the hcc patients were divided into twogroups according to their psi value median cut of eachdeas event according to univariate analyses a total of to investigate the prognostic significance of deasfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure protein“protein interaction ppi analysis of the identified differentially expressed alternative splicing deas interactions of the parent genes affectedby deas these genes were used to construct an intricate ppi network comprising nodes and edges the genes are denoted as nodes in the graph andthe interactions between them are represented as edges the shape size and color of the nodes respectively represent alternative splicing type value of logfcand change patterndeas events were significantly correlated with dfsand deas events were significantly correlatedwith os supplementary table s4 among these prognosisrelated deas events deas events were correlated withboth os and dfs p figure shows some of thedeas events for which the pvalue for both os and dfs waslower than to demonstrate the capability of as eventsfor prognostic prediction we randomly selected two prognosisrelated deas events and used them to draw survival curvesas shown in figure according to the psi value median ofnek2at and troptat the hcc patients could be stratifiedto form significant kaplan“meier curves by both os and dfssurvival analysis additionally the deas events that significantlycorrelated with survival in the univariate analysis were furtherassessed by multivariate analysis as shown in supplementaryfigure s4 there were five and six deas events that could berecognized as independent prognostic indicators for os and dfsrespectively these findings confirm that deas events possessnot only an important biological meaning but also a potentialclinical significanceconsidering the prognostic value of the aboveidentified asa prognostic model integrating multias was established so thatit can be easily applied to clinical practice based on the survivalrelated deas a relative regression coefficient was calculated bylasso analysis by forcing the sum of the absolute value ofthe regression coefficients to be less than a fixed value certaincoefficients were shrunk exactly to zero and the most powerfulprognostic marker of all the hcc survivalassociated deaswas selected including four as supplementary figure 5acombining the relative expression levels of the as in themodels and the corresponding lasso coefficients a riskscore was calculated for each patient obviously patientswith higher rs generally had a significantly worse overallsurvival than those with lower rs p supplementaryfigures 5bc as the majority of events occurred within years timedependent roc curves were used to assess theprognostic power based on os at and years respectivelysupplementary figure 5dclustering hcc patients using deasassociated with prognosisgiven our findings of significant heterogeneity among deas atan individual level which could reflect the diï¬erent outcomesfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure multiomics analysis of the splicing factors sfs in hepatocellular carcinoma hcc a cbioportal analysis of the sfs in the cancer genomeatlas hcc patients oncoprint was used to produce a landscape of genomic alterations legend in sfs rows at the individual level columns inframe deletionstruncated mutations and missense mutations with known or unknown significance are shown in orange blue green and gray respectively with onethird heightthe copy number variations are annotated with the full height amplification is shown in red and copy number loss is in blue heat map matrix shows the variation insfs at expression level the expression abundance from high to low is represented by color gradient from red to blue b expression of the sfs in tumortypes heat map color gradient depicts the normalized expression of sfs between different tumor types c differential expression analysis of representative sf tia1in hcc the expression of tia1 in hcc tissue was significantly higher than that in normal liver tissueof patients with hcc we conjectured that there might existdistinct patterns of as among diï¬erent hcc patients thishypothesis was verified using consensus unsupervised analysisbased on the deas the optimal number of clusters wasdetermined by combining the gap statistic and elbow method theoptimal balanced partition as suggested was k figure 8aaccordingly all the hcc patients were divided into four clustersas follows i n ii n iii n and iv n figures 8bc as illustrated bythe heat map the four clusters had a significant interconnectivityamong which cluster ii appeared as a wellindividualized clusterwhereas there was more classification overlap among clustersi iii and iv figures 8bc kaplan“meier survival analysisand logrank test were used to evaluate the associations betweenprognosis and the as clusters as illustrated in figures 8dethe stratification of hcc patients based on as clusters showeda significant correlation with distinct patterns of survival thevariation tendency that resulted in the as stratification betweenfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure specific regulatory network of hepatocellular carcinomarelated alternative splicing as events a correlation network of splicing factors sfs anddifferentially expressed alternative splicing the shape size and color of nodes respectively represent type as event or sf value of logfc and change patternupregulated or downregulated the breadth of the line represents the interaction strength b“g representative dot plots of the correlations between theexpression of sfs and percent spliced in values of as eventsfigure prognostic value of differentially expressed alternative splicing deas in hepatocellular carcinoma part deas events simultaneously associated withoverall survival os and diseasefree survival dfs univariate analysis of deas for os and dfs respectively unadjusted hazard ratios boxes and confidenceintervals horizontal lines
Colon_Cancer
"colorectal cancer crc remains the third most prevalent cancer type and leading cause of cancerrelated deaths with million cases and deaths worldwide during the occurrence and progression of crc result from a wide array of cellular transformation processes which include genetic and epigenetic mutations that drive uncontrolled cell proliferation and escape from apoptosis2“ chemotherapy and surgery remain the major therapeutic treatment for crc patients5 fluoropyrimidinebased chemotherapy eg 5fluorouracil has been used as the firstline systemic chemotherapy of treating advanced crc for over a half century6 however most patients receiving chemotherapy finally develop drug resistance which is considered to be the major reason for crc therapy failure7 furthermore even though chemotherapy has significant antitumor activity the side effects can affect the quality of a patient's life which makes the new therapeutic approaches urgentdrug design development and therapy “ sun this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution “ non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0csun dovepresstraditional chinese medicines such as dendrobium have been shown to exert anticancer activity in many kinds of cancers89 erianin 2methoxy5[2345trimethoxy phenylethyl]phenol figure 1a a natural compound derived from dendrobium candidum shows various pharmacological activities and therapeutic potential to inhibit multiple cancers in vivo and in vitro10“ li demonstrated that erianin inhibited the proliferation of acute promyelocytic leukemia hl60 cells by regulating the expression of bcl2 and bax10 in addition erianin caused moderate growth delay in xenografted human hepatoma bel7402 and melanoma a37511 furthermore erianin induced cell cycle g2mphase arrest and apoptosis via the jnk signalling pathway in osteosarcoma and bladder cancer1213 erianin can also inhibit cell invasion metastasis and angiogenesis in lung cancer and breast cancer by the figure erianin inhibited crc cells growth a chemical structure of erianin b and c sw480 and hct116 cells were treated with indicated concentration b and time c of erianin cell viability was assessed by cck8 assay p ˂ p ˂ d and e ncm460 cells were treated with indicated concentration d and time e of erianin cell viability was assessed by cck8 assay f sw480 and hct116 cells were performed colony formation assay after being treated with indicated concentration of erianinsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun regulation of ido mpp and timp expressions1415 interestingly besides the function on cell growth apoptosis and migration erianin was found to strongly affect the serum levels of cytokines and immune response in liver cancer16 more importantly in addition to the anticancer effects previous a study also suggested that erianin had no major anrelated toxicities12however to the best of our knowledge neither the mechanism nor the effect of erianin on colorectal cancer has been reported hence in this study we evaluate the antitumor potential and molecular mechanisms of erianin in human colorectal cancer sw480 and hct116 cells and provide a theoretical basis of erianin application for colorectal cancer therapymaterials and methodsmaterialsantibodies against cleaved parp cat bak cat bax cat bcl2 cat bclxl cat catenin cat cyclin d1 cat cmyc cat hdac2 cat and gapdh cat were purchased from cell signaling technologies danvers ma usa antibody against αtubulin cat t6199 was purchased from sigma aldrich co st louis mo usaerianin was purchased from shanghai yuanye bio technology co ltd china and dissolved in dmso wntcatenin signaling inhibitor wnt974 was purchased from medchemexpress monmouth junction nj usa and dissolved in dmsocell culturethe human colorectal cancer cell lines sw480 and hct116 were purchased from american type culture collection atcc manassas va usa cells were maintained in rpmi1640 medium supplemented with fbs thermo fisher scientific waltham ma usa uml penicillin and µgml streptomycin thermo fisher scientific and cells were cultured at °c with co2cell viability and colony formation assaycell viability was assessed with the cell counting kit cck8 dojindo japan according to the manufactorer™s instructionsfor the colony formation assay crc cells cells well were seeded in a sixwell plate and maintained in medium for “ days subsequently the colonies were fixed with paraformaldehyde and stained with crystal violet and the number of clones was counted using an inverted microscopekit quantitative realtime pcr qrtpcrtotal rna from crc cells was isolated using rna isolation kit omega norcross ga usa according to the manufacturer™s protocol total rna µg was used as the template for cdna synthesis by using iscripttm reverse transcription super mix biorad laboratories inc hercules ca usa before the samples were analyzed using sybr green master mix on a realtime pcr system biorad laboratories inc the primer sequences used were as follows cmyc forward 5ʹ aaacacaaacttgaaca gctac3ʹ reverse 5ʹ atttgaggcagtttacatt atgg3ʹ cyclin d1 forward 5ʹaggcggatgagaac aagcaga3ʹ reverse 5ʹcaggcttgactccagaag gg3ʹ cd47 forward 5ʹggcaatgacgaaggaggt ta3ʹ reverse 5ʹatccggtggtatggatgaga3ʹ and gapdh forward 5ʹcacccactcctccacctttg3ʹ and reverse 5ʹccaccaccctgttgctgtag3ʹ the 2δδcq method was used to calculate the relative expression levelswestern blottingfor western blotting μg cellular protein extracts were separated in sdspage gel and were then transferred to nitrocellulose membranes emd millipore burlington ma usa the membrane was blocked with nonfat milk and incubated with primary antibodies overnight at ° then the membranes were incubated with secondary antibody and the proteins were visualized using super signal west pico chemiluminescent substrate thermo fisher scientifictransit transfectionplasmid pegfpn1betacatenin was purchased from addgene watertown ma usa lipofectamine thermo fisher scientific carlsbad ca usa was used for transit transfection according to the instructionscatenin sirna was purchased from sigmaaldrich co lipofectamine rnaimax thermo fisher scientific was used for transfection according to the instructiondrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepresscell cycle analysisafter treated with vehicle or indicated drugs crc cells were harvested by trypsinization fixed with ethanol and retained at ˆ’°c overnight after cells were centrifuged and washed with pbs they were resuspended in propidium iodide pi solution containing rnase μgml in the dark at room temperature for min and then studied in a flow cytometercaspase37 activity assayapoone„¢ homogeneous caspase37 assay promega corporation madison wi usa was used to measure caspase37 activity briefly apoone® homogeneous caspase37 reagent μlwell was added to a 96well plate and the plate was then placed on a shaker for five minutes “ rpm before incubating for h at room temperature the reading of each well was measured by spectrofluorometerapoptosis assay by annexin vannexin vfitc staining was used to detect the extent of apoptosis induced by erianin briefly crc cells were treated with erianin for h and were then collected and resuspended in μl annexin vbinding buffer and μl pi for minat room temperature in the dark then the cells were finally analyzed by the flow cytometry bd facs calibur with an emission filter of nm for pi red and “ nm for fitc greenapoptosis assay by dapithe effect of erianin on apoptosis induction was evaluated by dapi staining assay crc cells × were seeded in a 96well plate after treatment the cells were washed three times with pbs and paraformaldehyde was added to each well for fixation after permeabilization with triton x100 solution dapi solution was added the cells with condensed and fragmented chromatin were analyzed by echo fluorescence microscopycellular thermal shift assayfor cellular thermal shift assay crc cells were pretreated with μm mg132 for one hour and then incubated with erianin for four hours after washing with icecold pbs cells were aliquot into pcr tubes μl each and incubated at different temperatures for four minutes after being frozen and thawed twice using liquid nitrogen cells were centrifuged and proteins were analyzed by western blottingtopfop luciferase reporter assaythe transcriptional activity of catenin was assessed using the topfop dualluciferase reporter system dual glo„¢ luciferase assay system promega the renilla luciferase plasmid prltk promega which controls for transfection efficiency was cotransfected with catenin responsive firefly luciferase reporter plasmid topflash emd millipore or the negative control fopflash emd millipore using the lipofectamine thermo fisher scientific cells were harvested after h in culture and the luciferase activity was determined by the luciferase assay system promega using a microplate luminometer berthold bad wildbad germanyflow cytometry analysiserianin treated crc cells were washed and resuspended in μl facs buffer and stained with fitcconjugated anticd47 bd biosciences san jose ca usa antibodies all samples were incubated for minutes at °c and then washed twice with facs buffer flow cytometry analyses were performed on bd facs canto iiin vitro phagocytosis assayfor phagocytosis assay thp1 derived macrophages were seeded in a sixwell tissue culture plate erianintreated crc cells were washed and labeled with μm of carboxyfluorescein succinimidyl ester cfse thermo fisher scientific after incubating macrophages in serum free medium for two hours cfselabeled crc cells were added to the macrophages for another two hours at °c macrophages were then washed and imaged with an inverted microscope the phagocytosis efficiency was calculated as the number of macrophages containing cfse labeled crc cells per macrophageschromatin immunoprecipitation chip assaychip assays were performed using the simplechip® enzymatic chromatin ip kit cell signaling technologies according to manufacturer's instructions using the antibodies against h3k27ac immunoprecipitated dna was analyzed by qrtpcr using the following primers cd47 promoter fragment f ²aggatgaatgatgtggcctgt3² and r ² caaacaggcattagcagcgt3² fragment f submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun ²ggggatgtgttggatacgct3² and r ² ctctg cgttcggctcgtcta3² fragment f ²agggaag agcagagcgagta3² and r ² ttgctttcactcc caccctc3² fragment f ²agagagaggacag tggggc3² and r ² ccagtcgcaggctccaga3² fragment f ² gccgcgtcaacagca3² and r ² aaaggcatcattcttggaaattgt3²with ¨° sw480 cells per mouse suspended in vivo xenograftnodscid shanghai slac laboratory animal co ltd china mice were injected subcutaneously in right flank in µl pbs and mixed with an equal volume of matrigel animals with tumors volume mm3 were divided into two groups n6 and treated with either placebo or mgkg erianin for continuously three weeks by intraperitoneal injection tumor size were measured at the indicated times all the animalrelated procedures were approved by the animal care and use committee of the changchun university of chinese medicine all animal experiments were conducted according to the nih guide for the care and use of laboratory animalsstatistical analysisdata were presented as mean ±sd from three independent experiments p value was determined using paired student™s ttest and a p value ˂ was deemed to indicate statistical significanceresultserianin inhibited crc cell growthfigure 1a illustrates the chemical structure of erianin to investigate the inhibitory effect of erianin on crc cell viability we treated two crc cell lines sw480 and hct116 with different concentrations of erianin and nm for and h as shown in figure 1b and c erianin treatment significantly inhibited the viability of crc cells in a dose and timedependent manner importantly erianin did not show cytotoxic effects on normal human colon mucosal epithelial cell line ncm460 figure 1d and e in addition consistent with the shortterm growth assay our colony forming unit assay also showed that erianin inhibited the colony formation ability of sw480 and hct116 cells figure 1ferianin elevated cell cycle arrest and apoptosisto verify the causal relation of cell viability inhibition the cell cycle distribution was analyzed erianin increased cell number at g2m phase but decreased cell number at s and g0g1 phases after 24h incubation with indicated concentration in sw480 and hct116 cells figure 2a and b to explore the effect of erianin on apoptosis we examined the activity of caspase the protein level of cleaved parp bax bak bcl and bclxl as shown in figure 2c“e the activity of caspase protein level of cleaved parp bak and bax pro apoptosis increased as the concentration of erianin increased in contrast the protein level of bcl2 and bclxl anti apoptotic decreased after erianin treatment figure 2e annexin v flow cytometry and dapi staining further confirmed that erianin could induce cell apoptosis figure 2f and gerianin inhibited catenin translocationincreasing evidence revealed that the wntcatenin pathway plays critical role in colorectal cancer tumorigenesis we hypothesized that erianin might have effect in modulating the wntcatenin pathway first we investigated the effect of erianin on catenin phosphorylation as shown in figure 3a no obvious change was observed on catenin phosphorylation level we then evaluated the effect of erianin on catenin translocation as shown in figure 3b“e catenin expression in cytoplasm was increased whereas expression in the nucleus was decreased with the treatment of erianin in a dose and timedependent manner to further explore the effect of erianin on catenin transcription activity we performed topfop dual luciferase assay we found that topfop relative luciferase activity was significantly decreased after erianin treatment both in sw480 and hct116 cells figure 3f and gerianin bound catenin directlysince erianin inhibited catenin translocation to the nuclear without changing its phosphorylation level we hypothesized that erianin might bind catenin directly to determine whether erianin physically binds catenin we performed a cellular thermal shift assay the results from this experiment indicated that erianin treatment increased the thermal stability of catenin when cells were pretreated with the proteasome inhibitor mg132 for one hour figure 4a and b in contrast erianin treatment had no effect on the thermal stability of gapdh a loading control figure 4a and b these results strongly suggested a specific physical interaction between erianin and catenindrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin elevated cell cycle arrest and apoptosis a and b sw480 and hct116 cells were treated with erianin for h and then analyzed by pi staining to determine cell cycle phase distribution c sw480 and hct116 cells were treated with erianin for h the relative caspase37 activity was measured using apoone„¢ homogenous caspase37 assay p ˂ p ˂ d and e the protein level of cleaved parp1 bak bax bcl2 and bclxl were analyzed by western blotting after treated with indicated concentration of erianin f and g sw480 and hct116 cells were treated with erianin for h apoptosis was assessed using annexinv flow cytometry analysis f or dapi staining gsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited catenin translocation a the protein level of indicated proteins was analyzed by western blotting after being treated with indicated concentration of erianin for h b“e the protein level of catenin in cytosol and nucleus was analyzed by western blotting after treated with erianin for indicated concentration b and c and time d and e f and g sw480 and hct116 cells were treated with erianin for indicated concentration f and time g the transcriptional activity of catenin was assessed by topfop luciferase reporter assay p ˂ p ˂erianin inhibited the expression of cmyc and cyclin d1as cmyc and cyclin d1 are the direct targets of the wnt catenin pathway we then evaluated the mrna and protein level of cmyc and cyclin d1 unsurprisingly both mrna and protein level of these two proteins were significantly decreased after erianin figure 5a“c interestingly no synergetic effect was observed when combining erianin with wntcatenin signaling inhibitor wnt974 which indicated that erianin regulates cmyc treatment drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin interacted with catenin a and b sw480 a and hct116 b cells were treated with μm mg132 for one hour followed by four hours incubation with nm erianin before performing thermal shift assay the lower panel shows the charts of percentages of nondenatured protein fractionand cyclin d1 via wntcatenin signaling figure 5d furthermore the inhibitory effect of erianin on cmyc and cyclin d1 expression and cell viability could be reversed by catenin overexpression figure 5e and f which indicated that erianin regulates crc cell growth via catenincd47 mediated phagocytosis we used an in vitro assay by coculturing thp1 derived macrophage with crc cell lines sw480 or hct116 as shown in figure 6g and h treatment of erianin markedly promote colorectal cancer cell phagocytosis by macrophages these results suggest that erianin treatment can attenuate cd47 expression and ultimately promote phagocytosis of crc cellserianin decreased cd47 expression and increased phagocytosisthe immune checkpoint protein cd47 is included in the list of wntcatenin target molecules with a role in immunity escape17 since catenin depletion by sirna inhibited the expression of cd47 figure 6a we then sought to know whether erianin regulates the expression of cd47 first we explored the effects of erianin on cd47 mrna protein and cell surface level in both sw480 and hct116 cells erianin treatment significantly decreased the mrna protein and cell surface level of cd47 figure 6b“d promoter analysis by ucsc genome browser demonstrates that h3k27 acetyl marks are enriched in cd47 promoter regions figure 6e next our chip assay demonstrated that h3k27ac enrichment specifically near promoter region f3f5 was significantly decreased with erianin treatment figure 6f to investigate the effect of erianin on erianin inhibited tumor growth in vivoto investigate the possibility of erianin as a potential therapy in crc we tested the function of erianin on tumor growth in a mouse model the mouse model was established by s c injection of sw480 cells into nodscid mice after three weeks treatment we analyzed the tumor size and weight as shown in figure 7a“c the tumor size and weight from the erianin treatment group were significantly lower than that from the control group in addition after days of bearing tumor the weight of the mice had no significant change figure 7dto examine the impact of therapy on catenin and its downstream signaling localization of catenin protein level of cd47 cmyc bcl2 and bax three representative tumors from each group were analyzed using western blotting as shown in figure 7e and f catenin expression in cytoplasm was increased whereas expression in nucleus was decreased with the treatment of erianin the submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited the expression of cmyc and cyclin d1 a“c after treated with indicated concentration and time of erianin mrna and protein level of cmyc and cyclin d1 were analyzed by qrtpcr and western blotting p ˂ d sw480 cells were treated with erianin orand wnt974 for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and f sw480 cells were treated with erianin for h followed by overexpression with catenin plasmid for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and cell viability was assessed by cck8 assay f p ˂protein level of cd47 cmyc and bcl2 decreased while bax increased after erianin treatment these data indicated that erianin inhibited tumor growth via catenin in vivodiscussioncrc is one of the most malignant and commonly diagnosed solid tumors all around the world18“ although crc incidence rates have declined somewhat chemotherapies are inefficient in most crc patients due to resistance2122 thus the development of acquired therapeutic drugs researching novel and safe treatment strategies is essential for improving the prognosis of crc patients in recent years natural medicinal plants are receiving more and more attention and considered to be important sources of treatment23 novel dendrobium is considered as one of the most important herbs in the orchidaceae family and shows diverse pharmacological functions including anticancer neuroprotective antidiabetic and immunemodulating activities24 erianin derived from dendrobium is one of the most for cancer drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin decreased cd47 expression and increased phagocytosis a sw480 cells were transfected with nontarget nt or catenin sirna for h protein levels of indicated protein weres measured by western blotting b“d sw480 and hct116 cells were treated with erianin for indicated dose the mrna level b protein level c and cell surface cd47 d were detected by qrtpcr and flow e the ucsc genome browser revealed the enrichment of h3k27ac on cd47 promoter f the enrichment of h3k27ac on cd47 promoter f1f6 was detected by chip assay g and h sw480 and hct116 were treated with indicated concentration of erianin for h representative images showed the effect of erianin on phagocytosis g and bar graphs showed quantitative analysis of phagocytosis h p ˂ p ˂submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited tumor growth in vivo a typical photos of tumors from the control and erianin treated groups b and c erianin decreased tumor volume and weight p ˂ d mice body weight of control and erianin treated groups was measure at indicated time e the protein level of catenin in cytosol and nucleus in three representative tumors from mouse to mouse of each group were analyzed by western blotting f the protein level of indicated protein in three representative tumors from mouse to mouse of each group were analyzed by western blottingdrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressnoteworthy constituents that have been used as an antipyretic and an analgesic in traditional chinese medicine25 recently several studies have proved that erianin shows significant antitumour activity in a variety of human cancer cells10ˆ’ consistent with literature in this study we found that erianin had a significant antiproliferative effect against crc cells the inhibitory effect caused by erianin may result from induction of apoptosis and arrest of cell cycle at g2m since the effect of erianin on crc cells has never been studied before we further confirm its antitumor activity in a mouse model which indicated that erianin significantly inhibited tumor growth in vivoseveral signaling pathways including egfrmapk pi3kakt or wntcatenin have been linked to crc genesis and progression26 as the aberrant activation is present in almost all crc cases wntcatenin signaling is prominent among these pathways27 inactivated mutations in the apc gene leads to stabilization and ensuing nuclear translocation of catenin to facilitate tcflef dependent transcription of wntcatenin signaling target genes such as cmyc and cyclin d1 to drive cell proliferation survival and metastasis28“ to understand the mechanisms of action of erianin we assessed the effect of erianin on wntcatenin pathway interestingly we found that erianin treatment had no effect on catenin phosphorylation but inhibited the translocation of catenin in the nucleus which suggested to us that erianin physically interacts with catenin our cellular thermal shift assay confirmed this hypothesis the thermal stability of catenin increased after erianin treatment as catenin downstream targets the expression level of cmyc and cyclin d1 significantly decreased after erianin treatmentcd47 a transmembrane glycoprotein expresses ubiquitously and mediates a œselfdonoteatme signal on normal cells however cd47 is often upregulated in tumor cells to evade innate immunity31“ anticd47 antibodies which block cd47 sirpα interactions and promote macrophage mediated phagocytosis of tumor cells has shown promise in several solid tumors31 in colorectal cancer cd47 promotes colon cancer cell migration and metastasis34 in addition upregulated immuneescape pathways such as cd47 sirpα are responsible for immune escape and survival in circulating tumor cells of colorectal cancer35 myc an oncogene identified as a wntcatenin target gene was reported to control cd47 transcription therefore mutations in components of the wntcatenin signaling pathway which induced
Colon_Cancer
"objective endoscopic full thickness resection eftr has shown efficacy and safety in the colorectum the aim of this analysis was to investigate whether eftr is cost effective in comparison with surgical and endoscopic treatment alternativesdesign real data from the study cohort of the prospective single arm wall resect study were used a simulated comparison arm was created based on a survey that included suggested treatment alternatives to eftr of the respective lesions treatment costs and reimbursement were calculated in euro according to the coding rules of and eftr r0 resection rate was used as a measure of effectiveness to assess cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were determined calculations were made both from the perspective of the care provider as well as of the payerresults the cost per case was ‚¬ for the eftr group ‚¬ for the standard endoscopic resection ser group ‚¬ for the surgical resection group and ‚¬ for the pooled alternative treatment to eftr from the perspective of the care provider the acer mean cost per r0 resection was ‚¬ for eftr ‚¬ for ser ‚¬ for surgical treatment and ‚¬ for all pooled and weighted alternatives to eftr the icer additional cost per r0 resection compared with eftr was ‚¬ for ser ‚¬ for surgical resection and ‚¬ for the pooled rate of alternatives results from the perspective of the payer were similar eftr is cost effective in comparison with surgical and endoscopic treatment alternatives in the colorectumintroductioncolorectal cancer is the third most common type of cancer and the second most common cause of cancer related deaths worldwide1 screening programmes for early detection of premalignant and malignant lesions led summary boxwhat is already known about this subject –º endoscopic full thickness resection eftr has shown clinical efficacy and safety in difficult to treat lesions in the colorectum –º the cost of the full thickness resection device is higher than the cost of standard endoscopic resection ser devices but lower than surgical devices –º cost effectiveness analyses on treatment with eftr compared with treatment alternatives do not existwhat are the new findings –º eftr leads to an almost reduction in cost per r0 resection average cost effectiveness ratio compared with surgery –º to achieve an additional r0 resection by surgical treatment compared with eftr incremental cost effectiveness ratio an additional cost of ‚¬ is necessary –º these findings are consistent both from the perspective of the care provider as well as the payerhow might it impact on clinical practice in the foreseeable future –º in terms of cost effectiveness eftr should be considered first before patients with difficult to treat lesions in the colorectum are sent to surgical treatmentto a significant reduction in cancer related mortality2 with more intense screening more lesions are detected which automatically creates the need for removal standard endoscopic resections ser such as endoscopic mucosal resection emr and endoscopic submucosal dissection esd are well established and sufficient for the vast majority of lesions however ser of non lifting lesions and lesions located at difficult anatomical to cite kuellmer a0a behn a0j beyna a0t et a0al endoscopic full thickness resection and its treatment alternatives in difficult to treat lesions of the lower gastrointestinal tract a cost effectiveness analysis bmj open gastro 20207e000449 101136bmjgast2020000449received may revised july accepted july authors or their employers re use permitted under cc by nc no commercial re use see rights and permissions published by bmjfor numbered affiliations see end of correspondence toarthur schmidt arthur schmidt uniklinik freiburg dekuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access locations eg appendiceal orifice is associated with increased complication rates or incomplete resection3“ these types of lesions are therefore often referred to surgery which is associated with significant morbidity and mortality and higher costs6 given the high number of polypectomies performed worldwide this is not only an issue of morbidity and mortality but also a huge economic challengethe efficacy and safety of endoscopic full thickness resection eftr of non lifting and other difficult to treat lesions have been demonstrated in multiple retrospective studies and in one prospective study7 the cost of the device is markedly higher than ser devices but lower than surgical treatment the aim of the present analysis was to evaluate whether eftr is cost effective in comparison with ser as well as surgical treatmentmethodsstudy populationto calculate the cost of eftr we analysed the study cohort of the wall resect trial nct02362126 in this single arm multicentre prospective study patients with ˜difficult to treat™ adenomas eg non lifting andor challenging anatomical location early adenocarcinomas and subepithelial tumours in the colorectum were treated with eftr the primary endpoints of the study en bloc and r0 resection rate were achieved in and respectively7 written informed consent was obtained from each patient included in the studysimulation second study arm based on a survey of endoscopistswith the wall resect study being single armed a second study arm was created based on treatment simulation in order to compare different treatment modalities a case report form crf was created and sent to each participating centre of the wall resect trial endoscopists at the respective location reviewed the endoscopic images and their case relevant data and decided which treatment modality they would have chosen if eftr were not available treatment alternatives included ser such as emr thermal methods and esd as well as surgical resection the crf was filled out in a pseudonymised fashioninformed consent had already been obtained within the wall resect studydetermination of case costs and reimbursementa certified online it tool the diagnosis related group drg web grouper was used to determine the reimbursement rate for each patient http drg uni muenster de index php therefore the code of the international classification of diseases icd10 and the specific code for the procedure performed operationen und prozedurenschl¼ssel ops code in each patient in both groups were put into the web grouper together with the predefined mean length of hospital stay ˜mittlere grenzverweildauer™ the drg which accounts for reimbursement was calculatedin the comparison arm the drg codes of were used because this was the year the wall resect study was performed for the eftr arm the drg codes of were used as reimbursement for eftr was increased that yearto calculate the cost per case another certified online it tool g drg report browser was used www g drg de g drg system_ abschlussbericht_ zur_ weiterentwicklung_ des_ g drg systems_ und_ report_ browser this was done by filling in the respective drg icd10 and ops code into the browser the data of the g drg report browser derive from the data that were sent in to inek authority managing the german drg system by certified hospitals ˜kalkulationshuser™ in grouping was performed following the rules of g drg version the main and secondary diagnoses are shown according to icd10 german modification gm version and the procedures according to ops version ˜g drg report browser inek gmbh™as reimbursement for the eftr group was taken from the cost per patient case would ideally also have been calculated from unfortunately these data will be first published by inek in to overcome this problem costs for eftr cases from the university of freiburg between and which were reported to inek were used for the analysiswith the cost of each patient case the mean cost for each treatment modality emr esd laparoscopic surgery transanal endoscopic microsurgery tem eftr could be calculated in the next step the mean cost for each treatment path ser surgical treatment and casemix alternative was determined this was done in the following fashion for ser the mean costs of emr and esd were used for calculation of the surgical treatment laparoscopic surgery and tem were taken together the mean costs of endoscopic and surgical treatments were subsumed as the casemix alternativedetermination of effectivenessthe r0 resection rate was defined as the efficacy parameter to determine cost effectiveness the r0 rate of eftr in the wall resect trial was to determine the efficacy of the therapeutic alternatives to eftr a selective literature review was performed in pubmed and cochrane databases identifying the largest studies comparing resection techniques and r0 rates the respective rates regarding ser found in the literature were for emr and for esd9 for the surgical oncological resection treatment as the gold standard a r0 resection rate was assumed for the tem a rate of had been reported10in order to compare all ser methods emresd all surgical resection methods laparoscopic surgerytem and all alternative methods endoscopic and surgical kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0ctable alternative treatment strategies to eftr with their respective efficacy based on literature review and calculationtreatmentefficacy n n180surgical oncological resection laparoscopictememresdsurgical treatment laparoscopic and temser emresdcasemix alternative assumed arezzo et al fujiya et al arezzo et al calculated calculated calculatedthe combined effectiveness of surgical treatment ser and casemix alternative was calculated by multiplication of the number of patients in each modality eg emr cases for ser with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group overall efficacy of surgical treatment and casemix alternative was performed in the same mannereftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgery˜casemix alternative™ with the eftr procedure a combined effectiveness of each treatment group was calculated this was done by multiplication of the number of patients in each modality eg emr cases with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group using this approach the ˜overall™ efficacy in the ser group was calculated as overall efficacy of surgical treatment and casemix alternative was performed in the same manner and was calculated as and respectively the respective r0 rates are shown in table calculation of costeffectivenessfor assessment of cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were calculated acer expresses the mean costs for the investigated outcome11 in our study acer describes the mean costs per successful r0 resection in the different treatment modalitiesacer is calculated with the following computational formula acer mean costseffect open accessicer expresses the additional costs of a treatment alternative for improvement in the investigated outcome12 in our study these are the incremental costs for the alternative treatment to eftr required to achieve an r0 resectionicer is calculated with the following computational formula icer mean costs interventionˆ’mean costs controleffect interventionˆ’ effect control the mean costs were the total costs of the respective treatment modality divided by the number of patients in each group for the calculation of cost effectiveness the ser methods emr and esd as well as the surgical resection methods laparoscopic resection and tem were taken together furthermore cost effectiveness was calculated for the casemix alternative to compare eftr with all alternativesresultscomparative study armendoscopist surveyfrom patients of the study cohort responses were included for further analysis in one patient the investigator recommended solely ˜thermal ablation™ as alternative treatment of choice thus the primary endpoint r0 resection could not be evaluated from the remaining patients the endoscopists recommended surgical treatment in of of cases thereof of were laparoscopic resections and of tem in of of cases an endoscopic resection was proposed thereof of were emr and of were esdcosts from the perspective of the care providercosts per case were derived from the drg report browser which represent costs of the respective treatment alternative for the year the costs for the eftr treatment were derived from university hospital freiburg and represent the mean costs from years to mid2019 the mean cost for eftr was ‚¬ the cost per surgical treatment laparoscopic surgery and tem was ‚¬ and for ser ‚¬ all alternative treatment strategies ˜casemix alternative™ op laparscopic surgery tem esd and emr were calculated as ‚¬ per case the results are shown in figure costs from the perspective of the thirdpartyaccording to the german drg system reimbursement for eftr is ‚¬ for surgical treatment ‚¬ was calculated the cost per case for ser is ‚¬ the cost for the casemix alternative is ‚¬ the results are shown in figure costeffectiveness analysis care provider viewpointaverage costeffectiveness ratiothe mean cost per r0 resection is ‚¬ in the eftr group and ‚¬ in the surgical group in the ser group the cost per r0 resection is ‚¬ in the casemix alternative group including all treatment kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access figure case costs ‚¬ for the different treatment modalities are shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryfigure incremental cost effectiveness ratio for the different treatment modalities compared with eftr is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryalternatives except eftr the mean cost per r0 resection is ‚¬ the results are shown in figure the casemix alternative ‚¬ the results are shown in figure incremental costeffectiveness ratioin comparison with eftr the incremental cost for an additional r0 resection is ‚¬ if ser is performed the cost for the surgical approach is ‚¬ and for figure average cost effectiveness ratio ‚¬ for the different treatment modalities is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgerycosteffectiveness analysis health insurance reimbursement viewpointaverage costeffectiveness ratiofrom the perspective of the health insurance the cost per r0 resection is ‚¬ in the eftr group in the ser group the cost is ‚¬ and in the surgical treatment group ‚¬ in the casemix alternative the cost per r0 resection is ‚¬ the results are shown in figure incremental costeffectiveness ratiothe icer of ser in comparison with eftr is ‚¬ the surgical approach costs an additional ‚¬ for the casemix alternative ‚¬ is necessary for an additional r0 resection the results are shown in figure discussionwith technical endoscopic progress patient care has constantly improved over the years however as with any technical innovation this is associated with higher costs therefore the efficacy of new methods and devices needs to be evaluated in relation to their costs13 to our knowledge this is the first cost effectiveness analysis cea for eftr our results demonstrate that eftr for difficult to treat lesions in the colorectum is cost effective in comparison with ser as well as surgical therapy furthermore the results are consistent when analysed from the perspective of the care provider as well as of the payerkuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0cfor our analysis a simulated control arm was created this was necessary as to date no randomised controlled trial rct investigating eftr versus alternative treatments has been published in our survey endoscopists proposed surgical treatment as the likely alternative to eftr in the majority of cases as opposed to ser via emr or esd all lesions within the wall resect trial were ˜difficult to resect™ lesions eg non lifting adenomas exhibiting a high risk of perforation or incomplete resection when treated with ser therefore it may be surprising that ser was suggested in of cases however the suggestions were made by expert endoscopists who might have decided towards an advanced endoscopic procedure more generouslyregarding the costs for each treatment modality it was not surprising that the cost of eftr is above ser ‚¬ vs ‚¬ this is due to the cost of the device in germany ‚¬ plus value added tax however the cost of eftr was roughly one third of the cost of surgery ‚¬ vs ‚¬ this reflects the minimally invasive nature of eftr compared with laparoscopic or open surgical operationswhile costs for endoscopic resection and surgical therapy were taken from official and certified tools web grouper and drg report browser the factual costs of the eftr procedure for the year that are determined in a representative cross section of hospitals have not been published yet by inek the administrator of the drg system reimbursement of the procedure changed in thus these data should have been used for calculation to overcome this problem the mean case costs for eftr per case in our home institution university hospital of freiburg germany in the time between and were used as a surrogate in an economic analysis of the cost of eftr in germany presented at the annual conference of the german society for digestive diseases obtained from different endoscopic centres reported ‚¬ per case as this is only above our number and therefore in the same range our calculated ‚¬ seems to be a realistic number14in our analysis we chose the r0 rate as a means to detemine effectiveness as this is the most objective parameter to assess curative resection and treatment success furthermore the r0 rate can be compared with the treatment alternatives of eftr as high quality meta analyses and therapeutic success rates exist for those procedures10 the r0 rate for surgical colonic resection was assumed to be however the patient cohorts of these studies are not equal the wall resect study included only ˜difficult to resect™ lesions mainly non lifting while the studies mentioned above included primarily treatment naive lesions larger studies on ser on non lifting lesions do not exist hence it is reasonable to assume that in these indications real r0 rates of ser would be lower and therefore cost effectiveness would be even worsefor measuring cost effectiveness acer and icer were determined the analysis was performed both open accessfrom the perspective of the care provider hospital as well as the reimbursement authority health insurance acer expresses the mean cost per r0 resection for both investigated perspectives our results reveal that costs are much lower for eftr compared with the surgical alternative although the effectiveness of the surgical approach in terms of radicality can be considered to be higher eftr is cost effective an r0 resection by eftr leads to nearly reduction in costs for the care provider ‚¬‚¬ and for the health insurance ‚¬‚¬ compared with ser eftr leads to marginally higher costs per r0 resection as explained above comparing eftr with ser has limitations as the investigated ˜difficult™ lesions in the wall resect study are not well studied for emr and esd however in comparison with all treatment alternatives ˜casemix alternative™ we calculated and reduction in costs similar to the surgical alternatives figure icer expresses the additional costs for an additional increase in the designated outcome in our analysis it expresses the additional costs that are necessary for an additional r0 resection as shown in figure all alternatives to eftr result in additional costs while ser results in a modest increase ‚¬ and ‚¬ additional ‚¬ and ‚¬ per r0 resection are required in the surgical group in the ˜casemix alternative™ group additional costs were ‚¬ and ‚¬ respectivelyan absolute threshold at which an icer is thought to be cost effective does not exist16 in the literature the willingness to pay threshold ranges from to “ and is highly subjective to the investigated outcome and the healthcare system for which the cea is made17“ for our analysis we assume that a more invasive treatment that produces at least ‚¬ more costs for an additional r0 resection cannot be regarded as being cost effectivefor our analysis we did not include costs of follow up endoscopies or further treatment arising from recurrency or from adverse events this was done due to the following reasons first reliable recurrence rates and long term follow up after eftr do not exist follow up in the wall resect trial was only weeks second the lesions of our patient cohort were heterogeneous including adenomas carcinomas and neuroendocrine tumours with different biological features and recurrent rates third treatment of recurrent lesions is not standardised and ranges from re eftr to snare polypectomy to removal with a biopsy forceps leading to highly variable costs fourth management of severe complications and consecutive morbidity differs in every patient and depends on severity of complication patients™ comorbidities and local expertise we do not have reliable data on costs for such treatment and a hypothetical model would have been highly speculative moreover in the wall resect trial of patients required consecutive surgery due to complications this rate is slightly kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access higher but still grossly comparable with the complication rates of emr and esd on the other hand complications after surgical resection eg anastomotic leakage are much more frequent up to “ and usually lead to higher morbidity hence even if costs related to complications were added icer is still likely to favour eftr compared with the group of treatment alternativesit is difficult to compare our results with other ceas as this is the first one for this indication the only previous cea on ser compared emr and esd in laterally spreading lesions irrespective of location or lifting sign in most analyses as in the study by bahin and colleagues19 a decision tree model was created to compare different outcome scenarios after each treatment path was filled with probabilities of occurrence costs per predefined outcome were calculated a potential bias of this approach is that the data for the probabilities of occurrence which influence the costs most are taken totally or at least in part from different studies19 22this harbours the risk of resulting in a very heterogeneous study population with uncontrolled confounders this risk can be minimised by deriving data from rcts with well balanced patient cohorts as recently published17 for our analysis we used a different approach than a decision tree factual variables and outcome data derived from the only prospective study on eftr treatment and not from assumptions the simulation of the control arm had to be performed due to the lack of rcts in this setting the strength of our study is that the very same clinician who actually performed the respective eftr could review the different lesions and decide on a solid basis which treatment alternative he or she would have used instead of eftr in our view this approach reflects the clinical situation more precisely than a decision tree modelin most ceas the costs per quality adjusted life years are calculated and taken for healthcare decisions neither survival nor quality of life measurements were part of the wall resect trial in line with most of the recently published cea we calculated costs per defined outcome as the primary endpoint17 our study has several limitations first the comparison arm of the study is based on simulation so there is always a risk of a bias second our analysis is specific to the german healthcare system and may therefore not be fully comparable with different healthcare systems in the world third the estimated r0 rate for the ser methods is very low and likely due to the piecemeal resection in the respective study if efficacy would have been measured as ˜freedom of recurrence™ efficacy would be higher as proven in the australian colonic endoscopic study24 nonetheless we used the published r0 rate because of the possibility to match this with the endpoint of the wall resect study furthermore as described above an endpoint such as freedom of recurrence cannot be determined reliably as such data do not exist for eftr fourth costs of complications and follow up were not included this is mainly due to lack of an rct and the short follow up period in an ideal cea all treatment related and hospital stay related costs would have been calculatedin our data indicate that eftr for difficult to treat lesions of the colorectum is cost effective compared with surgical and endoscopic treatment alternatives the results are consistent both from a care provider as well as from a third party payer perspective rcts and long term follow up are needed to further assess the cost effectiveness of eftrauthor affiliations1department of medicine ii medical center “ university of freiburg faculty of medicine university of freiburg freiburg germany2department of gastroenterology klinikum ludwigsburg ludwigsburg baden w¼rttemberg germany3department of gastroenterology evangelisches krankenhaus d¼sseldorf dusseldorf nordrhein westfalen germany4department of internal medicine and gastroenterology elisabeth hospital essen nordrhein westfalen germany5department of medicine ii interventional and experimental endoscopy inexen university hospital wurzburg wurzburg bayern germany6department of gastroenterology university hospital augsburg augsburg bayern germany7department of gastroenterology university hospital ulm ulm baden w¼rttemberg germany8department of gastroenterology klinikum dortmund dortmund nordrhein westfalen germany9department of gastroenterology helios klinikum krefeld krefeld nordrhein westfalen germany10department of gastroenterologyoncology klinikum sindelfingen b¶blingen sindelfingen baden w¼rttemberg germanycontributors as and kc invented and planned the present study and also the underlying wall resect study as assisted with data acquisition and analysis and revised the manuscript jb was responsible for data research and acquisition ak was responsible for data analysis and writing the manuscript kc tb bs am hm hn da mb ap mf tf mg and rt took part in the online survey to create the simulation comparison arm of the study furthermore they carefully read and revised the manuscriptfunding the authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorscompeting interests as and kc received lecture fees and study grants from ovesco endoscopy t¼bingen germany ak jb tb bs am hm hn da mb ap mf tf mg and rt have no conflicts of interest or financial ties to disclosepatient consent for publication not requiredethics approval the wall resect study was approved by the ethical board on january the study protocol conforms to the ethical guidelines of the declaration of helsinki as reflected in a prior approval by the institution's human research committee for the present study an additional approval by the institutional review board was not necessary since no additional personal data were collectedprovenance and peer review not commissioned externally peer revieweddata availability statement all data relevant to the study are included in the or uploaded as supplementary information data were derived from the wall resect trial nct02362126open access this is an open access distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idarmin a0kuellmer http orcid org kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0creferences who colorectal cancer fact sheet the global cancer observatory available http gco iarc fr today data factsheets cancers 10_ 8_ colorectum fact sheet pdf [accessed sep ] zauber ag winawer sj o'brien mj et a0al colonoscopic polypectomy and long term prevention of colorectal cancer deaths n engl j med “ hong sn byeon js lee b i et a0al prediction model and risk score for perforation in patients undergoing colorectal endoscopic submucosal dissection gastrointest endosc “ org mizushima t kato m iwanaga i et a0al technical difficulty according to location and risk factors for perforation in endoscopic submucosal dissection of colorectal tumors surg endosc “ agapov m dvoinikova e factors predicting clinical outcomes of endoscopic submucosal dissection in the rectum and sigmoid colon during the learning curve endosc int open 20142e235“ baum p diers j lichthardt s et a0al sterblichkeit und komplikationen nach viszeralchirurgischen operationen dtsch arztebl int “ schmidt a beyna t schumacher b et a0al colonoscopic full thickness resection using an over the scope device a prospective multicentre study in various indications gut “ aepli p criblez d baumeler s et a0al endoscopic full
Colon_Cancer
" the empirical dietary index for hyperinsulinemia edih score is a validated foodbased dietary scorethat assesses the ability of wholefood diets to predict plasma cpeptide concentrations although the edih hasbeen extensively applied and found to be predictive of risk of developing major chronic diseases its influence oncancer survival has not been evaluated we applied the edih score in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patterns after diagnosis and determine its influence on survivaloutcomesmethods we calculated edih scores to assess the insulinemic potential of postdiagnosis dietary patterns andexamined survival outcomes in a sample of stage iiii colorectal cancer patients in the nurses™ health studyand health professionals followup study cohorts multivariableadjusted cox regression was applied to computehazard ratios hr and confidence intervals ci for colorectal cancerspecific mortality and allcause mortalitywe also examined the influence of change in diet from pre to postdiagnosis period on mortalityresults during a median followup of years there were deaths which included colorectal cancerspecific deaths in the multivariableadjusted analyses colorectal cancer patients in the highest compared tolowest edih quintile had a greater risk of dying from colorectal cancer hr ci and a greater risk of allcause death hr 95ci compared to patients who consumed low insulinemicdiets from pre to postdiagnosis period patients who persistently consumed hyperinsulinemic diets were at higherrisk of colorectal cancer death hr151 95ci and allcause death hr 95ci our findings suggest that a hyperinsulinemic dietary pattern after diagnosis of colorectal cancer isassociated with poorer survival interventions with dietary patterns to reduce insulinemic activity and impactsurvivorship are warrantedkeywords colorectal cancer survival insulinemic dietary patterns insulin cpeptide correspondence fredtabungosumcedu1division of medical oncology department of internal medicine the ohiostate university college of medicine west 12th avenue 302b wisemanhallccc columbus oh usa2the ohio state university comprehensive cancer center arthur g jamescancer hospital and richard j solove research institute columbus oh usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctabung bmc cancer page of canceristhefourth most colorectalcommonlydiagnosed cancer in the united states while there ishigh potential for dietary patterns as a modifiable riskfactor for colorectal cancer development [ ] verylimited evidence exists among colorectal cancer survivors for example in a recent review we identified s published up to that reported on theassociation between dietary patterns and colorectalcancer development but only about five s onthe association between dietary patterns and outcomesamong colorectal cancer survivors [“] the evidenceshowed that the western dietary pattern often characterized by high intakes ofred andprocessed meats desserts and potatoesis associatedwith higher risk of allcause mortality but generally notwith colorectal cancerspecific mortality in patients withcolorectal cancer the prudent dietary pattern oftencharacterized by high intakes of fruits vegetables wholegrains and poultry showed similar results with inverseassociations for allcause mortality but no consistent association with colorectal cancerspecific mortality [“]higher adherence to other dietary patterns including themediterranean diet score dietary approaches to stophypertension meal plan american cancer society cancerprevention guidelines score healthy eating index scorewere generally associated with lower risk of allcausemortality but the associations were inconsistent acrossstudies [ ]refined grainschronic diseasesfurther research is therefore needed to clarify ifdietary patterns are importantfor colorectal cancerprognosis and if dietary changes can maximally impactoverall and cancerspecific survival biomarkerbaseddietary patterns may be helpfulin this regard forexample hyperinsulinemia and insulin resistance areconsidered important underlying mechanisms linkingpoor dietary patterns and lifestyle behaviors to the development of multipleincludingcolorectal cancer [“] studies have shown positiveassociations between circulating cpeptide concentrations a marker of beta cell secretory activity and colorectal cancer risk and prognosis [“] therefore adietary pattern associated with hyperinsulinemia may bemore predictive of outcomes following colorectal cancerdiagnosis than a dietary pattern not associated with thispathway we previously derived the empirical dietaryindex for hyperinsulinemia edih score to assess thepotential of dietary patterns to influence insulinemia which has been extensively applied in large cohortstudies and found to be predictive of nonfasting cpeptide concentrations [ ] longterm weight gain risk of developing colorectal cancer other digestive system cancers and other cancers however the influence of dietary insulinemic potentialon cancer survival outcomes has not yet been evaluatedthe objective of the current study is to apply the edihscore in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patternsafter diagnosis and determine its influence on survivaloutcomesmethodsstudy populationwe used data from the nurses™ health study nhs andthe health professionals followup study hpfs twoongoing cohorts in the united states hpfs was initiatedin and enrolled male health professionalsbetween the ages of and years nhs initiatedin enrolled registered female nurses aged to years fig data on medical lifestyleand other healthrelated factors was collected at baselineand have been updated every years thereafter ethicalapproval for our study was provided by the harvardth chan school of public health and those of participating registries as required and the institutional reviewboards of the brigham and women™s hospital studyparticipants provided consent by completing and submitting study questionnaires participants were free toterminate participation in the study at any timeassessment of diet and the empirical dietary index forhyperinsulinemia edih scorein both cohorts diet was assessed using a validated selfadministered food frequency questionnaire ffq thatassessed how often on average participants consumed astandard portion size of various foods in the past yearin the nhs diet was assessed in andevery years thereafter whereas in the hpfs diet wasassessed in and every years thereafter theedih score developed to empirically measure the insulinemic potential of whole diets using food groups hasbeen described in detail briefly thirtynine foodgroups were entered into stepwise linear regressionmodels to identify a dietary pattern most predictive ofplasma cpeptide levels the edih score represents aweighted sum of food groups with higher scores indicating hyperinsulinemic diets hyperinsulinemia andlower scores indicating low insulinemic diets the foodgroups contributing to lower edih scores are winecoffee fullfat dairy products whole fruit and green leafyvegetables whereas the food groups contributing to highedih scores arelowfat dairy products french frieslowenergy beverages cream soups processed meat redmeat margarine poultry nondark fish high tomatoesenergy beverage and eggs in the current study we calculated edih scores foreach participant based on the selfadministered ffqspostdiagnosis edih score was calculated based on the 0ctabung bmc cancer page of fig flow chart describing the flow of participants from the full cohorts to the final analytic sample in the nurses™ health study nhs andhealth professionals followup study hpfsfirst ffq returned at least months but not more than years after colorectal cancer diagnosis thus avoidingdiet assessment during active cancertherapy themedian time from diagnosisto postdiagnosis dietassessment was years prediagnosis edih score wascalculated based on the cumulative average of edihscores up to the last diet assessment before colorectalcancer diagnosis the median time from prediagnosisdiet assessment to diagnosis was yearspatients with colorectal cancer and mortality assessmentwhen a colorectal cancer diagnosis was reported duringthe previous years on the followup biennial questionnaires we requested permission to obtain hospital records and pathology reports blinded study physiciansthen reviewed these records and recorded data on tumorcharacteristics for nonrespondents the national deathindex was used to identify deaths and ascertain anydiagnosis of colorectal cancer that contributed to deathafter years of followup for disease diagnoses “ in nhs and to in hpfs we identified patients with pathologically confirmed colorectalcancer we excluded participants who died before in nhs or in hpfs had reported any cancerexcept nonmelanoma skin cancer before colorectalcancer diagnosis who died at diagnosis who did nothave prediagnosis diet or postdiagnosis diet patientswho did not complete a diet assessment between months and years after diagnosis or had diet assessedoutside of this period who had diabetes at colorectalcancer diagnosis and patients with stage iv or unknownstage at diagnosis therefore the current analysis included patients with stage i ii or iii colorectal cancerincluding participants from hpfs and from nhs fig deaths were ascertained throughreporting by family for persistent nonresponders wequeried the national death index with their names up 0ctabung bmc cancer page of to june for nhs and january for hpfs cause of death was assigned by blinded physicianscovariate assessmentboth cohorts assessed covariate data eg medical historylifestyle and health factorsthrough selfadministeredquestionnaires every years these factors included physical activity smoking habits alcohol intake multivitaminuse endoscopy status regular use of aspirin and othernonsteroidal antiinflammatory drugs nsaids familyhistory of colorectal cancer weight height menopausalstatus and postmenopausal hormone use only forwomenin both cohorts as previously described dietassessment was conducted every years [ ]statistical analysiswe categorized the edih score into quintiles withcohortspecific cutoffs then pooled the data for analysispersontime of followup was calculated from the dateof postdiagnosis diet assessment to death or to lastfollowup date january in hpfs or june innhs whichever was first we used the kaplanmeiermethod to generate survival curves by quintiles of edihscore and tested group differences highest vs lowestquintile using the logrank test for this test the edihscore was adjusted for total energy intake and bmi usingthe residual methodcox proportional hazards regression was used to calculate hazard ratios hrs of colorectal cancerspecificdeath or allcause death in edih quintiles quintile cutpoints were created separately by sex and applied in thepooled sample given that participants must survivefrom diagnosis until postdiagnosis diet assessment weused time since diagnosis as the underlying time scale toaccount for left truncation due to staggered entry thecox models were tested for the assumption of proportionality using timecovariate interaction terms andstratified by age sex and stage we fitted two models tothe data as follows model minimally adjusted modelincluded bmi demographic factors sex age at diagnosis and tumor characteristics stage subsite within thecolon grade of tumor differentiation model fullyadjusted model included all the covariates in model and postdiagnosisfactors packyears ofsmoking physical activity regular aspirin use pre topostdiagnosis weight change total alcohol intake andprediagnosis dietary pattern edih score testforlinear trend of risk across edih quintiles was performedusing the median postdiagnosis edih score in eachedih quintile as a continuous variable in the cox regression models and interpreting the pvalue of thisvariable as the pvalue for linear trendlifestyleto determine how changes in the insulinemic potential of diet before and after diagnosis influence survivalwe dichotomized pre and postdiagnosis edih scores atthe median and used to create a change variable withlow indicating a score below the median and high ascore above the median lowlow consistently lowdietary insulinemic potential before and after diagnosisie both scores below the median lowhigh patientsconsuming low insulinemic diets before diagnosis andmore hyperinsulinemic diets after diagnosis highlowpatients consuming hyperinsulinemic diets before diagnosis and then changed towards low insulinemic dietsafter diagnosis and highhigh patients who consistentlyconsumed hyperinsulinemic dietary patterns before andafter diagnosis we then applied these dietary patternchanges in multivariableadjusted cox models to examine risk of death from colorectal cancer and from othercauseschange preand postdiagnosisfrom postdiagnosis weightwe conducted exploratory subgroup analysesincategories of the following potential effect modifiers sexweightand prediagnosis edih score we categorized prediagnosisedih at the median median and ‰¥ median weightchange was calculated by subtracting prediagnosisweightthe continuousweight change variable was categorized as follows thosewho gained more than kg had a stable weight ˆ’ kg to kg or lost more than kg we also conducted subgroup analyses by time since diagnosis years ‰¥ years and age at diagnosis years ‰¥years tests of interaction between postdiagnosis edihscore and the potential effect modifiers were assessed byentering in the modelthe cross product of postdiagnosis edih score and the stratification variable andevaluated by the wald test all analyses were performedusing sas for unix all pvalues were two sidedresultscharacteristics of patients women with colorectal cancer after diagnosis are shown in table meanage at diagnosis was years and mean postdiagnosisbmi was kgm2 with of patients classified asoverweight or obese regarding disease stage hadstage i or ii and had stage iii disease during amedian followup of years there were allcausedeaths which included colorectal cancerspecificdeaths median overall survival by cancer stage was years for those with stage i disease years for thosewith stage ii and years for those with stage iiidisease fortyone percent of patients maintained astable weight between ˆ’ kg and kg between theprediagnosis and postdiagnosis period while lostmore than kg body weight and gained kgbody weight in the same period colorectal cancer patients with the most hyperinsulinemic dietary patterns 0ctabung bmc cancer page of table postdiagnosis characteristics of colorectal cancer patients by quintiles of postdiagnosis empirical dietary index forhyperinsulinemia edih score n characteristictotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile quintile ˆ’ to ˆ’ to n n quintile ˆ’ to ˆ’ n quintile ˆ’ to n quintile to n female age at diagnosisdage at diagnosis by sexdfemalemalecurrent smoker packyears of smokingbody mass index kgm2overweight bmi ‰¥ obese bmi ‰¥ physical activity methweekcphysical activity methweekcd by sex femalemale nonalcohol drinkers regular aspirin use location of cancer in the colon proximal colondistal colonrectumunspecifiedstage at diagnosis stage istage iistage iiimedian survival time yearsmedian survival time by staged years stage istage iistage iiiweight change categoriesd stable weight ˆ’ to kggained more than kglost more than kg avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intakecmetabolic equivalents from recreational and leisuretime activitiesdvalue is not age adjustedafter diagnosis quintile tended to have higher bodyweight and lower physical activity for example theaverage bmi among those classified in quintile was kgm2 and the average physical activity was methourweek compared with kgm2 and methourweek among those in quintile alsopatients consuming the most hyperinsulinemic dietarypatterns were less likely to have stage i disease and theyexperienced shorter survival times table patients consuming low insulinemic dietary patternshad higher intakes of wholegrains nuts vegetables wholefruits and coffee and lower intakes of refined grainscream soup eggs french fries butter margarine sugarsweetened beverages red meat and processed meat in 0ctabung bmc cancer page of table median 5th 95th percentile food and nutrient intake profiles of colorectal cancer patients by quintiles of postdiagnosisempirical dietary index for hyperinsulinemia edih scoretotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile n quintile n quintile n quintile n quintile n foods servingsweekprocessed meatred meat highenergy sugary beverages lowenergy sugary beverages margarinebutterfrench friesnondark fisheggslowfat dairycream souprefined grainstomatopoultrydark fishfullfat dairycoffeeteawhole fruitfruit juicepotatoesgreenleafy vegetablesdarkyellow vegetablesother vegetablesnuts total alcohol intake drinksweek whole grainsnutrient profile total carbohydrates gdtotal protein gd branchedchain amino acids gd total fat gdtotal fiber gd avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intaketerms of the nutrient profile resulting from this postdiagnosis dietary pattern patients consuming a lowinsulinemic dietary pattern had higher intakes of totalcarbohydrates and total fiber and lower intakes of total fattotal protein and branchedchain amino acids table kaplanmeier curves by quintiles of edih score areshown in fig with patients consuming a lowinsulinemic diet quintile experiencing better survivalfor colorectal cancerspecific and overall mortalitycompared to those consuming hyperinsulinemic dietsquintile in the multivariableadjusted analyses wefound that a hyperinsulinemic postdiagnosis dietarypattern was associated with higher risk of colorectalcancerspecific mortality and allcause mortality table 0ctabung bmc cancer page of fig kaplan“meier curves of a colorectal cancerspecific and b overall survival among patients with colorectal cancer by quintile of postdiagnosis empirical dietary index for hyperinsulinemia edih score logrank pvalues were calculated to test group differences quintile vs and adjusted for postdiagnosis total energy intake and postdiagnosis body mass indexcomparing colorectal cancer patients classified in the highestedih quintile to those in the lowest quintile there was a higher risk of colorectal cancerspecific death hr 95ci ptrend and a higher risk of allcause death hr 95ci ptrend afteraccounting for prediagnosis dietary insulinemic potentialamong other confounding variables table in relation to changes in the insulinemic potential ofthe diet before and after diagnosis patients whoconsumed a more hyperinsulinemic dietary patternconsistently before and after diagnosis were at higherrisk of dying from colorectal cancer hr ci and from other causes hr ci compared to patients who consistentlytable hazard ratios ci for colorectal cancerspecific and allcause mortality among patients with colorectal cancer byquintile of postdiagnosis edih scorestatistical modelcolorectal cancerspecific mortalityquintiles of the empirical dietary index for hyperinsulinemia edih scorequintile quintile quintile quintile ptrendquintile deathspatients aliveminimallyadjusted model reference fully adjusted model reference allcause mortalitydeathspatients aliveminimallyadjusted model reference fully adjusted model reference the minimallyadjusted models was adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stagegrade of tumor differentiation and location of primary tumor within the colon the fullyadjusted model was additionally adjusted for postdiagnosis physicalactivity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake andprediagnosis edih score 0ctabung bmc cancer page of fig hazard ratios for the association of change in dietary insulinemic potential between prediagnosis diet and postdiagnosis diet and risk ofdying form colorectal cancer crcsurvival and from all causes combined overall survival edih scores were dichotomized at the median lowlow the reference category represents participants who persistently consumed low insulinemic diets below the median edih from pre to postdiagnosis period lowhigh are those who changed from low insulinemic diets towards more hyperinsulinemic diets highlow represents thosewho changed from consuming hyperinsulinemic diets prior to diagnosis towards consuming low insulinemic diets after diagnosis whereas highhigh represents those who persistently consumed hyperinsulinemic diets prior to diagnosis and after diagnosis the number of deaths patientsalive in the four categories were as follows crcsurvival lowlow lowhigh highlow highhigh overallsurvival lowlow lowhigh highlow highhigh models were adjusted for age at diagnosis postdiagnosisbody mass index total energy intake sex race year of diagnosis cancer stage grade of tumor differentiation location of primary tumor withinthe colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake and prediagnosis edih scoreconsumed a low insulinemic dietary pattern before andafter diagnosis fig in subgroups of potential effect modifiers risk of colorectal cancerspecific mortality was significantly elevatedamong women and among those who lost body weightthose who were consuming a hyperinsulinemic dietarypattern before diagnosis and those younger than years for these subgroup analysesinteractions werestatistically significant only for sex in allcause mortalitytable discussionin the current study we showed that habitual consumption of hyperinsulinemic dietary patterns after colorectalcancer diagnosis or consumption of a hyperinsulinemicdietary pattern consistently before and after diagnosiswas associated with higher risk of dying from colorectalcancer and from all causes combinedthe insulinemic potential of diet was first estimatedby the insulin index which is based on a conceptsimilar to the more widely used glycemic index thatcharacterizes carbohydratecontaining foods accordingto their ability to raise blood glucose concentrationspostprandially compared with a reference food glucoseor white bread though carbohydrate content isone important factor influencing insulin response foodscan also stimulate insulin secretion in a carbohydrateindependent manner the insulin index directly quantifies the postprandial insulinemic potential of a food andtakes into account foods with a low or no carbohydratecontent it is important to understand that theinsulin index which was used in most previous studiesof dietary insulinemic potential and colorectal cancersurvival is conceptually and technically different fromthe edih and essentially uncorrelated spearman r ˆ’ the principle ofthe insulin index is how aparticularfood item stimulated insulin secretionindependent of underlying insulin resistance whereasthe edih is primarily driven by insulin resistance forcolorectal cancer the only other paper using the edihwas on cancer incidence both the insulin index and glycemic index assess thepostprandial shortterm effects of the diet unlike theedih score which predicts integrated insulin exposureie both fasting and nonfasting based on habituallongterm dietary intake postdiagnosis insulinindex and insulin load have been linked to higher risk ofdying from colorectal cancer [ ] higher dietary insulin load and insulin index after diagnosis of colorectalcancer were associated with increased risk of colorectalcancerspecific and overall mortality the association of postdiagnosis glycemic indices with colorectal 0ctabung bmc cancer page of table subgroup analyses of the association between dietary insulinemic potential and colorectal cancerspecific and allcausemortalitysubgroupptrendpinteractionquartile quartile quartile deathspatientsaliveedih quintilesquartile colorectal cancerspecific mortalitysexmenwomenweight change post minus prediagnosis weightstable weight ˆ’ to kgweight gain kgweight loss kgprediagnosis edih score median‰¥ medianage group at diagnosis years‰¥ yearstime since diagnosis years‰¥ yearsallcause mortalitysexmenwomenweight change post minus prediagnosis weightstable weight ˆ’ to kgweight gain kgweight loss kgprediagnosis edih score median‰¥ medianage group at diagnosis years‰¥ yearstime since diagnosis years‰¥ years ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref models were adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stage grade of tumordifferentiation and location of primary tumor within the colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirinuse weight change pre to postdiagnosis and postdiagnosis total alcohol intake and prediagnosis edih scorecancer prognosis has been inconsistent whereas onestudy found higher risk of colorectal cancer recurrenceand death associated with higher glycemic load but nothigher glycemic index another found no associationbetween glycemic load or glycemic index and colorectalcancer survival glycemic scores are primarilyreflective ofthe postprandial glucose responses ofcarbohydratecontaining foods whereas the edih scoredirectly reflects insulin increases induced by componentsof the dietary pattern that may or may not be contributing to calories eg coffee current study findingstherefore suggest that the direct effect of the diet on 0ctabung bmc cancer page of insulin may be more important than the effect of diet onglucose for colorectal cancer prognosis though theglycemic index is a measure of the shortterm postprandial effect of the diet on glucose concentrations it ispossible that such a habitual dietary pattern could overtime lead to sustained hyperinsulinemia and insulin resistance which could then mediate colorectal cancerprognosis however a previous study in these cohortsdid not observe an association between an overall lowcarbohydrate diet score and colorectal cancer or overallmortality although those who consumed a plantrichlowcarbohydrate diet which emphasized plant sourcesof fat and protein with moderate consumption of animalproducts had lower risk of colorectal cancerspecificmortality insulin is a growth factor and major regulator of cellmetabolism and its effects in target cells are mediatedby the insulin receptor a transmembrane protein withenzymatic activity evidence suggest that insulinstimulates growth mainly via its own receptor and notthe igf1 receptor and that in many cancer cells theinsulin receptor is overexpressed and the a isoformwhich has a predominant mitogenic effectis morerepresented than the b isoform the metabolicpathway stimulated by the activated insulin receptor toregulate glucose protein and lipid metabolism involvesthe pi3kakt pathwaycharacteristicsprovide a selective growth advantage to cancer cellswhen exposed to insulin therefore all conditions ofhyperinsulinemia both endogenousdiabetes metabolic syndrome obesity and exogenouseg hyperinsulinemic diets which also influence someof the endogenous conditions [ ] willincreasecancer risk and mortality theseegtypefor most ofalthough interactionsthe subgroupanalyses were not statistically significant some of thefindings merit some discussion the associations werestronger among women than among men which may berelated to severalfactors the larger sample size andstatistical power in our evaluation of women potentialconfounding with age as women were younger on average than men and a true biological interaction basedupon endocrine and associated metabolic factors wealso observed that there were worse outcomes amongpatients who lost weight than among those who maintained a stable weightto postdiagnosisperiod which may be consistent with complications ofprogressing disease leading to poor diet intakefrom premajor strengths of our study include the use of afoodbased edih score that is correlated with circulating cpeptide concentrations [ ] we had access tocomprehensive pre and postdiagnosis data on diet andimportant covariates which reduces the potential for residual confounding and recall bias our findings alsoaccounted for potential bias from staggered entry due todifferences between participants in the time betweendiagnosis and postdiagnosis diet assessment limitations to be considered in interpreting our findings include potential measurement error in the selfreporteddietary and lifestyle data though prior studies in thehpfs and nhs that evaluated the relative validity offfq data have shown reasonably good correlations between ffq and diet records [ ] though we adjusted for several potential confounding variables ahyperinsulinemic dietary pattern may be associated withother factors not included in the current study therefore we cannot completely rule out confounding byunmeasured variables given that we did not have information on cancer treatment which could influence dietary choices of cancer patients or modify the diet andsurvival association we adjusted all analyses by cancerstage at diagnosis which is the principal determinant ofcolorectal cancer treatmentin this large prospective study a higher edih scorereflecting higher insulinemic potential of the diet wasassociated with higher risk of death from colorectalcancer and from all causes taken together our resultssuggest that this association may be mediated partlythrough mechanisms involving hyperinsulinemia interventions with dietary patterns to reduce insulinemia mayenhance survivorship among colorectal cancer patientsabbreviationsbmi body mass index ci confidence interval edih empirical dietary indexfor hyperinsulinemia score ffq food frequency questionnairehpfs health professionals followup study hr hazard ratio methourweek metabolic equivalent hours per week nhs nurses™ health study nsaids nonsteroidal antiinflammatory drugs pi3kakt phosphatidylinositol kinaseprotein kinase b sas® statistical analysis software®acknowledgementswe would like to thank the participants and staff of the nurses™ health studyand health professionals followup study for their valuable contributions asw
Colon_Cancer
" clinical trials have been conducted to clarify the beneficial effects of vd3 1α25dihydroxy vitamind3 also known as calcitriol treatment in prostate cancer however the molecular mechanisms underlying theseeffects are not fully understood recent studies on igfbp3 have indicated its intracellular functions in cell growthand apoptosis the aim of this study was to confirm the benefits of lowdose vd3 treatment and clarify themolecular mechanisms underlying these beneficial effects in prostate cancer cellsmethods the molecular effects of simultaneous treatment of lncap cells and their genetically modified cell lineswith low concentration of docetaxel and vd3 were biologically and biochemically analyzed to further determinethe effects of vd3 treatment on igfbp3 induction system cells were temporarily treated with vd3 in combinationwith a transcriptional inhibitor or protein synthesis inhibitor bcl2 protein and its mrna behavior were also observed inigfbp3 expressionmodified lncap cells to determine the involvement of igfbp3 in the suppression of bcl2 by vd3treatmentresults changes in igfbp3 expression levels in lncap cells indicated that it mediated the inhibition of cell growthinduced by vd3 treatment igfbp3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancercells to vd3 in combination with the anticancer drug we further identified the distinct property of the igfbp3induction system wherein temporal vd3 stimulationinduced prolonged igfbp3 expression and vd3 treatmentinduced increase in igfbp3 expression were optimized based on the protein concentration rather than the mrnaconcentration meanwhile bcl2 expression was downregulated by vd3 treatment in an igfbp3independent manner these findings indicate the molecular mechanisms of igfbp3 induction stimulated by vd3 and igfbp3independent bcl2 suppression by vd3 treatment in prostate cancer cells the results could prompt a reevaluation ofvd3 usage in therapy for patients with prostate cancerkeywords vitamin d nonlinear igfbp3 induction bcl2 suppression prostate cancer treatment correspondence satorusasagawatokushukaijp1molecular biology laboratory research institute nozaki tokushukai hospitaltanigawa daito osaka japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cigarashi bmc cancer page of vitamin d has a central role in calcium and skeletalhomeostasis [ ] its pleiotropic role both in physiological and pathological phenomena such as cell growthimmune function and tumorigenesis has also been examined [“] which revealed that exposure of cancercells to vitamin d significantly reduces the cell growthrate in multiple cancer types [“] indeed recent epidemiologicalinvestigations have reported that highervitamin d concentration could prevent multiple types oftumorigenesis consistent with such finding for example an increase in colon cancer incidence with lowervitamin d dietary habits has been reported [ ]however suppressive effect on prostate cancer is stillunder discussion [ ]in turntraditional methodscurrently prostate cancer is one of the most commonthecancers in men worldwide clinical use ofprostatespecific antigen psatest dramatically improved the screening sensitivity of prostate cancer compared to that ofthenumber of patients with earlystage prostate cancer hasrapidly increased since the mid1990s [ ] unlikeother cancer types most cases of prostate cancer haveslow progression or have nonprogressive indolentsymptom and are localized in the prostate thus they areunlikely to cause poor physical condition or death therefore patients with prostate cancer need a less burdensome treatment in order to avoid potential harmfrom excessive treatmentalthough the impact of vitamin d as a single agent onprostate cancer has been investigated its significance remains under discussion [ ] meanwhile the synergistic or additive effects of vitamin d and its derivativeswith anticancer drugs on prostate cancer have beenclinically studied and encouraging results have been reported [ ] however the results of larger trials thatevaluated the synergistic effect of vitamin d in combination with docetaxel one of the firstline anticancerdrugs in prostate cancer chemotherapy showed limitedor nonsignificant benefit of vitamin d efficacy in castration or androgen deprivation therapy“resistant prostatecancer [ ] furthermore overconsumption of 1α25dihydroxy vitamin d3 vd3 also known as calcitriolthe biologically active form of vitamin d3 from food orprolonged treatment with vd3 derivatives could triggerhypercalcemia resulting in physiological side effects therefore to date vd3 has not been proactivelyused in the treatment of patients with prostate cancerthe biological function of vitamin d is mainly mediatedby vitamin d receptor vdr which acts as a transcriptional factor vitamin d receptor elements vdreon the promoter region of target genes are recognizedand transcriptionally activated by vitamin d“coupledvdr consistent with the diverse physiological functionof vd3 vdre was identified not only in the gene related to calcium and skeletal homeostasis but also in thegene related to fundamental cellular functions includingcell growth igfbp3 is one of the families of sixhigh affinity igfbps and was originally found in plasmaas a stabilizer and transporter of igfs in the bloodstream interestingly vdre was found on the prothe igfbp3 gene and recent studies havemoter ofrevealed that igfbp3 functions inside the cell as wellregulating cell growth and apoptosis [ ]methodsthis study aimed to investigate igfbp3 induction byvitamin d treatment and determine its role in prostatecancer treatment with vitamin d in combination withanticancer drugs in order to provide molecular biologicalevidence of benefit of vitamin d and to suggest effectivevitamin d usage in prostate cancer treatmentchemicals and reagentsdihydrotestosterone dht and calcitriol vd3 purchased from tokyo chemical industry tokyo japanwere resolved in ethanol as a stock solution pei maxmolecular weight was purchased from polysciences pa usa the other chemicals and reagentswere purchased from wako pure chemicalosakajapan and sigmaaldrich st louis mo usaincluding testosteronecharcoal stripping of fetal bovine serum fbsfbs was purchased from gibco waltham ma usato deplete hormonesin fbsdextrancoated charcoal powder was added to theserum and the mixture was incubated with rotation at degree overnight thereafter the mixture was centrifugedto pellet charcoal and the supernatant was filtered througha 022μm polyvinylidene difluoride membrane thecharcoalstripped serum was used for all experimentstotalthe concentrations oftestosterone and totalvitamin d in the serum were determined using a totaltestosterone test kit abbott japan chiba japan and atotal vitamin d test kit roche basel switzerland according to manufacturers™ instructions the concentrations of total testosterone in the pre and posttreatmentserum were nm and less than nm limit of detection respectively the concentrations of total vitamin d in the pre and posttreatment serum were and nm respectively thus the basal concentrationsin the culture medium supplemented with fbs wereless than nm total testosterone and approximately nm total vitamin dcell culturethe lncap cell line was obtained from american typeculture collection and cultured in dulbecco™s modified 0cigarashi bmc cancer page of eagle medium dmem sigmaaldrich supplementedwith 10charcoalstripping fbs the 293ft celllinewas purchased from invitrogen waltham ma usaand cultured in dmem supplemented with fbs mm of lglutamine sodium pyruvate and nonessentialamino acids the cells were cultured at a temperature of °c in co2“humidified condition the mycoplasma contamination was routinely checked and confirmed as negativecell growth assaydmem supplemented with charcoalstripping fbs was used for the cell growth assay the cells were seededat × cells per well in a 6well plate the next daythe medium was replaced with ml of fresh mediumand nm dht andor nm vd3 were added the cellculture was continued throughout the indicated periodthe cultured cells were trypsinized and the number ofcells was assessed using an automated cell countercountess iitm fl invitrogen each assay was repeatedat least three times and similar results were obtainedwestern blottingthe antibodies used for western blotting were mouseantiigfbp3 santa cruz biotechnology mouseantiβactin santa cruz biotechnology mouseantibcl2 santacruz biotechnology and horseradish peroxidase“conjugated secondary antibodies jackson immunoresearch laboratories westgrove pa usa the collected cells were resuspendedin ripa buffer supplemented with protease and phosphatase inhibitors roche basel switzerland and lysedusing bioruptortm ii sonicator cosmo bio tokyojapan cell lysates were resolved by “ nupagegels invitrogen and transferred onto polyvinylidenefluoride membrane millipore burlington ma usathe signals were developed using enhanced chemiluminescence reagent perkinelmer waltham ma usaand luminograph i atto tokyo japan was used forimage capture quantification of band signal wasanalyzed using cs analyzer software atto at leasttwo biological replicates of each experiment were performed with similar resultsrealtime reverse transcription“polymerase chain reactionthe total rna from the cultured cells was extractedusing trizol reagent invitrogen according to the manufacturer™s instruction the rna was reverse transcribedby the primescript rt reagent kit takara bio shigajapan using oligodt quantitative reverse transcription“polymerase chain reaction rtpcr reaction wasperformed using tb green premix ex taq ii takaraand genespecific primers supplementary table bycfx96 touch realtimepcr system bioradlaboratories hercules ca usa gene expression datawere normalized against glyceraldehyde 3phosphate dehydrogenase or hprt1 as internal control at leastthree biological replicates of each experiment were performed and similar results were obtainedflow cytometric analysis of cell cycle and apoptosisfor cell cycle analysis and apoptotic cell detection flowcytometric analysis was performed using the guavaeasycyte plus flow cytometry system millipore andguava cell cycle reagent and annexin v fitc apoptosiskit millipore according to manufacturer™s instructionas previously described at least three biologicalreplicates of each experiment were performed and similar results were obtainedlentiviral construction and transductionbackbone vectors plko1 puro plasmid andplenti cmv puro dest w118“ plasmid were provided by drs bob weinberg eric campeau andpaul kaufman respectively via addgene ref pentr1a plasmid and lentiviral packaging mix plp1plp2 and plpvsvg were purchased from invitrogenfulllength igfbp3 was cloned from lncap cell complementary dna cdna using kod fx neo toyoboosaka japan with a specific primer set supplementarytable and inserted into the pentr1a vector thenthe sequence was confirmed the igfbp3 cdna was introduced into the plenti cmv puro dest vector by recombinaseenzymeinvitrogen to generate the lentiviral expression vectorspecific short hairpin rnas shrnas were designedusing invitrogen or biosettia websites the selectedtarget sequence oligos supplementary table wereannealed and inserted into the plko1 puro vector according to addgene™s instruction the lentiviral expressionvector or shrna vector was cotransfected with the lentiviral packaging mix into the 293ft cells using pei maxinstead of lipofectamine according to invitrogen™sinstruction twentyfour hours posttransfectionthemedium was replaced with the culture medium forlncap cells one day later the lentiviruscontaining supernatants were collected and filtrated through a 045mmpolyvinylidene fluoride filter milliporereaction using lr clonaseiilentivirus infectionone day before infection × lncap cells wereplated into a 10cm dish then the culture medium wasreplaced with the lentiviruscontaining supernatant andculture was continued twentyfour hours post infectionreplaced with fresh culturemedium two days later the medium was replaced withfresh culture medium that contained μgml of puromycin and culture was continued until the noninfectedthe medium was 0cigarashi bmc cancer page of control cells were completely killed the puromycinselected cells were then subjected to each assaystatistical analysisresults are presented as mean ± standard deviation unless otherwise specified pairs of groups were comparedusing a twotailed unpaired student™s ttest onewayanalysis of variance was used for multiplegroup comparisons rather than specifying three a pvalue was considered statistically significant all statistical analyses were performed using excel software microsoftredmond wa usa and statcel3 addin for exceloms publishing tokyo japanresultsvd3 reduces cell growth rateconsistent with a previous report that treatmentwith vd3 inhibits growth of prostate cancer cells ourresults showed that vd3 treatment reduced the cellgrowth rate in a dosedependent manner and nm fig 1a left as shown below igfbp3 induction activity of vd3 was reached to plateau at nm concentration fig 4a on the other hand testosterone hasbeen reported to stimulate the growth of prostate cancer and the results of this study confirmed that dhttreatment from very low concentration nm stimulated cell growth rate and its activity was reached toplateau at less than nm concentration fig 1a centerthe purpose of our study was to investigate the role ofvd3igfbp3 induction system in cell growth inhibitionand to propose the potency oflowdose vd3 usagewhich could evade sideeffect of vd3 treatment such ashypercalcemia in therapy for patients with prostate cancer thus nm of dht and nm of vd3 concentrations were chosen as minimum but stably workingconcentration for following experiment previouslyithas been demonstrated that simultaneous treatmentwith vd3 and dht enhanced the reduction of cellgrowth rate compared to treatment with vd3 alone anda similar result was reproduced with lowdose dht nm and vd3 nm in this study fig 1a right tofurther characterize growth inhibitory effect with lowdose of dht nm and vd3 nm cell cycle andapoptotic analyses were performed with flow cytometrythe cell cycle analysis revealed that there was no significant change in the cell cycle phase distribution amongfig cellular response of lncap cells treated with vd3 and dht a effect of combined treatment of vd3 and dht on cell growth in lncapcells left vd3 treatment reduced cell growth in a dosedependent manner center dht treatment increased cell growth at lowerconcentrations right simultaneous treatment of vd3 and dht enhanced the reduction of cell growth compared to treatment with vd3 aloneb change in cell cycle phase by vd3 or dht treatment neither vd3 nor dht treatment significantly changed the cell cycle phase c induction ofapoptosis by vd3 or dht treatment neither lowdose vd3 nor dht treatment influenced apoptosis at shortterm d induction of igfbp3expression by vd3 or dht treatment vd3 treatmentinduced igfbp3 expression and cotreatment with dht enhanced the expression level ofigfbp3the ratio indicates the density of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium condition the uncropped fulllength blot images are presented in supplementary fig 5a 0cigarashi bmc cancer page of control no treatment dht nm vd3 nm anddhtvd3 treatment conditions fig 1b suggestingthat lowdose of vd3 or dhtvd3 treatment did notarrest the cell cycle at a specific phase previous reportshave shown that longterm vd3 treatment has apoptosisinducible activity and dht has apoptosis inhibitingactivity in a dosedependent manner in lncapcells however it was unclear or controversial whetherlowerconcentration of vd3 and dht at shorttermcould influence apoptosis respectively in lncap cellsthus the apoptosis assay was performed with nm ofdht and nm of vd3 for shortterm and found thatneither lowerdose dht vd3 nor dhtvd3 treatmentfor shortterm influenced apoptosis fig 1c these results suggested that the decrease in the cell number induced by lowdose vd3 or dhtvd3 treatment wasmainly due to a decrease in the cell growth rate to further address what was occurring at the molecular levelduring lowdose dht andor vd3 treatment the genesknown to regulate the cell cycle and inducible by dhtandor vd3 treatment were chosen and messengerrna mrna induction was quantitatively measuredfig 1d upper supplementary fig 1a the quantitativertpcr results showed that igfbp3 mrna inductionwas positively correlated to cell growth suppression inresponse to lowdose vd3 or dhtvd3 treatment andthe expression strength was dramatically sensitive tovd3 or dhtvd3 treatment consistent with thatigfbp3 protein was markedly induced by vd3 treatment and it was enhanced by simultaneous treatment ofvd3 with dht a similar response was observed in theexpression of ar the receptor of dht which wasknown to a one oftarget of vdr supplementaryfig 1bmultiple recent studies have revealed that igfbp3functions in cellular response including cell growth andapoptosisin an insulinlike growth factor igfindependent manner considering these findings we believethat igfbp3 can be a key molecule for vd3 treatmentin prostate cancer cellsigfbp3 was a dominant factor in cell growth suppressionto confirm ifigfbp3 dominantly suppresses cellgrowth in lncap cells we applied the gainoffunctionand lossoffunction approach using a lentivirus systemfirst we generated igfbp3“overexpressing lncapcells and found that the expression of igfbp3 mrnawas about higher compared to that by lowdosedhtvd3 treatment fig 2a as an infection controlegfpoverexpressing lncap cells were also generatedand it was confirmed that lentivirus infection per se didnot induce igfbp3 expression using these cell linesthe effect of igfbp3 on cell growth was observed fig2b results showed that the cell number of egfpoverexpressed cells treated with nm of dht and nm of vd3 for days was decreased to comparedwith that of untreated cells and the igfbp3“overexpressing cells showed comparable cell growth decreasewithout dhtvd3 treatment next we generatedshrna for igfbp3 shigfbp3“expressing lncap cellsthe knockdown of igfbp3 mrna and protein inducedby lowdose dhtvd3 treatment was confirmed inshigfbp3“expressing lncap cells fig 2c using thiscell line the effect of lowdose dht and vd3 treatmenton cell growth was observed as we expected the suppressive efficacy of lowdose vd3 on cell growth wasweakening and simultaneous treatment with nm ofdht and nm of vd3 increased cell growth fig 2dtaken together these data indicated igfbp3“dominantfactor of cell growth suppression induced by lowdosevd3 treatment in lncap cellsacceleration of anticancer drug effect by vd3as previously reported and we demonstrated abovevd3 alone is not cytotoxic at physiological and pharmacological concentrations meanwhile simultaneous treatment with vd3 has been reported to improve theefficacy of anticancer drugs including docetaxel howeverits molecular mechanisms were remained not fully uncovered here we supposed that igfbp3 might be amediator of vd3induced sensitization to anticancerdrugs in prostate cancer cells to confirm this hypothesis lncap cells were treated with lowdose of dhtvd3 in combination with several concentrations of docetaxel to determine the appropriate concentration ofdocetaxel for evaluating the vd3 effect we first screenedthe concentration of docetaxel based on cytotoxic activity and found that a docetaxel concentration nmkilled bulk of the cells treated fig 3a here ic50 ofdocetaxel was nm in our assay and it was consistent with previous reports “ nm thus a docetaxel concentration nm was chosen to observe theeffect of combinatoriallowdose dhtvd3 treatmentfor the following assay to evaluate the synergistic effectof dhtvd3 on cytotoxicity by docetaxel lncap cellswere treated with or nm of docetaxel withor without dhtvd3 and results showed that lowdosedhtvd3 with docetaxel reduced the living cell numberat the concentration range of “ nm but the effectwas masked when nm docetaxel was applied fig 3bsimilarlylowdose dhtvd3 with cisplatin reducedthe living cell number at the concentration range of “ nm supplementary fig to see if these enhancedcytotoxicity effects were dependent on igfbp3 igfbp“overexpressed or shigfbp3“expressed lncap cellswere analyzed in the same manner indeed in igfbp3“overexpressed cells the living cell number was reducedby docetaxel without dhtvd3 addition fig 3c 0cigarashi bmc cancer page of fig igfbp3 mediates the effect of vd3 on cell growth in lncap cells a overexpression of igfbp3 in lncap cells lncap cells were infectedwith lentivirus containing the igfbp3 gene and its overexpression was confirmed by quantitative reverse transcription“polymerase chainreaction b suppression of cell growth by igfbp3 igfbp3“overexpressed cells were cultured as they were and control cells were cultured withdhtvd3 for days and then the cell number was measured c knockdown of igfbp3 in lncap cells lncap cells were infected with lentiviruscontaining shrna for igfbp3 and igfbp3 knockdown was confirmed by quantitative reverse transcription“polymerase chain reaction andwestern blotting the ratio indicates the density of igfbp3 band normalized by corresponding βactin band d the igfbp3 knockdown cells weretreated with dht andor vd3 for days and then the cell number was measured the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5bcorrespondingly in the shigfbp3expressed cells reduction of living cell number by dhtvd3 addition wascanceled rather the cell living number was increasedliving cell number despitefig 3d the increase ofdhtvd3 addition in docetaxeltreated shigfbp3expressed cells was assumed by cancelation of vd3 effect and emerging of dht effect on cell growth basedthese findings lowdose dhtvd3“induced enhancedcytotoxicity by docetaxel on lncap cells was dependenton igfbp3 expressioncharacterization of the igfbp3 induction mechanismas demonstrated above igfbp3 had a pivotal role inlowdose dhtvd3induced enhanced cytotoxicity byantitumor drugs to further dissect the igfbp3 induction mechanism and to provide the molecular evidenceof vd3 treatment for clinical research mechanisms ofigfbp3 induction by dht and vd3 were analyzed aspreviously reported in prostate cancer cells vd3 treatment induces cyp24a1 as well an enzyme that catalyzes vd3 to its inactive form as a negative feedbackfactor the induced cyp24a1 limits the efficacy of vd3meanwhile activated ar induced by dht treatmentsuppresses cyp24a1 transcription thus cancelling thenegativefeedback loop to inactivate vd3 consistentwith that cyp24a1 induction by vd3 treatment and itssuppression by simultaneous treatment with dht wereconfirmed even when we applied lowdose dht and 0cigarashi bmc cancer page of fig vd3 enhanced cytotoxicity of docetaxel a dosedependent cytotoxicity of docetaxel lncap cells were treated with docetaxel for daysand then the living cell number was measured ic50 was nm b simultaneous treatment of vd3 with dosedependent docetaxelsimultaneous treatment of vd3 with docetaxel reduced the living cell number at “ nm range c igfbp3 overexpression was conducive todhtvd3 treatment on docetaxel treatment igfbp3“overexpressed or control cells were treated with dhtvd3 and nm of docetaxel andcultured for days and then the cell number was measured d igfbp3 knockdown canceled the effect of vd3 on docetaxel treatment in igfbp knockdown cells dhtvd3 treatment increased the cell number compared to that of the notreatment control and the cell number wasalmost equal to that of the dht treated sample the concentration of docetaxel used was nm the all experiments were performed in serumcontaining medium conditionvd3 which were enough to induce and suppresscyp24a1 expression supplementary fig meaningthat h lowdose dhtvd3 treatment could cancel thecyp24a1driven negativefeedback loop to further dissect the mechanism of igfbp3 expression in lncapcells the cells were treated with vd3 alone or dhtfixed in nm and vd3 in a dosedependent manner and nm when treated with vd3alone the induced igfbp3 reached a plateau at to nm in contrast when treated with vd3 together withdht the amount of induced igfbp3 was increased according to the increment of vd3 concentration fig 4athese results indicated that lowdose dht could improve igfbp3 induction activity of vd3 throughcyp24a1 suppressionclinically highdose vd3 or its derivatives for treatment can cause hypercalcemia thus its continual usageshould be carefully monitored to avoid sideeffect ofvd3 here we wondered if continual vd3 treatmentfig 4btop herewould be required for maintaining igfbp3 induction inprostate cancer cells to address this lncap cells weretreated with vd3 alone or lowdose dhtvd3 for or days followed by washout which was done by replacing the culture medium and continuing the culturefor days in totalintracellularigfbp3 protein was observed by western blottinginterestingly 1day treatment of vd3 or dhtvd3 induced stable igfbp3 expression fig 4b bottom although treatment of vd3 alone showed mild igfbp3induction compared to that by dhtvd3 note thatigfbp3 showed similar strength of expression between and days of vd3 or dhtvd3 treatment this resultclearly indicated that temporal vd3 treatment couldinduce prolonged stable igfbp3 expressionthis nonlinear response suggested the presence of aunique molecular property underlying the igfbp3 expression mechanism generally nonlinear cellular response such as sustained protein expression by temporal 0cigarashi bmc cancer page of fig characterization of igfbp3 expression induced by vd3 and dht treatments a effect of dosedependent vd3 treatment with or withoutdht on igfbp3 induction lncap cells were treated with vd3 alone at the indicated concentration or with vd3 and nm dht top westernblotting image of igfbp3 and bottom quantified graph of igfbp3 induction open circles vd3 alone filled circles dht vd3 b effect oftemporal treatment of dhtvd3 on igfbp3 induction top schematic time course of temporal treatment of vd3 and bottom western blottingimage of igfbp3 the ratio indicates the density of igfbp3 band normalized by corresponding βactin band c effect of mrna transcription andprotein synthesis on the stability of igfbp3 expression induced by dhtvd3 lncap cells were treated with vd3dht for day followed byactinomycin d actd μm or cycloheximide chx μm for another day after vd3dht was washed out or left as is top quantification graphof igfbp3 mrna induction middle western blotting image of igfbp3 and the combination of treatments bottom the ratio indicates thedensity of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium conditionthe uncropped fulllength blot images are presented in supplementary fig 5ctriggered by positivefeedback loopstimulation wasmechanismin which protein synthesis and transcriptional regulation was included as hysterical responsedriver thus to further dissect if protein synthesis ortranscriptional regulation or both are involved in nonlinear vd3igfbp3 induction actinomycin d and cycloheximide which are transcriptionalinhibitor andprotein synthesis inhibitor respectively were added withvd3 or dhtvd3 and behavior of igfbp3 proteinand its mrna was observed experimentally actinomycin d or cycloheximide was added day after treatmentwith vd3 alone or dhtvd3 and the culture was continued for another day when interfered with μm ofactinomycin d there was no change in igfbp3 mrnaor protein expression fig 4c by contrast when interfered with μm of cycloheximide the igfbp3 proteinwas immediately reduced howeverthe mrna wasunexpectedly increased several times higher than thosewithout cycloheximide interference fig 4c the unexpected mrna increase became stronger when dhtvd3 washout was performed ahead of the cycloheximideinterference these cellular responses on igfbp3 induction interfered by actinomycin d and cycloheximidesuggested that the cells had a protein abundance“basedpositivefeedback loop to maintain the total amount ofigfbp3 via transcriptional controligfbp3“independent bcl2 suppression by vd3as shown above although lowdose dht andor vd3treatment did not induce apoptosis fig 1d vd3 treatment rendered lncap cells sensitive to the antitumordrugs fig 3b and supplementary fig suggesting thatany apoptosisrelated factor might be influenced to addressinvestigated the behavior ofidea wethis 0cigarashi bmc cancer page of apoptosisrelated molecules in response to lowdosevd3dht treatment consistent with previous report although the concentration of vd3 was higher thanthat we used here bcl2 protein an antiapoptotic molecule was downregulated by lowdose vd3 treatmentfig 5a compared to that by vd3 alone it seemed thatbcl2 downregulation by dhtvd3 was not seemed tobe enhanced unlike to igfbp3 expression suggestingthat bcl2 downregulation by vd3 was igfbp3 induction independent manner to see whether bcl2 downregulation was igfbp3“dependent or not the behaviorof the expression of bcl2 protein and mrna was observed in shigfbp3expressedcells interestingly despitethe igfbp3 disappearance bcl2 downregulation wasobserved according to vd3 or dhtvd3 treatmentfig 5b bottom and it was not significantly different atinigfbp3expressionenhancedprotein and mrna expression level compared to that inshctrlexpressing cells moreover in order to confirmthatconditionshcyp24a1 expression cells were established in whichvd3 effect on igfbp3 induction was expected tostrengthen the knockdown of cyp24a1 under the vd3treatment condition was confirmed by qrtpcr supplementary fig indeed the amount of igfbp3 protein wasincreased in shcyp24a1expressing cellscompared to that in shctrl cells fig 5c bottom despiteigfbp3 expression downregulation bcl2 protein by lowdose vd3 or dhtvd3treatment was not observed compared to that in shctrlcells fig 5c bottom also the expression of bcl2mrna was not significantly changed fig 5ctopthese results suggested that the downregulation of bclenhancement offig igfbp3“independent reduction of bcl2 protein expression induced by vd3 a western blotting image of bcl2 reduction by vd3treatment the ratio indicates the density of bcl2 band normalized by corresponding βactin band b effect of igfbp3 suppression on bcl2reduction by vd3 treatment the cells were infected with lentivirus encoding of shrna for igfbp3 and then treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band c effect of igfbp3 overexpression on bcl2 reduction byvd3 treatment the cells were infected with lentivirus encoding of the cyp24a1 gene then the cells were treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5d e 0cigarashi bmc cancer page of protein by lowdose vd3 treatment was independentof igfbp3 induction besides the mrna of bcl
Colon_Cancer
it is hard to comprehend how much of their lives poor people spend waiting in the emergency room dealing with indifferent bureaucracies that are supposed to address basic needs in the laundromat where people must scramble for available machines and keep a close eye on their laundry waiting adds yet more pressure and pain on feet that may already be swollen ¦ many poor people spend years of their lives on waiting lists for a public housing unit a section voucher a bed in rehab a hearing in landlordtenant court they wait for erratic buses food at a food pantry or a bed in a shelter their names fill long lists of people in dire need williams p waiting is a pervasive aspect of poverty just as poor people are most likely to wait for laundry machines housing food so too they are most likely to wait for their healthcare and this can have especially grave consequences andaya 2018b oostrom the link between the timing of treatment and health outcomes is well documented in health disparities research it has become axiomatic that timely adherence to and receipt of medications screenings and followup health services is vital for decreasing inequities in health outcomes bickham and lim dickman as a result much public health intervention work aims to identify and rectify the sources of protracted times to diagnosis andor treatment diamant koopmanschap similarly healthcare anizations are increasingly attentive to time elements identifying time as a critical quality metric shorter wait times particularly for appointments or between diagnosis and the initiation of treatment reflect greater efficiency and improved patient satisfaction and outcomes bleustein michael nevertheless anizationlevel effort to reduce wait times is unevenly distributed bureaucratic interventions disproportionately advantage wealthier and privately insured patients the task of waiting corresponding author email address jeanhunlethwustledu jm hunleth 101016jsocscimed2020113296 received in revised form august accepted august socialsciencemedicine2642020113296availableonline19august2020027795362020publishedbyelsevierltd 0caa lee in and for healthcare”in waiting rooms for future appointments procedures and treatments”is performed overwhelmingly by people who are poor kennedy oostrom in this we focus on what we are calling active waiting a concept that we suggest more accurately describes the lived experience of gaps in healthcare action than does the more frequently used word delay when contemporary health disparities researchers examine patient behavior and action as it relates to the timing of treatment they often focus on patients™ delaying diagnostic tests or treatments while understanding why a person might delay a diagnostic procedure or treatment regimen can identify barriers eg long wait times on phones or for followup procedures a lack of transportation or money that discourage individuals from taking more immediate actions diamant thinking in terms of delays can also place excessive responsibility and blame on individuals for nonaction and nonadherence hunleth œwhy did she delay scheduling her followup appointment is not the same as œwhy did she wait to schedule her appointment and though waiting can lead to assumptions about passivity or nonaction social scientists have demonstrated that waiting is not a passive state auyero mulcahy we derive our own focus on active waiting as opposed to delay from narrative interviews we carried out with people who have chronic illnesses and who have difficulties affording their medications studies of chronic illness in the us offer important insights into waiting people living with illness and in poverty wait a lot during the many appointments needed to manage their conditions ie foron health service and treatment providers and for disability insurance and paychecks ie foron bureaucratic actors of key importance active waiting unlike delay remains true to the linguistic preferences of the individuals with whom we have worked participants in the study we describe below spoke of their experiences of temporality in healthcare as of waiting and not as of delaying further the concept of active waiting allows us and others working in and studying healthcare to move beyond analyses that either locate responsibility for wait times solely with the individuals ng the waiting or perpetuate overlydeterministic views of systemic barriers that erase experience and social action to frame our conceptualization of active waiting we bring together two scholarships one on the political economy of healthcare which includes the elucidation of bureaucratic time and the other on waiting as a feeling and an experience which gains meaning within power relations and through social interactions bureaucratic time and the agency of waiting patients who then comply with bureaucratic state demands auyero waiting may be imposed but these researchers show that it always entails a response understanding the experience of waiting in the us requires recognizing that the actions people take while waiting are interpreted using neoliberal ideologies that privilege among other things individual responsibility mulligan rylkobauer and farmer this focus on individual responsibility can lead to the blaming and shaming of people who time healthcare inappropriately or do not act appropriately within others™ expected timeframes holmes scholars have shown that this privileging of individual responsibility reinforces racialized and classed hierarchies of deservingness horton notions of individual responsibility and thus of individual failure place pressure on patients to do and in ng they avoid or mitigate the likelihood of blame and shame while they wait as we have previously shown the focus on individual responsibility can lead people to blame themselves and their loved ones for waiting too long to seek care hunleth sociologist andreas g¨ottlich suggests that waiting is an interaction that depends on œthe mutual interpretations of the actors”those who wait and who are waited upon”as well as others who behold the scenes of waiting g¨ottlich p that is people wait in relationship to and with others researchers who center the interpretive acts and emotions of ˜waiters™ in healthcare settings such as waiting rooms offer several insights into what we refer to as active waiting in a study of interactions in a waiting room strathman and hay suggested that œwhen patients are told that they cannot get an appointment within the desired”even physician recommended”timeframe it is like being told that their health and by extension they as persons are less important than bureaucratic schedules strathmann and hay p that is waiting can add to a sense of being devalued or neglected andaya sj¨oling and it can negatively influence peoples™ perceptions of their quality of care and confidence in their care provider bleustein given the above work we understand active waiting to be the experiences and responses that people devise to navigate shorten or otherwise survive waiting and also to anticipate and craft possible futures within the relationality and power dynamics of bureaucratic time resisting wait times by seeking information and other resources is part of active waiting as is delaying and complying active waiting is composed of such discrete actions that people might take as patients in one place or time but is irreducible to those actions and must be situated in how people manage multiple wait times at various scales further we include not only action but also how waiting on bureaucratic time feels carr teucher and casson have used the phrase œlived wait time to communicate the fact that feelings of time differ by circumstances researchers have shown that chronic illness lends itself to particular feelings of time and of life including those of time as suspended eg feelings that life is œon hold brown mulcahy sj¨oling we expand this by focusing on how chronic illness feels while waiting on powerful others to make life or death decisions while wait time is lived people also live during their wait times daily lives are not easily paused sj¨oling this means that the circumstances of individuals™ days and lives often change while they wait on healthcare less has been said about the waiting that goes on outside of clinical settings yet is ultimately tied to health in fact waiting is an implicit and normalized mechanism of bureaucratic systems in what follows we consider how these various aspects of time and waiting combine for people experiencing illness and financial strain we use active waiting”conceptualized as an action orientation relationship and feeling that is irreducible to a singular time and place and which shapes how individuals approach their health in particular times and places we do so in order to best examine how various aspects of time and waiting interweave in the lives of people who are chronically ill and poor though healthcare bureaucracy in the us requires most people to wait for care how long people wait how waiting is structured and the consequences of waiting vary people who do not have access to insurance or other resources must endure state and federal evaluations of their lowincome statuses abilities to work severities of illness citizenship and more to qualify for assistance tickamyer as funding for social programs like medicaid food stamps and disability are rolled back and work requirements become stricter demonstrating qualifications to obtain support is increasingly difficult and time consuming whittle while social welfare programs offer access to health services they often require long waits in waiting or exam rooms at facilities that accept medicaid or offer services on a sliding fee scale becker oostrom researchers have identified how people deal with the bureaucratic schedules that shape the course of their treatment mulcahy described how some people with cancer resisted the assumptions that they must be ˜patient™ patients by seeking information and resources to shorten their waiting periods still other researchers have shown that long waittimes for healthcare personnel in clinics and waiting rooms can lead to other forms of action waiting can push individuals out of the healthcare system or lead them to delay care becker and it can induce feelings of pervasive uncertainty and powerlessness among socialsciencemedicine26420201132962 0cthis research comes from a mixed methods study investigating cost related issues that affect adherence to medications recruitment occurred in and amidst political uncertainty about changes to healthcare legislation on the national stage a new and controversial presidential administration was beginning its term this administration had goals to repeal the affordable care act aca and introduce block grant funding to medicaid which remained a possibility throughout our data collection period for people with chronic illnesses the aca was significant it mandated that preexisting conditions be covered by insurance plans and that insurers offer at least a minimum prescription drug benefit while also creating platforms for uninsured individuals to purchase plans additionally national conversations about changes to prescription drug pricing were prominent in news coverage most of our narrative interviews were conducted in st louis missouri though a few occurred in east st louis illinois missouri did not expand their medicaid program with the rollout of the aca illinois did st louis is a metropolitan area with significant racial and economic health inequities to address the health needs of those without insurance in light of not expanding medicaid the gateway to better health program was created which provided basic coverage to people living below the poverty line this program was set to end in december during our study but its extension was later approved beyond that date many of the people who participated in this study were using or had used this program recruitment interviews our research team recruited people between the ages of and who had one or more chronic illnesses for which they were prescribed medication and who identified as having difficulties affording their prescriptions the study had a mixed methods design participants in the survey n were recruited from newspaper ads federally qualified health centers and a multispecialty clinic a subsample of the survey participants n also participated in a narrative interview focused on financial strain while we have presented the mixed methods results elsewhere this focuses solely on the narrative interviews we initially aimed to recruit interview participants stratified evenly based on prescription insurance coverage age and gender however this was adjusted based on the availability of participants the final number of interviews was table the interview guide was based on the mcgill illness narrative interview mini groleau we revised the guide and refer to it as the modified financial and illness narrative interview it is structured similarly to the mini to elicit different types of narratives but with an added focus on financial history two local nonprofit community anizations were selected as interview locations due to their proximity to and rapport with the participant population nonclinical environments available private interview spaces and anizational relationships with study team members trained research team members conducted interviews and a note taker was present in most cases the interviewer obtained informed consent and interviewees were compensated with a gift card interviews were audiorecorded and lasted about “ minutes immediately after the interview the interviewer and note taker wrote detailed field notes using a structured template that included their observations about body language any conversations not recorded a summary of the financial and illness narrative and reflective memos analysis interviews were professionally transcribed verbatim deidentified and checked for accuracy before being uploaded with their respective fieldnotes to nvivo we describe the codebook development below which was designed with the mixed methods study in mind during that codebook development the team identified the repetition of comments about waiting across the interviews the team developed a codebook using deductive and inductive codes deductive codes were based on questions of interest from the quantitative survey these focused primarily on medication cost coping access to basic needs and related concepts the team identified inductive codes while conducting interviews and refined and added to these codes while reviewing transcripts during the analysis and throughout the coding process we took note of a crosscutting theme in the narratives when asked to describe what it was like to deal with chronic illness participants said that chronic illness was about waiting we coded all discussions of waiting in the interviews defined as any mention of waits or delays and in the fieldnotes while waiting is a broad concept and was used to reference different processes we chose to include in our analysis all aspects of waiting described by participants rather than focus on just one eg waiting for health insurance coverage we did so to acknowledge that people may experience waiting that is interrelated and different from the temporal distinctions made by many healthcare practitioners four team members coded the interviews using the final codebook all transcripts were coded by at least two team members and discrepancies were reconciled through consensus between the two coders al memoed on the waiting code throughout this process focusing on issues such as what kinds of things people waited for how this influenced their decisions and emotions the outcomes of their waiting on their health and healthcare and how waiting shaped their aspirations for the future the team including coders interviewers and investigators compared how waiting was discussed within and across the transcripts and narrative types elicited by the guide as well as the intersection of waiting with other codes interpretations and exemplar quotes were evaluated by multiple team members to guard against selective use and bias in interpretation of the data and to encourage reflexivity all study activities were approved by washington university™s institutional review board results aa lee methods setting table interview participant characteristics raceethnicity nonhispanic black or african american nonhispanic white other gender female male had continuous health coverage in the past year had a gap in coverage in the past year age mean range health coverage n we anize our results to make clear the meanings and experiences of active waiting in the first section we outline the breadth of waiting that shapes how people wait and answers the question œwaiting for what the second section addresses the question œwaiting for whom and describes how participants ascribed meaning to waiting according to their social and economic positions and in relation to those in power what some participants referred to as the œwaiting game finally and building on the previous sections we focus on waiting as an active process that people manage through a variety of tactics in ng so we detail the ways in which people actively wait living their lives within structures of power that make them wait socialsciencemedicine26420201132963 0caa lee waiting for what big and small waits because we cover multiple forms of waiting we wish to be specific about what participants referred to when they talked about waiting the list was long and it included waiting in waiting rooms for transportation on the phone for surgery for paychecks and welfare checks for housing and for approval for disability and insurance benefits the things participants waited for varied according to their own unique health and social circumstances these waits were often predictable and included the spaces in which people wait eg waiting rooms the bureaucracies that enforce waiting eg disability claims the conditions that create waiting eg strained social safety net and the financial demands that when unmet lengthen wait times eg a ride when one does not have transportation a paycheck to afford a prescription the variation in chronic illnesses and in life circumstances of participants in the study however meant that they discussed the structures of waiting in different ways and put emphasis on different aspects within the predictability and variability there was an overarching theme of waiting for one or a succession of big things to happen what we term ˜big waits™ big waits were for things that had a hopedfor endpoint ones that might change an individual™s health situation for example many participants talked about waiting for disability assistance to come through or for health insurance coverage to start or for stable housing and they often centered their discussions of waiting around this primary event the endpoints could be a decision made by a social service agency or could be an anticipated and desired life change one woman told us that her big wait for insurance coverage was almost over because she would soon be eligible for medicare which multiple others also mentioned that they were awaiting œthe good news for me is“ a year from this august “ i turn but um i always feel like it™s all pending there is nothing concrete in any of the healthcare while participants talked about big waits in terms of their endpoints they also talked about how such endpoints led to other big waits take kate for example kate is a black woman in her early fifties who previously worked in home healthcare she has osteoarthritis graves™ disease hepatitis c and depression after both her mother and husband died a few years ago she struggled to afford housing and continue working through her arthritis pain when we interviewed kate she had been homeless for several months and was recently accepted off a waiting list for transitional housing kate had several big waits that came in succession and were contingent upon one another she waited for housing assistance she waited for medical release to work after sustaining injuries in a car accident she waited to consult with a doctor about surgeries she waited on a disability hearing scheduled months away though she had been diagnosed with hepatitis c her doctor would not begin treatment until her disability application was approved because of the high costs of the medication which would be covered if she were on disability kate worried that all of these big waits would have longterm repercussions on her health until the disability [benefits are approved] or something [else comes through] and september [the date of her disability hearing] comes they ain™t going to even give me the medicine ¦ we will just wait and see so i am just in limbo until until my hearing ¦ i know i ain™t going to die right away but i do want to get the medication kate found herself waiting a lot on a lot the interdependence of her waiting meant that one wait exacerbated other waits while taking care of her sick family members she put off her own health needs and bill payments leading to her eventual housing instability her lack of a consistent mailing address delayed her ability to submit a disability application even after she found more stable housing and was able to see a doctor the disability application delays led to postponed medical treatments without getting her health and injuries under control she could not work and get out of transitional housing for kate each big wait led to another big wait with worsening and compounding repercussions on her health and wellbeing other participants described having waited years for their disability applications to be approved or often denied in the meantime they incurred greater debt hired lawyers relied on family for support postponed treatments and endured these big waits “ waiting for disability benefits and for other significant changes in their financial or familial situations “ also created a number of smallerscale daytoday situations in which study participants waited one uninsured woman described waiting to fill her prescription and waiting to see her doctors while she searched for a full time job to afford both œyou wait to the extreme before you do anything about whatever the problem is because you don™t want to go to the hospital it costs too much many participants missed appointments or went without medications while waiting for new insurance benefits or payday or financial assistance paperwork to go through holding off on care was just one way to wait actively available to those whose actions were constrained by chronic illness and financial strain while big waits figured strongly into participants™ imaginations of their futures small waits were not insignificant allen™s diabetes had damaged his kidneys so severely prior to getting medicaid coverage that he needed dialysis he described the cycle of waiting while on dialysis allen “ whose big wait had been first getting medicaid coverage and then once covered getting on a kidney transplant list “ did not have a car or a steady way to get to his weekly dialysis appointments a few times a week he waited to be picked up by a medicaidcovered transportation company he had little control over when they would arrive and their arrival was often sporadic when they arrived early he had to wait at the clinic when they were late he either had to pay for his own cab or push back his dialysis appointment to later in the day and wait even more optimistic allen expressed gratitude that he was able to use medicaid and live with his brother while waiting for a kidney transplant his own big wait within multiple small waits while enduring the small waits he imagined a better future after the transplant describing to us what he would write in a book about his experiences œkidney transplant got him back to a better life now he™s back in the work force he hasn™t looked back to the sickness yet ¦ that™s my story the daytoday waiting was frustrating but the promise of this story along with having family able and willing to assist him an unmarried man without children shaped how he waited and his feelings while waiting big and small waits contribute to the dynamic aspect of active waiting where social ties and financial resources regulate how one is able to navigate and survive waiting waiting was drawn out for those without the economic resources to avoid institutions such as slidingscale clinics welfare agencies and financial assistance programs throughout the interviews participants described waiting as chronic but they also held onto a sense that once a waiting period passed then their future goals of health and financial stability would be that much closer for kate waiting was incremental and each waiting period required different tactics the big waits participants described both promised and suspended the future people grappled with the present while anticipating what might happen when and if the waiting ended some people imagined a time when they could afford healthcare holding off certain appointments until that time while others imagined a future œin the work force and attended appointments diligently even in the face of smaller or shorterterm waits such small waits too structured participants™ responses in how they waited based on what they were waiting for waiting for whom the œwaiting game many participants identified feeling like the systems in place to assist chronically ill and financially strained people were œplaying games with their lives by making them wait they were made to wait by social service programs appointment schedules hospital and clinic waiting rooms insurance company call centers the waiting punctuated their socialsciencemedicine26420201132964 0caa lee daytoday lives and left them guessing about the reasons behind their extensive chronic waiting they guessed at answering œwhy meœ”why they had to wait they also guessed at a course of action to shorten or otherwise deal with waiting the œwaiting game they described was one in which the odds were stacked against them the rules were opaque and endurance was necessary to win some study participants searched for ways to get around the game or to play it to their advantage how they played the waiting game “ which we recognize as active waiting “ depended on both their interpretations of the meaning of waiting and on the resources to which they had access in our interviews some people brought up suspicions about being forced to wait for appointments and in waiting rooms due to their economic status age race and other discriminatory factors one participant talked about how once in the examining room she felt as if her time waiting was not reciprocated by providers œthey do not want to spend a lot of time with you you know theythey™ve got to make a living too so they move on some speculated that doctors reserved time for people of a higher economic status these patientprovider dynamics that waiting exemplified left people feeling slighted by the medical system œah i sat there in that waiting room for hours and and they still wasn™t ready to see me ¦ no one had the respect ah even the courtesy to come out and say ˜well the doctor is running a little bit behind™ people felt that their lives were undervalued and unimportant when their time was not acknowledged or respected that bureaucratic systems seemed to withhold resources by imposing waiting suggested to some a denial of their belonging and deservingness as patients and as humans worthy of care experiences of medical racism and sexism frequently underpinned participants™ feelings that their time was being wasted rachel a black woman told us that she had been working and paying taxes since she was a kid and still the welfare system did not support her when she needed it recognized that high blood pressure was œblack peoples™ number one killer and explained that her waiting also stressed her body contributing to her anger these indignities played out in the clinic adding to the daily stresses she said she already felt as a black woman and grandmother living near the ferguson neighborhood where an officer murdered michael brown in and was not indicted for that murder participants also talked about waiting as a game that required endurance not just because of a lengthy office wait or the months or years it could take to get a disability application approved or denied but because of the opacity of the process they questioned application processes wondering if the wait to hear back about welfare and assistance services was a test of endurance œit is just a waiting game [for disability] until they get tired tired of you being in front of them and they decide to give it to you that is what they hope for is [you] giving up on it but if you keep with it sooner or later you will get it while a number of participants expressed optimism for a better future once a big wait was over waiting out bureaucratic processes wore down that optimism œi don™t believe i can keep going through this too much longer erica said she was exhausted from the strain of balancing utility payments with medical costs while waiting for her new insurance plan to begin and while coping with illness unexplained waiting on bureaucratic systems whether for an application to be approved or for insurance to kick in or to be seen by a busy doctor in a slidingscale clinic felt like an unfair game designed to keep them from receiving necessary medical care by framing waiting as a game the participants explained that there were rules that shaped the length and types of waiting and that they did not write them the rules were far from transparent and enforced differently based on race and social status as sherry made evident in her frustration with being made to wait waiting is not a passive state that happens without mental and physical consequences when people responded with patience and endurance through the game just like when they responded with anger and confrontation their waiting was active put differently participants described fashioning tactics in part based on their perceptions of the intentions of those for whom they waited they kept turning me down for disability and they kept saying i was too young to have problems i had you know but my body was breaking down and the doctors would never [help with the application] which was really so unfair and now that™s the part that really makes me feel a little sad like there is so much “ i hate to say racism “ but just unfair treatment because i™m a woman because of my age and then because of my race and so we™re talking like years [voice breaking] ¦ it made me be really poor rachel™s doctors held the power to demonstrate that she qualified for disability but as she recounted they were unwilling to fill out the documentation she needed to apply for disability she felt that the us had enough resources to
Colon_Cancer
glioma initiates from glial cells and contains several types such as astrocytoma and oligodendroglioma1 over a quarter of brain tumors are glioma which causes a large number of cancerrelated deaths every year around the world1 the current clinically therapeutic strategies are surgery combined with chemotherapy and radiotherapy2 however the prognosis of glioma patients remains not well post therapy3 hence it is urgently required to discover new molecular mechanism for glioma therapyboth long noncoding rna lncrna and microrna mirna belong to noncoding rnas which have no proteincoding ability lncrna is characterized with more than nucleotides while mirna is about nucleotides in length4 lncrna and mirna are involved in various cellular processes including cell division invasion and survival5 dysregulation of lncrna or mirna usually causes tumor initiation and progression67 for example lncrna linc00152 upregulation promotes gastric cancer growth and metastasis8 lncrna snhg6 overexpression facilitates lung cancer cell proliferation and metastasis9 mir3405p dysregulation promotes tumorigenesis of esophageal squamous cell carcinoma10 in addition mir126 cancer management and research “ du this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution “ non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphpcorrespondence jun wu weiwen qiu email wwwwjjjj924163com weiwenqhotmailcomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars262279 0cdu dovepresssuppresses colon cancer cell survival and induces apoptosis11 besides lncrna has been identified as potential competing endogenous rna cerna for mirna to function in cancer12 the potential roles underlying lncrna and mirna still require much investigation and the relationship between lncrna and mirna also needs to be definedlinc00173 is an oncogene in lung cancer and breast cancer1314 the function of linc00173 in glioma is unclear in the current study we found that linc00173 was upregulated in glioma tissues linc00173 high expression was associated with a low survival rate linc00173 depletion suppressed proliferation migration and invasion of glioma cells linc00173 was discovered to sponge mir765 to elevate nutf2 expression taken together our findings supported that linc00173 plays essential oncogenic roles in glioma through activating mir765nutf2 pathwaymaterials and methodsclinical samplesthirtyseven glioma tissues and normal tissues were collected from lishui city people™s hospital patients received no radiotherapy or chemotherapy before surgery all tissues were stored in liquid nitrogen association between linc00173 expression and clinical characteristics in glioma tissues was analyzed in table written informed consent was obtained from every patient this study was approved by the ethics committee of lishui city people™s hospital no and the table association between linc00173 expression and clinical characteristics in glioma tissuesfeaturesage years‰gendermalefemalegradei“iiiii“ivtumor size cm‰low n19high n18pvalueexperiments were conducted in accordance with the declaration of helsinkicell culture and treatmentthe normal human astrocyte nha and glioma cell lines were purchased from institute of biochemistry and cell biology of the chinese academy of sciences shanghai china cells were cultured using pmi1640 medium invitrogen carlsbad ca usa supplemented with fetal bovine serum fbs invitrogen shrnas against linc00160 mir6293p mimics mir6293p inhibitors and negative controls were obtained from genepharma and transfected into glioma cells using lipofectamine invitrogen according to the manufacturer™s instructions efficiency was validated using qrtpcr after hqrtpcrtotal rna was extracted from tissues and cell lines using trizol invitrogen carlsbad ca primescript rt reagent kit rr047a takara holdings inc tokyo japan was used to generate cdna from rna template qpcr was completed through sybr premix ex taq„¢ ii takara japan gapdh was the normalized control relative expression was calculated through the ˆ’δδct methodluciferase reporter assaythe fragment of linc00173 or nutf2 containing indicated mir765 binding site was constructed into pmir report vector for luciferase reporter assay glioma cells were transfected with report vector and mir765 mimics after h the luciferase reporter activity was measured through the dualluciferase reporter assay system promega madison wiwestern blot assaycells were lyzed using radioimmunoprecipitation buffer beyotime shanghai china protein concentration was determined by a bca protein assay kit thermo fisher scientific ma then proteins were separated using sdspage and transferred onto pvdf membranes after blockage using bsa for h the membrane was incubated the primary antibodies at °c overnight after washed times using pbst the membranes were incubated with horseradish peroxidaselabeled second antibody followed by detection the enhanced chemiluminescence reagent emd millipore usathrough submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du cck8 and colony forming assaysproliferation was measured using cck8 and colony formation assay cck8 assay was performed using the cck reagent dojindo kumamoto japan according to the manufacturer™s instructions and absorbance was determined at nm using a microplate reader biotek winooski vt for colony formation assay cells were seeded into 6well plates and cultured for days then the cells were fixed with methanol and stained with crystal violet for minutesedu assaycells were plated into 96well plates and incubated with edu μl at °c for h followed by detection using facstranswell migration and invasion assaystranswell plates corning ny were used to measure migration and invasion according to the manufacturer™s instructions in brief cells were suspended into μl serumfree medium and seeded into the upper chamber while the lower chamber was filled with µl of complete medium after incubation for cells in the lower chamber were fixed with methanol and stained with crystal violet for minutes migrated and invaded cells were counted through a light microscopestatistical analysisgraphpad prism graphpad ca usa was used to analyze results data were presented as means±standard deviation sd significant differences were analyzed using student™s ttest or oneway anova survival rate was analyzed by the kaplan“meier method and log rank test p005 was considered to be significantresultslinc00173 expression is elevated in gliomathe expression of linc00173 was firstly analyzed through qrtpcr we found that linc00173 level was elevated in glioma tissues compared with normal tissues figure 1a besides we found that linc00173 was also upregulated in glioma cell lines compared to nha cells figure 1b then according to the median value of linc00173 glioma tissues were classified into two groups after analysis we found that linc00173 high expression correlated with poor prognosis figure 1ctransfection of linc00173 enhanced glioma cell proliferation migration and invasionto explore the function of linc00173 u87 and u251 cells were chosen after shlinc00173 linc00173 expression was significantly downregulated figure 2a through cck8 assay we observed that linc00173 knockdown suppressed the proliferation capacity of glioma cells figure 2b and c which was validated by edu and colony formation assays figure 2d and e afterwards transwell assay was performed results indicated that linc00173 loss inhibited migration and invasion of glioma cells figure 2f and g thus linc00173 exerted oncogenic roles by affecting proliferation migration and invasionlinc00173 worked as the sponge for mir765linc00173 has been found to serve as cerna for several mirnas such as mir490 and mir2181314 to determine the mechanism of linc00173 in glioma we also figure linc00173 expression is elevated in glioma a the level of linc00173 in glioma tissues was measured b the expression of linc00173 in glioma cell lines and nhas c association between overall survival and linc00173 expression in glioma patients p005cancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 enhanced glioma cell proliferation migration and invasion a qrtpcr analysis of linc00173 expression in u87 and u251 cells b“e proliferation ability was measured using cck8 edu and colony formation assays f and g migration and invasion capacity was evaluated after linc00173 knockdown in glioma cells p005suppressed the supporting their direct performed bioinformatics analysis using mirdb we identified that mir765 was the most potential candidate because it scored the highest to validate it we constructed luciferase reporters figure 3a followed by luciferase reporter assay results showed that mir765 activity of linc00173wt only figure 3b interaction pulldown assay further demonstrated their interaction figure 3c qrtpcr found that linc00173 overexpression suppressed the level of mir765 figure 3d next bioinformatics analysis using mirdb and targetsan implied that nutf2 is the most potential target of mir765 the corresponding luciferase reporters were further constructed figure 3e luciferase reporter assay also demonstrated the interaction between nutf2 and mir765 figure 3f besides nutf2 expression was suppressed by mir765 mimics figure 3g moreover nutf2 level was decreased after linc00173 knockdown while mir765 inhibitors reversed it figure 3h finally we found that mir765 level was negatively correlated with linc00173 or nutf2 in glioma tissues figure 3i and jlinc00173 promoted glioma progression through mir765nutf2 pathwaywe noticed that nutf2 expression was upregulated in glioma tissues figure 4a and b suggesting an oncogenic role to investigate whether linc00173 regulates glioma progression through mir765nutf2 we restored the expression of nutf2 in shlinc00173 transfected cells cck8 and transwell assays demonstrated that nutf2 restoration successfully rescued the capacities of proliferation migration and invasion in glioma cells transfected with shlinc00173 figure 4c“f therefore linc00173 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du figure linc00173 worked as the sponge for mir765 a bioinformatics analysis indicated the binding sites between linc00173 and mir765 b u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter linc00173wt or linc00173mut then relative luciferase activity was determined c rna pulldown assay using biotinlabeled mirnas d relative expression of mir765 after linc00173 knockdown e bioinformatics analysis indicated the binding sites between mir765 and nutf2 f u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter nutf2wt or nutf2mut then relative luciferase activity was determined g qrtpcr analysis for nutf2 expression h western blotting analysis for nutf2 protein level i and j correlation analyses of linc00173 mir765 and nutf2 in glioma tissues using pearson™s correlation coefficient p005contributes to glioma progression through mir765nutf2 pathwaydiscussionas the most malignant brain tumor glioma leads to a huge number of deaths patients with glioma display a poor prognosis therefore it is of great significance to reveal the mechanism underlying glioma progression in this study we found that linc00173 was upregulated in glioma tissues and cells linc00173 overexpression predicted a poor prognosis moreover linc00173 knockdown the proliferation migration and invasion of glioma cells linc00173 was also found to inhibit mir765 and promote nutf2 expression summarily our research discovered that linc00173 is an important oncogenic lncrna in gliomasuppressed the potential roles of lncrna in glioma have been researched for a long time many lncrnas have been identified to participate in glioma development for example lncrna nck1as1 enhances growth and metastasis of glioma through targeting mir13823p to activate β catenin signaling2 lncrna ccat2 contributes to glioma progression by activating vegfa pathway15 lncrna linc00467 upregulation promotes glioma development through repressing p53 level16 previous study showed that linc00173 downregulation promotes nonsmall cell lung cancer cell growth and survival17 however another study showed that linc00173 enhances chemoresistance and facilitates tumor progression in small cell lung cancer13 besides linc00173 contributes to breast cancer development14 yet how linc00173 works in glioma remains undermined in our study we found that linc00173 was upregulated in glioma tissues linc00173 knockdown inhibited the proliferation migration and invasion of glioma cells therefore our data discovered that linc00173 is a new oncogene in glioma for the first timecancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 promoted glioma progression through mir765nutf2 pathway a and b nutf2 expression in glioma tissues and normal tissues according to tcga data using gepia tool and qrtpcr c and d proliferation was measured by cck8 assay e and f migration and invasion potential was determined by transwell assay p005lncrna has been found to serve as mirna sponge in tumor cells for instance lncrna ttnas1 sponges to promote breast cancer metastasis18 mir1405p lncrna cdkn2bas1 sponges mir3245p to regulate cellcycle progression in laryngeal squamous cell cancer19 previous studies also revealed linc00173 was a sponge for some mirnas such as mir4903p and mir2181314 in our study we did not observe linc00173 sponges above mirnas however through bioinformatics we identified linc00173 targeted mir765 in glioma we demonstrated their direct interaction and found that linc00173 overexpression inhibited mir765 expression mir765 has important roles in cancers mir765 was found to suppress tongue squamous cell carcinoma development20 mir765 also promotes myeloma and osteosarcoma progression2122 besides mir765 plays oncogenic or anticancer roles in gastric cancer and breast cancer2324 its role in glioma remains unclear our results suggested that mir765 was a tumor suppressor in gliomalncrnamirnamrna regulatory axis is widely observed in cancer for example linc00703mir181a klf6 axis suppresses the development of gastric cancer25 linc00312mir9cdh1 axis was found to promote breast cancer progression26 through bioinformatics we found that mir765 targeted nutf2 in glioma moreover we showed that nutf2 expression was regulated by linc00173mir axis the function of nutf2 in cancer is nearly unknown in our work we found that nutf2 expression was upregulated in glioma tissues compared to normal tissues moreover we found that nutf2 overexpression promoted the proliferation migration and invasion of glioma cells indicating nutf2 was an oncogene in gliomain conclusion our study showed that linc00173 acted as a sponge for mir765 to promote nutf2 expression and linc00173mir765nutf2 axis plays a critical function in promoting glioma progressionfunding this work was supported by zhejiang province analytical testing and experimental animal program lgd19h and zhejiang province welfare technology applied research project 2017c37111 disclosureall authors declare no conflicts of interest in this workreferences ostrom qt cioffi g gittleman h cbtrus statistical report primary brain and other central nervous system tumors diagnosed in the united states in neuro oncol 201921suppl 5v1“ v100 101093neuoncnoz150the of glioma huang l li x ye h et al long noncoding rna nck1as1 promotes sponging microrna13823p and activating the trim24wntbetacatenin axis j exp clin cancer res 101186s13046 tumorigenesis through chen w lei c liu p et al progress and prospects of recurrent glioma a recent scientometric analysis of the web of science in world neurosurg 2020134e387“e399 101016jwneu20 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du sun b meng m wei j wang s long noncoding rna pvt1 contributes to vascular endothelial cell proliferation via inhibition of mir190a5p in diagnostic biomarker evaluation of chronic heart failure exp ther med “ 103892etm20208599 feng s yao j chen y functional role of reprogrammingrelated long noncoding rna lincrnaror in glioma j mol neurosci “ 101007s120310140488z zhang d zhou h liu j mao j long noncoding rna asb16as1 promotes proliferation migration and invasion in glioma cells biomed res int sun l zhao m wang y neuroprotective effects of mir27a against traumatic brain injury via suppressing foxo3amediated neuronal autophagy biochem biophys res commun “ 101016jbbrc201612001 shi y sun h downregulation of lncrna linc00152 suppresses gastric cancer cell migration and invasion through inhibition of the erkmapk signaling pathway onco targets ther “ 102147otts217452 li k jiang y xiang x et al long noncoding rna snhg6 promotes the growth and invasion of nonsmall cell lung cancer by downregulating mir1013p thorac cancer wang x gu m ju y zhou j pik3c3 acts as a tumor suppressor in esophageal squamous cell carcinoma and was regulated by mir340 5p med sci monit 202026e920642 1012659msm923909 wei l chen z cheng n microrna126 inhibit viability of colorectal cancer cell by repressing mtor induced apoptosis and autophagy onco targets ther “ 102147 otts238348 chen y shen z zhi y long noncoding rna ror promotes radioresistance in hepatocellular carcinoma cells by acting as a cerna for microrna145 to regulate rad18 expression arch biochem biophys “ 101016jabb201803018 zeng f wang q wang s et al linc00173 promotes chemoresistance and progression of small cell lung cancer by sponging mir218 regulate etk expression oncogene “ to 101038s4138801909842 fan h yuan j li x et al lncrna linc00173 enhances triplenegative breast cancer progression by suppressing mir490 3p expression biomed pharmacother 1010 16jbiopha2020109987 sun sl shu yg tao my lncrna ccat2 promotes angiogenesis in glioma through activation of vegfa signalling by sponging mir424 mol cell biochem ““ 101007 s1101002003712y zhang y jiang x wu z et al long noncoding rna linc00467 promotes glioma progression through inhibiting p53 expression via binding to dnmt1 j cancer “ 107150 jca41942 yang q tang y tang c diminished linc00173 expression induced mir1825p accumulation promotes cell proliferation migration and apoptosis inhibition via agernfkappab pathway lung cancer am j transl res in nonsmallcell “ xue j zhang z li x ren q wang q long noncoding rna ttnas1 promotes breast cancer cell migration and invasion via sponging mir1405p oncol lett “ 1038 92ol201911222 liu f xiao y ma l wang j regulating of cell cycle progression by the lncrna cdkn2bas1mir3245prock1 axis in laryngeal squamous cell cancer int j biol markers “ 1011771724600819898489 ding j yang c yang s linc00511 interacts with mir765 and modulates tongue squamous cell carcinoma progression by targeting lamc2 j oral pathol med “ 101111 jop12677 long s long s he h chen g microrna765 is preregulated in multiple myeloma and serves an oncogenic role by directly targeting sox6 exp ther med “ 103892 etm20197473 lv db zhang jy gao k microrna765 targets mtus1 to promote the progression of osteosarcoma via mediating erkemt pathway eur rev med pharmacol sci “ 1026355eurrev_201906_18040 jiao y yuan c wu h li x yu j oncogenic microrna765 promotes the growth and metastasis of breast carcinoma by directly targeting ing4 j cell biochem yuan l ma t liu w et al linc00994 promoted invasion and proliferation of gastric cancer cell via regulating mir7653p am j transl res “ yang h peng m li y zhu r li x qian z linc00703 acts as a tumor suppressor via regulating mir181aklf6 axis in gastric cancer j gastric cancer “ 105230jgc2019 19e43 chen y qiu f huang l et al long noncoding rna linc00312 regulates breast cancer progression through the mir9cdh1 axis mol med rep “ 103892mmr201910895cancer management and research publish your work in this journal cancer management and research is an international peerreviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient the manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors dovepress submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalcancer management and research submit your manuscript wwwdovepresscom dovepress 0c'
Colon_Cancer
alcoholic liver disease ald is a chronic alcoholinduced disorder of the liver for which there are few effectivetherapies for severe forms of ald and for those who do not achieve alcohol abstinence in this study we used asystematic drugrepositioning bioinformatics approach querying a large compendium of geneexpression profiles toidentify candidate us food and drug administration fda“approved drugs to treat ald one of the top compoundspredicted to be therapeutic for ald by our approach was dimethyl fumarate dmf an nuclear factor erythroid related factor nrf2 inducer we experimentally validated dmf in liver cells and in vivo our work demonstrates thatdmf is able to significantly upregulate the nrf2 protein level increase nrf2 phosphorylation and promote nrf2nuclear localization in liver cells dmf also reduced the reactive oxygen species ros level lipid peroxidation andferroptosis furthermore dmf treatment could prevent ethanolinduced liver injury in ald mice our results provideevidence that dmf might serve as a therapeutic option for ald in humans and support the use of computationalrepositioning to discover therapeutic options for aldintroductionoxidative stress is implicated in the development ofdiverse liver disorders such as alcoholic liver diseaseald12 ald encompasses a variety of chronic liverdiseasesincluding liver steatosis fatty liver hepatitiscombined with ‚ammation fibrosis cirrhosis andultimately hepatocellular carcinoma hcc3 althoughalcohol abstinence is effective for patients with mild aldsteatosis there are few effective therapies for severeforms of ald and for those who do not achieve alcoholabstinence corticosteroid is the only treatment option toimprove the shortterm survival of severe alcoholiccorrespondence yongheng chen yonghenc163com orting liu liuting818126com1department of oncology nhc key laboratory of cancer proteomics statelocal joint engineering laboratory for anticancer drugs national center feriatrics clinical research xiangya hospital central south university changsha hunan china2department of gastroenterology xiangya hospital central south university changsha hunan chinathese authors contributed equally ye zhang shuang zhaoedited by m agostinihepatitis ah patients4 however many of these patientsdo not respond to this treatment and experience severeadverse effects such as infection5 therefore there is anurgent need to develop novel targeted therapeutics totreat severe forms of ald or patients who fail to achievealcohol abstinence the computational repositioning offood and drug administration fdaapproved drugs is apromising and efficient avenue for discovering new uses6given the high costs possible side effects high failurerate and long testing periods for developing new medicines an fdaapproved compound was known to begenerally safe in humans and available for clinical use7 itis possible to identify safe drugs with potentialforrepurposing in other conditions by using computationalstrategies which can eliminate the need for a phase isafety trial and expedite phase ii efficacy trials analysis ofinteractions between genes and fdaapproved drugsallow the pursuit of new indications for treating diseaseswith no fdaapproved pharmacotherapiesrecent advancements in computing and the dramaticexpansion of available highthroughput datasets have the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cenabled the development of drug repurposing to identifynovel treatment options for ald thus in this study weaimed to identify a new therapeutic option with potential forrepositioning in ald we used a systematic computationalapproach based on both public geneexpression patterns inald and the interactions between genes and fdaapproved drugs interestingly we identified nuclear factorerythroid 2related factor nrf2 as a novel therapeutictarget in ald8 nrf2 is a basic leucine zipper bziptranscription factor that regulates the expression of certainproteins which protect cells against oxidative stress underunstressed conditions nrf2 is kept in the cytoplasm bykelch likeechassociated protein keap1 and cullin3upon oxidative stress nrf2 is phosphorylated at ser40 andreleases from keap1 then translocates into the nucleus inthe nucleus nrf2 forms a heterodimer with one of thesmall maf proteins maff mafg and mafk binds tothe antioxidant response element are in the promoterregions of many antioxidative enzymes and regulates thetranscription of these enzymes such as glutamate“cysteineligase catalytic gclc and heme oxygenase1 ho1more surprisingly we found that the fdaapproved nrf2inducer9 dimethyl fumarate dmf which has not previously been described to have a therapeutic associationwith ald was determined to have a strong therapeuticpotential for repositioning in ald we evaluated the efficacy of dmf for ald in liver cells and in vivo using anethanolinduced mouse model concordant with our computational prediction the experimental results demonstratethat dmf is able to significantly ameliorate ethanolinducedliver injury compared to untreated groupsresultscomputational repositioning of fdaapproved drugs foraldto identify efficient therapeutic strategies for patientswith liver diseases we downloaded drug datasets thatcontain both clinical application and animal test fromgene expression omnibus wwwncbinlmnihgovgeogse accession number gse28619 and then weused a bioinformatics approach to testthe drugrepositioning potential of fdaapproved drugs for aldfrom this approach we computed the activity score ofcandidate drugs and compared geneexpression profiles inresponse to these drugs in ald then we annotated theknown gene targets of the topscoring candidates andqueried fdaapproved drugbank using gene targets as aninput which displayed an output of a list of chemicalcompounds notably ald cells are known to abnormallyexpress molecules in the antioxidant response pathwaythus we aimed to study one of the five topscored candidate genes nrf2 among nrf2compound interactionsthe main use of dmf is previously tested with some success in multiple sclerosis patients with relapsing formsofficial of the cell death differentiation associationfocused on thesuggesting that dmf used in the clinic may affect the aldgeneexpression signature this analysis led us to focus ondrugs targeting molecules fig 1a the majority of knownphysiologic or pharmacological nrf2 inducers are electrophilic molecules that covalently modify by oxidation oralkylation cysteine residues presentin the thiolrichkeap1 protein10 dmf is one of the known nrf2 inducers which has been tested for the treatment of multiplesclerosis and approved in for its drug bioavailabilityand efficacy11 currently mmf has been used to develop asecond generation of nrf2 inducers as prodrugs12therefore wefumarateregulationmechanism of nrf2 in liver disorders the generation oftoxic metabolites by ethanol such as lipidperoxidationproducts contributes to the pathogenesis of alcoholic liverinjury fumarates prevent ros accumulation via the nrf2pathway in liver cells therefore we used an ald mousemodel six mice a group and hepatic fibrosis rat modelnine rats a group to examine the role of fumarates in vivohepatic lipid accumulation was distinctively increased inethanolfed rats in order to address the role of dmf inhepatic lipid accumulation we administered ald micewith dmf at mgkgday or mgkgday for daysin order to address the role of dmf in hepatic fibrosis weadministered hepatic fibrosis rats with dmf at mgkgday or mgkgday for weeks dmf ameliorated thehepatic steatosis induced by ethanol as observed in liversections stained with hematoxylin and eosin he fig 1band supplementary fig s1a at the same time the highlycrosslinked collagen fraction increased significantly during ethanolinduced fibrosis progression while collagendeposition was partly reduced under dmf treatment fig1c and supplementary fig s1b to substantiate thefinding that dmf increases the activity of nrf2 pathwayto inhibit ald we collected liver sections from normaland ald mice and checked nrf2 and gclc proteinlevels in the mouse model we performed immunohistochemistry ihc and western blotting for nrf2 andgclc results revealed that dmf treatment significantlyincreased nrf2 and gclc protein levels in ald mouseliver when compared to the matched control groups fig1d“f and supplementary fig s1c ddmf and mmf activate the nrf2 pathway in liver cellsregulator ofnrf2 is an essentialthe antioxidantresponse pathway which promotes the expression of various genes in response to oxidative stress1314 fumaratesprotect neurons and astrocytes against ros damage15 todetermine whether dmf or mmf regulates the nrf2protein level in liver cells we cultured hepg2 and lo2cells under the treatment of μm dmf and mmf fordifferent lengths of time and found that both dmf andmmf increased the protein level of nrf2 in a timedependent manner fig 2a and supplementary fig s2a 0czhang et al cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0czhang et al cell death and disease page of see figure on previous pagefig computational repositioning of food and drug administration fdaapproved drugs for alcoholic liver disease ald a schematicrepresentation of the bioinformatics workflow for the repositioning approach used to identify potential candidate drugs and genes for the treatmentof ald b dimethyl fumarate dmf prevents ethanolinduced hepatic steatosis mice were fed with the control diet or ethanol diet containing vv ethanol respectively followed by treatment with mgkg dmf or mgkg dmf by oral gavage for days tissue sections from the mouseliver were prepared for hematoxylin and eosin he staining scale bars are μm c dmf decreases ethanolinduced hepatic fibrosis mice were fedas in b tissue sections from the mouse liver were prepared for collagen staining scale bars are μm d e dmf increases endogenous nrf2 andgclc to activate the nrf2 signaling pathway in the mouse liver immunohistochemical staining of nrf2 and gclc proteins in mouse liver tissuesliver tissue sections from different groups were stained immunohistochemically with antinrf2 antibody d or antigclc antibody e as indicateddata shown are from one mouse from each group scale bars are μm f nrf2 and gclc in mouse liver sections were compared against actb bywestern blotting the statistical analysis of all samples is shownfurther results revealed that the nrf2 protein level wasupregulated with increased dmf and mmf concentrationsfig 2b and supplementary fig s2b phosphorylationserine40 is required for nrf2 activation1617 to confirmthe activation of nrf2 we treated hepg2 or lo2 cellswith dmf and mmf respectively as indicated thendetermined the level of phosphorylated nrf2 protein bywestern blotting results showed that dmf and mmftreatment significantly increased the phosphorylation levelof nrf2 when we adjusted the sample loading to keep thenrf2 level constant fig 2c and supplementary fig s2cindicating that nrf2 was activatedin addition wechecked the protein levels of nrf2regulated genes15 ourdata showed that dmf and mmf treatment promoted theexpression of gclc and ho1 protein levels fig 2a bmoreover nrf2 knockdown dramatically decreasedgclc and ho1 protein upon either normal condition orfumarates treatment fig 2d and supplementary fig s2dcollectively our results demonstrate that fumarates activate the nrf2 pathway in liver cellsonce phosphorylated nrf2 can translocate into thenucleus and activate transcription of various detoxification and antioxidant enzymes upon exposure to stresses18to examine whether fumarates regulated nrf2 nuclearlocalization in liver cells we treated hepg2 or lo2 cellswith dmf and mmf at different concentrations for hfig 2e then cells were lysed and subjected to cytosolicand nuclear fraction extraction we found that dmf fig2e left pannel and mmf fig 2e right pannel promotednrf2 nuclear accumulation in a dosedependent mannermoreover we performed immunofluorescence in livercells confocal microscopy data showed that nrf2expression and nuclear localization were enhanced inhepg2 cells upon dmf and mmf treatment fig 2ftaken together our data provide evidence that fumaratesactivate nrf2 and promote its translocation from cytoplasm to the nucleusdmf and mmf reduce the ros level by activating nrf2 inliver cellsthe relative levels of gsh and gssg are associatedwith various disease aging and cell signaling events19“official of the cell death differentiation associationto illustrate the potency offumarates as antioxidantagents we performed the reaction to convert total glutathione and the oxidized form gssg to the reducedform gsh then we measured both total glutathioneand gssg in the luminescent reaction scheme with thegsh probe the results showed that dmf and mmfinduced a dosedependent increase of intracellular gshfig 3a b doxorubicin dox an effective anticanceragent can induce the generation of ros which then leadsto oxidative damage of cellular and mitochondrial membranes2223 27dichlorofluorescin diacetate dcfhdais a specific indicator of ros formation24 and has beenused widely as a fluorescence probe in cells2526 confocalmicroscopy data revealed that ros were accumulated inhepg2 cells with the presence of dox while dmf andmmf blocked the doxinduced accumulation of rosfig 3c and supplementary fig s3a then we performedsirna transfection in hepg2 cells to knock down nrf2and observed a significant increase of ros upon doxtreatment even in the presence of dmf and mmf fig3d and supplementary fig s3b moreover we usedmitotracker® red cmxros kit an agent which can bepassively transported through the cell membrane anddirectly gathered on the active mitochondria to test theeffect of fumarates on the mitochondrial ros level wefound a significant reduction of h2o2 or ethanolinducedmitochondrial ros under fumarates treatment fig 3eand supplementary fig s3cthese results suggest aresistant effect of fumarates in response to ros by activating the nrf2 pathwaydmf and mmf reduce rosinduced lipid peroxidation andferroptosis in liver cellsrecent studies showed accumulation of ros can lead tolipid peroxidation and ferroptosis27 therefore we speculated that fumarates regulate rosinduced ferroptosis toexamine ferroptosis in dox or ethanoltreated cells weexamined the levels of hepatic malondialdehyde mdaand nadpnadph content2829 consistent with rosinduced ferroptosis we found that dox or ethanoltreatment significantly increased lipid peroxidation fig4a b and decreased nadph content fig 4c we 0czhang et al cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0czhang et al cell death and disease page of see figure on previous pagefig dimethyl fumarate dmf and mmf activate the nrf2 pathway in liver cells a dmf or mmf treatment increases endogenous nrf2gclc and ho1 protein level in a timedependent manner hepg2 or lo2 cells were either untreated or treated with μm dmf or mmf for differentlengths of time followed by being lysed and subjected to western blotting with the indicated antibodies b dmf or mmf treatment increasesendogenous nrf2 gclc and ho1 protein level in a dosedependent manner hepg2 or lo2 cells were either untreated or treated with dmf ormmf at the indicated concentrations for h actb is shown as a loading control c dmf or mmf increases the nrf2 s40 phosphorylation levelhepg2 or lo2 cells were treated as in b analyzed by western blotting with nrf2 phospho s40 antibody and normalized against nrf2 proteinthe sample loading was adjusted to keep the nrf2 level constant d nrf2 knockdown decreases gclc and ho1 protein levels under normal orfumarates condition hepg2 or lo2 cells were transfected with sinrf2 or negative control nrf2 gclc and ho1 protein levels were determined bywestern blotting e dmf or mmf promotes nrf2 nuclear accumulation after treated with μm dmf left panel or mmf right pannel for hhepg2 or lo2 cells were subjected to cytosolic and nuclear fractionation and nrf2 protein levels were determined by western blotting histone3h3 and αtubulin were used as nuclear and cytoplasmic markers respectively while actb was used as a wholecell lysate maker f hepg2 cells weretreated with dmso μm dmf or μm mmf for h as indicated then paraformaldehyde fixed blocked and processed for immunofluorescencewith dapi blue or antibody against nrf2 green nrf2 staining is shown on the left and the merged nrf2 and dapi on the right bar μm relativenrf2 fluorescence intensity was calculated using imagej software the ratio was quantified mean values were calculated from the individualdistributions in ten cells per conditionobserved a decrease of mda levels and a restoration ofnadph when we added fumarates into liver cells pretreated with dox or ethanol fig 4a“c more evidencewas obtained when we detected the protein level of gpx4an important ferroptosis regulator which can inhibit cellmembrane phospholipid peroxidation results showedthat compared with dmso treatment gxp4 was substantially decreased under ethanolstimulated conditionindicating a promoting role of ethanol in liver lipid peroxidation and ferroptosis however we observed arestoration of the gxp4 protein level when we addedferrostatin1 an inhibitor of ferroptosis into hepg2 andlo2 cells pretreated with ethanol fig 4d and supplementary fig s4a a similar result was detected in mouseliver primary cells ethanol treatment lead to a significantdecrease offerrostatin1restored gpx4 protein pretreated with ethanol fig 4eand supplementary fig s4b in addition we treated livercells with erastin an inducer of ferroptosis which playsthe opposite role to ferrostatin1 in ferroptosis and foundfumarates led to an accumulation of gxp4 and nrf2protein even in the presence of ethanol or erastin fig 4ef we also detected lipid peroxidation with c11bodipy undecanoic acid by measuring the fluorescenceintensity in red color consistent with our previousresults an increase of ros production was observedunderthe treatment of ethanol and erastin whileferrostatin1 or fumarates can inhibit lipid peroxidationinduced by ethanol supplementary fig s4c suggestinga preventive effect of fumarates in rosinduced lipidperoxidation and ferroptosisendogenous gpx4 whiledmf inhibits ethanolinduced lipid peroxidation andferroptosis in vivothese results strongly suggest that dmf prevents rosinduced liver injury and ferroptosis via activating thenrf2 pathway we therefore studied the role of dmf inrosinduced ferroptosis in mice hepatocytes treated withofficial of the cell death differentiation associationethanol or not compared to the untreated group andferrostatin1 treated group groups treated by ferroptosisinducer erastin and ethanol had smaller ruptured mitochondria fig 5a these cellular morphological featuresare characteristic of ferroptosis however dmf ameliorated the ferroptosis induced by ethanol as observed bytransmission electron microscopymore evidence was obtained when we performed western blotting and ihc compared with the normal micethe protein levels of 4hne which indicated an increasedlipidperoxidationinduced ferroptosis were higher in aldmouse livers while the gpx4 protein level was lower incontrast dmf treatment could block lipidperoxidationinduced ferroptosis by decreasing the protein levels of4hne and increasing the protein levels of gpx4 in vivofig 5b“f these data further validate fumarates as inhibitors of the lipidperoxidationinduced ferroptosisdiscussionusing a computational repositioning of existing drugsbased on the publicly available geneexpression data todiscover therapies for ald we inferred that the nrf2inducer dmf could serve as a therapeutic option for aldand performed experimental validations which demonstrated the efficacy of dmf in ameliorating ald in livercells and in the mouse model the precise mechanism ofaction for dmf is unknown but it is known to activatethe nrf2 antioxidant pathway although dmf has notpreviously been suggested as a therapy for ald previousstudy has shown that nrf2 prevents alcoholinducedfulminant liver injury30 in this study we found thatfumarates activate the nrf2 signaling pathway promoting nrf2 phosphorylation and nuclear localization inliver cells nrf2 further activates the transcription ofgenes encoding various detoxification and antioxidantenzymes in response to rosoxidative stress is implicated in the development ofdiverse liver disorders such as ald nonalcoholic fatty 0czhang et al cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0czhang et al cell death and disease page of see figure on previous pagefig dimethyl fumarate dmf and mmf reduce the ros level by activating nrf2 in liver cells a b dmf or mmf enhances cellular redoxpotential by increasing gsh level hepg2 cells were treated with or without different concentrations of dmf a or mmf b for h and thenassessed for cellular gsh and gssg levels denotes p ns denotes no significance error bars represent mean ± sd for triplicate experiments cfumarates block dox or ethanolinduced ros accumulation hepg2 cells were pretreated with dox or ethanol for h followed by treatment with μm dmf or mmf for another h as indicated cells were loaded with dcfhda μm and incubated for min at °c in the darkfluorescence images were acquired by a confocal microscope bar μm d nrf2 knockdown accumulates ros damage in liver cells either with orwithout fumarates hepg2 cells were transfected with sinrf2 and treated as in c fluorescence images were obtained e fumarates block h2o2 orethanolinduced mitochondrial ros accumulation lo2 cells were pretreated with h2o2 or ethanol for h followed by the treatment with μmdmf or mmf for another h as indicated cells were incubated with mitotracker® red cmxros red at °c in the dark images were acquired byfluorescence microscope bar μmliver disease nafld and hcc2 elevated cellularstresses which are induced by alcohol hepatic viruses ordrugs play a vital role in the initiation and progression ofmultiple liver pathologies31“ certain stressed conditions can cause the accumulation of cellular rosuncontrolled production of ros results in oxidativestress on tissues and cells and causes lipid peroxidation34the nrf2 antioxidant pathway is a highly conservedsignal transduction pathway that allows cells tissues andans to survive under oxidative stress conditions35 ourstudy showed that fumarates activate the nrf2 signalingpathway reduce the cellular ros level and protect livercells from ethanolinduced oxidative injuryferroptosis is an iron and rosdependent form of celldeath which is characterized by the accumulation of lipidlevels3637 ros accumulationhydroperoxides to lethalcould directly react with unsaturated fatty acids whichmay lead to a destruction of the mitochondrial membrane a massive release of substances promoting apoptosisand increased ferroptosis dysregulation offerroptosis has been implicated in various pathologicalprocesses including cancer neurodegenerative diseasesacute renal failure druginduced hepatotoxicity ischemiareperfusion injury and tcell immunity3839 our studyshowed that fumarates upregulate the protein level ofgpx4 a gshdependent enzyme that reduces lipidhydroperoxides while decrease lipid peroxidation andferroptosis and thus ameliorate ethanolinduced liverinjury in the ald mouse model fig in addition thesefindings support that fumarates could also be effective inother ferroptosisassociated diseasesin recent years drug repurposing has gained more andmore attention for accelerating drug development40given the high costs possible side effects high failure rateand long testing periods for developing new medicines7drug repurposing provides an attractive approach to meetthe need for improved diseases treatment for exampledisulfiram an old alcoholaversion drug has emerged as acandidate for treating highrisk breast cancer7 hippeastrine hydrobromide hh which has been used to preventavian ‚uenza h5n1 has become a promising drug forinhibiting zika virus zikvinfection41 topiramate aofficial of the cell death differentiation associationsafe and effective drug for treating neurological diseasesis capable of ameliorating ‚ammatory bowel disease42in this study we demonstrate that computational repositioning of fdaapproved drugs by analyzing publicgeneexpression data can be used to infer drug therapiesfor ald and offer experimental evidence that the nrf2inducer dmf is capable of ameliorating disease pathophysiology in the ald mouse model dmf was alreadyestablished as a safe and effective drug for treating multiple sclerosis43 additional clinical investigation will beneeded to test whether dmf could benefit patients suffering from aldmaterials and methodscell culture and treatmentcell culture was performed as previously described44hepg2 or lo2 cells were cultured in dmemhigh glucose medium hyclone sh3002201 or rpmi mediummodified hyclone sh3080901 supplemented with fetal bovine serum gibco penicillin andstreptomycin gibco at °c in a humidifiedatmosphere containing co2 for fumarate treatmentcells were first cultured in the medium which containedfetal bovine serum then dmf sigmaaldrich and mmf sigmaaldrich of different concentrations were added into the medium the treatmentsto increase cell oxidative stress and ferroptosis wereperformed by adding ethanolsigmaaldrich e7023 mm doxorubicindox solarbio d8740 μm anderasitin selleck s7242 μm to the culture medium for hthen we treated liver cells with fumarates orferrostatin1 sigmaaldric sml0583 μm for another h all the concentrations are final concentrations in theculture mediumwestern blottingwestern blotting was performed as previously mentioned4546 hepg2 or lo2 cells were lysed in ripa lysisbufferbeyotime p0013b containing protease andphosphatase inhibitors cell debris was removed by centrifugation while celllysates were boiled for minand centrifuged at °c before loading on or 0czhang et al cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0czhang et al cell death and disease page of see figure on previous pagefig dimethyl fumarate dmf and mmf reduce rosinduced lipid peroxidation and ferroptosis in liver cells a b fumarates obviouslyreverse dox or ethanolinduced lipid peroxidation hepg2 a and lo2 b cells were pretreated with μm dox mm ethanol or μm erastinfor h followed by μm ferrostatin1 or μm fumarates for h thereafter cells were lysed and subjected to lipid peroxidation malondialdehydemda assay c fumarates reverse dox or ethanolinduced ferroptosis lo2 cells were pretreated with μm dox mm ethanol or μm erastinfor h followed by μm ferrostatin1 or μm fumarates for h thereafter cells were lysed and subjected to nadpnadph assay denotesp denotes p and ns denotes no significance error bars represent mean ± sd for triplicate experiments d“f fumarates block lipidperoxidation and ferroptosis in liver cells hepg2 cells lo2 cells d f or mouse liver primary cells e were pretreated with mm ethanol or μmerastin for h as indicated followed by μm ferrostatin1 or μm fumarates for another h thereafter cells were lysed and subjected to westernblotting for nrf2 and gxp4 with actb as loading control the statistical analysis of all samples is shown fsdspage gels then proteins were transferred ontopvdf membranes merck millipore ltd ipvh00010 forwestern blotting analysis the primary antibodies tophosphors40 nrf2 abcam ab76026 workingdilution nrf2 proteintech 163961ap working dilution gclc proteintech 126011ap working dilution ho1 proteintech 107011ap working dilution αtubulin proteintech 660311lg working dilution gxp4 abcam ab125066 working dilution histone3 proteintech 1ap working dilution actbβactin proteintech 205361ap working dilution werecommercially obtainedrna interferenceknocking down of nrf2 was performed by rnainterference following the manufacturer™s instructions forlipofectamine rnaimax reagent invitrogen the knockdown efficiency was determined by westernblotting synthetic sirna oligo nucleotides were obtainedcommercially from genepharma co ltd list of effectivesequences is as follows sinrf21 ²gguugagacuaccaugguutt3²sinrf22 ²ccagaacacucaguggaautt3²sinrf23 ²gccuguaaguccuggucautt3²negative control ²uucuccgaacgugucacgutt3²cytoplasmic and nuclear extractsfor nrf2 nuclear translocation experiments cells werecultured in the medium which contained fetal bovineserum then dmf and mmf of different concentrationswere added into the medium for h in all 10cmdiameter plates of hepg2 and lo2 cells were lysed andcytosolic and nuclear fractions were separated followingthe protocol provided by the nuclear and cytoplasmicextraction kit manufacturer active motif inc the nuclear pellets were washed three times with phosphate buffered saline containing freshly added proteaseand phosphatase inhibitors the cytosolic supernatantwas centrifuged to remove any nuclear contamination andtransferred to a new tube both the cytosolic and nuclearfractions were boiled separately in sds sample buffer andanalyzed by western blotofficial of the cell death differentiation associationgsh analysishepg2 cells were plated into white and flatbottom well plates and cultured for h at °c then we treatedcells with dmso or fumarates and incubated for another h for fluorescent gsh assay we first washed cells withhanks™ balanced salt solution solarbio h1045500 thendetermined the levels of reduced and oxidized gsh bygshgssg assay kit promega v6611 according to themanufacturer™s protocol totalrelative luminescenceunits rlu are graphed as means ± sd denotes p ns denotes no significance graphed data represents oneof three experimental repeatsmeasurement of cell lipid peroxidation and nadpnadphassayin thefumarates erasitin μm orliver cells were plated into 60mm dishes and culturedfor h at °c the treatments to increase cell lipidperoxidation were performed by adding ethanol mmdoxorubicindox μm and erasitin μm to theculture medium for h then we treated liver cells with orwithoutferrostatin1 μm for another h all the concentrations are finalconcentrationslipidperoxidation assay and nadpnadph assay we firstwashed cells with °c precooled phosphate bufferedsaline then determined the levels of cell lipid peroxidation by mda assay kit beyotime s0131 and nadpnadph quantitation colorimetric kit biovision k347according to the manufacturer™s protocol the totalhepatic mda content and nadpnadph levels aregraphed as means ± sd graphed data represent one ofthree experimental repeatsculture medium forimmunofluorescence staininghepg2 cells were plated into glass bottom cell culturedishes nest and pretreated with or withoutdox for h followed by addition of dmf and mmf intothe medium thereafter cells were first fixed with paraformaldehyde biosharp then permeabilized in triton x100 amresco blocked by bovine serum albumin amresco in pbs buffersigmaaldrich p5368 and lastly incubated with theindicated primary nrf2 antibody working dilution 0czhang et al cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0czhang et al cell death and disease page of see figure on previous pagefig dimethyl fumarate dmf inhibits ethanolinduced lipid peroxidation and ferroptosis in vivo a dmf prevents ethanolinducedferroptosis mice were fed as indicated on the final day morning the mice were given alcohol liquid gkg or maltodextrin control by gavageand sacrificed after h in addition ferrostatin1 mgkg and erastin mgkg were provided min before
Colon_Cancer
" natural orifice specimen extraction surgery is a novel technique of minimally invasive surgery thepurpose of this study was to compare the safety of laparoscopic anterior resection with natural orifice specimenextraction noselar and abdominal incision specimen extraction aiselar for sigmoid or rectum tumorsmethods medline pubmed embase central cochrane central register of controlled trials scopus andclinicaltrials databases were systematically searched for related s up to august the primary outcomesincluded postoperative complications overall postoperative complication incisionrelated complicationanastomotic fistula and severe complication and pathologic results lymph nodes harvested proximal resectionmargin and distal resection edge the statistical analysis was performed on stata softwareresults ten studies comprising patients were used for metaanalysis compared with aiselar noselar hadmore advantages in terms of overall postoperative complication odds ratio or ci to p incisionrelated complication or ci to p distal resection edge weighted meandifference wmd cm ci to cm p recovery of gastrointestinal function wmd ˆ’ day ci ˆ’ to ˆ’ day p pain scores in postoperative day wmd ˆ’ ci ˆ’ to ˆ’ p additional analgesics usage or ci to p and hospital stay wmd ˆ’ day ci ˆ’ to ˆ’ day p while the operation time of noselar was prolonged wmd min ci to min p the anastomotic fistula severe complication lymph nodes harvested proximalresection margin intraoperative blood loss and longterm outcomes in noselar were comparable with aiselars the safety of noselar was demonstrated and it could be an alternative to conventional surgery inlaparoscopic anterior resection for sigmoid and rectal tumors however further randomized and multicenter trials are requiredkeywords natural orifice specimen extraction nose laparoscopic anterior resection rectal tumor sigmoid tumor metaanalysis correspondence guangenyang163com1department of colorectal surgery hangzhou third hospital hangzhou zhejiang people™s republic of chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0che world of surgical oncology page of introductionover the past three decades laparoscopic surgery hasevolved incessantly especially in the field of colorectalsurgery it has been widely accepted by surgeons and patients in light of the better perioperative outcomes andanalogical longterm effectiveness compared with opensurgery for colorectal cancers [“]for conventional laparoscopic colorectal operation asmall laparotomy in the abdomen is required for specimen harvested and colorectal anastomosis because ofthe miniincision it causes many undesirable outcomessuch as incision pain wound infection scar and even incision hernia and the advantages of laparoscopic surgeryare reduced [“] to minimize those drawbacks anovel surgical variant known as natural orifice specimenextraction nose surgery with the features of naturalorifice specimen extraction and totalintraperitonealanastomosis has been introduced and is becoming ahotspot [“] some studies have reported the oncology and safety outcomes between nose surgery andconventional laparoscopic surgery are comparable [“] and the nose surgery therefore is supposed tohave a progress of minimally invasive surgeryrecently some metaanalysis studies had comparednatural orifice specimen extraction nose with abdominal incision specimen extraction aise in laparoscopiccolorectum resection for the colorectal disease [ ]however colorectum resection comprises right hemicolectomyleft hemicolectomy and anterior resection procedures among those surgeries were largely different and the surgical procedures and the excision extension between sigmoid and rectum resection aresimilar in addition several studies compared laparoscopic anterior resection with natural orifice specimenextraction noselar with abdominal incision specimen extraction aiselar for sigmoid or rectum tureleased [“] hence wemors wereconducted this metaanalysis to evaluate the safety ofnoselar in sigmoid and rectal tumorsrecentlymethodsstudy design and inclusion criteriathis metaanalysis followed the preferred reportingitems for systematic reviews and metaanalysis prisma statements the inclusion and selection criteria were determined before starting a literature searchonly when studies with fulltext on sigmoid or rectaltumors that compared noselar and aiselar andreported at least one of the endpoints of focus were retrieved and analyzed the most comprehensive researchwas recruited when overlapping researches was conducted by the same team no language restriction wasapplied conference s case reportsreviewsrobotic surgery and singleportwere not consideredlaparoscopic surgeryselection criteria conformed to the framework ofpico participant intervention comparison and outcome patients diagnosed with sigmoid or rectal tumorsbenign and malignant tumors requiring surgery wereincludedinterventions consisted of noselar andaiselar noselar was compared with aiselarin all eligible studies primary endpoint outcomes werepostoperative complications overall postoperative complication abdominal incisionrelated complication anastomotic leak and severe complication claviendindoclassification ‰¥ iii and pathologic results retrievedlymph nodes proximal resection edge distal resectionedge secondary outcomes included operation timeblood loss pain score numeric rating scale score additional analgesics gastrointestinalfunction recoveryhospitalization duration 5year diseasefree survivaldfs and 5year overall survival os search strategythe following databases had been searched up to august medline pubmed central cochrane central register of controlled trials embase scopus andclinicaltrials for a more accurate search the followingkeywords andor mesh terms were used œsigmoid neoplasms œrectal neoplasms œcolorectal neoplasmsœlaparoscopyœnatural orifice specimen extractionœtransvaginal specimen extraction œtransanal specimenextraction and œtransrectal specimen extraction thespecific search strategies among databases existed differences the search strategy of pubmed was presented inadditional text reference s of the eligible studies were reviewed to find the potentially relevant studiesstudy selection and quality assessmentretrieved studies were independently assessed for relevance by reviewers changjian wang and jinmingchen by screening the title and in order to enhance sensitivity studies were removed only when bothreviewers excluded the study subsequently a fulltextassessment was performed on the initial screening included studies the risk of bias was assessed by thenewcastleottawa scale nos for observational studies and studies achieving five or more stars were eligible cochrane collaboration™s tool for assessing risk forbias was used for randomized controlled trials [ ]all discrepancies were discussed before a final decisionwas madedata collection and statistical analysisdata from the recruited studies were extracted by tworeviewers changjian wang and jinming chen andused formeananalysis outcome valuesfurther 0che world of surgical oncology page of standard deviation and median interquartile rangewere extracted from each study considering potentialheterogeneity among studies we pooled the results byusing a randomeffects model the weighted mean difference wmd and confidence intervals cis wereapplied for continuous variables and the odds ratioor and cis were used for dichotomous variablesthe continuous outcomes were adopted the inverse variance method and dichotomous outcomes were adoptedthe mantel“haenszel statistical method when a studymerely offered the outcomes with median and interquartile range an estimation based on formulas designed byhozo was performed if a study did not provide the hazard ratio hr and cis of 5year dfsorand os the methods presented by tierney wereused for data extraction from survival curves thechisquare test and isquared value were used for measuring heterogeneity and i2 p was definedas significant heterogeneity sensitivity analyses basedon nos score ‰¥ and the sample size of noselargroup ‰¥ were conducted to assess the potentialsource of heterogeneity and the robustness of the resultspublication bias was examined with a funnel plot andharbord test p was considered statistically significant the statistical analysis was performed onstata softwareresultsliterature selection and characteristicsthe initial database search identified s ofwhich were removed based on the title and assessment the rest of the literature were evaluated byfulltext assessment and studies were excludedcharacteristics of the excluded studies were presented inadditional table ten studies were finally included forfurther qualitative and quantitative synthesis [ “ ] all of these were retrospective studies the process of the search and selection wasshown in fig a total of patients were recruitedin those studies with patients in the noselargroup and patients in the aiselar group themain characteristics of studies and patients were presented in table and details were shown in additionalfig flow chart of studies included in the metaanalysis 0che world of surgical oncology page of sunasunasunasunasunasunarosunaiangavsunasunasunarorororomutcermutceridomgsimutceridomgsimutceridomgsimutceridomgsiralesaipurnsonerocsetisinemcepsnoitcartxenoitacolromutleamefeamlrednegmutcer““idomgsimutcermutcermutcer““ralesonpuralesaipuraeyaegaralesonpuralesaipuralesonpugnitnuocisetapcitrapngisedydutsinogerraeyydutsisedutsdeducnliehtfoscitsiretcarahcinamelbatevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorternapajlateadasihianhclateuhianhclategnianhclategnahzianhclateuohzianhclategnxiianhclateuliydutsaissurydutsevitcepsorternawatihbaruaslateevitcepsorterecnarflatetsonedevitcepsorterydutsydutsianhclategnawrnleacsawattoeltsacwensonnoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaegnarinademronoitavedidradnats±naemsadrocertondetropera 0che world of surgical oncology page of euavlpytienegoretehieuapvldmwstluserldeoopicrororalesairalesonstneitapfoonˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’foonisedutsnoitacilpmocsemoctuollafostluserldeoopehtelbatsemoctuoyramirpsemoctuoevitarepotsopllarevonoitacilpmocnoitacilpmocdetaerlnoisicnilautsifcitomotsananoitacilpmocerevesdetsevrahsedonhpmylsemoctuoilcgoohtapingramnoitceserliamxorpegdenoitceserlatsidnoitcnufsemoctuoyradnocesemitnoitarepossoldooblevitarepoartnilanitsetnortsagifoyrevocerdopinapevitarepotsopegasusciseganallanoitiddayatslatipsohsoraeyevfisfdraeyevfiecnereffidnaemdethgewdmwinoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaliavvruseerfesaesidsfdliavvrusllarevosoyadevitarepotsopdopoitarsddoro 0che world of surgical oncology page of table the results of the pooled outcomes were summarized in table primary outcomesall included studies reported the overall postoperativecomplication the pooled data revealed that the postoperative complication in of patients whotreated with noselar and of patientswho treated with aiselar the or was ci to p with low heterogeneity i2 fig 2a among the studies eight studies reportedthe incisionrelated complication in of patients who underwent noselar and of patients who underwent aiselar the or was ci to p with no heterogeneity i2 fig 2b nine studies reported the anastomoticfistula of which ng reported zero events in bothgroups the remaining eight studies recorded anastomotic fistula in of patients sufferednoselar and of patients suffered aiselar or was ci to p withno heterogeneity i2 fig 2c a severe complication was defined based on the claviendindo classification two of the included studies recorded severecomplication claviendindo classification ‰¥ iii and thesevere complication in of patients withnoselar and of patients with aiselar or was ci to p withsignificant heterogeneity i2 fig 2da total of nine studies reported lymph node harvestthere was no significant difference in lymph node harvestbetween the two groups wmd ˆ’ ci ˆ’ to p no significant heterogeneity was observed i2 fig 3a the mean number of dissectedlymph nodes in the noselar group was and theaiselar group was the proximal resection marginwas reported in studies and the wmd in the upper resection edge was cm ci ˆ’ to cm p with no heterogeneity i2 fig 3b the distalresection margin was reported in studies and the wmdin the inferior resection edge was cm ci to cm p with no heterogeneity i2 fig3c the length of the distal resection margin in the twogroups was cm noselar group and cmaiselar groupsecondary outcomesa total of nine studies recorded operation time and intraoperative blood loss the pooled data revealed thatthe wmd of operative duration was min ci to min p heterogeneity i2 fig 4a the wmd of blood loss was ˆ’ ml ci ˆ’ to ml p heterogeneity i2 fig 4bfig forest plot comparing postoperative complications in thenoselar group and aiselar group a overall postoperativecomplication b incisionrelated complication c anastomotic fistulaand d severe complication 0che world of surgical oncology page of fig forest plot comparing pathologic outcomes in the noselar group and aiselar group a lymph nodes harvested b proximal resectionmargin and c distal resection edgesix studies provided data about the recovery of gastrointestinal function the wmd of bowel movement wasˆ’ day ci ˆ’ to ˆ’ day p heterogeneity i2 fig 5a the postoperative painwas recorded in studies and studies hisada and wang recorded the postoperative pain periodand the remaining reported the pain scores in postoperative day pod the wmd of pain score in pod 0che world of surgical oncology page of fig forest plot comparing intraoperative outcomes in the noselar group and aiselar group a operation time and b blood loss was ˆ’ ci ˆ’ to ˆ’ p heterogeneity i2 fig 5b the additional analgesicusage rate was also reported in those studies and theor of additional analgesics usage was ci to p heterogeneity i2 fig 5c theduration of hospital stay was reported in nine studiesthe wmd of hospital stay was ˆ’ day ci ˆ’ to ˆ’ day p heterogeneity i2 fig 5dfiveyear diseasefree survival dfs and 5year overallsurvival os were available in two studies the hazardratio hr in the 5year dfs was ci to p heterogeneity i2 fig 6a and thehr in the 5year os was ci to p heterogeneity i2 fig 6bsensitivity analysissensitivity analysis results based on the nos score ‰¥ and the sample size of noselar group ‰¥ were presented in additional table it showed a slight inconsistency in distal resection edge operation time andrecovery of gastrointestinal function and all the otheroutcomes showed a similar trend of results between thetwo groupspublication biasa funnel plot of overall postoperative complication wasperformed to detect publication bias it showed that allthe inclusive studies were within the confidenceinterval and no publication bias was found fig inaddition a harbord test confirmed there was no publication bias p discussionas a technique used for clinical treatment the safety ofnoselar should be efficiently proved morbidity isone of the most efficient indicators for assessing thesafety of an emerging technique postoperative complications may not only lead to failures of surgery but alsothreaten lives the overall postoperative complicationrate in noselar was lower than that in aiselar severe morbidity claviendindo ‰¥ iiiamong the two techniques was not a significant difference the operation involving digestive tract reconstruction anastomotic leakage is a potential risk once itoccurs reoperation is usually inevitable the incidence of anastomotic leakage in noselar wassimilar with that in aiselar in addition theincidence ofincisionrelated complications in noselar was significantly lower than that in theaiselar group obviously the reduction ofcomplications in noselar has largely attributed tothe decrease of incisionrelated complications althoughthe complications in noselar were reduced the riskof bacteria contamination in the peritoneal cavity shouldnot be neglected costantino had reported the peritoneal contamination in the nose group was higherthan that of the conventional group hence measures such as bowel preparation prophylactic antibioticsperitonealtransluminalwound retractor and abdominal drains are recommended to avoid the contamination of the peritonealcavity irrigationtransanallavagethe postoperative pathology results to some extentalso reflect the safety of a surgery this metaanalysisshowed lymph nodes harvested between the two groupswas comparable and it also conformed to the minimumrequirement of the guideline retrieved more than nodes in our metaanalysis the proximal marginin the noselar group was similar with the conventional group however the distal margin in the nosegroup was longer than that of the aiselar group the 0che world of surgical oncology page of potential cause of this difference was the use of transanalspecimen eversion and extraabdominal resection technique in the nose group [ ] because of thisprocedure the distal rectal resection is performed extraabdominally under direct vision moreover circumferential resection margin crm between the two groupshave no difference [ ] in addition accordingto our metaanalysis the longterm outcomes 5yeardfs and 5year os were comparable all of those indicated the nose technique was a safety procedure in thetreatment of sigmoid and rectal cancers nevertheless aconcern about tumor seeding was raised during the procedure of enterotomy and specimen extraction it is necessary to apply several measures such as the use ofprotection devices sterile specimen bags and avoidingoverpulling and compression during specimen extraction as a minimally invasive surgery noselar had moreadvantages in alleviating patient™s distress the reductionof pain scores in postoperative day pod was observed and this reduction could be attributed to thetrauma in noselar being further reduced owingto less pain the need for additional analgesics was alsoreduced in addition accelerating postoperative recoverywas also observed the recovery of gastrointestinal function and hospital duration in patients who sufferednoselar was much shorter besides some scholarsmay doubt if there have alterations in sexual urinary ordefecation function in the groups according to the included studies there were no differences in functionaloutcomes such as sexual urinary or defecation betweentwo groups [ ] even though a small part of patientsexperienced function alteration and the alternation wasreversible [ ] those all demonstrated thatnoselar was a safety surgery and to some extent ithad advantages in postoperative recoverynevertheless our study has several limitations firstlyintersphincteric resections were mixed with coloanalanastomoses with sigmoid cancer in our studies although there exist some differences we mixed the twotechniques and mainly considered there existing common procedures between sigmoid and rectum resectionin laparoscopic anterior resection and some studies didnot record the methods of outcome evaluation such asblood loss evaluation to some extent it reduces thecomparability of outcomes secondly the present metaanalysis relied solely on retrospective studies and someoriginal studies not presented how patients were selectedto be candidates for one technique or another the quality of all included studies was regarded as fair or good however this type of study cannot be comparedwith a randomized controlled trial and potential biascannot be ruled out thirdly this study only recruitedone multicenter research and some outcomes includedfig forest plot comparing postoperative recovery in the noselar group and aiselar group a recovery of gastrointestinalfunction b postoperative pain pod c additional analgesicsusage and d hospital stay 0che world of surgical oncology page of fig forest plot comparing longterm outcomes in the noselar group and aiselar group a 5year dfs and b 5year oslimited studies so further multicenter randomized controlled and more comprehensive studies containing adequate outcomes are needed fourthly the results ofsome pooled results among studies existed heterogeneity the sensitivity analysis could not be detected as thecause of heterogeneity although some results existedheterogeneity the major results were homogeneity andthe heterogeneity of outcomes such as operation duration blood loss and hospital stay can be explained byclinical heterogeneity such as the difference of patientssurgeons patient management and differences in surgical proficiency in nose technology in addition the results of the major parameters were robust all in all theresults of this analysis are convincedaccording to our metaanalysisthe advantages ofnose are reduced overall complications especiallyincisionrelated complications increased distal resectionedge enhanced recovery of gastrointestinal function reduced postoperative pain reduced additional analgesicsusage and shortened hospital stay and without an auxiliary patients operated by the nose technique achievebetter aesthetics however the operative time is prolonged although the nose technique has many advantagesthere are many requirements that should befollowed before the application of this technique in colorectal surgery firstly the nose should be operated byexperienced surgeons with conventionallaparoscopiccolorectal surgery secondly the indication of noseshould follow the indication of conventionallaparoscopic colorectal resection the depth of tumor invasionshould be within t3 and body mass index bmi shouldbe less than kgm2 for transanalnose and less than kgm2 for transvaginalnose transanal nose suitsfor male or female patients and the tumor diameter isfig funnel plot of the overall postoperative complications 0che world of surgical oncology page of recommended less than cm while transvaginal noseis only applied for female patients and the tumor diameter is limited within cm and the emergent conditionssuch as bowel obstruction perforation and massivebleeding are excluded all in all as surgeons follow appropriate indicationsthe noselar for sigmoid or rectal tumors is a safesurgery and the longterm outcomes between twooperations have no difference and the benefits of thenoselar in shortterm outcomes are noticeablethese findings promote enthusiasm in support ofnose surgery as an alternative approach forthetreatment of sigmoid and rectal tumorssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s1295702001982wadditional file additional file text the search strategy of pubmedadditional file additional table characteristics of the excludedstudiesadditional file additional table summary of the included studiesadditional file additional table the results of sensitivity analysisadditional file additional table quality assessment based on thenos for retrospective studiesabbreviationsnoselar laparoscopic anterior resection with natural orifice specimenextraction aiselar laparoscopic anterior resection with abdominal incisionspecimen extraction wmd weighted mean difference or odds rationos newcastleottawa scale dfs diseasefree survival os overall survivalcis confidence intervals hr hazard ratio pod postoperative day acknowledgementswe wish to thank the timely help given by junfeng hu in statistic analysisand mengdan zhou in language editingauthors™ contributionsguangen yang and zhong shen contributed to the conception and designof the study jun he performed the literature search and the writing of themanuscript changjian wang and jinming chen performed the data extraction haibo yao performed the data analysis qinyan yang and jianmingqiu participated in the writing of the manuscript guangen yang and haibo yao helped to revise the intellectual content the authors read and approved the final version of the manuscriptfundingthis work was funded by the zhejiang natural science foundation grantno lq19h160013 and the zhejiang medical health science and technologyproject grant no and the hangzhou health science andtechnology project grant no 2017a26availability of data and materialsall the data analyzed in this study was obtained from the included originals or related authorsethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of colorectal surgery hangzhou third hospital hangzhou zhejiang people™s republic of china 2departments ofgastroenterology pancreatic surgery zhejiang provincial people™s hospitalhangzhou zhejiang people™s republic of chinareceived february accepted july references wang cl qu g xu hw the short and longterm outcomes oflaparoscopic versus open surgery for colorectal cancer a metaanalysisint j color dis “ishibe a ota m fujii s suwa y suzuki s suwa h momiyama m watanabej watanabe k taguri m midterm followup of a randomized trial ofopen surgery versus laparoscopic surgery in elderly patients withcolorectal cancer surg endosc “allaix me giraudo g mistrangelo m arezzo a morino m laparoscopicversus open resection for colon cancer 10year outcomes of aprospective clinical trial surg endosc “lee l aboukhalil m liberman s boutros m fried gm feldman lsincidence of incisional hernia in the specimen extraction site forlaparoscopic colorectal surgery systematic review and metaanalysissurg endosc “hennessey db burke jp nidhonochu t shields c winter dc mealy k riskfactors for surgical site infection following colorectal resection a multiinstitutional study int j color dis “goto s hasegawa s hata h yamaguchi t hida k nishitai r yamanokuchis nomura a yamanaka t sakai y differences in surgical site infectionbetween laparoscopic colon and rectal surgeries subanalysis of amulticenter randomized controlled trial japanmultinational trialanization prev int j color dis “park js choi gs lim kh jang ys kim hj park sy jun sh clinical outcomeof laparoscopic right hemicolectomy with transvaginal resectionanastomosis and retrieval of specimen dis colon rectum “ooi bs quah hm fu cw eu kw laparoscopic high anterior resection withnatural orifice specimen extraction nose for early rectal cancer techcoloproctol “franklin mj kelley h kelley m brestan l portillo g torres j transvaginalextraction of the specimen after total laparoscopic right hemicolectomywith intracorporeal anastomosis surg laparosc endosc percutan tech “ wolthuis am de buck voa d'hoore a laparoscopic natural orificespecimen extractioncolectomy a systematic review world j gastroenterol“ xingmao z haitao z jianwei l huirong h junjie h zhixiang z totallylaparoscopic resection with natural orifice specimen extraction nose hasmore advantages comparing with laparoscopicassisted resection forselected patients with sigmoid colon or rectal cancer int j color dis “leung al cheung hy fok bk chung cc li mk tang cn prospectiverandomized trial of hybrid notes colectomy versus conventionallaparoscopic colectomy for leftsided colonic tumors world j surg “ hisada m katsumata k ishizaki t enomoto m matsudo t kasuya ktsuchida a complete laparoscopic resection of the rectum using naturalorifice specimen extraction world j gastroenterol “ ma b huang xz gao p zhao jh song yx sun jx chen xw wang znlaparoscopic resection with natural orifice specimen extraction versusconventional laparoscopy for colorectal disease a metaanalysis int j colordis “liu rj zhang cd fan yc pei jp zhang c dai dq safety and oncologicaloutcomes of laparoscopic nose surgery compared with conventionallaparoscopic surgery for colorectal diseases a metaanalysis front oncol zhou s wang x zhao c pei w zhou h liu q liang j zhou z wang xcomparison of shortterm and survival outcomes for transanal natural 0che world of surgical oncology page of orifice specimen extraction with conventional minilaparotomy afterlaparoscopic anterior resection for colorectal cancer cancer manag res“ xing j zhang c yang x wang h wang h yu e fu c comparison of shortterm outcomes of transrectal specimen extraction during laparoscopicsigmoid radical resection versus conventional laparoscopically assistedprocedure zhonghua wei chang wai ke za zhi “ wang r wei z liu q li w xiao l han hf yang s transanal versustransabdominal specimen extraction in laparoscopic rectal cancer surgery aretrospective analysis from china wideochir inne tech maloinwazyjne“ ng hi sun wq zhao xm jin l shen xx zhang zt wang j outcomes oftransanal natural orifice specimen extraction combined with laparoscopicanterior resection for sigmoid and rectal carcinoma an observational studymedicine baltimore 20189738e12347liu z efetov s guan x zhou h tulina i wang g tsarkov p wang x amulticenter study evaluating natural orifice specimen extraction surgery forrectal cancer j surg res “ hu jh li xw wang cy zhang jj ge z li bh lin xh shortterm efficacy ofnatural orifice specimen extraction surgery for low rectal cancer world jclin cases “ moher d liberati a tetzlaff j altman dg preferred reporting items forsystematic reviews and metaanalyses the prisma statement ann internmed “ w64 hawker ga mian s kendzerska t french m measures of adult pain visualanalog scale for pain vas pain numeric rating scale for pain nrs painmcgill pain questionnaire mpq shortform mcgill pain questionnaire sfmpq chronic pain grade scale cpgs short form36 bodily pain scalesf36 bps and measure of intermittent and constant osteoarthritis painicoap arthritis care res 201163suppl 11s240“ higgins jp altman dg gotzsche pc juni p moher d oxman ad savovic jschulz kf weeks l sterne ja the cochrane collaboration™s tool forassessing risk of bias in randomised trials bmj 2011343d5928 ga wells bsdo the newcastleottawa scale nos for assessing the qualityof nonrandomised studies in metaanalyses hozo sp djulbegovic b hozo i estimating the mean and variance from themedian range and the size of a sample bmc med res methodol tierney jf stewart la ghersi d burdett s sydes mr practical methods forincorporating summary timetoevent data into metaanalysis trials denost q adam jp pontallier a celerier b laurent c rullier elaparoscopic total mesorectal excision with coloanal anastomosis for rectalcancer ann surg “saurabh b chang sc ke tw huang yc kato t wang hm tzuliang cwfingerhut a natural orifice specimen extraction with single stapling colorectalanastomosis for laparoscopic anterior resection feasibility outcomes andtechnical considerations dis colon rectum “ dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg “ almazrou am suradkar k mauro cm kiran rp characterization ofreadmission by day of rehospitalization after colorectal surgery dis colonrectum “ costantino fa diana m wall j leroy j mutter d marescaux j prospectiveevaluation of peritoneal fluid contamination
Colon_Cancer
" triple negative breast cancer tnbc remains recalcitrant to most targeted therapy approaches however recent clinical studies suggest that inducing tumor damage can render tnbc responsive to immunotherapy we therefore tested a strategy for immune sensitization of murine tnbc 4t1 tumors through combination of focused ultrasound fus thermal ablation and a chemotherapy gemcitabine gem known to attenuate myeloid derived suppressor cells mdscsmethods we applied a sparse scan thermally ablative fus regimen at the tumor site in combination with systemically administered gem we used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity we also tested this combination in rag1ˆ’ˆ’ mice or t cell depleted wild type mice to determine the essentiality of adaptive immunity further we layered programmed cell death protein pd1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalresults the immune modulatory effect of fus monotherapy was insufficient to promote a robust t cell response against 4t1 consistent with the dominant mdsc driven immunosuppression evident in this model the combination of fusgem significantly constrained primary tnbc tumor outgrowth and extended overall survival of mice tumor control correlated with increased circulating antigen experienced t cells and was entirely dependent on t cell mediated immunity the ability of fusgem to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti pd1 thermally ablative fus in combination with gem restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic tnbc model this treatment strategy promises a novel option for potentiating the role of fus in immunotherapy of metastatic tnbc and is worthy of future clinical evaluationtrial registration numbers nct03237572 and nct04116320 metastatic breast cancer brca particularly the triple negative breast cancer tnbc phenotype is resistant to most chemical and molecularly targeted therapeutic approaches interestingly tnbc is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 early studies in the use of immunotherapies targeting the pd1programmed death ligand pd l1 checkpoint inhibitory axis showed some efficacy2“ in tnbc compared with other brca subtypes which are generally recalcitrant to checkpoint blockade activity in the tnbc subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in tnbc greater immunotherapy efficacy in tnbc has been recently observed with the use of antibodies targeting the pd1pd l1 checkpoint inhibitory axis in combination with nab paclitaxel5 this outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment tme found in tnbcamong the potential networks in tnbc that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets these have the capacity to impair adaptive immunity and promote tumor growth and metastasis among these cell types myeloid derived suppressor cells mdscs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both t cell activation and effector functions6 increased levels of this cell type have been demonstrated in tumor tissues of patients with primary brca while those with metastatic disease bear the highest abundance of circulating mdscs8 studies have sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate mdsc can improve antitumor immunity9“to this end the central premise put forth in this study is that focused ultrasound fus”a safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissues”can synergize with immunotherapy in a murine model of metastatic tnbc fus is capable of rapidly heating tumors to thermally ablative temperatures its extracorporeal application obviates the need for catheterization injection or implantation fus can be targeted with millimeter precision under mri or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues the bioeffects of fus hold distinct implications for tumor antigenicity immune cell activation and trafficking13 thermally active fus regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17“ colon20 kidney21 and brca23 pertaining to the challenge of myeloid cell immunosuppression in tnbc thermally ablative fus has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin il12 interferonÎ ifnÎ and tumor necrosis factorα tnfα from a variety of cancer cell lines and after in vivo treatment of tumors26 whether the ability of fus to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of brca is currently under debate with some studies showing activation of antigen presenting cells and t cell recruitment in patients with brca treated with thermally ablative fus28 while others show that additional innate stimuli are needed to support antitumor immunity23 notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells dcs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation fus regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine tnbc with extensive granulocytic mdsc involvement that is recalcitrant to anti pd1 while some activity is evident with the partial ablation approach significantly greater control was achieved by targeting mdsc inhibition in combination with thermally ablative fus this control was completely dependent on the adaptive immune responsemoreover we demonstrate that layering anti pd1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction these data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative fus once immunosuppressive myeloid cells are accounted for fus treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockademethodscell line maintenance4t1 and e0771 cell lines were maintained in rpmi l glut or dulbecco™s modified eagle™s medium dmem gl d glucose l glutamine respectively supplemented with fetal bovine serum fbs at °c and co2 thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments cells tested negative for mycoplasmaeight week old to week old female balbc or c57bl6 mice were obtained from nci charles river nci crl or the jackson laboratory female balbc rag1ˆ’ˆ’ mice were obtained from the jackson laboratory 4t1 or e0771 cells — were subcutaneously implanted into the right flank of mice mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum tumor outgrowth was monitored via digital caliper measurements tumor volume was calculated as follows volume length—width22 approximately days 4t1 or days e0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsin vivo ultrasoundguided fus partial thermal ablationmice were treated with fus either days 4t1 cohorts or days e0771 postimplantation on treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg zoetis and dexdomitor mgkg pfizer in sterilized saline mouse flanks were shaved and depilated following which ultrasound guided fus thermal ablation was performed using one of the two systems system and treatment details are provided in online supplementary materials and methods mice that did not receive fus treatment consistently underwent anesthesia and depilation of the flank additionally these mice underwent a ˜sham™ treatment consisting of exposure to the °c degassed water bath exposure for min following ˜sham™ or fus treatment all mice were moved to a heating pad and given antisedan for anesthesia reversal and recoverygemcitabine therapygemcitabine gem mgmouse in µl volume mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of fus treatment following which administration was repeated for an additional weeks administration of gem doses was based on existing literature demonstrating the use of gem for inhibition of mdscs in 4t112 the initial dose of gem was administered immediately prior to ˜sham™ or fus treatment sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cmice that did not receive gem received an intraperitoneal injection of ˜vehicle™ treatment µl of sterile saline at the time points specifiedpd1 blockade therapyfor checkpoint inhibitor therapy the rat anti mouse pd1 antibody αpd1 rmp114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse treatment was initiated on day ˜early αpd1™ or day ˜delayed αpd1™t cell depletionst cell depletion antibodies”anti cd8 clone bio x cell and anti cd4 gk15 clone bio x cell”were diluted in sterilized saline and administered intraperitoneally every to days starting at day days post fus for a total of seven doses µg of each antibody for a total µg per mouseimmunohistochemistryon day sham or fus exposed tumors were excised and fixed in neutral buffered formalin sigma fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin digital images of stained slides were acquired using the vectra automated quantitative pathology imaging system akoya biosciences whole slide screening and image capture were subsequently performed using phenochart akoya biosciencesflow cytometrymice were bled at days and via tail vein and samples were rbc lysed hybri max sigma and stained for flow cytometry analysis at days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment in order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse cd45 fitc clone f11 bd biosciences min prior to euthanasia 4t1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor dlns pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis additional details are provided in online supplementary materials and methodssamples were acquired on an attune nxt flow cytometer thermofisher scientific and data were analyzed with flowjo treestar or fcs express de novo software a representative gating strategy for granulocytic myeloid derived suppressor cell g mdsc and cd44 t cells is provided in online supplementary figure statistical analysisall statistical analyses were performed in graphpad prism graphpad software a detailed description of statistical methods for each experiment is provided in the corresponding figure legendopen accessanimal study approvalall animal work was performed under a protocol approved by the animal care and use committee at the university of virginia and conformed to the national institutes of health guidelines for the use of animals in researchresultspartial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsetsto achieve partial thermal ablation of 4t1 tumors we used an ultrasound guided fus system equipped with a single element therapeutic transducer driven at mhz figure 1a online supplementary figure a grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under b mode ultrasound guidance figure 1b“c the exceptionally small focus of this system rendered a low ablation fraction “ of total tumor volume immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1d one week following fus partial thermal ablation tumors and secondary lymphoid organs were excised for immunological characterization by flow cytometry figure 1b fus partial thermal ablation of 4t1 tumors conferred a significant increase fold in the absolute number of cd11c hi dcs within the axillary tumor draining lymph node adln of mice figure 1e while this was accompanied by a nearly threefold elevation in the absolute number of cd86 dcs within the adln figure 1f the percentage of dcs expressing cd86 did not change figure 1g increased numbers of dcs”and cd86 dcs in particular”suggest fus is promoting the maturation or trafficking of these cells in the dlns where they could encounter and activate t cells however this did not translate to tumor growth restriction data not shown we also did not observe significant differences in the absolute number of activated t cells in 4t1 tumors figure 1h or dlns data not shown following fus exposure suggesting limitations in the ability of fus activated dc to further drive an antitumor t cell responseimmune profiling by flow cytometry revealed that irrespective of fus exposure of the intratumoral cd45 immune cell population is comprised of cd11b myeloid cells figure 1i similarly approximately of the circulating immune cell population in 4t1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure notably ly6g granulocytic myeloid derived suppressor cells g mdscs significantly dominated the immune cell repertoire within 4t1 tumors relative to other myeloid including f480 macrophages ly6c cell subsets monocytic myeloid derived suppressor cells m mdscs and cd11c hi dcs figure 1j fus partial thermal ablation did not significantly alter the absolute number per sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure partial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsets a design overview of a custom ultrasound guided fus system consisting of a mhz single element transducer orthogonally co registered to an mhz linear ultrasound imaging array the tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °c ˜sham™ mice were similarly positioned but did not undergo sonications b schematic illustration of fus partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4t1 tumor bearing mice a grid of sonications was applied in a raster pattern onto the b mode ultrasound visible tumor in total two planes of sonication spaced mm apart were applied to each tumor grid points were spaced mm apart within a single plane one week following thermal ablation tumors and secondary lymphoid organs were excised for sham n6 or fus treated n5 mice and processed for flow cytometry c representative b mode ultrasound images of ectopic 4t1 tumors either before top or during bottom fus exposure sonication grid depicting targets red points is superimposed on b mode image during treatment subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed d representative he staining of either sham 4t1 tumors or those resected immediately following fus partial thermal ablation zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively e absolute number of cd11c hi dcs in the axillary tumor draining lymph node adln of 4t1 tumor bearing mice p00136 vs sham f absolute number of cd86 cd11c hi dcs in the adln p00063 vs sham g percentage of cd86 subset out of total cd11c hi dcs within adln h absolute number of intratumoral cd44 cd8 and cd44 cd4 t cells and regulatory t cells tregs per gram tumor i percentage of cd11b myeloid cells out of total cd45 immune cells across tumor spleen adln inguinal dln idln and nontumor draining axillary and inguinal lns ndlns p005 vs all other groups irrespective of fus exposure specifically tumor vs spleen p00226 tumor spleen vs all other organs p00001 j absolute number of intratumoral myeloid cells cd11c hi dcs f480 macrophages ly6c monocytic myeloid derived suppressor cells m mdscs ly6g granulocytic myeloid derived suppressor cells g mdscs per gram 4t1 tumor p00001 vs all other cell types irrespective of fus exposure all data represented as mean±sem significance assessed by unpaired t test f“h or two way analysis of variance followed by tukey multiple comparison correction i“k ˜ns™not significant dcs dendritic cells fus focused ultrasound hifu high intensityfocused ultrasoundsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets these observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4t1 tme must be addressed in order to facilitate the t cell response to fusfus partial thermal ablation in combination with gem constrains primary tnbc tumor outgrowth and extends overall survivalour observation of the overwhelming mdsc burden following 4t1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier to this end we tested a combinatorial paradigm incorporating gem a myelosuppressive chemotherapy demonstrated to inhibit mdscs transiently in the 4t1 model without consequence to t cell phenotype or function12to evaluate the efficacy of fus and gem in combination we used a preclinical ultrasound guided fus system to achieve partial thermal ablation of established 4t1 tumors 14d after tumor implantation average tumor volume of mm3 in combination with the single session of fus thermal ablation we initiated gem therapy mgmouse which was then readministered weekly for a total of three gem doses figure 2a combinatorial therapy synergized to produce significant constraint of 4t1 tumor outgrowth compared with sham and monotherapy groups figure 2b“cby termination of treatments at day 4t1 tumors exposed to fusgem combination saw nearly — and — reductions in average volume compared with sham or gem exposed tumors respectively figure 2b two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2d“e in a fraction of mice treated with fusgem we observed complete regression of 4t1 tumors although transient figure 2c tumor outgrowth eventually rebounded after termination of treatments 4t1 tumor bearing mice receiving fusgem treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and gem groups respectively hrs and for fusgem relative to sham and gem groups respectively figure 2f we additionally observed that fusgem significantly constrained outgrowth in a separate c57bl6 metastatic mammary carcinoma model e0771 online supplementary figure to further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided fus system theraclion echopulse that is already ce marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging fus thermal ablation in combination with cancer immunotherapy we observed that partial thermal ablation using the theraclion visualization and treatment unit mhz in combination open accesswith gem controlled 4t1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure these findings lend credence to the notion that the impact of combining gem with fus may be conserved across partial thermal ablation regimens moreover they demonstrate that the efficacy of fus partial thermal ablation in combination with gem can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallycombination of fus partial thermal ablation with gem increases the levels of circulating t cellslymphocytes”in particular cd8 and cd4 t cells”play an important role in responding to tumor antigen and generating a durable antitumor response based on the extended protective effect observed in mice treated with fusgem flow cytometry analysis was performed to evaluate the contribution of t cells in generating systemic and local tumor control we sampled the circulating immune cell repertoire in 4t1 tumor bearing mice via serial tail bleeds days and prior to readministration of gem and a terminal cardiac bleed at the time of spleen harvest day figure 3a combinatorial therapy significantly elevated absolute number of cd8 and cd4 t cells in the circulation at days and figure 3b“c and e“f moreover a trend threefold to fivefold increase in circulating t cells was noted in the fus group relative to sham figure 3b“c and e“f from days to systemic cd44 expressing antigen experienced t cell populations both cd8 and cd4 saw a steady significant increase after combinatorial therapy figure 3d and g a similar modest trend was noted for the fus monotherapy group relative to sham and gem figure 3d and g these changes were concordant with a decrease in circulating myeloid cd11b cells in gem recipient groups demonstrating the ability of gem to partially alleviate circulating myeloid burden figure 3hsplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4t1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression we observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6a“b consistent with this observation immunological characterization of spleens revealed a significant decrease in cd11b myeloid cells”a “ reduction in fusgem spleens relative to sham or monotherapy figure 3i while there appeared to be a trend toward more cd11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute cd11b cell numbers within the spleen data not shown the decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following fusgem treatment relative to these sham and gem groups combination therapy elevated splenic cd8 t lymphocytes by fold and sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem constrains primary triple negative breast cancer outgrowth and extends overall survival a overview of experimental design for evaluation combination of fus with serial gem treatment in murine mammary carcinoma b average 4t1 tumor outgrowth in sham n7 fus monotherapy n5 gem monotherapy n10 and combinatorial fusgem therapy groups n10 data are represented up to select time points corresponding with mouse dropout due to humane endpoints all data represented as mean±sem significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by tukey multiple comparison correction p005 vs all other groups specifically sham vs fusgem p00001 fus vs fusgem p00001 shamgem vs fusgem p00026 c 4t1 tumor outgrowth from individual mice in sham fus shamgem or fusgem groups data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint d representative images of 4t1 tumors excised at day scale bar1 cm e quantification of 2d tumor areas from images in previous panel f kaplan meier curve depicting overall survival of sham treatment n9 fus monotherapy n6 gem monotherapy n10 and combinatorial fusgem therapy n10 recipient mice significance assessed by log rank mantel cox test p005 vs all other groups specifically sham vs fus p02154 sham vs fusgem p00001 sham vs shamgem p00050 fus vs fusgem p00021 fus vs shamgem p00312 fusgem vs shamgem p00041sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen accessfigure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem increases the levels of circulating t cells a overview of experimental design to understand the impact of fus andor gem treatment on circulating immune cells b“c absolute number of circulating cd8 t cells at day b and day c d percentage of circulating cd8 t cells expressing cd44 from days to e“f absolute number of circulating cd4 t cells at day e and day f g percentage of circulating cd4 t cells expressing cd44 from days to h percentage of cd11b myeloid cells out of total cd45 immune cell in circulation from days to i“k percentage of myeloid cells i cd8 t cells j and cd4 t cells k out of total cd452 immune cells all data represented as mean±sem all data representative of sham n6“ fus monotherapy n4“ gem monotherapy n9 and combinatorial fusgem therapy n6“ groups significance assessed by analysis of variance followed by tukey multiple comparison correction for b c e f or fisher™s least significant difference lsd without multiple comparisons correction for i“k significance for d g and h assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by fisher™s lsd without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access fold figure 3j and cd4 t lymphocytes by fold and fold figure 3k these elevations were accompanied by a modest increase in percentage of foxp3 regulatory t cells tregs online supplementary figure 6e additionally increases in percentage of nk and b cells were noted twofold to fivefold online supplementary figure 6c“d these findings indicate that combinatorial therapy with fusgem promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasescombinatorial fusgem therapy does not promote robust local antitumor t cell responsesgiven the robust systemic immune signatures within the blood and spleen following fusgem we assayed 4t1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral t cell response figure 4a approximately hours prior to euthanasia mice received intravenous brefeldin a injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry immune characterization of tumors at days postimplantation”that is days subsequent to final gem administration”revealed no significant changes in absolute number of antigen experienced cd44 cd8 or cd4 t lymphocytes figure 4b“c moreover the polyfunctionality of these t cells as denoted by ifnÎ and granzyme b expression was not significantly altered figure 4d“e however intratumoral functional changes were noted in the myeloid compartment gem monotherapy modestly increased il 12p40 production by dcs fold but this was not conserved in the combinatorial therapy group figure 4f moreover while fus monotherapy generated a trend in elevated tnfα production by intratumoral g mdscs gem recipient groups saw a significant increase threefold relative to sham figure 4g these findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely interestingly intratumoral t cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionprotection conferred by combination of fus and gem is dependent on adaptive immunitysince our findings revealed no obvious advantage or function of adaptive immunity in the local tme we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with fusgem to this end we utilized an rag1ˆ’ˆ’ model that is deficient in t and b cells to address the hypothesis that mature t andor b cells play a role in the observed response wild type wt or rag1ˆ’ˆ’ mice bearing 4t1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes mice were subsequently treated with either gem monotherapy or the combination of fusgem the tumor growth inhibition offered by fusgem was entirely lost in rag1ˆ’ˆ’ mice relative to their wt counterparts with average 4t1 tumor volume in rag1ˆ’ˆ’ mice being over fivefold higher than that of wt mice on termination of treatments figure 5a of note despite a trend toward loss of protection in rag1ˆ’ˆ’ mice tumor outgrowth in response to gem monotherapy did not significantly stratify between wt and rag1ˆ’ˆ’ settings figure 5a we also observed a complete loss of fusgem mediated survival benefit over gem monotherapy in the rag1ˆ’ˆ’ setting figure 5b while these results demonstrate that an intact adaptive immune response is required for both the overall survival benefit and restriction of primary tumor offered by fusgem therapy they do not delineate the relative roles of t andor b cellsthus to address the hypothesis that the protective effect of fusgem is specifically dependent on cd4 and cd8 t cells we depleted these populations via serial coinjections of cd8 depleting and cd4 depleting antibodies in 4t1 tumor bearing wt mice on a fusgem figure 5c depletions were maintained between day and day and flow cytometry analysis of circulating immune cells at day confirmed that the target t cell populations were effectively depleted in all mice online supplementary figure consistent with the tumor escape observ
Colon_Cancer
" deregulated circular rnas circrnas are associated with the development of cancer and therapyresistance however functional research of circrnas mostly focus on potential mirna or protein binding and morepotential regulation of circrna on host gene dna in cancers are yet to be inspectedmethod we performed total rna sequencing on clinical breast cancer samples and identified the expressionpatterns of circrnas and corresponding host genes in patient blood tumor and adjacent normal tissues qpcrnorthern blot and in situ hybridization were used to validate the dysregulation of circrna circsmarca5 a series ofprocedures including rloop dotblotting dnarna immunoprecipitation and mass spectrum etc were conductedto explore the regulation of circsmarca5 on the transcription of exon of smarca5 moreoverimmunofluorescence and in vivo experiments were executed to investigate the overexpression of circsmarca5with drug sensitivitiesresults we found that circrnas has average higher expression over its host linear genes in peripheral bloodcompared to adjacent normal tissues circsmarca5 is decreased in breast cancer tissues contrary to host genesmarca5 the enforced expression of circsmarca5 induced drug sensitivity of breast cancer cell lines in vitro andin vivo furthermore we demonstrated that circsmarca5 can bind to its parent gene locus forming an rloopwhich results in transcriptional pausing at exon of smarca5 circsmarca5 expression resulted in thedownregulation of smarca5 and the production of a truncated nonfunctional protein and the overexpression ofcircsmarca5 was sufficient to improve sensitivity to cytotoxic drugs our results revealed a new regulatory mechanism for circrna on its host gene and provided evidencethat circsmarca5 may serve as a therapeutic target for drugresistant breast cancer patientskeywords breast cancer circrna dna damage repair rloop host gene correspondence kechenhusteducn chewhueducnleiweifrhotmailcom xiaolong xu jingwei zhang yihao tian and yang gao contributed equallyto this work3department of urology tongji hospital tongji medical college huazhonguniversity of science and technology wuhan china1school of basic medical sciences wuhan university wuhan hubeichinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cxu molecular cancer page of introductioncircular rnas circrnas are novel rnas that havebeen ubiquitously discovered in many species by highthroughput sequencing in recent years [ ] circrnasare generated by the backsplicing of intronic exonic orintergenic regions circrnas are resistant to rnase rand the stability of their structures makes these molecules ideal candidates for disease extensive studieshave revealed that dysregulated circrnas are involvedin the development of various cancers in gastric cancercircrnassuch as circpvt1 circlarp4 has_circ_ and circ_100269 have been shown to play arole in promoting tumor growth and their expression iscorrelated with high tnm stage and poor prognosis [“]in colon and hepatic carcinoma cirs7 promoted tumordevelopment and progression by activating the egfr andpi3kakt pathway [ ] circrnas such as circkif4ahsa_circ_0001944 hsa_circ_0001481 and circrna_0025202have been implicated in molecular typing brain metastasisand drug resistance in breast cancer [“] although greatprogress has been made the roles of circrna and relevantmolecular mechanisms remain largely unknownprevious studies have shown that circrnas exert theirfunctions in different ways as noncoding rnas circrnas regulate the expression of other genes by servingas sponges for microrna and rnabinding proteins[ ] in addition some circrnas have been shownto be translated into functional proteins [ ] inaddition circrnas have also been shown to directlyinteract with the genomic dna of the host gene inplant which results in altered parent gene expression however the interaction of circrnas and hostgene dna were less studied in human cancerssmarca5 is a member of the swisnf complex withatpdependent chromatin remodeling activity [“]in the process of dna damage repair smarca5 isinvolved in chromatin remodeling in dna damageregions providing a structural basis for the recruitmentof dna damage repair factors [ ] in tumorssmarca5 is highly expressed in hepatic carcinoma andprostate cancer and its expression levelis inverselyrelated to tumor radiosensitivity [ ]in this study we established circrnas have averagehigher expression than their host genes in peripheralblood comparing to tissues then we identified acircrna derived from smarca5 circsmarca5 issignificantly decreased in breast cancer cell lines andbreast cancer samples differentto previous worksrevealing circsmarca5 can also function as a competing endogenous rnas by binding with mirnamoleculesour mechanism explorationdisplayed circsmarca5 is involved in regulating dnarepair capacity by binding exon dna directly andfurther functionalinvestigation of this circrna may[“]contribute to the therapeutic implications for cytotoxicdrugresistant breast cancer patientsresultsidentification of expression of circrnas in breast cancerwe performed high throughput sequencing on tumort and adjacent normal tissue an and peripheralblood b of six breast cancer patients total rna withrrnadepleted library wereconstructed and thencircrnas expressed in those samples were identifiedcompared to tumor and adjacent normal tissue we observed average higher circscore expression of circrna linear host genes in blood than both tumor andadjacent normal tissue in all circrnas which wereexpressed across all six patients we observed averagecircscores from to in blood which is higherthan tumor to and adjacent normal tissue to in six patients fig 1a this result indicated average higher expression of circrnas than theirhost genes in peripheral blood comparing to tissueswhich might contribute to the exploration of diagnosticbiomarker for breast cancer we then selected sixcircrnas with high circscores average to in patients and performed further experimental validation in patients realtime pcr results establishedtwo circrnas circhipk3 and circsmarca5 weresignificantly differentially expressed between tumor andadjacent normal tissue fig 1b and figure s1a especially circsmarca5 was lower expressed in tumorsamples and less studied in previous work furthermorethe ratio of circtolinear expression of circrna linearhost genes of circsmarca5 in blood sample of healthvolunteers were significantly higher than those of breast cancer patients p fig 1c and figure s1bwe nextcirctolinear ofcircsmarca5 and clinical relevance in patients withbreast cancer and observed significant difference in thedistribution of the patients according to pathologic t p table s1 together these results indicating thepotential function and candidate biomarker attributes ofcircsmarca5 in breast cancerexamined theratio ofand functionallyinvestigatefound thatto characterizecircsmarca5 we firstly detected the expression of circsmarca5 in cell lines circsmarca5 is derived fromthe backsplicing of exon and exon of smarca5fig 1d as expected endogenous circsmarca5 butto rnase r digestionnot premrna was resistantfig 1dthe ntcircsmarca5 was further confirmed by northern blotassaycircsmarca5 was mainly present in the nucleus whereasits parent mrna was present exclusively in the cytoplasm as evidenced by qpcr northern blotting andrna in situ hybridization fig 1fh and figure s2fig 1e furthermore wethe existence ofin addition 0cxu molecular cancer page of fig see legend on next page 0cxu molecular cancer page of see figure on previous pagefig identification of circrnas in breast cancer a heatmap of circscore fbpcircfbplinear in tumor t adjacent normal tissue an and bloodsample b from six breast cancer patients b expression of six circrnas with high circscore were validated by rtqpcr assay in breast tumor andadjacent normal tissue represents p circrnas ids are according to circbase through their genomic coordinates c the ratio of circtolinear ofcircsmarca5 in blood sample of breast cancer patients and health volunteers total rna from blood sample of breast cancer patients and healthvolunteers was extracted and detected by rtqpcr the expression level was normalized with βactin as reference p was consideredstatistically significant d schematic illustration showing the genomic region of circsmarca5 derived from exons and of the smarca5 geneconvergent gray and divergent black primers were designed to amplify the linear or backsplicing products upper total rna from mcf7 cellswith or without rnase r treatment was subjected to rtpcr lower and further validated by sanger sequencing right e northern blot using ajunctionspecific probe or an exon probe showing the endogenous existence of circsmarca5 and smarca5 mrna from mcf7 cells with orwithout rnaser treatment r or r the bp marker indicates the smarca5 fulllength transcript transcribed in vitro the bp markerindicates exon and exon of smarca5 transcribed in vitro f the nucleus and cytoplasm mrna of mcf7 were extracted and smarca5 andcircsmarca5 expression levels were quantitated by rtpcr gapdh and hu6 serve as internal references of the cytoplasm and nucleus respectivelyœ indicates p g the nucleus and cytoplasm mrna of mcf7 were extracted smarca5 and circsmarca5 were examined by northernblotting and the smarca5 exon probe was applied in this experiment h subcellular localization of circsmarca5 and smarca5 in mcf7 cellsthe signals were examined by indirect rna fish and confocal microscopy the nucleus was counterstained with dapi the circsmarca5 probe waslabled by biotin while the smarca5 probe was labled by dig they were stained with red and green fluorescent secondary antibodies respectively ithe expression of circsmarca5 detected by northern blot mdamb231 bt474 mcf7 skbr3 are breast cancer cell lines mcf10a are normal breastcell line n1n2n3n4n5 are adjacent normal tissues t1t2t3t4 are breast cancer tissues œ indicates p next we examined the expression of circsmarca5 invarious breast cancer cell lines mcf7 skbr3 bt474mdamb231 and immortalized but nontransformedbreast epithelial cells mcf10a as well as in adjacentnormal tissues and breast cancer tissues northern blotresultsthe expression levels of circsmarca5 in mcf10a and normal adjacent tissuesare higher than breast cancer cell lines and cancer tissues fig 1i these results indicated that circsmarca5is downregulated in breast cancer tissues and cellsrevealed thatsequencecircsmarca5 decreases the expression of smarca5 incancer cellsto clarify the mechanisms of circsmarca5 we investigated its effects on the expression of its parent genesmarca5 the expression levels of circsmarca5 andsmarca5 were detected by the primers of junctionsequence and “ exonsrespectivelyknockdown of circsmarca5 increased both mrnaand protein levels of smarca5 while converselycircsmarca5 overexpression decreased smarca5levels fig 2ac and figure s3 consistently the proteinof smarca5 was high expressed in breasttumorsamples as compared with the corresponding controlsfigure s4 moreoverthe ratio of circtolinear ofcircsmarca5 was significantly lower in breast andrenaltumor tissue than the corresponding adjacenttissue specimens fig 2de and figure s1c besides asignificant negative correlation was also found betweencircsmarca5 and smarca5 expression in various celllines and primary cancer tissues fig 2df and figures5 which corroborates our observation that circsmarca5 decreased the expression of smarca5 incancer cellscirsmarca5 terminates the transcription of smarca5 atexon we further investigated the mechanism of circsmarca5in regulating the expression of smarca5 interestinglywe found that the overexpression of circsmarca5 indeed decreased the expression of smarca5 exons “but had minimal effects on the expression of exons “fig 3a next we designed a primer location in exon for the amplification of ² cdna ends by rapid amplification of cdna ends race pcr fig 3b left as shownin fig 3b smarca5 can give rise to multiple isoformsimportantly we found a decrease in a band of bpupon circsmarca5 overexpression while an bpband displayed the opposite phenomenon fig 3b rightsanger sequencing showed that the bp band andthe bp band are derived from fulllength and truncated mrna exons to respectively of the smarca5 gene fig 3c consistent with the race resultsnorthern blot assay further demonstrated that ectopiccircsmarca5 expression decreased smarca5 levelsand promoted truncated mrna levels fig 3d the observations gathered thus far have led us to hypothesizethat circsmarca5 prevents transcription from exon of smarca5 indeed chip analysis indicated that thebinding of pol ii to exons “ of smarca5 was higherthan that to exons “ fig 3e left and the ectopic expression of circsmarca5 decreased the binding of pol iito exons “ of smarca5 fig 3e right to furtheraddress whether circsmarca5 could terminate the transcriptional elongation of smarca5 we cloned a series ofexons of smarca5 in a luciferase plasmid reporterfig 4a upper the transient transfection of these luciferase reporters containing the “ exon sequence revealed that luciferase activity was significantly decreasedwhen circsmarca5 was overexpressed fig 4a lower 0cxu molecular cancer page of fig circsmarca5 decreases the expression of smarca5 in cells a generation of circsmarca5knockdown and circsmarca5overexpressingcells mcf7 cells were infected with lentiviruses expressing shrna against circsmarca5 shcircsmarca5 three different oligonucleotides orcircsmarca5 plcdhcircsmarca5 rtqpcr was performed to evaluate the expression of circsmarca5 gapdh was used as an internal controlb rtqpcr showing the levels of circsmarca5 and smarca5 in mcf7 cells stably expressing shnc shcircsmarca5 plcdhcir control orplcdhcircsmarca5 c western blot showing the levels of smarca5 in mcf7 cells stably expressing shnc shcircsmarca5 plcdhcircontrol plcdhcircsmarca5 plcdhcircsmarca5δ without splicinginducing sequence gapdh was used as an internal control df theratio of circtolinear of circsmarca5 in tumor tissue were significantly lower than normal tissue in breast cancer samples d and rcc samplese p a negative correlation between circsmarca5 and smarca5 expression was observed in breast cancer samples d rcc samplese and various cell lines fto further confirm the effect of circsmarca5 on thetranscriptional elongation of smarca5 we insertedexons of smarca5 between dsred and egfp as indicated fig 4b upper the egfp level was significantly decreased by circsmarca5 when exons “were present fig 4b lower we further investigatedthe role of circsmarca5 in the regulation of smarca5 at the protein level as expected circsmarca5overexpression downregulated the protein levels ofsmarca5 and upregulated truncated smarca5δsmarca5 protein levels fig 4c and figure s6which was confirmed by mass spectrometry fig 4dmoreover we found that δsmarca5 is more susceptible to proteolysis by the proteasome than smarca5 fig 4e together these results show the roleof circsmarca5 in the termination of transcriptionalelongation at exon of smarca5circsmarca5 can form rloops with its parent geneto further dissect the mechanism of smarca5 transcriptional termination mediated by circsmarca5 weinvestigated whether circsmarca5 can bind genomic 0cxu molecular cancer page of fig cirsmarca5 terminates the transcription of smarca5 at exon a rtqpcr analysis of the expression of smarca5 in mcf7 cells using aseries of paired primers œ indicates p b rapid amplification of cdna ends race pcr analysis of smarcac5 transcripts the pcrproducts were readily identified by agarose gel electrophoresis each set of samples was repeated three times c sanger sequencing of twotranscripts of smarcac5 that are regulated by circsmarca5 overexpression d northern blotting using the junctionspecific probes for exons and to show the expression levels of the transcripts of smarcac5 mrna from mcf7 cells stably expressing control vector or plcdhcircsmarca5 circoe e circsmarca5 prevents transcription from exon of smarca5 chipseq analysis showing that the binding of pol ii toexons of smarca5 was higher than that to exons chipqpcr showed that the ectopic expression of circsmarca5 decreased thebinding of pol ii to exons of smarca5smarca5 dna to form an rloop dotblotting withrloopspecific s96 antibody supported our hypothesisthat circsmarca5 can bind exons “ of smarca5genomic dna fig 5a additionally we performeddnarna immunoprecipitation drip qpcr and confirmed the interaction between circsmarca5 andexons “ pretreatment with rnase h ablated thisinteraction confirming that the interaction is rloopspecific fig b and figure s7 the interaction of circsmarca5 with the dna of smarca5 was directlyverified by fluorescence in situ hybridization fig 5cconsistent with previous findings dotblotting ofthe genome without rna digest revealed that the binding of circrna to genomic dna may be widely presentin cancer cells fig 5d we next determined the specificsequence of exons “ required for rloop formationa series of fragments from exons “ were hybridizedwith circsmarca5 for the dotblotting assay as shownin fig 5e the bp fragment of the ² end of exon plays important role in interacting with circsmarca5moreoverthe secondary structure of circsmarca5was determined by the software mfold whichrevealed the sequence ²aacaaaauugggaaagaugaaaugcuucaaau3² from the ² end of exon located in the loop region of circsmarca5 fig 6awe thus hypothesized that this sequence might play akey role in mediating the circsmarca5dna interaction to this end we synthesized the wildtype andmutant phosphorylated dna fragments ant andantmut respectively corresponding to this sequencefig 6b dotblotting demonstrated that wildtypeoligonucleotides ant can bind to circsmarca5 but 0cxu molecular cancer page of fig see legend on next page 0cxu molecular cancer page of see figure on previous pagefig cirsmarca5 blocks the transcription of smarca5 and promotes the generation of a truncated smarca5 protein δsmarca5 aschematics of luciferase reporter constructs containing the smarca5 exon sequence as indicated upper the smarca5 exon sequenceplays an important negative role in mediating the effect of circsmarca5 overexpression on luciferase activity lower b schematics offluorescence reporter constructs containing the smarca5 exon sequence as indicated upper mcf7 cells were transiently transfected withthese fluorescence reporters along with or without circsmarca5 cooverexpression after transfection for hours the reporter transcriptionactivities were measured by flow cytometry assay c circsmarca5 overexpression downregulated the protein levels of smarca5 whileupregulating truncated smarca5 δsmarca5 protein levels mcf7 cells stably overexpressing circsmarca5 or control cells were treated withdmso or mg132 western blot analysis was performed using an antibody targeting the nterminus of smarca5 to evaluate the expression ofsmarca5 and δsmarca5 gapdh was used as an internal control d the δsmarca5 protein was identified by mass spectrometry and detectedsmarca5 peptides were showed in the map the redlabeled portion is the amino acid sequence of the translated defective transcript e mcf7cells expressing flagsmarca5 and flagδsmarca5 were treated with cycloheximide chx μgml the cell lysates were subsequentlyharvested at sequential time points or h after treatment and then the cell lysates were immunoblotted with antiflag or antiactin antibodymutant oligonucleotides antmut cannot bind to circsmarca5 fig 6c as expected dripqpcr showedthat ant inhibited circsmarca5 binding to the dnaat exons “ whereas antmut had no effect on thisinteraction fig 6d furthermore the transfection ofant prevented the decrease in smarca5 proteinlevels in mcf7 cells stably expressing circsmarca5whereas antmut had no effect on smarca5 proteinlevels fig 6e importantly the mutation of the keysequence in circsmarca5 impaired the interactionwith its parent gene which was confirmed by dotblotting and dripqpcr assays fig 6fh and figures8 unlike circsmarca5 circsmarca5mut had littleeffect on smarca5 protein levels fig 6i theseresults suggested that circsmarca5 formed rloopswith its parent gene to inhibit the expression of smarca5 in cancer cellscircsmarca5 inhibits dna damage repair functionto explore the roles of circsmarca5 in cancer progression we overexpressed and depleted circsmarca5in mcf7 cells by lentiviral vectors and then examinedthe effect of circsmarca5 on cell proliferation migration and apoptosis however the results showed thatboth overexpressed and depleted circsmarca5 had noeffect on these three activities figure s9 previousstudies have indicated that smarca5 plays an important role in regulating the dna repair process and main[ “]taining theconsistent with previous reports smarca5 overexpression improved dna repair capacity and reducedthe expression of chk1 and chk2 after dna damagerepair figure s10a given that circsmarca5 canpromote the production of the truncated δsmarca5protein we tested whether the truncated protein is alsofunctional the overexpression of flagδsmarca5had a minimal effect on the expression of chk1 andchk2 after dna damage repair figure s10b suggesting that δsmarca5 isa nonfunctional proteinproduct we next assessed whether circsmarca5 canthe genomestability ofshowedlowerthan thatsignificantlyformation assaysaffect the function of dna damage repair capacitycck8 and clonerevealed thatcircsmarca5 overexpression increased sensitivity tocisplatin or bleomycin in mcf7 cells fig 7a b nextmcf7 cells were treated with the indicated concentration of cisplatin or bleomycin for h and then thedna damage was evaluated by single cell gel electrophoresis scge at and h mcf7 cells expressingcircsmarca5repaircapacity than did control cells fig 7c in paralleldna damage was examined after h of treatmentwith cisplatin or bleomycin by using an antiγh2axantibody consistent with the scge results the γh2axin mcf7 cells expressing circsmarca5 wassignalsignificantly higherin mcf7 cells asevidenced by immunostaining fig 7d consistentlycisplatin significantly enhanced the levels of dnadamage response proteins chk1 and chk2 in mcf7cellsexpressing circsmarca5 fig 7e whereasseveral key cellcycle genes were reduced specificallyupon circsmarca5 overexpression fig 7f to testwhether circsmarca5 rloop formation is necessaryfor its dna repair function we transfected ant orantmutinto circsmarca5expressing cells thescge assay and γh2ax measurement showed thatant significantly enhanced the dna repair capacitywhile antmut had no effect on this activity fig 7gand figure s11 furthermore ant significantlydecreased the degree of colocalization between circsmarca5 and its cognate dna locus figure s12in addition unlike circsmarca5 the overexpressionof circsmarca5mut had little effect on the dnarepair rate fig 7h and figure s13a b next we determined whether smarca5 could mediate the effects ofcircsmarca5 in preventing dna damage repair asshown in fig 7i the γh2ax signal was much lower incircsmarca5expressing cellscomplemented withsmarca5 than that in cells expressing circsmarca5alone as expected δsmarca5 could not rescue theinhibition of dna damage repair function induced by 0cxu molecular cancer page of fig see legend on next page 0cxu molecular cancer page of see figure on previous pagefig circsmarca5 interacts with its site of transcription a circsmarca5 interacts with the exon sequence of the smarca5 locus a seriesof exon dna fragments were hybridized with circsmarca in vitro the dnarna hybridization strength was quantified by dotblot with rloopspecific s96 antibody hybridization stringency was altered by decreasing ionic strength mm nacl b dripqpcr analysis of the exon sequence of smarca5 to detect the association of circsmarca5 in mcf7 cells rnase htreated andor dripqpcr analysis of the exon sequence as a control c circsmarca5 partially localized at its site of transcription double fish of circsmarca5 red and its parent dnaregion green the nucleus was stained by dapi d dotblot of rloops in mcf7 cell genomic dna preparations treated with dnase i rnase hor rnase r the dnarna hybrids in genome dna were analyzed by s96 antibody e mapping of the rloop formation region of circsmarca5a series of exon deletion mutants were hybridized with circsmarca5 for the dotblotting assay the dnarna hybridization intensity wasanalyzed by dotblot with an s96 antibody targeting the dnarna hybrid strand hybridization stringency was altered by decreasing ionicstrength mm naclcircsmarca5 figure s13c d moreover the overexpression of smarca5 could significantly rescue thegrowth defects of cells expressing circsmarca5 asdemonstrated by a colony formation assay fig jtogetherthese results demonstrated the roles ofcircsmarca5 in regulating the dna repair process inmcf7 cellstheevaluatecircsmarca5 overexpression enhances the cisplatinresponse in breast cancerto furthertherapeutic potential ofcircsmarca5 in breast cancer in vivo we establishedcircsmarca5 overexpression clones in mcf7 cells asshown in fig 8a the overexpression of circsmarca5efficiently enhanced the sensitivity of mcf7 xenograftsto concurrent cisplatin treatment fig 8a b the overexpression of circsmarca5 was confirmed by in situhybridization and qpcr analysis fig 8c along with decreased smarca5 protein levels and increased γh2axlevels fig 8d in addition qpcr analysis demonstratedthat circsmarca5 can be detected in the bloodsuggesting that circsmarca5 is a secretory moleculecollectively these data demonstrate that circsmarca5could serve as a potential therapeutic target to restoresensitivity to cisplatin therapy in breast cancerdiscussionprevious studies have indicated that circrnas have multiple functions in cancer development and progression[“] in this study we identified multiple expressedcircrnas in breast cancer samples and observed averagehigher abundance of circrnas over their host genes inperipheral blood than tissues which might contribute tothe exploration of diagnostic biomarkerfor breastcancer we then identified that circsmarca5 is significantly decreased in breast cancer tissues using rnaseqmore importantly we define a critical role for circsmarca5 in the regulation of dna damage repaircapacity and the drug sensitivity of breast cancer cellsin vitro and in vivo through the negative regulation ofits parent gene smarca5 these findings are of highclinical relevance because chemotherapy with cisplatinand bleomycin remains the standard of care in breastcancer [“] hence the restoration of circsmarca5levels provides an approach to overcome treatmentresistance in breast cancer patientssmarca5 also known as snf2h is a member of theswisnf chromatinremodeling complex during dnadamage repair processes smarca5 is recruited todna damage sites where it induces the ubiquitinationand phosphorylation of histone h2a which facilitateschromatin remodeling and dna damage repair [ ]in this study we show that circsmarca5 expressionresulted in the downregulation of smarca5 and theeffect of circsmarca5 overexpression on dna repaircapacity was reversed by concomitant smarca5 overexpression suggesting that the effect of circsmarca5on dna repair capacity is mediated through smarca5circrnas exert functions in various ways such as forming an rloop with dna to regulate splicing and transcriptional pausing for example circsepallata3regulates the splicing of its parent mrna through rloop formation in addition circrnas are a novelclass of cernas that sponge mirnas thus positivelyregulating gene expression [ ] additionally circrnas such as exonintron circrnas regulate geneexpression through specific rnarna interactions withu1 snrna furthermore circrnas also exertfunctions by binding to proteins and regulating theiractivities we identified one mechanism by whichcircsmarca5 regulates the drug sensitivity of breastcancer cells to cisplatin and bleomycin through thedownregulation of smarc5 circsmarca5 is recruitedto its parent gene locus leading to rloop formationtranscriptiontruncatedδsmarca5 protein upregulation and decreased smarca5 expression this regulatory mechanism has alsobeen verified in cervical cancer hela cells figure s14however our evidence demonstrates that circsmarca5has no significant effect on the proliferation migrationand apoptosis of breast cancer cells suggesting that thismolecule functions in a celltype and contextdependentmanner notablythatnonfunctionalterminationweprovideevidence 0cxu molecular cancer page of fig circsmarca5 can form an rloop with its parent gene a secondary structure prediction for circsmarca5 using the mfold program thesequence key shared by the minimum free energy structure and the thermodynamic ensemble structure is marked by red b thethiophosphorus nucleic acid analog ant complementary to key and its mutant antmut were synthesized in vitro c dotblot verifying theinteraction between circsmarca and ant or antmut d dripqpcr analysis on exon or exon sequences of smarca5 to detect theassociation of circsmarca5 in mcf7 cells overexpressing ant or antmut rnase htreated andor dripqpcr analysis of the exonsequence as a control œ indicates p e western blot analysis shows that transfection of ant into circsmarca5overexpressing cells canrestore smarca5 protein levels but antmut cannot f g dotblot analysis quantifying rloop strength between the smarca5 locus andcircsmarca5 or circsmarca5mut guanine converted to cytosine of the key sequence h dripqpcr in mcf7 cells transfected withcircsmarca5 or circsmarca5mut rnase htreated genomic dna and qpcr of exon1314 were treated as controls œ indicates p iwestern blot analysis shows that overexpression of circsmarca5 to mcf7 cells can decrease smarca5 protein levels but circsmarca5mutcannot 0cxu molecular cancer page of fig circsmarca5 decreases dna repair capacity a circsmarca5 increases sensitivity to cisplatin or bleomycin in mcf7 cells mcf7 cellsstably expressing control vector or plcdhcircsmarca5 were treated with cisplatin or bleomycin for h and cck8 was used to measure cellviability b relative colony formation units of mcf7 cells stably expressing control vector or plcdhcircsmarca5 treated with μm cisplatin or μgml bleomycin after hours the drugs were replaced by fresh medium the number of colonies was quantified c d e single cell gelelectrophoresis scge assay indicating that circsmarca5 overexpression inhibits cell recovery from dna damage mcf7 cells stably expressingcontrol vector or plcdhcircsmarca5 treated with μm cisplatin or μgml bleomycin after incubation for h the cells were recoveredwith fresh medium for or hours and then collected for scge analysis c immunofluorescence assay using an antiγh2ax antibody d andwestern blot assay with the indicated antibodies e f rtqpcr assay showing the relative levels of several key cell cycle genes in mcf7 cellsstably expressing control vector or plcdhcircsmarca5 treated with dmso or μm cisplatin for h and replaced with fresh medium for hœ indicates p g scge assay showing that the cotransfection of ant in circsmarca5overexpressing cells can restore the dna repaircapacity but the cotransfection of antmut cannot h scge assay showing that the overexpression of circsmarca5 in mcf7 cells can decreasedna repair capacity but the overexpression of circsmarca5mut cannot i smarca5 abrogates γh2ax levels induced by circsmarca5 mcf7cells were infecte
Colon_Cancer
" natural orifice specimen extraction surgery is a novel technique of minimally invasive surgery thepurpose of this study was to compare the safety of laparoscopic anterior resection with natural orifice specimenextraction noselar and abdominal incision specimen extraction aiselar for sigmoid or rectum tumorsmethods medline pubmed embase central cochrane central register of controlled trials scopus andclinicaltrials databases were systematically searched for related s up to august the primary outcomesincluded postoperative complications overall postoperative complication incisionrelated complicationanastomotic fistula and severe complication and pathologic results lymph nodes harvested proximal resectionmargin and distal resection edge the statistical analysis was performed on stata softwareresults ten studies comprising patients were used for metaanalysis compared with aiselar noselar hadmore advantages in terms of overall postoperative complication odds ratio or ci to p incisionrelated complication or ci to p distal resection edge weighted meandifference wmd cm ci to cm p recovery of gastrointestinal function wmd ˆ’ day ci ˆ’ to ˆ’ day p pain scores in postoperative day wmd ˆ’ ci ˆ’ to ˆ’ p additional analgesics usage or ci to p and hospital stay wmd ˆ’ day ci ˆ’ to ˆ’ day p while the operation time of noselar was prolonged wmd min ci to min p the anastomotic fistula severe complication lymph nodes harvested proximalresection margin intraoperative blood loss and longterm outcomes in noselar were comparable with aiselars the safety of noselar was demonstrated and it could be an alternative to conventional surgery inlaparoscopic anterior resection for sigmoid and rectal tumors however further randomized and multicenter trials are requiredkeywords natural orifice specimen extraction nose laparoscopic anterior resection rectal tumor sigmoid tumor metaanalysis correspondence guangenyang163com1department of colorectal surgery hangzhou third hospital hangzhou zhejiang people™s republic of chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0che world of surgical oncology page of introductionover the past three decades laparoscopic surgery hasevolved incessantly especially in the field of colorectalsurgery it has been widely accepted by surgeons and patients in light of the better perioperative outcomes andanalogical longterm effectiveness compared with opensurgery for colorectal cancers [“]for conventional laparoscopic colorectal operation asmall laparotomy in the abdomen is required for specimen harvested and colorectal anastomosis because ofthe miniincision it causes many undesirable outcomessuch as incision pain wound infection scar and even incision hernia and the advantages of laparoscopic surgeryare reduced [“] to minimize those drawbacks anovel surgical variant known as natural orifice specimenextraction nose surgery with the features of naturalorifice specimen extraction and totalintraperitonealanastomosis has been introduced and is becoming ahotspot [“] some studies have reported the oncology and safety outcomes between nose surgery andconventional laparoscopic surgery are comparable [“] and the nose surgery therefore is supposed tohave a progress of minimally invasive surgeryrecently some metaanalysis studies had comparednatural orifice specimen extraction nose with abdominal incision specimen extraction aise in laparoscopiccolorectum resection for the colorectal disease [ ]however colorectum resection comprises right hemicolectomyleft hemicolectomy and anterior resection procedures among those surgeries were largely different and the surgical procedures and the excision extension between sigmoid and rectum resection aresimilar in addition several studies compared laparoscopic anterior resection with natural orifice specimenextraction noselar with abdominal incision specimen extraction aiselar for sigmoid or rectum tureleased [“] hence wemors wereconducted this metaanalysis to evaluate the safety ofnoselar in sigmoid and rectal tumorsrecentlymethodsstudy design and inclusion criteriathis metaanalysis followed the preferred reportingitems for systematic reviews and metaanalysis prisma statements the inclusion and selection criteria were determined before starting a literature searchonly when studies with fulltext on sigmoid or rectaltumors that compared noselar and aiselar andreported at least one of the endpoints of focus were retrieved and analyzed the most comprehensive researchwas recruited when overlapping researches was conducted by the same team no language restriction wasapplied conference s case reportsreviewsrobotic surgery and singleportwere not consideredlaparoscopic surgeryselection criteria conformed to the framework ofpico participant intervention comparison and outcome patients diagnosed with sigmoid or rectal tumorsbenign and malignant tumors requiring surgery wereincludedinterventions consisted of noselar andaiselar noselar was compared with aiselarin all eligible studies primary endpoint outcomes werepostoperative complications overall postoperative complication abdominal incisionrelated complication anastomotic leak and severe complication claviendindoclassification ‰¥ iii and pathologic results retrievedlymph nodes proximal resection edge distal resectionedge secondary outcomes included operation timeblood loss pain score numeric rating scale score additional analgesics gastrointestinalfunction recoveryhospitalization duration 5year diseasefree survivaldfs and 5year overall survival os search strategythe following databases had been searched up to august medline pubmed central cochrane central register of controlled trials embase scopus andclinicaltrials for a more accurate search the followingkeywords andor mesh terms were used œsigmoid neoplasms œrectal neoplasms œcolorectal neoplasmsœlaparoscopyœnatural orifice specimen extractionœtransvaginal specimen extraction œtransanal specimenextraction and œtransrectal specimen extraction thespecific search strategies among databases existed differences the search strategy of pubmed was presented inadditional text reference s of the eligible studies were reviewed to find the potentially relevant studiesstudy selection and quality assessmentretrieved studies were independently assessed for relevance by reviewers changjian wang and jinmingchen by screening the title and in order to enhance sensitivity studies were removed only when bothreviewers excluded the study subsequently a fulltextassessment was performed on the initial screening included studies the risk of bias was assessed by thenewcastleottawa scale nos for observational studies and studies achieving five or more stars were eligible cochrane collaboration™s tool for assessing risk forbias was used for randomized controlled trials [ ]all discrepancies were discussed before a final decisionwas madedata collection and statistical analysisdata from the recruited studies were extracted by tworeviewers changjian wang and jinming chen andused formeananalysis outcome valuesfurther 0che world of surgical oncology page of standard deviation and median interquartile rangewere extracted from each study considering potentialheterogeneity among studies we pooled the results byusing a randomeffects model the weighted mean difference wmd and confidence intervals cis wereapplied for continuous variables and the odds ratioor and cis were used for dichotomous variablesthe continuous outcomes were adopted the inverse variance method and dichotomous outcomes were adoptedthe mantel“haenszel statistical method when a studymerely offered the outcomes with median and interquartile range an estimation based on formulas designed byhozo was performed if a study did not provide the hazard ratio hr and cis of 5year dfsorand os the methods presented by tierney wereused for data extraction from survival curves thechisquare test and isquared value were used for measuring heterogeneity and i2 p was definedas significant heterogeneity sensitivity analyses basedon nos score ‰¥ and the sample size of noselargroup ‰¥ were conducted to assess the potentialsource of heterogeneity and the robustness of the resultspublication bias was examined with a funnel plot andharbord test p was considered statistically significant the statistical analysis was performed onstata softwareresultsliterature selection and characteristicsthe initial database search identified s ofwhich were removed based on the title and assessment the rest of the literature were evaluated byfulltext assessment and studies were excludedcharacteristics of the excluded studies were presented inadditional table ten studies were finally included forfurther qualitative and quantitative synthesis [ “ ] all of these were retrospective studies the process of the search and selection wasshown in fig a total of patients were recruitedin those studies with patients in the noselargroup and patients in the aiselar group themain characteristics of studies and patients were presented in table and details were shown in additionalfig flow chart of studies included in the metaanalysis 0che world of surgical oncology page of sunasunasunasunasunasunarosunaiangavsunasunasunarorororomutcermutceridomgsimutceridomgsimutceridomgsimutceridomgsiralesaipurnsonerocsetisinemcepsnoitcartxenoitacolromutleamefeamlrednegmutcer““idomgsimutcermutcermutcer““ralesonpuralesaipuraeyaegaralesonpuralesaipuralesonpugnitnuocisetapcitrapngisedydutsinogerraeyydutsisedutsdeducnliehtfoscitsiretcarahcinamelbatevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorterydutsevitcepsorternapajlateadasihianhclateuhianhclategnianhclategnahzianhclateuohzianhclategnxiianhclateuliydutsaissurydutsevitcepsorternawatihbaruaslateevitcepsorterecnarflatetsonedevitcepsorterydutsydutsianhclategnawrnleacsawattoeltsacwensonnoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaegnarinademronoitavedidradnats±naemsadrocertondetropera 0che world of surgical oncology page of euavlpytienegoretehieuapvldmwstluserldeoopicrororalesairalesonstneitapfoonˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’foonisedutsnoitacilpmocsemoctuollafostluserldeoopehtelbatsemoctuoyramirpsemoctuoevitarepotsopllarevonoitacilpmocnoitacilpmocdetaerlnoisicnilautsifcitomotsananoitacilpmocerevesdetsevrahsedonhpmylsemoctuoilcgoohtapingramnoitceserliamxorpegdenoitceserlatsidnoitcnufsemoctuoyradnocesemitnoitarepossoldooblevitarepoartnilanitsetnortsagifoyrevocerdopinapevitarepotsopegasusciseganallanoitiddayatslatipsohsoraeyevfisfdraeyevfiecnereffidnaemdethgewdmwinoitcartxenemicepsnoisicnilianmodbahtiwnoitceserroiretnacipocsorapalralesainoitcartxenemicepsecifirolarutanhtiwnoitceserroiretnacipocsorapalralesonisnoitaverbbaliavvruseerfesaesidsfdliavvrusllarevosoyadevitarepotsopdopoitarsddoro 0che world of surgical oncology page of table the results of the pooled outcomes were summarized in table primary outcomesall included studies reported the overall postoperativecomplication the pooled data revealed that the postoperative complication in of patients whotreated with noselar and of patientswho treated with aiselar the or was ci to p with low heterogeneity i2 fig 2a among the studies eight studies reportedthe incisionrelated complication in of patients who underwent noselar and of patients who underwent aiselar the or was ci to p with no heterogeneity i2 fig 2b nine studies reported the anastomoticfistula of which ng reported zero events in bothgroups the remaining eight studies recorded anastomotic fistula in of patients sufferednoselar and of patients suffered aiselar or was ci to p withno heterogeneity i2 fig 2c a severe complication was defined based on the claviendindo classification two of the included studies recorded severecomplication claviendindo classification ‰¥ iii and thesevere complication in of patients withnoselar and of patients with aiselar or was ci to p withsignificant heterogeneity i2 fig 2da total of nine studies reported lymph node harvestthere was no significant difference in lymph node harvestbetween the two groups wmd ˆ’ ci ˆ’ to p no significant heterogeneity was observed i2 fig 3a the mean number of dissectedlymph nodes in the noselar group was and theaiselar group was the proximal resection marginwas reported in studies and the wmd in the upper resection edge was cm ci ˆ’ to cm p with no heterogeneity i2 fig 3b the distalresection margin was reported in studies and the wmdin the inferior resection edge was cm ci to cm p with no heterogeneity i2 fig3c the length of the distal resection margin in the twogroups was cm noselar group and cmaiselar groupsecondary outcomesa total of nine studies recorded operation time and intraoperative blood loss the pooled data revealed thatthe wmd of operative duration was min ci to min p heterogeneity i2 fig 4a the wmd of blood loss was ˆ’ ml ci ˆ’ to ml p heterogeneity i2 fig 4bfig forest plot comparing postoperative complications in thenoselar group and aiselar group a overall postoperativecomplication b incisionrelated complication c anastomotic fistulaand d severe complication 0che world of surgical oncology page of fig forest plot comparing pathologic outcomes in the noselar group and aiselar group a lymph nodes harvested b proximal resectionmargin and c distal resection edgesix studies provided data about the recovery of gastrointestinal function the wmd of bowel movement wasˆ’ day ci ˆ’ to ˆ’ day p heterogeneity i2 fig 5a the postoperative painwas recorded in studies and studies hisada and wang recorded the postoperative pain periodand the remaining reported the pain scores in postoperative day pod the wmd of pain score in pod 0che world of surgical oncology page of fig forest plot comparing intraoperative outcomes in the noselar group and aiselar group a operation time and b blood loss was ˆ’ ci ˆ’ to ˆ’ p heterogeneity i2 fig 5b the additional analgesicusage rate was also reported in those studies and theor of additional analgesics usage was ci to p heterogeneity i2 fig 5c theduration of hospital stay was reported in nine studiesthe wmd of hospital stay was ˆ’ day ci ˆ’ to ˆ’ day p heterogeneity i2 fig 5dfiveyear diseasefree survival dfs and 5year overallsurvival os were available in two studies the hazardratio hr in the 5year dfs was ci to p heterogeneity i2 fig 6a and thehr in the 5year os was ci to p heterogeneity i2 fig 6bsensitivity analysissensitivity analysis results based on the nos score ‰¥ and the sample size of noselar group ‰¥ were presented in additional table it showed a slight inconsistency in distal resection edge operation time andrecovery of gastrointestinal function and all the otheroutcomes showed a similar trend of results between thetwo groupspublication biasa funnel plot of overall postoperative complication wasperformed to detect publication bias it showed that allthe inclusive studies were within the confidenceinterval and no publication bias was found fig inaddition a harbord test confirmed there was no publication bias p discussionas a technique used for clinical treatment the safety ofnoselar should be efficiently proved morbidity isone of the most efficient indicators for assessing thesafety of an emerging technique postoperative complications may not only lead to failures of surgery but alsothreaten lives the overall postoperative complicationrate in noselar was lower than that in aiselar severe morbidity claviendindo ‰¥ iiiamong the two techniques was not a significant difference the operation involving digestive tract reconstruction anastomotic leakage is a potential risk once itoccurs reoperation is usually inevitable the incidence of anastomotic leakage in noselar wassimilar with that in aiselar in addition theincidence ofincisionrelated complications in noselar was significantly lower than that in theaiselar group obviously the reduction ofcomplications in noselar has largely attributed tothe decrease of incisionrelated complications althoughthe complications in noselar were reduced the riskof bacteria contamination in the peritoneal cavity shouldnot be neglected costantino had reported the peritoneal contamination in the nose group was higherthan that of the conventional group hence measures such as bowel preparation prophylactic antibioticsperitonealtransluminalwound retractor and abdominal drains are recommended to avoid the contamination of the peritonealcavity irrigationtransanallavagethe postoperative pathology results to some extentalso reflect the safety of a surgery this metaanalysisshowed lymph nodes harvested between the two groupswas comparable and it also conformed to the minimumrequirement of the guideline retrieved more than nodes in our metaanalysis the proximal marginin the noselar group was similar with the conventional group however the distal margin in the nosegroup was longer than that of the aiselar group the 0che world of surgical oncology page of potential cause of this difference was the use of transanalspecimen eversion and extraabdominal resection technique in the nose group [ ] because of thisprocedure the distal rectal resection is performed extraabdominally under direct vision moreover circumferential resection margin crm between the two groupshave no difference [ ] in addition accordingto our metaanalysis the longterm outcomes 5yeardfs and 5year os were comparable all of those indicated the nose technique was a safety procedure in thetreatment of sigmoid and rectal cancers nevertheless aconcern about tumor seeding was raised during the procedure of enterotomy and specimen extraction it is necessary to apply several measures such as the use ofprotection devices sterile specimen bags and avoidingoverpulling and compression during specimen extraction as a minimally invasive surgery noselar had moreadvantages in alleviating patient™s distress the reductionof pain scores in postoperative day pod was observed and this reduction could be attributed to thetrauma in noselar being further reduced owingto less pain the need for additional analgesics was alsoreduced in addition accelerating postoperative recoverywas also observed the recovery of gastrointestinal function and hospital duration in patients who sufferednoselar was much shorter besides some scholarsmay doubt if there have alterations in sexual urinary ordefecation function in the groups according to the included studies there were no differences in functionaloutcomes such as sexual urinary or defecation betweentwo groups [ ] even though a small part of patientsexperienced function alteration and the alternation wasreversible [ ] those all demonstrated thatnoselar was a safety surgery and to some extent ithad advantages in postoperative recoverynevertheless our study has several limitations firstlyintersphincteric resections were mixed with coloanalanastomoses with sigmoid cancer in our studies although there exist some differences we mixed the twotechniques and mainly considered there existing common procedures between sigmoid and rectum resectionin laparoscopic anterior resection and some studies didnot record the methods of outcome evaluation such asblood loss evaluation to some extent it reduces thecomparability of outcomes secondly the present metaanalysis relied solely on retrospective studies and someoriginal studies not presented how patients were selectedto be candidates for one technique or another the quality of all included studies was regarded as fair or good however this type of study cannot be comparedwith a randomized controlled trial and potential biascannot be ruled out thirdly this study only recruitedone multicenter research and some outcomes includedfig forest plot comparing postoperative recovery in the noselar group and aiselar group a recovery of gastrointestinalfunction b postoperative pain pod c additional analgesicsusage and d hospital stay 0che world of surgical oncology page of fig forest plot comparing longterm outcomes in the noselar group and aiselar group a 5year dfs and b 5year oslimited studies so further multicenter randomized controlled and more comprehensive studies containing adequate outcomes are needed fourthly the results ofsome pooled results among studies existed heterogeneity the sensitivity analysis could not be detected as thecause of heterogeneity although some results existedheterogeneity the major results were homogeneity andthe heterogeneity of outcomes such as operation duration blood loss and hospital stay can be explained byclinical heterogeneity such as the difference of patientssurgeons patient management and differences in surgical proficiency in nose technology in addition the results of the major parameters were robust all in all theresults of this analysis are convincedaccording to our metaanalysisthe advantages ofnose are reduced overall complications especiallyincisionrelated complications increased distal resectionedge enhanced recovery of gastrointestinal function reduced postoperative pain reduced additional analgesicsusage and shortened hospital stay and without an auxiliary patients operated by the nose technique achievebetter aesthetics however the operative time is prolonged although the nose technique has many advantagesthere are many requirements that should befollowed before the application of this technique in colorectal surgery firstly the nose should be operated byexperienced surgeons with conventionallaparoscopiccolorectal surgery secondly the indication of noseshould follow the indication of conventionallaparoscopic colorectal resection the depth of tumor invasionshould be within t3 and body mass index bmi shouldbe less than kgm2 for transanalnose and less than kgm2 for transvaginalnose transanal nose suitsfor male or female patients and the tumor diameter isfig funnel plot of the overall postoperative complications 0che world of surgical oncology page of recommended less than cm while transvaginal noseis only applied for female patients and the tumor diameter is limited within cm and the emergent conditionssuch as bowel obstruction perforation and massivebleeding are excluded all in all as surgeons follow appropriate indicationsthe noselar for sigmoid or rectal tumors is a safesurgery and the longterm outcomes between twooperations have no difference and the benefits of thenoselar in shortterm outcomes are noticeablethese findings promote enthusiasm in support ofnose surgery as an alternative approach forthetreatment of sigmoid and rectal tumorssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s1295702001982wadditional file additional file text the search strategy of pubmedadditional file additional table characteristics of the excludedstudiesadditional file additional table summary of the included studiesadditional file additional table the results of sensitivity analysisadditional file additional table quality assessment based on thenos for retrospective studiesabbreviationsnoselar laparoscopic anterior resection with natural orifice specimenextraction aiselar laparoscopic anterior resection with abdominal incisionspecimen extraction wmd weighted mean difference or odds rationos newcastleottawa scale dfs diseasefree survival os overall survivalcis confidence intervals hr hazard ratio pod postoperative day acknowledgementswe wish to thank the timely help given by junfeng hu in statistic analysisand mengdan zhou in language editingauthors™ contributionsguangen yang and zhong shen contributed to the conception and designof the study jun he performed the literature search and the writing of themanuscript changjian wang and jinming chen performed the data extraction haibo yao performed the data analysis qinyan yang and jianmingqiu participated in the writing of the manuscript guangen yang and haibo yao helped to revise the intellectual content the authors read and approved the final version of the manuscriptfundingthis work was funded by the zhejiang natural science foundation grantno lq19h160013 and the zhejiang medical health science and technologyproject grant no and the hangzhou health science andtechnology project grant no 2017a26availability of data and materialsall the data analyzed in this study was obtained from the included originals or related authorsethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of colorectal surgery hangzhou third hospital hangzhou zhejiang people™s republic of china 2departments ofgastroenterology pancreatic surgery zhejiang provincial people™s hospitalhangzhou zhejiang people™s republic of chinareceived february accepted july references wang cl qu g xu hw the short and longterm outcomes oflaparoscopic versus open surgery for colorectal cancer a metaanalysisint j color dis “ishibe a ota m fujii s suwa y suzuki s suwa h momiyama m watanabej watanabe k taguri m midterm followup of a randomized trial ofopen surgery versus laparoscopic surgery in elderly patients withcolorectal cancer surg endosc “allaix me giraudo g mistrangelo m arezzo a morino m laparoscopicversus open resection for colon cancer 10year outcomes of aprospective clinical trial surg endosc “lee l aboukhalil m liberman s boutros m fried gm feldman lsincidence of incisional hernia in the specimen extraction site forlaparoscopic colorectal surgery systematic review and metaanalysissurg endosc “hennessey db burke jp nidhonochu t shields c winter dc mealy k riskfactors for surgical site infection following colorectal resection a multiinstitutional study int j color dis “goto s hasegawa s hata h yamaguchi t hida k nishitai r yamanokuchis nomura a yamanaka t sakai y differences in surgical site infectionbetween laparoscopic colon and rectal surgeries subanalysis of amulticenter randomized controlled trial japanmultinational trialanization prev int j color dis “park js choi gs lim kh jang ys kim hj park sy jun sh clinical outcomeof laparoscopic right hemicolectomy with transvaginal resectionanastomosis and retrieval of specimen dis colon rectum “ooi bs quah hm fu cw eu kw laparoscopic high anterior resection withnatural orifice specimen extraction nose for early rectal cancer techcoloproctol “franklin mj kelley h kelley m brestan l portillo g torres j transvaginalextraction of the specimen after total laparoscopic right hemicolectomywith intracorporeal anastomosis surg laparosc endosc percutan tech “ wolthuis am de buck voa d'hoore a laparoscopic natural orificespecimen extractioncolectomy a systematic review world j gastroenterol“ xingmao z haitao z jianwei l huirong h junjie h zhixiang z totallylaparoscopic resection with natural orifice specimen extraction nose hasmore advantages comparing with laparoscopicassisted resection forselected patients with sigmoid colon or rectal cancer int j color dis “leung al cheung hy fok bk chung cc li mk tang cn prospectiverandomized trial of hybrid notes colectomy versus conventionallaparoscopic colectomy for leftsided colonic tumors world j surg “ hisada m katsumata k ishizaki t enomoto m matsudo t kasuya ktsuchida a complete laparoscopic resection of the rectum using naturalorifice specimen extraction world j gastroenterol “ ma b huang xz gao p zhao jh song yx sun jx chen xw wang znlaparoscopic resection with natural orifice specimen extraction versusconventional laparoscopy for colorectal disease a metaanalysis int j colordis “liu rj zhang cd fan yc pei jp zhang c dai dq safety and oncologicaloutcomes of laparoscopic nose surgery compared with conventionallaparoscopic surgery for colorectal diseases a metaanalysis front oncol zhou s wang x zhao c pei w zhou h liu q liang j zhou z wang xcomparison of shortterm and survival outcomes for transanal natural 0che world of surgical oncology page of orifice specimen extraction with conventional minilaparotomy afterlaparoscopic anterior resection for colorectal cancer cancer manag res“ xing j zhang c yang x wang h wang h yu e fu c comparison of shortterm outcomes of transrectal specimen extraction during laparoscopicsigmoid radical resection versus conventional laparoscopically assistedprocedure zhonghua wei chang wai ke za zhi “ wang r wei z liu q li w xiao l han hf yang s transanal versustransabdominal specimen extraction in laparoscopic rectal cancer surgery aretrospective analysis from china wideochir inne tech maloinwazyjne“ ng hi sun wq zhao xm jin l shen xx zhang zt wang j outcomes oftransanal natural orifice specimen extraction combined with laparoscopicanterior resection for sigmoid and rectal carcinoma an observational studymedicine baltimore 20189738e12347liu z efetov s guan x zhou h tulina i wang g tsarkov p wang x amulticenter study evaluating natural orifice specimen extraction surgery forrectal cancer j surg res “ hu jh li xw wang cy zhang jj ge z li bh lin xh shortterm efficacy ofnatural orifice specimen extraction surgery for low rectal cancer world jclin cases “ moher d liberati a tetzlaff j altman dg preferred reporting items forsystematic reviews and metaanalyses the prisma statement ann internmed “ w64 hawker ga mian s kendzerska t french m measures of adult pain visualanalog scale for pain vas pain numeric rating scale for pain nrs painmcgill pain questionnaire mpq shortform mcgill pain questionnaire sfmpq chronic pain grade scale cpgs short form36 bodily pain scalesf36 bps and measure of intermittent and constant osteoarthritis painicoap arthritis care res 201163suppl 11s240“ higgins jp altman dg gotzsche pc juni p moher d oxman ad savovic jschulz kf weeks l sterne ja the cochrane collaboration™s tool forassessing risk of bias in randomised trials bmj 2011343d5928 ga wells bsdo the newcastleottawa scale nos for assessing the qualityof nonrandomised studies in metaanalyses hozo sp djulbegovic b hozo i estimating the mean and variance from themedian range and the size of a sample bmc med res methodol tierney jf stewart la ghersi d burdett s sydes mr practical methods forincorporating summary timetoevent data into metaanalysis trials denost q adam jp pontallier a celerier b laurent c rullier elaparoscopic total mesorectal excision with coloanal anastomosis for rectalcancer ann surg “saurabh b chang sc ke tw huang yc kato t wang hm tzuliang cwfingerhut a natural orifice specimen extraction with single stapling colorectalanastomosis for laparoscopic anterior resection feasibility outcomes andtechnical considerations dis colon rectum “ dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg “ almazrou am suradkar k mauro cm kiran rp characterization ofreadmission by day of rehospitalization after colorectal surgery dis colonrectum “ costantino fa diana m wall j leroy j mutter d marescaux j prospectiveevaluation of peritoneal fluid contamination
Colon_Cancer
" introduction it is well known that immune system is highly specific to protect the body against various environmental pathogens the concept of conventional vaccination has overcome the pandemic situation of several infectious diseases outbreak area covered the recent idea of immunization through oral route edible vaccine is vital alternatives over conventional vaccines edible vaccines are composed of antigenic protein introduced into the plant cells which induce these altered plants to produce the encoded protein edible vaccine has no way of forming infection and safety is assured as it only composed of antigenic protein and is devoid of pathogenic genes edible vaccines have significant role in stimulating mucosal immunity as they come in contact with digestive tract lining they are safe costeffective easytoadminister and have reduced manufacturing cost preproofhence have a dramatic impact on health care in developing countries expert opinion the edible vaccine might be the solution for the potential hazard associated with the parenteral vaccines in this review we discuss the detailed study of pros cons mechanism of immune stimulation various outbreaks that might be controlled by edible vaccines with the possible future research and applied application of edible vaccine keywords edible vaccine outbreak transformation mucosal immunity adjuvants conventional vaccine probiotic 0c preproof introduction the immune system is a dynamic structure in our body that protects us from various pathogens immune system continuous tracking of molecules which circulate within the body to detect substances which negatively affect our health once the foreign body pathogens are identified the immune system attacks to neutralize them with the help of antibodies1 human immune cells are extremely complex and quickly adjusted to overcome every day™s challenges literarily the immune system can be described as a complex collection of cells tissues ans and process working together to prevent disease the immune system targets microanism like a virus bacteria pathogen parasitic worm toxins allergens and even sometimes own cell that show unusual characteristics as microanism rapidly evolves in a very short period the immune system has to be prepared to handle massive diversity of antigens which are commonly identified as an agent that activates immune system2 generally each molecule may be an antigen but researchers have found that carbohydrate and proteins offer the best response whereas lipids and nucleic acids are poor antigens the preproofsensitivity and the specificity of the immune system should be taken in to account for the developments of highly specific chemical and cellular tools as it is known that immune system is complex but well anized as a result this destroys or kills the invading pathogens ensuring longterm protection against pathogens and immunological memory for the body that immediately reacts to subsequent encounters with the same antigen3sometimes body immune system is unable to combat with these pathogens or microanisms may be due to the resistance causes in the microbe over the long period in which they modified their internal external structure modify receptors that present on their surface or due to the new strain that does not attack before to the human body to overcome this problem scientists develops vaccines usually contains whole microbe either killed or as a live form microbe small parts a protein molecule or toxins that mimic the diseasecausing pathogen these vaccine elements are used to activate the body's immune responses to identify kill or prevent further attacks of pathogens such as viruses bacteria fungi or other foreign bodies4 in both infectious and noninfectious disease vaccines play a crucial role to prevent it nowadays conventional approaches have successfully developed a vaccine against many infectious diseases particularly by attenuating the pathogen inactivation of microbes or by subunits of microbes still the effectiveness stability cost and safety of these current conventional vaccines remain important considerations in the production of vaccine delivery storage and availability of vaccine therefore it is important to create a new vaccine that is more costeffective and safer for the benefits of people relative to the current vaccine hence 0c preproofa new alternative approach comes into the picture nowadays that is œedible vaccine refer to the use of edible plantvegetable parts or probiotics5 live microanisms as a vaccine which is taken orally as whole or parts of plantvegetable the concept of œedible vaccine first given by œcharles arntzen in as per the new research the probiotics and plant may be genetically modified with the help of gene transfer or transformation and plant virus that contains the human pathogen proteins for the development of an edible vaccine against various lifethreatening disease such as cholera chickenpox aids malaria foot and mouth disease fmd hepatitis b and c etc some of these diseases require booster vaccination or multiple antigens to promote and retain protective immunity the plants can express more than one transgene which allows delivery of multiple antigens for repeated inoculation8 mostly the oral vaccine is associated with degradation of proteins in the stomach due to the action of the gastric enzyme and low ph and gut which restricts immune response but due to the rigid protective plant cell wall these proteins get protection from the stomach acids and enzymes as human enzymes are incapable of breaking down glycosidic bonds of plant cell wall carbohydrates9but subsequently release proteins at the gut lumen due to the action of microbial enzymes which break the plant cell wall bioencapsulation lactobacillus species preproofare considered intrinsically resistant to acid and the survival rate of probiotic lactobacilli increased in acidic condition when given with glucose10the resistant of probiotic and by encapsulation of antigensin plants stomach acid or at low ph will provide extra future prospects and tools to over come the challenges that will be faced by edible vaccines regarding its degradation in the stomach due to gastric enzyme now fda has approved plant cells for costeffective production of protein drugs pds in large scale current good manufacturing practice cgmp hydroponic growth facilities11 in freezedry lyophilized plant cells proteindrugs pds are more stable at ambient temperature for a longer period and maintain their folding and efficacy11 outbreak of diseases an outbreak occurs when several cases of a particular disease increase more than expected in a specific region over a specific period in the last three decades numbers of highly transmissible or pathogenic infectious disease like zika ebola monkeypox sars measles polio cholera diphtheria cases have increased in many parts of the globe12 this trend further continues with zoonotic spillover events expected to continue as a result of population expansion or overcrowding of cities unhygienic condition pollution and migration to uninhabited areas and effect of global warming on vector distribution due to the presence of these factors it decreases the ability of the individual to combat these diseases in recent 0c preproofyear's demand for pharmaceutical products vaccine increase over the world to treat these diseases however some counties are unable to manufacture these vaccines due to poor infrastructure or buy these vaccines at higher prices in these countries the edible vaccine may be a better option to combat diseases as edible vaccines are cheap and stable for a longer time table criteria for the selection of plant or plant part for the development of an edible vaccine edible vaccines are the form of medication taken as food by the humans and animals to boost their immunity thus the criteria needed in selecting plants for the production of edible vaccine must meet specific requirements the far most important criteria of selecting plant is its life cycle13 it shouldn™t be longer as if it takes long time for maturation then their production and maintenance cost increase the next criteria are the choice of plant for antigen incorporation which should not contain any biomolecule that will interact with it next many plants have been identified and studied for the edible vaccine which was transformed to express antigen for rotavirus gastroenteritis cholera autoimmune diseases and rabies moreover several experiments have used vegetable potato but potatoes may not be the ideal choice for edible vaccines since frying or boiling will degrade certain antigenic proteins certain foods such as bananas tomato carrots peanuts corn and tobacco have a more promising potential as edible vaccines as it can be eaten raw not only because they are commonly available but since genetic engineering is efficiently developed these kinds of vegetable plants the following plant list contains edible vaccines previously studied in animals and which are required to be approved in both human and animal use potato preproofimportant one is the stability of the antigen under high temperature as all vegetable or plant parts are not eaten raw eg potato rice pea sometime they are boiled or fried at higher temperature plant and plant parts that can be consumed raw are preferable for human immunization however banana might be the ideal source of edible vaccines because they are consumed raw even by infants and are a major crop in many developing counties8 the one disadvantage of both banana and tomato is the low protein content of the fruit which might limit the amount of antigen that can be expressed13 in contrast seeds of the plants such as maize and soybean have higher protein content and have been reported to express very high level of foreign proteins edible vaccine from the plant source 0c preproofmason et al was the first person who conducts the vaccinebased assay produced in potato solanum tuberosum to fight against ltb stain produced by e coli in mice14 in that same year in rats and human volunteers in the same year the effectiveness of the antigens produced from potatoes solanum tuberosum towards the nontoxic subunit of vibrio cholera endotoxin and the norwalk virus capsid pathogen was identified in rats and human volunteers1215thanavalas'group proposed in that potatoes could play a role in human hepatitis b as an oral reinforcement since injectable vaccine cause redness swelling or itching at the site of administration also the edible vaccine for the animal has now been developed to replace the injectable vaccine for animal protection12 tomatotobacco model species for the edible vaccine1 cherry tomatillos tomato solanum lycopersicum an appropriate candidate for vaccine development for coronavirus that causes a highly acute respiratory syndrome sars for the development of recombinant sarscoronavirus cov vaccine sspike protein sprotein and its truncated fragment are considered as the best choice912 the genome of tomato and tobacco is incorporated with nterminal fragment of sarscov protein s1 used to develop the safe effective and inexpensive vaccine when these plantbased vaccines for sars give to mice shows significant increase level of sarscovspecific iga after oral ingestion of tomato expressing s1 protein whereas tobaccoderived s1protein indicate the presence of sarscovspecific igg detect by elisa analysis and western blot16tobacco is not an edible plant but play a major role in the development of the vaccine as it is used as a proofofconcept preprooffor hbsag gene of hepatitis b lines of transgenic cherry tomatillos have been grownthe expression of genes was seen through the whole plant but it was maximum in the fresh leaves weight of ngg and with fresh fruit weight of ngg12 lettuce lactuca sativa express the bsubunit of the thermolabile protein of e coli responsible for both human and animal enteric disease show the possibility of this vegetable as an edible vaccine in the typical swine fear hog pest virus glycoprotein e2 was expressed by lettuce in poland the transgenic lettuce that shows effect against hepatitis b virus is in the development stage17 soybean in the study e coli bacteria bsubunit of thermolabile toxin expression was performed in the endoplasmic reticulum er of soybean glycine max which yielded a total antigen level 0c preproofof up to of the total soybean seed protein without any problem during drying for further processing moreover when this protein is given orally to rats leads to a rise in systemic igg and iga1819 algae chlamydomonas reinhardtii green algae has been used as a tool to achieve a large number of proteins specific to both animal and humans for therapeutic purpose1819the use of algae for the production of vaccines is optimistic as algae have a very high growth rate the entire system can be used as a raw material for the development of edible vaccines besides to facilitate the already rapidly growing algae can be cultivated in bioreactors c reinhardtii contains one chloroplast which facilitates the stability of the desired antigens in the algal line notably the effectiveness of algal vaccines after lyophilization is unchanged which might based on the expression of the capsid protein norwalk virus the transgenic plant was developed protein deposition in the unripened fruit with a lower accumulation in red fruit was reported up to of soluble protein expression in seeds allowed the storage of antigenic peptides thus creating a plant with a high yield of proteins with an average protein content of about which would preclude intensive purification procedure by pharmaceutical industries1220in addition to the expression of hemagglutinin protein h a pa against rinder pest virus rpv pea plants were used the total soluble protein level of expression in leaves was observed to be determined by western blot even more preproofpromote global delivery of edible algae vaccine12 pea studies are also required to improve the expression of a protein in transgenic peas banana in banana plants hbsag expression was reported with four distinct cassettes pher phb pefeher and pefehbs at the different level expression of hbsag were studied with pcr reverse transcription pcr and southern hybridization method expression levels reached a height of ngg in the plant and the antigen was found in banana leaves however because of the long period required the shrub needs to grow the use of this vaccine was denied21 papaya in a papaya carica papaya vaccine was developed to counter cysticercosis caused by taenia solium by expressing synthetic peptides in transgenic papaya clones vaccine was tested in rats with an immunogenic response of per cent in vaccinated animals these 0c preproofedible vaccines may offer good relief both in humans and in pigs which are the two major carriers of the disease22“ carrot in an experiment carrot along with a thaliana was utilized to develop an edible vaccine for surface hiv antigen expression and in the study it was reported that rats showed more positive effect compared to those nontreated animals25 carrot daucus carota has a positive effect in the treatment of hiv not only because carrots are nutritious and tasty but because of carrot main chemical constituent carotenoids which on consumption by rats increases monocytes lymphocytes and other immune defence thus people with a weakened immune system might benefit from the use of this potential edible antihiv vaccine26 the efficacy of this antihiv vaccine must be confirmed by a clinical trial in it has been reported that the ureb subunit of helicobacter pylori was used in transgenic carrots as a potential vaccine transgenic carrot expressing the b subunit from e coli thermolabile toxininduced iga and igg production and occurred at the intestinal and systemic level in the rat2728 rice preproofa research in found that transgenic rice oryza sativa plants expressing the b subunit of e coli induces significant number of antibodies to this subunit in the same year an immune response was found to be caused in chicken by transgenic rice that is a result of the vp2 antigenic protein from infectious bursitis in pcr and southern blot analysis confirmed the functional expression of hbsag in rice seeds2930 in addition transgenic rice was developed in in parallel to express the subunit b of the e coli thermolabile toxin used to convert plant cells using bioballistic approach pcr verified the expression india and china both are the world two biggest rice producer and have the capability to export these modified rice vaccine plant all over the globe31 selected gene encoding antigen genes from a pathogenic anism bacteria viruses or parasites that have already been identified and for which antibodies are easily accessible can be controlled in a twofold fashion in one scenario the whole structural gene is inserted inside the functional elements of the ' to ' plant transformation vector allowing the transcription and aggregation of the coding sequence in the plant32 for the second scenario the epitope is identified within the antigen dna fragment encoding may be used to create genes through fusion with coated protein gene with the plant virus eg cmv or tmv then the recombinant virus is then introduced into suitable healthy plants which produce several new plants 0c preprooftable method for the transformation of genedna into selected plants essentially there are two types of plant transformation methods but many other approaches have also been utilized to transformation vectorplasmid carrier system agrobacterium tumefacien mediated gene transfer agrobacterium a soil bacterium naturally occurring has been used to transfer a small fragment of dna into the plant genome and is called transformation5051 in this method the appropriate recombinant dna is inserted into the tregion of disarmed tiplasmid of agrobacterium tumefaciens plant pathogens which is cocultured with the plant cells andor the tissue that will be transformed the insertion of the exogenous genes and the infection of such a plant tissue into sufficiently modified agrobacterium tdna cell contributed to studies of the stable gene incorporation in the genome of the plant as well as transgenic protein production32 however this technique is sluggish and yield are lower but the application of this transformation is first limited to tobacco plant and too few other plant species which extend to most preproofvegetable species including leguminose and graminae microprojectile bombardment biolistic method this is a sophisticated method based on the microprojectile bombardment the selected dna sequence is precipitated on microparticles of metals eg tungsten gold and bombarded by a particle gun at an accelerated rate toward selected plant tissue5253 these metallic microparticles penetrate the cell walls and the exogenous dna is emitted into the cell where it is integrated into the nuclear genome through a process known for the photosynthetic role of cytoplasmic anelles called chloroplast comprising chlorophyll particle gun shoots adequately charged metallic particles with selected and processed dna which penetrate chloroplast and merge with its genome32 transformation of the chloroplast is an effective alternative to nuclear transformation5455 pds in chloroplast are more stable when plant cells lyophilized and when preserved at ambient temperature therefore the freezedrying method improves pds concentration and prevents bacterial contamination11 electroporation in this method dna is introduced into the cells to which the electrical pulses of high voltage are released which are intended to create transitory pores in the 0c preproofplasmalemma it requires the extra effort to weaken the cell wall as it serves as an efficient barrier to the entrance of dna into the cell cytoplasm32 mechanisms of action of edible vaccines for mucosal and peripheral immune figure response mucosal and peripheral immunity a critical issue for oral vaccination immune response to the vaccine is affected by the route of immunization the form of antigen and the active content of vaccine mediate specific tissue tropism there is now substantial evidence supporting the existence of at least two immune systems a peripheral immune system and a mucosal immune system20 these systems operate separately and simultaneously in most species including human protective immunity acquired during recuperation is usually referred to as systemic immunity but the fact is that it might be dominated by an incomplete form of immunity dictated by a specific pathogen as the systemic immunity might be a combination of mucosal and peripheral immunity lymphocyte traffic patterns regulated by selective expression of adhesion proteins in peripheral or mucosal lymphatic tissues affects the outcome of an immune response for example the same antigen may produce qualitatively different immune responses in lymph nodes spleen or peyer's patches the antigens in the lymph are presented over the fixed antigenpresenting cells in lymph nodes results in peripheral immunity characterized by the appearance of specific igg in the blood the antigen in the blood is presented in strategic preprooftissue interface in the spleen this also results in peripheral immunity however the microenvironment of the spleen is somewhat more complicated as it also accommodates circulating antigenpresenting cells and immunoreactive t and b cells from other tissues committed to either peripheral or mucosal immunity triggers of antigen in the lumens of enteric ans presented on payer's patches commitment to mucosal immunity characterized by the release of specific iga into the secretions2056 the mucosal surfaces are a popular site for delivering therapeutic small molecules due to the ease of administration and speed of uptake across the large surface areas efficacy of the mucosal route of immunization is largely based on the fact that mucous membranes constitute the largest immunogenic an of the body this interface is endowed with the wellanized lymphatic structure called malt mucosaassociated lymphoid tissue which constitute t and b cells innate and adaptive arms of the immune system oral vaccines stimulate the generation of immunity in gutassociated lymphoid tissue galt which includes lymph nodes payer's patches in which lymphocytes are the major component 0c preproof are b cells while are t cells and isolated lymphoid follicles in the gastrointestinal tract git a significant hurdle impacting protein delivery to the git that the antigens are rapidly degraded within the harsh environment of the digestive tract is the remarkable challenge for vaccine development it will also be important to consider the characteristics of the git in which several factors including proteolytic enzymes acidic ph bile salts and limited permeability that may hinder the induction of a protective immune response20 mucosal immunity system described above have a clear image that induction of mucosal immune response starts with the recognition of an antigen by specialized cells called mcells located in the mucosal membranes of lymphoid tissues such as peyer™s patches within the small intestine57 then the apc internalize and process the antigen as soon mcell channel antigens into the underlying tissues causing activation of cd4cells57 which leads to the pathogen this whole process is elicited in figure the antigen bioencapsulation by the plant cell which avoids degradation and conformational alterations and the enhancement of mcells uptake of the conjugation of the vaccine antigen with specific ligands will overcome the challenge faced by the conventional vaccine for mucosal immunity stimulation458 second generation edible vaccine figure the secondgeneration edible vaccines are multiplecomponent protective vaccines against multiple pathogens which can produce more than one antigenic protein by crossing two cell lines containing different antigens in the same plant the adjuvant is coexpressible with the same antigen a trivalent edible vaccine against cholera enterotoxigenic escherichia germinal center development bcells maturation and class switching to iga through cd40cd40 ligand interaction and cytokine secretion the antigenic epitopes present on apc then activate bcells with the help of tcells12 due to the expression of chemokine hormone receptors like cxcr5 or cxcr10 the bcells migrate to the mesenteric lymph nodes where they mature into plasma cells and finally migrate to the mucosal membrane and differentiate into plasma cells causing secretion of dimeric and polymeric iga12 on passing through the mucosal epithelial layer toward the lumen the iga molecules complex with membranebound secretary components to form secretary iga siga transported into the lumen the siga interacts with specific antigenic epitopes and neutralize the invading preprooffigure 0c preproofcolietec and rotavirus are the examples that could effectively initiate an immune response59 factor affecting the efficacy of an edible vaccine antigen loaded in the specific plant tissue is the principle of edible vaccines thus the efficacy and the potency of the vaccines are significantly affected by the nature of œadjuvants œadjuvants are the biomolecules lectins saponins that do not exhibit immunogenic response but potentiate the immune response when coadministered with an antigen adjuvants can improve immune and potentiate responses by acting as a depot to guide antigens to relevant sites protect them from degradation control release and activate apcs20 immunogenically inert biodegradable adjuvants like lipids proteins starch polysaccharides or polyesters act as the delivery vehicles for efficient availability at antigen presenting cells apcs there are major two methods to associate antigen and particles ie encapsulated by the particle by entrapment and linked to the surface by chemical conjugation or physical adsorption the choice of carrier particles is critical to maximizing the bioavailability of its complex with antigens encapsulated antigens are afforded protection against extracellular proteases at the time of transport to the target immune responsive sites20 preproofthe erosion of the carrier particles leads to the exposure of antigens to apcs including dendritic cells dcs macrophages and monocytes thus a fine balancing act is needed to avoid overprotection of the encapsulated antigen inhibiting the release of the therapeutic or premature release if protection is inadequate the above case leads to the reduced bioavailability that weakened the host immunity alternatively the conjugation or linked to the surface by chemical bonding or physical adsorption have efficient therapeutic action as it can facilitate delivery too specific immune responsive sites or cells the structures like liposomes virus like particles vlps virosomes and immunostimulatory complexes iscoms are well recognized by apcs because they have characteristics including size shape and surface properties that are similar to viral and bacterial pathogens that the immune system has evolved to attack20 beyond the factors regarding adjuvants and vehicles for the efficacy of an edible vaccine these are some factors that significantly affect the efficacy of edible vaccines \uf0b7 antigen selection \uf0b7 efficacy in the model system \uf0b7 choice of plant species \uf0b7 delivery and dosing frequency 0c preproof\uf0b7 release patterncontrolled and sustained \uf0b7 public perceptions and attitudes to genetic modification the advantage over the conventional vaccine figure due to the use of the oral route the administration of the edible vaccine is less complicated than the conventional methods that are given through im sc and intradermal thus it removes the needs of trained medical personnel and decreases the risk of infection as there is no need for the sterilization of premises and the manufacturing areas \uf0b7 such oral immunization will become a practical key strategy for effective disease prevention in lowincome countries in general \uf0b7 processing purification sterilization packaging or delivery does not require rigorous structure in edible vaccines minimizing longterm costs in relation to conventional vaccines32 \uf0b7 maintenance and distribution of edible vaccine are easier than the conventional vaccines as it enabling the preservation without the constant cold chain storage \uf0b7 improved storage possibilities for edible vaccines become possible as transgenic plant seeds have lesser moisture content heat stable and can quickly be dried6032 \uf0b7 a plant containing therapeutically active edible vaccine protein is free of toxins pathogens and do not have the risk of proteins to reform into the infectious anism8 \uf0b7 improved compliance is particularly related to children's who refuse to take injections preproof\uf0b7 most specifically in this case the immunity is activated on the mucosal surfaces of the gi including those which are the first line of defence on the mouth mucosal of the vaccine immunity \uf0b7 sophisticated equipment and machinery are not required in edible vaccines since they can easily be cultivated on rich soils and are costeffective relative to fermenters where the recombinant cell is cultured in a controlled manner32 limitation beyond the several advantages and the convenience over the conventional vaccines edible vaccines have certain challenges that have to be overcome for efficient and pure beneficial edible vaccination some of them are pointed below 0c preproof\uf0b7 uncertainty in calculating the appropriate oral dosage which might take multiple rounds for a patient to get the effective therapeutic action and raises the final expense of its application32 \uf0b7 the concentration of peptide or protein of edible vaccine varies from generationtogeneration planttoplant and fruitto fruit32 \uf0b7 patientrelated factor such as patient age and weight also affected the dose to be administered \uf0b7 repeated intakes of these antigenbearing plants which stimulate the immune system might over stimulate the immune system itse
Colon_Cancer
cancer is the second leading cause of death in the united states cancer screenings candetect precancerous cells and allow for earlier diagnosis and treatment our purpose was tobetter understand risk factors for cancer screenings and assess the effect of cancer screenings on changes of cardiovascular health cvh measures before and after cancer screenings among patientsmethodswe used the guideline advantage tga”american heart association ambulatory qualityclinical data registry of electronic health record data n patients to investigateassociations between timeseries cvh measures and receipt of breast cervical and coloncancer screenings long shortterm memory lstm neural networks was employed to predict receipt of cancer screenings we also compared the distributions of cvh factorsbetween patients who received cancer screenings and those who did not finally we examined and quantified changes in cvh measures among the screened and nonscreenedgroupsresultsmodel performance was evaluated by the area under the receiver operator curve aurocthe average auroc of curves was for breast for cervical and for coloncancer screening distribution comparison found that screened patients had a higher prevalence of poor cvh categories cvh submetrics were improved for patients after cancerscreeningsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation guo a drake bf khan ym langabeer iijr foraker re timeseries cardiovascularrisk factors and receipt of screening for breastcervical and colon cancer the guidelineadvantage one e0236836 101371 pone0236836editor antonio palazo´nbru universidad miguelhernandez de elche spainreceived april accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236836copyright guo this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement the data are ownedby a third party and the authors do not havepermission to share the data requesting access tothe guideline advantage tga data must be done one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsby contacting the american heart association viaemail qualityresearchheart the python coderelated to the analyses can be found in githubrepository githubcomaixiaguopythoncodefunding the authors received no specificfunding for this workcompeting interests the authors have declaredthat no competing interests existdeep learning algorithm could be used to investigate the associations between timeseriescvh measures and cancer screenings in an ambulatory population patients with moreadverse cvh profiles tend to be screened for cancers and cancer screening may alsoprompt favorable changes in cvh cancer screenings may increase patient cvh healththus potentially decreasing burden of disease and costs for the health system eg cardiovascular diseases and cancersintroductioncancer is the second leading cause of death for both men and women in the united statesus breast cancer is the second leading cause of cancer death among women colorectal cancer ranks second among men and third among women while cervical cancer ranksas a major cause of cancer death among women regular cancer screenings for breast cervical and colorectal cancers can help to diagnose cancers early and reduce cancer deaths for example in the past years the number of deaths caused by cervical cancer has significantly decreased thanks to pap tests which can find abnormal cervical cells before they turn tocancer similarly colonoscopy removes noncancerous colon polyps before becomingmalignant and regular mammography screening can identify breast cancer in an earliermore treatable stage thus breast cancer screening bcs cervical cancer screening cecsand colorectal cancer screening cocs are very important for early detection and treatmentfactors associated with cancer screenings include demographic factors health insurancecoverage education level smoking status obesity and cholesterol testing for example receiptof mammography is associated with modifiable factors such as weight smoking and other lifestyle factors [“] receipt of cecs is associated with healthier weight lower cardiovascular disease occurrence and lower cholesterol some studies suggest that smokingsedentary lifestyle high body mass index and high comorbidity are associated with a higherpercentage of cocs participation [“] traditionally data for such studies originate fromquestionnaires claims data and telephone surveys and statistical analysis methods such aslogistic regression models are applied to examine the associations between the risk factors andcancer screenings electronic health records ehr contain longitudinal healthcare information and data including diagnoses medications procedures lab tests and images andtherefore can be used to discover new patterns and relationships from the rich data deeplearning algorithms have been widely and successfully used in bioinformatics and healthcarefields as they can effectively capture features and patterns in longitudinal data in this study we investigated associations between longitudinal cvh risk factors and thereceipt of cancer screenings using ehr data by the long shortterm memory lstm model we then studied the distribution of cvh factors between patients who did and did notreceive cancer screenings to further investigate the associations finally we compared measures of cvh longitudinally within those who did and did not receive screening to betterunderstand the effect of cancer screenings on cvh measuresmaterials and methodsethics statementall the data were fully anonymized before we accessed them our study was approved by theinstitutional review board at the washington university school of medicine in st louis we one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsobtained a written acknowledgement of proprietary rights and nondisclosure and data useagreement from the american heart association the washington university_nda_dua_contractid 158065_20190426_kdata source and study populationthe guideline advantage tga is a clinical data registry established in by the americancancer society the american diabetes association and the american heart associationaha ehr data has been collected from over clinics across the us by the tga totrack and monitor disease management and outpatient preventative care we used longitudinal tga data to predict three types of cancer screenings among unique patientswe used a 6year range “ to identify female patients in the “ year oldage group who received bcs female patients in the “ year old age group who receivedcecs and patients in the “ year old age group who received cocs if patientsreceived multiple types of cancer screening we only considered the first using the same criteria for gender and age we randomly selected a comparison group of patients who did notreceive cancer screenings for bcs for cecs and for cocswe utilized the following cvh measures defined by the aha smoking status body massindex bmi blood pressure bp hemoglobin a1c a1c and cholesterol lowdensitylipoprotein ldl in our dataset we then classified them into three categories ideal intermediate or poor according to table we utilized the multum drug database as a template to convert the drug names in our dataset to their corresponding drug classes thelevenshtein distance algorithm was employed for the conversion by comparing the drugnames in our dataset to the multum drug database template the conversion was consideredsuccessful and medications were considered as treatments for bp a1c or ldl table if thedistance between the two compared strings was less than five all cvh measurements prior tothe date of cancer screening were considered in the analysis for those who received screeningand all cvh measurements in the data set were considered in the analysis for those who didnot receive screeningfor the primary analysis we selected patients who had at least one measure of cvh for bcs for cecs and for cocs in the comparison groups there were availabledata for bcs cecs and cocsstatistical analysiswe first studied the lstm prediction of cancer screening from timeseries cvh factors wedivided each cvh factor into its submetric of œideal œintermediate or œpoor according totable for example if a patient had a measure of œideal blood pressure then that featuretable measures of cvh which are available in the tga adapted from lloydjones poor healthintermediate healthideal healthhealth behaviorssmoking statusyesformer � monthsbody mass index� kgm2“ kgm2health factorsnever or quit months kgm2ldl� mgdl“ mgdl or treated to goal mgdlblood pressurefasting plasmaglucosesystolic � mm hg or diastolic � mmhgsystolic “ mm hg or diastolic “ mm hg or treated togoalsystolic mm hgdiastolic mm hg� mgdl“ mgdl or treated to goal mgdl101371 pone0236836t001 one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningswas called blood pressure ideal all features were then embedded to a 32dimensional vectorspace by word2vec for each type of cancer screenings the python genism word2vecmodel used the following hyperparameters size embedding dimension was window themaximum distance between a target word and all words around it was min_count theminimum number of words counted when training the model was sg the training algorithm was cbow the continuous bag of words time information for each measure wasadded and was calculated by the difference in days between each visit date and the most recentvisit date thus each feature was associated with its own time point in the unit of daysthe resulting embedded vectors and associated time points were fed to the lstm modeldue to the comparison group being much larger than the number of patients with cancerscreening we randomly selected patients for bcs patients for cecs and patientsfor cocs and repeated this process for times to account for the imbalance betweenscreened and unscreened groups each time the data set for each type of cancer screening wassplit into a training data set and a test data set we trained the lstm model onthe training data and tested the trained model on the test data we utilized the average of thearea under the receiver operator curve auroc to evaluate the performance of our lstmmodel for each type of cancer evaluatedour lstm model comprised an input layer one hidden layer with dimensions andan output layer the hyperparameter used in the model was as follows a sigmoid function wasused as the activation function in the output layer a binary crossentropy was used as the lossfunction adam optimizer was used to optimize the model with a minibatch size of sampleswe then investigated whether distributions of cvh“counts and percentages for each submetric“differed between patients who did and who did not receive cancer screenings by chisquared test finally we studied changes in cvh factors within screening group for the samepatients who received screening and for those who did not within screening group we compared cvh measures from before and on the day of the screening to the cvh measures collected after the screening for the patients who did not receive screening we compared cvhmeasures before and after the midpoint of the visit dates if patients only had a single visitthen they were not included in the before and after analysis analyses were conducted by usingthe libraries of scikitlearn scipy matplotlib with python version in resultsthe majority of our study population was white with a mean of age of approximately yearsfor bcs years for cecs and years for cocs table the nonwhite study populationwas predominantly africanamerican the average number of measures avg amongpatients who received screening was higher than that of patients who were not screened forexample the average number of bp measurements for patients with bcs was for cecsand for cocs compared to for bcs for cecs and for cocs for patients who werenot screenedfig displays the performance of lstm cancer screening predictions in terms of repeated aurocs for each type of screening the average auroc of curves was forbcs for cecs and for cocstable lists the numbers and proportions of patients in ideal intermediate and poor categories for each submetric for the comparison between patients who received cancer screeningand those who did not we applied a chisquared test to check if the frequencies herepercentages between screening groups were significantly different from one other within eachcvh submetric as shown in table patients who received cancer screening had a higher one 101371 pone0236836 august one 0ctable characteristics [mean sd or n ] of the study population by receipt of cancer screeningcardiovascular risk factors and receipt of cancer screeningscancer screeningsbcsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cecsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cocsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n n n n yesnon � n n avg avg avg avg avg avg � the percentages may not add up to due to rounding101371 pone0236836t002prevalence of poor a1c for bcs for cecs and for cocs compared topatients who did not receive screening for bcs for cecs and for cocsfig shows changes in cvh submetrics within the same patient screening groups fig a“2c show the changes in cvh submetrics for the patients who were screened while fig2e and 2f show the changes in cvh for patients who were not screened one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the area under the curve auc are shown for lstm cancer screening predictions from timeseries cvh factors which were repeated times withdifferent comparison patients for bcs a cecs b and cocs c101371 pone0236836g001from the first column of fig we can see that the prevalence of œpoor submetricsdecreased after cancer screenings for example all five submetrics improved after bcs fig a while bp and a1c improved after cecs fig 2b and bp a1c and smoking improvedafter cocs fig 2c notably for the prevalence of poor a1c decreased for all patients whoreceived cancer screenings in bcs in cecs and in cocs on the other handfrom the second column of fig we can see that the prevalence of œpoor a1c increased forall comparison patientsdiscussionin this study we demonstrated associations between timeseries cvh risk factor measuresand receipt of three types of cancer screenings ie breast cervical and colon cancer screenings by using a nationally representative dataset“tga data the tga data enabled us toexamine multiple sites cvh submetrics and types of cancer screenings using advanced deeplearning models an advantage of our study was that all cvh submetrics were investigatedsimultaneously for an association with different cancer screenings on a unique nationallyrepresentative dataset of patients ie the large tga data set which contains longitudinaltable comparison cvh factors between patients with cancer screening or without [n ]patients with bcs n chisquared pvalueidealintermediatepoorpatients without bcs n idealintermediatepoorpatients with cecs n chisquared pvalueidealintermediatepoorpatients without cecs n idealintermediatepoorpatients with cocs n chisquared pvalueidealintermediatepoorpatients without cocs n idealintermediatepoorbmi bmi bmi 101371 pone0236836t003bp bp bp a1c a1c a1c ldl ldl ldl smoking smoking smoking one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the plots of percentages for poor cvh factors for the same patients before and after time points of cancer screening for patients with screeningsa“c and before and after middle time points for patients without cancer screenings d“f the first row is for bcs second row is for cecs andthe third is for cocs101371 pone0236836g002cvh measurements and cancer screening patterns from more than different clinics in theusthe comparison of different cvh measure distributions between patients who receivedcancer screenings and those who did not showed that patients with poorer cvh especiallypoor a1c were more likely to receive cancer screenings specifically patients with poorera1c were more likely to receive cancer screenings some recent studies have showed that individuals with diabetes had higher incidence of certain cancers and also were more likely tobe diagnosed with advancedstage tumors [“] thus providers might be more likely torecommend patients with diabetes to uptake cancer screenings for early prevention of developing cancers which may lead to more individuals with diabetes to participate in cancerscreeningsmoreover we investigated the effects of cancer screenings on the changes of cvh measuresof the patients to better understand if the screenings had potential associations with theimprovement of cvh measures our results indicated that patients who received cancerscreenings appeared to have better control of cvh factors especially a1c than patients whodid not receive cancer screenings specifically a1c levels were improved after patientsreceived any type of screening while a1c levels worsened among patients who did not receivecancer screening a similar trend could be observed for bmi it became better after patientsreceived any type of screening while bmi became worse among patients without bcs orcocs levels of bp were improved after patients received bcs or cocs screenings and worsened among patients without bcs or cocs poor levels of ldl decreased among patientsafter receipt of bcs and among those without bcs however ldl improvements were muchgreater among patients after receipt of bcs decrease in ldl than those without bcs decrease in ldl after receipt of bcs and cocs current smoking declined comparedto the increase observed among those without the screeningsin summary our analyses showed that patients with poor cvh measures were more likelyto receive cancer screenings patients with receipt of cancer screenings appeared to haveimproved cvh measures after the screening as compared to before one possible reason forthis was that patients might receive more attention and through care from providers to detectand manage cvh by virtue of reviewing cancer screening and other risk factor data at thepopulation level better cvh is associated with a lower risk of cardiovascular disease cvdand cancers thus cancer screenings may indirectly decrease burden and cost on thehealth system eg cvd and cancers by improving patient cvh healthlimitationsthere were some limitations in our analyses we used values of auroc to evaluate associations between timeseries cvh measurements and receipt of cancer screenings higherauroc values indicated stronger associations between predictors and the binary outcomes however our observed auroc values were relatively low and thus have limited clinicalutility at this time cancer screenings are potentially affected by cvh and other factors weacknowledge that we had relatively few patients with receipt of cancer screening specificallythere were relatively few patients who received cancer screenings compared to patients whodid not within the same age and gender groups this limitation likely affected the accuracy of one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsour prediction models the prediction accuracy of our models could be improved if morepatients in our data set had received cancer screeningswe demonstrated that deep learning lstm models can effectively predict the associationsbetween timeseries cvh measures and receipt of cancer screening poor cvh especiallypoor a1c may prompt providers to recommend cancer screening for their patients andpatients who received cancer screening may also receive better care for andor have improvedselfmanagement of cvh especially a1c overall these findings suggest that unhealthierpatients are screened for cancers and that cancer screening may also prompt favorablechanges in cvhauthor contributionsconceptualization randi e forakerformal analysis aixia guosupervision randi e forakerwriting “ original draft aixia guowriting “ review editing bettina f drake yosef m khan james r langabeer ii randi eforakerwwwmedicalnewstodaycoms282929phpreferences humphrey ll helfand m chan bks woolf sh breast cancer screening a summary of the evidencefor the us preventive services task force annals of internal medicine 107326000348191375_part_120020903000012 american cancer society cancer facts figures am cancer soc 10 pmid wwwcdcgovcancercervicalstatisticsindexhtmwwwcdcgovcancerdcpcpreventionscreeninghtmwwwcdcgovcancercervicalstatisticsindexhtm1 edwards qt li ax pike mc kolonel ln ursin g henderson be ethnic differences in the use ofregular mammography the multiethnic cohort breast cancer res treat 101007s1054900800497 pmid bynum jpw braunstein jb sharkey p haddad k wu aw the influence of health status age andrace on screening mammography in elderly women arch intern med 101001archinte165182083 pmid lipscombe ll hux je booth gl reduced screening mammography among women with diabetesarch intern med 101001archinte165182090 pmid berz d sikov w colvin g weitzen s œweighing in on screening mammography breast cancer restreat 101007s105490080037y pmid cook nr rosner ba hankinson se colditz ga mammographic screening and risk factors for breastcancer am j epidemiol 101093ajekwp304 pmid fontaine kr heo m allison db body weight and cancer screening among women j womenshealth gend based med 101089152460901300233939 pmid hsia j kemper e kiefe c zapka j sofaer s pettinger m the importance of health insurance asa determinant of cancer screening evidence from the women™s health initiative prev med baltim 101006pmed20000697 pmid hueston w stiles m the papanicolaou smear as a sentinel screening test for health screening inwomen arch intern med “ pmid one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings robb ka miles a wardle j demographic and psychosocial factors associated with perceived risk forcolorectal cancer cancer epidemiology biomarkers and prevention robb ka miles a wardle j perceived risk of colorectal cancer sources of risk judgments cancer epidemiol biomarkers prev 10115810559965epi060151 pmid gimeno garca az factors influencing colorectal cancer screening participation gastroenterologyresearch and practice 1011552012483417 pmid jensen pb jensen lj brunak s mining electronic health records towards better research applications and clinical care nature reviews genetics 101038nrg3208 pmid goodfellow i bengio y courville a deep learning mit press 101533 rajkomar a oren e chen k dai am hajaj n hardt m scalable and accurate deep learning withelectronic health records npj digit med 101038s4174601800291 pmid hochreiter s s long shortterm memory neural comput “ bufalino v bauman ma shubrook jh evolution of œthe guideline advantage lessons learnedfrom the front lines of outpatient performance measurement ca cancer j clin “103322caac21233 pmid wwwscrippssparkleassetsdocumentsheart_rhythm_factspdf shickel b tighe pj bihorac a rashidi p deep ehr a survey of recent advances in deep learningtechniques for electronic health record ehr analysis ieee j biomed heal informatics 101109jbhi20172767063 pmid levenshtein vi binary codes capable of correcting deletions insertions and reversals sov phys dokl citeulikeid311174lloydjones dm hong y labarthe d mozaffarian d appel lj van horn l defining and settingnational goals for cardiovascular health promotion and disease reduction circulation “ 101161circulationaha109192703 pmid mikolov t corrado g chen k dean j word2vec proc int conf learn represent iclr kingma dp ba j adam a method for stochastic optimization corr 2015abs14126 pearson k on the criterion that a given system of deviations from the probable in the case of a correlated system of variables is such that it can be reasonably supposed to have arisen from random sampling philos mag “tsilidis kk kasimis jc lopez ds ntzani ee ioannidis jpa type diabetes and cancer umbrellareview of metaanalyses of observationlal studies bmj online 101136bmjg7607 pmid lipscombe ll fischer hd austin pc fu l jaakkimainen rl ginsburg o the associationbetween diabetes and breast cancer stage at diagnosis a populationbased study breast cancer restreat 101007s1054901533235 pmid bhatia d lega ic wu w lipscombe ll breast cervical and colorectal cancer screening in adults withdiabetes a systematic review and metaanalysis diabetologia 101007s00125 pmid wilkinson je culpepper l associations between colorectal cancer screening and glycemic control inpeople with diabetes boston massachusetts “ prev chronic dis wang yq wang cf zhu l yuan h wu lx chen zh ideal cardiovascular health and the subclinicalimpairments of cardiovascular diseases a crosssectional study in central south china bmc cardiovasc disord 101186s1287201706979 pmid foraker re abdelrasoul m kuller lh jackson rd van horn l seguin ra cardiovascularhealth and incident cardiovascular disease and cancer the women™s health initiative am j prev med “ 101016jamepre201507039 pmid yin j using the roc curve to measure association and evaluate prediction accuracy for a binary outcome biometrics biostat int j 1015406bbij20170500134 one 101371 pone0236836 august one 0c'
Colon_Cancer
" camp responsive element binding protein creb5 is a transcriptional activator in eukaryotic cells that canregulate gene expression previously we found that creb5 was involved in the occurrence and development of colorectalcancer crc using bioinformatics analysis however the biological roles and underlying regulatory mechanism of creb5 incrc remain unclearmethods realtime pcr western blotting and immunohistochemistry were used to examine creb5 expression in vitroexperiments including migration assay woundhealing assay chicken chorioallantoic membrane assay and human umbilicalvein endothelial cells tube formation assay were used to investigate the effects of creb5 on crc cell migration and tumorangiogenesis ability additionally an orthotopic implantation assay was performed in nude mice to confirm the effects ofcreb5 in vivo furthermore gene set enrichment analysis was performed to explore the potential mechanism of creb5 incrcresults we found that creb5 expression was highly upregulated in crc creb5 overexpression was positively correlatedwith advanced who stages and tnm stages and shorter survival in crc patients moreover creb5 overexpressionpromoted while creb5 silencing reduced the invasiveness and metastatic capacity of crc cells both in vitro and in vivofurthermore creb5 directly interacted with the met promoter and activated the hepatocyte growth factormet signallingpathway importantly inhibition of met reduced the invasion and metastasis of creb5overexpressing crc cells suggestingthat creb5 promotes metastasis mainly through activation of met signalling our study demonstrates a crucial role for creb5 in crc metastasis by directly upregulating met expressioncreb5 may be both a potential prognostic marker and a therapeutic target to effectively overcome metastasis in crckeywords colorectal cancer creb5 invasion metastasis met correspondence llifimmucom liaowt2002gmailcom shuyang wang junfeng qiu and lei liu contributed equally to this work1department of pathology nanfang hospital southern medical universityguangzhou guangdong chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang of experimental clinical cancer research page of colorectal cancer crc is one of the most commoncancers ranking third in morbidity and tumorrelatedmortality among both men and women worldwidemoreover approximately of crc patients have metastases at the time of diagnosis metastasis is theleading cause of death among crc patients although systemic treatment of metastatic crc hasimproved the 5year survival rate is only “ andbecause ofthis poor prognosis understanding theunderlying mechanism of the metastatic process in crcis criticalfor both early detection of metastases andmore effective treatment the gene camp responsive element binding protein creb5 which is located on chromosome 7p151encodes a transcription activator in eukaryotic cells creb5 belongs to the atfcreb family the membersof which are characterized by a high affinity for campresponse elements cres the targets of the atfcreb family include transcriptional regulators including chromatinmodifying enzymes coactivators and corepressors genes involved in mitochondrial homeostasisand protein import and genes associated with proliferation and cell cycle entry metabolism proteases transporters and chaperones as an atfcreb familymember the creb5 protein contains several importantfunctional domainsincluding nterminal zinc fingerand cterminal bzip domains the latter of which includes a dna binding region and leucine zipper creb5 is a transcription factor that specifically binds tocre as a homodimer or a heterodimer with cjun orcrebp1 and functions as a credependenttransactivator creb5 is physiologically required for embryonic development in mice recent studies revealedthe roles of creb5 in the development and progressionof cancers examination of tcga pan cancer datasetsrevealed frequent creb5 amplification and overexpression in kidney cancers sarcomas lymphomas and lungadenocarcinomas as well as glioblastomas and gliomas experimentalinvestigations showed that creb5was upregulated in ovarian cancers hepatocellularcarcinoma and prostate cancer high creb5expression correlated with a poor prognosis in epithelialovarian cancer and hepatocellular carcinoma creb5 overexpression increased the proliferation of hepatocellular carcinoma moreover overexpression oramplification of creb5 promoted proliferation and mediated resistance to ar inhibition in metastatic castrationresistant prostate cancers in silico analysis showedthat the creb5 regulatory network was involved in crcmetastasis in addition qrtpcr assay revealed thatcreb5 mrna was upregulated in crc tissues and cells in vitro assays revealed that overexpression of creb5resulted in enhanced proliferation and migration andapoptosis inhibition in crc cells these findings suggest that creb5 may play an essential role in the progression of crc howeverthe specific function andmolecular mechanism of creb5 in crc metastasis remain largely unclearactivation of the hgfmet signalling pathway hasbeen reported to lead to the occurrence and metastasisof a variety of tumorsincluding crc breast cancerovarian cancer lung cancer and liver cancer as atyrosine kinase receptor met can be activated bydimerization multimerization and phosphorylation afterbinding to its ligand hepatocyte growth factor hgf activation of hgfmet can initiate downstreamsignalling pathways that drive malignant progression inmany types of tumors met is considered an essentialfactor for early invasion and metastasis of crc and canbe regarded as an important prognostic indicator in the present study we found that creb5 promotes crc invasion and metastasis by increasing metexpression to activate hgfmet signalling these results uncover a new molecular mechanism for cancermetastasis and suggest that creb5 may be a promisingtarget for crc treatmentmaterials and methodspatients and specimensa total of pathological specimens were obtainedfrom colon cancer patients between and atthe department of pathology nanfang hospital southern medical university the medical records of these patients provided information on sex age and thefollowing essential factors tumor pathological characteristics pathologic stage t stage dukes stage lymph nodemetastasis and distant metastasis ten pairs of fresh biopsies collected from crc patients and matched noncancerous mucosaltissue were obtained from theoperating room of nanfang hospital the fresh biopsieswere stored in liquid nitrogen before usein addition a tissue microarray no co802 containing colon cancer tissue specimens and adjacentnoncancerous tissue specimens was purchased fromailina biotechnology company approval for the use ofclinical materials for research purposes was obtainedfrom the southern medical university institutionalboard guangzhou china all samples were collectedand analysed with the prior written informed consent ofthe patientscell culturesthe human crc celllines sw480 ht29 hct15hct116 sw620 ls174t sw837 lovo dld1 andrko were purchased from the american type culturecollection sw620 ht29 and lovo cells were culturedin dmem medium gibco supplemented with 0cwang of experimental clinical cancer research page of foetal bovine serum fbs gibco sw480 hct116hct15 ls174t sw837 and dld1 cells were culturedin rpmi medium gibco with fbs gibcoplasmidscreb5 constructs were generated by cloning pcramplifiedfulllength human creb5 cdna into psinef2puro thefollowing primers were used for cloning including enzymesforward primer ² cgcgaattcatgatttatgaggaatccaa3² ecor i reverse primer ²ccggctagcttaaagaatcggattcaggt3² nhe i for deletion ofcreb5 short hairpin rna shrna sequences creb5²aacaagtcatccagcataa3² creb5shrna1shrna2 ²ggaatatctcgatgcataa3² were separately cloned into a gv248 vector psinef2puro and gv248were purchased from addgene incthe intensity of staining was graded according to thefollowing criteria no staining weak staining lightyellow moderate staining yellow brown and strong staining brown the staining index was calculated as the staining intensity score × the proportion ofpositive tumor cells using this method of assessmentwe evaluated the expression of creb5 in benign breastepithelium and malignant lesions by determining thestaining index with scores of and cutoff values for creb5 were selected on the basis of ameasure of heterogeneity with the logrank statisticaltest with respectto overall survival optimal cutoffvalues were identified a staining index ‰¥ was used todefine tumors with high creb5 expression and anindex ‰¤ was used to define tumors with low creb5expressionrna isolation reverse transcription rt and realtimepcrtotal rna samples from cultured cells and primarytumor tissues were extracted using trizol reagent invitrogen usa according to the manufacturer™s instruction realtime rtpcr was performed at least threetimes in triplicate using sybr green mix toyobojapan and the abi prism sequence detectionsystem applied biosystems usa the data were normalized to the geometric mean of the housekeeping genegapdh and calculated using the 2δδct method primerexpress was used to design the realtime pcr primersand primer sequences for amplification are shown insupplementary table s2luciferase reporter assaygenomic dna extracted from sw480 cells was used as atemplate to amplify met promoter fragments met promoter fragments were obtained by pcr and constructedinto pgl3basic plasmid using a fast ligation kit followingmanufacturer™s instructions sangon b620511“ thesequences of the pcr primers are listed in supplementarytable s4 cells at confluence in a 24well plate weretransfected using lipofectamine fortyeight hoursafter transfection luciferase activity was measured usingthe dualluciferase reporter assay system promega corpmadison wi usa and normalized to renilla luciferasegene expression all the experiments were performed intriplicatewestern blotting analysiswe carried out western blotting as previously described using anticreb5 abcam ab168928 antimetcell signaling technology antipmetcellsignaling technology antiakt cell signalingtechnology antipaktcell signaling technology antierkcell signaling technology antiperk cell signaling technology and antisnail cell signaling technology antiantiαtubulin antibodybodies mouse monoclonalsigma was used as the internal controlimmunohistochemistryimmunohistochemistry ihc staining was performed aspreviously described using creb5 antibody abcamusa ab168928 the degree of ihc staining wasreviewed and scored independently by two observersbased on both the proportion of positively stained tumorcells and the intensity of staining [ ] the proportion of tumor cells was scored as follows no positivetumor cells positive tumor cells “positive tumor cells and positive tumor cellschromatin immunoprecipitation chip assaychip assays were carried out as previously described precleared lysates were incubated with creb5 antibody abcam ab168928 or normal mouse immunoglobulin g cst as a negative control overnightat °c with rotation the human met promoter wasamplified by pcr all chip assays were performed threetimes and the sequences of the pcr primers are listedin supplementary table s3migration assaya boyden chamber with an 8μmpore filter membranewas used for the in vitro migration and invasion assaybriefly cells × in culture medium containing fbs were seeded in the upper chamber and culturemedium with fbs was added in the lower chamberas a chemoattractant the upper side of the filter wasfirst coated with matrigel bd biosciences sanjose ca usa after incubation for h cells on theupper side of the filter were removed with cotton swabscells that migrated to the lower surface of the filter werefixed in paraformaldehyde and stained with giemsa 0cwang of experimental clinical cancer research page of the migratory cells were counted random ×fields per well three independent experiments wereperformed and the data are presented as the mean ±semwoundhealing assaycells were seeded in 6well plates and incubated underpermissive conditions until confluence after serumstarvation for h wounds were created in the confluent cells using a pipette tip wound healing within thescrape line was then observed and photographed at indicated time points each experiment was repeated at leastthree timeschicken chorioallantoic membrane assaya chicken chorioallantoic membrane cam assay wasperformed on the sixth day of development of fertilizedchicken eggs as previously described human umbilical vein endothelial cell tube formationassayfirst μl of matrigel was pipetted into each well of24well plates and polymerized for min at °c human umbilical vein endothelial cells huvecs × in μl of conditional medium were added to eachwell and incubated at °c in co2 for h imageswere obtained under a brightfield microscope and thecapillary tubes were quantified by the counting lengthorthotopic mouse metastatic model to 6weekold balbc athymic nude mice nunuwere obtained from the animal center of southernmedical university guangzhou china all mice werehoused in a sterile environment cells × permouse were orthotopically inoculated into the caecumof anaesthetized nude mice the mice were sacrificedwithin weeks after surgery individual ans were excised and metastases were observed by histological analysis tissues were then fixed with formaldehyde andparaffinembedded and then 5mm sections were cutand stained with haematoxylin and eosin he thenumbers of gross metastatic foci were determined usinga dissection microscope all the mice used in this studywere maintained under specific pathogenfree conditions and all animal experiments were conducted in accordance with standard procedures and approved by theinstitutional use committee for animal carestatistical analysisall statistical analyses were carried out using spss version pearson correlation analysis was used for expression correlation analysis the survival curves of crcpatients in low and highcreb5 expression groups wereanalysed by the kaplanmeier method and the logranktest was used to compare differences p was considered significantresultscreb5 is upregulated in crc and associated with a poorprognosisthe expression of creb5 was analysed in differenttypes of malignant tumors in the public database oncomine wwwconominecom revealing that creb5was upregulated in crc tissues in of crc datasetssupplementary fig s1a additionally a gene set enrichment analysis gsea plot showed significant enrichment of tumorrelated genes and crcrelated genesets in the highcreb5 expression group supplementary fig s1b realtime pcr and western blotting analyses showed that creb5 was differentially expressed incrc cell lines supplementary fig s1cd in additioncreb5 was significantly upregulated in ten crc tissuescompared with adjacent normal intestinal epithelial tissues fig 1a and b ihc showed that creb5 proteinwas weakly expressed in normal tissue but markedly increased in adenocarcinoma cells and was mainly localized in the nucleifig 1c kaplanmeier survivalanalysis showed that crc patients with higher creb5protein expression levels had a poorer prognosis fig1d in addition creb5 expression levels were significantly correlated with the t classification lymph nodemetastasis and distant metastasis p supplementary table s1 creb5 expression was substantiallyhigher in tumors from patients with distant metastasismoreover high creb5 expression was also positivelycorrelated with who stages p supplementarytable s1 these data suggested that creb5 expressionis significantly correlated with advanced stages of crccreb5 activates met signallinggseas of creb5regulated gene signatures revealed thathigher creb5 expression was positively correlated withenrichment of an met signalling pathway signaturegse17538 fig 2a to validate this result we establishedstable creb5overexpressing and creb5knockdowncrc cell lines fig 2b upregulation of creb5 significantly increased while knockdown of creb5 decreasedthe expression of total met at both translational andtranscriptional levels fig 2c and d in addition creb5overexpression markedly increased but creb5 downregulation significantly attenuated the expression of phosphorylated met perk pakt and snail fig 2c moreovermet expression was increased in a dosedependent manner at both translational and transcriptional levels by transiently transfecting sw480 cells with a creb5 expressionvector fig 2e 0cwang of experimental clinical cancer research page of fig creb5 is upregulated in crc and associated with a poor prognosis a and b realtime pcr and western blotting analysis of creb5 expression inpaired human colon cancer tissues and adjacent noncancerous tissues p quantity one software was used to quantify the protein expressionlevels c ihc representative images of creb5 expression in normal intestinal epithelium and crc tissues scale bar μm d the paraffin samples of crc patients were divided into a lowcreb5 expression group n and a highcreb5 expression group n based on ihc results thekaplanmeier method was used to analyse survival curves and the logrank test was used to compare differences p fig creb5 activates the met signalling pathway a gsea of gse17538 in met signalling pathways es p b stable overexpressionand interference cell lines were detected by western blotting and realtime pcr c the expression of met and downstream signalling moleculesin creb5knockdown or creb5overexpressing cells was observed by western blotting d creb5 had an effect on met by realtime pcr in theindicated cells e after transient transfection of different amounts of the creb5overexpression plasmid in sw480 cells the protein and mrnalevels of met were detected by western blotting and realtime pcr respectively p 0cwang of experimental clinical cancer research page of creb5 associates with the met promotergiven that creb5 is a transcriptional factor and upregulatesmet at the transcriptional level we performed a luciferasereporter assay to investigate whether creb5 can increasemet promoter activity a 27kb fragment of the fulllengthmet promoter region was subcloned into a luciferase vector met promoter activity wasincreased by cotransfection with a creb5 expression vector in sw480 cellsbut decreased in hct116 cells expressing creb5 shrna ina dosedependent manner compared with empty vectorsfig 3a to determine the effective regions of the met promoter that creb5 may affect we transfected met promotertruncations fig 3b into hct116 cells expressing eithercreb5 shrna or a scramble control sequence as shown infig 3c luciferase activity was increased in cells carrying thefulllength met promoter and truncations ˆ’ and ˆ’ bp upstream of the transcription start site but not incells carrying truncations ˆ’ to ˆ’ bp or ˆ’ to ˆ’ bp knockdown of creb5 expression bycotransfection of creb5 shrna significantly decreased metpromoter activity fig 3c furthermore we performed chromatin immunoprecipitation chip assays and identified thatthe ˆ’ to ˆ’ 223bp region of the met promoter whichcontains an ap1 motif was a creb5 protein binding sitefig 3d these data identify met as a direct transcriptionaltarget of creb5downregulation of creb5 represses invasiveness andreduces the metastatic potential of crc cellsnext we investigated the role of creb5 in invasivenessand metastasis in crc cells silencing of creb5 significantly compromised the migratory and invasive abilitiesof crc cells fig 4a and b supplementary fig s2a andb the tubule formation and chicken cam assays revealed that knockdown of creb5 strongly inhibited theformation of tubules by huvecs and inhibited angiogenesis in cams fig 4c and d supplementary figs2c orthotopic inoculation assay showed that knockdown of creb5 inhibited liver metastases fig 4eknockdown of creb5 also obviously extended the overall survivaltime of nude mice inoculated with thecrc cell lines fig 4f these results indicate that silencing creb5 inhibits the invasiveness and metastasis of crc cellsinhibition of met attenuates the invasion and metastasisof crc cells by creb5 in vivo and in vitroto determine the functional relationship between creb5and met in the invasion and metastasis of crc weknocked down met using two met shrnas or suppressed met activation using the met inhibitor crizotinib emd silencing or inhibition of met insignificantlysw480creb5or ht29creb5cellsfig creb5 regulates met and binds directly to the met promoter a the met promoter sequence was cloned into pgl3basic vector containing theluciferase reporter gene and then transfected into crc cells with the indicated treatments b schematic diagram of the full and truncated met promoterc the fulllength met promoter or its truncations were cloned into pgl3basic vector containing the luciferase reporter gene and then transfected intohct116 cells with creb5 shrna or empty vector d chip analysis of creb5 binding to the met promoter in sw480 cells p 0cwang of experimental clinical cancer research page of fig downregulation of creb5 inhibits the invasion and metastasis of crc cells in vivo and in vitro a and b woundhealing assay and transwellmigration assay were performed to evaluate the invasive and migratory abilities of crc cells with different treatments in vitro c huvec tubeformation after stimulation with the indicated conditioned medium d representative images of the cam assay histograms show the formationof secondary and tertiary blood vessels after stimulation with the indicated conditional medium scale bar mm e orthotopic transplantationwith the indicated hct116 cells in nude mice n in each group was performed and representative gross images of the livers and intestinesare shown the arrows indicated the tumors liver sections were stained with haematoxylin and eosin he scale bar μm f the kaplanmeiermethod was used to analyse survival curves in the specified treatment groups and the logrank test was used to compare differences p decreased the expression of phosphorylated met perkand pakt as well as snail supplementary fig s3a inaddition the invasive and migratory abilities of sw480creb5 or ht29creb5 cells were partially diminished byinhibition of met fig 5a and b supplementary fig s3band c moreover upregulation of creb5 expression enhanced the capacity of crc cells to induce tube formationand angiogenesis in cams in contrast angiogenesis ability was partially diminished by met inhibition fig 5cand d supplementary fig s3d orthotopic inoculationassay showed that creb5 significantly promoted liver metastases and decreased the overall survival of mice fig 5eand f conversely inhibition of met significantly attenuated the formation of metastatic foci by sw480creb5cells and extended the survival time of mice inoculatedwith sw480creb5 cells fig 5e and fcreb5 expression positively correlates with metexpression in crcto assess a potential link between creb5 and met expression in human crc we analysed tcga crc dataand identified a strong positive correlation between highexpression levels of creb5 and met p r fig 6a in addition analyses of fresh crc tissuesshowed that creb5 expression was positively correlatedwith met expression at both mrna p r and protein levels p r fig 6b and c 0cwang of experimental clinical cancer research page of fig overexpression of creb5 promotes the invasion and metastasis of crc cells but inhibition of met weakens these effects a and b theinvasive and migratory abilities of crc cells in vitro with different treatments were evaluated by woundhealing assay and transwell migrationassay c huvec tube formation after stimulation with the indicated conditional medium d representative images of the cam assay histogramsshow the formation of secondary and tertiary blood vessels after stimulation with the indicated conditional medium scale bar mm eorthotopic transplantation with the indicated sw480 cells in nude mice n in each group was conducted and representative gross images ofthe livers and intestines are shown the arrows indicate the tumors liver sections were stained by he scale bar μm f the kaplanmeiermethod was used to analyse the survival curves of different treatment groups and the logrank test was used to compare differences p p p furthermore ihc revealed that creb5 expression waspositively correlated with met fig 6d p discussionmetastasis of crc is a multistep process requiring the accumulation of genetic andor epigenetic alterations andabnormal expression of genes involved in signal transduction pathwaysincluding oncogenic mutation of krasand activation of the erkmapk pathway wntβcatenin signalling and tgfβ signalling ap1 dnabinding sequences act as crucial response elements fortranscriptional activation by the raserk pathway creb5 is a credependent transactivator downstream ofthe raserk signalling pathway since it interacts with cjun which is one of the ap1 subunits to form a homodimer or a heterodimer along with foxd1 and atf3creb5 formed a transcription factor regulatory networkthat negatively regulates mapk signalling which is suppressed by fzd3 in melanoma previous studies haverevealed that creb5 mrna was upregulated in crc asdemonstrated by bioinformatics analysis or qrtpcrexamination in human cancer tissues [ ] in thecurrent study we demonstrated that creb5 was highlyupregulated in crc at both the mrna and protein levelsoverexpression of creb5 was significantly associatedwith aggressive cellular characteristics of crc eg an 0cwang of experimental clinical cancer research page of fig creb5 positively correlated with met expression in crc a correlation analysis of creb5 and met in tcga crc data r p band c correlation analysis of creb5 and met at the mrna r p and protein levels r p in fresh crc tissues dthe expression of creb5 and met protein in specimens including colon cancer tissue specimens and adjacent normal tissue specimens wasdetected by ihc representative ihc images left and correlation analysis right of creb5 and met expression scale bar μm high expressionof creb5 n high expression of met n low expression of creb5 n low expression of met n p advanced who stage and an advanced tnm stage andpoorer patient outcomes suggesting that creb5 might bean oncogene and a prognostic marker of crc progressionconsistent with our results upregulation of creb5 hasbeen reported to be responsible for poorer outcomes inpatients with epithelial ovarian cancer and hepatocellular carcinoma in prostate cancers creb5 overexpression occursthrough both copy number gain and increased gene expression however examination of tcga colorectalcancer datasets via cbioportal revealed rare amplificationof creb5 suggesting that overexpression of creb5 iscontrolled attranscriptional and posttranscriptionallevels creb5 has been shown to be a downstream target of lncrna snhg5mir1323p and circularrna circvapamir125a in addition fzd3 inhibits transcriptional networks controlled by creb5 however the alternative mechanisms involved inupregulation ofthe creb5 gene and activation ofcreb5mediated signalling require further investigationthe effects of creb5 overexpression on promotingcell proliferation and migration have been demonstratedusing in vitro assays in human hepatocellular carcinomacells and crc cell lines [ ] in the current studywe showed that creb5 overexpression promoted whilecreb5 silencing reduced the invasiveness and metastaticcapacity of crc cells both in vitro and in vivo mechanistically creb5 directly interacted with the met promoter and activated the hgfmet signalling pathwayimportantlyinhibition of met reduced the invasion 0cwang of experimental clinical cancer research page of and metastasis of creb5overexpressing crc cells suggesting that creb5 promotes metastasis mainly throughactivation of met signalling our data provide solid evidence that upregulation of creb5 plays an essential rolein crc metastasis recently overexpression or amplification of creb5 was reported to promote proliferationand mediate resistance to ar inhibition in metastaticcastrationresistant prostate cancers these datasuggest that creb5 may function as a multitaskingregulator in cancer progression and clinical outcomessignallingtransportimmunegrowth factorscreb family members can be phosphorylated via various intracellular signal transduction pathways such asprotein kinase a pka calmodulindependent proteinkinasecamk mitogenactivated protein kinasesmapks and other kinases upon phosphorylationcreb recruits crebbinding protein cbp and binds tothe cres of the promoters of its target genes target genes containing consensus sites for creb bindinginclude those related to metabolism transcription neuropeptidesneurotransmitters cell cyclecellsurvivalregulationdna repairreproductiondevelopmentandstructure specifically knockdown of creb1creb5increased tumor necrosis factor alpha tnfα levelsenhanced the expression of phosphonfκb p65 andnfκb p65 and induced immunosuppression in monocytes in prostate cancer creb5 could improve resistance to enzalutamide with the help of foxa1 andselectively enhance the interaction of ar with targetgenes critical for survival however little is knownabout the downstream targets of creb5 involved in theprogression of crc our results showed that creb5 directly interacted with the met promoter and activatedthe hgfmet signalling pathway which in turn increased the expression of downstream erk and pi3ksignalling cascades meanwhile the expression of snailan essential emt transcription factor was also upregulated via the creb5hgfmet axistranscriptional factor that interacts with the met promoter at the ap1 motif and activates met expressionin our data suggest that creb5 has an essential role in crc metastasis by regulating the protooncogene met interfering with creb5 may representan alternative therapeutic target to prevent or reducemetastasis in crcsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s13046020016730additional file table s1 the relationship between creb5 expressionand clinicopathological parameters table s2 primer sequences used forrealtime pcr ² to ² table s3 sequence of primers for chip assaytable s4 sequence of primers for luciferase reporter assayadditional file figure s1 bioinformatics analysis of creb5expression the expression of creb5 in crc and other malignant tumorswas analyzed by oncomine database the inclusion criteria were that thedifference of creb5 expression between tumor tissue and normal tissuewas more than times and the arrangement of gene position was lessthan with p the outliers in the red and blue boxes representthe number of data sets with high and low expression of creb5respectively the right table of a represents the copa score of creb5 in crc data sets b the two crc chips gse17538 n andgse35896 n from the public database of geo was analyzed bygsea the plot showed significant enrichment of tumorrelated gene setkegg_pathway_in_cancer and colorectal cancerrelated gene setkegg_colorectal_cancer in the creb5 high expression group cand d realtime pcr and western blotting analysis of creb5 endogenous expression in indicated crc cellsadditional file figure s2 representative images of woundhealingassay a transwell migration assay b and huvec tube formation assayc with i
Colon_Cancer
curative therapeutic options for a number of immunological disorders remain to be established and approaches for identifying drug candidates are relatively limited furthermorephenotypic screening methods using induced pluripotent stem cell ipscderived immunecells or hematopoietic cells need improvement in the present study using immortalizedmonocytic cell lines derived from ipscs we developed a highthroughput screening htssystem to detect compounds that inhibit il1 secretion and nlrp3 inflammasome activation from activated macrophages the ipscs were generated from a patient with neonatalonset multisystem inflammatory disease nomid as a model of a constitutively activatednlrp3 inflammasome hts of compounds including fdaapproved drugs and compounds with known bioactivity identified compounds as predominantly il1 inhibitorssince these compounds are known inflammasome inhibitors or derivatives of these resultsprove the validity of our hts system which can be a versatile platform for identifying drugcandidates for immunological disorders associated with monocytic lineage cellsintroductionone of the main cell types affected by immunological disorders are white blood cells such aslymphocytes monocytes and neutrophils although our understanding of the cellular pathophysiology of immunological disorders has greatly benefited from in vitro studies usingpatientderived primary hematopoietic cells or in vivo animal models these approaches haveseveral limitations patientderived hematopoietic cells cannot be obtained in sufficient quantities and their in vitro phenotypes can be affected by the in vivo conditions of the patient suchas the cytokine milieu or the administration of therapeutic agents while animal models haveprovided important insights into these disorders species differences in the immunologicala1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation seki r ohta a niwa a sugimine y naitoh nakahata t induced pluripotentstem cellderived monocytic cell lines from anomid patient serve as a screening platform formodulating nlrp3 inflammasome activity one e0237030 101371 pone0237030editor xiaoping bao purdue university unitedstatesreceived march accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237030copyright seki this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfunding this work was supported by the grant forthe core center for ips cell research of researchcenter network for realization of regenerativemedicine from the japan agency for medicalresearch and development amed [tn and mks] the program for intractable diseases researchutilizing diseasespecific ips cells of amed and [tn and mks]practical research project for allergic diseasesand immunology research on allergic diseasesand immunology of amed [mks]practical research project for rareintractablediseases of amed [mks] and thedevelopment causes discrepancies in the function and phenotype of the immune cells [“]overall highthroughput screening hts of therapeutic compounds using patientderivedcells or animal models is usually not feasiblethe establishment of disease or patientspecific induced pluripotent stem cells ipscs has led to the development of a new field of disease modeling owing to their pluripotencyand capacity for selfrenewal ipscs can function as an unlimited source of patientderivedsomatic cells and progenitor cells ipscs have also been used as a source of phenotypebasedhts [“] however several roadblocks remain for ipscbased hts as follows obtaining alarge number of differentiated progenies from pscs is cost and laborintensive and theyield and function of the differentiated cells often vary among clones and experimentalbatchesjapan society for the promotion of science jspswe have established ipscs from patients with autoinflammatory syndromes including neokakenhi grant number [mks]nippon shinyaku co ltd provided support inthe form of salaries to the authors [rs and hn]the specific roles of rs and hn are articulated inthe ˜author contributions™ section the funders hadno role in study design data collection andanalysis decision to publish or preparation of themanuscriptcompeting interests rs and hn are employeesof nippon shinyaku co ltd this employmentdoes not alter our adherence to one policieson sharing data and materialsnatalonset multisystem inflammatory disease nomid also known as chronic infantile neurological cutaneous and articular [cinca] syndrome nakajonishimura syndrome and blau syndrome for disease modeling in these studies ipscderived myeloid cells wereimmortalized by transducing lentiviral vectors that encoded myc bmi1 and mdm2 anddisease phenotypes were recapitulated in vitro thus ipscderived immortalized myeloid celllines ipsmls can be expanded from one experimental batch with reduced financial and laborcosts they also can be stored and differentiated into terminally differentiated progenies therefore ipsmls can overcome the roadblocks associated with ipscbased hts nomid is the most severe form of cryopyrinassociated periodic syndrome caps anautoinflammatory disease caused by heterozygous mutations in the nlrp3 gene [ ]nacht lrr and pyd domainscontaining protein nlrp3 is expressed mainly in myelomonocytic lineage cells and acts as a sensor of cellular stress induced by various pathogens andsterile stimuli in normal macrophages a œpriming stimulus such as lipopolysaccharidelps induces the expression of nlrp3 and prointerleukin il1 an inactive form of theproinflammatory cytokine il1 then an œactivating stimulus such as adenosine triphosphate atp enhances the assembly of a protein complex known as nlrp3 inflammasomethis inflammasome contains the protease caspase1 which processes proil1 to the matureform on the other hand lps stimulation of monocytic cells obtained from untreated capspatients induces robust il1 secretion without secondary activating signals due to autoactivation of nlrp3 inflammasome indeed antiil1 therapy for caps patients has beenproven effective [ ] however antiil1 therapy has several weak points the efficacy ofantiil1 therapy is often inadequate for patients with severe phenotypes il1 maturation is mediated not only by nlrp3 inflammasome but also other inflammasomes and proteases [ ] thus a complete blockade of il1 may result in excessive immunosuppressionmoreover the cost and lifelong injection of biologics worsen the patients™ quality of life therefore other therapeutic approaches such as the direct inhibition of nlrp3 inflammasomeactivity are under considerationnlrp3 inflammasome is an attractive drug target because nlrp3 inflammasome activation is associated with the pathogenesis of various chronic inflammatory conditions recently several selective nlrp3 inhibitors entered the clinical phase their chemicalstructures are undisclosed but presumed to be sulfonylureas or their derivatives mcc950 asulfonylureabased potent selective inhibitor of nlrp3 inflammasome activation wasalso recently identified as a direct nlrp3 inhibitor by binding to the walker b atphydrolysismotif of the nacht domain [ ] given that capsrelated mutations frequently occursin the nacht domain it is not surprising that most capsrelated nlrp3 mutantsescape efficient mcc950 inhibition therefore novel nlrp3 inflammasome inhibitorseffective for diseases including caps are sought one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningin the present study we developed an hts system to identify compounds that regulate theactivity of nlrp3 inflammasome using ipscs generated from a nomid patient we established ipsmls which we used as a prototype of nlrp3activated immune cells by immortalizing ipscderived monocytic progenitor cells we conducted hts of approximately compounds and validated their inhibitory effect on the secretion of il1resultsfunctional monocytic cells on feeder and serumfree monolayer culturewe previously reported that macrophages derived from ipscs carrying diseaseassociatedmutations in the nlrp3 gene showed excessive secretion of il1 without secondary signals these macrophages were considered functional based on compatible appearances withprimary macrophages under an electron microscope the secretion of proinflammatory cytokines and the phagocytosis of listeria monocytogenes macrophages in that study wereobtained via a differentiation protocol using op9 feeder layers recently we have updated ourmonocytemacrophage differentiation system to a defined condition without feeder cells andserums which can also produce functional macrophages we therefore examined whethermonocytes obtained with the feeder and serumfree monolayer differentiation system exhibited a similar in vitro phenotypefor this a nlrp3mutated ipsc clone derived from a nomid patient with a somaticnlrp3y570c mutation was differentiated into monocyticlineage cells since thenlrp3 mutation was a somatic mosaicism the nlrp3nonmutated wildtype clonederived from the same donor was used as an isogenic control ipsc line the differentiatedcells showed a mononuclear and slightly foamy appearance consistent with the appearance ofin vitro differentiated monocytes fig 1a and expressed the hematopoietic cell marker cd45the myeloid cell marker cd11b and the monocytic cell marker cd14 fig 1bwe collected floating monocytic cells from the culture supernatant and evaluated theircytokine production we used lps as a priming signal and silica as a secondary inflammasomeactivating signal as expected the wildtype ipscderived monocytes required twosequential signals to secrete il1 fig 1c while the monocytes carrying the nlrp3 mutationshowed excessive il1 secretion without a secondary signal fig 1d both clones showedrobust secretion of il6 confirming the appropriate downstream signal transduction of lpsstimulation fig 1c and 1d overall the monocytic cells differentiated under feeder andserumfree condition showed the abnormal il1 secretion associated with the in vitro phenotype of nomidestablishment of ipsmls from monocytic progenitor cellswe next established an ipsml line from the ipscderived monocytic cells for this we recovered monocytic lineage cells and introduced lentiviral vectors encoding myc bmi1 andmdm2 after “ days culture in the presence of macrophage colonystimulating factormcsf the cells started to continuously proliferate in an exponential manner fig 2a theipsmls showed a small mononuclear appearance consistent with the appearance of monocyticlineage cells fig 2b the integration of transgenes into the genome of ipsmls was confirmed fig 2c the ipsmls expressed cd45 cd11b and cd14 similar to the originalipscderived monocytic cells fig 2d karyotype analysis of the ipsml demonstrated thatmost of the cells maintain normal karyotype except for some normal variations s1 tablebecause monocytes are heterogeneous progenitor cells that can differentiate into macrophagesand dendritic cells [ ] they might be composed of a heterogeneous population potentiallymaking their cytokine production insufficient we therefore compared the cytokine profiles of one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig functional monocytic cells on feeder and serumfree monolayer culture a a representative maygiemsa staining image of monocytic cells derived fromipscs b a flow cytometric analysis of monocytic cells the staining profiles of specific antibodies thick lines and isotypematched controls gray areas are showncd il1 and il6 secretion from monocytic cells with wildtype c and mutant d nlrp3 cells were stimulated with lps ngml for hours and silica μgml for an additional hour bars show the mean ± sem of four experiments � p paired ttest101371 pone0237030g001ipsmls and terminallydifferentiated ipsmlderived macrophages the ipsmls carrying anlrp3 mutation nomidmls were differentiated into macrophages mlmps fig 2esince mlmps produced an increased amount of il1 and il6 compared with nomidmlsfig 2f and 2g and showed less variability than ipscderived monocytes fig 2h wedecided to use mlmps to construct our htsestablishment of an hts platform using mlmpsto establish our hts system we differentiated nomidmls into mlmps and disseminatedthem onto 384well plates we added the compounds at the same time as when we disseminated the cells into the culture wells then the mlmps were cultured with appropriate concentrations of compounds for hours in order for the cells to firmly adhere to the plate surfaceand they were stimulated with lps for another hours fig 3a after incubation the supernatant was collected and the relative concentrations of cytokines were measured using ahomogeneous time resolved fluorescence htrf assay in this protocol a caspase1 inhibitor one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig establishment of ipsmls from monocytic cells a cumulative growth curve of ipsmls the number of cells was counted and cumulated in every passageculture the day of lentiviral transduction was regarded as day b a representative maygiemsa staining image of ipsmls c a reverse transcriptionpcr analysisof ipsmls transgenespecific primer pairs were used my myc b bmi1 md mdm2 as a positive control lentiviral expression vectors were used d a flowcytometric analysis of ipsmls the staining profiles of specific antibodies thick lines and isotypematched controls gray areas are shown e a phase contrastimage of mlmps f g il1 f and il6 g secretion from nomidmls and mlmps cells were stimulated with lps ngml for hours the bars show themean ± sem of four experiments �� p paired ttest h the coefficient of variation cv of lpsstimulated ipscderived monocytes fig 1c and 1dnomidmls and mlmps f g101371 pone0237030g002acyvadcho specifically inhibited the secretion of il1 in a dosedependent mannerfig 3b in contrast the proteasome inhibitor mg132 which inhibits the nfκb pathway byblocking the degradation of iκb decreased the production of both il1 and il6 in a one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig establishment of an hts platform using mlmps a schematic illustration of the compound screening b dosedependent inhibition of il1 closedcircles and il6 open circles secretion by acyvadcho mg132 and mcc950 bars show the mean ± sem of five experiments c the quality of the assaysystem was evaluated signaltobackground ratios and z™factors of plates for il1 closed and il6 open are shown d criteria and number of hitcompounds [il1] and [il6] indicate the percent inhibition of il1 and il6 secretion respectively e doseresponse curves of the hit compounds bars showthe mean ± sd of four wells101371 pone0237030g003dosedependent manner fig 3b these data proved the specificity of the hts system wealso evaluated the inhibitory effect of mcc950 mcc950 inhibited the secretion of il1 withan approximate ic50 at μm without inhibiting the secretion of il6 fig 3b overall theipsmlbased cytokine assay in combination with the htrf system successfully detected the one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeninginhibitory effect of previously reported compounds proving the application of our system tohtshts using annotated compoundsto identify compounds that inhibit the activation of mutant nlrp3 we next performed htsusing compounds including fdaapproved drugs and compounds with known bioactivity to evaluate the quality of the assay system the z™factor and signalbackground sb ratio of each plate were calculated using the data from dmso controls with or without stimuli s1 fig regarding il1 the z™factor and sb ratio were consistently high for each384well plate z™factor ± and sb ratio ± mean±se fig 3c since a z™factor over indicates an excellent assay these data proved the appropriateness of our hts system the z™factor and sb ratio for il6 were slightly lower than those for il1 z™factor± and sb ratio ±in the first screening we assessed the inhibitory effects of all compounds influencing cytokine secretion at μm s2 table and s2 fig we first selected the compounds thatinduced a more than reduction in il1 secretion fig 3d of these the compoundsthat also nonspecifically inhibited or enhanced il6 secretion were excluded we adopted amore relaxed criterion for il6 modulators because the z™factors of the il6 assay were lowerthan those of the il1 assay we also excluded duplicate compounds and known steroidswith broad antiinflammatory effects for the remaining compounds we evaluated thereproducibility of the inhibitory effects at different dose twelve compounds showed ic50values at less than μm and predominant il1 inhibition at least at two different dosesfinally seven compounds consistently showed predominant il1 inhibition fig 3e allseven compounds have previously been reported to inhibit nlrp3 inflammasome [“]indicating their inhibitory effects on both wildtype and mutant cellsvalidation of hit compounds with primary human cellswe also wondered if the effect of these compounds could be recapitulated in primary humancells we used primary peripheral blood mononuculear cells pbmcs obtained from healthyvolunteers for the validation to obtain prompt il1 secretion from nonmutant cells westimulated the pbmcs with lps and atp compounds modulating the activity of hsp9017aag and herbimycin a showed a selective inhibitory effect on il1 secretion comparable to the effects seen in mlmps fig on the other hand the remaining compoundsaside from the pancaspase inhibitor zvadfmk inhibited both il1 and il6 fig indicating that they had offtarget effects or their il1 inhibitory effects were nonspecificdiscussionwe established a diseaseassociated phenotypic hts system for nomid using ipsmls ourhts platform showed a stable z™factor and sb ratio for the amount of il1 to avoid falsepositives such as nonspecific inhibitors and cytotoxic compounds we also measured theamount of il6 an inflammasomeindependent cytokine we identified compounds as predominant inhibitors of il1 secretion from among candidates the compounds identified here have already been reported as inhibitors of nlrp3 inflammasome proving thevalidity of our strategy screening a larger number of compounds may help identify novel andmore specific compoundswe could efficiently immortalize monocytic progenitor cells derived from human ipscsmoreover these ipsmls could differentiate into terminally differentiated macrophagesmlmps as previously reported [ ] mlmps showed enhanced cytokine secretion and one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig hit validation with pbmcs dosedependent inhibition of il1 closed circles and il6 open circles secretion from pbmcs of a healthy donor cellswere stimulated with lps ngml for hours and atp mm for an additional hour bars show the mean ± sd of four wells101371 pone0237030g004therefore were deemed suitable for hts in this manner the financial and labor costs of htscan be dramatically decreased our strategy can be applied to other immunological disordersin which monocytemacrophagelineage cells play critical pathological roles in addition theimmortalization of ipscderived hematopoietic cells for hts can be expanded to otherhematopoietic disorders one concern associated with the immortalization of monocytic cellsis that the immortalization can affect the characteristics of the cells especially regarding thecell cycle and death which may affect the immunological functions to avoid any potentialchanges in the cellular phenotype we first expanded the ipsmls generated a larger numberof frozen stocks and then used the same passage number of the stocks for a series of htssessionsmodulating the function of the inflammasome by small compounds is a promising frontierfor controlling diseases associated with inflammasomemediated chronic inflammation somecompounds such as mcc950 have already been shown to be markedly effective without anysignificant side effects in animal models nevertheless identifying more candidates to modulate nlrp3 inflammasome is desirable as mcc950 inefficiently inhibits capsrelated nlrp3mutants interestingly we identified several compounds effective in mutant cells that were previously reported to inhibit the activation of nlrp3 inflammasome indicating that the activation of mutant nlrp3 shares activating signals to some degree with wildtype nlrp3however several compounds that exerted predominantly il1 inhibitory effects on mutantcells were nonselective on pbmcs which could be explained by offtarget effects or false positives offtarget effects including cytotoxicity could be the result of a higher sensitivity bypbmcs to the true positives the clear reason is difficult to elucidate however because of theheterogeneity of the pbmc population on the other hand ipsmls consist of relativelyhomogenous cell populations and may therefore be more useful than primary cells in in vitro one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningexperiments as for nonspecific inhibitors and false positives in hts the upregulation of certain signaling pathways irrelevant to the inflammasome in ipsmls might be one cause cytotoxic compounds may also suppress cytokine secretion in a nonspecific manneradditionally our hts system relies on the amount of secreted cytokines which can varybetween cells and only indirectly describes the inflammasome activity thus a combination ofmeasuring the cytotoxicity and direct detection of the inflammasome activity should improvethe accuracy of our hts systemto conclude although phenotypic screenings have been performed with various cell typesdifferentiated from human pscs the number of screenings with immune cells is still limitedour hts system provides a large number of functional monocytic lineage cells and a versatileplatform for compound screening to modify diseaseassociated phenotypes therefore it canbe used to screen candidate compounds for the treatment of congenital immunological disorders associated with monocytic lineage cellsmaterials and methodsstudy approvalthis study was approved by the ethics committee of kyoto university r0091g0259 andwritten informed consent was obtained from the patient™s guardians in accordance with thedeclaration of helsinkimaintenance and differentiation of human ipscswe previously established ipscs from a nomid patient cira188ai with nlrp3 somaticmosaicism y570c undifferentiated ipscs were maintained on mitotically inactivatedsnl feeder cells with primate es cell medium reprocell japan supplemented with ngml bfgf wako pure chemicals industries japan feeder and serumfree monocytic celldifferentiation was performed in accordance with previously described protocols with somemodifications [ ]lentivirus productionlentiviral constructs encoding myc bmi1 and mdm2 in the csiiefrfa vector and twoplasmids for lentiviral vector packaging pcmvvsvgrsvrev and pcaghivgp werekindly provided by dr satoru senju kumamoto university kumamoto japan and hiroyukimiyoshi riken bioresource center tsukuba japan plasmids were transfected into 293tcells crl3216 atcc usa by lipofection lipofectamine ltx thermo fisher scientificusa and days later viral ps in the culture supernatants were concentrated by centrifugation at rpm for hours at ˚cgeneration of ipsmlsipsmls were generated as previously described with some modifications [ ] briefly onday of the monocytic cell differentiation from ipscs floating cells were collected andinfected with lentiviruses the cells were cultured in stempro34 serumfree medium thermofisher scientific containing mm lglutamine in the presence of gmcsf ngml andmcsf ngml rd systems usa after approximately days proliferating ipsmlsappeared for macrophage differentiation ipsmls within passages after thawing were cultured in rpmi1640 medium sigmaaldrich usa containing fetal bovine serum equitechbio usa and mcsf ngml for days with a medium change on day adherent one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningcells were collected by treatment with accumax innovative cell technologies usa and usedfor the subsequent experimentsmaygiemsa stainingcells were seeded onto glass slides by cytospin thermo fisher scientific and stainedwith maygrunwald and giemsa staining solution merck kgaa germany in accordancewith the manufacturer™s instructions the slides were examined using a biorevo bz9000keyence japan a planapo × objective nikon japan and the bzii viewer software program keyence were used for the image acquisitionflow cytometrycells were treated with fcr blocking reagent miltenyi biotec germany and stained with primary antibodies cd45fitc becton dickinson and company usa cd11bpe andcd14apc beckman coulter usa at dilutions of for negative controls primary antibodies were replaced with mouse igg1 bd dapi sigmaaldrich was used to exclude deadcells the flow cytometric analysis data were collected using a macsquant analyzer miltenyibiotec and analyzed using the flowjo software program treestar usareverse transcriptionpolymerase chain reaction pcrrna samples were prepared using an rneasy mini kit qiagen germany total rna ng was reverse transcribed into cdna using a primescript rt master mix kit takarajapan pcr was performed on a veriti thermal cycler thermo fisher scientific withtakara ex taq hs polymerase using approximately ng cdna the forward primers targeting the coding region of the genes were ™gatcagcaacaaccgaaaat3™ myc™ccattgaattctttgaccagaa3™ bmi1 and ™gctgaagagggctttgatg3™mdm2 the reverse primer targeting wpre was ™gttgcgtcagcaaacacagt3™measurement of cytokinescells were seeded at × cellswell onto a 96well cell culture plate in rpmi1640 mediumcontaining fbs the cells were stimulated with ngml lps from e coli k12 invivogen usa for hours and μgml silica us silica usa for an additional hour aftercentrifugation the supernatants were collected the concentrations of cytokines in the culturesupernatants were analyzed using a flowcytomix kit ebioscience usa and a macsquantanalyzer in accordance with the manufacturer™s instructioncompound screeningcells were seeded at × cellswell onto 384well cell culture plates in rpmi1640 mediumcontaining fbs and compounds four hours later the cells were stimulated with ngmllps for hours after centrifugation the supernatants were transferred to 384well smallvolume white plates using a biomek nxp beckman coulter the relative cytokine levels weremeasured using an htrf kit cisbio bioassays france and a powerscan4 microplatereader ds pharma biomedical japan with excitation at nm and emission at and nm the htrf signals were calculated as the htrf ratio ×em nmem nmand then the percent inhibition was calculated using dmso controls cell seeding and reagentdispensation were performed with a multidrop combi thermo fisher scientific thereagents purchased are as follows acyvadcho mg132 merck kgaa mcc950sigmaaldrich fdaapproved drug library iccb known bioactives library enzo life one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningsciences usa usdrug collection international drug collection microsource discoverysystems usa lopac1280 sigmaaldrich tocriscreen mini tocris bioscience uk andkinase inhibitor libraries enzo life sciences merck kgaa selleck chemicals usahit validation with pbmcscryopreserved human pbmcs were purchased from cellular technology limited usa thecells were thawed in accordance with the manufacturer™s instructions and seeded at ×cellswell onto 384well cell culture plates in rpmi1640 medium containing fbs andcompounds four hours later the cells were stimulated with ngml lps for hours and mm atp sigmaaldrich for an additional hour the relative cytokine levels were measuredusing the htrf kit as described abovestatistical analysesstatistical analyses were performed using the excel and graphpad prism software programsgraphpad software usa statistical significance was evaluated using the paired ttestp was considered statistically significant the z™factor and sb ratio were calculated asfollowsz0 ¼ 00 ð3shc þ 3slcþðmhc 00 mlcþsb ¼ mhcmlcwhere σ is the standard deviation sd μ is the mean hc is the high control and lc is the lowcontrolsupporting informations1 fig htrf ratios of the assay controls each of the plates contained high stimulated and low not stimulated controls treated with dmso values falling outside of themean ± 3sd were excluded as outliers red crosses except when z™factors and sb ratios werecalculatedtifs2 fig scatter plot of the compound screening percent inhibitions for il1 xaxis and il yaxis within a range of to are shown seven hit compounds are markedtifs1 table karyotype analysis of ipsmlspdfs2 table a total list of compounds with the assay dataxlsxs1 raw imagepdfacknowledgmentswe thank drs takayuki tanaka mitsujiro osawa and yohei nishi cira kyoto universitykyoto japan for fruitful discussions and technical assistance dr peter karagiannis cirakyoto university kyoto japan for proofreading the paper ms harumi watanabe cira one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningkyoto university kyoto japan for providing administrative assistance and dr akitsu hottacira kyoto university kyoto japan for providing 293t cellsauthor contributionsconceptualization ryosuke seki megumu k saitodata curation akira ohta megumu k saitofunding acquisition megumu k saitoinvestigation ryosuke seki akira niwa yoshinori sugiminemethodology akira ohtaresources akira ohtasupervision akira niwa haruna naito tatsutoshi nakahata megumu k saitowriting “ original draft ryosuke sekiwriting “ review editing megumu k saitoreferences mestas j hughes cc of mice and not men differences between mouse and human immunology jimmunol “ epub 104049jimmunol17252731 pmid holsapple mp west lj landreth ks species comparison of anatomical and functional immune system development birth defects res b dev reprod toxicol “ epub 101002bdrb10035 pmid geissmann f manz mg jung s sieweke mh merad m ley k development of monocytes macrophages and dendritic cells science “ 101126science pmid takahashi k tanabe k ohnuki m narita m ichisaka t tomoda k induction of pluripotent stemcells from adult human fibroblasts by defined factors cell “ 101016jcell200711019 pmid takahashi k yamanaka s induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors cell “ 101016jcell200607024pmid lo cicero a jaskowiak al egesipe al tournois j brinon b pitrez pr a high throughput phenotypic screening reveals compounds that counteract premature osteogenic differentiation of hgpsipsderived mesenchymal stem cells sci rep epub 101038srep34798 pmid pubmed central pmcid pmc5064407thorne n malik n shah s zhao j class b aguisanda f highthroughput phenotypic screening of human astrocytes to identify compounds that protect against oxidative stress stem cellstransl med “ epub 105966sctm20150170 pmid pubmed central pmcid pmc4835244 darville h poulet a rodetamsellem f chatrousse l pernelle j boissart c human pluripotentstem cellderived cortical neurons for high throughput medication screening in autism a proof ofconcept study in shank3 haploinsufficiency syndrome ebiomedicine “ epub 101016jebiom201605032 p
Colon_Cancer
dysregulation of ribosome production can lead to a number of developmental disorderscalled ribosomopathies despite the ubiquitous requirement for these cellular machinesused in protein synthesis ribosomopathies manifest in a tissuespecific manner with manyaffecting the development of the face here we reveal yet another connection between craniofacial development and making ribosomes through the protein paired box pax9pax9 functions as an rna polymerase ii transcription factor to regulate the expression ofproteins required for craniofacial and tooth development in humans we now expand thisfunction of pax9 by demonstrating that pax9 acts outside of the cell nucleolus to regulatethe levels of proteins critical for building the small subunit of the ribosome this function ofpax9 is conserved to the anism xenopus tropicalis an established model for humanribosomopathies depletion of pax9 leads to craniofacial defects due to abnormalities inneural crest development a result consistent with that found for depletion of other ribosomebiogenesis factors this work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic developmentauthor summarywe are only beginning to understand the complex process of making human ribosomesthe cellular machines critical for all protein synthesis in humans making a ribosomerequires hundreds of regulatory factors to ensure proper cellular growth and development dysregulation of this process can lead to a number of tissue specific disorderstermed ribosomopathies here we have discovered a new role for the protein pairedbox pax9 in making human ribosomes while pax9 has traditionally been known toa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation farleybarnes ki deniz e overton mmkhokha mk baserga sj paired box pax9 the rna polymerase ii transcription factorregulates human ribosome biogenesis andcraniofacial development genet e1008967 101371 pgen1008967editor paul a trainor stowers institute formedical research united statesreceived february accepted june published august copyright farleybarnes this is anopen access distributed under the terms ofthe creative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all rnaseq andrnapii chipseq files are available from the geneexpression omnibus geo database and areaccessible through the geo series accessionnumber gse154764 wwwncbinlmnihgovgeoqueryacccgiaccgse154764 allnumerical data that underlies graphs or summarystatistics is available in the supporting informations4 table genetics 101371 pgen1008967 august genetics 0cfunding this work was supported by the nationalinstitutes of health r01gm115710r01gm122926 and r35gm131687 to sjbr01hd081379 to mkk t32gm007223 to sjb andkif f31de026946 to kif and a pilot grant from theyale cancer center to sjb the funders had no rolein study design data collection and analysisdecision to publish or preparation of themanuscriptcompeting interests the authors have declaredthat no competing interests existpaired box pax9 regulates human ribosome biogenesisplay a role in regulating the levels of proteins required for craniofacial and tooth development in humans we expand this function of pax9 by showing that pax9 acts outside ofthe cell nucleolus to regulate the levels of proteins critical for building the small subunit ofthe ribosome in addition we show that this function is conserved to the model anismxenopus tropicalis this link between pax9™s role in craniofacial development and inribosome biogenesis may lead to new insights into the pathogenesis of these pax9 mutations in humansintroductionwhen ribosome biogenesis is genetically disrupted in humans a number of surprisingly tissuespecific disorders called ribosomopathies arise for example genetic disruption of thetcof1 polr1c or polr1d genes in the ribosomopathy treacher collins syndrome tcsomim results in reduced preribosomal rna prerrna transcription [“] interestingly while these mutations each affect the global process of prerrna transcriptionpatients have specific defects in craniofacial development tcs patients present with hypoplasia of the facial bones micrognathia with or without cleft palate narrowing of the ear canaland bilateral conductive hearing loss [“] modeling the disease in mice has shown that thistissue specificity arises from differential tissue susceptibility to p53 levels p53 levels are stabilized upon disruptions in ribosome biogenesis when free ribosomal proteins bind to mdm2the e3 ligase for p53 [ ] this stabilization of p53 leads to apoptosis of the developing neural crest cells ultimately resulting in the mandibulofacial dysostosis of tcstcs is not the only ribosomopathy to affect craniofacial development for example diamond blackfan anemia dba omim is characterized by anemia low reticulocytecount and elevated erythrocyte adenosine deaminase activity [ ] however dba patientsoften also have craniofacial anomalies and cleft palate reviewed in additionally theribosomopathy acrofacial dysostosis cincinnati type omim is caused by mutationsin polr1a that inhibit prerrna transcription resulting in craniofacial defects amore precise understanding of the role of the factors involved in human ribosome biogenesiscan therefore shed light on the molecular mechanisms underlying aberrant craniofacialdevelopmentthe process of making the cellular machines required for protein synthesis called ribosomebiogenesis includes a large number of factors that work together to control cell growth anddevelopment in humans these proteins are still being defined previous studies have shownthat ribosome biogenesis begins with the transcription of the tandemly repeated ribosomaldna rdna into the 47s polycistronic prerrna fig 1a the prerrna is further modified and processed to create the mature 18s 58s and 28s rrnas these rrnas are incorporated along with the 5s rrna and ribosomal proteins into the small ssu and large lsusubunits of the ribosome the 47s prerrna is transcribed by rna polymerase i rnapiwhile the 5s rrna is transcribed by rna polymerase iii rnapiii various assembly factorsand the ribosomal proteins are transcribed by rna polymerase ii rnapii in addition torequiring all rna polymerases synthesis and assembly of functional ribosomes integrates anumber of cellular signaling pathways to ensure proper regulation of this essential process inresponse to stimulithe complex development of the face is controlled by a number of proteins including several rnapii transcription factors such as paired box pax9 pax9 belongs to a family oftranscription factors that play key roles in anogenesis and neural crest cell development by genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisfig pax9 is required for human ribosome biogenesis a ribosome biogenesis at a glance the tandemly repeated ribosomal dna rdna istranscribed into the 47s polycistronic preribosomal rna prerrna by rna polymerase i rnapi this 47s prerrna is processed throughmultiple steps to form the mature 18s 58s and 28s rrnas which are incorporated into the small and large subunits of the ribosome along with the5s rrna and ribosomal proteins ribosomes perform cytoplasmic cellular protein synthesis through the translation of mrnas b pax9 depletionreduces nucleolar number from “ to only in mcf10a cells left panel nuclei stained in hoechst are shown in blue nucleoli are shown in pinkand stained with antifibrillarin antibody as in sigfp top was used as a negative control “ nucleolinucleus and siutp4 middle was usedas a positive control nucleolusnucleus sipax9 is shown at the bottom right panel quantitation of the number of nucleoli per nucleus for sigfptop siutp4 middle or sipax9 bottom c pax9 is not required for rnapi transcription in mcf10a cells a dualluciferase reporter assay wasused to quantify luminescence after sirna depletion of pax9 the plasmids are phrdiresluc firefly to report rnapi transcription and arenilla transfection control as in the ratio of firefly to renilla luciferase was normalized to the sint control n sinol11 was used as apositive control data were analyzed by student™s t test using graphpad prism ���� p � d pax9 is required for pre18s rrnaprocessing in mcf10a cells left schematic of prerrna processing steps in human cells intermediates detected by probe p3 are indicated with ablack box below center northern blot with probe p3 a probe for the 7sl rna was used as a loading control intermediates detected by probe p3 areshown to the right of the northern blot negative controls were mock no sirna and sint nontargeting siutp4 was used as a positive control right quantitation by ramp of probe p3 upper and 7sl lower northern blots graph is mean ± sem n data were analyzed by2way anova using graphpad prism ���� p � ��� p � �� p � and � p � ptp indicates the 47s 45s and 43s processingintermediates e pax9 sirna depletion in mcf10a cells results in an increased ratio of 28s18s by agilent bioanalyzer significance was calculatedby student™s t test in graphpad prism where �� p � f pax9 sirna depletion in mcf10a cells results in decreased global protein synthesis asassessed by the puromycin incorporation assay a representative western blot using an antipuromycin antibody with a β actin loading control isshown to the left protein was harvested after knockdown for hours using the indicated sirnas mock indicates no sirna and mock μmindicates no sirna and half the concentration of puromycin sint nontargeting was used a positive control quantitation of replicates using cellsof different passage numbers is shown to the right significance was calculated by oneway anova in graphpad prism where ���� p � and��� p � g pax9 depletion in mcf10a cells results in decreased 40s 60s and 80s ribosome subunit levels representative polysome profile ofmcf10a cells depleted using sirnas targeting either pax9 red or a nontargeting sint blue control equal amounts of protein were loaded oneach gradient this experiment was performed times using cells of different passage numbers101371 pgen1008967g001controlling gene expression [ ] in humans mutations in pax9 cause tooth agenesis aswell as hair loss [“] and reviewed in indeed pax9 mutations are the most prevalent mutation in patients with nonsyndromic tooth agenesis including oligodontia additionally mice homozygous for a partial deletion of pax9 likely null have craniofacialmalformations including cleft palate skeletal abnormalities and arrested tooth developmentand die a few hours after birth pax9 mice also exhibit a range of cardiac malformations another commonly impacted tissue in ribosomopathies while some research hasbeen done to identify the signaling pathways regulated by pax9 attempts to correct the developmental defects have been only partially successful [“] therefore further studies areneeded to identify all factors regulated by pax9 in order to understand pax9™s role in craniofacial developmentour work ties together pax9 and ribosome biogenesis filling in some gaps in our knowledge of the many cell growth and signaling pathways influenced by pax9 depletion we originally identified pax9 as a potential regulator of ribosome biogenesis in an sirna screen forproteins required to maintain nucleolar number probing pax9™s specific role in makingribosomes in human tissue culture cells we discovered that pax9 is required both for the prerrna processing that produces the small subunit 18s rrna and for global protein synthesiswe employed the genomewide transcriptomics analysis rnaseq to define pax9 dependentmrnas necessary for making ribosomes several of the differentially expressed mrnasincluding several ribosomal proteins were further examined to pinpoint roles for these proteins in prerrna processing and global protein synthesis finally given an established rolefor pax9 in human craniofacial development we sought to model this disease in xenopus tropicalis x tropicalis embryos an established model of human ribosomopathies depletion ofpax9 in x tropicalis did indeed alter craniofacial patterning as well as neural crest development a migratory cell population that plays a major role in establishing craniofacial structurex tropicalis embryos depleted of pax9 also show defective prerrna processing these resultsshed light on the plethora of factors whose expression is regulated by pax9 and open the doorto likely connections between pax9™s role in craniofacial development and human ribosomebiogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisresultspax9 depletion disrupts small subunit ribosome biogenesispreviously we performed an sirna screen for new regulators of nucleolar number in humanmcf10a cells we identified pax9 by this screening approach as a protein that whendepleted reduced the number of nucleoli per nucleus from “ to only fig 1b as in cells were fixed after hours of knockdown using pools of sirnas targeting sigfp as a negative control siutp4 as a positive control or sipax9 this transient knockdown approach successfully depleted pax9 s1a and s1b fig the mcf10a cells were then stained with anantibody to fibrillarin fbl a nucleolar protein to detect nucleoli and with hoechst todetect nuclei a cellprofiler pipeline was used to quantify the number of nucleoli per cellnucleus which shifted from “ in sigfp control cells to only in sipax9 and siutp4treatedcells we had previously used oligonucleotide deconvolution to additionally confirm thatpax9 depletion leads to reductions in nucleolar number using this approach individualdepletion of pax9 using of the sirnas from the original pax9 pool reduced the numberof nucleoli from “ to only per cell nucleus [ ] as the sirna screen served as a phenotypic readout of nucleolar function we hypothesized that pax9 plays a role in humanribosome biogenesis through its function as an rnapii transcription factorwe sought to investigate the extent to which pax9 depletion affects human ribosome biogenesis using a panel of assays the first assay probes pax9™s role in rnapi transcriptionusing a dualluciferase reporter system previously published by our laboratory and others [ ] as pax9 is a known rnapii transcription factor it was relevant to test it for a possibleadditional role in rnapi transcription in this reporter assay the ratio of firefly luciferasewhich is under the control of the rdna promoter and measures rnapi transcription wasquantified relative to a renilla luciferase transfection control relative to a nontargeting control sirna sint sirnas targeting pax9 had no significant effect on rnapi transcriptionlevels after hours of knockdown in mcf10a cells fig 1c mock no sirna and sinol11were used as negative and positive controls respectively pax9 is therefore not required forrnapi transcription in mcf10a cellsnorthern blotting was used to define pax9™s role in prerrna processing in humans prerrna processing occurs via a number of different pathways fig 1d left and requires a number of transacting factors we therefore employed different probes to pinpoint any prerrna processing defects occurring after pax9 depletion in mcf10a cells s2a fig after hours of sirna knockdown pax9 depletion resulted in a significant increase in the 30s prerrna intermediate as well as a decrease in the levels of its 21s processing product relative tothe nontargeting sirna sint fig 1d middle and right and s2 fig additionally 41s levelswere decreased relative to the primary processing transcript 47s plus the 45s and 43s processing intermediates herein termed the primary transcript plus or ptp fig 1d middle andright and s2 fig quantitation of the ratios of each intermediate relative to its precursor in theprocessing pathway by ratio analysis of multiple precursors ramp confirmed the statistical significance of these results fig 1d right and s2 fig these effects were also significant relative to a 7sl loading control fig 1d right and s2 fig because the 30s and 21sintermediates are both precursors to the 18s rrna pax9 is required for ssu biogenesis thesame prerrna processing defect was also detected in human embryonic kidney hek293ftand colon carcinoma rko cells depleted of pax9 indicating that pax9™s role in ribosomebiogenesis is conserved among diverse human cell lines s1 figbecause the prerrna processing defects indicate aberrant ssu biogenesis we sought todetermine the extent to which pax9 depletion affects the production of the mature 18s rrnaagilent bioanalyzer quantitation shows an increase in the ratio of 28s to 18s fig 1e genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesiscombined with the northern blot results indicating defects in the processing of the precursorsto the 18s rrna this result is consistent with a predicted reduction in 18s rrna levels takentogether these results argue that pax9 is required likely indirectly for the biogenesis of thesmall subunit of the ribosome which contains the 18s rrnawe also utilized a puromycin incorporation assay to test the extent to which pax9 depletion alters the final product of ribosome biogenesis global cellular translation fig 1f cellswith or without pax9 depletion are treated with a low dose μm of puromycin which isincorporated into all nascent peptides produced during a hour pulse western blottingfor incorporated puromycin shows decreased protein synthesis after hours of pax9 depletion relative to a nontargeting sirna control sint fig 1f mock μm puromycin withno sirna and mock at a halfdose of puromycin μm were used as negative controls fig1f these results confirm that pax9 depletion leads to reduced protein synthesis consistentwith a role for pax9 in ssu biogenesisbecause the above assays indicated a role for pax9 in small subunit prerrna processingand global protein synthesis we also tested the extent to which pax9 depletion is required forribosomal subunit biogenesis and assembly using polysome profiling we determined thatpax9 depletion does result in significantly decreased ssu 40s levels in mcf10a cells fig1g consistent with the reduction in 18s rrna levels seen in fig 1e additionally 60s and80s polysome fractions also showed significant decreases after pax9 knockdown fig 1ginterestingly both here and in previous experiments mcf10a cells do not demonstraterobust polysome fractions using this technique regardless we conclude that pax9 depletion results in decreased ssu biogenesis that impacts the assembly and function of theribosomeas disruptions in ribosome biogenesis result in interruptions in the cell cycle [“] wealso examined the extent to which pax9 depletion changed the distribution of mcf10a cellswithin the cell cycle using flow cytometry s3 fig relative to a sint control depletion ofpax9 for hours resulted in a minor increase in the proportion of cells in g1 phase of thecell cycle but this was not statistically significant s3 fig sirnas targeting the ribosome biogenesis factor nol11 were used as a positive control and depletion of this protein resulted inan increase in the proportion of cells in g2 s3 fig consistent with previous findings inall these assays allowed us to conclude that pax9 regulates human ribosome biogenesisrnaseq analysis upon pax9 depletion reveals decreased levels ofnucleolar mrnas responsible for small subunit maturationas pax9 is a known rnapii transcription factor we hypothesized that pax9 works indirectlyto modulate ssu biogenesis fig 2a this is consistent with a nuclear but not nucleolar localization of pax9 in existing databases [“] pax9 may act directly as a transcription factorfor nucleolar proteins or indirectly for proteins that affect the expression or function of nucleolar proteins to test the hypothesis that pax9 affects nucleolar protein expression through itsfunction as a rnapii transcription factor we used rnaseq in mcf10a cells to define the setof mrnas that were differentially expressed after pax9 depletion relative to a nontargetingcontrol sirna sint pax9 depletion resulted in the differential expression of over mrnas fold change � or � q � fig 2b and s1 table approximately half of these were reduced in their levels consistent with the hypothesis that pax9 acts as a transcription factor to drive their expressionwhen considered as a whole the rnaseq dataset is enriched for several pathways knownto be regulated by pax9 s4a fig ingenuity pathway analysis of the differentiallyexpressed mrnas reveals enrichment of both wntca2 signaling differential expression of genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisfig rnaseq transcriptomics analysis in human tissue culture cells reveals changes in the expression levels of over nucleolar mrnas afterpax9 knockdown a schematic of how pax9 would act as a rnapii transcription factor to drive the levels of mrnas required for making the smallsubunit ssu of the ribosome in the cell nucleus pax9 binds to dna to affect the transcription of mrnas that either encode nucleolar proteinsdirect solid arrow or to transcribe mrnas that affect the levels of mrnas encoding nucleolar proteins indirect dotted arrows the resulting mrnasare translated in the cytoplasm into proteins that function in ssu prerrna processing in the nucleolus b rnaseq analysis after pax9 sirnadepletion in mcf10a cells reveals decreased levels of mrnas encoding nucleolar proteins relative to a nontargeting sirna control sint pax9depletion resulted in differential expression of mrnas fold change � or and fdr � of these mrnas had a decreased foldchange � and of those mrnas code for proteins designated as nucleolar in at least one of three databases [“] of the mrnas whoselevels were decreased and that also code for nucleolar proteins were chosen as candidates for followup studies c qrtpcr confirms reduced mrnalevels of the rnaseq candidates after pax9 sirna knockdown in mcf10a cells after depletion using sirnas targeting either pax9 or a nontargeting control sirna sint the levels of the indicated mrnas were quantified by qrtpcr using primers to each target gene relative to a 7slcontrol and sint data are shown as mean ± sem three replicates using cells of different passage numbers with technical replicates each wereperformed significance was calculated by oneway anova using graphpad prism where ���� p � d depletion of of the candidatemrnas rps6es6 rps9us4 rps28es28 and fbl individually results in the same prerrna processing defect as pax9 sirna depletion in mcf10acells representative northern blot after knockdown of the indicated sirnas using probe p3 a probe for the 7sl rna was used as a loading control prerrna processing intermediates detected by probe p3 are shown to the right of the northern blot ptp indicates the 47s 45s and 43s prerrnaprocessing intermediates e quantitation of northern blots using probe p3 as shown in fig 2d using ramp graph is mean ± sem n datawere analyzed using 2way anova in graphpad prism where ���� p � ��� p � and �� p � quantitation relative to the 7sl loadingcontrol is shown in s5 fig f sirna depletion of rnaseq candidates in mcf10a cells results in decreased global protein synthesis after hoursof knockdown with the indicated sirnas mcf10a cells were pulsed with puromycin for hour and protein was harvested western blotting with anantipuromycin antibody as well as a β actin loading control was carried out representative western blots shown to the left mock mock at half theconcentration of puromycin μm and sint nontargeting sirnas were used as negative controls g quantitation of three replicates usingmcf10a cells of different passage numbers of the puromycin incorporation assays following depletion with the indicated sirnas relative to the sint andβ actin loading controls is shown as mean ± sem n significance was calculated by student™s ttest using graphpad prism where ���� p � ���p � and � p � 101371 pgen1008967g002 pathway members p x ˆ’ s4b fig and wntβcatenin signaling differentialexpression of pathway members p x ˆ’ s4c fig in cells depleted of pax9expression levels are increased for many of the mrnas in the wnt signaling pathway this isconsistent with previous results suggesting a role for pax9 in the negative regulation of wntsignaling [ ]as an additional validation of the rnaseq dataset we determined that the genomesequences kb upstream of the start sites of the differentially expressed mrnas containmultiple potential pax9 binding sites making these mrnas candidates for direct transcriptional regulation by pax9 scanning for known pax9 binding sequences [™sgtcacgcwtgantgma3™ ™cgcgtgaccg3™ cd192ains ™gcgtgacca3™ and e5 ™gcggaacgg3™] in the kb upstream of the mrnas using centrimo analysis reveals and potential pax9 binding sites respectively in the kbupstream of the mrnas this number of potential binding sites upstream of the mrnas sites per gene is similar to that observed in pax9 chip experiments in the vertebral column of e125 mice sites per gene additionally centrimo enrichmentanalysis of the sequence kb upstream of each of the differentially expressedmrnas reveals significant enrichment of different dna binding sequences including thepax3 pax5 pax6 and pax7 dna binding domains as multiple pax proteins can bindthe same dna sequence this provides further evidence for pax9 regulation of these mrnas these analyses therefore support the hypothesis that pax9 regulates the levels of themrnas identified in our rnaseq datasetin the rnaseq dataset many of the differentially expressed mrnas have knownroles in nucleolar function for example have appeared in other genomewidesirna screens for nucleolar function s1 table [ ] additionally of the differentially expressed mrnas code for proteins designated as nucleolar in at least of nucleolar databases s1 table [“] this is a significant enrichment in the expected number of nucleolar proteins assuming that nucleolar proteins make up only of the proteins inhuman cells surprisingly expression of most of the mrnas encoding nucleolar proteins genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis was downregulated upon pax9 depletion indicating that pax9 is requiredto maintain normal levels of many mrnas whose protein products are destined for functionin the nucleolus s1 tableto determine the mechanism of pax9™s function in ssu biogenesis we chose candidatesfrom the rnaseq dataset to follow up on in greater detail the mrna levels for the candidates were all downregulated after pax9 depletion and all code for nucleolar proteins fig 2band s1 table [“] four of the candidates rps6es6 rps9us4 rps28es28 and fblwere chosen on the basis of literature suggesting a role for these proteins in ssu prerrnaprocessing in hela cells [ ] additionally it was pertinent to analyze rpl5ul18 as it hasa known role in the p53dependent nucleolar stress response qrtpcr confirmed thernaseq results with reduced levels of each mrna when pax9 is depleted in mcf10a cellsfig 2c interestingly depletion of either rps9us4 or rps28es28 resulted in a decrease innucleolar number from “ to only in our original sirna screen therefore it is possible that the mechanism through which pax9 depletion results in decreased nucleolar numberrelies upon reduced expression of rps9us4 andor rps28es28 fig 2awe were able to confirm that depletion of of the tested candidates rps6es6 rps9us4 rps28es28 and fbl in mcf10a cells resulted in prerrna processing defects similarto that of pax9 depletion fig 2d and 2e s5 fig only rpl5ul18 did not give the 30sincrease characteristic of pax9 depletion although this was expected given its known role inlsu prerrna processing additionally depletion of several of the candidates individually resulted in significantly decreased global protein synthesis by the puromycin incorporation assay similar to the effect seen after pax9 depletion fig 2f and 2g puromycinincorporation was also reduced after rps6es6 depletion although it was not statistically significant fig 2f and 2g therefore pax9 may function as a transcription factor to directly orindirectly increase the expression of rps6es6 rps9us4 rps28es28 andor fbl fig 2aas the proteins encoded by these mrnas are required for ssu ribosome biogenesis fig their depletion after pax9 knockdown is a plausible mechanism through which pax9 regulates prerrna processing and global protein synthesisrnapii chipseq analysis reveals decreased transcription of mrnasencoding nucleolar proteins after pax9 depletionas the rnaseq analysis confirmed that levels of nucleolar mrnas were decreased afterpax9 depletion fig we sought to map how rnapii distributes on genes using rnapiichipseq as a readout of transcription through this approach we aimed to untangle the effectsof pax9 depletion on rnapii transcription from its effects on mrna stability since rnapiichipseq is able to detect genomewide changes in rnapii occupancy as pax proteins areable to both activate and repress target protein expression we have included genes withboth increased and decreased rnapii occupancy in this analysis approximately mrnaswere differentially occupied by rnapii upon pax9 knockdown compared to the nontargeting control sirna sint in mcf10a cells fold change cutoff � or � and maxtags � s2 table of these had decreased rnapii occupancy consistent with pax9 acting as a transcriptional driver of these mrnasto assess the validity of the rnapii chipseq dataset we again used centrimo to identifypotential pax9 dnabinding sites in the kb upstream of the genes with differentialrnapii occupancy searching for the ™ cgcgtgaccg ™ pax9 binding motif definedin revealed possible sites in the bp upstream of the differentially occupiedgenes also the known pax9 dna binding motifs cd192ains ™gcgtgacca3™ ande5 ™gcggaacgg3™ had and binding sites in these sequences respectively genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisadditionally analysis of motif enrichment ame identified the pax5 and pax6 dnabinding motifs as being significantly enriched in the kb of sequence upstream of the genes p � since multiple pax proteins can bind the same motif this suggests thatthis dataset does contain mrnas that are regulated by pax9 of the differentially occupied genes have also been shown to be differentially regulated by pax9 directly in pax9chipseq experiments on e125 wt vertebral column murine tissue fig 3a and s2 table these analyses confirm the ability of rnapii chipseq to detect changes in rnapiimediated transcription after pax9 knockdownbased on the hypothesis that pax9 acts as an rnapii transcription factor for regulators ofnucleolar function fig we expected to detect changes in the rnapiimediated transcription of a number of mrnas encoding nucleolar proteins after pax9 depletion indeedmrnas coding for nucleolar proteins were enriched with of the genes with differentialrnapii occupancy coding for nucleolar proteins in at least one of three databases s2table [“] this is again higher than would be expected assuming that nucleolar proteins account for approximately of all cellular proteins additionally depletion of onegene with differential rnapii occupancy anln resulted in decreased nucleolar number inour sirna screen similar to the phenotype seen after pax9 depletion fig 1b notably of the genes with differential rnapii occupancy appeared in other genomewide screens for ribosome biogenesis factors s2 table [ ] another targeted screeninvestigated the effects of of the genes top2a and cdca8 on prerrna processingwhen depleted by sirna in hela cells however depletion of neither gave the 30s prerrna increase on northern blots characteristic of pax9 depletion in al
Colon_Cancer
inanic arsenic ias is the chemical form of as commonlyfound in drinking water atsdr and in some foodscubadda chronic exposure to ias has been associatedwith risk of skin bladder lung and liver cancers iarc aswell as with other diseases naujokas including diabetes maull cardiovascular abhyankar moon saquib states respiratorysanchez and neurological diseases caito andaschner parvez humans and most other mammalian species have developed a mechanism for detoxifying iasthat involves the sequential conversion of ias to monomethylasmas and mas to dimethylas dmas thomas both methylation steps are catalyzed by orthologs of a singleenzyme arsenic oxidation state methyltransferase as3mtlin the methylation of ias by as3mt not onlyaddress correspondence to miroslav st½blo department of nutritionuniversity of north carolina at chapel hill chapel hill nc usa telephone email styblomeduncedu or beverly hkoller department of genetics university of north carolina at chapel hillchapel hill nc usa telephone emailbkolleremailuncedusupplemental material is available online 101289ehp6943this document was reviewed by the center for computational toxicology andexposure office of research and development us environmental protectionagency and approved for publication approval does not signify that thecontents reflect the views of the agency nor does mention of trade names orcommercial products constitute endorsement or recommendation for usethe authors declare they have no actual or potential competing financialinterestspublished august received february revised july accepted july note to readers with disabilities ehp strives to ensure that all content is accessible to all readers however some figures and supplementalmaterial published in ehp s may not conform to standards due tothe complexity of the information being presented if you need assistanceaccessing content please contact ehponlineniehsnihgov our staï¬will work with you to assess and meet your accessibility needs within working dayspromotes wholebody clearance of as drobn¡ hughes but also produces methylated intermediates that contain highly reactive and toxic trivalent as asiii watanabe andhirano masiii and dmasiii unlike their pentavalent counterparts masvand dmasv exceed ias in potency as cytotoxinsgenotoxins and enzyme inhibitors thomas hencemasiii and dmasiii may be critical determinants of toxic and carcinogenic eï¬ects associated with chronic exposure to ias alteredcapacity to methylate ias has been linked to an increased risk ofdiseases associated with ias exposure ahsan pierce vahter in most mammals the gene encoding as3mt is present asa single copy ˆ¼ kb from the gene encoding bloc1 relatedcomplex subunit borcs7 to date the methylation of ias isthe only known function of as3mt however recent studies haveidentified the as3mtborcs7 locus as conferring risk for schizophrenia duarte compared with healthy individualsexpression of borcs7 and of the humanspecific splicing variantof as3mt as3mtd2d3 has been found to be consistently higher inthe brains of patients with schizophrenia li notablyexpression levels of as3mt and borcs7 were found to be weaklycorrelated li suggesting that the two genes may sharetranscriptional regulatory elements although published datashowed that ias exposure can aï¬ect as3mt expression in micest½blo the role of ias exposure in the expression ofborcs7 or as3mtd2d3 which is thought to lack ias methylationactivity has never been studiedlaboratory studies of the mechanistic basis of iasassociateddiseases have been hindered by substantial diï¬erences between laboratory animals and humans in their capacity to metabolize anddetoxify ias rats unlike humans sequester significant portions ofingested ias in erythrocytes in the form of dmasiii lu mice the species most commonly used in mechanistic studies diï¬erfrom humans displaying very high rates of as methylation thisspecies diï¬erence is associated with faster rates of urinary clearanceof methylated metabolites and the predominance of dmas as themain urinary metabolite of ias in mice vahter interspeciesdiï¬erences in as metabolism may contribute to difficultiesenvironmental health perspectives august 0cencountered replicating some of the eï¬ects of ias exposure reportedin humans including the carcinogenic eï¬ects in laboratory micethus producing an ias methylation phenotype in mice that resembles the phenotype found in humans could create a better animalmodel to study the role of ias metabolism in adverse health eï¬ectsof chronic ias exposure to generate such a model we have humanized the entire borcs7as3mt locus in 129s6 mice by syntenicreplacement in this process the segment of mouse dna carryingthese two genes was removed and replaced with the syntenic regionof human dna in mouse embryonic stem es cells using homologous recombination this ensured that the junctions between thehuman and mouse dna are known to the base pair level mice weregenerated from the es cells with the expectation that they wouldexpress the human as3mt and borcs7 proteinsthe present study describes in detail the creation of thehumanized mouse strain expression of human as3mt andborcs7 in tissues of the humanized mice and metabolism ofias in these mice after a single dose and during a subchronic exposure to ias in drinking watermethodsrationale for humanizing the borcs7as3mt locuswe chose to excise the 61kb segment of mouse dna carrying theas3mt and borc7 genes and replaced it with the correspondingpromoter of as3mt abuts59kb segment of human dna the untranslated region of borc7 and the weak correlation inthe the expression pattern of the genes suggests possible shared transcriptional regulatory elements li in addition the current human transcript databases for example the university ofcalifornia santa cruz genome browser ucsc lists a putative readthrough borcs7as3mt transcript lacking the finalexon of borcs7 and the initial exon of as3mt this suggests thepossibility that at least some as3mt activity may be linked to transcription driven by the borcs7 promoter thus the inclusion ofborcs7 in the humanized region increases the likelihood that theelements directing the tissue species diï¬erences in the expressionof as3mt are included in the humanized regionassembly of borcs7as3mt displacerthe borcs7as3mt displacer construct was assembled using astandard recombineering approach figure the mouse arms ofhomology were derived from the 129s7ab22 bmq bac librarysource bioscience plc nottingham uk the short homologyarm was derived from bac bmq455l14 and the long arm ofhomology from bac bmq345l20 the segment of humangenomic dna containing the borcs7 and as3mt loci wasderived from the human tile path bac rp11753c18 bacpacresources center children™s hospital oakland research instituteoakland ca the single nucleotide polymorphisms snps previously associated with interindividual diï¬erences in ias metabolism apata or schizophrenia risk li which were included in the this borcs7as3mt segment aredescribed in table s1 a dna segment bp extendingfrom chr1946683032 to were deleted from the mousegenome and replaced with a 59261bp segment of human dnaextending from chr10102845563 to the haplotypeof the as3mt gene carried in the displacer construct is 1a wood the resistance marker gene used for selection of escells in which the displacer construct underwent genomic integration consisted of a phosphoglycerate kinase pgkneo cassetteflanked by mutant loxp sites the marker gene can be excised leaving only a nonfunctional lox site in its placeall polymerase chain reactions pcrs carried out during thedisplacer assembly used taq polymerase bio basic with an initial denaturation temperature of °c for s followed by cycles of denaturation for s at °c annealing for s at a°c and extension at °c with an extension time of min per bp of product length all redet recombination reactionswere carried out using a commercially available kit gene bridgescat no k001 all predesigned primers used during the construction of the displacer vector were from sigmaaldrich and arelisted in table s2 in descriptions of specific assays each of theseprimers is listed by the corresponding number as stated above the short arm of the displacer construct wasderived from the bmq455l15 bac library this was accomplished by first replacing the borcs7as3mt region of the bac withan ampicillin resistance marker by redet recombination theampicillin resistance marker was amplified from the pbluescriptii sk cloning vector stratagene primers and the homology arms referred to as the red arm primers and and the ds arm primers and respectively were generated by pcr amplification ofsegments of the bmq455l15 bac the homology arms werefused to the ampicillin resistance gene by overlap pcrabhuman locuscyp17a1borcs7as3mtmouse locuscyp17a1borcs7as3mt kb deletedcnnm2cnnm2cborcs7 as3mt displacerddisplaced mouse locuscyp17a1floxedneofloxedneoborcs7as3mtcnnm2homologousrecombinationborcs7as3mtcnnm2 kb insertedfigure scheme for humanization of the mouse borcs7as3mt locus a human borcs7as3mt locus showing the relative positions of the borcs7 andas3mt genes as well as the closest flanking genes b mouse borcs7as3mt locus with flanking genes c schematic of the borcs7as3mt displacer construct d humanized locus prior to cremediated marker excision the names of human genes are capitalizedenvironmental health perspectives august 0cwas used to insert the human borcs7as3mt segment and neomycin resistance cassette by redet recombination into thebmq354l20 bac carrying the short arm and ampicillin genesimultaneously displacing the ampicillin gene this reactionyielded the final borcs7as3mt displacer vector which wasdigested with noti to release the bac backbone before transfection into es cellsgeneration of mouse es cellsthe parental es cell line phnx43 used in these studies was generated as follows male and female 129s6svevtac mice taconicbioscience were mated females were checked every morning forcopulatory plugs indicative of successful mating three days laterfemales were euthanized by lethal carbon dioxide co2 exposurefollowed by physical euthanasia and the 35d embryos blastocysts were collected by flushing the uterus with co2independentmedium gibco cat no supplemented with bovine serum albumin sigmaaldrich cat no a3675 individualembryos were collected from the lavage fluid with a pipette andplaced in 2cm2 wells that had been seeded h earlier withprimary mouse embryo fibroblasts mefs gibco cat nogsc6005m blastocysts and mefs were cultured in gibcoknockout„¢ ko medium cat no supplementedwith esqualified fetal bovine serum fbs gibco cat no and 2mm lglutamine gibco cat no after “ d in culture when the inner cell mass of the embryosreached ˆ¼ mm in diameter the embryos were removed and disrupted with a pipette and placed in a new well seeded with mefsthis process of serial expansion of a single inner cell mass wascontinued until a single inner cell mass was expanded to at least colonies of cells cells were then further expanded by disruptionand exposure to trypsin for min at °c after expansionand cryopreservation in dimethyl sulfoxide sigmaaldrichcat no d2660 a sample of the cell line was subjected to karyotype analysis in karyologic inc rtp nc the cell line used inthis study had a xy normal male mouse karyotypeexpression of human borcs7as3mt in mouse es cellssinglecell suspensions of the parental es cell were prepared bytreatment with trypsin as described above in a volume of ml — cells were electroporated in the presence of lgof dna the apparatus used was a btx electro cell manipulator with cuvette btx cheshire analytical the settingsused were v lf r8 ohms after electroporation the cells were distributed onto seven 100mm tissue cultureplates corning cat no in gibco ko medium supplemented with esqualified fbs gibco cat no and mm lglutamine gibco cat no after hselection was initiated with the addition of geneticin„¢ final concentration mgml gibco cat no after d individual neomycin resistant colonies became visible cells fromˆ¼ colonies were pooled and used for rna isolation to confirmthat the human genes were expressed see details below once thiswas established individual colonies from the remaining plateswere transferred individually by pipette to a 96cell plate each colony was trypsinized and a portion used for pcr analysis to identify those in which the dna had integrated by homologousrecombination as described below the remaining cells wereallowed to grow and cultures carrying a targeted allele as determined by the assay detailed below were transferred sequentially to and 60cm2 tissue culture plates at this point some cellswere cryopreserved while a portion of each was used for karyotypeanalysis and to confirm the expression of human as3mt by probebased droplet digital pcr ddpcr using commercially availablethe displacer short arm together with the inserted ampicillingene was subcloned from the modified bmq455l15 bac into aplasmid vector by redet recombination the plasmid vectorwas prepared by ligation of four pcr products the vector backbone carrying a cole1 origin of replication and a spectinomycingene was derived by amplification of the pfloxxerx plasmid previously assembled in koller™s laboratory see table s3 primers and the bmq455l15 bac was used as atemplate for the other three pcrs us arm primers arm primers and andand short ds arm primers and all pcr productswere gel purified on a agarose gel using a commerciallyavailable kit qiagen cat no digested with bbsi nebcat no r3539s and mlui neb cat no r0198s and columnpurified qiagen cat no fifty nanograms of each pcrproduct was combined and ligated neb cat no m0202s in a20ll reaction for h at °c the ligation reaction was heatinactivated at °c for min and then ll of the reaction wastransformed into chemically competent e coli dh5afcellszymo research cat no t3002 according to the manufacturer™s directions and plated on luria broth agar plates supplemented with spectinomycin at lgml the resulting plasmidvector was digested with mlui and used for redet recombination with the modified bmq455l15 bac the resulting plasmidwas in turn digested with bbsi to release the displacer short armand ampicillin resistance gene flanked by the us and dshomology arms this bbsi restriction fragment was introducedinto the bmq345l20 bac by redrecombination to create abac vector in which the ampicillin resistance marker wasflanked by the displacer arms of homologythe segment of the human borcs7as3mt locus included inthe displacer vector was prepared for excision from the rp11753c18 bac in two steps in the first step a neomycin resistancegene was inserted into the bac upstream of the borcs7 gene toaccomplish this the neo gene flanked by mutant loxp sites wasligated to homology arms referred to as blackred and purplerespectively the neomycin resistance gene was excised from thevector psfiijt15neojtz17sfii previously assembled in koller™slaboratory see table s4 by digestion with sfii neb cat nor0123s the blackred homology arm was amplified primers and using the bmq455l15 bac as a templateand the purple homology arm was amplified primers and using the rp11753c18 bac as a template all fragments were gel purified as described above and the blackred andpurple homology arm pcrs were digested with bgli neb catno r0143s and then column purified as described above theloxpflanked neomycin resistance gene was ligated to the homology arms as described above in a 20ll reaction containing a totalof lg of dna with the three fragments at an equimolar ratiotwo microliters of the ligation reaction was used for a redetreaction with the rp11753c18 bac in the second step an ampicillin gene was inserted downstream of the as3mt gene in therp11753c18 bac carrying the neomycin resistance marker toaccomplish this homology arms referred to as bluegreen andyellow respectively were added to the ampicillin resistance genethe bmq455l14 bac was used as the template for the bluegreen pcr primers and pbluescript ii sk was used as the template for the ampicillinyellow pcr primers and the two pcr products were gel purifiedand then combined in an overlap pcr primers and the resulting pcr product was gel purified and ngwas used for a redet reaction with the rp11735c18 bac carrying the neomycin resistance cassetteapproximately ng of the modified rp11753c18 bacwas digested with mlui in a 20ll digest and ll of the digestenvironmental health perspectives august 0cprimers applied biosystems hs00960526 here a 20ll reaction mixture was pipetted into a dg8„¢ cartridge along with llof droplet generation oil for probes bio rad and loaded into theqx200 droplet generator bio rad according to the manufacturer™s instructions after droplet generation the droplets weretransferred to an eppendorf twintec® 96well plate and placed in ac100 touch thermal cycler bio rad using the manufacturer™srecommended cycling conditions after cycling the plate was readin the qx200 droplet reader bio rad quantasoft„¢ softwareversion a portion of each of the es colonies ˆ¼ cells was disruptedby incubation for min at °c in lysis buï¬er consisting of trisedta [ mm trisma base thermo fisher scientific cat nobp152 mm ethylenediaminetetraacetic acid edta thermofisher scientific cat no volvol triton„¢ x100millipore sigma cat no and mgml proteinase kroche cat no ] lysates from all colonies werescreened for homologous recombination by pcr using primersscreen f and see table s4 with gxl polymerase takarabio according to the manufacturer™s suggested protocol the pcrprogram used was °c initial denaturation for s followed by cycles with s denaturation at °c s annealing at °c minat °c and a final 10min extension at °c pcr products wereanalyzed by agarose gel electrophoresis agarose bio basiccat no d0012 in — trisacetateedta running buï¬er the sizeof the pcr product was determined by comparison with 2logladder neb cat no n3200ltwentyseven clones with homologous recombination wereidentified and were subjected to further analysis because of thishigh recombination frequency it was not necessary to use clustered regularly interspaced short palindromic repeats and crisprassociated protein 9crisprcas9 to stimulate recombinationevents rna was prepared from pools of the cells and examinedfor the expression of the human genes rna was also preparedfrom a number of the individual clones after expansion brieflycells were lysed directly in the culture dish and homogenized byrepeated pipetting rna was isolated using rna bee or stat60teltest according to the manufacturer™s instructions rna yieldand quality were assessed using a nanodrop thermofisher one to two micrograms total rna was reverse transcribedwith the high capacity reverse transcription kit appliedbiosystems expression of the human as3mt was examined byqualitative pcr qpcr on a c100 touch thermal cycler biorad using commercially available primers applied biosystemshs00960526generation of mice expressing human borcs7as3mtfor injection into blastocysts the es cells carrying the humanborcs7as3mt locus were again trypsinized to generate singlecell suspensions collection of recipient blastocysts blastocystinjection and transfer to pseudopregnant dams was carried out bythe unc chapel hill animal models core following proceduresdescribed by longenecker and kulkarni and by koller to maintain the mutation on the 129s6svevtac background the chimeric mice were bred to 129s6svevtac micetaconic bioscience the resulting f1 mice were identifiedby taq as wildtype wt homozygotes for the mouse as3mtborcs7 locus wtwt or heterozygous wths or homozygoushshs for the human as3mtborcs7 locus the primers usedwere common and endo for the endogenous locus and common anddisplaced for the displaced locus see table s2 the pcr assayswere carried out using taq polymerase bio basic with an initialdenaturation temperature of °c for s followed by cycles ofdenaturation for s at °c annealing for s at a °c andextension at °c for s with a final extension of min the f2generation”consisting of hshs homozygotes hswt heterozygotes and wtwt homozygotes”was produced by mating of f1hswt heterozygotes the hshs hswt and wtwt oï¬springfrom the f2 generation were housed together one litter per cagecunder controlled conditions with 12h lightdark cycle at ± and ± relative humidity the parental mice and mice in thef1 and f2 generations were fed purina labdiet 5v5r and drankdeionized water ad libitumall proceduresinvolving mice were approved by theuniversity of north carolina institutional animal care and usecommittee 0ecollection of tissues for mrna expression analysistissues for mrna expression analysis were collected from f2hswt mice both males and females measurement of rnaexpression in hswt mice which were heterozygous for thehuman and mouse gene allowed direct comparison of expressionin the same tissuerna sample minimizing the eï¬ects of physiological variation between animals or quality of sample whichcould result in subtle diï¬erences in rna quality and cdnaformationmice were euthanized by exposure to co2 followed by physical euthanasia the following tissues were collected whole brainparts of brain cortices pons medulla hippocampus cerebellumpituitary spinal cord adrenals liver spleen kidney duodenumjejunum colon stomach uterus ovaries testes heart and skinfrom back of neck neuronal and glial cells were also dissectedbrain parts and spinal cord were collected from male mice at dof age neuronal cells were isolated from embryonic day e17embryos and glial cells from postnatal day p1 pups all othertissues were collected from to 12wkold mice three mice wereused for each tissue blood was collected from lethally anesthetized mice via cardiac puncture with an edtacoated syringe justprior to thoracotomy to isolate mononuclear cells from blood ml of whole blood were layered onto ml of histopaque® sigma and centrifuged at room temperature for min with nobrakes at — g the upper layer was discarded and the interfacecontaining the mononuclear cells was transferred to a 15ml conical tube the cells were then washed twice with phosphatebuï¬ered saline and then lysed with rna beeneuronal cell isolation and culture brains were harvestedfrom eight e17 embryos and placed in a petri dish containinghanks™s balanced salt solution hbss gibco meninges wereremoved cortices dissected and placed in a new petri dish containing hbss cortices were transferred to a 15ml conical tube containing ml hbss and centrifuged at — g for min at °cthe tissue was digested with ml tryple express gibco catno and dnase i roche cat no for min at °c the digested cortices were centrifuged at — gfor min at °c the tryple dnase i was aspirated and thecortices were washed for min with ml fbs to inactivate thetrypsin the tissue was centrifuged again and resuspended in complete dulbecco™s modified eagle medium dmem gibco with fbs vwr glutamax gibco with penicillinstreptomycingibco and 2mercaptoethanol sigma the cortices were titurated times with a 10ml pipette and then times with aflamedtip pasteur pipette the cells were then passed through a100lm cell strainer into a clean conical tube cells were then pelleted by centrifugation at — g for min the cell pellet wasresuspended in ml complete dmem and plated in mm culture dishes coated with lgml poly dlysine sigma — cells per dish after h 1lm cytosine bdarabinofuranosidearac sigma cat no c6645 was added using a mediachange without exposing the cells to air the cells were harvestedfor rna isolation on day environmental health perspectives august 0cmixed glial cell culture brains were harvested from six p1pups and placed in a dish containing hbss meninges wereremoved cortices dissected and placed in a conical tube containing cold dmem cortices were centrifuged at — g for min at°c and media replaced with ll cold dmem the corticeswere titurated times with a flamedtip pasteur pipette coated inserum the cells were then passed through a 100lm cell strainerinto a fresh conical tube cells were pelleted by centrifugation at — g for min the cell pellet was resuspended in ml complete dmem and ml of the suspension was plated on three100mm culture plates the medium was changed h later andthen daily the cells were harvested for rna on day isolation of adrenal cortex and medulla to obtain samplesenriched for each of these two distinct functional regions of thegland the adrenal cortex was removed from medulla of male andfemale adrenal gland by microdissection enrichment was quantitated by qpcr analysis of genes known to be expressed in onlyone of these two regions chgb in the medulla and cyp11b1 inthe cortex the human protein atlas using appliedbiosystem taqman probes mm00483287 and mm01204952respectively rna was isolated from cells and tissues using themethod described for es cells however the tissues were first homogenized in stat60 in a desktop homogenizer fastprep mp bio using ceramic beadsanalysis of as3mt as3mtd2d3 borcs7 as3mt andborcs7 expression in collected tissues and cellsboth ddpcr and qpcr were used to detect and quantify as3mtas3mtd2d3 borcs7 as3mt and borcs7 mrna the specificmethod is indicated in each figure legend ddpcr is minimallyinfluenced by diï¬erences in the efficiency of the mouse and humanprimer sets quan and therefore provides a means ofdirect comparison of expression between the endogenous mouselocus and the humanized locus the distribution of the as3mttranscript in the tissues was compared with as3mt protein distribution described by the human protein atlas for the human tissues sybr green qpcr was used to compare rna expression inwhole brain cortex hippocampus pituitary cerebellum pons medulla spinal cord glial cells neurons adrenals ovaries testesheart kidney liver colon jejunum spleen and blood ddpcr wasused to assess rna expression in es cells whole brain liver adrenals spinal cord ovaries testes spleen and heart for all assays“ lg total rna was reverse transcribed with the high capacityreverse transcription kit applied biosystemsfor sybr green qpcrthe relative expression of fulllength as3mtthe d2d3 variant and the borcs7as3mtfusion transcript were assessed on a quantstudio6 flex„¢ realtime pcr system applied biosystems sybr green reactions were run using itaq universal sybr green supermixbio rad nm of each primer and ngll of cdnaprimers used for quantification of as3mt isoforms were d2d3fand d2d3r for the d2d3 isoform and fullf and fullr for thefulllength isoform see table s2 for analysis of borcs7as3mt readthrough transcripts expression in mouse tissues lgrna was reverse transcribed and sybr green quantitative pcrwas run using primers gaacagtcatcggatctacagga3and and itaq sybrgreen universal supermix bioradgaacagtcatcggatctacagga3for ddpcr absolute concentration of as3mt as3mt borcs7and borcs7 was acquired using the ddpcr supermix forprobes no dutp bio rad and taqman gene expressionassays as3mt mm00491075_m1 as3mt hs00960526_g1borcs7 mm01205060_m1 and borcs7 hs00376014_m1all from applied biosystems the ddpcr analysis was carriedout as described aboveevaluation of the metabolism of a single dose of iasthe metabolism of ias was examined in to 22wkold hshsmale n and female n f2 oï¬spring and their wtwtmale n and female n littermates the mice weregiven a single dose of ias sodium arsenite pure sigmaaldrich in deionized water diw by gavage lg askg ofbody weight immediately after dosing each mouse was placedin a metabolic cage mousecage and urine and feces were collected in 24h intervals for d during these d all mice drankdiw ad libitum the mice were fasted during the first h buthad free access to purified laboratory diet envigo teklad catno ain93g during the second and the third days our published data showed that ias content in this type of diet rangesfrom to ˆ¼ lg askg douillet huang murko the 24h urine and feces samples werefrozen in dry ice and stored at ˆ’c 0eevaluation of the metabolism of ias during subchronicexposureafter collection of urine and feces following the singledoseadministration the hshs and wtwt mice were again cagedtogether one litter per cage and maintained on the purified dietand diw for wk to allow for clearance of as from the bodywhich was confirmed by measuring total as tas levels inurine see the œresults section for details both wt andhshs mice now to 27wkold were then exposed to iassodium arsenite pure sigmaaldrich in drinking water lg asl for wk spot urine samples ˆ¼ to llwere collected weekly body weights were recorded before andafter the exposure after wk all mice were sacrificed by cervical dislocation without anesthesia and tissues were collectedincluding the liver kidneys pancreas spleen heart lung adrenals kidneys bladder visceral fat calf muscle testes or ovaries and cecum and colon all tissues were flashfrozen in dryice and stored at ˆ’c 0eanalysis of as species in urine feces and tissuesspeciation analysis of as was carried out in 24h urines and fecescollected after the single dose of ias and in spot urine samplescollected during subchronic ias exposure the speciation analysis was also performed in livers and kidneys collected after subchronic exposure at sacrifice ten percent homogenates of liverand kidney were prepared in icecold diw wtvol usingwheaton potterelvehjem“style tissue grinders with a ptfepestle and wheaton overhead stirrer apparatus dwk lifesciences feces were snap frozen in liquid nitrogen and pulverized to powder using a steel mortar and pestle placed in dry icethe powder was mixed with 2n ultrapure phosphoric acid thermo fisher and digested in a mars5 microwave cemcorp for h at °c this method eliminates the biologicalmatrix but does not alter as speciation currier the digestates were neutralized by sodium hydroxide sigmaaldrich to ph the urines tissue homogenates and neutralized digestates were treated with lcysteine sigmaaldrich at room temperature for h to reduce pentavalent asspecies to their trivalent counterparts prior to the analysiscurrier the cysteinetreated samples were analyzed by hydridegeneration atomic absorption spectrometrycoupled with a cryotrap hgctaas as previously describedcurrier hern¡ndezzavala this analysis determined the concentrations of ias mas and dmas tasconcentration in urine feces and tissues was calculated as thesum of ias mas and dmas for assessment of the efficiencyof ias metabolism after a single oral dose the amount of tasenvironmental health perspectives august 0cwas expressed as the percentage of the dose by comparing theamount of elemental as administered as ias to each mousewith the amount of tas in 24h urine and feces samples collected from that mousethe instrumental limits of detection lod for ias mas anddmas using this method are and pg as respectivelyhern¡ndezzavala an imputed value of was usedfor measurements below the lod hgctaas is also capableof detecting and quantifying trimethylarsine oxide tmasohern¡ndezzavala a product of ias metabolism byrat as3mt waters however because tmaso israrely detected in human urine and because tmaso was not aproduct of ias methylation by recombinant human as3mt inour published study ding we did not includetmaso analysis in this studystatistical analysisoneway analysis of variance with studentnewmankeuls ortukey™s multiple comparison posttest was used to assess diï¬erences in gene expression and in the concentrations and proportions of as species among wtwt and hshs mice student™s ttest was applied for comparison of mouse and human geneexpression in a single tissue of hswt mice and for comparisonof as species concentrations or proportions between male andfemale mice and between hshs and wtwt mice of the samesex the statistical analyses were performed using prism software graphpad software diï¬erences with p were considered statistically significantresultsexpression of borcs7 and as3mt in hswt miceexpression of as3mt and borcs7 were directly compared withthose of endogenous mouse genes in tissues collected from f2male hswt mice that carried one copy of the mouse locus andone copy of the human locus figure 2a using both conventionalqpcr and when appropriate ddpcr expression of humanas3mt and borcs7 was easily detected in all tissues expressingthe corresponding mouse genes although the level of expressionof the human and mouse genes diï¬ered figure 2bc notable inthis analysis was
Colon_Cancer
neutrophils account for “ of circulating leukocytes and are the first immune cells recruitedto an ‚ammatory site they play an important role in the innate immune response topathogens as patients with neutropenia are highly susceptible to bacterial and fungal infections neutrophils perform numerous functions that target microbes including phagocytosis the releaseof antimicrobial peptidesproteases and netosis interestingly neutrophils have garneredconsiderable interest for their emerging and prominent roles in modulating cancer growth andmetastatic progression the roles played by neutrophils in the cancer setting are diverse andcomplex leading to the concept of neutrophil heterogeneityplasticity and the notion that distinctneutrophil subsets might existgranulopoiesisdiï¬erentiationand mobilization of maturefrom the bone marrow intocirculation this process begins with the commitment of granulocytemonocyte myeloidsegmented neutrophilsregulatedprocessthatinvolvestheisatightlyedited bybrahm segaluniversity at buffalo united statesreviewed byye liuniversity of texas md andersoncancer center united statesconnie jackamancurtin university australiacorrespondencepeter m siegelpetersiegelmcgillcaspecialty sectionthis was submitted tocancer immunity and immunotherapya section of the frontiers in immunologyreceived may accepted july published august citationhsu be shen y and siegel pm neutrophils orchestrators of themalignant phenotypefront immunol 103389fimmu202001778frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypegmps which progressthrough a series ofprogenitorsneutrophil progenitors myeloblast promyelocyte myelocytemetamyelocyte band cell untilthey become a matureneutrophil in cancer dysregulated granulopoiesis hasled to theidentification of diï¬erent neutrophil subsets that play a role intumor progression preneus comprise a neutrophil precursorpopulation that retain their proliferative capacity and expandin the bone marrow and spleen of tumor bearing mice preneus diï¬erentiate into immature and mature neutrophilswith the former found to accumulate in growing tumors anearly stage committed unipotent neutrophil precursor nep hasalso been identified and their adoptive transfer into humanizedmice promoted solid tumor growth by inhibiting t cellactivation two neutrophil subsets highdensity neutrophilshdns and lowdensity neutrophils ldns were identified invarious tumor models by diï¬erential density centrifugation hdns represent mature segmented neutrophils whereas ldnscomprise a heterogeneous mixture of mature and immatureneutrophils increasing mobilization of ldns into theperipheral blood was associated with enhanced tumor growthand metastasis “functionsand antitumorigenicin addition to the identification of distinct neutrophil subsetsneutrophils exhibit plasticity in response to tumorderivedfactors in a manner similar to macrophages neutrophils havebeen classified into two categories n1 and n2 to describetheir prorespectively in vivo evidence has shown that tumorassociatedneutrophils tans can change their function from a protumor phenotype n2 to an antitumor n1 phenotypewith the addition of a tgf inhibitor arguing that tgfis an important factor driving the n2 phenotype incontrast signals associated with an antitumor n1 phenotypeinclude type iinterferons and those propagated by themet receptor however this categorization is likelyto represent an oversimplification of neutrophil diversityneutrophil polarization similar to macrophages could alsorepresent a continuum of diï¬erent neutrophil phenotypespresent in the tumor microenvironment these advancesregarding the degree of neutrophil heterogeneityplasticityobserved in the cancer setting have sparked an intense andrenewed interestin this cell population while there areongoing discussions in the field regarding the relationshipsubsets we directbetween pmnmdscs and neutrophilto excellentthe readerthatfully discussthese we will briefly discuss antitumorrelationshipsneutrophilreview will primarilyroles of neutrophils and neutrophildiscussassociated functionsgrowth andmetastatic progressionfunctions howeverin promotingthe recentreviewstumorthisantitumor neutrophil functionscan participateantitumorneutrophilsmechanisms thattumor growth or eliminate cancercells a wellstudied neutrophilassociated function isin a variety oflimittheir ability to generate reactive oxygen species ros tolimit tumor progression upon tumor cell contact mousederived neutrophils can release hydrogen peroxide to eliminatemetastatic cancer cells in vitro subsequentlyit wasdemonstrated that expression of trpm2 transient receptorpotential cation channel subfamily m2 on tumor cells increasedtheirsensitivity to neutrophilmediated h2o2dependentcytotoxicity this occurred through a mechanism that involveda transient increase in ca2 mobilization within cancer cells trpm2 upregulation in tumor cells occurred followingan epithelialtomesenchymaltransition emt and cancercells that have undergone an emt were more susceptible toneutrophilmediated killing more recently an interactionbetween the receptor for advanced glycation end productsrage which is expressed on tumor cells and cathepsin gpresent on murine neutrophils was shown to mediate in vitrotumor cell cytotoxicity in a h2o2dependent manner the release of neutrophil ros is also dependent on the tumormicroenvironment in hypoxic tumor microenvironments theability of murine neutrophils to kill tumor cells in vivo throughthe release of ros is greatly diminished thus neutrophilshave the capacity to mediate rosdependent direct tumorcell killingcausingthe interplay of neutrophils with otherimmune celltypes can also indirectly limittumor progression tumorassociated neutrophils suppress the protumorigenic role ofil17 secreting Îδ t cells by inhibiting their proliferationlow glutathione levels in Îδ17 t cells rendered them sensitiveto neutrophilderived rosenhanced oxidativestress and reduced proliferation in earlystage humanlung cancer a subset ofimmature neutrophils have beenidentified as having antigenpresenting functions and act topromote antitumor immunity by stimulating the secretionofinaddition to neutrophilt cellinteractions communicationbetween neutrophils and monocytes can also elicit antitumoreï¬ects nonmetastaticifnÎproducing monocytes to the lungs ifnÎ release activatestmem173sting within neutrophils whichstimulatesneutrophilmediated killing of disseminated cancer cells in thelungs ‚ammatory cytokinesfrom t lymphocytescan mobilizecancercellsneutrophils have been shown to ltrate deposits of prostatecancer cells within bone metastases importantly neutrophilsimpaired bone metastasis progression by inhibiting stat5signal transducer and activator of transcription functionwithin prostate cancer cells resulting in their apoptotic cell death recently neutrophils have been reported to be involvedin antibodymediated trogocytosis a process that mechanicallydisrupts the plasma membrane of antibodyopsinized cancercells leading to a lyticnecrotictype cell death iga antibodiesagainst receptors expressed by cancer cells her2 egfr couldenhance neutrophilmediated trogocytosis of cancer cells if thecd47sirpα innate immune cell checkpoint was simultaneouslyblocked taken together these results demonstrate thatneutrophils can impair tumor growth and metastasis using acombination of direct and indirect cancer cell killing mechanismssupplementary table frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure neutrophil functions that promote tumor growth and metastasis to support primary tumor growth neutrophils can mediate t cell suppression and altermacrophage differentiation neutrophil release of timp1 enhances tumor cell invasion by inducing epithelialtomesenchymal transition once in circulation circulatingtumor cells interact with neutrophils which enables tumor cell proliferation secretion of various pro‚ammatory markers such as il8 il1 or mmps can mediateincreased tumor cell extravasation in addition neutrophils can inhibit intraluminal nkmediated killing of circulating cancer cells leading to increased extravasation atthe metastatic site various systemic and microenvironmental factors can promote neutrophil ltration neutrophils can awaken dormant cancer cells by promotingecm remodeling and angiogenesis lastly continued growth of the metastatic lesion is facilitated by key neutrophildependent mechanisms which includeangiogenesis proliferation immune suppression and immune exclusion csf1 colony stimulating factor timp1 tissue inhibitor of matrix metalloprotease pdl1programmed death ligand tgf transforming growth factor ros reactive oxygen species mmp matrix metalloproteinases gmcsf granulocyte macrophagecolony stimulating factor angptl2 angiopoetin like2 fgf2 fibroblast growth factor ltb4 leukotriene b4 inos inducible nitric oxide synthase net neutrophilextracellular trap caf cancerassociated fibroblastneutrophil functions thatpromote primary tumor growthneutrophils promote primary tumor growth by variousmechanisms figure netosis is a process that involvesthe extrusion of neutrophilderived chromatin structures thatare decorated with neutrophil granule constituents whichform extracellular structures called neutrophil extracellulartraps nets normally netosis and net productionhave been described in the context of a neutrophil™s ability tocapture and kill bacteria extracellularly however netshave been shown to play an important role in the growth of aprimary tumor tumor microenvironmental changes includingtumorassociated coagulation and enhanced thrombosishave been linked to enhanced tumor growth several recentstudies suggest that netosis may play an important role inthese processes lps stimulation was shown to increase c3arexpression within neutrophils enhance netosis and increasecoagulation these events were correlated with n2 neutrophilpolarization and increased tumor growth interestingly ithas recently been shown that immature neutrophils preferentiallyrespond to cancer cell derived c3a to promote their migration subsequently it was shown that breast cancer cells thatexpressed high levels of gcsf and il1 exhibited highneutrophil counts and tumorassociated thrombosis which wasdependent on net formation pharmacological blockadefrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeof il1 receptor signaling reduced net formation attenuatedtumorassociated thrombosis and impaired tumor growth nets can also directly ‚uence cancer cell proliferationneutrophil elastase ne present within nets activates tumorcells to increase mitochondria biogenesis and atp productionthereby further enhancing the growth of cancer cells in addition to the impact of nets neutrophils canalso interact with other immune cells through additionalmechanisms to promote tumor growth neutrophilderived roscan inhibit t cell proliferation creating an immunosuppressiveenvironment that is supportive of tumor growth phenotypiccharacterization and singlecell rna sequencing identified aneutrophil subset that is cd84hi which exhibited potent t cellsuppressive activity and increased ros production in amodel of gastric cancer neutrophils were activated by tumorderived gmcsf that resulted in elevated programmed deathligand pdl1 expression these pdl1 neutrophils wereable to suppress t cell function and promote tumor growth secretion of mmp9 matrix metalloproteinase fromltrating neutrophils activates latent tgf and induces tcell suppression and tumor growth in a colorectal cancer model siglecfhigh neutrophils in lung adenocarcinoma createdan immunosuppressive environment by promoting macrophagediï¬erentiation causing the release of high levels of rosand enabling tumor progression together these findingsindicate that neutrophils that ltrate diverse primary tumorscan modify the local environment in diï¬erent ways to favortumor growthneutrophil functions thatpromote metastasisthe ability of cancer cells to leave the primary tumor anddisseminate to distant ans represents the deadliest aspectof cancer progression indeed the emergence of metastaticcancer accounts for ˆ¼ of cancer related deaths themetastatic cascade represents a series of barriers to cancercells and neutrophils have been found to assist cancer cells insuccessfully navigating several of these distinct steps figure supplementary table local invasionintravasationltrating neutrophils within primary tumors are associatedwith an increase in emt enhanced metastasis and pooroutcomes mechanisticallyof matrixmetalloprotease timp1 secreted by neutrophils induced anemt and consequently increased the migration and invasion oftumor cells cancer cells that had undergone an emt expressedcd90 which enhanced timp1 secretion by neutrophils in acontactdependent manner inhibitortissuesurvival in circulationextravasationthe ability of circulating tumor cells ctcsto surviveis criticalfor metastasis formation the formation ofheterotypic cancer cell”neutrophil clusters was found to greatlyincrease metastatic fitness using a 4t1 breast cancer modelit was demonstrated that ctcneutrophil interactions reliedon vcam1 dependent adhesion which enhanced cancercell proliferation and increased metastasis indirectlyneutrophils can also inhibit nk cellmediated tumor clearancein circulation thereby increasing the intraluminal survival ofdisseminated tumor cells in this study 4t1 breast cancer cellswere injected subcutaneously to mobilize murine neutrophilsly6gfollowing which d2a1 breast cancer cells wereinjected intravenously mice bearing 4t1 cells exhibited reducedclearance of d2a1 cells from the lungs when compared to micethat were not injected with 4t1 cells depletion of nk cellsresulted in enhanced d2a1 cancer cell accumulation in the lungswhile neutrophil depletion had the opposite eï¬ect cancer cells that have survived in circulation must exitthe bloodstream and extravasate into tissue parenchyma neutrophils have been shown to regulate the extravasationprocessthrough several mechanisms neutrophilderivedfactors can diminish the integrity of the endothelial barrierpermitting cancer cellsil8il1 and matrix metalloproteasesmmp8 and mmp9released from neutrophils activated endothelial cells reducedendothelialtransendothelialmigration and accelerated the rate of cancer cell extravasation to extravasate more easilyincreasedfunctionbarriersites“netosis and net constituents can support cancer cellextravasation through enhanced trapping of ctcs withinmetastaticimportantly blocking netosisdecreases cancer cell adhesion and inhibits metastatic spread tothe lung and liver furthermore changes within specificmetastatic microenvironments such as exposure to ozoneor redox imbalance triggered netosis and led to increasedentrapment of cancer cells in the lung and enhanced metastasis collectively these studies show that neutrophils play animportant role in enhancing tumor cell survival and increasedextravasation which promote cancer metastasisrecruitmentearly seedingsurvivalsystemic and tumorderived factors have been implicatedin neutrophilin the premetastatic nichetumorderived il1 induces Îδ t cells to produce il17aand granulocytecolony stimulating factor gcsf whichresults in the recruitment of immunosuppressive neutrophilsto the lung gmcsf and il5 have been shown topromote the expansion and recruitment of prometastaticneutrophils in the lungs of obese mice which promotes lungmetastasis angiopoetinlike2 angptl2 secreted byosteosarcoma cells implanted in the tibia stimulates lungepithelial cells which led to the accumulation of neutrophilsin the lung and enhanced lung metastatic burden in the lung neutrophils secrete ltb4increases theinitiating cellsproliferation of ltb4rpositive metastasis activation of notch1 in colorectalcellsdrives tgf2dependent recruitment of immunosuppressiveneutrophils within the liver which enabled the formation of livermetastases cancerthatnets also support early cancer cell seeding and colonizationof metastases induction of nets by ovarian tumorderivedfactors has been shown to be important in promoting metastasisfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeto the omentum in the liver nets have also beenshown to promote metastasis by activating cancerassociatedfibroblasts growth in the metastatic siteneutrophils have been shown to promote the growth ofmetastases after seeding minor subclones of breast cancer cellsthat secrete il11 and figf cfosinduced growth factor cansupport the formation of polyclonal metastases composed ofdriver and passenger subpopulations these il11 producingsubclones activated il11 responsive mesenchymal stromal cellswhich induced chemokine secretion and subsequent recruitmentof prometastatic neutrophils tumor cellderived gmcsfwas shown to stimulate neutrophils to synthesize and secretetransferrin an iron transport protein which has mitogenicactivity that promotes lung metastatic growth when taken up bycancer cells a recurring function of prometastatic neutrophils is theirability to create an immunosuppressive microenvironmentthat support metastasis within lung metastasesinduciblenitric oxide synthase inos producing neutrophils havebeen shown to limit cd8 t cell dependent antitumorresponses by promoting immune suppression recently p53deficient cancer cells were found to increase the expressionof wnt ligands which in turn upregulated il1 productionfrom tumorassociated macrophages high il1 levelsengaged Îδ17 t cells which subsequently enhanced neutrophilrecruitment that promoted the formation of lung metastases furthermore loss of elf5 e74like transcription factorexpression in triplenegative breast cancer led to increased ifnÎ signaling resulting in the expansion of immunosuppressiveneutrophils in addition to tumorderived factors a lackof systemic testosterone levels can lead to an impairmentof antitumor neutrophil functions a shift toward immatureneutrophils was observed in castrated male mice leading toincreased neutrophilderived ros and suppression of nk cellactivation that promoted increased lung metastatic burden intwo melanoma models recently a role for net formationhas been described for the continued growth of establishedmetastases nets released during cancer progressionwas shown to limitthe ability of nk and cytotoxic tcells to eliminate cancer cells specifically net formationimpaired direct contact between the cancer cells and cytotoxicimmune cells nk and t cells inhibition of netosis with aprotein arginine deiminase pad4 inhibitor synergized withimmune checkpoint inhibitors to control tumor growth andmetastasis proangiogenic functions have long been ascribed forneutrophils which revealed that neutrophilderived proteasessuch as mmp9 could release stored angiogenic factors vegffgfs that were stored in the local environment to enable bloodvessel formation recently a diï¬erent mechanism bywhich neutrophils enhance angiogenesis has been describedthe synthesis and secretion of fibroblast growth factor fgf2 by neutrophils in the liver microenvironment drivesangiogenesis and growth of nascent colorectal cancerderivedhepatic metastases dormantresidual disease andtherapy resistanceneutrophils have also been implicated in awakening dormantcancer cells lpsinduced tissue ‚ammation led to metastaticoutgrowth of dormant tumor cells in a neutrophildependentmanner mmp9 produced by neutrophils can trigger thegrowth of dormant cancer cells by remodeling extracellularmatrix and releasing potent angiogenic factors ne andmmp9 which are enzymes associated with nets can cleavethe extracellular matrix ecm leading to integrinmediatedsignaling which awakens dormant cancer cells and promotescancer cell growth severalstudies have shown that neutrophils promoteresistance to therapy doxorubicin and paclitaxel resistant breastcancer cells express more il17 and cxcr2 ligands whichincreases neutrophil recruitment a neutrophilenrichedsubtype characterized in triple negative breast cancer tnbcdetermined that neutrophils were largely immunosuppressiverendering these tumors resistant to immune checkpoint blockadetherapy in a genetically engineered mouse model ofsarcoma neutrophils promote resistance to radiation therapyby activating mitogenactivated protein kinase mapk pathway in addition cd177 neutrophil ltrates in colorectalcancer patients are associated with adverse outcome in patientsreceiving bevacizumab [antivascular endothelial growth factora vegf a] furthermore lysyl oxidaselike loxl4expressing neutrophils that ltrated colorectal cancer livermetastases were found to identify patients that were resistant toantiangiogenic therapy metabolic programming inneutrophilsrecentinteresthasbeenconceptin thethereofimmunometabolism and the realization that altered cellularmetabolism in ltrating immune cells can have a significantimpact on tumor growth and metastasis neutrophilsare typically viewed as a cell type that is heavily reliant onglycolysis to perform their eï¬ector functions consistentwith this notion neutrophils have very few mitochondria andinhibitors of oxidative phosphorylation oxphos do notalter their rates of oxygen consumption howeverduring tumor progression neutrophils have been shown toundergo a metabolic switch which involves the upregulationof genes associated with oxphos fatty acid metabolism andglycolysis figure neutrophils isolated from lewis lungcarcinoma exhibit increased flux through oxphos glycolysisand increased atp production compared to naïve neutrophilssuggesting that multiple metabolic strategies are engaged intumor ltrating neutrophils recently upregulation offatp2 fatty acid transport protein in neutrophils was shownto increase lipid accumulation in these cells fatp2 regulated theuptake of arachidonic acid which was subsequently convertedto prostaglandin e2 neutrophilderived prostaglandin e2 wasfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure metabolic changes in cancerassociated neutrophils neutrophils which possess few mitochondria are reliant on glycolysis to generate atp to fueleffector functions such as phagocytosis generation of reactive oxygen species and netosis in cancer neutrophils upregulate oxidative phosphorylation oxphosand fatty acid transporters to mediate many neutrophil functions including migration and t cell suppression under nutrient limiting conditions such as low glucoseneutrophils can reprogram their metabolism to break down fatty acids or utilize certain amino acids glutamate proline to fuel protumorigenicprometastaticfunctions ppp pentose phosphate pathway glut glucose transporter mct monocarboxylate transporter tca tricarboxylic acid cycle fatp2 fatty acidtransport protein aa arachidonic acid pge2 prostaglandin e2found to be important or neutrophilmediated cd8 t cellsuppression and tumor growth metabolic flexibility refers to the ability of a cell to shiftbetween one metabolic program to another in response tochanging metabolic demands or nutrient supply high metabolicflexibility increases the cell™s ability to survive various andeverchanging metabolic microenvironments neutrophilsubpopulations can also exhibit metabolic flexibility figure in breast cancer splenic neutrophils can engage mitochondrialdependent fatty acid oxidation as a predominate fuel sourceto support ros production and maintain t cell suppression under glucoselimiting conditions similar to certain tumormicroenvironments immature ldns have been shown to utilizeoxphos to generate atp that is required to support theirprotumorigenic functions indeed immature ldns can supportnetosis under nutrient limiting conditions via mitochondrialdependent amino acid catabolism which is importantforefficient breast cancer liver metastasis in addition thelongevity of neutrophils could also be altered due to the enhancedmetabolic flexibility the ex vivo halflife of mouse circulatinghdns and ldns was and h respectively suchobservations raise the intriguing possibility that under certainconditions distinct neutrophil subsets may not be as shortlived as previously thought these studies argue that increasedmetabolic flexibility in distinct neutrophil populations may beimportant for cellular functions that can ‚uence tumor growthand metastatic progressionclinical importance futureperspectives on treatmentin keeping with their protumorigenicmetastatic functions thepresence of neutrophils across diï¬erent cancers was shownto be strongly associated with adverse patient outcomes frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeamong certain subtypes of breast cancer er the presence ofa neutrophil ltrate in the primary tumor is also indicativeof worse patient outcomes furthermore in patients withadvanced cancers serum il8 levels and neutrophil ltrationare associated with worse overall survival and diminishedresponse to immune checkpoint inhibitors the mobilization of neutrophils into circulation also hasprognostic significance the neutrophiltolymphocyte rationlr is an important risk stratification and treatment selectiondiagnostic tool for cancer patients a high nlr is associated withpoor prognosis in many solid human cancers “ a highnlr is also associated with decreased overall survival in patientswith tnbc or metastatic breast cancer an important and unanswered question with respect to thenlr is the type of neutrophil that is being detected in thesepatients are they high or lowdensity neutrophils interestinglyldns have been identified in patients with breast cancer lungcancer head and neck cancers urologic cancers and lymphoma “ in patients with advanced lung cancerit wasreported that higher proportion of ldns predictedpoorer survival these observations are in keeping withthe protumorigenic and prometastatic functions associatedwith ldnn2 neutrophils while most studies reveal a negativeprognostic impact of neutrophils in cancer there was one studythat associated the presence of a cd16high cd62dim neutrophilsubset with increased survival of head and neck squamous cellcarcinoma patients these observations highlight the needfor better markers that are capable of discriminating betweenneutrophils that exert antitumor vs those that mediate protumormetastatic eï¬ectsmechanistic insights have greatly advanced our knowledgeoftumorderived factorstumor growth andmetastasis in a neutrophildependent manner additional studiesimpactthatreferences sipsas nv bodey gp kontoyiannis dp perspectives for the management offebrile neutropenic patients with cancer in the 21st century cancer “ 101002cncr20890 kolaczkowska e kubes p neutrophil recruitment and function in healthand ‚ammation nat rev immunol “ 101038nri3399inde visser ke neutrophilssb wellenstein md coï¬eltcancer neutral no more nat rev cancer 101038nrc201652 “ cowland jb borregaard n granulopoiesis and granules of humanneutrophils immunol rev “ 101111imr12440 evrard m kwok iwh chong sz teng kww becht e chenbone marrow neutrophilstraffickinganalysisspecializedpopulationsexpansioninofal developmentaljetrevealsand 101016jimmuni201802002functionseï¬ectorimmunity“79e8 zhu yp padgett l dinh hq marcovecchio p blatchley a wu r identification of an early unipotent neutrophil progenitor with pro tumoralactivity in mouse and human bone marrow cell rep “41e8 101016jcelrep201807097 sagiv jy michaeli j assi s mishalian i kisos h levy l phenotypicdiversity and plasticity in circulating neutrophil subpopulations in cancercell rep “ 101016jcelrep201412039focused on characterizing the phenotypic and functional role ofneutrophils in cancer it may be possible to develop strategies thatspecifically target those neutrophil subsets that actively promotetumor growth and metastasis while sparing those neutrophilsthat possess antitumor and antimicrobial functions finallythe emerging concept of metabolic flexibility that is exhibited bycertain neutrophil subsets may aï¬ord new ways of targeting theseprotumorigenicmetastatic neutrophilsauthor contributionsbh ys and ps wrote the review and prepared the figuresall authors contributed to the and approved thesubmitted versionfundingwork from the authors laboratory cited in this review wassupported by an operating grant to ps from the cancer researchsociety and the terry fox research institute and québecbreast cancer foundation grant bh acknowledgessupport from the charlotte and leo karassik foundation phdfellowship and the rolande and marcel gosselin graduatestudentship ys holds an entrance studentship from thegoodman cancer research centre ps is a mcgill universitywilliam dawson scholarsupplementary materialthe supplementary materialfor this can be foundonline at httpswwwfrontiersins103389fimmu202001778fullsupplementarymaterial coï¬elt sb kersten k doornebal cw weiden j vrijland k hau cs il17producing gammadelta t cells and neutrophils conspireto promote breast“ 101038nature14282cancer metastasis nature hsu be tabaries s johnson rm andrzejewski s senecal j lehuede c immature lowdensity neutrophils exhibit metabolic flexibility thatfacilitates breast cancer liver metastasis cell rep “15e6 101016jcelrep201905091 fridlender zg sun j kim s kapoor v cheng g ling l polarizationof tumorassociated neutrophil phenotype by tgfbeta œn1 versus œn2tan cancer cell “ 101016jccr200906017 ohms m möller s laskay t an attempt to polarize human neutrophilstoward n1 and n2 phenotypes in vitro front immunol 103389fimmu202000532jablonska j leschner s westphal k lienenklaus s weiss s neutrophilsresponsive to endogenous ifnbeta regulate tumor angiogenesis andgrowth in a mouse tumor model j clin invest “ 101172jci37223 finisguerra v di conza g di matteo m serneels j costa s thompsonaa met is required for the recruitment of antitumoural neutrophilsnature “ 101038nature14407 ostuni r kratochvill f murray pj natoli g macrophages and cancer frommechanisms to therapeutic implications trends immunol “ 101016jit201502004frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotype brandau s moses k lang s the kinship of neutrophils and granulocyticmyeloidderived suppressor cells in cancer cousins siblings or twins semincancer biol “ 101016jsemcancer201302007 vols s sionov rv granot z always look on the bright side antitumor functions of neutrophils curr pharmac design “ granot z henke e comen ea king ta norton l benezra r tumorentrained neutrophils inhibit seeding in the premetastatic lung cancer cell “ 101016jccr201108012 gershkovitz m caspi y fainsodlevi t katz b michaeli j khawaled s trpm2 mediates neutrophil killing of disseminated tumor cells cancer res “ 10115800085472can173614 gershkovitz m fainsodlevi t khawaled s shaul me sionov rvcohendaniel l microenvironmental cues determine tumor cellsusceptibility to neutrophil cytotoxicity cancer res “ 10115800085472can180540 sionov rv fainsodlevi t zelter t polyansky l pham ct granotz neutrophil cathepsin g and tumor cell rage facilitate neutrophilanti8e1624129 1010802162402x20191624129cytotoxicity oncoimmunologytumor mahiddine k blaisdell a ma s crequergrandhomme a lowell caerlebacher a relief of tumor hypoxia unleashes the tumoricidal potentialof neutrophils j clin invest “ 101172jci130952 mensurado s rei m lanca t ioannou m goncalvessousa n kubo h etal tumorassociated neutrophils suppress protumoral il17 gammadeltat cells through induction of oxidative stress plos biol 16e2004990 101371 pbio2004990 singhal s bhojnagarwala ps o™brien s moon ek garfall al rao as etal origin and role of a subset of tumorassociated neutrophils with antigenpresenting cell features in earlystage human lung cancer cancer cell “ 101016jccell201606001et hagerling c gonzalez h salari k wang cy lin c roblescooperationtumor prevents metastatic progressionaliinduced byof breast cancer proc natl acad sci usa 101073pnas1907660116eï¬ector monocyteneutrophilimmunethe primary “ costanzogarvey dl keeley t case aj watson gf alsamraae myu y neutrophils are mediators of metastatic p
Colon_Cancer
"objective endoscopic full thickness resection eftr has shown efficacy and safety in the colorectum the aim of this analysis was to investigate whether eftr is cost effective in comparison with surgical and endoscopic treatment alternativesdesign real data from the study cohort of the prospective single arm wall resect study were used a simulated comparison arm was created based on a survey that included suggested treatment alternatives to eftr of the respective lesions treatment costs and reimbursement were calculated in euro according to the coding rules of and eftr r0 resection rate was used as a measure of effectiveness to assess cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were determined calculations were made both from the perspective of the care provider as well as of the payerresults the cost per case was ‚¬ for the eftr group ‚¬ for the standard endoscopic resection ser group ‚¬ for the surgical resection group and ‚¬ for the pooled alternative treatment to eftr from the perspective of the care provider the acer mean cost per r0 resection was ‚¬ for eftr ‚¬ for ser ‚¬ for surgical treatment and ‚¬ for all pooled and weighted alternatives to eftr the icer additional cost per r0 resection compared with eftr was ‚¬ for ser ‚¬ for surgical resection and ‚¬ for the pooled rate of alternatives results from the perspective of the payer were similar eftr is cost effective in comparison with surgical and endoscopic treatment alternatives in the colorectumintroductioncolorectal cancer is the third most common type of cancer and the second most common cause of cancer related deaths worldwide1 screening programmes for early detection of premalignant and malignant lesions led summary boxwhat is already known about this subject –º endoscopic full thickness resection eftr has shown clinical efficacy and safety in difficult to treat lesions in the colorectum –º the cost of the full thickness resection device is higher than the cost of standard endoscopic resection ser devices but lower than surgical devices –º cost effectiveness analyses on treatment with eftr compared with treatment alternatives do not existwhat are the new findings –º eftr leads to an almost reduction in cost per r0 resection average cost effectiveness ratio compared with surgery –º to achieve an additional r0 resection by surgical treatment compared with eftr incremental cost effectiveness ratio an additional cost of ‚¬ is necessary –º these findings are consistent both from the perspective of the care provider as well as the payerhow might it impact on clinical practice in the foreseeable future –º in terms of cost effectiveness eftr should be considered first before patients with difficult to treat lesions in the colorectum are sent to surgical treatmentto a significant reduction in cancer related mortality2 with more intense screening more lesions are detected which automatically creates the need for removal standard endoscopic resections ser such as endoscopic mucosal resection emr and endoscopic submucosal dissection esd are well established and sufficient for the vast majority of lesions however ser of non lifting lesions and lesions located at difficult anatomical to cite kuellmer a0a behn a0j beyna a0t et a0al endoscopic full thickness resection and its treatment alternatives in difficult to treat lesions of the lower gastrointestinal tract a cost effectiveness analysis bmj open gastro 20207e000449 101136bmjgast2020000449received may revised july accepted july authors or their employers re use permitted under cc by nc no commercial re use see rights and permissions published by bmjfor numbered affiliations see end of correspondence toarthur schmidt arthur schmidt uniklinik freiburg dekuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access locations eg appendiceal orifice is associated with increased complication rates or incomplete resection3“ these types of lesions are therefore often referred to surgery which is associated with significant morbidity and mortality and higher costs6 given the high number of polypectomies performed worldwide this is not only an issue of morbidity and mortality but also a huge economic challengethe efficacy and safety of endoscopic full thickness resection eftr of non lifting and other difficult to treat lesions have been demonstrated in multiple retrospective studies and in one prospective study7 the cost of the device is markedly higher than ser devices but lower than surgical treatment the aim of the present analysis was to evaluate whether eftr is cost effective in comparison with ser as well as surgical treatmentmethodsstudy populationto calculate the cost of eftr we analysed the study cohort of the wall resect trial nct02362126 in this single arm multicentre prospective study patients with ˜difficult to treat™ adenomas eg non lifting andor challenging anatomical location early adenocarcinomas and subepithelial tumours in the colorectum were treated with eftr the primary endpoints of the study en bloc and r0 resection rate were achieved in and respectively7 written informed consent was obtained from each patient included in the studysimulation second study arm based on a survey of endoscopistswith the wall resect study being single armed a second study arm was created based on treatment simulation in order to compare different treatment modalities a case report form crf was created and sent to each participating centre of the wall resect trial endoscopists at the respective location reviewed the endoscopic images and their case relevant data and decided which treatment modality they would have chosen if eftr were not available treatment alternatives included ser such as emr thermal methods and esd as well as surgical resection the crf was filled out in a pseudonymised fashioninformed consent had already been obtained within the wall resect studydetermination of case costs and reimbursementa certified online it tool the diagnosis related group drg web grouper was used to determine the reimbursement rate for each patient http drg uni muenster de index php therefore the code of the international classification of diseases icd10 and the specific code for the procedure performed operationen und prozedurenschl¼ssel ops code in each patient in both groups were put into the web grouper together with the predefined mean length of hospital stay ˜mittlere grenzverweildauer™ the drg which accounts for reimbursement was calculatedin the comparison arm the drg codes of were used because this was the year the wall resect study was performed for the eftr arm the drg codes of were used as reimbursement for eftr was increased that yearto calculate the cost per case another certified online it tool g drg report browser was used www g drg de g drg system_ abschlussbericht_ zur_ weiterentwicklung_ des_ g drg systems_ und_ report_ browser this was done by filling in the respective drg icd10 and ops code into the browser the data of the g drg report browser derive from the data that were sent in to inek authority managing the german drg system by certified hospitals ˜kalkulationshuser™ in grouping was performed following the rules of g drg version the main and secondary diagnoses are shown according to icd10 german modification gm version and the procedures according to ops version ˜g drg report browser inek gmbh™as reimbursement for the eftr group was taken from the cost per patient case would ideally also have been calculated from unfortunately these data will be first published by inek in to overcome this problem costs for eftr cases from the university of freiburg between and which were reported to inek were used for the analysiswith the cost of each patient case the mean cost for each treatment modality emr esd laparoscopic surgery transanal endoscopic microsurgery tem eftr could be calculated in the next step the mean cost for each treatment path ser surgical treatment and casemix alternative was determined this was done in the following fashion for ser the mean costs of emr and esd were used for calculation of the surgical treatment laparoscopic surgery and tem were taken together the mean costs of endoscopic and surgical treatments were subsumed as the casemix alternativedetermination of effectivenessthe r0 resection rate was defined as the efficacy parameter to determine cost effectiveness the r0 rate of eftr in the wall resect trial was to determine the efficacy of the therapeutic alternatives to eftr a selective literature review was performed in pubmed and cochrane databases identifying the largest studies comparing resection techniques and r0 rates the respective rates regarding ser found in the literature were for emr and for esd9 for the surgical oncological resection treatment as the gold standard a r0 resection rate was assumed for the tem a rate of had been reported10in order to compare all ser methods emresd all surgical resection methods laparoscopic surgerytem and all alternative methods endoscopic and surgical kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0ctable alternative treatment strategies to eftr with their respective efficacy based on literature review and calculationtreatmentefficacy n n180surgical oncological resection laparoscopictememresdsurgical treatment laparoscopic and temser emresdcasemix alternative assumed arezzo et al fujiya et al arezzo et al calculated calculated calculatedthe combined effectiveness of surgical treatment ser and casemix alternative was calculated by multiplication of the number of patients in each modality eg emr cases for ser with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group overall efficacy of surgical treatment and casemix alternative was performed in the same mannereftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgery˜casemix alternative™ with the eftr procedure a combined effectiveness of each treatment group was calculated this was done by multiplication of the number of patients in each modality eg emr cases with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group using this approach the ˜overall™ efficacy in the ser group was calculated as overall efficacy of surgical treatment and casemix alternative was performed in the same manner and was calculated as and respectively the respective r0 rates are shown in table calculation of costeffectivenessfor assessment of cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were calculated acer expresses the mean costs for the investigated outcome11 in our study acer describes the mean costs per successful r0 resection in the different treatment modalitiesacer is calculated with the following computational formula acer mean costseffect open accessicer expresses the additional costs of a treatment alternative for improvement in the investigated outcome12 in our study these are the incremental costs for the alternative treatment to eftr required to achieve an r0 resectionicer is calculated with the following computational formula icer mean costs interventionˆ’mean costs controleffect interventionˆ’ effect control the mean costs were the total costs of the respective treatment modality divided by the number of patients in each group for the calculation of cost effectiveness the ser methods emr and esd as well as the surgical resection methods laparoscopic resection and tem were taken together furthermore cost effectiveness was calculated for the casemix alternative to compare eftr with all alternativesresultscomparative study armendoscopist surveyfrom patients of the study cohort responses were included for further analysis in one patient the investigator recommended solely ˜thermal ablation™ as alternative treatment of choice thus the primary endpoint r0 resection could not be evaluated from the remaining patients the endoscopists recommended surgical treatment in of of cases thereof of were laparoscopic resections and of tem in of of cases an endoscopic resection was proposed thereof of were emr and of were esdcosts from the perspective of the care providercosts per case were derived from the drg report browser which represent costs of the respective treatment alternative for the year the costs for the eftr treatment were derived from university hospital freiburg and represent the mean costs from years to mid2019 the mean cost for eftr was ‚¬ the cost per surgical treatment laparoscopic surgery and tem was ‚¬ and for ser ‚¬ all alternative treatment strategies ˜casemix alternative™ op laparscopic surgery tem esd and emr were calculated as ‚¬ per case the results are shown in figure costs from the perspective of the thirdpartyaccording to the german drg system reimbursement for eftr is ‚¬ for surgical treatment ‚¬ was calculated the cost per case for ser is ‚¬ the cost for the casemix alternative is ‚¬ the results are shown in figure costeffectiveness analysis care provider viewpointaverage costeffectiveness ratiothe mean cost per r0 resection is ‚¬ in the eftr group and ‚¬ in the surgical group in the ser group the cost per r0 resection is ‚¬ in the casemix alternative group including all treatment kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access figure case costs ‚¬ for the different treatment modalities are shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryfigure incremental cost effectiveness ratio for the different treatment modalities compared with eftr is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryalternatives except eftr the mean cost per r0 resection is ‚¬ the results are shown in figure the casemix alternative ‚¬ the results are shown in figure incremental costeffectiveness ratioin comparison with eftr the incremental cost for an additional r0 resection is ‚¬ if ser is performed the cost for the surgical approach is ‚¬ and for figure average cost effectiveness ratio ‚¬ for the different treatment modalities is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgerycosteffectiveness analysis health insurance reimbursement viewpointaverage costeffectiveness ratiofrom the perspective of the health insurance the cost per r0 resection is ‚¬ in the eftr group in the ser group the cost is ‚¬ and in the surgical treatment group ‚¬ in the casemix alternative the cost per r0 resection is ‚¬ the results are shown in figure incremental costeffectiveness ratiothe icer of ser in comparison with eftr is ‚¬ the surgical approach costs an additional ‚¬ for the casemix alternative ‚¬ is necessary for an additional r0 resection the results are shown in figure discussionwith technical endoscopic progress patient care has constantly improved over the years however as with any technical innovation this is associated with higher costs therefore the efficacy of new methods and devices needs to be evaluated in relation to their costs13 to our knowledge this is the first cost effectiveness analysis cea for eftr our results demonstrate that eftr for difficult to treat lesions in the colorectum is cost effective in comparison with ser as well as surgical therapy furthermore the results are consistent when analysed from the perspective of the care provider as well as of the payerkuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0cfor our analysis a simulated control arm was created this was necessary as to date no randomised controlled trial rct investigating eftr versus alternative treatments has been published in our survey endoscopists proposed surgical treatment as the likely alternative to eftr in the majority of cases as opposed to ser via emr or esd all lesions within the wall resect trial were ˜difficult to resect™ lesions eg non lifting adenomas exhibiting a high risk of perforation or incomplete resection when treated with ser therefore it may be surprising that ser was suggested in of cases however the suggestions were made by expert endoscopists who might have decided towards an advanced endoscopic procedure more generouslyregarding the costs for each treatment modality it was not surprising that the cost of eftr is above ser ‚¬ vs ‚¬ this is due to the cost of the device in germany ‚¬ plus value added tax however the cost of eftr was roughly one third of the cost of surgery ‚¬ vs ‚¬ this reflects the minimally invasive nature of eftr compared with laparoscopic or open surgical operationswhile costs for endoscopic resection and surgical therapy were taken from official and certified tools web grouper and drg report browser the factual costs of the eftr procedure for the year that are determined in a representative cross section of hospitals have not been published yet by inek the administrator of the drg system reimbursement of the procedure changed in thus these data should have been used for calculation to overcome this problem the mean case costs for eftr per case in our home institution university hospital of freiburg germany in the time between and were used as a surrogate in an economic analysis of the cost of eftr in germany presented at the annual conference of the german society for digestive diseases obtained from different endoscopic centres reported ‚¬ per case as this is only above our number and therefore in the same range our calculated ‚¬ seems to be a realistic number14in our analysis we chose the r0 rate as a means to detemine effectiveness as this is the most objective parameter to assess curative resection and treatment success furthermore the r0 rate can be compared with the treatment alternatives of eftr as high quality meta analyses and therapeutic success rates exist for those procedures10 the r0 rate for surgical colonic resection was assumed to be however the patient cohorts of these studies are not equal the wall resect study included only ˜difficult to resect™ lesions mainly non lifting while the studies mentioned above included primarily treatment naive lesions larger studies on ser on non lifting lesions do not exist hence it is reasonable to assume that in these indications real r0 rates of ser would be lower and therefore cost effectiveness would be even worsefor measuring cost effectiveness acer and icer were determined the analysis was performed both open accessfrom the perspective of the care provider hospital as well as the reimbursement authority health insurance acer expresses the mean cost per r0 resection for both investigated perspectives our results reveal that costs are much lower for eftr compared with the surgical alternative although the effectiveness of the surgical approach in terms of radicality can be considered to be higher eftr is cost effective an r0 resection by eftr leads to nearly reduction in costs for the care provider ‚¬‚¬ and for the health insurance ‚¬‚¬ compared with ser eftr leads to marginally higher costs per r0 resection as explained above comparing eftr with ser has limitations as the investigated ˜difficult™ lesions in the wall resect study are not well studied for emr and esd however in comparison with all treatment alternatives ˜casemix alternative™ we calculated and reduction in costs similar to the surgical alternatives figure icer expresses the additional costs for an additional increase in the designated outcome in our analysis it expresses the additional costs that are necessary for an additional r0 resection as shown in figure all alternatives to eftr result in additional costs while ser results in a modest increase ‚¬ and ‚¬ additional ‚¬ and ‚¬ per r0 resection are required in the surgical group in the ˜casemix alternative™ group additional costs were ‚¬ and ‚¬ respectivelyan absolute threshold at which an icer is thought to be cost effective does not exist16 in the literature the willingness to pay threshold ranges from to “ and is highly subjective to the investigated outcome and the healthcare system for which the cea is made17“ for our analysis we assume that a more invasive treatment that produces at least ‚¬ more costs for an additional r0 resection cannot be regarded as being cost effectivefor our analysis we did not include costs of follow up endoscopies or further treatment arising from recurrency or from adverse events this was done due to the following reasons first reliable recurrence rates and long term follow up after eftr do not exist follow up in the wall resect trial was only weeks second the lesions of our patient cohort were heterogeneous including adenomas carcinomas and neuroendocrine tumours with different biological features and recurrent rates third treatment of recurrent lesions is not standardised and ranges from re eftr to snare polypectomy to removal with a biopsy forceps leading to highly variable costs fourth management of severe complications and consecutive morbidity differs in every patient and depends on severity of complication patients™ comorbidities and local expertise we do not have reliable data on costs for such treatment and a hypothetical model would have been highly speculative moreover in the wall resect trial of patients required consecutive surgery due to complications this rate is slightly kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access higher but still grossly comparable with the complication rates of emr and esd on the other hand complications after surgical resection eg anastomotic leakage are much more frequent up to “ and usually lead to higher morbidity hence even if costs related to complications were added icer is still likely to favour eftr compared with the group of treatment alternativesit is difficult to compare our results with other ceas as this is the first one for this indication the only previous cea on ser compared emr and esd in laterally spreading lesions irrespective of location or lifting sign in most analyses as in the study by bahin and colleagues19 a decision tree model was created to compare different outcome scenarios after each treatment path was filled with probabilities of occurrence costs per predefined outcome were calculated a potential bias of this approach is that the data for the probabilities of occurrence which influence the costs most are taken totally or at least in part from different studies19 22this harbours the risk of resulting in a very heterogeneous study population with uncontrolled confounders this risk can be minimised by deriving data from rcts with well balanced patient cohorts as recently published17 for our analysis we used a different approach than a decision tree factual variables and outcome data derived from the only prospective study on eftr treatment and not from assumptions the simulation of the control arm had to be performed due to the lack of rcts in this setting the strength of our study is that the very same clinician who actually performed the respective eftr could review the different lesions and decide on a solid basis which treatment alternative he or she would have used instead of eftr in our view this approach reflects the clinical situation more precisely than a decision tree modelin most ceas the costs per quality adjusted life years are calculated and taken for healthcare decisions neither survival nor quality of life measurements were part of the wall resect trial in line with most of the recently published cea we calculated costs per defined outcome as the primary endpoint17 our study has several limitations first the comparison arm of the study is based on simulation so there is always a risk of a bias second our analysis is specific to the german healthcare system and may therefore not be fully comparable with different healthcare systems in the world third the estimated r0 rate for the ser methods is very low and likely due to the piecemeal resection in the respective study if efficacy would have been measured as ˜freedom of recurrence™ efficacy would be higher as proven in the australian colonic endoscopic study24 nonetheless we used the published r0 rate because of the possibility to match this with the endpoint of the wall resect study furthermore as described above an endpoint such as freedom of recurrence cannot be determined reliably as such data do not exist for eftr fourth costs of complications and follow up were not included this is mainly due to lack of an rct and the short follow up period in an ideal cea all treatment related and hospital stay related costs would have been calculatedin our data indicate that eftr for difficult to treat lesions of the colorectum is cost effective compared with surgical and endoscopic treatment alternatives the results are consistent both from a care provider as well as from a third party payer perspective rcts and long term follow up are needed to further assess the cost effectiveness of eftrauthor affiliations1department of medicine ii medical center “ university of freiburg faculty of medicine university of freiburg freiburg germany2department of gastroenterology klinikum ludwigsburg ludwigsburg baden w¼rttemberg germany3department of gastroenterology evangelisches krankenhaus d¼sseldorf dusseldorf nordrhein westfalen germany4department of internal medicine and gastroenterology elisabeth hospital essen nordrhein westfalen germany5department of medicine ii interventional and experimental endoscopy inexen university hospital wurzburg wurzburg bayern germany6department of gastroenterology university hospital augsburg augsburg bayern germany7department of gastroenterology university hospital ulm ulm baden w¼rttemberg germany8department of gastroenterology klinikum dortmund dortmund nordrhein westfalen germany9department of gastroenterology helios klinikum krefeld krefeld nordrhein westfalen germany10department of gastroenterologyoncology klinikum sindelfingen b¶blingen sindelfingen baden w¼rttemberg germanycontributors as and kc invented and planned the present study and also the underlying wall resect study as assisted with data acquisition and analysis and revised the manuscript jb was responsible for data research and acquisition ak was responsible for data analysis and writing the manuscript kc tb bs am hm hn da mb ap mf tf mg and rt took part in the online survey to create the simulation comparison arm of the study furthermore they carefully read and revised the manuscriptfunding the authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorscompeting interests as and kc received lecture fees and study grants from ovesco endoscopy t¼bingen germany ak jb tb bs am hm hn da mb ap mf tf mg and rt have no conflicts of interest or financial ties to disclosepatient consent for publication not requiredethics approval the wall resect study was approved by the ethical board on january the study protocol conforms to the ethical guidelines of the declaration of helsinki as reflected in a prior approval by the institution's human research committee for the present study an additional approval by the institutional review board was not necessary since no additional personal data were collectedprovenance and peer review not commissioned externally peer revieweddata availability statement all data relevant to the study are included in the or uploaded as supplementary information data were derived from the wall resect trial nct02362126open access this is an open access distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idarmin a0kuellmer http orcid org kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0creferences who colorectal cancer fact sheet the global cancer observatory available http gco iarc fr today data factsheets cancers 10_ 8_ colorectum fact sheet pdf [accessed sep ] zauber ag winawer sj o'brien mj et a0al colonoscopic polypectomy and long term prevention of colorectal cancer deaths n engl j med “ hong sn byeon js lee b i et a0al prediction model and risk score for perforation in patients undergoing colorectal endoscopic submucosal dissection gastrointest endosc “ org mizushima t kato m iwanaga i et a0al technical difficulty according to location and risk factors for perforation in endoscopic submucosal dissection of colorectal tumors surg endosc “ agapov m dvoinikova e factors predicting clinical outcomes of endoscopic submucosal dissection in the rectum and sigmoid colon during the learning curve endosc int open 20142e235“ baum p diers j lichthardt s et a0al sterblichkeit und komplikationen nach viszeralchirurgischen operationen dtsch arztebl int “ schmidt a beyna t schumacher b et a0al colonoscopic full thickness resection using an over the scope device a prospective multicentre study in various indications gut “ aepli p criblez d baumeler s et a0al endoscopic full
Colon_Cancer
"melatonin is a sleepregulating hormone created by the pineal glandand is released at night it has been found to have biological activityin almost all living anisms including plants animals and microbes itcan quickly enter cells through the bilipid bilayer and exhibit scavenging activity towards oxygen free radicals as well as antioxidant properties due to its low molecular weight and amphiphilic nature peripheral tissues have been found to show a high affinity towardsthis hormone melatonin and hence act on receptors and bindingsites studies reported by tan show that melatonin can alsobe produced in the mitochondria and hence tissue melatonin levelsare more than that of serum levels hence can be used as a moleculethat targets mitochondria the main physicochemical and biologicalproperties of melatonin are sleepinducing effects antioxidant behavior [“] anti‚ammatory activity [“] antiapoptotic effects and neuroprotective effects it also regulates variousŽ corresponding authoremail address renjithsbcollegeacin r thomas101016jmolliq2020114082 elsevier bv all rights reservedphysiological functions of the brain as melatonin can diffuse quicklythrough the bloodbrain barrier it is effectively used in the treatmentof brain injuries rapid eyemovement sleepbehavior disorder patients are managed by a combination treatment of melatoninand clonazepam [“] the sensing of bacteria through tolllikereceptor4 and regulation of bacteria through altered goblet cells andantimicrobial peptides are all involved in the anticolitic effects of melatonin in ‚ammatory bowel disease melatonin is involved in theaging process growth towards puberty and modulation of blood pressure this versatile compound blocks proangiogenic andantiangiogenic effects caused by docetaxel and vinorelbine which areantitumor drugs and it enhances their tumorfighting behavior this molecule can modify the redox state of the rat pancreaticstellate cellmelatonin is an endogenous hormone that is involved in circadianrhythm control it is inexpensive and safe as it has a significant effectas an antioxidant and anti‚ammatory melatonin chemically nacetyl5methoxytryptamine is a tryptophan derivative has multiplephysiological effects and can be used to treat many diseases related tovirus infections especially respiratory diseases in covid19 patientswith digestive complications melatonin has positive effects 0cn alzaqri of molecular liquids melatonin can thus be used as an adjunctive or even as a regular therapyas no antiviral treatment is currently available electrochemical measurements of melatonin overflow demonstrate that melatonin secretiondecreases with age melatonin treatments result in the enhancement of essential oil production in salvia species in the context of the recent covid pandemic melatonin can beresearched as a potential molecule to control the dangerous effect ofthis disease rising patients' tolerance and decreasing the mortality infatal virus infections would control the innate immune response and reduce ‚ammation during this period melatonin is a molecule with respective properties as it decreases the overreaction of the innateimmune response and overshoots ‚ammation but also facilitatesadaptive immune function even though melatonin is a critical biomolecule few works havebeen reported on the electronic structure and reactivity of this moleculeexcept for a preliminary work reported by turjanski and coworkers in using semiempirical methods in this manuscript we describe a detailed investigation into the quantum mechanical propertiesof melatonin its spectral features reactivity preferences and the resultsof docking studies with three known structural protein receptors of thenovel coronavirus2 we found that melatonin docks strongly with thethree proteins we hypothesise that this compound can be used as anadjuvant medicine for the treatment of covid19 also significant restby a person peacefully sleeping in dark surroundings will enhance theproduction of this hormone which could help in the management ofcurrent patients or as a preventive measure in the vulnerablepopulation material and methodsthe melatonin molecule was optimised using the gaussian09 software package with the dftb3lyp functional and the 6311g 2dp basis set b3lyp is a commonly used functional and 63112dpbasis set is mediumsized basis set with diffused functions over heavyatoms and polarization functions to bring accuracy we performed frequency calculations to ensure that no imaginary frequency exists suchthat the geometry determined would correspond to a global minimumfor reaching the optimised geometry we used the same geometry for calculating frontier molecular analysis natural bonding orbitals and nonlinear optical studies for uv“visible spectrum simulation we usedtimedependent density functional theory tddft with longrangecorrected camb3lyp functionals with 6311g 2dp as the basis setbecause electronic transitions are timedependent phenomena tddftcalculations are done using the optimised geometry obtained fromb3lyp6311g2dp simulations the frontier molecular orbitals wereviewed from the checkpoint file generated during the optimisation calculations a wavefunction file was generated during a single point groundstate calculation job using which the subsequent analysis performedthe melatonin molecule has more than two reaction sites for examplemethoxy carbonyl“amide and purine ring reaction sites of melatonincalculated using the multiwavefunction software for calculatingtotal electrostatic potential average localised ionization energies and noncovalent interactions as it is reported that melatonincan be used as an adjuvant therapeutic material to fight covid19 we decided to dock the molecule with three ncov2019 protein's rcsb site melatonin is effective in critical care patients by decreasing vessel permeability anxiety use of sedation and increasing the quality ofsleep which may also be beneficial to covid19 patients for improvedclinical outcome melatonin especially has a high health profile significant data indicate that melatonin reduces virusrelated diseases and willpossibly also be effective in patients with covid19 the target proteinswere downloaded cleaned removed alien atoms and molecules andthen used for docking the energy received from the swissdock software and the score values received from patchdock as well as thedocked results collected from biodiscovery studio software arepresented results and discussion geometry of melatoninthe geometry of the molecule explains its rigid structure thestructure of melatonin can be explained based on its physical parametersof bond lengths and bond angles between important atoms or groups asshown in fig the bond lengths of and arebonds of 1c14n 7c14n and 12h14n respectively and the corresponding bond angles are1090343° ° and ° for 1c14n7c 1c14n12h and 7c14n12h respectively the bond angle of° for 4c25o26c having bond lengths of and for 4c\\\\25o and 25o\\\\26c respectively the bond lengths of 18c21n21n22h 21n23c 23c\\\\24o and 23c\\\\30c are and respectively with the corresponding bondangles of ° ° ° ° ° and° for 18c21c22h 18c21c23c 22h22n23c 21n23c24o21n23c30c and 24o23c30c respectively frontier molecular orbital fmo properties of melatoninthe frontier molecular orbitals are highly reactive orbitals of othermolecules and some chemical descriptors are shown in table the higher occupied molecular orbital homo lower unoccupied molecular orbital lumo and energy gap of melatonin are ˆ’ˆ’ and kcalmol respectively the energygap is significant indicating that the molecule is inherently stable theionization energy electron affinity hardness softness chemical potential electronegativity electrophilicity index and nucleophilicity indexof melatonin are ˆ’ and kcalmol respectively interactionof the melatonin with the biological target can be explained by the softness value the softness value is high kcalmol indicatingthat the molecule can positively interact with biological systems andshow the desired effect electron transition study and excitedstate properties of melatonin insolutionthe electron transition study explains electrontransfer excitedstates we used the td“dft formalism using camb3lyp functionalsand 6311g 2dp basis sets in an implicit solvation atmosphere ofmethanol using the iefpcm model as transmission occurs some energyis also emitted melatonin electron transitions to homo having a pyrrole ring and oxygen in methoxy homo1 over the pyrrole ring andethyl carbons and homo2 over acetamide oxygen nitrogen andethyl carbons with energies of ˆ’ ˆ’ and ˆ’ ev respectively melatonin electron transitions to lumo which is over the pyrrolering lumo which is over the pyrrole ring oxygen in methoxyethyl carbons and acetamide nitrogen and carbon and lumo2 having acetamide carbons acetamide nitrogen and carbons with energiesof and ev respectively the electronic spectral datausing td“dft simulations indicate a significant λmax of nm in amethanol solvent the transitions are due to the movement of electronsfrom homo1 to lumo and homo to lumo with an oscillator strength of the electronic transitions are due to chargetransfer transitions from one region of the molecule to another whichindicates its inherent stability due to electronic excitations nonlinear optical behavior of melatoninscientists and technologists working in the molecular electronics fieldare continuously searching for compounds with substantial nonlinearoptical nlo activity such compounds find immense application in electronic displays surveillance equipment and consumer electronic gadgetscomputationally the ability of a molecule to act as an nlo material can be 0cn alzaqri of molecular liquids fig geometry of melatonindetermined from the polarizability and hyperpolarizability data [“]the nlo properties of melatonin are shown in table this is an essentialbehavior of melatonin that has a light absorption nature movement ofelectrons or protons as compared with a standard nlo material such asurea the dipole moment of melatonin is d which is times greater than urea hyperpolarizability mean polarizabilityand anisotropy of the polarizability of melatonin are and esu and which are and times greater than urea respectively the compound is not centrosymmetric hence generates secondorder spherical harmonics and betahyperpolarizability functions this compound can hence be used as an anic nonlinear optically active substance in anic electronicappliances nature of nbo study of melatonina molecule especially one with profound biological activity may havemany intramolecular electron delocalisation and hyperconjugativestabilisation regions natural bond orbital analysis which is a quantummechanical method is useful for this type of study the molecular orbitaltable frontier molecular orbital properties for melatoninchemical descriptorshomolumoionization energy i ɛhomo ˆ’homoelectron affinity a ɛlumo ˆ’lumoenergy gap homo ˆ’ lumoglobal hardness η i ˆ’ a global softness s ηchemical potential μ i a electronegativity χ ˆ’μelectrophilicity index ω μ2 2ηnucleophilicity index n ωenergy in kcalmolˆ’ˆ’ˆ’properties of melatonin for the occupancy of the natural orbitals wereperformed by the nbo suite embedded in the gaussian softwarefrom donorbonding orbital σ c1c2 with occupancy is toacceptor antibonding orbitals σ c3c4 σ c5c6 and σ c7c8exhibiting the transition the energies are and kcalmol respectively from σ c3c4 with occupancy is to σ c5c6 and the rydberg orbital r c30 with the energies are and kcalmol respectively from σ c5c6 having an occupancy is to σ c3c4 r and r h33 with the energies are and kcalmol respectively from σ n21c23 has occupancy to rydberg orbital r c30 and r h33 with the energies are and kcalmol respectively from σ c23o24 having the occupancy is to σ c1c2 σ c2c3 r c23 and r h33 withthe energies are and kcalmol respectivelyfrom σ c23c30 has occupancy is to r h17 r c18 rh19 r c23 r o24 r c30 r h33 σ c1c2 σ c1c6σ c2c3 σ c26h27 σ c30h31 σ c30h32 and σ c30h33 having the energies are and kcalmol respectively from σ o25c26 having occupancy is to r c30 andσ c1c2 with the energies are and kcalmol respectivelyfrom σ c26h27 to σ c1c2 having the energy kcalmol withthe occupancy is from σ c30h31 with the occupancy is to r h17 r c18 r h19 r c23 r o24 r c30 rh33 σ c1c2 σ c2c3 σ c23o24 σ c30h31 and σc30h32 having the energies are and kcalmol respectively from σ c30h32 with occupancy is to r h17 rc18 r h19 r c23 r o24 r c30 r h33 σ c1c2 σc2c3 σ c30h32 and σ c30h33 having the energies are and kcalmol respectively and from σ c30h33 with the occupancy is to r h17 r c18 r c19 r c23 r c24 ro25 r c26 r h28 r c30 r h33 σ c1c2 σ c1c6σ c2c3 σ c2c4 σ c18h19 σ c23o24 σ c26h27 σ 0cn alzaqri of molecular liquids table nonlinear optics property for melatoninnonlinear propertydipole moment μhyperpolarizability βmean polarizability α0anisotropy of the polarizability δαmelatonin d esu esu esuurea d esu esu esucomparison of melatonin with urea times greater than urea times greater than urea times greater than urea times greater than ureac30c31 σ c30h32 and σ c30h33 having the energies are and kcalmol respectivelyfrom core bonding orbital c c23 which has the occupancy electrons move to antibonding r c23 r c30 and r h33 withthe transition energies are and kcalmol respectively from c c23 with the occupancy is to r h33 σ c2c8 and σ c26h28 having the energies are and kcalmol respectively from c o24 to σ c26h29 has the energy is kcalmol with occupancy is and from c c30 having the occupancy is to r h17 r c23 r c30 r h33 σc2c8 σ c26h28 σ c30h31 and σ c30h32 with the energies are and kcalmol respectively from lone pair orbital n n14 with the occupancy is to σ c7c8 with the energy kcalmol from nn21 has the occupancy is to σ c23o24 having the energy kcalmol and from n o24 having the occupancy is to rc23 r c30 r h33 σ c1c2 and σ c2c3 with the energiesare and kcalmol respectively fromantibonding orbital σ c1c2 having the occupancy to rc30 and σ c2c3 with the energies are and kcalmolrespectively from σ c5c6 has occupancy is to r c30 withthe energy is kcalmol and from σ c23o24 has occupancy is to r c30 with the energy is kcalmol the inherentstabilisation offrom the series ofhyperconjugative interactions presented above these interactions canalso be between the melatonin and the surrounding solvent moleculeswhich reveals its stabilisation in biological medium and also betweenthe molecule and the target proteins used in the dockingthe molecule is evident total electrostatic potentials esp and average localised ionization energy alie of melatoninthe electrostatic potential explains how reactive sites canundergo nucleophilic or electrophilic addition or substitution reactionsof melatonin shown in fig within bohr and a color changefrom blue to red indicates charges on elements from ˆ’ to the blue color appears in both methoxyoxygen and acetamideoxygen these are electronrich sites and electrophiles can quickly attack them the red color appears on all of the hydrogens these areelectronpoor sites and nucleophiles can quickly attack themthe alie clarifies the stability of any molecule based on saturatedand unsaturated bond electron movements which are localised ordelocalised the number of the resonance structure is proportionalto the stability of the molecule the alie of melatonin shown in fig iswithin the range of ± bohr color is from indigo to red and the numerical value is from to the blue color of protons in themethoxy group three protons in the sixmembered ring in the indolegroup methyl protons and both adjacent carbons in the acetamidegroup are all sites that act as electrophiles the red color ofacetamide‘carbon conjugated carbon with oxygen atoms and bothacetamideamide and methoxy groups are all sites that act as nucleophiles these blue and red regions represent saturated bonds thebluishgreen regions are on indole rings to methoxy carbons via oxygenand acetamide chains this indicates that delocalised electrons and unsaturated bonds lead to several resonance structures and explains thestability of melatonin the electrophilic and nucleophilic reactive centres identified above interact with the covid virus proteins and providevarious electrostatic and noncovalent interactions and increases drugaffinity noncovalent interaction nci properties of melatoninnoncovalent interactions are a valuable biological property of molecules and are nonbonded directly but are bound by some forces suchas hydrogen bonding van der waals bonding andor steric constraintsnoncovalent interactions of melatonin are shown in fig plotted as agraph with energy plotted versus a reduced density gradient hydrogen bonds appear from ˆ’ to ˆ’ au between oxygenand protons from the acetamide group the van der waals force rangesfig electrostatic potentials of melatoninfig average localised ionization energy for melatonin 0cn alzaqri of molecular liquids fig noncovalent interactions of melatoninfrom ˆ’ to au between acetamideoxygen and its adjacentprotons in both methyl and methoxy groups the steric force rangesfrom to au between the indole ring the methyl groupand the carbonylamide group noncovalent interactions are a groupof interactions like hydrogen bond pistacking hydrophobic interactions van der waal's forces iondipole interactions and dipoledipoleinteractions responsible for the stabilisation of the molecule and thedocking between melatonin and the covid proteins molecular dockingmolecular docking is one of the essential functions of biologically active molecules this is the theoretical evidence to design the structureand reactivity relationship of a molecule at present the covid19 pandemic caused by a new strain of the coronavirus is creating havocthroughout the world we made efforts to dock the melatonin withthe three proteins isolated from the virus represented through thepdb id 6lu7 6m03 and 6w63 were deposited in the database as mentioned in the methodology sectionwith the rapid spread of the novel coronavirus globally the designof vaccines is of great importance sarscov2 is an enveloped nonsegmented and single stranded positive sense rna virus the bestdrug target among coronaviruses is the main protease mpro also called3cl protease this is a key coronavirus enzyme and plays a vitalrole in mediating viral replication and transcription it is identified ashaving a mechanismbased inhibitor the main targeting protease protein pdb 6lu7 is widely studied a series of frontier molecular orbital based interaction analyseswere performed on the complex between the main protease ofcovid19 and the peptidelike inhibitor whose fundamental structure was obtained from the protein pdb 6lu7 anothertargeted protease protein in an apo form pdb 6m03 shows themost stable form after binding with the selected drug threonine residue with the help of several covalent bond interactionwith a ˆ’ kcalmol docking affinity lasinavir brecanavirtelinavir rotigaptide 13bis2ethoxycarbonylchromon5yloxy2lysyloxypropane and pimelautide can be consideredas the main protease inhibitors of covid19 by docking them tothe binding cavities of apo pdb 6m03 and holo pdb 6lu7 another protease protein pdb 6w63 is a reversible inhibitorthe flavonoid narcissoside is reported to have a high affinity towards the protease protein pdb 6w63 according to moleculardocking studies thus these three protease proteins pdb 6lu7pdb 6m03 and pdb 6w63 can be included in the category ofnonstructural proteins in the structure of sarscov2 from table the result from swissdock explains the biological activity of melatonin with coronavirus proteins pdb id 6lu7 6m03and 6w63 in general the total δg is more than ˆ’ kcalmol isright active luckily melatonin has a total δg of ˆ’ ˆ’ andˆ’ kcalmol with coronavirus2 proteins pdb id 6lu7 6m03 and6w63 respectively and the total δg is directly proportional to the fullfitness energy values which are ˆ’ ˆ’ and kcalmol respectively it can also be seen from this table theinterfull fitness intrafull fitness full solvent fitness full surface fitnessδg complex polar solvent δg complex nonpolar solvent δg proteinpolar solvent δg protein nonpolar solvent δg ligand polar solventδg ligand nonpolar solvent δg van der waals force and δg electricforce relationships between melatonin and coronavirus2 proteins asreferredthe result from patchdock are as follows the score values are and total surface interacting area and and the minimum atomic contact energies are ˆ’ˆ’ and ˆ’ kcalmol for melatonin with coronavirus2 proteins pdb id 6lu7 6m03 and 6w63 respectively figs and s1show the skeletal structure and protein residue interactions betweenmelatonin and coronavirus2 protein pdb id 6lu7 6m03 and 6w63table explains what protein residues are interacting with melatoninand details the residue names labels hydrophobic values pka valuesaverage isotropic displacements secondary structures residue solventaccessibility sidechain solvent accessibility percent solvent accessibility and percent sidechain solvent accessibility values of coronavirus2proteinstable and fig s2 show the residue structure of the favorable nonbond interactions between melatonin and coronavirus2 proteinstable lists favorable nonbond interactions of 6lu7 having conventional hydrogen bonds carbonhydrogen bonds pidonor hydrogenbonds pisulfur and pialkyl with melatonin 6m03 has pisigmapipi tshaped and pialkyl with melatonin while 6w63 has pipi tshaped pialkyl and pialkyl with melatonin along with the bond distance from chemistry fig s2 and table show the residue structure 0cn alzaqri of molecular liquids table swissdock result for melatonin with coronovirus2 proteins pdb id 6lu7 6m03 and 6w63energysimple fitnessfull fitnessinter full fitnessintra full fitnesssolvent full fitnesssurface full fitnessextra full fitnessδg complex solvent polarδg complex solvent nonpolarδg protein solvent polarδg protein solvent nonpolarδg ligand solvent polarδg ligand solvent nonpolarδg van der waals forceδg electric forcetotal δg6lu7ˆ’ kcalmolˆ’ kcalmolˆ’ kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol 6m03ˆ’ kcalmolˆ’ kcalmolˆ’ kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol6w63ˆ’ kcalmolˆ’ kcalmolˆ’ kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmolof the unfavorable nonbond interactions between melatonin and coronavirus2 proteins protein 6lu7 does not have any unfavorablestericinteractions protein 6m03 having three unfavorable nonbond interactions and protein 6w63 having unfavorable bumpnonbond interactions fig s2 and table show unsatisfied bonds within melatonininteracting with coronavirus proteins when interacting protein 6lu7has one hydrogen donor and one oxygen acceptor protein 6m03 hastwo hydrogen donors and two oxygen acceptors and protein 6w63has one hydrogen donor and two oxygen acceptors with melatonin table shows noncovalent interactions between melatonin and coronavirus2 proteins hydrophobic groups of protein 6lu7 residues areahis41 aleu141 acys145 ahis164 amet165 and aglu166those of protein 6m03 residues are ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu167 and those ofprotein residues 6w63 are ahis41 acys44 amet49 aleu50 amet165 aglu166 aleu167 and agln189 with melatonin as shownin fig s2 and table the hydrophilic groups of protein 6lu7 residuesare ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189 those of protein 6m03 residues are ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189 andthose of protein 6w63 residues are ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192 with melatonin as shownin fig s3 and table neutral groups of protein 6lu7 residues are atyr54 agly143 and aser144 those of protein 6m03 residues are agly143 aser144 and apro168 and those of protein 6w63 residuesare apro52 atyr54 aarg188 and athr190 with melatonin asshown in fig s4 and table acidic groups of protein 6lu7 residuesare aglu166 and aasp187 that of protein 6m03 residue is aglu166 and protein 6w63 residues are aglu166 and aasp187 withmelatonin as shown in table and fig s5 basic group interactions ofprotein 6lu7 residues are ahis41 ahis164 ahis172 and aarg188those of protein 6m03 residues are ahis41 ahis163 ahis164 andahis172 and those of protein 6w63 residues are ahis41 ahis164and aarg188 as shown in table and fig s6 tan etal has shownthat melatonin and derivatives has excellent biological responses likeacting against oxidative stress and free radical scavenging [“]our studies show that melatonin molecule can interact with differentproteins present in the ncov19 virus and inhibit their proliferationthese results need further clinical follow up and could assist in the management of covid pneumonia conclusionswe conducted a detailed quantummechanical investigation of thehormone melatonin and regulation of the sleepwake cycle naturalbonding orbital studies revealed the intensity of several intramolecularinteractions the various frontier molecular orbital data explain the nature and physical parameters of melatonin and the nonlinear opticalproperties are compared with urea which is a standard materialfig skeletal structure of interactions between melatonin and 6lu7 a 6m03 b and 6w63 c coronavirus2 protein residues 0cn alzaqri of molecular liquids table interactions between melatonin and coronavirus2 protein residuesnamelabelhydrophobicitypkapdbidsavg isotropicdisplacementsecondarystructureresidue solventaccessibilitysidechain solventaccessibilitypercent solventaccessibilitypercent sidechainsolvent accessibility6m03 histidine6lu7 histidineahis41amet49methionineatyr54tyrosinealeu141leucineaasn142asparagineagly143glycineaser144serineacys145cysteineahis164histidinemethionineamet165glutamic acid aglu166ahis172histidineaasp187aspartic acidaarg188arginineglutamineagln189ahis41methionineamet49phenylalanine aphe140aleu141leucineaasn142asparagineglycineagly143aser144serineacys145cysteineahis163histidineahis164histidinemethionineamet165glutamic acid aglu166aleu167leucineapro168prolineahis172histidineagln189glutamine6w63 histidineahis41cysteineacys44methionineamet49leucinealeu50prolineapro52tyrosineatyr54histidineahis164methionineamet165glutamic acid aglu166aleu167leucineapro168prolineaasp187aspartic acidarginineaarg188agln189glutamineathr190threonineglutamineagln192ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’““““““““““““““““““““““““““helixturnhelixcoilturnturncoilturnsheetsheetsheetsheetcoilcoilcoilhelixhelixcoilcoilturnturncoilturnsheetsheetsheetsheetcoilturnsheetcoilhelixcoilcoilcoilcoilhelixsheetsheetsheetcoilturncoilcoilcoilcoilcoilwavefunction studies gave information about electrostatic potentialsaverage localised ionization and noncovalent interactions these datahelped to predict the reactivity and identify the active site of the reactivity of the molecule melatonin docks with novel coronavirus proteinsand shows a variety of interactions with an excellent docking scorewhich leads to inhibition of the virus proteins leading to its destructionhence clinicians can consider incorporating melatonin also in thecovid19 treatment regime after further studiestable favorable nonbond interactions between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63hydrogen bondhydrogen bondhydrogen bondhydrogen bond otherhydrophobichydrophobichydrophobichydrophobichydrogen bondhydrophobichydrophobichydrophobicconventional hydrogen bondconventional hydrogen bondcarbon hydrogen bondpidonor hydrogen bond pisulfurpialkylpisigmapipi tshapedpialkylconventional hydrogen bondpipi tshapedpialkylpialkylaglu166nunk0hunk0hacys145sgunk0amet165caahis41unk0unk0hahis41unk0unk0hdonorhdonorhdonorhdonor sulfurpiorbitalschpiorbitalspiorbitalshdonorpiorbitalspiorbitalspiorbitalsunk0oahis164oaglu166ounk0acys145unk0unk0acys145aglu166ounk0amet165amet165hacceptorhacceptorhacceptorpiorbitals piorbitalsalkylpiorbitalspiorbitalsalkylhacceptorpiorbitalsalkylalkyl 0cn alzaqri of molecular liquids table unfavorable nonbond between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63nilunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorable bumpunfavorable bumpunfavorable bump carbon hydrogen bondunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpacys145sgaglu166ounk0hagln189caagln189caagln189cbagln189cgagln189cgagln189cgagln189cgagln189cdagln189cdagln189cdagln189cdagln189oe1agln189oe1agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189haagln189haagln189haagln189hg1agln189hg2agln189hg2agln189he21agln189he21agln189he21agln189he21agln189he22agln189he22agln189he22stericstericsteric hdonorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericunk0cunk0caglu166ounk0cunk0hunk0cunk0cunk0hunk0nunk0hunk0cunk0cunk0hunk0hunk0cunk0hunk0nunk0cunk0ounk0cunk0hunk0hunk0hunk0cunk0hunk0hunk0hunk0cunk0hunk0nunk0cunk0cunk0hunk0cunk0cunk0hstericstericsteric hacceptorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericsterictable unsatisfied bonds in melatonin with coronavirus2 proteinspdb ids6lu76m036w63nameunk0hunk0ounk0hunk0hunk0ounk0ounk0hunk0ounk0oatomunsatisfied typehohhoohoodonoracceptordonordonoracceptoracceptordonoracceptoracceptorrenjith thomas conceptualization formal analysis investigation methodology project administration resources softwaresupervision validation visualization writing review editingdeclaration of competing interestthe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to ‚uence the work reported in this paperacknowledgementsresearchers supporting project number rsp202078 king saudcredit authorship contribution statementuniversity riyadh saudi arabianabil alzaqri cenceptualization funding acquisition tpooventhiran investegation methodology original draft alialsalme investegation original draft ismail warad resourcesreview methods athira m john methodology writing draftappendix a supplementary datasupplementary data to this article can be found online at 101016jmolliq2020114082table noncovalent interactions between melatonin and coronavirus2 proteinshydrophobicityhydrophilicityneutral groupacidic groupbasic grouppdbids6lu7ahis41 aleu141 acys145 ahis164 amet165 and aglu1666m03 ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu1676w63 ahis41 acys44 amet49 aleu50 amet165aglu166 aleu167 and agln189ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192atyr54 agly143 andaser144agly143 aser144 andapro168apro52 atyr54 aarg188 and athr190aglu166 andaasp187aglu166aglu166and aasp187ahis41 ahis164 ahis172 and aarg188ahis41 ahis163 ahis164 and ahis172ahis41 ahis164 andaarg188
Colon_Cancer
" in the current research we have developed silver and iron nanops of isolated proanthocynidinpac from grape seed by green synthesis and evaluated for antimicrobial antioxidant activity and in vitrocytotoxicity against colon cancer cell linesresults one percent solution of isolated proanthocynidin in water was vigorously mixed with silver nitrate and ferric chloride solution and kept for h to yield pacagnp and pacfenp the synthesized nanops werecharacterized by uv ftir xrd and sem analysis and evaluated for antimicrobial potential against selectedmicrobes moreover the synthesized nanops were studied for dpph assay and in vitro cytotoxicity usingcolon cancer cell lines colo320dm and ht29 mtt srb and trypan blue assay uv spectroscopy confirmed thedevelopment of nanops sem analysis showed that the ps were aggregated in the size range of to nm antimicrobial potential was found to be less against staphylococcus aureus pseudomonas aeruginosa andescherichia coli whereas cytotoxicity of pacagnp and pacfenp against colo320dm and ht29 exhibited promisingresults as compared to the pure pac pacagnp and pacfenp exhibited ± and ± inhibition respectively against dpph radical whereas pure pac showed ± inhibition and standardascorbic acid exhibited ± inhibition of dpph radical the silver and iron nanops were successfully developed by green synthesis method usingisolated proanthocynidin which is economical and ecofriendly the use of metal nanops may open up a newopportunity for anticancer therapies to minimize the toxic effects of available anticancer drugs specifically intargeting specific sitekeywords proanthocynidin silver and iron nanops antioxidant activity cytotoxicity colorectal cancercolo320dm ht29 correspondence randivedheerajgmailcom1department of pharmaceutics rajarambapu college of pharmacy kasegaonwalwa sangli maharashtra indiafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 0cshejawal of genetic engineering and biotechnology page of highlights 0f the present research work is focused on greensynthesis of silver and iron nanops of isolatedproanthocynidin from grape seed extract 0f this is the first attempt to assess scientifically theanticancer efficacy of the developed metalnanops on in vitro colon cell lines ht29 andcolo320dm 0f the results of the study revealed that developednanops are having significant cytotoxic activityagainst colon cancer as compared with pureproanthocynidin phytoconstituents 0f this is an ecofriendly economical easy and rapidmethod of development of metal nanops withminimum usage of hazardous chemicals 0f use of isolated phytoconstituents which are devoidof toxic effects of all other anticancer drugs andbetter absorption as well as cellular uptake due tonanosize for targeting specific site will be a newerresearch area a foremost reason of cancer death in men and womenis colorectal cancer crc and it affects nearly millionpeople throughout the world every year it is thethird most frequently diagnosed cancer in both men andwomen about new cases and around deaths have been estimated to have occurred in inthe usa the occurrence of crc in china is lower thanthat of western countries but it has increased in thecurrent years and has become a substantial cancer burden in china mainly in the more developed areas epidemiologicalthe dailyconsumption of fresh fruits and vegetables is connectedwith a reduced risk of cancer the special effects may bemoderately attributed to the presence of several polyphenolic compounds which are known to exhibit antioxidant and free radicalscavenging properties literature hasshown thatit is very essential to deliver anticancer drug at the siteof action and the dosage form must exhibit high cellpenetration or permeability and drug solubility nanops may enter the human body via several routesthe probability of penetration depends on the size andsurface properties of ps and on the anatomicalstructure of the specific sites of the exposure routes nanotechnology can be termed as the exploitation ofmatter through specific chemical andor physical processes to produce materials with specific propertieswhich can be utilized in particular applications [ ] ananop can be a microscopic p or materialthat has at least one dimension less than nm in size[“] different bulk materials exhibit sole optical electrical thermal physical and chemical properties andhence they find a range of applications in the areas ofenvironment medicine chemistry energy agricultureinformationandheavy industry [ ] recently there has been a keeninterest in the green synthesis of nanops consumer goodscommunicationowing to the characteristic catalytic and optical properties of the metal nanops as compared to the bulkmaterial they have fetched greater attention to date inmultidisciplinaryin thepharmaceutical and cosmetic which position onwardgrowth in commercial interest and calling for effectualsynthesis procedures to match the growing demand ofsilver iron and gold nanops especiallyscientificareasayurvedic and herbalformulations available in themarket diverge in quality and therapeutic efficiencyowing to the differences in composition of the plantphytoconstituents [“] advancement in the ayurvedic herbal medicine has been revolutionized from theshowing of phytochemicals and pharmacological activities to elucidating their mechanisms of action and sitesof action also currently there is an increased interest in the herbal drugs and remedies for the treatmentof chronic diseases proanthocyanidins are the naturally available polyphenolic compounds which varied in chemical structure pharmacological action and characteristics andextensively available in fruits seeds vegetables nutsflowers and bark grape seed is an abundant source of proanthocyanidinpacs which is known to be a powerful inhibitor ofaromatase activity it is an enzyme expressed in higherlevels in cancerous than in usual breast tissues pacs belong to a bigger class of abundant plantderivedcompounds flavonoids which offers numerous valuablehealth effects largely because of its antioxidant properties [ ] it has been shown to defend against oxidative stress and tobaccoinduced dna damage andexhibited selective prominent cytotoxicity against somehuman cancers including colon lung breast prostateand gastric carcinomas [“]they are secondary plant metabolites available inmany diverse kinds of fruits vegetables and plantbasedbeverages and also available in cocoa apple grapes teaand red wine pac classes of condensed tannins areoligomers and polymers of catechin and ˆ’epicatechin and other related flavonoids chiefly linked by eitherbtype c4 †’ c6 or c8 or atype linkages c2 †’ o7grape seed extract gse falls into the btype categoryand grape seed extractrich diets have been connectedwith a reduced risk of chronic cardiac diseases andalso a variety of common cancers including colorectalcancer [“]proanthocyanins are strong free radical scavengers andare supposed to be contributors to the health benefits of 0cshejawal of genetic engineering and biotechnology page of fruits and vegetables [ ] proanthocyanidins obtained from grape seeds have been proven to protectagainst uv lightinduced carcinogenesis stop immunesuppression enhance interleukin il12 and decreaseil10 apple pacs have established synergistic effects with lysosomotropic compounds in increasing theanticancer properties targeting human colon cancerderived metastatic cells [ ]an environmentally friendly option is to prepare nanops dependent on three important parametersnamely solvent medium reducing and stabilizing or capping agent for nps therefore the present study isimportant with respect to the development of metalnanops of phytoconstituent specifically for targeting the cancer it has many advantages like being ecofriendly and costeffective and mainly the isolated phytoconstituents have prominent activity as compared withextract of plants and their parts moreover the side effects of the synthetic drugs can be avoided furtherin vivo animal study of the metal nanopbased formulation is the new area of researchmethodschemicalsproanthocynidin pac was obtained as a gift samplefrom influx healthcare mumbai maharashtra all thechemicals used in the study were of analytical grade silver nitrate agno3 and ferric chloride fecl3 were purchased from loba chem kolhapur ciprofloxacin wasobtained from okasa pharmaceutical satara celllinecolo320dm and ht29 were procured from nccspune maharashtramicroanism usedthe test anisms used in this study were staphylococcus aureusatcc pseudomonas aeruginosaatcc and escherichia coli atcc theculture was obtained from yashawantrao chavan college of science saidapur karad ms india”synthesis of metallic nanopssynthesis of proanthocyanidin silver nanopspacagnpspacagnps were synthesized using proanthocyanidinsolution and agno3 solution in accordance with theprocedure mentioned by phull with minor modification equal volumes of proanthocyanidinaqueous solution and silver nitrate solution were incubated at ambient temperature for “ h to obtainpacagnps synthesis of pacagnps was detected bynaked eye with a change of color from dark red to faintbrown which was confirmed by uv spectroscopy thecollected pacagnps were centrifuged for min at rpm and dried in vacuum chamber at °c shownin fig synthesis of proanthocyanidin iron nanopspacfenpsfor the synthesis of profenps proanthocyanidin aqueous solution and ferric chloride solution were used inaccordance with the procedure mentioned by raju with minor modification ferric chloride andproanthocyanidin solution were vigorously combined in a ratio of the mixture was then placed onan orbital shaker for h at an ambient temperature toobtain pacfenps the synthesis of pacfenps was observed with a color change from dark black to darkbrown which was also confirmed by uv spectroscopythe collected pacfenps were centrifuged for min at rpm and dried in vacuum chamber at °cshown in fig characterization of nanopsuvvis absorbance of pac pacagnp and pacfenpthe development of metal nanops of pacagnpand pacfenp by the proanthrocyanidin was recordedperiodically using a uv spectrophotometer shimadzuthe samples were diluted with ml of deionized waterand measured for uvvis spectrum after the formationof nanops that change in color deionized waterwas used as a blank for correction all samples were scanned from to nmsem and histogram analysis of pacagnp and pacfenpscanning electron microscopy sem is a normally usedmethod of evaluation and morphological analysis ofnanops at the nanometer to micrometer scale developed pacagnp and pacfenp were characterizedusing a highresolution scanning electron microscopeschottky field emission scanning microscope su5000the samples were prepared by a simple drop coating ofsuspended gold solution on to an electric clean glass andallowed the solvent to evaporate and the samples weredried atand analyzed in amicroscoperoom temperatureftir spectroscopy analysis of pac pacagnp and pacfenpto recognize the different biomolecules present in theproanthocynidin and the phytocompounds in ag and fenanops after synthesis were analyzed by ftirbruker alpha echo atr spectrum was recorded inthe range of “ cmˆ’xrd analysis of pacagnp and pacfenpthe green synthesized silver and iron nanops ofpac were evaluated by xrd analysis using an xrd xray diffractometer bruker d8 discover operated 0cshejawal of genetic engineering and biotechnology page of fig uv and synthesis npat a voltage of kv and ma with cu kα radiation inθ“2θ configurations the crystalline size was determinedfrom the width of the xrd peaks by assuming that theywere free from nonuniform strainsantimicrobial activity of pac pacagnp and pacfenpin vitro antimicrobial activity was performed using theagar well diffusion technique the sterile agar wasinoculated with the bacterial culture s aureus p aeruginosa and e coli for h at °c antimicrobial activities were tested on nutrient medium against s aureusp aeruginosa and e coli which are representative typesof grampositive and gramnegative anisms wellswere bored by using a sterile borer standard solutionciprofloxacin and test samples pac pacagnp andpacfenp mgml was prepared by dissolving the testsample in dmso were placed into the wells μlplates were then kept for h in the refrigerator to enableprediffusion of the extracts into the agar finally theplates were incubated overnight h at °c theantimicrobial activity was determined by measuring thediameter of zone of inhibition [ ]in vitro cytotoxicity studies of pac pacagnp andpacfenp by using mtt assayhuman ht29 cell and colo320dm were obtainedfrom the national center for cell sciences pune msindia the cell cultures were maintained indmem supplemented with fetal bovine serum thecells were plated at a density of × cells per well in a96well plate and cultured for h at °c the cellswere subsequently exposed the plates were incubatedfor h and cell proliferation was measured by adding μl of mtt thiazolyl blue tetrazolium bromide dye mgml in phosphatebuffered saline per well theplates were incubated for a further h at °c in a humidified chamber containing co2 formazan crystalsformed due to reduction of dye by viable cells in eachwell were dissolved in μl dmso and absorbancewas read at nmfinally the percent cytotoxicity of the compounds wascalculated by using the following formulapercent cytotoxicity ¼ reading of controlreading of treated cellsreading of control 02 since the absorbance was directly associated with thenumber of viable cells the percent viability was determined from the absorbancein vitro cytotoxicity studies of pac pacagnp andpacfenp by using srb assayhuman ht29 cells and colo320dm were maintainedin dmem supplemented with fetal bovine serumthe cells were plated at a density of × cells perwell in a 96well plate and cultured for h at °cthe cells were subsequently exposed to μgml compound after drug incubation μl tca waskept for h at °c then the plate was washed with 0cshejawal of genetic engineering and biotechnology page of tdw triple distilled water and air dried thereafter μl srb dye was added in each well and kept for min at room temperature again the plate was washedthree times with acetic acid and air dried finally μl tris buffer was added and the absorbance wasread at nmthe percent cytotoxicity of the compounds was calculated by using following formulapercent cytotoxicity ¼ reading of controlreading of treated cellsreading of control 02 in vitro cytotoxicity studies of pac pacagnp andpacfenp by using trypan blue assaythe dye exclusion test is used to find out the number ofviable cells present in a cell suspension it is based onthe principle thatlive cells possess undamaged cellmembranes that exclude certain dyes such as trypanblue eosin or propidium whereas dead cells do not exclude in this test a cell suspension is simply mixed withtrypan blue dye and then visually examined to determine whether cells take up or exclude dye in the studypresented here a viable cell will have undamaged a clearcytoplasm whereas a nonviable cell will have a bluecytoplasmfifty microliters of celllines of human ht29 cellsand colo d was taken in microcentrifuge tubethey were incubated for min and then added μlof all samples of nanops in concentration of μg mlˆ’ which were prepared by dissolving inphosphate buffer ph and dmso they were incubated in co2 incubator for min and thereafter trypan blue μl was added in each tube theywere further incubated for min in co2 incubatorand analyzed for total viable cells and nonviable cellsby using nubars slide antioxidant activity of pac pacagnp and pacfenp bydpph 22diphenyl2picryl hydrazyl hydrate assaythe scavenging ability of pac pacagnp and pacfenp on the stable free radical was calculated with themethod expressed by mensor twenty microliters of pac pacagnp and pacfenp solutions wasseparately added in three labeled testtubes subsequently ml of methanolic solution of dpph and ml of methanol were added to each test tube afterwhich all tubes were allowed to react at an ambienttemperature for min the control was prepared as described above without any extract and nanopsmethanol was used to correct the baseline after minof incubation the discoloration of the purple color wasmeasured under a uvvisible spectrophotometer theradicalscavenging activity was determined by the following formula scavenging activity ððþ ¼ aabs controlðþaabs sampleðaabs controlþþ 02 where a control is the absorbance of control samplepacagnp and pacfenp measured at nm and nm respectively and a control is the absorbance ofpac measured at nmstatistical analysisstatistical data ofthe cytotoxicity were assessed ongraphpad prism for windows bit with version results were analyzed by oneway anova withdunnett™s posttest analysis of variance the meanstandard error mean sem of all calculated values wasshown in each group a value of p or was considered statistically significantresultsuvvis spectroscopy of pac pacagnp and pacfenpwhen the aqueous pac was mixed with aqueousagno3 and fecl3 solution the color of the solutionchanged which indicated the formation of silver and ironnanops this change in color was due to the collective coherent oscillation of conduction electrons atthe surface of the nanops that interact with the oscillatingaphenomenon called surface plasmon resonance sprthis change in color indicated the reduction of ag andfe ions which was traced with uvvis spectroscopythe pac showed max at nm and silver nanop pacagnp possesses specific wavelength that canabsorb atmax nm whereas iron nanopspacfenp exhibit max at nm as shown in fig incidentelectriclightfieldoftheftir spectrum of pac pacagnp and pacfenpin the ftir of proanthocynidin fairly sharp peaks at and cmˆ’ were observed which indicate thepresence of the functional group present in the compound however the aromatic at ccvalencecmˆ’ the oh phenolic at ohvalence and chvalence arene alkane och3 the methoxylic at cmˆ’ and co stretching at appeared in the ir spectrum of complex as shown infig 2apacagnpthese characteristic vibrations after reduction of agions were shifted to new peaks at and cmˆ’which indicated the presence of the functional grouppresent in the compound however the aromatic at ccvalencethe oh phenolic atohvalencechvalencecmˆ’and 0cshejawal of genetic engineering and biotechnology page of fig a ftir b sem histogram c xrdarene alkane och3 the methoxylic at cmˆ’and co stretching at appeared in the irspectrum of complex as shown in fig 2ain addition bioreduction showed that the and cmˆ’ bands were suppressed in theagnp proanthocynidin and nps showed similar absorption bands indicating that nps might be stabilized byproanthocynidin on the basis of the orange yellow andbrownish green color of the biomass and the groups suggested by ftir analysisit was hypothesized thatproanthocynidin may be involved in silver nanopsynthesispacfenpthese characteristic vibrations after reduction of fe3ions were shifted to new peaks at and cmˆ’which indicated the presence of the functional grouppresent in the compound however the aromatic at ccvalencethe oh phenolic atohvalencechvalencecmˆ’and arene alkane och3 the methoxylic at cmˆ’and co stretching at appearin the irspectrum of complex as shown in fig 2a in additionbioreduction showed that the and cmˆ’ bands were suppressed in the fenpproanthocynidin and nps showed similar absorptionbandsindicating that nps might be stabilized byproanthocynidin on the basis of the orange yellow andblackish color of the biomass and the groups suggestedby ftir analysis it was hypothesized that proanthocynidin may be involved in iron nanop synthesissem and histogram analysis of pacagnp and pacfenpa scanning electron microscope was used to analyze thestructure of pacagnp and pacfenp nanops thatare developed and represented in fig 2b the nanops formed were aggregated having a size range of to nm this aggregation of the nanops can beminimized or prohibited by increasing the concentrationof the proanthocynidin extract histograms of both thenanops have been represented in fig 2b exhibiting 0cshejawal of genetic engineering and biotechnology page of average p size pacfenp μmand pacagnp μmxrd spectrum of pacagnp and pacfenpthe xrd pattern of the synthesized silver and ironnanops formed using proanthocynidin is shown infig 2c the diffraction peak at 2θ ° and subsequenthigher order reflections can be indexed to the ag andother facets of silver nanops by comparing jcpdsfile no in case of pacfenp the peak shown at2θ ° corresponds to the iron compared with standard xrd for iron jcpds data pdf no the xrd spectrum also revealed a weak peak around2θ ° which can be attributed to the phytochemicalcomponents it thus confirmed that the nanopsformed on the membrane consisted of crystalline xrdindicated possible multicomponent product formation athigher energyantibacterial activity of pac pacagnp and pacfenpthe zones of inhibition revealed that there is very little antimicrobial potential of pac pacagnp andpacfenp against pseudomonas aeruginosa staphylococcus aureus and e coli the results are highlightedin table and the zone of inhibition is depicted infig results of cytotoxicity of pac pacagnp and pacfenpthe results of cytotoxicity assay by mtt and srb assayhave been presented in table with respect to two different colon cancer cell lines namely colo320dm andht29 a variation in the results of different assay wasobserved howeverthe silver nanops exhibitedbetter activity than pure pac in both the methods theresults of mtt assay against ht29 demonstrated thatpacagnp showed maximum ± inhibitionrepresented in fig 4x a in case of colo320dm silver nanops exhibited ± inhibition asrepresented in fig 4x b whereas the srb assay ofpac pacagnp and pacfenp against ht29 revealed ± inhibition by pacagnp as shown in fig4x c in case of colo320dm srb assay a maximumof ± inhibition was exhibited by silver nanops as depicted in fig 4x dthe results of trypan blue assay revealed that silvernanopaticles pacagnp showed ± nonviability of colo320dm cell line when observed on amotic microscope whereas iron nanops pacfenpexhibited ± inhibition of ht29 cell lines asrepresented in table results of dpph 22diphenyl2picryl hydrazyl hydrateassay of pac pacaunp and pacfenpthe dpph activity results demonstrated effective freeradical percent scavenging potential of pac pacaunpand pacfenp as depicted in fig 4y as compared to ascorbic acid standardthe concentration responsecurves of dpph radicalscavenging activity of pacpacaunp and pacfenp are shown in table it wasobserved that pacagnps were more effective than pacextract and pacfenps at a concentration of μgmlˆ’ the scavenging activity of pacagnps was observed to be ± while at similar concentration for the pacfenps it was reported to be ± the outcome of antioxidants on dpph is thoughtto be due to their hydrogen donating abilitydiscussionengineered nanomaterials showed imperative benefitsdue to their unique nanostructure along with their significant properties for the designed applications metallic nps have been synthesized using several different methods such as chemical reduction electrochemical microbiological reduction ultrasonication methodand microwave radiation the present researchwork is focused on green synthesis of silver and ironnanops ofisolated proanthocynidin from grapeseed extract it is an ecofriendly economical easy andrapid method of development of metal nanopswith minimum usage of hazardous chemicals the developed silver and iron nanops were characterized byuv spectroscopy which showed the change in absorbance after development of nanops ftir spectrumprovides the information about the chemical change ofthe functional groups involved in bioreduction [ ]the ftir spectra of developed nanops confirmedthe formation of silver and iron nanops as characteristic vibrations after reduction of ag ions weretable results of antibacterial activity of pac pacagnp and pacfenp against selected microbial strainssrnosample namepac purepacagnppacfenpciprofloxacin stdvalues are expressed in triplicate mean sdzone of inhibition diameter mm against the selected microanismspseudomonas aeruginosa ± staphylococcus aureus ± ± ± ± ± ± e coli ± ± 0cshejawal of genetic engineering and biotechnology page of fig antimicrobial activityshifted to new peaks at and cmˆ’ and characteristic vibrations after reduction of fe3 ions wereshifted to new peaks at and cmˆ’ alsocharacteristic color change can be attributed to the surface plasmon resonance of deposited agnps and itclearly indicated the development of nanops the size of nanops and its morphology wereclearly observed in the sem images the average size fortable results of cytotoxicity of pac pacagnps and pacfenp by mtt and srb assay using colo320dm and ht29 cell linescompoundmtt assay against ht29 control”percent inhibitionmean odpercent viabilityproanthocynidin plainproanthocynidin agnpproanthocynidin fenpmtt assay against colo320dm control”proanthocynidin plainproanthocynidin agnpproanthocynidin fenpsrb assay against ht29 control”proanthocynidin plainproanthocynidin agnpproanthocynidin fenpproanthocynidin plainsrb assay against colo320dm control”proanthocynidin plainproanthocynidin agnpproanthocynidin fenpvalues are expressed in triplicate mean sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0cshejawal of genetic engineering and biotechnology page of fig graph of x cytotoxicity and y antioxidant dpphpacfenp was observed to be μm and μm for pacagnp the xrd resultsrevealed weak peak at 2θ ° for phytoconstituentwhereas diffraction peak at 2θ ° was observed for silver nanops and iron nanops showed thepeak at 2θ °in case of antimicrobial activity the developed silver andiron nanops exhibited little antimicrobial potential ascompared to pure proanthocynidin metal nps increase theantibacterial potential due to the formation of reactive oxygen species ros developed from different types of ironoxide nanops like feo fe2o3 and fe3o4 the freeradical produced in the reaction causes intracellular stressesthat can damage the dna of the cell the in vitro cytotoxicity results against ht29 andcolo320dm showed that pacagnp exhibited ± inhibition mtt assay and ± inhibitionofcolo320dm pacagnp demonstrated ± inhibition mtt assay and ± inhibition srbassayagainst ht29assaysrbincasein dpph assay pacfenp and pacagnp exhibited ± and ± inhibition respectivelythe obtained parameters of the characterization andevaluation of the nanops clearly revealed that ascompared to proanthocynidin the silver and iron nanops possess better antioxidant and anticancer potential against colorectal cancer thusthe use ofisolated phytoconstituents which are devoid of side effects and promoting better absorption and cellular uptake owing to their nanosize will certainly achieveeffective targeting and has to be provided greater attention as a newer research area also several review papershave been published about the synthesis of silver nanops using natural polymers like kcarrageenan andsynthetic polymers like poly vinyl pyrrolidone and polyethylene glycol to improve the strength and stability ofnanops as per the study of moustafa the use ofnatural and synthetic polymer for the development ofsilver nanops opened up new research area in medicinal and biological field along with food industry we have successfully synthesized pacagnps and pacfenps using proanthocynidin isolated from grape seedextract by employing a green synthesis method which istable results of cytotoxicity of pac pacagnps and pacfenp by using trypan blue assaysrnodrugcolo320dmpercent viability ± percent nonviable ± proanthocynidin plainproanthocynidin agnpproanthocynidin fenpvalues are expressed in triplicate mean sd ± ± ± ± ht29percent viability ± ± ± percent nonviable ± ± ± 0cshejawal of genetic engineering and biotechnology page of table antioxidant activity of dpph radicalscavenging activityconcentrations μg mlˆ’standard percent inhibition ± pac percent inhibition ± pacagnps percent inhibition ± pacfenps percent inhibition ± ic50 ± ± ± ± “ ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± values are expressed in triplicate mean sdconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of pharmaceutics rajarambapu college of pharmacy kasegaonwalwa sangli maharashtra india 2department of pharmaceuticalchemistry rajarambapu college of pharmacy kasegaon walwa sanglimaharashtra india 3department of pharmaceutics bharatividyapeeth college of pharmacy kolhapur maharashtra indiareceived may accepted august availability of data and materialsall the data required for the processing of the s are presented inthe œresults section supporting data was included separatelyethics approval and consent to participatenot applicable biao l tan s zhang x gao j liu z fu y synthesis andcharacterization of proanthocyanidinsfunctionalized ag nanopscolloids surf b biointerfaces “ gurushankar k gohulkumar m prasad nr rishnakumar n synthesischaracterization and in vitro anticancer evaluation of hesperetinloadednanops in human oral carcinoma kb cells adv nat sci nanoscinanotechnol “nanops j nanomater1“andsimpleenvironmentallybenignrelativelypacagnps and pacfenps were obtained with ps between and nm in size it is easy and costeffective and does not involve any harmful and poisonous chemicals all the other characterization like uvftir xrd and sem confirmed the development ofnanops we have observed significant antioxidantactivitycapacitiespacagnps and pacfenps exhibited a little antibacterial activity against the selected strains of microbes in anutshell the study showed that the developed nps fromthe isolated pac exhibit beneficial antioxidant and anticancer potential when assessed by three different in vitroassay methods with specifically colon cancer cell linesthus it may open up a new opportunity for anticancertherapies that need further researchradicalscavengingfreeandabbreviationspac proanthocynidin pacagnp proanthocynidin silver nanoppacfenp proanthocynidin iron nanop uv ultraviolet visiblespectroscopy ftir fourier transform infrared spectroscopy xrd xraydiffraction sem scanning electron microscope dmem dulbecco™s modifiedeagle™s medium dpph 22diphenyl2picryl hydrazyl hydrate mtt dimethylthiazol2yl25diphenyltetrazolium bromide srb sulforhodaminebacknowledgementsthe authors are thankful to the secretary of kes society kasegaon andprincipal of rajarambapu college of pharmacy kasegaon sanglimaharashtra for providing facilities we are also thankful to the managingdirector influx healthcare mumbai for providing pure phytoconstituentmarkers dr sandip patil md biocyte institute of research and developmentbird sangli mhauthors™ contributionsthe work was c
Colon_Cancer
t he gut is the largest immune an of the humanbody and also an important target an for variouskinds of stress caused by severe insults such as infectiontrauma and shock12 these stresses are considered to haveinstitution at which the work was performed department oftraumatology and acute critical medicine osaka universitygraduate school of medicine yamadaoka suita osaka japancorresponding yasutaka nakahori md division of trauma andsurgical critical care osaka general medical center bandaihigashi sumiyoshi ward osaka japan emailp_olysyahoocojpreceived apr accepted jul funding informationno funding information providedan important role in promoting infectious complications andmultiple an dysfunction syndrome from the viewpointsof deteriorated intestinal epithelium the immune systemand commensal bacteria gut dysfunction is now recognizedas a cause for the promotion of diseases34lipids have been a focus not only as energy sources butalso as immunemodulating substrates5 unlike long andmediumchain fatty acids shortchain fatty acids scfaswhich mainly consist of acetate propionate and butyratewith two to four carbon atoms are principally derived fromthe fermentation of carbohydrates and amino acids by anaerobic microanisms shortchain fatty acids are usedmainly by intestinal epithelial cells as energy substrates andthey ‚uence the motility of the intestinal tract and increaseintestinal blood flow we previously reported on altered gutflora and fecal anic acids in critically ill patients andshowed that these patients had significantly lower levels of the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicinethis is an open access under the terms of the creative commons attributionnoncommercial licensewhich permits use distribution and reproduction in any medium provided the original work is properly cited andis not used for commercial purposes of 0c of y nakahori acute medicine surgery 20207e558fecal anic acids especially scfas than healthy volunteers67despite the evidence implicating the importance ofscfas in the gut the effect of scfas on prognosis in critically ill patients has not yet been clarified in this study wemeasured eight kinds of fecal anic acidsincludingscfas in critically ill patients and investigated the effect ofscfas on their prognosismethodspatientst his retrospective study enrolled patientswho i fulfilled the criteria of systemic ‚ammatoryresponse syndrome sirs according to the american college of chest physicians and the society of critical caremedicine8 ii had a serum creactive protein crp levelgreater than mgdl iii were admitted to the departmentof traumatology and acute critical medicine osakauniversity graduate school of medicine osaka japan during the period november “january iv were treated in the intensive care unit for more than days we usedcrp ‰¥ mgdl to focus on cases with severe ‚ammationbecause crp is mainly used as a marker of ‚ammationwe excluded patients from whom we could not collect anyfecal samples during hospitalization continuous enteralfeeding was initiated with kcalday and graduallyincreased depending on the patient™s condition we usedimpact novartis nutrition minneapolis mn usa as anenteral nutritional product for all patients in our intensivecare unit and used “ kcalkg ideal body weight as thegoal for the administered dose fourteen healthy volunteersprovided fecal samples as controls for examinations of fecalanic acids concentrations this study was approved bythe institutional review board of osaka university approval no and informed consent was obtained fromeach patient™s familyfecal samples and determination of fecalanic acid concentrationswe collected fecal samples serially from all patients thefecal samples were transported at 00°c to the yakult central institute tokyo japan we measured eight kinds ofanic acid in the feces by highperformance liquid chromatography feces were homogenized in ml distilledwater the homogenate was then placed in an eppendorftube and centrifuged at g for min at °c a mixture of ml of the resulting supernatant and ml of moll perchloric acid was mixed well in a glass tubeand allowed to stand at °c for h the suspension wasthen passed through a filter with a pore size of lmnihon millipore tokyo japan the sample was analyzedfor anic acids by highperformance liquid chromatography as previously described9 using a waters conductivity detector waters milford ma usa equipped withtwo columns shodex kc811 showa denko tokyojapan the concentrations of fecal anic acids were calculated with the use of external standards the reproducibilityand stability of these measurements were shown previously10 we retrospectively evaluated the minimum andmaximum value of each fecal anic acid measured duringhospitalization and determined prognostic factors by classification and regression tree cart analysissurveillance and definition of complicationsbacterial infection was diagnosed in accordance with thedefinitions of the centers for disease control and prevention11 body temperature was measured continuouslysurveillance cultures from blood and sputum were undertaken routinely for each patient in cases of suspected infectionand computedtomography scanning were carried out as necessary bacteremia was defined as a positive blood culturelaboratory testingchest xraystatistical analysiscontinuous variables expressed as the median 25th and 75thpercentiles were compared by the mann“whitney utestcategorical variables expressed as number were comparedby the v2test unless the expected counts in any of the cellswere below in which case the fisher exact test was usedwe used cart analysis which is a binary recursive partitioning using nonparametric approaches to identify keyfecal anic acids and their cutoff values for mortality12we also used multivariate logistic regression analysis toquantitatively evaluate the effect of the covariates that weresuggested by cart analysisthe most important prognostic factors were selected anda predictive model was developed as follows first the minimum and maximum values of each fecal anic acid wereselected from all patients the maximum value was the highest value and the minimum value was the lowest value measured during hospitalization second the mann“whitney utest was applied to discover variables with potential prognostic value and covariates with p were excludedfinally cart was used to create a tree using the minimumand maximum data values of all fecal anic acids theeffects of the minimum and the maximum values of the fecalanic acids that were selected as key prognostic factors by the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of table characteristics of critically ill patients included in this studytotal n survivors n nonsurvivors n pvalueage yearssex maleicu stay daysapache ii score on admissionorigins of sirssepsistraumaburnunknownbacteremianumber of antibiotic typesduration of antibiotic use days “ “ “ “ “ “ “ “ “ “ “ “ “ “ “categorical values are expressed as number continuous values are expressed as median 25th“75th percentiles bold values indicatestatistical significanceapache acute physiology and chronic health evaluation icu intensive care unit sirs systemic ‚ammatory response syndromecart analysis and age sex and acute physiology andchronic health evaluation apache …± score on admission were analyzed with the multivariable logistic regressionmodelsall reported pvalues were twosided and a pvalue of was considered to indicate statistical significance allstatistical analyses were undertaken using ibm spss statisticsversion armonk ny usaresultspatient characteristicsc haracteristics of the patients are shown intable there were survivors and nonsurvivors the two groups did not differ significantly in termsof sex or apache ii score age in the nonsurvivors wassignificantly older than that in the survivors p the principal origin of sirs was sepsis in of the patients in the two groups significantly more types ofantibiotics were used in the nonsurvivors than in the survivors during hospitalization p and the durationof antibiotic use in the nonsurvivors was significantlylonger than that in the survivors p the incidenceof bacteremia was significantly higher in the nonsurvivorsthan that in the survivors vs p anic acid profilestiming of fecal sample collection is shown in table wecollected fecal samples for scfa analysis fecalsamples were collected evenly throughout hospitalization inboth the survivors and the nonsurvivors the minimum andmaximum values of each fecal anic acid and the resultsof univariate analysis are shown in tables and in termsof the minimum values total anic acids acetate propionate and isovaleric acid were significantly decreased in thenonsurvivors in terms of the maximum values lactate formic acid and succinic acid were significantly increased inthe nonsurvivors otherwise isovaleric acid and acetatewere significantly decreased in the nonsurvivorsadditionally cart and multivariate logistic regressionanalyses of the predominant covariates were carried out fortable timing of fecal sample collection in critically illpatientssurvivorsnonsurvivorstotalweek week week week week week week week week week weekstotal the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558table minimum values of fecal anic acids in eachgroup of critically ill patientssurvivorsnonsurvivorspvaluetotal anic acidsacetatepropionatebutyratesuccinic acidlactateformic acidisovaleric acidvaleric acidvalues are expressed as mean 06 se lmolg of feces bold val 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 ues indicate statistical significancetable maximum values of fecal anic acids in eachgroup of critically ill patientssurvivorsnonsurvivorspvalue 06 06 06 06 06 06 06 06 06 total anic acidsacetatepropionatebutyratesuccinic acidlactateformic acidisovaleric acidvaleric acidvalues are expressed as mean 06 se lmolg of feces bold val 06 06 06 06 06 06 06 06 06 ues indicate statistical significancethe minimum and maximum values of the fecal anicacids for the minimum values the primary split variablewas determined to be the minimum value of propionate andthe cutoff value was lmolg of feces the secondarysplit variable was determined to be the minimum value ofacetate and the cutoff value was lmolg of feces themortality in each partition and the diagnostic characteristicsof this tree are shown in figure for the maximum values of fecal anic acids the primary split variable was determined to be the maximumvalue of lactate and the cutoff value was lmolg offeces the secondary split variable was determined to bethe maximum value of formic acid and the cutoff valuewas lmolg of feces the mortality in each partitionand the diagnostic characteristics of this tree are shown infigure the diagnostic characteristics of the tree for minimumvalue showed a sensitivity of and specificity of whereas those ofthe tree for maximum valueshowed a sensitivity of and specificity of for the minimum values of fecal anic acids multivariate logistic regression analysis revealed that age and theminimum values of propionate and acetate were significantprognostic factors for mortality table for the maximumvalues age and maximum lactate were identified as significant prognostic factors for mortality table discussiont his study provides the first in vivo evidence toour knowledge that the altered balance of fecal anicacids is associated with mortality in critically ill patients arecent study has shown that scfas particularly acetate andpropionate bind the gproteincoupled receptor gpr43in adipose tissue stimulation of gpr43 by scfas was necessary for the anti‚ammatory responses because gpr43deficient mice showed exacerbated ‚ammation in modelsof colitis arthritis and asthma the scfa“gpr43 interactions profoundly affected intestinal and systemic ‚ammatory responses13 the low concentrations of acetate andpropionate observed in nonsurvivors in the present studycould reduce the anti‚ammatory effect fukuda 14showed that acetate produced by protective bifidobacteriaimproves intestinal defense mediated by epithelial cells andthereby protects the host against lethal infection furthermore asahara 15 showed a positive correlation betweenfecal acetic acid level and tightjunctionrelated gene expression in the intestinal epithelium in their experiment using aninfected mouse modelthe maximum values of lactate and formic acid were alsoselected as prognostic factors fig our group previouslyshowed that when patients had severely acidic feces fecallactate succinic acid and formic acid were significantlyincreased over those of patients with normal feces we alsoshowed that abnormal fecal ph was associated with the incidence of bacteremia and mortality16 the increase of lactateand formic acid can make feces severely acidic and severelyacidic feces could injure intestinal epithelial cells and worsen the patient™s condition the present results indicate thatnot only a lack of scfas but also an accumulation of lactateand formic acid might be associated with disruption of thegastrointestinal environmentin critically ill conditionswhich leads to bacterial translocationthere was a significant difference between the two groupsin age number of antibiotic types and duration of antibioticuse in this study previous studies have suggested thatchanges in the composition of bacterial species and genes in the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of fig charts showing mortality in critically ill patients partitioned by the minimum value of fecal anic acids using classificationand regression tree analysis min minimumfig charts showing mortality in critically ill patients partitioned by the maximum value of fecal anic acids using classificationand regression tree analysis max maximum the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558table results of multivariate logistic regression analysis in critically ill patients using the minimum value of fecal anic acidscoeff bsepvalueodds ratio confidence intervallower limitupper limitmortalityagesexapache iipropionate minacetate min 00 00 00bold values indicate statistical significanceapache acute physiology and chronic health evaluation coeff b coefficient min minimum se standard errortable results of multivariate logistic regression analysis in critically ill patients using the maximum value of fecal anic acidscoeff bsepvalueodds ratio confidence intervallower limitupper limitmortalityagesexapache iilactate maxformic acid max 00bold values indicate statistical significanceapache acute physiology and chronic health evaluation coeff b coefficient max maximum value se standard errorthe intestinal flora occur with aging17 it is also reported thatthe genes related to scfa production were lost from theintestinal flora and that glycolytic ability was decreased withaging18 these changes could be one of the reasons whyaging was detected as an important factor related to mortality in the present study there is no doubt that antibiotics areimportant butthe intestinal florasome systemically administered antibiotics are excreted inthe bile and secreted in the upper gastrointestinal tract andupon reaching the colon they cause serious damage to theintestinal flora19 shortchain fatty acids could also bereduced with the destruction of the intestinal flora whichmight have worsened the prognosis20they adversely affectshortchain fatty acids are mainly used by intestinalepithelial cells as energy substrates and some are absorbedinto the portal flow to the liver and used as systemic energysources they can also ‚uence the motility of the intestinaltract absorption of electrolytes and pancreatic secretion andcan increase intestinal blood flow in the present study totalanic acids including scfas were significantly decreasedin the critically ill patients compared with those in thehealthy controls data not shown one reason would be thealtered gut flora that produce scfas the commensal gutflora in humans plays an essential role in homeostasis andprotection from injury in the gut2122 under critically illconditions it is difficult to maintain normal gut flora6 notonly does the stress derived from diseases such as traumaand burns affect the balance of the gut microbiota but alsothe stress caused by the treatment with various therapeuticagents such as histamine h2 receptor blockers catecholamines and broadspectrum antibiotics can ‚uence the gutflora we reported that critically ill patients had to times fewer total anaerobes bifidobacterium andlactobacillus and times greater staphylococcus whencompared with counts in healthy volunteers6 these dataindicate that the balance of gut flora is significantly disturbed in critically ill patients the concentrations of fecalanic acids decreased significantly in the nonsurvivors asshown in table and we believe thatthis was likelybecause the number of good bacteria bifidobacterium andlactobacillus had decreased similarly the cause of thereduction of shortchain fatty acids in the present study may the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of have been the reduction of the scfaproducing bacteriasuch as clostridium eubacterium peptococcusandfusobacterium2324synbiotics are the combination of probiotics and prebiotics the clinical effects of synbiotics have been reported inthe fields of pediatric surgery abdominal surgery and intensive care25 in our preliminary report critically ill patientstreated with synbiotics maintained the necessary amounts offecal anic acids including scfas and had fewer incidences of enteritis pneumonia and bacteremia than thosewithout synbiotics26 these reports suggest that the maintenance of gut flora and scfas would be a promising intestinal therapy synbiotics were given to patients in thisstudy in the synbioticstreated group maximum values offecal anic acids acetic acid propionic acid succinicacid lactic acid and formic acid increased significantly butminimum values did not increase only the minimum valueof lactic acid decreased significantly however there wasno fixed protocol for the treatment with synbiotics in thisstudy the treatment start date was variable and the averagehospital day of starting treatment was day “ so itwas difficult to evaluate the effects of synbiotics in thisstudy evaluating the impact of synbiotics on fecal anicacids and prognosis in critically ill patients will be a futuretaskour study has some limitations this was a retrospectivestudy and thus the timing of feces sampling was differentbetween patients the number of samples was too small tomake conclusions about the temporal patternsin conclusion the minimum values of fecal acetic acidand propionate in the nonsurvivors were significantly lowerthan those in the survivors the altered balance of fecalanic acids was associated with mortality in critically illpatients thus the maintenance of scfas could be a targetfor future intestinal therapyacknowledgementsw e thank all the staff of the department of traumatology and acute critical medicine osakauniversity school of medicinedisclosureapproval of the research protocol this study was approvedby the institutional review board of osaka universityinformed consent informed consent was obtained from eachpatient™s familyregistry and the registration no of the studytrial naanimal studies naconflict of interest nonereferences meddings j the significance of the gut barrier in disease gut “ macfie j o™boyle c mitchell cj buckley pm johnstoned sudworth p gut origin of sepsis a prospective studyinvestigating associations between bacterialtranslocationgastric microflora and septic morbidity gut “ clark ja coopersmith cm intestinal crosstalk a new paradigm for understanding the gut as the motor of critical illness shock “ deitch ea bacterial translocation or lymphatic drainage oftoxic products from the gut what is important in humanbeings surgery “ kreymann kg berger mm deutz ne espen guidelines on enteral nutrition intensive care clin nutr “ shimizu k ogura h goto m altered gut flora andenvironment in patients with severe sirs j trauma “ yamada t shimizu k ogura h rapid and sustainedlongterm decrease of fecal shortchain fatty acids in criticallyill patients with systemic ‚ammatory response syndromejpen j parenter enteral nutr “ american college of chest physicianssociety of criticalcare medicine consensus conference definitions for sepsisand an failure and guidelines for the use of innovative therapies in sepsis crit care med “ sugawara g nagino m nishio h perioperative synbiotic treatment to prevent postoperative infectious complications in biliary cancer surgery a randomized controlled trialann surg “ kikuchi h yajima t correlation between waterholdingcapacity of different types of cellulose in vitro and gastrointestinal retention time in vivo of rats j sci food agr “ horan tc andrus m dudeck ma cdcnhsn surveillancedefinition of health careassociated infection and criteria forspecific types of infections in the acute care setting am jinfect control “ shimizu k ogura h hamasaki t altered gut flora areassociated with septic complications and death in critically illpatients with systemic ‚ammatory response syndrome digdis sci “ maslowski km vieira at ng a regulation of ‚ammatory responses by gut microbiota and chemoattractantreceptor gpr43 nature “ fukuda s toh h hase k bifidobacteria can protectfrom enteropathogenic infection through production of acetate nature “ asahara t takahashi a yuki n kaji r takahashi tnomoto k protective effect of a synbiotic against multidrug the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558resistant acinetobacter baumanniimodel antimicrob agents chemother “in a murine infection guarner f malagelada jr gut flora in health and diseaselancet “ osuka a shimizu k ogura h prognostic impact of gill sr pop m deboy rt metagenomic analysis of thefecal ph in critically ill patients crit care r119human distal gut microbiome science “ o™toole pw jeffery ib gut microbiota and aging science macfarlane s macfarlane gt regulation of shortchain fatty “ rampelli s candela m turroni s functional metagenomic profiling of intestinal microbiome in extreme ageingaging albany ny “ de gunzburg j ghozlane a ducher a protection ofthe human gut microbiome from antibiotics j infect dis “ p 13erezcobas ae gosalbes mj friedrichs a gutmicrobiota disturbance during antibiotic therapy a multiomic approach gut “acid production proc nutr soc “ pryde se duncan sh hold gl stewart cs flint hj themicrobiology of butyrate formation in the human colonfems microbiol lett “ shimizu k ogura h asahara t probioticsynbiotictherapy for treating critically ill patients from a gut microbiotaperspective dig dis sci “ shimizu k ogura h goto m synbiotics decrease theincidence of septic complications in patients with severesirs a preliminary report dig dis sci “ the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c'
Colon_Cancer
" curcumin is herbal compound that has been shown to have anticancer effects in preclinical andclinical studies the anticancer effects of curcumin include inhibiting the carcinogenesis inhibiting angiogenesisand inhibiting tumour growth this study aims to determine the clinical effects of curcumin in different types ofcancers using systematic review approachmethods a systematic review methodology is adopted for undertaking detailed analysis of the effects of curcuminin cancer therapy the results presented in this paper is an outcome of extracting the findings of the studiesselected from the s published in international databases including sid magiran iranmedex irandoc googlescholar sciencedirect scopus pubmed and web of science isi these databases were thoroughly searched andthe relevant publications were selected based on the plausible keywords in accordance with the study aims asfollows prevalence curcumin clinical features cancerresults the results are derived based on several clinical studies on curcumin consumption with chemotherapydrugs highlighting that curcumin increases the effectiveness of chemotherapy and radiotherapy which results inimproving patient™s survival time and increasing the expression of antimetastatic proteins along with reducingtheir side effects the comprehensive systematic review presented in this paper confirms that curcumin reduces the sideeffects of chemotherapy or radiotherapy resulting in improving patients™ quality of life a number of studiesreported that curcumin has increased patient survival time and decreased tumor markers™ levelkeywords prevalence curcumin clinical feature cancer systematic review research over the past years has significantly increased our understanding of the molecular genetic basisof cancer it is now well known that cancer is caused bya set of molecular genetic changes that lead to loss ofgrowth control and cellular differentiation resulting in correspondence nsalarikumsacir masoudmohammadi1989yahoocom5department of biostatistics school of health kermanshah university ofmedical sciences kermanshah iran7department of nursing school of nursing and midwifery kermanshahuniversity of medical sciences kermanshah iranfull list of author information is available at the end of the uncontrollable cell growth that eventually leads to tumorformation more than half of all cancers occur in developingcountries including those located in southern americaand asia nearly threequarters of people of these countries are classified into low or middleincome categoriesthe cancer survival rates in developing countries aregenerally onethird ofthe patients in the developedcountries there are million new cases of cancereach year with million new cancer cases in the developed countries and million in developing countries in the next decades cancer will be one of the leading the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cmansouri bmc cancer page of causes ofillness worldwide and the number of newcases of various types of cancer is expected to rise to million by furthermore cancer is predicted to bethe leading cause of death by given that cancerstatistics are on the rise and their treatments are costlyit is very crucial to find effective and economically viablemethods for patients in low and middle income countries therefore this study is motivated by using effectiveand relatively cheap treatments for cancer therapy asystematic review of the clinical studies on curcumin useand its effectiveness in inhabiting and treating varioustypes of cancer is carried out to obtain comprehensiveinformation aboutthe curcumin effects on cancertherefore a structured review of all published sand other relevant documents on the use of curcuminfor cancer therapy creates a more complete picture ofcurcumin effects on cancer patients from different angles in the process of this review only evidence from thestudies with highest quality are selected to gather information and derive on curcumin effects andeffectiveness at various stages of cancer therapyamong the medical herbs flavonoids are a large subgroup of the family of natural polyphenolic compoundsthat are the result of secondary metabolism in plants in recent years research has shown that flavonoids havebeen very effective in the prevention and control of common diseases complex such as cancer cardiovasculardiseases alzheimer™s stroke diabetes osteoporosisand rheumatoid arthritis furthermore there are robustevidence of antiviral antiinflammatory and antiallergic effects of flavonoidsin the recent years the use and effectiveness of medicinal herbs in treatment of various diseases has been received enormous attention huge research efforts weremade on extraction and examination of the properties ofthe herbal compounds in the treatment of different typesof diseases eg cancers and providing detailed mechanisms of drug performance of these compounds amongst the wide range of the medical herbs curcuminis an effective ingredient of turmeric plant with the scientific name of œlonga curcuma chemical name ofœdiferuloylmethane and the chemical formula of c21h20 o6 as illustrated in fig curcumin makes up between to of turmericcompounds and is considered as the main cause of yellowgolden colour of turmeric and it has also been identified as responsible for many ofthe properties ofturmeric [ ] however curcumin has low inherenttoxicity and various properties with great impact and applications on a wide range of pharmacological developmentsantiinflammatoryantimicrobial and anticancer drugs [“]antioxidantincludingcurcumin has been shown to have preventive and therapeutic effects on various types of cancers the findingsfrom several studies suggest that curcumin compound canprevent the formation and spread of tumors or reduce theirsize it was shown that curcumin can inhibit the formationof cancer and spread the cancerous cells by exerting antiangiogenic effects inducing apoptosis and interfering withthe cell proliferation cycle [ ] curcumin exerts itsanticancer effects through a variety of mechanisms curcumin inhibits and suppresses the proliferation of a widerange of cancer cells which exerts its effects by reducingthe modulation of antiapoptotic gene products activatingcaspase and upregulating cancersuppressive genes such asp53 [“] recent studies confirm the preventive andtherapeutic effects of curcumin on various types of cancersindicating that it can prevent or reduce the formation orspread of tumors curcumin inhibits tumor invasion by reducing the modification of matrix metalloproteasesmmps the cell surface adhesive molecules nfκ ap1tnfα lox and cox2 chemokines growth factorsher2 and egfr inhibits nterminal activity and tyrosine kinase protein [ ] curcumin inhibits angiogenesisin some tumors by suppressing angiogeniccytokines such as il6 il23 and il1 [“] due tothe strong relationship between inflammation and cancerthe antiinflammatory effects of curcumin would well resultin its antitumor effects it was reported that curcumin hasprevented the development of several types of cancer by reducing the production of mediators of the inflammatoryprocess such as cox2 lipoxygenase inos and relatedcytokines one of the possible mechanisms for suppressing tumor proliferation is the chemical inhibitor effectof curcumin as a result topical use of curcumin considerably inhibits inflammation due to tetradecanoylphorbol13fig the chemical expansion of curcumin by coreldraw graphics suite 0cmansouri bmc cancer page of acetate 12o tpa hyperplasia cell proliferation odcactivity production of active oxygen species oxidativedna changes and papillomavirus formation [“] multiple human gastrointestinal cell interactions with curcumininhibit lipid peroxidation inhibit cox2 expression inhibitpge2 production and increase glutathionestransferaseenzyme levels [ ] the other mechanism of the anticancer effects of curcumin is due to its interference in thecell cycle and reduction in cdk expression cdks are actually serine threonine kinases that control cell cycle progression furthermore curcumin inhibits the stat3phosphorylation which is responsible for signalling carcinogenic pathways given that cancer statistics are on the rise and theirtreatments are quite costly it is very crucial to find someeffective methods and economically viable for low andmiddleincome patients therefore this paper providesupto date evidence and findings of clinical studies onthe effects of curcumin contributions in tumor cells survival and metastasis using a systematic review approachmethodssystematic review approach is adopted for undertakingthis study by extracting the findings of the relevant studies selected from the s published in national andinternational databases including sid magiran iranmedex irandoc google scholar sciencedirect scopuspubmed and web of science isi these databases werethoroughly searched and the relevant publication records were selected based on the plausible keywords inaccordance with the aim of this study as follows prevalence curcumin clinical features cancerthe selection of relevant studies for the systematic review and the output quality control process involvedseveral steps first all related s were collectedbased on the search keywords mentioned in the nextstep the specifications including the name of thejournal and authors were hidden and the full text of thes were made available to the reviewers each was investigated independently by two reviewersmm shr and if an was excluded in the studyfig the flowchart on the stages of including the studies in the systematic review prisma 0cmansouri bmc cancer page of detailed rationale were give accordingly in the case ofdisagreement between the two reviewers the wasjudged by a third reviewer in this paper all studies related to clinical investigations of curcumin use and impacts at varioustreatment weresystematically examined without any time constraintsand according to prisma guidelines fig stages of cancer selection criterias with the following characteristics were selectedfor metaanalysis original research s clinical trialstudies s that their full text and data are availableand studies that examined the clinical effects of curcumin in various types of cancers we prepared a list of s specifications based on prisma includingthe researcher™s name the title the year and placeof the study sample size and number of patients duration of study dosage of the drug and the result of theintervention table including review papers exclusion criteriastudiessystematic reviewmetaanalysis cohort casecontrol crosssectional descriptive and those which didn™t present samples fromcancer patients and those which conducted with secondary data were excluded from the review duplicate publication and multiple publicationssamepopulation will be removed using citation managementsoftware endnote version x7 for windows thomsonreutersfrom thequality assessmentthe quality of the selected s was evaluated basedon criteria outlined by the consort checklist the lastconsort statement published in included items the consort statement has been shown to improve the scientific quality of rct reporting [ ]each was blindly assessed by two independentevaluators mm shr the result of each item wasassessed by yes point or no point and some itemswere assessed as not applicable due to the features ofstudies accordingly the maximum quality score of was considered and papers with a score of less than were considered to have low quality and thus they wereexcluded from the studycurcumin role in the prevention of cancersfree radicals and toxic products resulted from oxidativestress play an important role in the early stages of cancerformation therefore compounds that have antioxidanteffects can be helpful in preventing cancer formationcurcumin has the property of trapping free radicals andthus can play a crucial role in inhibiting the onset ofcancer several cellular and preclinical studies havereported that curcumin inhibits dna damage caused byoxidative factors such as ionizing radiation by inhibitingfree radicals and active oxygen species the nfkappab plays an important role in the formation of nitricoxide synthase and oxidative stress and as a resultcauses cancer curcumin suppresses the onset ofcancer by inhibiting nfkappab from formation [ ]curcumin was reported to be effective on liver enzymescytochrome p450 which has an imminent role in theoxidation and detoxification of toxins it also inhibits thephase i enzymes that is involved in the production oftoxic metabolites and carcinogens furthermore curcumin activates the phase ii enzymes which plays a crucialrole in detoxification of toxic metabolites curcumin prevent tumor formation and growth by inhibitingand activating these two enzymes phase i ii effect of curcumin on metastasis angiogenesis andinflammation in cancer cellsangiogenesis is the process of new blood vessel formation from preexisting vessels that is dependent on aprice equilibrium between antiangiogenic and angiogenicfactors however under pathological conditions for example tumor growth this tight regulation becomes lostwhich can result in tumor metastasis many gene products that are produced by different cells have a role inangiogenesis process hypoxia usually occurs in tumorsites in order to overcome to hypoxia tumor cells regulate and control the expression of genes related to angiogenesis cell cycle metastasis and drug resistance usinghypoxiainducible factor hif1 hif1 was first recognized as a transcription factor involved in hypoxiainduced erythropoietin expression this factor has beenpresented as a main transcription regulator for thesemolecules [ ] several studies have shown thathif1 activation of genes including vascular endothelialvegf angiopoietin1 ang1 andgrowth factorangiopoietin2 ang2 nfkb etc induced angiogenesis in the tumor cells furthermore the activation ofgenes such as insulinlike growth factor igf2 transforming growth factor a tgfa and mapk and pi3ksignalling pathway will also enable the survival proliferation and metastasis of tumor cells hif1 by activating genes involved in angiogenesis and also activatessignalling pathways associated with cell survival and proliferation plays an important role in the stability andgrowth of tumors as above mentioned hif1α is apotent activator of angiogenesis and curcumin inhibitsits expression ap1 is a transcription factor that is activated in response to hypoxia which is the principlephysiological stimulus that induces angiogenesis it isalso involved in the conversion of epithelial cells to mesenchymal cells which is the primary stage of metastasisand causes the expression of mmp and upa urokinase 0cmansouri bmc cancer page of table examines the characteristics extracted in the studiesauthor™s name yearcountry duration ofdosage of the drughejazi2013 belcaro2014 iranitalystudy years“ g per daynumber ofpatientsresultscurcumin reduces the severity of urinary symptoms mg with soy lecithin curcumin reduced side effectsbayetrobert2010 france months to mgryam2013 kanai2011 hemati2011 garcea2005 sharma2001 sharma2004 usajapaniranukukukcruzcorrea m2006 usa years months months“ months months months g g g to mg per day“ g per day“ g per day g per dayyu he2010 china“ days g per daydurgaprasad2005 india weeks g per daydhillon2008 usa“ g per dayide2010 japan months g per daygolombick2009 australia months g per daypolasa1992 hastak1997 indiaindia days g per day months g per daycheng2001 taiwan months g per dayrai2010 uk days g per daymarcia cruz correa2018 richard greil2018 newyorkusa month weeks mg orally twice adaydoses between and mg per minutelynne m howells2019 unitedkingdom days g per dayplasminogen activator genes that are involved in tumorangiogenesis and its invasion curcumin inhibits the expression of this transcription factor curcumin mayinhibit angiogenesis directly by regulating angiogenicgrowth factors growth factors as well as the genes including angiopoietin1ˆ’ hif1 ho1 and transcription factors such as nfkappab fig [“] it isknown that hypoxic stress and activation of betagrowthfactor tgf stimulate vegf expression by activatingap1 and the hypoxiainducible factors hif1 curcumin is an important inhibitor in ap1 activationand it has recently been shown that curcumin is a directinhibitor of hif1 transcription factor activity whichcauses the transcription of many genes associated withcurcumin lowers the concentration of the cea markertumorcurcumin reduces some skin complicationscurcumin increased patient survivalcurcumin reduces some skin problemscurcumin increased the effectiveness of the coloncurcumin reduced glutathione stransferase activitycurcumin reduces prostaglandin e2 productionreduces the number and size of polyps without anysignificant toxicitycurcumin has been shown to improve the overallhealth of patients with colorectal cancercurcumin reduced lipid peroxidation and increasedglutathione content in patientswelltolerated limited absorption and showedactivity in some patientsreduced the serum prostatespecific antigen contentin combination with isoflavonesdecreased para protein load and urinary n telopeptideof type i collagenreduced the urinary excretion of mutagens in smokersreduced the number of micronuclei in mucosal cellsand in circulating lymphocytesimproved the precancerous lesionsincreased vitamins c and e levels decrease dmalondialdehyde and 8hydroxy deoxy guanosine contents inthe serum and salivano difference in polyp size and number betweenplacebo and curcuminno variation in tumor size according to recist criteriacurcumin was a safe and tolerable adjunct to folfoxchemotherapy in patients with metastatic colorectal cancerangiogenesis in tumors [ ] it is also shown thatcurcumin will reduce the expression of membrane surface moleculesadhesionmolecule1 vascular cell adhesion molecule1 and eselectin which play a role in cellular adhesion fig intracellularincludingcurcumin affects a number of adhesive cellular molecules involved in tumor growth and metastasis processes curcumin caused reduction in the expression ofadhesive molecules inside the cells of icam1 vcamvcam or vascular cell adhesion molecule and mmpswhich play an important role in cellular adhesion andmetastasis furthermore curcumin results in increase ofthe expression of various antimetastatic 0cmansouri bmc cancer page of fig the effect of curcumin on angiogenesis and metastasis in cancer cells by coreldraw graphics suite including tissue inhibitor metalloproteinaseproteinstimp the nonmetastatic gene nm23 and ecadherin lack of ecadherin would increase the possibility of metastasis because ecadherin are essential tomaintain cellular adhesion angiogenesis is alsolinked with neoplasia angiogenesis means the formationof new blood vessels which is generally a major step intumor survival and growth curcumin inhibits cancer invarious ans [“ ]easyto assess whetherthe antiinflammatory effects of curcumin have beenproven in many studies since oxidative stress leads tochronic inflammatory diseases antioxidant compoundscan be useful in the prevention and treatment of inflammatory disorders [ ] on the other handsince curcumin has a high antioxidant activity it willnot beantiinflammatory activity is also dependent on its antioxidant activity [ ] since many of the antioxidantsthat have been already identified do not have antiinflammatory properties it seems unlikely that the antiinflammatory effects of curcumin are due solely to itsantioxidant properties curcumin as a potent antiinflammatory factor expresses its own effects throughseveral mechanisms first curcumin inhibits the activation of the nfκ factor [ ]curcumin™sthe lab based studies have revealed that curcuminneutralizes oxidative stress caused by tumor and restorestnfαnfkappabcurcuminactivityinhibitsproduction thus tcell apoptosis caused by tumor willbe minimised resultsin the initial screening of databases s wereidentified after deleting duplicate s studieswere obtained after deleting unrelated s studies were obtained17 s were also deleted due tolack of access to their fulltext or falling into the lowquality category at the end studies entered the finalphase and analysis as illustrated in fig the specifications and details of the studies considered in this systematic review are summarized in table according to the studies presented in table curcumin has reduced side effects including skin complications depending on the different doses of curcuminprescribed for the patients suffering from cancer theirsurvival rate was increased and their symptoms ofchemotherapy were reduced in studies examining theeffect of curcumin on colorectal cancer curcumin hasincreased efficacy in the large intestine reduced glutathione stransferase activity and reduced prostaglandin e2production curcumin also reduces the number and sizeof polyps without any significant toxicity curcumin inpancreatic cancer reduces lipids™ peroxidation and increases glutathione content in the patients with this typeof cancer in prostate cancer curcumin reduces theserum levels of prostatespecific antigen in combination 0cmansouri bmc cancer page of with isoflavones and also reduces the severity of urinarysymptoms according to the published studies the useof curcumin during radiation therapy for breast cancerpatients improved treatment outcomes for these patients such as preventing skin symptoms reducing painand suffering of patients improving their quality of lifeduring treatment and reducing delays or unwantedstops during the course of radiation therapy curcumincan regulate multiple signalling pathways and affect different moleculartargets low cost pharmacologicalsafety efficiency and multiple molecular targets makecurcumin a promising product for the prevention andtreatment of various human diseases table after collecting various s from reputable databases and deleting duplicate s and removing unrelated s to the main aim of this paper we finallyconsidered s for further investigation and analysis the main aim of this paper is to review the clinicalstudies about curcumin and its various purposeseffectson cancer the results reported from numerous clinicalstudies that have examined the effects of curcumin onthe patients who are suffering from cancer and undergoing radiotherapy and chemotherapy were very promising here we briefly describe some of these studieswhich are summarised in table garcea evaluated patients in the ukin this study each patient received to mg ofcurcumin per day at the same time that these patientsreceived curcumin they were treated with radiotherapyand chemotherapy the results of this study revealedthat curcumin increased the effectiveness of the treatment plan for colorectal cancer in the patients receivedwith curcumin importantin bayetrobert conducted a study consists offourteen patients with advanced breast cancer who werebeing treated with docetaxel chemotherapy and simultaneously received curcumin at different doses up to amaximum dose of g per day for days per each treatment cycle finally patients participated in this studywere able to complete this treatment plan nutropeniaand leukopenia were the mosttoxicitiescaused by docetaxel administration after days two patients refused to continue treatment because they received curcumintreatment continued by reducing the dosage to a maximum of g per day nine patients were screened fortumor response six weeks after completing the courseof treatment patients partially responded well butthree patients still suffered from the disease in thisstudy the ca tumor marker did not decrease butthe cea tumor marker decreased compared to the initial value prior to the treatment in patients the vegfvascular endothelial growth factor as a tomur markerwhichandcurcumin capsules howeverindicatestumrowth metastasismalignancy was reduced by compared to the baseline before treatment in the effect of curcumin on reducing the sideeffects of radiotherapy and chemotherapy in patientswith ovarian lung colon liver kidney and stomach cancers was investigated eighty patients received mg ofcurcumin simultaneously with radiotherapy the duration of this study was days the incidence of side effects such as nausea diarrhea constipation and weightloss decreased in patients who treated with both radiotherapy and curcumin in the patients who are simultaneously under radiotherapy and received curcumin theprevalence of side effects such as skin lesions mouthand throat ulcers swallowing problems nausea vomitingfatigue weakness and common medications required for treating side effects were statistically lowerthan the control group in a study conducted by hemati in iran patientsreceiving radiation therapy to the breast area due tobreast cancer from days before the start to the end ofradiotherapy mgcapsules containing curcuminwere taken orally times a day yu he evaluated patients in their study andstated that a dose of g of curcumin per day for “ days improved the general health of patients withcolorectal cancer through increasing the expression ofp53 molecules in tumor cells in a study conductedby cruzcorrea it was stated that a dose of g of curcumin per day will reduce the number and size of polypswithout any significant toxicity discussionin the recent years several studies have been conductedon the biological effects of curcumin in more than studies have been recently published curcumin hasshown to have various effects in cancer treatments curcumin has antioxidant antibacterial antifungal antiviralantiinflammatory antiproliferative proapoptotic effects etc curcumin has tremendous potential for treatment of neurodegenerative diseases arthritis diabetespsoriasis allergies intestinal inflammation kidney poisoning alzheimer™s depression aids multiple sclerosis cardiovascular disease and especially cancer [“] the numerous and multifaceted effects of curcuminin determining the cellular targets and molecular mechanisms involved in curcumin pathways have attractedmuch attention from researchers curcumin is a multifaceted molecule and has many therapeutic effects themultifaceted effects of curcumin are due to its capacityto interact with different molecules and to regulate multiple molecular pathways and their targets one of the compelling properties of curcumin whichmakes it appropriate for therapeutic use is its low toxicity so that even its consumption up to a dose of g 0cmansouri bmc cancer page of per day does not cause any side effects consumption of curcumin in highdose prevents cancer cells frommultiplying although it does not damage healthy cells[ ]minimaltoxicity alongside with possessing manytherapeutic effects have led to the widespread use of natural plantderived compounds in the treatment of cancer the compounds found in nature target various cellular and molecular aspects of cancer cells the researchers have demonstrated that curcumin regulatessignalling pathways in cancer cells reduces the expression of proteins related to drug resistance and increasesthe performance of antitumor drugs at various levelscurcumin reverses drug resistance mechanisms and results in increasing the sensitivity of chemotherapyresistant cells in the research conducted by keyvanighamsari they demonstrated that curcumin is aneffective chemical in cancer treatment in laboratory studies which have been performed onthe cellular categories of colorectal cancer the derivedresults show that curcumin inhibits cell growth and alsostimulates apoptosis by interacting with several molecular targets furthermore curcumin has been used as partof dietary formulations to prevent colon cancer in vitroand in vivo these compounds have been shown to haveanticancer properties for colon cancer and its inflammation the results of this study show that curcuminwould be effective in preventing colorectal cancer in animals this property offers promising expectations inhumans due to the limited number of the human clinical studiesthe corresponding results are somehowcontradictory on the other hand there exist several unanswered questions about dosage bioavailability optimalsigns and potential toxicity which should be investigatedin future studies using sufficiently large samples inaddition curcumin can induce autophagy apoptosisand cell cycle arrest in order to reduce the survival andproliferation oflung cancer cells curcumin has thispromising capability to increase the effectiveness ofradiotherapy in the treatment of lung cancer by targetingdifferent signalling pathways such as epidermal growthfactor receptor and nf κb curcumincontaining nanocarriers increase bioavailability cell uptake and curcumin antitumor activity [ ]in a study conducted by cruzcorrea oral curcumin was prescribed to the patients with adenomatouspolyposis this research was implemented to determinethe safety and efficacy of curcumin in patients with adenomatous polyposis in this study mg of oral curcumin was administered twice per day over monthsto patients with adenomatous polyposis the resultsshowed that there was no significant difference betweenthose who received oral curcumin and those receivingplacebo in another study conducted by grell patients were subjected to receive doses between and mg per minute the main aim of their studywas to evaluate the safety of curcumin locally in patientswith advanced or metastatic cancer the results obtainedfrom their study showed that no change in tumor sizewas observed based on the recist criteria in howells evaluated patients with the age over and with metastatic colorectal cancer using the histological diagnosis quality oflife and neurotoxicity ofthese patients were assessed using questionnaires thederived results showed that curcumin is a safe and tolerable adjunct for folfox chemotherapy in patients withmetastatic colorectal cancer overall the results suggest that curcumin can be usedas an effective combination in inhibiting and controllingcancersimproving clinical symptoms and preventingtumor spread and metastasis this compound wouldaffect various molecular pathways and inhibits vasodilation cell proliferation and metastasiscurcumin is a natural product found in turmeric thathas a unique chemical structure with particular biological and medicinal properties through various cellular and molecular mechanisms curcumin inhibits thecarcinogenesis and their growth due to the fact that nospecific toxic effects of this natural product have beenreported its use has been considered as a drug supplement in therapeutic diets of cancer patients in a number of studies considered in this systematic review haveshown that taking curcumin would increase the expression of antimetastatic proteins in several other studiesit was reported that curcumin has also increased patientsurvival and decreased tumor marker concentrationabbreviationsmmps matrix metalloproteases vcam vascular cell adhesion moleculewho world health anization sid scientific information database prisma preferred reporting items for systematic reviewsacknowledgementsthe authors thank the faculty members of the faculty of nursing andmidwifery kermanshah university of medical sciencesauthors™contributionsshr and ns and km contribute
Colon_Cancer
" the cell proliferative markers are very important in breast cancer since spag5 and numa proteinsplay a significant role in the mitosis regulatory network and cell division we aimed to study their mrna levels aswell as spag5 gene amplification correlated to clinicopathological status in ductal carcinoma of the breastmethods spag5 and numa gene expressions were investigated in breast cancer tissues and normal adjacenttissues via realtime pcr pum1 was selected as the reference gene qmf pcr method was applied to study spag5gene amplification and agbl2 bod1l and por were designated as internal control genes gene amplification wasdetermined by calculating a dosage quotient for each dna fragmentresults increased spag5 mrna expression was detected in breast cancer tissues p and related to tumorsize no significant difference was observed between numa gene expression level in tumor tissue and the normaladjacent tissue p however we observed that numa expression was significantly increased in erpositivetumor tissues there was no clear correlation pattern between spag5 and numa mrna levels r seventeenpercent of tissues showed complete amplification in spag5 gene fragments our results were consistent with the previous publications regarding spag5 gene expression andamplification in breast cancer with an emphasis on the prominent role of this protein in tumor pathogenesis ourresults failed to yield any correlation between spag5 and numa mrna levels which implies independence of thesegenes in breast cancer pathogenesiskeywords spag5 numa gene expression gene amplification breast ductal carcinomaintroductionbreast cancer is the most common cancer type and the second leading cause of cancer death in women worldwidenumerous studies have been conducted to find the cellularand molecular biomarkers to improve early detection andmanagement of the disease and reduce its mortality ratethe study published by abdelfatah has drawn attention to spermassociated antigen spag5 as a new correspondence shshahbazimodaresacir1department of medical genetics faculty of medical sciences tarbiatmodares university tehran iranfull list of author information is available at the end of the prognostic biomarker in breast cancer using the interactome map they showed a prominent role for spag5 in patient™s response to chemotherapy and survival spag5 gene is located on chromosome 1717q112comprising exons with nine reported mrna splicevariants it is categorized among conserved genes as ithas been present since the common ancestor of chordates the aminoacids protein of spag5 is a homodimer which binds to microtubules in the mitoticspindle and regulates its dynamic function spag5 isone of the essential components required for chromotiming of sister chromatidsome alignment normal the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cmohamadalizadehhanjani world of surgical oncology page of segregation and spindle pole formation spag5 proteinis expressed in a cell cycledependent manner with thehighest level at telophase in nonmitotic cells various stimuli upregulate spag5expression spag5 protein plays multiple roles in different pathways of cell growth and differentiation includingpi3kakt upon cell oxidative stress spag5 preventsapoptosis via mtorc1 suppression significant evidenceconfirms thatthe pi3kaktmtor pathway is themost common defected signaling cascade in breast cancer increased spag5 mrna expression in tumortissues was reported in different cancers such as cervixlung and bladder [“] indicating theprostatehigher spag5 levels the poorer prognosis and patientsurvivalincreasedspag5 expression in cancer the cellular and molecularevents affected by this increase are still unclear it is notwell elucidated whether spindlassociated proteins areinvolved as much as cell signaling cascades[ ] despite several reports ofliverone of the important spag5 interactors in cytokinesisis the nuclear mitotic apparatus numa protein whichdirectly binds and recruits spag5 to the mitotic spindlethis interaction is reciprocal in a way that the lack ofspag5 interferes with the appropriate numa spindledistribution and function numa gene is located on11q13 with exons that encodes a large aminoacids protein it acts as a microtubulebinding proteininvolved in the arrangement of the spindle poles andproper separation of the chromosomes in a complexcomposed of dyneindynactinnuma ddn dynein is an energy supplier for eukaryotic cellular movements and dynactin serves as its cofactor studies havedemonstrated numadynein role in dna doublestranded break repair by homologous recombination related to brca proteins numa is also involved in different cellular pathways including plk1 activated celldivision and caspasemediated apoptotic response our study aimed to investigate the association betweenspag5 and numa mrna levels in breast cancer tumors since spag5 gene amplification was also involvedin the pathogenesis ofthe breast cancer we alsointended to identify any association between the amplified genome and spag5 numa mrna levelsmethodspatientspatients were selected between january and october by random sampling from farmaniyeh hospitaland sina hospital affiliated to tehran university ofmedical sciences inclusion criteria were as follows anew case of biopsyconfirmed breast invasive ductal carcinoma idc with or without ductal carcinoma in situdcis exclusion criteria were receiving any chemotherapy radiotherapy and hormone therapy the patientssigned a consent form approved by the ethics committeeof tarbiat modares university with the registrationnumber of irtmurec1396681 eighty tissue samplesincluding forty tumors and forty normal tissues adjacentto the tumor nat were collected under the supervision of the breast surgeon patient™s clinicopathologicalcharacteristics comprising age family history of cancerlymph node involvement tumor size tumor grade estrogen receptor er progesterone receptor pr andhuman epidermal growth factor receptor her2 statuses were recorded clinical stage was determined bytnm system including tumor size lymph node involvement and metastasis lymph node involvement wasconfirmed histologically according to ajcc guidelinethe molecular stage was defined using tnm tumrade and er pr and her2 status we further classified patients to breast cancer subtypes luminal a luminal b her2 positive and basallike ki67 index as amarker for tumor proliferation was applied to subtypeluminal tumors by the cutoff value of since nats were taken from the nontumorous regionof the same patient™s breast for a more accurate comparison we also included three samples of healthy breasttissues from individuals with no history of breast cancerthe reason for surgery in these healthy individuals wascosmetic restorations and they did not have any historyof cancer in 1st and 2nddegree family relativesmcf7 and mdamb231 cell line culturebreast cancer cell lines were used as the control of realtimepcr and quantitative multiplex fluorescent pcr qmfpcr mcf7 ibrc c10082 and mdamb231ibrcc10684 were obtained from the national cell bank of iranboth cell lines were invasive breast ductal carcinoma bearing an epitheliallike morphology cell lines were culturedin the medium containing dulbecco™s modified eaglemedium dmem and fetal bovine serum fbsdna and rna extractionafter homogenization of mg breast tissue dna andrna were simultaneously extracted by allspintm minikit geneall pishgam biotech iran dna and rnaquantities were determined by spectrophotometry at and nm the observation of 18s rrna and28s rrna bands in gel electrophoresis was in favor ofappropriate rna quality cdna synthesis was performed by 2x rt premix kit biofact noavaran tebiran mmlv reverse transcriptase oligodt and random hexamer were simultaneously applied to increasecdna synthesis efficacyrealtime pcrthe expression of spag5 and numa genes were quantified by realtime rt pcr to normalize the results 0cmohamadalizadehhanjani world of surgical oncology page of pum1 was selected as the reference gene pum1 is ahousekeeping gene with high and constant expression innormal and tumor breasttissue primer design forspag5 numa and pum1 genes was conducted by primer3 and oligo analyzer software and checked onblast basic local alignment search tool websites toprevent genomic dna amplification all realtime pcrprimers spanned exonexon junctions the detailedcharacteristics of realtime primers are provided in table realtime pcr was performed by applied biosystemstep one system using sybr green dye pcr efficiencywas calculated by linreg pcr software through linearregression analysis1bod1l atpgtpbinding protein likeqmf pcrspag5 gene amplification was examined through qmfpcr and biorientation of chromosomes in cell division likeagbl2 and cytochrome p450 oxidoreductase porwere selected as internal control genes the primerswere designed for three parts of spag5 gene ² endmiddle and ² end of the gene all internal controls andthree spag5 primers comprised similar tm table to fluorescently label pcr fragments universal primerwas opted for as an efficient and costeffective methoduniversal primer sequence published by roche companywhich does not have any specific complementary site inthe human genome was selected each fragmentwas amplified by primers tailed locusspecific forwardprimerlocusspecific reverse primer and 6famlabeled universal primer in a concentration ratio of due to the lower starting concentration of the tailedlocusspecific primer as pcr progress it was graduallydepleted and the 6fam labeled universal primer wassubsequently included amplitaq goldtm dna polymerase ampliqone pishgam biotech iran with hotstart activity was applied for a multiplex pcr reactioncapillary electrophoresis was performed via μl of thepcr products μl offormamide and μl ofgenescan500 liz size standard applied biosystemscourtaboeuf france the fluorescent pcr productswere heatdenatured chilled on ice and separated on an8capillary sequencer genetic analyzer appliedbiosystems france the results were processed withgenemarker software the peak area values wereimported into an excel microsoft file and the copynumber of each fragment was calculated by the dosagequotient dq methodstatistical analysisthe sample size was calculated by epi info„¢ softwarespss statistic version and graphpad prism version were used for realtime data analysis to determine thetype of appropriate statistical method data were analyzed by kolmogrovosmirnov for distribution normaltheity since the distribution of data was normalparametric test was used pairedsample ttest wasemployed to compare spag5 and numa gene expression level between tumors and nats the links betweenclinicopathological status and mrna levels were calculated via independenttest and onewayanova for qmf pcr result analysis spss statisticversion was used to determine the frequency of samples with gene amplificationsample tresultspatient™s characteristicsthe mean age of the patients was ± years theminimum and maximum ages of the patients were and years thirteen patients had a family history of cancer mainly breast or colon cancer detailedclinicopathological features of the patients are given intable clinical stage was categorized by tnm system and theresults revealed that stage i stage ii and stages iiiivassigned to and of the samples respectively as indicated in table of the sampleswere categorized in the molecular stage i in stageii and in stages iiiiv further classification ofbreast cancer subtypes showed that luminal her2 positive and basallike comprised and of tumorsrespectively using ki67 index analysis weobserved that luminal a allocated to and luminal bto of the tumorstable specific primers designed for quantitative realtime pcrgenepum1sequence ²_3²fcctaccaactcatggtggatgtlengthspag5numaragccagcttctgttcaagactfaaggagaagactgaacaagagaccrtcatctgccactgctgtcaagfagagagcaaactaagcaggtggrcctggacagccttcagcttcttmgcproduct bppcr polymerase chain reaction tm melting temperature gc guanine cytosine bp base pair pum1 pumilio rnabinding family member f forward r reversespag5 spermassociated antigen numa nuclear mitotic apparatus protein 0cmohamadalizadehhanjani world of surgical oncology page of table specific primers designed for qmf pcrgenebod1lsequencefaatgcctccgctttcaggcchagbl2porspag55²spag5middlespag53²ratcacttggcaactcacacatggfgcgagctgcattccatgcgrtcccagctttggaaacgcacfagccactttgtgccagatcartccagcacgtgttcacatcafccctaagaagcccaaaatgcgrtcctggaaagttgggtcgagfaggctgctcatctgattcatgcrcaagctaccatctgcccacgfgttaggaaagggtcgaaagggcracaccctatcaaaagtctgttcclengthtmgcproduct bpqmf pcr quantitative multiplex fluorescent polymerase chain reaction ch chromosome tm melting temperature gc guanine cytosine bp base pair bod1lbiorientation of chromosomes in cell division like agbl2 atpgtpbinding protein like por cytochrome p450 oxidoreductase spag5spermassociated antigenspag5 gene expression levelsmelting curve analysis of the pcr products showed a single peak indicating specific amplification of the desiredgene fragment to normalize the results the ct differenceδct of spag5 and pum1 was computed to comparespag5 gene expression in tumor with nats δδct wasdetermined the fold change was calculated by ˆ’δδctmethod the spag5 and pum1 gene pcr efficiency was obtained by linregpcr softwarethe statistical analysis revealed a significant differencebetween spag5 gene expression levels in tumor relativeto nats p spag5 gene expression was upregulated in tumor tissues with a mean fold change of and the mean difference of fig using independent sample t test the spag5 mrna levelswere analyzed in correlation to age tumor grade er pr andher2 status as indicated in table the results showed nostatistically significant association between spag5 gene expression and these clinicopathological featureshowever correlation studies revealed a significant linkbetween spag5 gene expression and clinical stage p since the clinical stage was defined by tumor sizelymph node involvement and metastasis we analyzedthese parameters separately the patients™ tumors werecategorized according to the size “ cm “ cm “cm and spag5 gene expression was evaluated in the patients™ groups by oneway anova analysis the resultsdemonstrated a significant link between spag5 mrnalevels and tumor size p spag5 gene expressionlevel was higher in larger tumors lymph node involvement and metastasis revealed no significant correlation tospag5 mrna levels the statistical analysis regardingmolecular stage and molecular subtypes failed to showany significant association table numa gene expression levelsthe mean of ct values for numa and pum1 were analyzed for duplicate samples and modified based on reaction efficiency the results of linreg analysis indicatedpcr efficiency of for numa gene the specificity ofthe pcr amplification was confirmed by melting curvegraphs of each reaction the mean fold change of numagene expression in tumors relative to nats was which was not statistically significant p056 furtherdata analysis disclosed a significant association betweennuma gene expression and er status p0006 ernegative tumors showed eight times less numa gene expression than erpositive tumors er is one of the mainparameters of molecular subtype classification so as expected this significant difference was also observed regardinglink between numa expression andmolecular subtypes p however numa expression did not show any significant difference in the molecular stage or the clinical stage table thethe correlation study to other clinicopathological status revealed no significant link with age tumor gradelymph node involvement and tumor size no significantconnection was observed regarding pr or her2 statustable the correlation between spag5 and numa geneexpression in tumor tissuethe study of the correlation was conducted betweenspag5 and numa genes expression using the pearsontest in fig the x and yaxes show δct of spag5 andnuma genes respectively the test results showed thatthere was no statistically significant correlation betweenthe two mrna levels r 0cmohamadalizadehhanjani world of surgical oncology page of table statistical analysis of clinicopathological variables related to spag5 and numa mrna expressionsspag5agegradelnfherprher2numaagegradelnfherprher2n t years yearsi and iiiiipositivenegativepositivenegativepositivenegativepositivenegativepositivenegative years yearsi and iiiiipositivenegativepositivenegativepositivenegativepositivenegativepositivenegativeˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ssigtailedstderror cilowerˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ upperˆ’ ˆ’ ˆ’ ˆ’ independent sample t test was applied to compare the spag5 and numa gene expression levels according to clinicopathological features normal distribution ofvariables and equality of variances were checked by kolmogorovsmirnov and levene™s test respectively the significance level was selected at n number t t test sig significance p value std standard ci confidence intervals spag5 spermassociated antigen numa nuclear mitotic apparatus protein lnlymph node involvement fh family history er estrogen receptor pr progesterone receptor her2 human epidermal growth factor receptor qmf pcr resultsthe fragment analysis was performed following verifiedmultiplex pcr pcr products should have at least bplength differences to be distinguishable in capillary electrophoresis fig we applied universal primer toavoid nonspecific pcr products and amplify multiplelociin one pcr reaction the copy number of eachspag5 fragment was determined by calculating the dqobtained by dividing the fragment peak area by the meanpeak area of control genes as demonstrated in fig the x and yaxes represents dna fragments length inbase pair bp and signal intensity as a relative fluorescent unit rfu the 3sd and ˆ’ 3sd values were considered as cutoff points values greater than wasconsidered amplified and values less than as deletionaccording to previous publications mcf7 and mdamb231 cell lines showed completespag5 gene amplification while three normal breasttissues and two healthy donor blood samples which usedas normal controls did not show any gene amplificationwe calculated mean dq of and for mcf7and mdamb231showed complete gene amplification in all three spag5gene fragments with mean dq of comparing samples which had no amplification in any fragment of spag5 gene mean dq the rest of thesamples showed amplification in one or two parts of thegene with a mean dq of respectively six samples 0cmohamadalizadehhanjani world of surgical oncology page of table analysis of staging related to spag5 and numa mrna expression clinical stage was determined by the tnm systemtumor size lymph node involvement metastasis according to ajcc guideline the molecular stage was defined using tnm tumrade and er pr and her2 statusspag5clinical stagemolecular stagemolecular subtypesnumaclinical stagemolecular stagemolecular subtypesstage istage iistages iii and ivstage istage iistages iii and ivluminal aluminal bher2 positivebasallikestage istage iistages iii and ivstage istage iistages iii and ivluminal aluminal bher2 positivebasalliken sum of squaresdfmean squarefp valueoneway anova was employed to analyze the difference between spag5 and numa gene expression levels related to clinical stage molecular stage andmolecular subtypes normal variable distribution and equality of variances were checked by kolmogorovsmirnov and levene™s test respectively our chosensignificance level was n number df degree of freedom spag5 spermassociated antigen numa nuclear mitotic apparatus proteinfig fold changes of spag5 and numa genes calculated by the relative expression software tool rest spag5 mrna expression wasincreased times in breast tumors than the normal adjacent tissues p the mean fold change of numa gene expression in tumorrelative to normal adjacent tissue was 0cmohamadalizadehhanjani world of surgical oncology page of fig pearson correlation of spag5 and numa expression levels in tumor samples the x and yaxes show δct of spag5 and numagenes respectivelydiscussionthe main purpose ofthis study was to investigatespag5 gene expression and amplification related tonuma mrna levels in breast cancer tissues as thenormal control sample we used the same patient™s nontumor breastthisit can be assumed thattissuefig gel electrophoresis of multiplex pcr first well is loaded byladder bp and wells “ are loaded with dna of tumor tissuesbands arrangement from biggest to smallest are ² end of spag5 bp middle of spag5 bp por bp agbl2 bpbod1l bp and ² end of spag5 bpseemingly healthy tissue which was adjacent to a tumormay have altered gene expression or amplification toprevent this drawback we attempted to sample at thefarthest possible distance from the tumor as recommended by the cancer genome atlas tcga tcga mentioned that nat sampling must be performed cm from the tumor margin it has been reported that genetic changes could be detected in thenormal margin of the tumor but are rarely evident atdistances greater than cm in our study we further evaluated the nats histopathologically and we observed no morphological or molecular alterations toevaluate possible changes in spag5 and numa geneexpression in the nats three healthy breast tissuesfrom women referred for cosmetic surgery were included in the study the δct of spag5 and numagenes in these three samples were the same as the meanof δct of spag5 and numa genes in nats indicatingthatthe nats did not bear noticeable expressionchanges in the studied genesour results showed that spag5 mrna expressionwas increased times in breast tumors than thenats correlation studies revealed a significant link between spag5 gene expression and tumor size withhigher levels in larger tumors our results also showed asignificant correlation between spag5 gene expressionand clinical stagespag5 knockdown has shown cell growth suppressionand apoptosis enhancement while the increased spag5expression promotes cell proliferation and inhibits apoptosis as previously mentioned spag5 preventsapoptosis through pi3kaktmtor pathway and issimilarly involved in wntβcatenin signaling pathwayactivation via stimulation of wnt and βcatenin expression clinical studies have confirmed the role ofspag5 in breast cancer prognosis and survival it has 0cmohamadalizadehhanjani world of surgical oncology page of fig dna fragments electropherograms in genemarker® software the x and yaxes represent dna fragments length in bp and signals intensity as arelative fluorescent unit rfu respectively the orange peaks were generated by standard size fragments and the blue peaks by spag5 dna fragmentsand control genes the order of the peaks from the largest to the smallest corresponds to ²spag5 bod1l agbl2 por middle spag5 and ²spag5been shown that patients with higher spag5 gene expression were at high risk of cancer spag5 was also proposed as a proliferative marker in earlystage of cancer li reported spag5 as an oncogene in basalliketriplenegative breast cancer by direct interaction withmycbinding protein mycbp which results in an increased cmyc transcriptional activity cmyc regulatesthe expression of several genes involved in the process ofcell growth and dna repair exhibiting high expression inbasallike tumors based on the obtained results they considered spag5mycbpcmyc axis as a potential therapeutic target in the triplenegative patients howeverour study did not reach similar results and we observedno significant difference in spag5 gene expression inbasallike tumorsthe previous study had suggested a link between spag5and numa during cell division we also analyzed thecorrelation between spag5 and numa gene expression inpairwise comparisons our results yielded no significantcorrelation between spag5 and numa indicating independent expression of these two genes in the process ofbreast tumorigenesis although no link was found betweenthe expression levels of these two genes further studiescould investigate the association of their proteinswe investigated numa transcript levels expression inbreast cancer tissues relative to nats and we observeddecreased numa expression however it was not statistically significant an interesting finding of our studywas that numa expression in erpositive was higherthan ernegative tumor specimens it can be justifiedthat numa expression increases in erpositive cellssince the er requires nuclear matrix components suchas numa for its transcriptional activity previous studieshave suggested the association between er intranuclearmobility and the dynamics of the nuclear matrix the main function of the er is localized in the nucleuswhere it acts as a liganddependent transcription factorit interacts with multiple intranuclear components including nuclear matrix proteins to regulate transcriptionof the estrogenresponsive genes in the presence of aligand er distribution shifts from the diffused to thediscrete state indicating the binding of the receptor tothe nuclear matrix elements since numa is a part ofthe nuclear matrix components it could be concludedthat it plays a pivotal role in the mobility of er our results showed a significant association betweennuma mrna levels and molecular subtypes which areclassified based on er pr and her2 expression 0cmohamadalizadehhanjani world of surgical oncology page of recently using machine learning algorithms kothari attempted to find potential genes discriminatingbasallike from other subtypes they reported tbc1d9as a candidate gene that its expression was linked to improved patient™s survival further analysis revealed differentroles for tbc1d9 in anelle biogenesis andlocalization as well as recruitment of numa to the mitotic centrosome it has been shown that brca deficiency causes cortical asymmetry of numadyneincomplex in dividing cells which provoke genomic instability and aneuploidy a new role for numa wasrevealed related to diffusion regulation of p53bindingprotein 53bp1 which plays a prominent role in dnarepair higher numa mrna levels were significantlylinked to increased breast cancer patients survival evenwhen specifically analyzed in basallike tumors association study using snps in nearly genesshowed that a 300kb region on chromosome 11q13encompassing the numa gene influences breast cancerrisk they suggested a794g variant as a susceptibility allele for breast cancer however additional investigations did not support the role of numa variants asbreast cancer susceptibility alleles the other phenomenon of interest in our study wasthe spag5 gene amplification applying qmf pcr toevaluate gene copy number variation cnv we observed complete amplification in of tumor samplesthese results were in line with the results of the abdelfatah that reported “ spag5 gene amplification in breast tumors defects in dna replicationor telomere dysfunction promote gene amplificationwhich can lead to tumor progression in any stage ofbreast cancer gene amplification confers a selectiveadvantage during earlystage of cancer by increasing oncogenes expression in amplified regions it has beenshown that gene amplification is associated with moreaggressive tumors as well as resistance to chemotherapywhich can predict disease prognosis despite the significant results our study also has particular limitations that should be addressed in the futurethe association of spag5 and numa at the proteinlevels and the molecular mechanism of their interactionneed to be clarified in vitro and in vivo furthermoredue to the limited sample size we included idc samplesin the study without considering the presence or absenceof dcis it would be interesting to study the gene expression differences between pure idc and idcdcisour study highlights the role of increased spag5 geneexpression and gene amplification in breast cancer theresults showed that in largersized tumors the spag5mrna wasthatspag5 may be further studied as a therapeutic target inincreased this evidence suggeststhe treatment of breast cancer therefore complementary studies can be suggested to investigate the role ofthe proteins that interact with spag5 we did not observe any significant correlation between spag5 andnuma mrna levels however our results indicated anassociation between numa and tumor er expressionstatus this finding reported for the first time in ourstudy could be the starting point for further studies oner and cell division apparatus in breast canceracknowledgementsthe authors would like to thank the patients who participated in this surveyauthors™ contributionsauthors™ contributions are as follows zmh project development datacollection and management data analysis and manuscript writingeditingss project development data collection and management data analysismanuscript writingediting lg data collection and management dataanalysis manuscript writingediting all authors read and approved the finalmanuscriptfundingnot applicableavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding authors upon reasonable requestethics approval and consent to participatethis study was approved by the ethics committee of tarbiat modaresuniversity all patients provided written informed consent and the studywas conducted in accordance with the declaration of helsinkiconsent for publicationnot applicablecompeting intereststhe authors declare no competing interestsauthor details1department of medical genetics faculty of medical sciences tarbiatmodares university tehran iran 2department of surgery sina hospitaltehran university of medical sciences tehran iranreceived may accepted august referencesabdelfatah tma agarwal d liu dx russell r rueda om liu k spag5 as a prognostic biomarker and chemotherapy sensitivity predictor inbreast cancer a retrospective integrated genomic transcriptomic andprotein analysis lancet oncol “ mack gj compton da analysis of mitotic microtubuleassociated proteinsusing mass spectrometry identifies astrin a spindleassociated protein procnatl acad sci u s a “thein kh kleyleinsohn j nigg ea gruneberg u astrin is required for themaintenance of sister chromatid cohesion and centrosome integrity j cellbiol “thedieck k holzwarth b prentzell mt boehlke c klasener k ruf s inhibition of mtorc1 by astrin and stress granules prevents apoptosis incancer cells cell “yang yf zhang mf tian qh fu j yang x zhang cz spag5 interactswith cep55 and exerts oncogenic activities via pi3kakt pathway inhepatocellular carcinoma mol cancer valk k vooder t kolde r reintam ma petzold c vilo j geneexpression profiles of nonsmall cell lung cancer survival prediction andnew biomarkers oncology “liu jy zeng qh cao pg xie d yang f he ly spag5 promotesproliferation and suppresses apoptosis in bladder urothelial carcinoma by 0cmohamadalizadehhanjani world of surgical oncology page of upregulating wnt3 via activating the aktmtor pathway and predictspoorer survival oncogene “liu h hu j wei r zhou l pan h zhu h spag5 promoteshepatocellular carcinoma progression by downregulating scara5 throughmodifying betacatenin degradation j exp clin cancer res chu x chen x wan q zheng z du q nuclear mitotic apparatus numainteracts with and regulates astrin at the mitotic spindle j biol chem “seldin l poulson nd foote hp lechler t numa localization stability andfunction in spindle orientation involve and cdk1 interactions mol biolcell “ vidi pa liu j salles d jayaraman s d
Colon_Cancer
" mutations in the exonuclease domain of pole a dna polymerase associated with dna replicationand repair lead to cancers with ultrahigh mutation rates most studies focus on intestinal and uterine cancers withpole mutations these cancers exhibit a significant immune cell infiltrate and favorable prognosis we questionedwhether loss of function of other dna polymerases can cooperate to pole to generate the ultramutatorphenotypemethods we used cases and data from cancer types in the cancer genome atlas to investigate mutationfrequencies of different dna polymerases we tested whether tumor mutation burden patient outcomediseasefree survival and immune cell infiltration measured by estimate can be attributed to mutations in polqand polzrev3lresults thirty six percent of colorectal stomach and endometrial cancers with pole mutations carried additionalmutations in polq eq polzrev3l ez or both dna polymerases ezq the mutation burden in thesetumors was significantly greater compared to poleonly e mutant tumors p in addition eq ez and eqz mutant tumors possessed an increased frequency of mutations in the pole exonuclease domain p colorectal stomach and endometrial eq ez and eqz mutant tumors within tcga demonstrated diseasefree survival even if the pole mutations occurred outside the exonuclease domain p however immunescores in these tumors were related to microsatellite instability msi and not pole mutation status this suggeststhat the host immune response may not be the sole mechanism for prolonged diseasefree survival ofultramutated tumors in this cohort results in this study demonstrate that mutations in polq and rev3l in pole mutant tumors shouldundergo further investigation to determine whether polq and rev3l mutations contribute to the ultramutatorphenotype and favorable outcome of patients with pole mutant tumors correspondence beatriceknudsenpathutahedu1department of biomedical sciences cedarssinai medical center losangeles ca usa2samuel oschin cancer research institute socci cedarssinai medicalcenter los angeles ca usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chuang bmc medical genetics page of the analysis of thousands of cancers by the cancergenome atlas tcga consortium and academic institutions revealed a small group of cancers with mutationsin pole and an ultramutator phenotype [ ] multiplestudies have found significantly improved survival in patients with pole mutated endometrial cancers [ “]while the survival benefit was not as profound in polemutated colorectal carcinoma the pole geneencodes the catalytic subunit of dna polymerase epsilon which catalyzes the leading strand synthesis duringdna replication pole possesses highfidelity dnapolymerization proofreading and ²² exonuclease activities which promote accurate dna synthesis firstidentified and reported in “ of colorectal carcinomas[ ] pole mutations were also noted at frequencies of “ amongst uterine corpus endometrialcancers [ ] and in gastric adenocarcinoma mutations can be found across the entire pole genebut those in the pole exonuclease domain are mostprevalent in cancers with ultrahigh mutation rates mutmb these cancers exhibit higher mutationrates than microsatellite instable msi tumors associated with mismatch repair abnormalities in additionpole ultramutant cancers also possess a high frequency of ctoa transversions [ ] a tumor associated inflammatory response similar to thatin msitumors has been reported to occur early on duringdevelopment of pole mutated endometrial and colorectal cancers it is thought to be caused by neoantigens that are generated as a result of the high mutation burden [ ] and render pole mutant cancersresponsive to immunotherapy polymerase theta polq is a lowfidelity dna polymerase lacking a ² to ² exonuclease function theenzyme is involved in the alternative nonhomologousendjoining pathway altnhej which is a backupmechanism of double stranded dna break repair thispathway predominates in cancer cells when other dnarepair pathways are missing or when telomere ends aredeprotected [ ] the loss of polq sensitizes cellsto ionizing radiation and polqdeficient mice exhibit increased dna instability and genomic rearrangementssuggesting a role for polq as a guardian of the genome both overexpression and loss of polq increasemutation frequencies [ ] multiple structuralmotives in polq can interact with dna rad51 andbrca1 in addition polq forms a complex withparp1 in a pathway of synthetic lethality with brca1and is thus considered a therapeutic target [ ]however which domains in polq should be targetedremains to be determined rev3l rev3 like dna directed polymerase zetapolz catalytic subunit is involved in dna synthesisthat reads through damaged dna translesion dnasynthesis tls the high efficiency of polz bypassing a broad spectrum of dna lesions led to its recognition as a master tls polymerase polzrev3l has been linked to carcinogenesis in breastlung gliomas and gastric cancers and modulatescisplatin sensitivity [“]because studies describe overlapping functions andsynergy among the over dna polymerases [ ] we undertook an unbiased approach to identifymutations in polymerases within the tcga tumorcompendium this analysis revealed a greater frequencyof mutations in polq and polzrev3l compared toother polymerases of similar gene length therefore wepropose that polq and polzrev3l may cooperatewith pole in generating ultrahigh mutation ratesmethodsdata acquisitionthe data used in this study are based upon the wholeexome sequence data sets generated by the tcgaresearch network httpcancergenomenihgov thelocations and frequencies of somatic mutations msistatus and clinical stage and follow up information intcga provisional datasets were obtained from cbioportal httpwwwcbioportal [ ] up to supplementary table mutation data were obtainedby first selecting the œquery tab from cbioportal thecancer type specific data were chosen from the œtcgapancancer atlas studies we entered the genes ofinterest pole polq and rev3l to retrieve mutationdata from the genes when the result page wasdisplayed we accessed the information underneath theœmutations tab we downloaded the number of mutations in each sample the annotation of protein aminoacid change and the type of mutation the survivalinformation for each case months after diagnosis wasobtained through the œcomparisonsurvival link functional domains of the proteins were provided by pfamdatabase data visualization for mutations wasperformed with mutationmapper in cbioportalcase selection criteriawe searched all cancer types in tcga for those thatpossess or more cases with pole mutations thisyielded tumor types that we named the pancandata set in this study we then determined the frequencyof mutations within additional polymerase within pancan we selected the three adenocarcinomas uterinecorpus endometrial adenocarcinoma colorectal adenocarcinoma and stomach adenocarcinoma with the highesttriple mutated dnapolymerase status for more detailed analysisfrequency of double or 0chuang bmc medical genetics page of studies performedkaplan meier survival plots were generated using clinicalfollowup data available within the tcga database todetermine the global mutational spectrum we classified types of nucleotide transitions or transversions thefrequency of each mutation type was calculateddigital images of all eq mutant tumors and representative cases of tumors with neither pole nor polqmutations and of msi tumors were assessed by one author jr for the amount of tumor immune infiltratethe combination of tumor infiltrating lymphocytes andthe peritumorallymphoid infiltrate was graded on ascale between and tumor associated lymphocyteswere graded as none minimal rare to per highpowered field hpf to per hpf and perhpf peritumoral lymphocytes were assessed at thedeepest advancing tumor front graded at low powernone minimal mild moderate andmarked these visual semiquantitative scores werecompared with the immune scores evaluated using estimate estimation of stromal and immune cells inmalignant tumor tissues using expression data estimate score were obtained from the website ofestimate at md anderson httpsbioinformaticsmdandersonestimatediseasehtml theplatformtype selected was rnaseqv2 data for colorectalstomach and endometrial cancer types were downloaded next we separated the colorectal stomach andendometrial cancer cases into quartiles defined by thehighest intermediate and lowest of mutational counts or msi status and calculated the immunescores for each groupstatistical data analysisall statistical analyses were conducted in r v313 plotswere generated using ggplot2 package in r datavisualization methods were described previously the horizontal lines in the boxplots represent the 1st2nd and 3rd quartiles and whiskers outside the boxshow the interquartile range the significance of thedifferences of data illustrated in the boxplots wascalculated using the wilcoxon ranksum tests the chisquare test was performed to test the significance ofdifferences in frequencies of all tables the significancein the kaplan“meier survival plot was calculated usingthe log rank test statistical significance was accepted atp resultsof the cancer types in the tcga database weidentified cancer types pancan with or morecases that possessed mutations in the pole proteincoding region fig 1a and supplementary table these cancer types contained cases with polemutations anywhere in the exome of these polemutant tumors carried mutations in one or more of the other dna polymerases we observed dna polymerases polq and polzrev3l to be most frequentlymutated fig 1b these two polymerases were mutatedin of tumors with pole mutations in fact thesetwo polymerases were even more commonly mutatedthan pole in our pancan cohort supplementary figure altogether cases with pole and polq mutations eq cases with pole and polzrev3lmutations ez and cases with pole polq andpolzrev3l eqz mutations were identified in thepancan cohort fig 1c mutations in the exonucleasedomain of pole are responsible for causing the ultramutator phenotype in colorectal and uterine corpuscancers [ ] in order to determine the contribution of polq and rev3l to the ultramutator phenotype we compared the mutation frequencies of tumorswith mutations in only pole to eq ez and eqzmutant tumors mutation frequencies in the cellulargenome increased in the following order no polemutations poleonly mutations anywhere with thepole exome eq ez eqz mutations fig 1dthe median mutation count of eqz tumors was morethan 10fold higher p than that of tumors withonly pole mutations e q and e z mutant tumorsalso displayed significantly higher mutation countscompared to eonly mutant tumors suggesting a contribution of mutationally altered polq or polzrev3l tothe overall cancer mutation rates next we determinedthe number of mutations in the exonuclease and polymerase domains of pole in the cancer types withinour pancan compendium fig 1e the percentage ofpole mutations in the exonuclease domain was greaterin eq ez and eqz mutant tumors compared topoleonly mutant tumors p in contrast thepercentage of mutations in the dna polymerase domainwas similar thus our data confirm the notion thatmutations in the exonuclease domain of pole areresponsible for ultrahigh mutation rates in additionmutations in polq and rev3l may further increasethe mutation burden however why mutations in polqand rev3l preferentially increase tumor mutationfrequencies remains elusive endometrialto further investigate a potential role of these mutantdna polymerases in the ultramutator phenotype wefocused on colorectalucec andstomach stad cancers these cancer types containthe highest numbers of tumors with eq ez and eqz amongst the cancer types included in pancanfig 1a supplementary table among these cancertypes we identified cases with eq cases with ezand cases with eqz mutations fig 2a in thesecancers the mutation burden in pole eq ez and e 0chuang bmc medical genetics page of fig cancer types pancan with poleqz mutations in tcga a number of cases with pole only eq ez and eqz mutations in cancertypes cohort referred to as pancan within tcga the xaxis shows the actual number of cases with pole green eq orange ez pink and eqz blue mutations the yaxis displays the cancer types uterine corpus endometrial carcinoma ucec stomach adenocarcinoma stadcolon and rectum adenocarcinoma skin cutaneous melanoma skcm lymphoid neoplasm diffuse large bcell lymphoma dlbc lungadenocarcinoma luad breast invasive carcinoma brca sarcoma sarc cervical squamous cell carcinoma and endocervical adenocarcinomacesc pancreatic adenocarcinoma paad lung squamous cell carcinoma lusc head and neck squamous cell carcinoma hnsc bladderurothelial carcinoma blca kidney renal clear cell carcinoma kirc and liver hepatocellular carcinoma lihc b case numbers with mutationsin polymerase genes the number of cases in pancan with mutations in the following polymerases is displayed on the yaxis dntt pola1polb pold1 polg polh poli polk poll polm poln polq rev1 rev3l c venn diagram displaying the number of cases in pancan withmutations in or pol genes d mutations per mb yaxis of pancan cases without pole mutations other or with pole eq ez and eqzxaxis mutations number of cases in each group are listed in parenthesis e mutation frequencies in pole exonuclease and polymerase domainsas a percentage of total number of mutations in the pole exome œother refers mutations in the entire exome outside the exonuclease orpolymerase domains the cases are grouped by their polymerase mutation status on the yaxis and the number in parenthesis represents thetotal number of pole mutations within each group 0chuang bmc medical genetics page of fig poleqz mutations in colorectal endometrial ucec and stomach stad cancers a venn diagram displaying the number of caseswith mutations in or pol genes b mutation groups of cases without polymerase mutations other or with mutations in pole only eq ez and eqz the number of cases in each group is listed in parenthesis the pvalue for comparison of pole and eq groups is not significantp c number of cases with mutations in pole exonuclease domain in various mutation groups d percentages the ratio of transitions tiand transversions tv are shown on the yaxis for core stad and ucec the xaxis shows the mutation groupsqz mutant tumors paralleled the mutation burden inthe whole pancan cohort compare fig 2b and fig1c supplementary figure 2ad eqz mutant tumors demonstrated on average an 8fold increase inmutation frequencies compared to tumors with onlypole mutations amongst pole mutant tumors tumors carried mutations outside the pole exonuclease domain while tumors carried mutations withinthe exonuclease domain the frequency of poleexonuclease mutationsin fig 2cprovides a valid explanation forthe difference inmutation rates and potential association of poleexonuclease domain mutations with mutationsinpolq and polzrev3l which are the other twomost frequently mutated dna polymerases casesoverall exonuclease domain mutations were identifiedin cases of pole only mutant tumors eqcases ez cases and of eqz cases fig 2cstratified by cancer types pole exonuclease domainmutations occurred in colorectal endometrial and stomach tumors demonstrating cancertype specific frequencies supplementary figure 3a incontrast to the pole gene that demonstrates mutationalhotspots in the exonuclease domain mutational hotspotsin the polq gene are not associated with a functionalprotein domain supplementary figure 3b c dwhile rev3l does not reveal mutational hotspotsapproximately of mutations lead to truncated protein expression supplementary figure 3e f anothercharacteristic of pole mutant tumors are c to a and g 0chuang bmc medical genetics page of to t transversions we observed the greatest increaseof nucleotide transversions in cancers with eqz mutafig 2d consistent with the loss of poletionsexonuclease activity in these tumorssince mutations in pole confer increased disease freesurvival dfs in patients with uterine cancer even inthose patients with highgrade tumors [ ] we investigated the prognostic role of polq and rev3l mutations in pole mutanttumors kaplanmeier curveswere constructed for colorectal endometrial and stomach cancer cases with followup data fig 3a using thetcga annotations of dfs in individual patients nocancer recurrences were observed in the eq ez andeqz mutant groups pole exonuclease domain mutations were observed in cases in the good survivalgroup and case in the poor survival group consistentwith the expected long dfs periods of patients withpole exonuclease domain positive tumors in additionanalysisin the pancan cohort cases with mutations in pole outside the exonuclease domain were in the good survival group of those had concurrent mutations in polq or rev3l orin both polymerases fig 3b furthermore a kaplanmeierrevealedimproved dfs associated with mutations in polq andrev3l the favorable survival outcome was observed incolorectal endometrial and stomach cancers howeverno favorable outcome was observed in diffuse bcelllymphoma p these data provide preliminaryevidence of cancertype specificfavorable survivaloutcomes in tumors with pole mutations that arelocated outside the pole exonuclease domain if concurrent mutations in polq rev3l or in both polymerasesare presentcompared to microsatellite stable tumorsmssmicrosatellite instability msiin colorectal cancerconfers a better prognosis to determine whetherfig survival and clinical characteristics of patients with polymerase mutations colorectal endometrial ucec and stomach stad cancers akaplanmeier curves of diseasefree survival dfs for groups of patients pole only n median followup months green line eqn median followup months orange line ez n median followup months pink line and eqz n median followup months blue line tumors without mutations in pole polq or rev3l exomes grey line the overall pvalue is p individual pvalues eqz vrs pole “ p eqz vrs none “ p eq or ez or eqz vrs pole “ p b polymerase mutation analysis ofcases in the good survival group in panel a the red bar indicates cases with pole exonuclease mutations c cancer typespecific illustration ofmutation count pole polq and rev3l mutations microsatellite instability msi and tumor stage 0chuang bmc medical genetics page of the favorable outcome of eq ez and eqz mutantcancers can be explained by msi or tmn stage we examined the relationship between msi statustumorstage and polymerase mutations in colorectal endometrial and stomach cancers fig 3c and supplementaryfigure and supplementary table despite improveddfs rates the full range of tumor stages was observedamongst eq ez and eqz tumors p supplementary table 2a comparing the poleonly and eqz mutant cancers did not reveal a significant difference in tumor stage but differed in the frequency ofmsi cases p pole mutant tumors were morefrequent in the msi group than in themss group in addition the frequency ofmsi cases in pole mutant tumors differed between the cancer types p supplementary table 2b c although msi is enriched in samples with highmutation levels supplementary table 2d as expectedp were10fold higher mutation countsobserved in cancers with eqz mutations compared tomsi without eqz mutations supplementary figure these results suggest that mutations in eq ez and eqz confer a better prognosis independent of msi statusand tmn stage in colorectal endometrial and stomachadenocarcinomaswe next examined the amount ofthe cancerassociated immune infiltrate the immune scoreobtained through estimate corresponded tothe categorical score of the immune infiltrate derivedfrom digital he images supplementary fiure therefore we used the estimate immune scoresfor further analysis of colorectal endometrial andstomach cancers as shown in fig 4a a significantdifference was observed in the median immunescores between groups with lowintermediate andhigh mutation burden grouped based on mutationburden and not on e q z mutantseemethods and as expectedthe median immunescores increased with total mutation levels surprisingly the immune scores in eqz mutant tumorsdid not differ significantly from tumors with a lowlevel of mutationsfig 4b as expected msitumors possessed higher immune scores than msstumors p immunescores of msi and eqz mutation tumors weresimilar to those in the msi group and higher thanmss and eqz mutation tumors fig 4d withinthe group of tumors with pole exonuclease domainmutations mss tumors possessed lowerimmunescores than msi tumors but the difference was notsignificanttogether results in this tcga cohort demonstratethat the immune response is driven by msi ratherthan pole exonuclease domain mutationssupplementary fig 4c finallystatusp figurediscussionan analysis of dna polymerases in tumors withmutations in the pole revealed additional mutations inspecific polymerases most commonly in polq andpolzrev3l among the cancer types colorectaluterine and stomach cancer were mostfrequentlyafflicted by these mutations cancers with mutations inpole and polq eq pole and polzrev3l ezand in all polymerases eqz were associated withthe highest mutation burden and an excellent prognosisindependent of msi status and tumor stage mutationsin the exonuclease domain were observed in of eqz mutant tumors but only in of poleonly mutant tumors or in of eq ez tumorshowever despite harboring 10fold more mutationsthan msi tumors and 8fold more mutations than themutation frequencies associated with poleonly mutanttumors eqz mutant tumors did not display significantly more inflammationthe main result from that analysis is that patients withcolorectal stomach and endometrial cancers bearing eq ez and eqz mutations have disease free survival dfs at a median follow up time of months incontrast patients with tumors bearing mutations inpole only most of which outside the pole exonucleasedomain had a dfs of at follow up of monthsthe favorable dfs in eq ez and eqz mutated tumors occurred even in tumors with mutations in polethat are located outside the pole exonuclease domainthe contribution of mutations in pole to tmb cž”asubstitutions and cancer type associations are describedin table of raynor using a larger resourceof cases and should be used to interpret the mutationsin the current study listed in supplementary figure as a whole the current study expands the spectrum ofpole mutant tumors with an excellent prognosis thefavorable prognosis included patients with high tumorstage which echoes prior studies demonstrating a favorable outcome of uterine tumors with pole exonucleasemutations despite adverse standard clinicopathologicindicators including high grade high stage and lymphovascular invasion [ ] while the high mutationfrequencies may cause an early growth advantage as tumors evolve they may succumb to high mutationburden as new mutations can no longer be tolerated andcause tumor cell death [ ] or increased sensitivityto therapeutic agentsthe prevailing hypothesis for the favorable prognosisof cancers displaying the hypermutator phenotype is theincreased attack by the immune system evidence insupport of this theory is the observation that tumorinfiltrating and peritumorallymphocytes are increased and that cytotoxic activities in cd8 and cd4are heightened in polelymphocyte populations 0chuang bmc medical genetics page of fig estimate immune scores by mutation frequency quartiles eqz mutation groups and msi in colorectal endometrial ucec and stomachstad cancers a estimate immune scores in cancers within high intermediate and low overall mutation quartiles b immune scores of sampleswith eq ez and eqz mutations compared to the low mutation quartile from panel a c immune scores in groups of cancers separated by msistatus d immune scores in msi and mss eqz cases compared to all other msi cases for each panel the number of cases within each group isincluded in parentheses on the xaxismutated endometrial cancers [“] similar to hypermutated msi tumors this observation has led tothe hypothesis that immune checkpoint inhibitors maybe efficacious in pole ultramutated tumors ourresults question a direct relationship between mutationburden tumor immune response and pdl1 expressionalso raised in a larger study across cases from cancer types in tcga while we observed a concordance between the computational and histological assessments of the immune infiltrate the immune score intumors with eqz mutations depended on msi statusthis result suggests that the immune infiltrate attributable to mutations in eqz mutant tumors may be lessor that its composition may involve immune cells otherthan lymphocytes lesser cd8 and gammainterferongene expression signatures have also been observed ingastrointestinal tumors with a large single nucleotidevariantsnv burden that was attributed largely topole exonuclease mutations perhaps eqz mutations occur at a later point in tumor evolution whenimmunosuppressive factors already dominate we alsocannot rule out the possibility of increased numbers ofcytotoxic lymphocytes intermixed with eqz mutanttumor cells because computational methods and inspection of he images are not sensitive enough to detectsmall differencesinfiltrating lymphocytestils that may have large antitumoral effectsin tumora significant limitation of the study lies in the relatively small number of tumors this limitation cautionsthe generalization of results and seemingly novel insights 0chuang bmc medical genetics page of into the hypermutator phenotype studies by othergroups attributed hypermutator phenotype to specificmutations primarily within the pole exonucleasedomain given the proofreading function of the exonuclease domain it makes sense that mutations outsidethe domain have a lesser effect on tmb in agreementwith this concept our study reveals that compared totumors harboring only a mutation in pole polesinglemutant tumors of cases pole exonucleasedomain mutations are more common in tumors withboth double ez and eq and triple eqz dnapolymerase mutations of cases and doubleand triple mutant tumors have higher mutation countsthan pole single mutant tumors while it cannot befully excluded that polq and polzrev3l mutationsare bystander events in pole mutanttumors weobserve a higher tmb in cases with mutations in allthree polymerases supplementary figure 2a and bmechanistically polq and polz are thought to function in different repair processes polq in alternativemicrohomologymediated nonhomologous dna repair pathway and polz in translesion dna synthesishow these dna repair processes cooperate with thereplicative dna polymerase pole to prevent genomeinstability remains unknown this will be an importantsubjectthe mechanismfor further understanding ofunderlying the hypermutator phenotypesif validated in additional cohorts our findings may haveimportant clinicalimplications they build upon andexpand the previously well documented good prognosticimpact of pole exonuclease mutationsin uterinecancer that have generated intense interest in part dueto the paradox of a favorable prognosis in tumors withpathologic indicators of poor prognosis while in thisstudy prolonged dfs is observed in colorectal endometrial and stomach cancers with eqz mutations thisis not the case in other noncarcinoma cancer typeswithin tcga thus we find that the positive outcomeprediction is cancer type specific altogether resultsfrom this study provide a rationale for including polqandor polzrev3l mutations in clinical outcomestudies of tumors with pole mutations however future validation is required to confirm the concept that isrevealed in the current studycolorectal core stomach stad and endometrial ucec cancers byspecific polymerase mutated groups in tcga data sets supplementaryfigure locations of mutations in pole polq and rev3l exomes inindividual colorectal core stomach stad and uterine cancers ucecsupplementary figure relationships between mutation spectrumand mutation counts pole polq and rev3l exome mutations msi andtumor stage of individual cases supplementary figure mutationrates per mb yaxis of core stad and ucec cases with msi and eq ez and eqz xaxis mutations supplementary figure relationshipbetween pathology inflammation score and estimate immune scores forcore stad and ucec supplementary figure estimate immunescores in colorectal core endometrial ucec and stomach stadcancers supplementary table number of cases with pole polq zrev3l or multiple exome mutations in the pancan cohortsupplementary table contingency tables showing number of casesof colorectal endometrial and stomach cancers in each categoryabbreviationspole polymerase epsilon polq polymerase theta polzrev3l rev3 likedna directed polymerase zeta polz catalytic subunit tcga the cancergenome atlas msi micro satellite instability mss micro satellite stabilitytmb tumor mutation burden tnm tumor lymph node metastasis estimate estimation of stromal and immune cells in malignant tumor tissuesusing expression dataacknowledgementsnot applicableauthors™ contributionsall authors have read and approved the content of this manuscript fhstudy design and data analysis ht data interpretation jr data analysis andmanuscript writing bsk data interpretation and manuscript writingfundingthe prostate cancer foundation funded salaries of fh and bsk forcomputational analysis steven spielberg team science award andr01ca131255 bsk funded salaries for data analysis and paper writing of fhand bsk we also acknowledge the institutional support of salaries throughthe nih g20 rr030860 to the cedarssinai biobank and translational research core for salaries of bsk and fh the content of this publication doesnot necessarily reflect the views or policies of the department of health andhuman services nor does any mention of trade names commercial products or anizations imply any endorsement by the us governmentavailability of data and materialssequencing data can be obtained from national cancer institue gdc dataportal and are published in raw genomic and clinical data can befound at the nci genomic data commons httpsportalgdccancergovlegacyarchive the mc3 mutation annotation file can be accessed at httpsgdccancergovaboutdatapublicationsmc32017 and the processed datafiles can be viewed at httpsgdccancergovaboutdatapublicationspancanatlasethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors do not declare any competing interestssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12881020010899additional file supplementary figure number of cases polemutations n and mutations in the exomes of dna polymerasegenes in pancan supplementary figure mutation counts forauthor details1department of biomedical sciences cedarssinai medical center losangeles ca usa 2samuel oschin cancer research institute soccicedarssinai medical center los angeles ca usa 3surgerycedarssinai medical center los angeles ca usa 4pathology andlaboratory medicine cedarssinai medical center los angeles ca usa 5department of pathology university of utah salt lake city ut usa 0chuang bmc medical genetics page of received january accepted july referenceskandoth c schultz n cherniack ad akbani r liu y
Colon_Cancer
cancer stem cells cscs are important factors contributing to tumorigenesiswe examined whether cscs isolated from colorectal cancer crc cells possessmetastatic properties that can be transferred to noncscs via the deliveryof mir200c enclosed in extracellular vesicles evs the inhibitory effectof atractylenolide i atl1 a traditional chinese medicinal compound onmir200c activity and metastatic transfer was investigated evs were isolatedfrom colorectal cscs the expression of mir200c was evaluated in cscs andcscderived evs and horizontal transfer of metastatic properties via evs tononcscs was investigated in terms of cell behavior and phosphatidylinositol45bisphosphate 3kinase pi3kprotein kinase b aktmammalian target ofrapamycin mtor signaling cscs isolated from metastatic crc cells exhibitedhigher levels of mir200c than those in nonmetastatic crc cells overexpressionof mir200c in cscs enhanced metastatic potential by promoting proliferationand inhibiting apoptosis in turn leading to the release of evs carrying an excessof mir200c noncscs cocultured with mir200ccontaining evs exhibitedenhanced invasion and stemness maintenance associated with pi3kaktmtoractivation demonstrating successful metastatic transfer via ev delivery furthermore atl1 impaired the evmediated transfer of metastatic propertiesby suppressing mir200c activity and disrupting ev uptake by noncscsevs are critical signal transducers that facilitate intercellular communicationand exchange of metastatic properties which can be controlled by atl1 theabbreviations akt protein kinase b anova analysis of variance atl atractylenolide crc colorectal cancer csc cancer stem cell evextracellular vesicle fbs fetal bovine serum fitc fluorescein isothiocyanate mir microrna mtor mammalian target of rapamycin mtt345dimethylthiazol2yl25diphenyltetrazolium bromide oct4 octamerbinding transcription factor pbs phosphatebuffered saline pi3kphosphatidylinositol45bisphosphate 3kinase sd standard deviation sox9 sexdetermining region ybox tem transmission electron microscopythis is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided theoriginal work is properly cited the authors clinical and translational medicine published by john wiley sons australia ltd on behalf of shanghai institute of clinical bioinformaticsclin transl med 202010e139101002ctm2139wileyonlinelibrarycom ctm2 of 0c of tang findings are useful in the development of micrornabased anticancer strategiesby targeting evmediated activity especially using natural compoundsk e y wo r d satractylenolide i extracellular vesicles metastasis pi3kaktmtor stemnessintroductioncolorectal cancer crc is one of the most prevalent cancers worldwide with an estimated new cases and deaths in in the united states alone1 mostcrcrelated deaths are caused by metastasis of malignant tumours2 and research on crc treatment has thusfocused on controlling the occurrence of metastasis thefactors that affect crc metastasis are manifold and it ischallenging to gain a comprehensive view of the mechanisms involved within micrornas mirs have beena recent highlight in diverse areas of medical researchincluding targeted cancer therapy because of their effectson a variety of biological responses such as oncogenesis and tumor metastasis the identification of specificmirs as oncomirs or tumor suppressors has advancedour knowledge of cancer development and contributedto the emergence of mirbased therapeutic schemes forexample targeting the oncogenic mir155 delayed tumrowth3 and a deliverable therapeutic formulation againstlung cancer has been developed based on the tumor suppressor mir344 interestingly mir200c has been identified as both an oncomir5 and a tumor suppressor6 in crcwith these contrasting reports it is necessary to furtherinvestigate the role of mir200c in crc development andmetastasisthe metastatic properties of mir200c have beendemonstrated to be transferrable via extracellular vesicles evs as carriers between tumor cells with differentmetastatic abilities7 evs are spherical ps that arecategorized based on their size and origin for examplethose with diameters of nm are known as exosomesand originate from endosomes whereas those with diameters of nm to µm are known as microvesicles oroncosomes in cancer cells originating from the plasmamembrane8 evs derived from cancer stem cells cscsreportedly mediate the crosstalk between cells via the horizontal transfer of tumorigenic factors between cells9 suchas oncogenes and proteins although cscs form only asubset of the cancer cell population they are believed tobe a key determinant of tumorigenesis and play important roles in regulating the tumor environment and metastasis the maintenance of stemlike properties in cscsis critical in promoting disease onset and thus studieshave focused on therapeutic means of disrupting or impairing the maintenance of stemness1011 whether the protumorigenic potential of cscs such as metastatic andstemlike traits can be conferred to nonmetastatic crccells via the transfer of mircontaining evs remains thusfar unknownaside from conventional anticancer schemes such assurgical intervention and chemotherapy natural herbaland plantbased compounds have gained attention as adjuvant or complementary therapies in cancer treatmentbaizhu or atractylodes macrocephala koidz is a dryperennial rhizome in the asteraceae family and has beenapplied as a typical component of many traditional chinese medical formulations1213 among the components ofbaizhu three forms of atractylenolides atls i ii andiii have been identified to exert pharmacological properties and atl1 is the main active ingredient that has exhibited therapeutic effects against crc1417 despite this studies on atl1 in contemporary cancer treatment are scarceas are those on its effects on cscs and the dynamics ofmircarrying evswe are interested in investigating whetherthemetastatic potential of crc cells can be amplifiedwhen they are cultured with cscderived evs carryingmir200c which is presumed to be oncogenic we alsoexamined whether atl1 possesses the ability to suppresscrc metastasis by exploring its effect on the transferof mir200c by evs and provide a speculation on themechanisms involved in its antitumorigenic actionmaterials and methodscell culture transfection andtreatmenttwo human crc cell lines highmetastasis lovo cellsand lowmetastasis ht29 cells were obtained from thecell bank of the chinese academy of sciences shanghai branch shanghai china lovo cells were culturedin f12k medium gibco waltham ma containing fetal bovine serum fbs gibcoand ht29 cells were cultured in mccoy™s 5a medium gibco containing fbs at —¦c in an 0ctang of atmosphere containing co2 to isolate cscs from lovoor ht29 cells denoted as lovocscs and ht29cscsrespectively four eppendorf tubes were prepared and µl of cell suspension — cellsml were added to eachtube then µl of cd44 antibody ebioscience waltham ma was added to tube µl of epithelial cell adhesion molecule epcam antibody ebioscience was added to tube cd44 and epcam antibody µl of each were added to tube and no antibodywas added to tube empty label after min of incubation at —¦c the cells were subjected to flow cytometricsorting the isolated cscs were stored in sterile eppendorftubes until use to overexpress or silence mir200c mimics and inhibitors genepharma shanghai china weretransfected into crc cells or colorectal cscs transfectionwas performed by incubating cells with mir200c mimicsor inhibitors at nm for h for experiments involving atl1 treatment mg of atl1 purity ‰¥ hplcbatch no dst170606014 dsiter chengdu china wasprepared in µl of dimethyl sulfoxide to prepare a stocksolution with a concentration of approximately mmthe stock solution was then diluted in culture medium andadministered at µm for h phosphatebuffered salinepbs was used as a controlcscderived evsisolation and characterization ofevs were isolated from nontreated or transfected lovocscs or ht29cscs using the exoquicktc exosome precipitation solution for culture media spinal fluid andurine exotc50a1sbi system biosciences palo altoca the cells were cultured in fbsfree lowglucose dulbecco™s modified eagle medium for h after which themedium was collected and centrifuged at — g for min the supernatant was collected and centrifuged at — g for min and the supernatant was collectedagain and ultracentrifuged at — g for min theprecipitate was resuspended in ml of pbs and ultracentrifuged at — g for min after which the precipitate was resuspended in pbs at a ratio of the mixturewas centrifuged at — g for min and the supernatantwas subjected to sucrose density gradient purification ofevs after the gradient was ultracentrifuged at —g for min the ev fraction ˆ¼ sucrose was carefullycollected using a long pipette tip the collected fraction wasultracentrifuged at — g for min and the resultingprecipitate containing isolated evs was collected all subsequent experiments involving coculture with evs exceptfor pkh labeling were performed with µgml evs for htransmission electron microscopytransmission electron microscopy tem was performedto identify the isolated evs the evs were fixed with glutaraldehyde in m pbs ph and the fixed evswere added dropwise to a treated nickel mesh for minafter the mesh was washed with pbs glutaraldehydewas added dropwise and incubated for min after whichthe mesh was washed several times with doubledistilledwater then filtered uranyl acetate was added to thesample dropwise and incubated for min excess liquidwas blotted with filter paper and the sample was dried themorphology of the evs was observed using tem345dimethylthiazol2yl25diphenyltetrazolium bromideassaycrc cells or colorectal cscs in the logarithmic growthphase were collected for 345dimethylthiazol2yl25diphenyltetrazolium bromide mtt assay the cells wereseeded in 96well plates at — cellswell and culturedovernight at —¦c the cells were subjected to transfectionor atl1 treatment as described in section if applicable after or h of culture µl of mgmlmtt reagent pab180013 bioswamp wuhan china wasadded to each well and the cells were further cultured for h then the mtt solution was removed and µl ofdimethyl sulfoxide was added to each well the plate wasgently shaken for min and the absorbance of the wellswas measured using a plate reader at nmand invasiontranswell assay of cell migrationtranswell chambers corning inc corning ny wereplaced in the wells of a 24well plate and immersed inpbs for min before the experiment after cells were subjected to µgml ev andor µm atl1 treatmentfor h they were cultured in fbsfree medium for hfor the migration assay18 the cells were trypsinised resuspended in fbs and seeded into the upper transwellchambers at — cellsml mlwell in the bottomtranswell chambers ml of medium containing fbs was added in each well the plates were incubatedat —¦c for h and the cells were fixed with ml of paraformaldehyde in each well for min at room temperature the fixative was removed and the cells were washedonce with pbs then ml of crystal violet solutionpab180004 bioswamp was added to each well and after 0c of tang min of staining the cells were washed three times withpbs cells that did not migrate were removed using a cotton swab and migrated cells were observed and countedat — using an inverted microscope dm il led leicamicrosystems wetzlar germany the invasion assay wasperformed following the same procedure except that eachchamber was coated with µl of matrigel bdbiosciences franklin lakes nj at —¦c for min priorto cell seedingcell markers and apoptosisflow cytometry detection of stemthe purity of isolated cscs was evaluated by flow cytometry using cd44 and epcam19 cscs — weresuspended in µl of flow cytometry buffer in aneppendorf tube and µl of fluorescein isothiocyanatefitcconjugated cd44 or phycoerythrinconjugatedepcam antibody was added to each tube the tubes wereincubated at —¦c for in the absence of light then thecells were washed with ml of buffer and centrifuged at — g at —¦c for min and the supernatant was removedthe cells were resuspended in µl of buffer and subjected to flow cytometry using a cytoflex s apparatusbeckman coulter brea ca data were analyzed usingnovoexpress software acea biosciences inc san diegoca for apoptosis flow cytometry was performed usinga fitcannexinv apoptosis detection kit bdbiosciences h after crc cells or colorectal cscs weretreated with µm atl1 or subjected to transfectiontrypsinised cells were centrifuged at — g for minand the supernatant was removed approximately — cells were resuspended in pbs and centrifuged at —g at —¦c for min the previous step was performed threetimes and the cells were resuspended in µl of bindingbuffer thereafter µl of annexin vfitc and µl ofpropidium iodide were added to the cells after min ofincubation at —¦c in the dark µl of binding bufferwas added and the cells were subjected to flow cytometrythe data were analyzed using novoexpress softwarepolymerase chain reactionquantitative reverse transcriptionstemloop quantitative reverse transcription polymerasechain reaction was performed to quantify the expressionof mir200c in crc cells colorectal cscs and cscderived evs after h of culture with µgml evsandor µm atl1 rna was extracted using trizol ambion inc foster city ca according to themanufacturer™s instructions and reverse transcriptionta b l e polymerase chain reactionprimer namemir200c stemloopmir200c forwardmir200c reverseu6 forwardu6 reverselist of primers for quantitative reverse transcriptionsequencectcaactggtgtcgtggagtcggcaattcagttgagtccatcatgggtaatactgccgggtaaactggtgtcgtggagtcggcctcgcttcggcagcacaaacgcttcacgaatttgcgtof the extracted rna into cdna was carried out usingthe advantage rtforpcr kit takara dalianchina quantitative polymerase chain reaction was conducted using the sybr green pcr reagent kit km4101kapabiosystems with the primers listed in table pcrproceeded as follows initial denaturation for min at—¦c cycles of denaturation for s at —¦c annealingfor s at —¦c and extension for s at —¦c and finalextension for s at —¦c and s at —¦c data wereacquired using qbase plus software and analyzed by theˆ’δδct method western blotphosphatidylinositol45bisphosphateto confirm that ev isolation was carried out successfully western blot of exosomal markers cd63 cd81and tsg101 was performed in cscs cscderivedevs and the lysates of cells from which the evs wererelated to stemness maintenanceisolated proteinsand3kinasepi3kprotein kinase b aktmammalian target ofrapamycin mtor signaling were also evaluated bywestern blot after h of culture with µgml evsandor µm atl1 proteins were extracted fromcells or evs radioimmunoprecipitation assay bufferpab180006 bioswamp containing protease and phosphatase inhibitors was added to lyse the cells at —¦c thelysates were transferred to an eppendorf tube heatedfor min at —¦c and centrifuged at — g for min the protein content in the supernatant was quantified using a bicinchoninic acid assay kit pab180007bioswamp for western blot µg of protein was subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis the electrophoresed proteins were transferredto a polyvinylidene fluoride membrane ipvh00010millipore burlington ma and blocked using skimmilk for h at room temperature thereafter the membranes were incubated overnight at —¦c with rabbitprimary antibodies againstthe following proteinsoctamerbinding transcription factor oct4 0ctang of ab18976 abcam cambridge uksexdeterminingregion ybox sox9 ab185966 abcam nanog ab106465 abcam cd63 pab33929bioswamp cd81 pab33928 bioswamp tsg101 pab32949 bioswamp pi3k ab191606abcam phosphopi3k ppi3k ab182651abcam akt ab8805 abcam pakt ab38449 abcam mtor ab32028 abcampmtor ab84400 abcam and gapdh pab36264 bioswamp after three washes withpbstween for min each the membranes wereincubated with goat antirabbit igg secondary antibody sab43711 bioswamp for h at room temperature and washed again three times with pbs for mineach for visualization of the proteins the membraneswere incubated with an enhanced chemiluminescenceagent wbkls0010 millipore in the dark and proteinband gray values were analyzed using an automaticscanner tanon5200 tanon shanghai china the dataare presented as the ratio to the gray value of the controlgroup taken as a value of in arbitrary units aufluorescence labelingevaluation of ev uptake via pkh67evs were labeled following the instructions of the pkh67green fluorescent cell linker midi kit midi671ktsigmaaldrich st louis mo isolated evs were mixedwith ml of diluent and µl of pkh67 solution both provided in the kit and incubated for min then ml of bovine serum albumin in pbs was added for quenching the labeled evs were ultracentrifuged twice at — g at —¦c for min and resuspended in µl of pbs toexamine ev uptake lovo or ht29 cells were seeded in a24well plate at — cellswell and incubated at —¦cfor h in an atmosphere containing co2 then µg of pkh67labeled evs were added to each well and theplate was further incubated at —¦c in co2 for or h the cells were fixed for min with paraformaldehyde and the nuclei were stained with hoechst afterwhich they were observed under a leica dm il led fluorescence microscope using a — objective lens for evuptake the microscopy imaging parameters were set atinitial acquisition and were kept constant between acquisitions green fluorescence represents pkh67 labelingof evsstatistical analysisstatistical analysis was performed using originpro thedata are expressed as the mean ± standard deviation sdof three technical replicates n differences betweentwo groups were compared using onesample ttest oneway analysis of variance anova followed by tukey™spost hoc test was conducted for comparisons between multiple groups more than two p indicates statisticalsignificanceresultsproperties of colorectal cscseffect of atl1 on the behavior andcolorectal cscs were successfully isolated from lovo andht29 cells as evidenced by the flow cytometric profilesof cd44 and epcam figures 1a and s1 which aremarkers of cscs15 we first evaluated the influence of µm atl1 on the behavior of colorectal cscs figures 1band 1c demonstrate that in cscs isolated from both lovohigh metastatic potential and ht29 low metastaticpotential cells proliferation was inhibited by atl1 forup to h atl1 also disrupted stemness maintenance inboth lovocscs figure 1d and ht29cscs figure 1eas revealed by the decreased protein expression of stemness indicators oct4 sox9 and nanog next themigration and invasion of cscs was investigated usingtranswell assays ht29cscs exhibited low metastaticpotential and their limited migratory and invasive abilitiesresulted in unsuccessful trials of transwell assay for thisreason only results for lovocscs are shown evidentlyatl1 at µm significantly decreased the migrationfigure 1f and invasion figure 1g of lovocscs theseresults are complemented by flow cytometry observations showing that the administration of atl1 inducedremarkable apoptosis in cscs compared to that in nontreated cells figure 1h corresponding flow cytometricprofiles of cell cycle progression in cscs are provided infigure s2relationship between mir200c andthe metastatic potential of colorectal cscsto elucidate whether there is a correlation betweenmir200c and the metastatic potential of crc cells andcolorectal cscs we detected the expression of mir200cin lovo and ht29 cells we anticipated that the highlymetastatic lovo cells would exhibit higher expression ofmir200c than ht29 cells and the results confirmed ourspeculation figure 2a the same trend was observedin the cscs derived from the respective crc cells wethen proceeded to compare the expression of mir200cin colorectal cscs with that in the crc cell line from 0c of tang f i g u r e characterization of colorectal cscs isolated from lovo and ht29 cells a flow cytometric sorting of cscs using cd44 andepcam as markers the percentage of parental lovo and ht29 cells expressing both cd44 and epcam was approximately representingthe proportion of cscs cscs isolated from parental lovo and ht29 cells denoted as lovocscs and ht29cscs respectively exhibited highexpression of both markers relative proliferation of b lovocscs and c ht29cscs was inhibited by atl1 at µm for up to h compared to that of control pbs cscs the protein expression of stemness markers oct4 sox9 and nanog relative to gapdh 0ctang of which they were isolated comparing the same numberof lovo or ht29 cells and lovocscs or ht29cscsthe cscs showed a clear increase in mir200c expressionfigure 2b administration of µm atl1 to cscssignificantly attenuated the expression of mir200c compared to that in nontreated cscs figure 2c from theseresults it is speculated that the high metastatic potentialof lovo cells may be associated with the expression ofmir200c in the csc subpopulationwe further investigated the role of mir200c in regulating crc cell behavior in particular proliferation andapoptosis lovo and ht29 cells were treated with mir200c mimics mir200cmim or inhibitors mir200cinh the transfection efficiency was excellent in lovocells wherein mir200cmim and mir200cinh significantly upregulated and downregulated the expressionof mir200c respectively however in ht29 cells theinhibitor demonstrated high efficiency whereas the efficiency of the mimic was suboptimal figure 2d aftermimic or inhibitor treatment crc apoptosis and cell proliferation were examined using flow cytometry and mttassay respectively in terms of apoptosis mir200cinhinduced an increase in the percentage of apoptotic lovoand ht29 cells figure 2e corresponding flow cytometric analysis of cell cycle progression is demonstrated in figure s3 in addition in both lovo figure 2f and ht29figure 2g cells proliferation was enhanced by mir200cmim but suppressed by mir200cinh together with theprevious results these findings suggest that mir200c confers crc cells with metastatic traits by promoting cellproliferation and inhibiting apoptosis subsequently weinvestigated whether mir200c overexpression and interference exert similar effects in colorectal cscs as theydid in crc cells lovocscs and ht29cscs were transfected with mir200cmimics or inhibitors and the transfection efficiency was validated figure 3a similar tothe crc cells flow cytometric analysis showed a markedincrease in apoptosis in both types of cscs transfected withmir200cinh figure 3b corresponding flow cytometric analysis of cell cycle progression in transfected cscsis demonstrated in figure s4 this was supported by themtt assay which revealed that the proliferation of bothtypes of cscs was enhanced by mir200cmim but inhibited by mir200cinh over a period of h figures 3cand 3d collectively the results demonstrate that transfection of mir200c mimics and inhibitors had a directeffect on csc behavior thus the effects of mir200cobserved previously in crc cells may be a result of changesin the properties of the colorectal csc subpopulationfound withinfrom mir200ctransfected colorectal cscscharacterization of evs derivedwe then explored whether the properties conferred bymir200c can be horizontally transferred between cellsvia a carrier agent to do this we isolated evs fromlovocscs and ht29cscs lovocscsevs and ht29cscsevs respectively western blot of exosomal markers cd63 cd81 and tsg10120 confirmed successful evisolation as these markers were exclusively expressed inevs and were almost nonexistent in the lysates of cscsfrom which the evs were derived figures 4a and 4bevs were also identified by tem figure 4c next weexamined whether the cscderived evs acted as a carrier for mirnas in particular mir200c we first confirmed that the basal level of mir200c was lower in ht29cscsevs than that in lovocscsevs figure 4d thencscs isolated from lovo or ht29 cells were transfectedwith mir200c mimics or inhibitors as described and evswere isolated from the transfected cscs evs from nontransfected cscs acted as controls we observed that mir200cmimtransfected lovocscs and ht29cscs produced evs that exhibited significantly elevated mir200cexpression than did the nontransfected cscs contrarilytransfection of mir200cinh in cscs resulted in the isolation of evs with attenuated mir200c expression compared to that in control evs figure 4e to determinewhether evs carrying mir200c mediated the horizontal transfer of metastatic traits we cultured lovo cellswith evs derived from either lovocscs or ht29cscstransfected with mir200c mimics or inhibitors lovo cellsshowed higher expression of mir200c when mir200coverexpressing evs were added whereas correspondinglyevs from mir200cinhtransfected cscs led to lowermir200c expression figure 4f the migratory and invasive capabilities of the cultured lovo cells were thenassessed using a transwell assay with evs from nontransfected cscs used as controls in the case of both lovocscs and ht29cscs the transfection of mir200cmimin the cscs resulted in evs that greatly enhanced thein d lovocscs and e ht29cscs was downregulated by atl1 at µm after h of culture compared to that of control pbscscs transwell assay of the f migration and g invasion of lovocscs demonstrated decreased cell counts in both cases when atl1 wasadministered at µm compared to those of control pbs cscs h the percentage of apoptotic lovocscs and ht29cscs was elevatedin the presence of atl1 at µm compared to that of control pbs cscs the data represent the mean ± sd of three independent technicalreplicates ttest p p at h 0c of tang f i g u r e mir200c expression in parental crc cells and colorectal cscs a relative basal expression of mir200c in lovo cells highmetastatic potential and ht29 cells low metastatic potential as well as isolated cscs lovocscs and ht29cscs demonstrates a possiblerelationship between mir200c and metastatic property b mir200c expression in lovo and ht29 cells relative to that of their correspondingcscs the same number of colorectal cscs clearly exhibited higher mir200c expression than did crc cells c mir200c expression wasattenuated by atl1 at µm in both lovocscs and ht29cscs d transfection efficiency of mir200c mimics mir200cmim inhibitorsmir200cinh and their corresponding negative controls mir200cmimnc and mir200cinhnc in lovo and ht29 cells mir200cmimand mir200cinh respectively induced significant upregulation and downregulation of mir200c in lovo and ht29 cells e the percentageof apoptotic lovo and ht29 cells was increased by mir200cinh but remained unchanged in the case of mir200cmim relative proliferationof f lovo and g ht29 cells subjected to transfection of mir200c mimics or inhibitors or their corresponding nc mir200cmim andmir200cinh respectively enhanced and inhibited the proliferation of both types of parental crc cells the data represent the mean ± sd ofthree independent technical replicates ttest or anova p p at hmigratory and invasive capabilities of lovo cells interference of mir200c on the other hand produced cscderived evs that limited the migration and invasion oflovo cells figures 4g and 4h we also examined whether µm atl1 had an inhibitory effect on migration andinvasion as it did on cell proliferation demonstrated previously as anticipated atl1 impaired the migration andinvasion of lovo cells in the presence of evs as confirmed by the significant decrease in cell count in thecase of transfected and nontransfected cscs figures 4gand 4hwith cscderived evscoculture of lovo and ht29 cellswe were interested in the specific effect of cscderivedevs on the stemness maintenance of noncscs and theinvolvement of mir200c therein lovo or ht29 cellswere cultured with evs derived from lovocscs or ht29cscs that were not transfected control or transfectedwith mir200c mimics or inhibitors and the expressionof stemness markers oct4 sox9 and nanog was evaluated compared to control evs from nontransfected 0ctang of f i g u r e transfection of mir200c mimicsinhibitors in colorectal cscs a transfection efficiency of mir200c mimics mir200cmim inhibitors mir200cinh and their corresponding negative controls mir200cmimnc and mir200cinhnc in lovocscs andht29cscs mir200cmim and mir200cinh respectively induced significant upregulation and downregulation of mir200c in both lovocscs and ht29cscs b the percentage of apoptotic lovocscs and ht29cscs was increased by mir200cinh but remained unchangedin the case of mir200cmim relative proliferation of c lovocscs and d ht29cscs subjected to transfection of mir200c mimics orinhibitors or their corresponding nc mir200cmim and mir200cinh respectively enhanced and inhibited the proliferation of both typesof colorectal cscs the data represent the mean ± sd of three independent technical replicates anova p p at hcscsthose isolated from mir200cmimtransfectedlovocscs and ht29cscs induced stronger expressionof stemness markers whereas evs isolated from mir200cinhtransfected cscs suppressed their expressionthis was the general trend observed in lovo cells cocultured with lovocscsevs figure 5a or ht29cscsevs figure 5b as well as in ht29 cells coculturedwith lovocscsevs figure 5c or ht29cscsevs figure 5d additionally we observed that the incorporation of µm atl1 in the coculture of crc cellsand evs had an inhibitory effect on stemness as demonstrated by the decrease in oct4 sox9 and nanog inall caseswe also investigated whether mir200c carried by isolated evs affected the activation of the pi3kaktmtorsignaling pathway in crc cells the same cocultures wereperformed and the expression of phosphorylated pi3kakt and mtor was detected relative to the respectivetotal protein content similar to stemness maintenancepi3kaktmtor activation was promoted in crc cells 0c of tang f i g u r e isolation and characterization of cscderived evs as a carrier of mir200c a expression of exosomal markers cd63 cd81and tsg101 was detected in lovocscs evs isolated from lovocscs and cell lysates after isolation b expression of exosomal markers cd63cd81 and tsg101 was detected in ht29cscs evs isolated from ht29cscs and cell lysates after isolation in both cases the lysates of thecscs showed negligible expression of these markers compared to that in cscs and cscderived evs c transmission electron microscopy ofevs derived from lovocscs scale bar nm d comparison of the relative basal expression of mir200c in evs derived from nontransfected lovocscs and ht29cscs lovocscsevs expressed higher levels of mir200c than did ht29cscsevs e expression of mir200cin evs derived from lovocscs or ht29cscs that were first transfected with mir200c mimics mir200cmim or inhibitors mir200cinhevs derived from nontransfected cscs are denoted as control mir200cmim and mir200cinh respectively induced significant upregulation and downregulation of mir200c in both lovocscsevs and ht29cscsevs relative to control levels f expression of mir200c inlovo cells cocultured with lovocscsevs and ht29cscsevs cscs were subjected to various transfections transfection of cscs withmir200cmim and mir200cinh resulted in the secretion of evs that respectively induced significant upregulation and downregulation ofmir200c in lovo cells relative to control levels g transwell assay of the migration and invasion of lovo cells cocultured with lovocscsevs cscs were subjected to various transfections with or without atl1 administration at µm relative to control levels migration andinvasion were enhanced by evs derived from mir200cmimtransfected cscs but suppressed by those derived from mir200cinhtransfectedcscs in all cases atl1 reduced the degree of migration and invasion scale bar µm h quantification of the data in f by cell countthe data represent the mean ± sd of three independent technical replicates ttest or anova p cocultured with evs from mir200cmimtransfectedcscs but decreased by mir200c interferencethis was the case for lovo cells cocultured with lovocscsevs figure 6a or ht29cscsevs figure 6b aswell as in ht29 cells cocultured with lovocscsevsfigure 6c or ht29cscsevs figure 6d the additionof µm atl1 induced a decrease in the phosphorylation of the abovementioned proteins signifying thatpi3kaktmtor activation was inhibited the resultssuggest that evs derived from cscs carried different 0ctang of f i g u r e effect of ev coculture on stemness maintenance in lovo and ht29 cells lovocscs and ht29cscs were first transfectedwith mir200c mimics mir200cmim or inhibitors mir200cinh lovo cells were cocultured for h with evs isolated from nontran
Colon_Cancer
coronaviruses covs belong to a family of envelopedviruses with a positive sense singlestranded rna genomecovs cause illness ranging from upper respiratory tractinfections urtis resembling the common cold to lowerrespiratory tract infections lrtis such as bronchitis pneumonia and even severe acute respiratory syndrome sarswith most serious disease outcomes in the elderly immunocompromised patients and infants [ ] hcovoc43oc43 hcov229e 229e hcovnl63 nl63 andhcovhku1 hku1 were the first documented humancovs hcovs which usually cause urtis and less frequently are associated with ltri diseases in the lastdecades two human coronaviruses created great concernfor the world medical community due to significant diseaseand mortality [ ] in severe acute respiratorysyndromecoronavirus sarscov was characterized byacute atypical pneumonia and diï¬use alveolar damagedad in roughly patients and with almost deathsrepresenting a nearly mortality rate more recentlyin a new human coronavirus designated as middle eastrespiratory syndromecoronavirus merscov was identified and the global ongoing outbreak of mers with over official cases and deaths represented approximately case fatality rate to date in humans over the last few months a new strain of human coronavirus sarscov2 also known as 2019ncov hascaught the world™s seven continents™ attention with its rapidglobal spread aï¬ecting at least countries and territoriesinfecting more than and claiming more than lives worldwide the coronavirus pandemichas promoted isolation and uncertainly fear and panicworldwide in additionlikely lead to changes inpolitical and economic power in ways that can be determined only later it willit is important to note that there are many similaritiesamong diï¬erent coronavirus species but not in all aspects 0coxidative medicine and cellular longevitydepending on the molecular mechanism of viral inhibitionpromoted by an antiviral agent the analysis of the data andcomparison between animal and human covs must be donevery carefully in fact it is important to note that there arediï¬erences between human and animal cov receptorswhich will likely result in diï¬erent affinities or unlikely interactions of an antiviral agent with the diï¬erent cov receptors howeverif the antiviral agent interferes with thereplication andor assembly of the covs there is a higherprobability of obtaining similar antiviral activity results inhuman cov tests [ ] following this line our searchin specialized literature was focused mainly on studies thatinvestigated the anticoronavirus eï¬ects of natural antioxidants by inhibiting proteases for viral replication materials and methodsthe present study was carried out based on a search of the literature of natural antioxidants and coronavirus the searchperformed in the pubmed database included studies published until march and used the following keywordscoronavirusstressmerscov sarscov 229e nl63 oc43 hku1merscov virus infection and middle east respiratorysyndrome virus the scientific publications were selectedfrom studies published in the english languageantioxidants flavonoids oxidative pathogenic mechanism of coronavirusinduced cell damagethe high mortality rate associated with the three pathogenichcovs has been mainly attributed to the development ofdigestive and respiratory tract injuries observed followinginfection acute atypical pneumonia and diï¬use alveolardamage that progress to deposition of fibrous tissue denudedairways haemorrhage and elevated macrophage ltrationare sometimes accompanied by watery diarrhoea dehydration and vomiting [ ]despite the molecular mechanisms of coronavirusinduced intestine and lung pathogenesis not fully elucidatedand still unclear studies have suggested that lateterm diseaseprogression is unrelated to viremia it is now believed morelikely to be associated with the immunopathological mechanism [ ] viral clearance and subsequent recovery frominfection require activation of an eï¬ective host immuneresponse however many immune eï¬ector cells may alsocause injury to host tissues together with ‚ammatoryand immune response signaling the presence of oxidativecompounds such as reactive oxygen species ros playsimportant roles in the pathogenic mechanism of cell damageinduced by covs through oxidative stress oxidative stress is defined as an interruption andorderegulation of the signaling and redox system that can becaused by an imbalance in the production of oxidant andantioxidant species among the main oxidant agentsros and reactive nitrogen species rns stand out in orderto counterbalance the oxidant species there is an antioxidantsystem formed by enzymes and nonenzymatic molecules however during pathological events such as viral infections there may be an increase in the production of oxidantspecies not neutralized by the antioxidant system resultingin oxidative stress that promotes cellular damage throughprotein denaturation changes in the functions of nucleicacids lipid peroxidation and cell death [“]in addition during viral infection oxidative stress contributes to viral pathogenesis through stimulating ‚ammation loss of immune function and increased viral replicationthat may occur due to the activation of the nuclear factorkappa b nfκb transcription pathway [“] currentevidence suggests that cytokine dysregulation”also calledcytokine storm”contributes to severe disease caused by thepathogenic covs [ ] the exact mechanisms are notclear yet but research on ‚uenza a virus shows that infection causes a rapid ‚ux of ‚ammatory cells this isfollowed by an increase in reactive oxygen species productionand cytokine expression and release which ultimately leadsto acute lung injury in general rna viruses promotechanges in the body™s antioxidant defense system aï¬ectingenzymes such as superoxide dismutase sod and catalasecat in addition to reducing the levels of antioxidantmolecules such as ascorbic acid carotenoids and reducedglutathione gsh [“] wu reported that glucose6phosphate dehydrogenasean important antioxidantenzyme that produces nadph knockdown cells were moresusceptible to infection by hcov229e than normal cells interestingly ye and colleagues have reported that theinhibition of ros production alleviates ‚ammation causedby ‚uenza a virus infections in an experimental model of sarsinduced acute lunginjury in mice it was noted that phospholipid oxidationdue to oxidative stress is one of the main triggering factorsof acute lung injury this happens through the activation ofthe innate immune response culminating in the activationof pulmonary macrophages via tlr4triftraf6nfκbsignaling furthermore hypoxia caused by acute lunginjury can cause myocardial injury due to the production ofros aggravating infections caused by coronavirus disease covid19 mitochondria have an essential function in energy generation and for this reason their function and integrity arestrictly regulated in order to respond to varying energyrequirements and environmental conditions mitochondria are known to function as the control point in apoptoticpathways releasing proapoptotic factors mainly ros whichfunction as a signaling molecule that may result in cell death[ ] some studies have shown a relationship betweencoronavirus infection and dysfunctional or damaged mitochondria leading to the release of ros and other proapoptotic substances [ ] in a recent study xu reportedthat ros and p53 play key roles in regulating many kindsof the cell process during coronavirus infection in vero cellsaccording to the authors coronavirus infection appears toinduce a timedependent ros accumulation which in turnis linked to regulatory mechanisms of p53 activation andapoptosis in infected cells antioxidant substances promote improvement in casesof disease caused by coronaviruses such as apolipoproteind”a lipocalin that promoted a neuroprotective eï¬ect against 0coxidative medicine and cellular longevityencephalitis induced by human coronavirus oc43”in micethis protective eï¬ect occurred through the reduction of oxidative stress cerebral lipid peroxidation and regulation of‚ammation [ ] also the treatment with antioxidantssuch as pyrrolidine dithiocarbamate or nacetylcysteine significantly inhibits moreover melatonin promotes downregulation of acute lungoxidative injury due to its anti‚ammatory and antioxidantactions making it a possible compound in the treatment ofcovid19 based on these studies compounds thathave antioxidant actions can be helpful in the treatment ofinfections promoted by coronaviruscoronavirusinduced apoptosisin general antioxidant properties of polyphenolic compounds such as some flavonoids have been associated withthe presence of aromatic phenolic rings that promote theelectron donation and hydrogen atom transfer to free radicals acting as free radical scavengers reducing agents andquenchers of single oxygen formation thus the aimof this study was to investigate the antioxidant capacity andantiviral activity of natural antioxidants against coronavirusthe compounds are illustrated in figure occurrence and antioxidant properties ofanticoronavirus compoundsquercetin can be found in plants such as rubus fruticosus land lagerstroemia speciosa l pers [ ] also quercetinshows antioxidant activity at a concentration of μmoll inhepg2 cells inhibiting oxidative stress promoted by h2o2 promotes an increase in sod cat and glutathioneperoxidase gpx and reduces lipid peroxidation in rats withchronic prostatitischronic pelvic pain syndrome moreover quercetin improves sepsisinduced acute lung injury inrats by reducing lipid peroxidation and ‚ammation andincreasing sod and cat levels in addition quercetin glycosides with antioxidant activity such as quercetin 3βglucoside have already been isolated from plants such as passiflora subpeltata ortega andchamomilla suaveolens pursh rydb [ ] the administration of quercetin 3βglucoside mgkg poinstreptozotocininduced diabetic rats promotes an increasein the levels of antioxidant enzymes sod cat andgpx and nonenzymatic antioxidants vitamins c and eand gsh and a reduction of lipid peroxidation quercetin 3βgalactoside hyperoside is found mainly in plantsof the hypericum genus such as hypericum perforatum l[ ] moreover it showed cardioprotective activity inhigh glucoseinduced injury of myocardial cells throughdecreased apoptosis and ros production and increasedsod levels quercetin 7ramnoside is also found inplants of the hypericum genus such as hypericum japonicum thunb ex murray this flavonoid shows hepatoprotective activity against carbon tetrachloride in mice bydecreasing lipid peroxidation and increasing cat andgsh levels in addition to presenting values of μmand22diphenyl1picrylhydrazyldpph and ²azinobis3ethylbenzthiazoline6sulphonic acid abts assays respectively μm intheepigallocatechin gallate is present in parkia roxburghii gdon and is one of the main metabolites found in green teaand liubao tea camellia sinensis lo kuntze also gallocatechin gallate can be found in this plant [“] literaturedata reveal that the administration ip of mg100 g ofepigallocatechin gallate in rats with streptozotocininduceddiabetes mellitus promotes a reduction in oxidative stressthrough reductions in parameters such as indirect nitricoxide synthesis and status total oxidative as well as anincrease in levels of cat and total antioxidant capacity ofplasma furthermore it promotes cardioprotection byantioxidant mechanisms green tea has a high antioxidant capacity due to the highlevels of catechins present he and collaborators compared the antioxidant activities of catechins and reported thatepigallocatechin gallate has greater antioxidant activity viaradical scavenging activity μm with values of ± ± and ± compared to its epimergallocatechin gallate with values of ± ± and ± in the dpph abts and ferric reducingantioxidant power frap respectively amentoflavone is a biflavonoid present in leaves ofginkgo biloba l garcinia brasiliensis l and nandinadomestica l [“] this biflavonoid has a high antioxidantcapacity “ demonstrated in scavenging tests ofdpph abts superoxide and hydroxyl radicals moreover amentoflavone prevents acute lung injury induced bysepsis in rats by decreasing thiobarbituric acid reactive substance tbars levels and by increasing levels of sod andgsh apigenin is mainly present in flowers and leaves beingabundantly found in apium graveolens l petroselinum crispum mill fuss and matricaria chamomilla l sánchezmarzo and collaborators evaluated the antioxidantcapacity of apigenin using the trolox equivalent antioxidantcapacity teac oxygen radical absorbance capacityorac and frap assays the results show that apigeninhas good antioxidant activity with values of ± μmol teammol ± μmol teammol and ± μmol fe2mmol respectively in addition oraladministration of apigenin mgkgday for days in anexperimental model of cardiotoxicity induced by doxorubicin in rats promoted cardioprotection by reducing levels ofmalondialdehyde mda increasing sod levels and preventing cardiomyocyte apoptosis luteolin is present in foods such as carrot cabbage teaand apple and is found in ugni molinae turcz [ ] datashow that luteolin μgml increases the levels of gsh theexpression of gsh synthetase and the activity of sod andcat in human colon cancer cells ht29 furthermoreluteolin attenuates the sepsisinduced acute lunginjury in mice by reducing lipid peroxidation and increasingsod and cat activity in addition to suppressing the nfκbpathway herbacetin is ubiquitous in plants of the genus rhodiolasuch as rhodiola rosea l herbacetin glycosides are alsopresent in the roots of r sachalinensis a bor and show antioxidant activity veeramani reported that theadministration of herbacetin mgkg po in mice with 0coxidative medicine and cellular longevityohohohohhoohooohohhooohoh oquercetinohohoooohohohquercetin 3𝛽galactosideohohooohoohoohohhoohohohoh oquercetin 7ramnosideohooohohoohoohoohohohquercetin 3𝛽glucosidehooohooohohohohohohepigallocatechin gallateohgallocatechin gallateohhooohoh ohooluteolinhooohoooohoohohoohrhoifolinhohohoohohohohohohoohoohooh oamentoflavoneohhooohohohoherbacetinhohoooohooohohohoapigeninooohooohhopectolinarinooopsoralidinhooohohohohcatechinohhoohoooh ohelichrysetinohohomyricetinohohohhohoohoohoisobavachalconeohhooohscutellareinohresveratrolfigure chemical structures of bioactive antioxidants against coronavirusobesityassociated insulin resistance promotes an increasein the activity of the enzyme glucose6phosphate dehydrogenase which is directly related to the production ofnadph pectolinarin is present in plants of the genus cirsiumsuch as cirsium setidens nakai and cirsium japonicum dcthe administration of pectolinarin and mgkg pofor two weeks in rats promotes antioxidant eï¬ects in hepatic 0coxidative medicine and cellular longevitytable antioxidant properties of natural inhibitors of coronaviruscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencetype of cells μmoll mgkg po mgkg poinhibiting oxidative stress promoted byh2o2promoted an increase in sod cat andgpx and reduced lipid peroxidationreduces lipid peroxidation and increasessod and cat levelsincreases levels of sod cat gpx mgkg povitamins c and e and gsh and reduces nmoll mgkgic50 μm dpphec50 μm abtsrats with streptozotocininduced diabetes mellitus mg100 g iprats with streptozotocinnicotinamideinduceddiabetes mellitus mgkg po μm mgkg μgml ± μmol teammol ± μmol teammol and ± μmol fe2mmolrespectively mgkg pomodelshepg2 cellsrats with chronicprostatitischronic pelvicpain syndromesepsisinduced acute lunginjury in ratsstreptozotocininduceddiabetic ratshigh glucoseinducedinjury of myocardial cellsccl4induced liver damagemodel in micedpphabtsquercetinquercetin 3βglucosidequercetin 3βgalactosidequercetin ramnosideepigallocatechingallateepigallocatechingallatedpphabtsfrapgallocatechingallateamentoflavoneacute lung injury inducedby sepsis in ratsdpph abts superoxideand hydroxyl radicalsteacoracfrapcardiotoxicity induced bydoxorubicin in ratshuman colon cancer cellsht29acute lung injury inducedby sepsis in micemice with obesityassociated insulinresistance inductionhepatic injury induced bydgalactosamine in ratsoracapigeninluteolinherbacetinpectolinarinrhoifolincatechinlipid peroxidationdecreases apoptosis and ros productionand increases sod levelsdecreases lipid peroxidation and increasescat and gsh levelsscavenging of free radicalsreduces indirect nitric oxide synthesis andtotal oxidative statusincreased levels of cat and totalantioxidant capacity of plasmaincreased levels of cat sod and gshreduced levels of superoxide and proteincarbonyl pco and prevented dnadamage ± ± and ± ± and ± respectively ± respectivelydecreases tbars levels and increaseslevels of sod and gshscavenging of free radicalsscavenging of free radicalsreduces levels of mda increases sodlevels and prevents cardiomyocyteapoptosis μgmlincreases levels of gsh expression of gshsynthetase and the activity of sod and mgkg ip mgkg po and mgkg poapproximately troloxequivalents μmcatreduces lipid peroxidation increases theactivity of sod and cat and suppressesthe nfκb pathwayincreases the activity of glucose6phosphate dehydrogenaseincreases levels of sod gsh glutathionereductase and glutathione stransferasescavenging of free radicalsscavenging of free radicals 0coxidative medicine and cellular longevitytable continuedtype of cellscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencemodelsabtsfrap ± mol troloxequivalentsmol ± mol trolox equivalentsmoldihydrorhodamine oxidation assayandic50 ± μmisobavachalconedpph sc50frapabts sc50psoralidinelectron spin resonancemyricetinhelichrysetinscutellareinresveratroldpphchinese hamster lungfibroblast cells v794treated with h2o2oracdpphabtssuperoxide radicalsdpph sc50r2rats with obstructive lungdiseasehypercholesterolemicapoeko mouserespectively μm ± mmic50 μmequivalent to feso4 mm respectively·7h2o and μgml and μgml μgml ± trolox equivalents ± μm ± μm and ± μm respectivelyscavenging of free radicalsscavenging of free radicalsscavenging of free radicals and respectivelyprevents dna damage and lipidperoxidationincreases the activity of sod cat andgpxscavenging of free radicalsscavenging of free radicals μmoldmscavenging of free radicals mgkg mgkgincreases sod activity and reduces mdalevelsinhibits the activity and expression ofnadph oxidasesincreases sod gpx and cat levels injury induced by dgalactosamine by increasing levels ofsod gsh glutathione reductase and glutathione stransferase [ ]rhoifolin is found in citrus fruits such as citrus limettarisso studies have indicated that its radical peroxyl scavenging capacity is higher than trolox in orac assays approximately trolox equivalents μm [ ]meanwhile the catechin is a flavonoid present inleaves of green tea wine and fruits [ ] grzesik investigated the antioxidant action of catechinthrough the abts scavenging activity and frap teststhe results show values of ± mol troloxequivalentsmol and ± mol trolox equivalentsmol respectively in addition catechin shows greaterprotective properties in the dihydrorhodamine oxida ± μm than gsh and ascorbiction assay ic50acid and μm respectively psoralidin is a prenylated coumestan which is found inplants of the fabaceae family such as psoralea corylifolia lxiao and collaborators investigating the antioxidant potential of compounds isolated from p corylifolia observed thatpsoralidin shows the best antioxidant activity by the methodof electron spin resonance spectroscopy with an ic50 value of μm the compound isobavachalcone has been isolated fromplants of the fabaceae and moraceae families [ ] isobavachalcone shows a strong antioxidant activity in dpphsc50 frap and abts sc50 assays with values of μm ± mm equivalent to feso4·7h2o and mm respectively in addition the compound has beenreported to inhibit the nfκb pathway in sephadexinducedlung injury in rats [ ]helichrysetin is a chalcone that is found in plants of thehelichrysum genus such as helichrysum odoratissimum l in a study investigating the antioxidant activity of natural and prenylated chalcones vogel found that helichrysetin is the substance that shows the highest antioxidantactivity in the orac test with values of ± troloxequivalents myricetin is widely found in the plant families myricaceae and anacardiaceae and is widely used as health foodsupplement due to its antioxidant properties [ ]bennett demonstrated that myricetin reacts withoxygencentered galvinoxyl radicals more than timeshigher than vitamin e dalphatocopherol furthermoremyricetin was able to scavenge and on the dpphassay μgml and μgml respectively interestinglythe compound prevents dna damage by lipid 0coxidative medicine and cellular longevity2h2o2 2gsh2o2h2h2o2vit evit cgshnadphnonenzymatic antioxidantsgpxsodcatsemyzne tnadixoitna2h2o gssgo2 h2o2o2 h2omdapcotbarsbio m arkers of oxidative stressgeneration of rosoxidative stresscell damagenaturalantioxidants sesadixohpdan2o2 nadphnadp 2o2 hfigure the main antioxidant mechanisms of natural compounds reported in this review dashed line inhibition full line activationperoxidation and increasing the activity of sod cat andgpx in chinese hamster lung fibroblast cells v794treated with h2o2 scutellarein is found in scutellaria barbata d don andpolygonum viscosum buchham [ ] liu investigated the antioxidant activity of scutellarein through thedpph abts and superoxide scavenging assays they notedthat the compound shows good antioxidant activity withvalues of ± μm ± μm and ± μmrespectively while the trolox a standard antioxidant compound presented ± μm ± μm and ± μm respectively resveratrol is found in grapes peanuts and blueberriesand can be isolated from veratrum grandiflorum o loes literature shows that resveratrol has good antioxidantvalues of μmoldmactivity with dpph sc50r2moreover it is able to reduce the production of ros by inhibiting the activity and expression of nadph oxidases byeliminating oxidant agentsincluding radical hydroxylsuperoxide hydrogen peroxide and peroxynitrite the treatment of resveratrol mgkg po in ratsreduces oxidative stress in obstructive lung disease byincreasing sod activity and reducing mda levels indicatinga decrease in lipid peroxidation table shows themain actions of natural antioxidants discussed in this studyand figure illustrates these activities effect of natural antioxidants incoronavirus infectionsthis review focused on studies reporting on the anticoronavirus activity of natural antioxidants based on exclusioncriteria data from nineteen compounds were discussedthe oxidative stress pathway could potentially be a keyelement in coronavirusinduced apoptosis and pathogenesis for this reason it is interesting to investigate the useof antioxidants as potential therapeutic tools”either as analternative or as an adjuvant to conventional therapies”inthe treatment of coronavirus infections among the antioxidant compounds evaluated as for coronavirus infectionsare the flavonoids which are compounds widely found infruits vegetables and certain beverages in fact researchgroups have reported that antioxidant flavonoids includingcatechin luteolin apigenin quercetin and quercetin rhamnosideinhibit ros accumulation and apoptosis ofcells infected with diï¬erent coronavirus including porcineepidemic diarrhoea coronavirus pedv and transmissiblegastroenteritis coronavirus tgev [“]as shown with the recent covid19 pandemic thesearch for alternative or new antiviral therapies for theremainstreatment of coronavirus diseasesimportantbased on the literature antioxidanttherapies oï¬er anattractive option 0coxidative medicine and cellular longevitytable natural antioxidants tested in in vitro coronavirus infection models and their main results and mechanism of actionantioxidanttype of cells testedconcentrationic50antiviral eï¬ectmechanism of actionreferencecatechintgevinfected st cellscatechin“ μminhibition of tgevinduced apoptosissuppression of the tgevinducedbcl2 reduction baxredistribution cytochrome crelease and caspase3 activation resveratrolmersinfected vero e6cellsresveratrol μmquercetinepigallocatechingallategallocatechingallate gcgquercetin rhamnosideq7rrecombinant 3clprowas expressed in pichiapastoris gs115quercetin μmepigallocatechingallate μmgallocatechingallate μmpedvinfectedvero cellsq7r μminhibition ofmersinducedreduction of the expression ofinfectionapoptosis andnucleocapsid n protein essential prolonged cellular survivalfor merscov replicationafter virus infectioninhibition of coronavirusreplicationreduction of the formationof a visible cytopathiceï¬ect cpe without dnafragmentationgcg displayed a binding energyof kcal mol1 to the active siteof 3clpro and the galloyl moietyat 3oh position was required for3clpro inhibition activitynot specificity amentoflavoneapigeninluteolinquercetinquercetin3βgalactosideherbacetinrhoifolinpectolinarinsarscov 3clproinhibition usingfluorescence resonanceenergy transfer analysismolecular dockingsprfretbasedbioassays andmutagenesistryptophanbasedfluorescence methodherbacetinisobavachalconequercetin3βdglucosidehelichrysetintryptophanbasedfluorescence methodamentoflavone3βgalactoside μmapigenin μmluteolin μmquercetin μmquercetin μmherbacetin μmrhoifolin μmpectolinarin μmherbacetin μmisobavachalcone μmquercetin3βdglucoside μmhelichrysetin μminhibition of sarscovreplicationflavonoids exhibited sarscov3clpro inhibitory activity[ ]inhibition of merscovreplicationflavonoids exhibited merscov3clpro inhibitory activity isobavachalconepsoralidinlineweaver“burk anddixon plotsmyricetinscutellareinsprfretbasedbioassaysisobavachalcone μmpsoralidin μmmyricetin μmscutellarein μminhibition of sarscovreplicationisobavachalcone and psoralidinexhibited sarscov papainlikeprotease inhibitory activitymyricetin and scutellareininhibition of sarscovpotently inhibit the sarscovreplicationhelicase protein in vitro byaï¬ecting the atpase activity the high number of deaths and clinical complicationsobserved in sars and merscov epidemics motivatedthe search for eï¬ective therapeutic agents this was necessitated when many of the tested conventional drugs andtherapies proved ineï¬ective in treating sarsantiviralcov infections for exampletreatment ofthe initial 0coxidative medicine and cellular longevitycell culture coronavirusnaturalantioxidants suppress bcl2 reductionsuppress bax redistributionsuppress cytochorome c releasesuppress caspase3 activationreduce formation of a visiblecytopathic effect without dnafragmentationreduce nucleocapsid n protein expressioninhibit 3clike proteaseinhibit 3clike proteaseinhibit papainlike proteaseinhibit helicase protein by affectedatpase activitytgevpedvmers“covsars“covfigure inhibitory actions of natural antioxidants against coronavirussarscov with antiviral agents such as ribavirin and corticosteroids did not achieve very satisfactory resultsmainly because corticosteroids exertimmunosuppressoreï¬ects on the humoral and cellular immune systems[ ] other drugs such as pentoxifylline were considered for the treatment of sars due to its interestingtherapeutic propertiesanti‚ammatoryantiviral immunomodulatory and bronchodilatory eï¬ectshowever it too was not successful in the clinical treatment of sarscov infection includethatinhibition ofmany antioxidant compounds show antiviral activityagainst sarscov the antiviral activity has been mainlyattributed to thethe 3clike protease3clpro of sarscov a vital enzyme for sarscov replication as an example multiples studies havereported that quercetin and quercetinderived compoundssuch as quercetin 3βgalactoside display potent 3clproinhibitory e5ï¬ect and consequent reduction of sarscovreplication other antioxidants such as epigallocatechin gallate gallocatechin gallate amentoflavone apigeninluteolin herbacetin rhoifolin and pectolinarin are alsofound to efficiently block the enzymatic activity of sarscov 3clpro [ ]moreover some natural antioxidants exhibit promisingantiviral activity against sarscov infection by interferingwith diï¬erent targets involved in sarscov replication inparticular the sarscov papainlike protease plpro andsarscov helicase protein kim reported that isobavachalcone and psoralidin inhibit plpro in a dosedependentmanner with ic50 ranging between and μm previously yu reported that myricetin and scutellareinpotently inhibit the sarscov helicase protein in vitro byaï¬ecting the atpase activity merscov is another zoonotic coronavirus transmitted between animals and human beings that causes severemorbidity and mortality no antiviral medicines with satisfactory efficacy for the treatment of merscovinfectedpatients have been identified to date similar to sarscov natural antioxidant libraries have been probed forpotentialinhibitory compounds against merscov 3clike protease jo showed that herbacetin isobavachalcone quercetin 3βdglucoside and helichrysetinfourcompounds with recognized antioxidant activity can blockthe enzymatic activity of merscov 3clpro using atryptophanbased fluorescence method furthermore theexperimental and computational studies show that flavonoland chalcone are favourite scaï¬olds to bind with the catalytic site of merscov 3clpro in a study performed by lin the antiviral activitiesof resveratrol were investigated in mersinfected vero e6cells the authors reported a significantinhibition ofmerscov infection and prolonged host cell survival aftervirus infection which they speculate was promoted by resveratrol in addition they also found that the expression ofthe nucleocapsid n protein which is essential for merscov replicationis decreased after resveratrol treatment it is important to mention that in vitro models of coronavirus infection also show antiviral activity of flavonoidsextracted from flowering cherry cultivars and black tea[ ] finally antioxidants such as resveratrol alsoare able to block infection produced by herpesvirus the discovery of antiviral compounds from a bioactivecompound against other viruses is an interesting strategy forobtaining new antiviral drugs table shows the mainactions of the natural antioxidants against the coronavirusand figure summarizes these activities 0c conclusionsin conclusion this review shows that antioxidant compounds prominently flavonoids exhibit antiviral action inmodels of coronavirus infections in general the antiviralactivity might be attributed at least in part to the inhibitoryeï¬ect on the enzymatic activity of targets involved in coronavirus replication including sarscov 3clpro sarscovpapainlike protease plpro sarscov helicase proteinand merscov 3clpro in addition some studies provideevidence that the reduction of ros accumulation retardsthe coronavirusactivated apoptotic signaling thereforethe mechanisms of oxidative stress could be the key elementto be studied in coronavirus infectionsincluding thoserelated to ‚ammatory processes arising from the action ofthis virus obviously further investigations are needed toelucidate other pharmacological mechanisms by which natural antioxidants play an antiviral eï¬ect despite the findingsreported in this reviewthey cannot be generalized tocovid19 however the data provided support to the investigation of natural antioxidants as a potential therapeuticapproach in the treatment for covid19 and its severe clinical complications either as an alternative or as an adjuvantto conventional therapies and contribute to the search fornew prototypes in the development of drugs against coronavirus infectionsabbreviations229e3clproabtshuman coronavirus229e3clike protease²azinobis3ethylbenzthiazoline6sulphonic acidcoronavirusescatalasediï¬use alveolar damage22diphenyl1picrylhydrazylferric reducing antioxidant powerreduced glutathioneglutathione peroxidasehuman coronaviruseshuman coronavirushku1lower respiratory tract infectionsmalondialdehydecovscovid19 coronavirus disease catdaddpphfrapgshgpxhcovshku1lrtismdamerscov middle east respiratory syndromecoronavirusnfκbnuclear factor kappa bhuman coronavirusnl63nl63human coronavirusoc43oc43oxygen radical absorbance capacityoracprotein carbonylpcoporcine epidemic diarrhoea coronaviruspedvplpropapainlike proteasereactive nitrogen speciesrnsreactive oxygenated speciesrossarssevere acute respiratory syndromesarscov severe acute respirator
Colon_Cancer
" pharmacology and toxicology laboratory csirinstitute of himalayan bioresource technology preproof 0c preproofinfection f0b7 systemic oxidative stress and inflammation are significant outcomes of sarscov2 highlights f0b7 activated gsk3 following sarscov2 infection provoke the oxidative stress and inflammation in the host f0b7 gsk3 phosphorylates nucleocapsid protein of sarscov2 and helps in disease progression f0b7 inhibition of gsk3 can be a suitable target in curbing of covid19 pandemic 0cwith the host defence mechanism by the help of gsk3 protein the virally infected cells show the coronavirus disease covid19 outbreak caused by severe acute respiratory syndrome coronavirus sarscov2 had turned out to be highly pathogenic and transmittable researchers throughout the globe are still struggling to understand this strain's aggressiveness in search of putative therapies for its control crosstalk between oxidative stress and systemic inflammation seems to support the progression of the infection glycogen synthase kinase3 gsk3 is a conserved serinethreonine kinase that mainly participates in cell proliferation development stress and inflammation in humans nucleocapsid protein of sarscov2 is an important structural protein responsible for viral replication and interferes activated gsk3 protein that degrades the nuclear factor erythroid 2related factor nrf2 protein resulting in excessive oxidative stress activated gsk3 also modulates crebdna activity phosphorylates nfκb and degrades catenin thus provokes systemic inflammation preproofinteraction between these two pathophysiological events oxidative stress and inflammation enhance mucous secretion coagulation cascade and hypoxia which ultimately leads to multiple ans failure resulting in the death of the infected patient the present review aims to highlight the pathogenic role of gsk3 in viral replication initiation of oxidative stress and inflammation during sarscov2 infection the review also summarizes the potential gsk3 pathway modulators as putative therapeutic interventions in combating the covid19 keywords covid19 gsk3 nfκb nucleocapsid protein oxidative stress sarscovpandemic list of abbreviations ace2 angiotensinconverting enzyme ad alzheimer™s disease adp adenosine diphosphate aiibb3 glycoprotein iibiiia ards acute respiratory distress syndrome 0care antioxidant response elements asc apoptosisassociated specklike protein containing a card atp adenosine triphosphate balf bronchoalveolar lavage bzip basic leucine zipper cats “ catalase cbp creb binding protein covid19 coronavirus disease creb camp response elementbinding protein cul3 cullin gpx glutathione peroxidase gsh intracellular glutathione gsk3 glycogen synthase kinase3 damp death associated molecular pattern gcsf granulocyte colony stimulating factor hcv hepatitis c virus hdac3 histone deacetylase ho1 heme oxygenase1 ifnÎ interferongamma preproofnfκb nuclear factorκb nlrp3 nodlike receptors protein mcp1 monocyte chemoattractant peptide mip1α macrophage inflammatory protein 1α myd88 myeloid differentiation primary response nadph nicotinamide adenine dinucleotide phosphate hydrogen ikk ikb kinase il6 interleukin iraks interleukin il 1rassociated kinase iκb inhibitor of kappa b keap1 kelchlike ech associated protein licl lithium chloride nlrp3 nucleotidebinding domain nodlike receptor protein nox nadph oxidase nprotein nucleocapsid protein nrf2 nuclear factor erythroid 2related factor 0cntd nterminal domain o superoxide anion o2 oxygen molecule oxpls oxidized phospholipids pamp pathogen associated molecular pattern par proteaseactivated receptors pd parkinson™s disease pedv porcine epidemic diarrhea virus ros reactive oxygen species sarscov2 severe acute respiratory syndrome coronavirus tak1 transforming growth factor tgfactivated kinase tf tissue factor tirap tirdomaincontaining adaptor protein sgmrna sub genomic messenger rna sods superoxide dismutase ppr pattern recognition receptor psgl pselectin glycoprotein ligand1 rigi retinoic acidinducible gene i preproofvwf von willebrand factor xo xanthine oxidase xor xanthine oxidoreductase tlr3 toll like receptor3 tnf tumor necrosis factor tnfr tumor necrosis factor receptor tnfα tumour necrosis factoralpha traf6 tumour necrosis factor receptor associated factor trs transcription regulating sequence introduction in late december wuhan china got attention worldwide after getting several patients diagnosed with pneumonia following a viral infection on 11th february the pathogenic strain of the virus was taxonomically designated as severe respiratory syndrome coronavirus sarscov2 by the international committee on taxonomy of viruses ictv the 0cassociated diseased condition was termed covid19 by the world health anization who the who announced sarscov2 virus infection a pandemic as it infected nearly million persons and engulfed more than worldwide sarscov2 is a member of coronaviruses consisting of kb singlestranded positivesense rna as genetic material it shows genetic similarity between another human coronavirus ie sarscov while similarity with bat coronavirus ratg1 and shares a high similarity index with pangolin coronavirus respiratory droplets are the primary source of viral transmission either through nasopharyngeal or oral route dry cough and high fever are the sarscov virusassociated respiratory disease replication within the host cell in disease progression the present review provides an inthe infected cells however in the case of sarscov2 infection aggressive inflammation significant symptoms observed in patients within days following viral infection the disease pathophysiology of covid19 also shows a close resemblance with previous reported and oxidative stress help in viral replication and damage the airway epithelium cell that results in acute respiratory distress syndrome ards which makes the condition worst glycogen synthase kinase3 gsk3 is a serinethreonine evolutionary conserved central molecule that the majority of respiratory viral infections are associated with the recruitment of immune cells the release of proinflammatory cytokines oxidative stress and finally phagocytosis of mainly participates in cell proliferation migration development apoptosis and immune regulation acquired and innate activation of gsk3 is associated with suppression of host immunity and inhibition of antioxidant response it is also supporting viral genome preproofdepth knowledge of oxidative stress inflammation and viral replication related to gsk3 during sarscov2 infection further the review highlights the gsk3 pathway modulators' gsk3 is a versatile serinethreonine kinase that regulates glycogen metabolism it consists of two isoforms gsk3α and gsk3 encoded by two separate genes both the isoforms share sequence similarity between kinase domains despite they never compensate for each other's' loss of function gsk3 has two prime functional domains a substratebinding domain which acquires substrates to gsk3 while the other kinase domain is responsible for phosphorylation of the substrate the nterminal region of gsk3 contains atp binding domain whereas the cterminal region consists of a large conserved activation loop responsible for the enzyme's full activation activation of gsk3 depends on the siteputative role as therapeutic interventions in combating the covid19 pandemic gsk3 structure 0cspecific phosphorylation that is controlled by various kinases gsk3 prefers prephosphorylate substrate by recognizing consequence sequences stxxxphosphost on substrate gsk3 is also involved in wntcatenin and sonic hedgehog cell signalling pathways mediating in cell proliferation differentiation maturation and cell adhesion transcription factors cjun creb stat3 cebpα nfat myc nfκb and p53 are the major substrate of gsk3 that can manipulate the expression of several other genes impaired activity of gsk3 has recognized in several clinical conditions such as metabolic disorders cancers alzheimer's disease ad parkinson's disease pd bipolar disorders and various other neurodegenerative diseases sarscov2 infection and inflammation covid19 patients' systemic cytokine profile shows a close resemblance with cytokine release syndrome characterized by macrophage activation an elevated level of cytokines like tumour necrosis factoralpha tnfα interleukin6 il6 and interferongamma ifnÎ further elevated levels of these cytokines trigger ards characterized by a low level of oxygen in the severity of symptoms and death in sarscov2 infected patients depends on the viral infection and is greatly affected by the aggressive behaviour of the host immune system blood and difficulty in breathing leading to the death of the infected patients previous data on sarscov demonstrated that the virus predominately affects the endothelium cells of preproofin counterdefence the virus encodes numerous immunesuppressive proteins that help employs the same host receptor angiotensinconverting enzyme ace2 for infection like sarscov indicating that both the viruses target the same set of cells for infection the as an antagonist of interferon signalling interruptions in interferon signalling happened at various stages preventing the recognition of viral rna through pattern recognition receptor expression of the ace2 receptor is reduced in the lungs following sarscov infection disrupting the reninangiotensin system that affects fluidelectrolyte balance blood pressure it to evade from host immune response and helps in replication similarly to counter such problem sarscov2 evolves with numerous structural and nonstructural proteins that act the airway alveoli vascular system and macrophages in the pulmonary an sarscov2 increases the vascular permeability and inflammation in the airway ppr inhibiting the synthesis of type i interferon protein via interrupting the tolllike receptor1 tlr1 and retinoic acidinducible gene i rigi signalling disturbing stat signalling and initiating the host mrna degradation and interrupting host translation machinery fig1 0cat the time of replication cytopathic viruses including sarscov2 show a massive death and injury of the infected epithelial and endothelial cells triggering the excessive release of cytokines and chemokines in addition to this inflammationinduced cell deathpyroptosis also observed in sarscov2 patients that further provoke the systemic inflammatory response pyroptosis signalling proceeds via nodlike receptors protein nlrp3 present on the cell membrane activate caspase1 through asc apoptosisassociated specklike protein containing a caspase recruitment domain adaptor protein activated caspase1 further triggers the synthesis of proinflammatory cytokines such as il1 and il6 fig1 these cytokines further attract the other immune cells mostly tlymphocytes and monocytes at the site of infection bronchoalveolar lavage balf fluid from the sever lymphocyte and immune cells' requirement at the site of infection in most of the patients these recruited cells clear the infection recedes the inflammatory response and leads to recovery however some patients show cytokine storms because of an imbalance in the population of monocytederived fcn1 macrophage in addition to these responses sever cases of sarscov2 infection also disclose a significant expansion in the population of proinflammatory monocytes cd14 and cd16 in the peripheral blood as compared to mild covid19 patients showed ccl2 and ccl7 chemokines which require the recruitment of ccr2 monocytes further balf analysis also revealed a highly inflammatory around of sarscov2 infected patients show lymphopeniainfiltration of preproofsevere hospitalized covid19 patients' blood plasma exhibits a higher level of alleviation in the t cell population which is more noticeable in severe cases the level of helper t cell cd4 cytotoxic t cell cd8 and regulatory t cell were below the average level in severe cases of covid19 as compared to mild cases cd8 t cells directly attack and kill the virusinfected cells while cd4 participates in the production of cytokines to recruit other immune cells at the same time regulatory t cell maintains the normal immune homeostasis along with inhibition of proliferation the proinflammatory activity of maximum immune cascade that further inflames the lungs sarscov2 infected patients also show cases lymphocytes natural killer cells and bcells fig1 granulocyte colonystimulating factor gcsf il2 il6 il10 monocyte chemoattractant peptide mcp1 macrophage inflammatory protein 1α mip1α and tnfα the blood plasma of the infected patients shows a significantly higher level of il6 in severe cases compared to mild or nonsevere cases which further contributes to macrophage activation syndrome pulmonary infiltrationbased assessment in ards patients also revealed that a 0cmore significant portion of lung injury is associated with a higher level of il6 in peripheral blood all of this evidences suggest that sarscov2 infection is responsible for dysregulation of the host immune system with the abnormal synthesis of cytokines chemokines and a decrease in the level of lymphocytes that ultimately leads to cytokine storm responsible of multian failure role of nuclear factorκb in disease progression nuclear factorκb nfκb is the leading player that responds immediately following the a pathogenic stimulus provoked by a bacteria or a virus invasion exposure of mitogen proinflammatory cytokines growth factors and stress activates ikb kinase ikk which relb and crel are grouped in firstclass characterized by the presence of transactivation domain while nfkb1 p50 and nfkb2 p52 belongs to the second group that is devoid of transcriptionalmodulation activity so both the classes of proteins need to be heterodimerized with each other to perform their functions under normal physiological conditions rela and p50 the heterodimer's predominant form is inactivated in the cytoplasm by ikb protein pathogen's invasion by promoting inflammation controlling cell proliferation and survival nfκb is a heterodimeric transcription factor that belongs to the rel protein family there are 05rel proteins present in mammalian cells that further divided into two classes rela p65 preproofmembranelike tolllike receptor tlr pathogen associated molecular pattern pamp and death associated molecular pattern damp are inflammatory stimulating molecules suggested that the nucleocapsid protein of sarscov directly interacts with nfκb translocate it to the nucleus and finally upregulates il6 gene expression ample of shreds of evidence is there that shows sarscov directly or indirectly activates nfκb protein excessive cytokine release especially il6 plays a crucial role in sarscov2 infection and further progression of pathogenic conditions nfκb is a transcription factor that controls the expression of proinflammatory genes responsible for the cytokine storm a study following infection nfκb also activated by receptors present on the cell surface further phosphorylates and degrades ikb protein via ubiquitination process released by virusinfected cells which act as ligands for tlr subsequently activating nfκb protein via myd88dependent pathway oxidative stress is another important factor responsible for cytokine storm generation via crosstalk between nuclear factor erythroid related factor nrf2 and nfκb pathway nfκb suggested as a negative regulator of nrf2 driven genes either by recruiting histone deacetylase hdac3 which promote local histone hypoacetylation or deprive the cbp creb binding protein fig1 0c sarscov2 infection and oxidative stress oxygen is a crucial molecule in the aerobic system to maintain normal life processes under normal cellular conditions the oxygen molecule utilized to generate chemical energy in the form of atp in a very tight and controlled manner the oxygen molecule combustion generates a small number of reactive oxygen species ros which utilized for usual cell signalling cascades ros are oxygen molecules with an unpaired electron that behaves as free radicals and reactive metabolites several ros forms were discovered so far such as peroxidase oxygenfree radicals nitrogen oxide and singlet oxygen molecules generally ros associated cellular damage is processed via sophisticated antioxidant machinery involving both enzymatic catalase cats superoxide dismutase sods and glutathione peroxidase gpx and nonenzymatic glutathione and nicotinamide adenine dinucleotide phosphate mitochondrial dna get degraded under the influence of oxidative stress subsequently hydrogen [nadph] mechanisms in normal physiological conditions the antioxidant systems can work simultaneously to combat the exceeded levels of ros however in a pathological state ros overwhelmed the antioxidant mechanism and generated œoxidative stress in cells all the crucial cellular components such as proteins lipids nuclear and the available literature of clinical and preclinical experiments proposed that oxidative preproofensures the clearance of the virus but due to imbalanced host immune system they also start to release excessive cytokines that further aggravate to cytokine storm the recruited phagocytic cell participates in ros generation along with inflammatory response nicotinamide adenine dinucleotide phosphate oxidases nadph oxidase and xanthine cov2 infection activates the host airway epithelium and alveolar macrophage further releasing cytokines to attract another immune cell from the blood neutrophils and monocyte that further differentiate into macrophage at the site of injury recruitment of these cells burst is another prompting factor for mortality following sarscov infection sarstriggering the process of cell death oxidase xo are the two wellknown enzymes responsible for oxidative stress in respiratory viral infections nadph oxidase nox is an evolutionary conserved membranebounded enzyme complex that catalyzes the molecular oxygen into superoxide human™s nadph oxidase family consists of members nox15 duox1 and duox2 its cterminal region comprises nadph binding site flavin adenine dinucleotide binding domain while the nterminal region consists of transmembrane α helical domains with four conserved hemebinding sites nox2 is predominantly expressed in the recruited 0cphagocytes neutrophils and macrophages at the viral infection site and contributes to oxidative stress a study reported that alveolar macrophage depended oxidative stress is responsible for acute lung injury progression following h5n1 viral infection in mice mostly via oxidized phospholipid and superoxide however the same pathological events reduced following the suppression of p47phox a regulatory subunit of nox2 in a study influenza a virusinfected nox2y mice showed reduced oxidative stress improved alveoli epithelium condition less production of superoxide and reduced airway inflammation compared to wild type mice fig inflammation xor is converted into xo by oxidation of cysteine amino acid or calciumin superoxide synthesis via nox2 enzyme complex xanthine oxidase xo is another dependent proteolysis xo shows more affinity toward molecular oxygen resulting in the transfer of a univalent and divalent electron to oxygen that further generates superoxide and ros generating enzyme that participates in oxidative stress following respiratory viral infection in the mammalian system this enzyme is existing in interchangeable form between hydrogen peroxide respectively fig2 in vitro rhino virus™s infection in primary bronchial and a549 respiratory epithelial cell lines decreased the intracellular glutathione xo to xanthine oxidoreductase xor xor is predominantly distributed in healthy tissues and reduces nad to nadh by utilizing electron form substrate while during similarly ex vivo influenza a virusinfected alveolar macrophage exhibited an increase preproofdecreased superoxide generation thus revealed that xo also participates in oxidative stress during infection in vivo analysis also revealed that xo is the main contributor to and serum analysis however allopurinol and chemical modified superoxide dismutase decreased the oxidative stress and mortality rate this evidences revealed that xo also superoxide synthesis during a respiratory viral infection mouse infected with influenza viral showed a higher mortality rate which found to be associated with xo and superoxide in balf gsh level leading to oxidative stress via enhanced superoxide production serine protease inhibitor or xo inhibitor oxypurinol treatment enhanced the intracellular levels of gsh and participates in the viral associated disease progression via oxidative stress a part of these activated phagocyte releases prooxidant mediators such as tnf and il1 which further enhances the oxidative stress in host cells during viral infection tnf binds with the complex ii of the mitochondrial respiratory chain hampering oxidative phosphorylation via restricting electrons transport as a result the electron transport chain becomes leakier and lastly it enhances superoxide production tnf also helps in detachment of nfκb protein from ikb complex resulting in suppression of antioxidant gene expression via binding to their 0c1keap1 binding domain keap1 is a cystine rich and cytoplasmic protein whose npromoter region following translocation from the cytoplasm to the nucleus fig during stress condition neutrophils also release lactoferrin along with lysosomal protein under the influence of il1 which further binds to iron and start to accumulate in the reticuloendothelial system when an ironbinding threshold reached superoxide ions combine with free iron to generate hydroxyl radicals via fenton reaction and enhances oxidative stress nrf2 a key regulator of antioxidant genes nrf2 is the main transcription factor that plays an important role to overcome oxidative stress it is a basic leucine zipper bzip protein that belongs to the cap ˜n™ collar family of transcription factors nrf2 consist of highly conserved functional domain termed as neh nrf2ech homologies “ neh1 is a leucine zipper domain through which nrf2 interact with other transcription factors whereas neh2 is the kelchlike ech associated protein however during stress conditions nrf2 detached from the keap1 protein translocate to the terminal domain binds with cul3dependent e3 ubiquitin ligase complex while cterminal domain binds with nrf2 protein under normal physiological conditions keap1 protein nucleus heterodimerize with small musculoaponeurotic fibrosarcoma mafs proteins and finally initiate or supress the transcription of genes that consists of electrophile response elements ere or antioxidant response elements are in their promoters nrf2 regulates preproofbecause of its highly vascular nature and indirect contact with environmental oxidant which had already proven in numerous of respiratory disease it was found that lungspecific nrf2 conditional knockout rodents showed pulmonary protective behaviour in respiratory disorders more than genes expression belonging to oxidative stress inflammation autophagy metabolism and excretion the pulmonary system is more exposed to oxidative stress ubiquitinates the nrf2 resulting in its proteasomal degradation infection systemic oxidative stress and inflammation linked thrombus formation in sarscovabnormal coagulation a higher level of ddimers and low platelet count are the signs of poor prognosis and significant reasons for multiple an failure and death in severe cases of covid19 microthrombus had reported in the lungs the heart the kidneys and the brain of covid19 patients cytokine storm induces aberrant coagulation by expressing the tissue factor tf pathway tf is a member of cytokine receptor 0csuperfamily and type i integral membrane glycoprotein which is highly abundant in the vasculature subendothelium especially in the brain lungs gut skin as well as in the monocytes in response to proinflammatory cytokines especially il6 the expression of tf is upregulated in the monocytes and the perivascular cells resulting in tf exposure to circulation the exposed portion of tf forms a complex with circulating factor vii thus enhance its catalytic activity that further activates downstream circulating factors such as ix and x activated factor x participates in the transformation of prothrombin into thrombin that finally leads to the formation of blood clots by conversion of fibrinogen into fibrin fig main consequences of the cytokine storm that also provokes thrombin production via proteasediphosphate adp thromboxane a2 translocate cell adhesion molecule pselectin and cd40 ligand on the surface of platelet along with activation of the integrin aiibb3 receptor the released thromboxane a2 and adp trigger activation of neighbouring platelets via activated receptors par signalling pathway par is a unique gprotein coupled cell surface receptor that carries its ligand and remains inactive until unmasked by proteolytic cleavage by the tffactorviia complex thrombin mediated par activated platelets undergo a morphological transformation release platelet activators such as serotonin adenosine thromboxane receptor and p2y12 respectively activated aiibb3 on platelets' surface binds with von willebrand factor vwf and fibrinogen that contributes to platelet aggregation platelet activation different proinflammatory events and fibrin clot formation are the preproofalong with cytokine storm oxidized phospholipids oxpls also participate in the recognition receptors in an experimental model of acute lung injury oxpls triggered cytokine storm release via tlr4triftraf6nfκb pathway il6 further promoted tf platelets during viral infection adherent leukocyteplatelets interaction provides positive feedback to amplify the overall inflammatory response and procoagulation events these activated endothelium cells following par signalling also exposes cell adhesion molecules eselectin pselectin icam1 and vcam1 and expresses monocyte chemo coagulation cascade via the tlr4 receptors present on the monocytes and endothelium cells attractant proteini that facilitates recruitment adhesion and migration of leukocytes and events are prothrombotic which further contributes to blood clot formation fig oxpls concerned as pams patterns that are recognized by numerous conserved patternexpression on monocytes and activated the endothelium cell to express monocyte adherent protein for their requirement which finally participated in inflammatory events thrombotic complications can be reduced in preexisting metabolic and cardiovascular disorders in covid19 patients by interfering with the oxpls activated monocyte or 0cwhich consists of domains including the nterminal domain ntddomain cterminal domain ctddomain and linker region lkrdomain nterminal domain enriched with endothelial cell additionally during inflammation the natural anticoagulant pathways such as tf pathway inhibitors or antithrombin are nearly diminished subsequently facilitating coagulation cascade gsk3 and sarscov2 infection the virus has to undergo many complex processes that are tightly regulated to infect a host cell it begins with viral genomic rna entry into the host cytosol transcription and finally budding off as viral progeny these viral progenies are similar to their parent in morphology and function that consists of structural proteins spike s protein envelop e protein matrix m protein and nucleocapsid n protein the n protein of severe acute respiratory syndrome coronavirus is the most abundant protein existing in an infected host cell among all be responsible for nuclear localization signal the cterminal domain of the n protein is also responsible for protein dimerization both the domains of n protein ie domain and protein sequencing also revealed that n protein is highly conserved among the species preproofinterestingly to perform such activity nprotein should recognize the viral genomic nucleocapsid protection of viral genome timely replication and proper transmission is the capsid's primary function nprotein also inhibits host cell proliferation and cytokines by rna associate with it and finally oligomerize by selfassociation to form capsid or terminal domain mapped between and amino acids is enriched with lysin thought to arginine motif responsible for the multimerization of the nprotein and predicted as a hot spot region for phosphorylation in brief nprotein divided into three main domains that play diverse functions during different stages of the virus life cycle nprotein is a type of capsid protein whose primary function is to pack the virus's genomic rna into the protective positive charge amino acids which is responsible for binding with viral rna whereas cother proteins covering domain are linked to each other through linker region domain that consists of serineinteracting with elongation factors 1α resulting in a halt of translation mechanism moreover the nprotein of sarscov also hijacks the host innate immune system for the progress of new viral progeny and associated disease development posttranslational phosphorylation of the virus nprotein is essential for their activity which results in an increased affinity of nprotein toward virus rna rather than nonviral rna gsk3 found 0cto be an essential kinase responsible for the phosphorylation of nprotein on the serine residue in linkerregion fig sarscov infected veroe6 cells showed an reduction in viral titer and cytopathic effects following the treatment of gsk3 inhibitor kenpaullone and lithium chloride thus suggested phosphorylation by this kinase be strongly linked with the viral replication several subgenomic mrnas synthesized due to discontinued transcription mechanisms during the coronavirus replication which encodes major structural proteins transcription regulating sequence trs responsible for the discontinuous transcription process exists in front of each gene body trs and after the leader sequence leader trs templateswitching events happen via base pairing between the body trs and leader trs to synthesize the discontinuous minusstranded rnas discontinuous nested plusstrand this discontinuous transcription mechanism tightly controlled for the successful compilation participate in discontinues to a continuous process of virus replication moreover of the virus life cycle among all the structural proteins nprotein tightly regulates the discontinued transcription mechanism as the synthesis of subgenomic mrna is reduced subgenomic mrna transcribed from the previously generated minusstranded rnas phosphorylation of nprotein at the serinearginine motif also inhibits the tra
Colon_Cancer
cancer is a complex and heterogeneous disease with many possible genetic and environmentalcauses the same treatment for patients of the same cancer type often results in different outcomes in terms ofefficacy and side effects of the treatment thus the molecular characterization of individual cancer patients isincreasingly important to find an effective treatment recently a few methods have been developed to constructcancer samplespecific gene networks based on the difference in the mrna expression levels between the cancersample and reference samplesmethods we constructed a patientspecific network with multiomics data based on the difference between areference network and a perturbed reference network by the patient a network specific to a group of patients wasobtained using the average change in correlation coefficients and node degree of patientspecific networks of thegroupresultsnetworks with multiomics data the main differences of our method from previous ones are as follows networksare constructed with multiomics mrna expression copy number variation dna methylation and micrornaexpression data rather than with mrna expression data alone networks are constructed with bothnormal samples and cancer samples of the specified type to extract cancerspecific gene correlations and bothpatient individualspecific networks and patient groupspecific networks can be constructed the results of evaluatingour method with several types of cancer show that it constructs more informative and accurate gene networks thanprevious methodss the results of evaluating our method with extensive data of seven cancer types show that thedifference of gene correlations between the reference samples and a patient sample is a more predictive feature thanmrna expression levels and that gene networks constructed with multiomics data show a better performance thanthose with single omics data in predicting cancer for most cancer types our approach will be useful for finding genesand gene pairs to tailor treatments to individual characteristicskeywordsindividualspecific gene network groupspecific gene network cancer multiomics datacorrespondence khaninhaackr1department of computer engineering inha university incheon southkoreafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the ™s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0clee bmc medical genomics 13suppl page of for the past years we have witnessed the rapid development of targeted cancer therapy targeted therapies forcancer work by targeting specific genes proteins or tissues that contribute to cancer growth and survival manytargeted therapies are effective only for patients with specific genetic alterations known as driver mutations thathelp cancer cells form and grow [ ] thus identifying genetic mutations specific to individual patients is ofutmost importance to determine targeted therapies thatcan effectively cure cancer patients while minimizing sideeffects motivated by a massive amount of data generated byhighthroughput technologies several cancer studies usedgene networks to explore gene expression characteristics [“] however constructing a patientspecific genenetwork with a single sample obtained from a patient isdifficult because a gene network requires many samples tocompute genegene relationsrecently a few methods have been proposed to construct cancer samplespecific gene networks based onthe difference in the mrna expression levels betweenthe cancer sample and reference samples for exampleliu proposed a method to construct a samplespecific network by computing the difference between areference network from multiple reference samples anda network perturbed by a new sample however a slightchange to the reference samples can result in a significantly different samplespecific network for the samesample due to the small number of reference samples furthermore their samplespecific networks cannot reflectposttranslational modification and epigenetics becausethe networks are built using mrna expression data onlythis paper presents a new method for constructingcancer patientspecific and groupspecific gene networkswith multiomics data using a samplespecific networkand network propagation method network propagation strategies are widely used in recent cancerrelatedresearch li presented a synergy prediction algorithm using network propagation and predicted the drugsynergy in various cancers zhang introduceda propagation algorithm which learns the mutated subnetworks underlying tumor subtypes using a supervisedapproach and classified tumors to known subtypes onbreast and glioblastoma tumors peng identifiedbladder cancerrelated genes by propagating informationfrom seed genes to candidate genes the primary focusof our method is to construct a gene correlation network specific to cancer with multiomics data thus it isdifferent from a typical gene coexpression network thatrepresents coexpression relations between genes frommrna expression data our gene network is not a generegulatory network because our network does not showregulatory relations between genesthe main differences of our method from previous onesare as follows networks are constructed with multiomics mrna expression copy number variation dnamethylation and microrna expression data rather thanwith mrna expression data alone networks are constructed with both normal samples andcancer samples of the specified type to extract cancerspecific gene correlations and both patient individualspecific networks and patient groupspecific networks canbe constructed as shown later in this paper the resultsof evaluating our method with several types of cancershow that it constructs more informative and accuratetargetspecific networks than previous methodsmethodsat the top level our method consists of the followingsteps data processing constructing individualspecific gene networks and constructing a groupspecific gene networks a highlevel description of themethod is given in fig data collection and preprocessingfrom the broad institute tcga gdac firehose we obtained multiomics data of cancer samples of seventypes breast invasive carcinoma brca colon adenocarcinoma coad head and neck squamous cell carcinomahnsc pankidney cohort kipan liver hepatocellular carcinoma lihc lung adenocarcinoma luad andlung squamous cell carcinoma luscthe multiomics data used in this study includemrna expression mrnaseq copy number variationcnv dna methylation and mature mirna expression mirseq data the mrnaseq data were processedusing quartile normalized rsem and then log2transformed the segmented cnv data were convertedto genelevel data using the ensembl api and thecntools package of bioconductor the methylationdata were filtered to select the probe with the mean signal values for each gene the mirseq data were processedby rpm and log2transformed mrnas and mirnas thatwere not expressed in more than of the total sampleswere excluded in further analysis missing expression values of mrnas and mirnas were replaced by the smallestpositive normalized floatingpoint number realmin ofmatlab the number of samples and genes used in thisstudy are available in additional file individualspecific gene networkin each group of tumor samples and normal samples wefirst computed genegene relations by the pearson correlation coefficient pcc selected highly correlated genepairs ie those with pcc and constructed twosample networks one for each group from the tumorsample network we removed edges common to both 0clee bmc medical genomics 13suppl page of fig overview of constructing an individualspecific network and a groupspecific network with multiomics datatumor and normal sample networks and obtained a template reference network for cancer fig 2a the templatereference network consists of highlycorrelated gene pairsthat are specific to cancerwith n reference samples which may be different fromtumor samples used in the template network we computed pcc for every pair of genes in the template reference network and constructed a reference network forthe reference samples for a patient of interest we constructed a network which is a perturbed network byadding a single sample of the patient to the n reference samples a patientspecific network was obtainedby subtracting the reference network from the perturbednetwork 01pcc pccn1 ˆ’ pccnwe computed the difference in the absolute value ofpcc between the perturbed reference network and reference network by eq we also carried out a ztest ofpccn1ˆ’pccn by eq for a large n we can approximatethe mean μ and standard deviation σ of pccn1 ˆ’pccn as and ˆ’ pcc2nn ˆ’ respectively pccchange pccn1 ˆ’ pccnz ˆ’ score pccchange ˆ’ μpccchangeσ pccchange pccchangeˆ’pcc2nnˆ’the edges of the patientspecific network were classified into four types correlationgained edges fene pairs whose pccs are increased from the referencenetwork to the patientspecific network correlationlost edges for gene pairs whose pccs are decreased fromthe reference network to the patientspecific network correlationreversed edges for gene pairs whose signs ofpccs are changed from positive to negative or negativeto positive and correlationinvariant edges for genepairs with little change in pccs between the reference andpatientspecific networks ie those with zscore fig 2bthe edges were classified in the following way we firstselected gene pairs with zscore as correlationinvariant type and then selected gene pairs which havedifferent signs of pccs between the reference networkand the patientspecific network as correlationreversedtype the remaining gene pairs were classified into eithercorrelationgained or correlationlost type depending onwhether their pccs are increased correlationgained ordecreased correlationlost from the reference network tothe patientspecific network thus zscore ‰¥ in bothcorrelationgained and correlationlist gene pairsgroupspecific gene networka groupspecific gene network is useful when analyzinga large number of patientspecific gene networks afterconstructing patientspecific gene networks we obtained 0clee bmc medical genomics 13suppl page of fig process of constructing a patientspecific gene network a template of the cancer reference network obtained by removing edges common toboth networks b patientspecific gene network and four types of edges in the network edges with a zscore represent correlationinvariantgene pairs and edges with zscore ‰¥ and different signs of pccn and pccn1 represent correlationreversed gene pairs edges with zscore ‰¥ and 01pcc are correlatedgained gene pairs and those with zscore ‰¥ and 01pcc are correlationlost gene pairsa gene network specific to a group of patients based onthe average 01pcc and node degree of the patientspecificnetworks fig if the dominant type for a particular edge is ˜correlationgained™ positive 01pcc in thepatientspecific networks the edge is represented in redin the groupspecific network in contrast if the dominant type for a particular edge is ˜correlationlost™ negative 01pcc in the patientspecific networks the edgeis represented in blue in the groupspecific network inthe groupspecific network only the dominant type isshown for each edge if nondominant types are shownin addition to the dominant type for each edge the network becomes cluttered and unreadable the node sizeof a groupspecific gene network is proportional to theaverage degree of the nodeintegration of multiomics datato integrate multiomics data we first computed intergene correlations by pcc with four different types ofsingle omics data mrna expression cnv dna methylation and mirna expression separately and selectedsignificant intergene correlations only in mrna expression cnv and dna methylation data we select thetop pcc with pvalue in mirna expression data we selected the top pcc with pvalue due to a smaller number of mirnas inthe data the intergene correlations selected in eachsingle omics data are represented in four correlationmatrices mexpr mcnv mmethyl and mmirna andnormalizedusing the proteinprotein interactions ppis of thestring database we constructed separate weightednetworks from each omics data by eq in eq wexprwcnv and wmethyl denote the weighted networks andppiexpr ppicnv and ppimethyl are subnetworks of a ppinetwork consisting of genes present in each omics datasince the ppi network does not contain information onmirna a weighted network for mirna was not constructedcid3wexpr ˆ’ cid2 ˆ’ ppiexprwcnv ˆ’ ˆ’ mcnv × ˆ’ ppicnv wmethyl ˆ’ cid2 ˆ’ ppimethylwe then integrated the multiomics data by linear xmethylregression using eq in eq yi xcnvand xmirnadenote gene i™s expression level cnv levelmethylation level and mirna regulator expression levelrespectively βcnvdenote the regression coefficients of gene i™s expression level on cnv and methylation respectively βmirnais the regression coefficientof gene i™s expression level on its mirna regulator j™sexpression leveland βmethyl ˆ’ mexpr ˆ’ mmethylcid3 × cid2cid3 × cid2cid3iiijiiijyi βcnvii βmethylxcnvixmethyli ncid4j1βmirnaijxmirnaij 01 0clee bmc medical genomics 13suppl page of fig process of constructing a groupspecific gene network from multiple patientspecific gene networks in the groupspecific gene network thenode size and node color are proportional to the average degree and average 01pcc of the node respectively if the dominant type for a particularedge is ˜correlationgained™ positive 01pcc in the patientspecific networks the edge is represented in red in the groupspecific network if thedominant type for a particular edge is ˜correlationlost™ negative 01pcc in the patientspecific networks the edge is represented in blue in thegroupspecific networkfrom the regression coefficients and the weighted networks a weight matrix w was derived and normalizedinto w eqs and the weight matrix w is symmetricso wij wji w11 w22 w33 and w44 represent wexprwcnv wmethyl and mmirna respectively the submatrices w21 and w31 contain regression coefficients βcnvand βmethylfor every gene i respectively w41 representsiβmirnaij the submatrices w32 w42 and w43 are emptyiŽ¡Ž¢Ž¢Ž£w w11 w12 w13 w14w21 w22 w23 w24w31 w32 w33 w34w41 w42 w43 w44Ž¤Ž¥Ž¥Ž¦eq and updated iteratively by eq in this iterative process the influence of the seed is propagated tothe neighbors until a mean squared error of st and stˆ’‰¤ × ˆ’Ž§ŽªŽªŽªŽªŽ¨ŽªŽªŽªŽªŽ©if v is a nonseed nv ‰¥ αif v is a nonseed nv αif v is a seednvnvenvˆ’α × nvnvdv st λ × stˆ’ × w ˆ’ λ × d wheres1 d where nv is the number of neighbors of node v and nv isthe number of seeds in the neighbors the parameter αwhich is a threshold for nv was set to and λ was set to genes with the top st were used in findingcancerrelated genes and in classifying tumor samples andnormal samplesw i j w i jcid11cid12cid12cid13mcid4k1w i k × mcid4k1w k jin network propagation seed genes have greater impactthan nonseed genes on their neighbors thus only thegenes with a high average 01pcc were selected as seedgenes for a groupspecific network and their mirnasregulators extracted from mirtarbase were used asseed mirnas we calculated the cancerrelevance st ofeach gene to reflect the effect of the seed genes and mirnas on neighbors the initial score d was calculated byresultspatientspecific and groupspecific gene networksin this study we constructed patientspecific genenetworks for seven cancer types additional file foreach cancer type we also constructed groupspecific genenetworks as an example fig shows a groupspecificgene network derived from lung squamous cell carcinoma lusc patients 0clee bmc medical genomics 13suppl page of there are three distinct subnetworks in the networkfor the lusc group the subnetwork enclosed in box aof fig contains many hub genes large green nodesthe subnetwork in box b consists of correlationgainededges dark red edges whereas the subnetwork in box ccontains many correlationlost edges dark blue edgescomparison of multiomics data and singleomics datawe performed leaveoneout cross validation loocvto evaluate cancerrelevance score st of a gene and thecontribution of multiomics data to finding cancerrelatedgenes for comparison the cancerrelevance scores werecomputed with multiomics data and single omics dataseparately each seed gene was regarded as a nonseed anda new cancerrelevance score was calculated for the geneseed genes and nonseed genes were considered as positive and negative respectively seed genes included in thetop n genes were considered as true positives and seedgenes not included in the top n genes were consideredas false negatives similarly nonseed genes included infig groupspecific gene network for lung squamous cell carcinoma lusc patients a subnetwork of multiple hub genes large greennodes b subnetwork of correlationgained edges dark red edges c subnetwork with many correlationlost edges dark blue edges 0clee bmc medical genomics 13suppl page of the top n genes and nonseed genes not included in thetop n genes were considered as false positives and truenegatives respectivelywe carried out loocv with different ratios of seedgenes to nonseed genes figure shows the receiver operating characteristic roc curve and the area under thecurve auc of loocv of the cancer relevance of geneson data of breast cancer samples with various seedratios ranging from to enlarged plots of fig are available in additional file for comparative purposes we also computed the cancer relevance of geneswith single omics data as shown in fig multiomicsdata consistently exhibited better performance than singleomics data with any seed ratio between to forlater analysis the seed ratio was set to by default theaverage 01pcc and class label of each gene are available inadditional file indeed the superiority of multiomics data over singleomics data in determining the cancer relevance scoreof genes was observed in all seven types of canceradditional file in seven types of cancer the cancerrelevance score of genes computed with multiomics dataexhibited a good performance auc ˆ¼ thecancer relevance score of genes computed with mrnaexpression data showed the second best performanceauc ˆ¼ in particular the cancer relevancescore computed with mrna expression data showed avery similar performance to that with multiomics inbreast cancer brca the performance of the cancer relevance score computed with cnv auc ˆ¼and dna methylation data auc ˆ¼ alonewas lower than that with mrna expression data auc ˆ¼fig roc curves of the leaveoneout cross validation of the cancer relevance score st of genes with different ratios of seed genes breastcancer samples were used in the leaveoneout cross validation the performance of multiomics data is always better than that of single omics data 0clee bmc medical genomics 13suppl page of evaluation of gene correlations and networksmany networkbased approaches to cancer research havefocused on finding genes that show differential expressions between tumor samples and normal samples genegene correlations ie intergene correlations may bemore helpful than individual genes because intergenecorrelations depend on the expression of neighbor genesin a gene regulatory network to compare the effect ofusing individual genes to that of intergene correlationsie 01pcc we constructed a support vector machinesvm model for classifying cancer samples and normalsamples the svm model was implemented using csvcand rbf kernel and the parameter values of the modelwere determined by the grid search algorithm mrnaexpression levels and 01pccs were used as features ofthe svm models for rigorous validation the test dataused in testing the models were not used in training themadditional file as shown in fig 6a 01pcc showed a better performance than mrna expression levels for six cancertypes except lusc the classification model with 01pccshowed mcc above in six cancer types except hnscwe also examined the effect of different networks on individualspecific networks in the workby liu ppi data with high confidence scoresin the string database were used to construct a network however the ppi data of stringdoes not reflect cancer typespecific characteristicsfigure 6b shows the performance of the classificationmodel with two different networks network from ppi data of string the approachby liu and cancer network ourapproach 01pcc was used as a feature of the classification model except for coad the performance ofthe classification model with the cancer network was better than the model with the string reference network in particular the classification model showed a significant difference for breastcancer brca mcc of vs mcc of detailed results of the classification model are available inadditional file discussionin the analysis of finding cancerrelated genes and genepairs we focused on a subnetwork of genes with a 01pcctable shows the top genes with a high average 01pcc in each groupspecific network of seven cancertypes in breast invasive carcinoma brca fam171a1showed the highest average 01pcc in the groupspecificnetwork fam171a1 is known as a potential biomarkerin triplenegative breast cancer foxc1 is involvedin tumor development and metastasis and associated withpoor prognosis in basallike breast cancer il33 isoverexpressed in various cancers and the serum concentration of il33 is a valuable indicator of poor prognosis inbreast cancer mamdc2 is significantly correlatedwith diseasefree survival of breast cancer patients mterfd1 is closely related to breast cancer recurrencefig results of evaluating features and networks by a validation set a comparison of mrna expressions of genes and 01pcc of genepairs b comparison of the cancer network with the network from ppi data 0clee bmc medical genomics 13suppl page of table top genes with a high average 01pcc in a groupspecific network for seven cancer typesbrcakipanlihccoadhnscfam171a1foxc1il33mamdc2mterfd1hoxa7cttnbp2znf204pjam3frem1gfra2scnn1bddx27rnps1ube2imdficctnnbl1cdk5rap1esf1trmt6cyp2j2barx2znf135ppfia1parlfaddcst6cobloraov1xpo7tfcp2l1kcnq1arl15kctd1oaz2tmem45bhpcal1tmem91sema5begln3slc19a3ecm1cyp2b6fbp1gpaa1f9pahagxt2hsd17b6rnase4luadcldn18adamts8pecam1sftpa1gimap6akr1c1mmeatad2cyp3a5chaf1bluscnsun2cct5fbxo45gpr116slc39a8fxr1inmtveph1crtamwdr53 and hoxa7 plays a critical role in regulating theproliferation of erpositive cancer cells in colon adenocarcinoma coad gfra2 showed thehighest average 01pcc in the groupspecific network itis known to be crucial for enteric neuron survival scnn1b and ddx27 are significantly related to colorectal cancer [ ] no direct relation of rnps1 withcolorectal cancer is known but rnps1 is essential tononsensemediated mrna decay that plays complexfunctions in cancer knockdown of sumo conjugating enzyme ube2i also known ubc9 or e2 inhibitsmaintenance and selfrenewal of colorectal cancer stemcell while overexpression of ube2i increases colorectalcancer cell stemness among the top genes with a high average 01pccin lung adenocarcinoma luad several genes such ascldn18 adamts8 pecam1 and sftpa1 have beenknown to be associated with luad in previous studies[“] no direct relation of nsun2 and slc39a8 withlung squamous cell carcinoma lusc has been known sofar however recent studies [ ] reported that nsun2is correlated with survival in other types of squamous cellcarcinomas gao also showed that the epigeneticsilencing of slc39a8 expression by dna methylation isinvolved in the acquisition of resistance against cadmiumin lung cells and the relation between cadmium andlung cancer has received much attention many othergenes in table found in the groupspecific networksfor head and neck squamous cell carcinoma hnscpankidney cohort kipan and liver hepatocellular carcinoma lihc are also directly or indirectly related tocancerin addition to individual genes we identified genepairs of the same type ie either correlationgained orcorrelationlost in most patientspecific networks of thesame type table shows the most frequent gene pairs in breast cancer samples the most frequent gene pairsin other types of cancer are listed in additional file it isinteresting to note that all the gene pairs shown in table include at least one gene in the gene pair mamdc2hoxa7 and that they are correlationgained edges in thegroupspecific network for breast cancer figure showsa subnetwork containing mamdc2 and hoxa7 in thegroupspecific network of breast cancer the subnetworkwas obtained by selecting the edges for which the proportion of the same edge type ie correlationgained or lostis above in the total individualspecific networks ofbreast cancer patients it is interesting to note that all thegene pairs in table are included in the subnetworkto date the actual role of the mamdc2 gene in cancer is not clear but meng reported mamdc2as one of three genes mamdc2 tshz2 and cldn11that are significantly correlated with diseasefree survival of breast cancer patients mamdc2 is known as atarget of mir196a in head and neck squamous cell carcinoma as a member of the family of homeoboxgenes hoxa7 is associated with cell proliferation nerveinvasion distant metastasis and degree of tumor differentiation in several cancers [ “] hoxa7 is regulatedtable the most frequent gene pairs in breast cancersamples all the gene pairs are of a correlationgained type thegenes of table are shown in bold the proportion represents theratio of the gene pairs of the same type ie correlationgained orlost to the total number of patientspecific networksgene pairgene pairsproportion of the gene pairsin total cancer samplesmamdc2hoxa7mamdc2ccl14mamdc2znf204pmamdc2klmamdc2svep1mamdc2coro2bhoxa7meox2hoxa7hoxa9mamdc2sobpmamdc2hoxa9 0clee bmc medical genomics 13suppl page of fig a subnetwork of the groupspecific network of brca which contains mamdc2 and hoxa7 genes in the most frequent gene pairs shown intable are enclosed by a circleby several mirnas including mir196 [“] thusboth mamdc2 and hoxa7 are related with mir196but a clear relation among them is to be uncoveredso far most approaches to constructing individualspecific gene networks have been constructed based onthe differential expressions between a small number ofreference samples and a sample of interest howeversuch networks cannot reflect posttranslational modification and epigenetics and are not reliable because a slightchange to the reference samples can result in a significantly different samplespecific network for the samesamplein this paper we presented a new approach to constructing cancer patientspecific and groupspecific networks with multiomics data the main differences ofour method from previous ones are as follows gene networks are constructed with multiomics mrnaexpression copy number variation dna methylationand microrna expression data rather than with mrnaexpression data alone networks can beconstructed with cancer samples of the specified type and both patient individualspecific networks and patientgroupspecific networks can be constructed the resultsof testing our method with several cancer types showedthat it constructs more informative and accurate genenetworks than existing methodsevaluation of our method with extensive data of sevencancer types showed that changes in gene correlations 01pcc between the reference samples and a patient sample is a more predictive feature than mrna expressionlevels and that gene networks constructed with multiomics data are more powerful than those with singleomics data in predicting cancer for most cancer typesmore work is required to validate the genes and genepairs identified in the cancer patientspecific and groupspecific networks however the method for constructingnetworks specific to individual patients or patient groupswith multiomics data should be useful aids in determining effective treatments to individual characteristicssupplementary informationsupplementary information accompanies this paper athttpsdoi101186s12920020007367additional file number of samples and genes in types of canceradditional file roc curve and auc of the cancerrelevance score ofbrca by various seed ratiosadditional file average 01pcc and class label of each gene in typesof canceradditional file roc curve of the cancerrelevance score of each cancertype with the seed ratio of additional file performance of classification of tumor samples andnormal samplesadditional file top gene pairs for each cancer type 0clee bmc medical genomics 13suppl page of abbreviationsauc area under the curve brca breast invasive carcinoma cnv copynumber variation coad colon adenocarcinoma gdac genome dataanalysis center hnsc head and neck squamous cell carcinoma kipan pankidney cohort lihc liver hepatocellular carcinoma loocv leaveoneoutcross validation luad lung adenocarcinoma lusc lung squamous cellcarcinoma mcc matthews correlation coefficient pcc pearson correlationcoefficient ppi proteinprotein interactions roc receiver operatingcharacteristic svm support vector machine tcga the cancer genome atlasacknowledgementsthe authors thank the anonymous reviewers for the constructive commentsthat contributed to improving the final version of the paperabout this supplementthis has been published as part of bmc medical genomics volume supplement selected s from the 15th international symposiumon bioinformatics research and applications isbra19 medical genomicsthe full contents of the supplement are available online at httpsbmcmedgenomicsbiomedcentralcomssupplementsvolume13supplement6authors™ contributionswl designed and performed all about this study and prepared the initialmanuscript dh helped integrating multiomics data of breast cancer khsupervised the work and wrote the manuscript all authors read and approvedthe final manuscriptauthors™ informationwook lee is currently working toward his phd degree at the department ofcomputer engineering inha university korea deshuang huang is a directorof the institute of machines learning and systems biology tongji universitychina kyungsook han is a professor at the department of computerengineering inha university koreafundingthis work was supported by the national research foundation of korea nrfgrant funded by the ministry of science and ict nrf2017r1e1a1a03069921and inha university research grant publication of this was funded bythe nrf grant nrf2017r1e1a1a03069921 the funders had no role in thedesign of the study data collection and analysis or writing the manuscriptavailability of data and materialsadditional files are available at httpbclabinhaackrcancernetworkethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of computer engineering inha university incheon southkorea 2institute of machine learning and systems biology school ofelectronics and information engineering tongji university shanghaichinareceived may accepted june published august references widakowich c de castro g de azambuja e dinh p awada a reviewside effects of approved molecular targeted therapies in solid cancersoncologist “liu s kurzrock r toxicity of targeted therapy implications for responseand impact of genetic polymorphisms cancer treat rev “verma m personalized medicine and cancer j personalized med“barabasi al gulbahce n loscalzo j network medicine a networkbasedapproach to hum
Colon_Cancer
"circadian clocks have important physiological and behavioral functions in humans and other anisms whichenable anisms to anticipate and respond to periodic environmental changes disturbances in circadian rhythmsimpair sleep metabolism and behavior people with jet lag night workers and shift workers are vulnerable tocircadian misalignment in addition non24h cycles influence circadian rhythms and cause misalignment anddisorders in different species since these periods are beyond the entrainment ranges in certain special conditionseg on submarines and commercial ships non24h watch schedules are often employed which have also beendemonstrated to be deleterious to circadian rhythms personnel working under such conditions suffer fromcircadian misalignment with their onwatch hours leading to increased health risks and decreased cognitiveperformance in this review we summarize the research progress and knowledge concerning circadian rhythms onsubmarines and other environments in which non24h watch schedules are employedkeywords circadian rhythm circadian clock entrainment range metabolism alertness submarine most anisms living on this planet possess circadianclocks that orchestrate their daily physiological and behavioral rhythms to the 24h rotation period of the earth however under special conditions in which thecycling periods do not extend over h the circadiansystems may be challenged in the laboratory manualnon24h conditions are established to study the basicfeatures of circadian rhythms although such conditionsare very rare in nature however in human societies asubstantial group of people lives or works under non24h schedules the non24h conditions were observedto lead to a decrease in adaptability to the environment correspondence guojinhumailsysueducn1key laboratory of gene engineering of the ministry of education state keylaboratory of biocontrol school of life sciences sun yatsen universityguangzhou chinafull list of author information is available at the end of the in all tested anisms across different kingdomscluding bacteria fungi plants and animalsinthe circadian clocks are well conserved across speciesthus we will first introduce the knowledge derived fromcircadian studies in model anisms and the next focuson summarizing studies in humans the former partwould help to elucidate circadian misalignment in circadian rhythms metabolism sleep and the cognitive andbehavioral consequencescircadian clock and circadian rhythmscircadian clocks govern life processes at multiple layersincluding the molecular cellular anism and integrallevels at the anism level circadian clocks are hierarchically composed of master clocks located in thepacemakers and peripheral clocks located in other tissues in mammals the pacemaker is the suprachiasmaticnucleus scn in the anterior of the hypothalamus inbirds and reptiles the eyes pineal gland or scn are the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cguo military medical research page of range circadian rhythms can be adjusted to match theenvironmental periods a process that is termed entrainment or synchronization circadian rhythms are both robust and flexible within a certain range tcycles thatdeviate from the natural 24h cycles can also inducerhythms with the same periodicity as the environmentin this scenario the endogenous circadian periodicitymay be masked by the non24h tcycles the factorsthat can entrain circadian rhythms are known as zeitgebers synchronizers or time givers [ ]to experimentally determine the entrainment rangesanisms are exposed to a series of fixed tcycles radually changing tcycles in addition the entrainment range to an environmental cue can be inferredby the phaseresponse curve prc information of thespecies given that according to the prc of a speciesthe maximum phase delay and maximum phase advanceare x hours and y hours respectively the entrainmentrange of this species to the indicated cue would rangefrom h “ y to h x hours prc representsthe nonparametric effects of zeitgebers on entrainmenthowever parametric components are also involved indetermining the entrainment range the parametricmodel assumes that light changes the velocity of the circadian rhythms for entrainment while the nonparametric model emphasizes the phase shift theentrainmentranges differ between specieshumans have a circadian clock with an endogenousperiod τ of approximately h [ ] with upper andlower limits of and h respectively in conjack bean canavalia ensiformis displayed leaftrastmovement rhythmicity synchronized to lightdark ld but not ld suggesting that the lower entrainment limit is between and h fig 2a somespecies such as bakery mold neurospora crassa andhouse sparrow passer domesticus show very wide entrainment ranges in banding rhythms of conidia releaseconidiation fig 2b and locomotor rhythms respectively [ ]circadian rhythms run free when the t cycles are beyond the entrainment ranges under ld squirrelmonkeyscore bodytemperature cbt rhythm with a period of hsaimiri displaycebidaeacircadian pacemakers depending on the species [“]the peripheral clocks have their own circadian rhythmsbutthey can also be synchronized by pacemakersthrough the nervous and endocrine pathways at the molecular level circadian rhythms are generated by a set of core circadian genes which constitutetranscriptiontranslational negative feedback loops despite differences in circadian clock genes the regulatory mechanisms are highly conserved across kingdoms[ ] in mammals including humans positive circadianelements include brain and muscle arntlike protein bmal12 and clock and the negative elements include period per “ and cryptochromecry reverbs ˆ’α and and retinoic acidreceptor rarrelated orphan receptor ror ˆ’α ˆ’and Î are additional regulators in mammalian circadiancircuits which act as ancillary loops to regulate bmail1expression reverb proteins inhibit while ror proteins activate the transcription of bmal1 [ ] at thetranscriptomic level circadian clocks regulate the expression of approximately “ of proteinencodinggenes in mice [ ] via the output pathways circadianclocks control most of the physiological and behavioralprocesses in mammals circadian clocks govern thecircadiandiurnal rhythms in sleepwake cycles feedingfasting control metabolism hormone secretion and immunity function fig although circadian rhythms are endogenous they areto the influence of environmental geneticsubjectphysiological and pathological factors fig [ ] ithas been demonstrated that disruptions in circadianrhythms impair adaptability in a variety of anisms in humans compromised circadian rhythms causechronic health consequences including an increased riskof gastrointestinal illness loss of bone mineral densitycoronary artery disease endocrine disruption metabolicsyndrome and cancer moreover disrupted circadianrhythms also affect emotions cognition and performance fig [“]entrainment range of circadian rhythmsthe cycling periods of the environment such as lightand temperature are defined as tcycles in a certainfig circadian rhythm disturbance and their consequences 0cguo military medical research page of fig circadian rhythms of different anisms in non24h conditions a leaf movement alternatively opening and closing rhythms ofcanavalia ensiformis c ensiformis b conidiation banding rhythms of n crassa neurospora is inoculated in a long and hollow glass tube withmedium inside which is called a race tube in the race tube neurospora grows towards the other end and releases conidiation bands coloredorange the release of conidiation is controlled by the circadian clock only the left part of the race tubes is shown c rectal temperature rhythmsof c saimiri neurospora conidiation rhythms are entrained in ld ld and ld while canavalia leaf movement rhythms andsubmarine crew rectal temperature rhythms were not entrained in ld therefore they have different entrainment ranges panel a was drawnaccording to the study by panel b was drawn according to the study by and panel c was drawn according to the study by instead of h suggesting the occurrence of a nearlyfreerunning cbt rhythm fig 2c an approachhas been proposed to measure the freerunning periodbased on the displayed periods under non24h cyclesbeyond the entrainment ranges [ ] importantly therhythms beyond the entrainment ranges should be calledsuperposition or superimposition [ ] superposition may result in a period different from the one measured in a constant condition despite the different entrainment ranges differentanisms exhibit rhythms with endogenous periodsafter transfer to constant conditions suggestingthe endogenous characteristics of the sustainability ofcircadian rhythms in a study the pregnant mice werekept in ld or ld and their progenies werealso raised in these tcycles until they reached puberty the progeniesshowed freerunning periodssoon after they were placed in constant conditions the sole recorded exception was that hydrodictyon maintains a period of h in the constantdark for at least days after having been retrievedfrom the ld condition different physiological and behavioral rhythms mayalso have different entrainment ranges within one species or even within individuals suggesting the existence of independent oscillators in addition to affectingthe pacemaker of the circadian clock zeitgebers alsobypass it directly impacting the overt rhythms whichcausesinternal desynchronization between differentphysiological processes as a consequence when t cyclesare out of the entrainment ranges eg the sleepwakecycles and the cbtthe molecular mechanisms determining the entrainment range have not been elucidated the only clue regarding this mechanism was that in mice clock mutantsexhibit a wider entrainment range ld to ld compared to wildtype mice suggesting that circadianclock genes play critical roles in regulating entrainment mutants with less robust rhythms may be moreprone to environmental entrainment although such passive rhythms have no anticipating property unlike circadian rhythmscompromised adaptive fitness in non24h cyclingconditionsthe circadian clocks derive from the past longterm evolution which confers the fitness of the cycling environment and it is plausible that lives on earth cannotadapt to non24h cycling conditions in a short periodof time two strategies were employed to test this hypothesis one strategy is to study the effects of abnormalcycles of environmental cues on anisms and theother strategy is to compare the competitiveness of mutants with different endogenous circadian period lengthsor with no circadian rhythmicity the adaptive fitness ofa number of model anisms has been investigatedconcerning the circadian clock for the environmenttable all the available evidence suggests that living undercircumstances with cycling periods beyond the entrainment ranges is harmful to reproductive fitness cyanobacteriaendogenouscircadian periods were mixed and after a month in culturethe strain with a period resonating with thepossessingstrainsdifferent 0cguo military medical research table selected studies changes in circadian rhythms and adaptation of model anisms under non24h conditionsspeciescyanobacteria synechococcus elongates ld ld adaptabilitydecreased growth competence and adaptabilitynon24h periods hpage of references[ ]bakery mold neurospora crassatemperature cycles t12conidiation rhythms ran freeld ld ld conidiation rhythms were entrainedld ld ld conidiation rhythms were entrainedtomatold ld significantly decreased growththale cress arabidopsis thalianald ld decreased leaf chlorophyll content decreased photosyntheticcarbon fixation decreased vegetative growth and survivalld ld the mutants showed a strong positive correlation in competitionunder the tcycle growth conditionsjack bean canavalia ensiformisld ld ld leaf movement rhythms were entrained under ld ran freeunder ld and ld fruit fly drosophila melanogasterld ld reduced life spanblowfly phormia terraenovaet26 t28decreased life spans under t26 and t28house sparrow passer domesticusld ld ld locomotion rhythms were at least partially entrained orshowed superpositioncommon chaffinch fringilla coelebsld ld ld ld activity rhythms were entrained to ranges from ld to ld mouse mus musculustemperature cycles t20 superposition of luciferase rhythms in lung and scn tissuespartially entrainednile grass rat arvicanthis niloticusrat rattus rattust22“feeding cycles t20 t28locomotion rhythms were entrained between t and t decreased food intake and weight gain squirrel monkey cebidae saimiristudies under asymmetric tcycles are not includedl denotes œlight d denotes œdark ld xy denotes cycling conditions with light for x hours and dark for y hours alternatively scn suprachiasmatic nucleuscore body temperature ran free lower amplitudeld ld environment prevailed while the strain with a perioddifferent from the environment failed in the competition [ ] several models have been proposedto explain the clockcontrolled fitness in cyanobacteria including the limited resource model diffusiblefactor model celltocell communication model andthe escapefromlight hypothesis which may alsohelp to characterize the enhanced fitness by the circadian clock in other anisms [ ] the escapefromlight hypothesis postulates that in the early stageof evolutionthe circadian clock acts to segregatelightsensitive reactions temporally to nighttime egcell division and dna damage repair to avoid thedeleterious effects of radiation and oxidation fromsunlight [ ]the growth ofgrowth and flowing time were extensively affected although the results varied in different species a comparison oftomato under different ldconditions demonstrated thattomato grows mostquickly in ld but very slowly in either ld or ld similar experiments have been conducted in a variety of species including house sparrowjack bean canavalia ensiormis n crassa and squirrelmonkey saimiri sciureus which indicate that light regimens with periods that deviated dramatically from hhampered growth or survival [ ] in a previousstudy several plants were tested in a series of ld cyclesranging from ld s5 s to ld for instance soybean soja max limper under ld min1 minshowed a markedly chlorotic phenotype and a notablereduction in size contained numerous spots of dead tissue and showed a tendency to die prematurely ofcourse the day length also contributes to the comprehensive influences on plantsit has been demonstrated that in many anismsnon24h cycles have extensive negative or deleteriouseffects on survival and competitive fitness lesion of thescn in antelope ground squirrels ammospermophilusleucurus led to the loss of circadian rhythmicity in locomotor activity and an increased ratio of loss caused bypredation the tau mutation in the enzyme caseinkinase 1ε ck1ε had a dramatic influence on circadianperiods in mice with the heterozygous mice showing atwohour shorter period while the homozygous miceshowed a fourhour shorter period in the seminaturalenvironment the relative frequency of the tau allele decreased to from the initial half in months due todisadvantages in both adult survival and the recruitmentof juveniles into the cohorts in addition to fitness misalignment of circadianrhythms with non24h cycling conditions impairs manyphysiological and metabolic processes moreover the 0cguo military medical research page of disruption of circadian rhythms leads to abnormalities incognition in animals the circadian control of cognitionand behavior has been demonstrated in many animalspecies disruption of mouse circadian rhythms by ldcycles led to extensive ramifications in metabolism brainfunction and behavior for instance in ld miceandchanges in emotionality which were consistent withchanges in neural architectureincluding shorter dendritic length and reduced complexity of neurons in themedial prefrontal cortex exhibited decreased cognitiveflexibilityin addition non24h cycles shorten life spans in manytested anisms for instance pittendrigh and minisraised drosophila under the conditions of t h andt h and the life spans of drosophila in both conditions were considerably shorter than those lived in t h a hypothesis proposed that when the cycling zeitgeberperiod is close to a demultiplicative period of the freerunning period the oscillator may also be entrainedwhich is known as frequency demultiplication however frequency demultiplication is more likely torepresent a passive response to cycling environmentalstimuli than circadian oscillations [ ]effects of non24h conditions on human healthand performancenon24h regimens affect circadian rhythms and sleepkleitman and richardson were the first persons who recorded their experiences under a 28h condition in acavern of mammoth national park in kentucky usain the constant dark of mammoth cave the sleepwakecycles and body temperature of richardson adapted tothe 28h cycles but kleitman failed in forced t21and t27 schedules simulated in spitzbergen the bodytemperatures in of subjects adapted immediatelywhile the excretory rhythms adapted in only three subjects suggesting the occurrence of desynchronization[ ] even periods with a slight difference from hcan be harmful to the growth or fitness of an individualfor instance a study of the sleepwake rhythms andsleep time in mars lander phoenixin which participants lived and worked on a 2465h schedule whichwas in accordance with the daily cycling period of marsrevealed inadaptation in the sleepwake rhythms in someof the subjects and loss of sleep in almost all of the subjects since the thirteenth century the 4h on8h off watchschedule has been employed in maritime crews which isdramatically different from a 24h day in the militarythe watch schedule of the us board vessel crews shiftedin the last century from the 4h on8h off routine tothe 6h on12h off routine to accommodate the threeshift sections [ ] approximately of enlistedmen serving aboard us navy submarines stand watchon the latter schedule in contrast most officers andsome senior enlisted men stand watch on a foursectionrotation ie h on and h off in studies of the circadian rhythms of submarine crewmembers some physiological variables including bodytemperature serum or urine melatonin cortisol andurine catecholamine have been used as hallmarks [“] in a 4h on8h off schedule the oral temperatureof the subjects exhibited an 24h period instead of a12h period kelly investigated circadianrhythms of salivary melatonin in crew members during a prolonged voyage on a trident nuclear submarineand found that under 18h duty cycles the endogenousmelatonin rhythm of the crew members showed an average period of hin non24h cycles the circadian parameters including period amplitude and phase are all subject tochange during a prolonged voyage under a 4h on8h off schedule altered circadian rhythms in cbt occurred among the watchers the mostcommonphenomenon was the dampened amplitude one subject even showed a freerunning pattern naitoh found that a 6h on12h off routine duringa nuclear submarine patrol caused a loss of 24hrhythmicity in oral temperature which was due notonly to a decreased circadian amplitude but also to adispersion of the time of the peak a different watchtime in three shift groups who used alternative 4hon8h off regimens induced changes in the phase ofrectalandadrenaline excretion noradrenalinetemperatureexcretionchanges in circadian variables suggest that under non24h schedules circadian rhythms are prone to alteration and desynchronization may occur as a consequence in schaefer analyzed the rhythms ofbody temperature pulse rate and respiration rate of crew members under two different schedules an 18hwatch schedule 6h on12h off and a 24h schedule the results showed that crew members using theformer schedule but not the latter one exhibit superposition of 18h and 24h periods in all tested variables asimilar superposition was also observed in other studies[ ] as the schedule continues the impact oncircadian rhythms becomes more pronounced naitoh revealed that in a longterm voyage the groupsynchronization in oral temperature rhythmicity disappeared after approximately daysearly in it was noticed that sleep insufficiencywas caused by a 6h on12h off schedule in the submarine crew in schaefer measured theparameters of cbt heart rate and respiration rate every h for subjects living under different schedules some ofwhom employed a 6h on12h off the roster other 0cguo military medical research page of subjects employed the normal 24h schedule theformer schedule caused superposition on the rhythmsshowing both 24h and 18h periods sleep insufficiencywas also recorded in these subjects a survey of submarine crew members indicated that over ofthem had medical complaints and most complaintswere about sleeping difficulty however prevalentstudies have demonstrated that under non24h conditions both the quantity and quality of sleep are altered [ ] in real missions the sleep time ofthe submarine crew varies in a wide range extendingfrom h to h which may be influenced by manyenvironmental factors almost all studies indicated that non24h scheduleselicit misalignment with the environmental cycles anddeficiency in sleep quantity due to split sleep patternswhich means that sleep is separated into two or moreparts during a day usually both sleep quantity and sleepquality at night are better than those in the daytime [“] in contrast kosmadopoulos reportedthat a split sleep pattern showed no detrimental effectson sleep quality and performance suggesting the necessity of conducting more extensive and systematicanalysesmelatonin regulates sleep in a circadian fashionfor subjects living in an 18h routine or a combinedroutine of h and hthe melatonin levels ofthese subjects showed periods between h and h suggesting thatinfree running occurred tothethiscyclesleepwakeendogenouscontrastphysiological parametersincluding melatonin andtemperature have considerably narrower entrainmentcausedesynchronization in physiology psychology and behavior at the systemic level discrepancy mayrangesin submarines apart from non24h adherence to arotating watch roster other physical and chemicalenvironmental cues in the cabin also influence the physiology and behavior of the crew members these cues include confinement deleterious atmospheres sunlightdeprivation and insufficient lighting hypercarbia noiseelectromagneticlightspectrum and intensity high temperature and humidityfig [ “] the effects of many of thesecues on human circadian rhythms have not been fullyelucidatedradiationradiationionizingmoreover in a real mission jet lag may further contribute to the effects of circadian desynchronizationcondon found that the sleep length of watchkeepers was shorter when they crossed four to five timezones eastbound compared to crossing the time zoneswestboundshowedslower adaptation on eastbound travel this findingmay be explained by the fact that the human freerunning period is h which facilitates westboundadaptation a 3day shift regimen is widely employedin submariners which may cause frequent shiftssuch as social jet lagsubjectiveand thealertnessfig disturbed circadian clock and impaired physiology and behavior in the submarine crew some connections between the components arenot shown 0cguo military medical research page of non24h regimens lead to changes in metabolismthe circadian clock governs metabolism by modulating the expression and function of essential geneswithin severalimportant metabolic pathways in atemporal fashion conversely misalignment in circadian systems disturbs metabolic homeostasis [ ]the homeostasis of metabolism is also tightly associated with sleep [ ] in mice decreased food intake and weight were observed in mice raised underld or ld suggesting that non24h cyclingconditions may influence metabolic homeostasis in an anchoredsleep study subjects were exposed toa schedule that enabled them to sleep for h fixedfrom to and another h unfixed duringthe day the subjects showed altered acrophases inincludingbody temperature and urinary variablespotassium and sodium suggesting thattheirmetabolisms were affectedthe level ofsleep consistentlyinsulin is under the control of thecircadian clock which peaks at nightin humans ma found that saliva insulin dramatically increased late at night and early in the morning in subjects under a schedule of h for work andrest and h forthe insulinlevels and insulin glucose ratios were increased inmice under ld conditions metabolic syndrome is a constellation associated with metabolicriskincluding abdominal obesity abnormal bloodlipid levels hypertension and impaired fasting glucose kang and song reported that submarine crew members had higher risks of metabolicsyndrome and changed levels oflow highdensitylipoprotein cholesterol and impaired fasting glucoseunder a non24h onwatch schedule taken together these data suggest that metabolism may bedisturbed which might be largely attributed to thenon24h schedulesin recent years the involvement of the gut microbiome in animal and human metabolism has beenincreasingly recognized changes in the category andthe abundance of gut and oral microbiota also display a circadian pattern [“] changes in alimentary tract microbiota account for alterations in hostmetabolism relative to the fecal microbiomethesalivary microbiome has rarely been studied despiteits higher accessibility the oral microbiome containsbacteria viruses and fungi which associate and formbiofilms environmental changes might convert themto be pathogenic and trigger intestinal diseases orother inflammatory diseases recently ma found that disruption of the composition of theoral microbiome occurred in subjects living under anon24h schedule some bacterial species showedspecific change patterns eg tenericutes and sr1corresponding to sleep deprivation and circadiandesynchronization respectivelynon24h regimens affect mood cognition andperformancefatigue is an important cause of accidents and incidents at sea one requirement of safe submarineoperations is to ensure that sailors work at their highest level of readiness the non24h onwatch schedules cause misalignment in circadian rhythms whichin turnleads to sleep deprivation [ ] impairments in circadian rhythms and sleep have extensivephysiological and cognitive effects including increasedfatigue and changes in mood vigilance and workproductivity figs and it is critical to pay closeattention to the circadian rhythms and performancein submarineas mooreede pointed outœthere should be some global concern aboutthehealth and performance of these men since they arethe ones with their fingers directly on the nuclearbutton crewsnaitoh assessed the mood changes in agroup of nuclear submariners by using subjectivequestionnaires of thayer™s activation mood œactivity ma and mood œhappiness mh and revealedthat during a 10week submerged patrol the 24hrhythmicities of mood œactivity and mood œhappiness werecontrolthe results from moodperiodscale questionnaires also indicated that compared tothe patrol period significantly more feelings of œactivity and œhappiness were observed in the postpatrol period abolished compared to thein another studyscheduletheresults ofin humansthe circadian nadir trough of cbtand alertness is late at night and early in the morning and changes in body temperature reflect the dynamics of metabolism which is also one ofthedeterminants of alertness [ “] under the 4hthe letteron8h offcancellation test and vector testfrom watchkeepers demonstrated in the case of the former thatthe œbest time is forenoon while in the latter it islate afternoon the slowest performance occurred either at the start of watches beginning immediatelyafter wakening or near in the morning which isthe nadir of alertness under submarine watchroutines both misalignments in circadian rhythmsand decreases in sleep length and quality imposenegative effects on mood cognition and performance[ ] during the existingwatch schedules used in canadian forces were evaluated revealing thatthe cognitive efficacy in thesubmarine crew dramatically decreased to a dangerously low level showed that ma the effects of 0cguo military medical research page of under a 4h on8h off routine the alertness of thesubjects decreased dramatically in the morning aschronic sleep deprivation may occur during non24h cyclesit is unclear whether misaligned circadianrhythms or sleep loss contributed more to the decreased alertnessthe fatigue avoidance scheduling toolfast„¢program has been developed to optimize the scheduling and performance of the air force consideration and integration of moresuch asindividual differences might help to improve theprediction accuracy and practicability in not only theair force but also for personnel working under conditions with non24h periodsincluding submarinecrew members factorstheseand most ofto date many studies have documented the impairment of non24h schedules on circadian rhythms andcognitionstudies have beenconducted at the physiologicallevel by contrast themolecular mechanisms governing the effects of disruptedcircadian rhythms have not been elucidated which maythethuslimitphysiologicalofcountermeasuresunderstandingtheofdevelopmentindepthandthechangesin militaryand commercialcountermeasures conclusions and perspectivescircadian disruption profoundly affects health andperformancevesselcrews during a 90day voyage which is the longestvoyage recorded to date disturbance in circadianrhythms was reported in many chinese crew members most of the crew members experienced sleepdisordersflagging dysphoria and faintness somecrew members attempted to overcome these conditions by chewing dry peppers or daubing mentholcamphorate on their temples in maritime missions circadian misalignment causes serious consequences including high personnel turnover “per voyage and requirements to train the new entrant howeverthe question remains of how we canovercome circadian misalignment and avoid deleterious consequences the entrainment range needs tobe considered if the environmental period is withinor close to the entrainment rangeit may be usefulto usecircadianrhythms if the environmental period is far beyondthe entrainment range it may be useful to constructa manmade 24hperiod microenvironmentisalso possible to combine both approaches someefforts have been made to lessen the deleterious effects of disrupted circadian rhythms and to improvehealth and performance which primarily involvedcountermeasuresto adjusttheitoptimization of the watch schedules and modifications of the zeitgebersoptimized watch schedules may improve circadianrhythms and performance duplessis compared the impacts of different watch schedules 6hon8h off and a compressed close6 h watch schedule and the wrist activity results showed that sleepduration was longer under the 6h on8h off schedule sleep discontinuity appears to be considerablyless severe on the 6h on12h off watch schedulethan that of the 4h on8h off watch schedule incontrast the 6h on12h off schedule allowed morecontinuous sleep which might be explained bythe fact that t12 is shorter than t18 compared tohuman frp the best approach is to use a 24h roster instead of a non24h roster a 24h schedule wasused durin
Colon_Cancer
"high mobility group box hmgb1 is a nonhistone chromatinassociated protein widely distributed in eukaryoticcells and is involved in dna damage repair and genomic stability maintenance in response to stimulus like bacteriaor chemoradiotherapy hmgb1 can translocate to extracellular context as a danger alarmin activate the immuneresponse and participate in the regulation of inflammation and cancer progressionkeywords hmgb1 rage tlr damp inflammation cancerchromatinassociated proteinit washigh mobility group box hmgb1 is a highly conservative nucleoprotein and belongs to the group of nonhistonefirstextracted from calfthymus chromatin in andnamed for its high mobility in gel electrophoresis subsequentinvestigations found that hmgb1 couldtranslocate from the nucleus to the cytoplasm after posttranslational modifications including acetylation phosphorylation and methylation hmgb1 can be expressedat the neuron membrane as well in response to chemoradiotherapy or hypoxia hmgb1 could be transferredto the extracellular context mainly through two waysactive secretion from immunocompetent cells or passiverelease from apoptotic or necrotic cells extracellularhmgb1 transmits danger signals to surrounding cellsby interacting with its classical receptors such as thereceptor for advanced glycation end products rageand tolllike receptors tlr249indepth studies implicated that hmgb1 was a multifunctional protein involved in a variety of cellularsubcellularbiological properties depending on itslocalizationandbinding receptors fig posttranscriptional modification correspondence yizhangzzueducn1biotherapy center cancer center the first affiliated hospital ofzhengzhou university zhengzhou china2state key laboratory of esophageal cancer prevention treatmentzhengzhou university zhengzhou chinafull list of author information is available at the end of the in the nucleus hmgb1 plays a key role in the processof dna replication transcription chromatin remodeling and vdj recombinationthus regulating dnadamage repair and the maintenance of genome stabilityas a dna chaperone cytoplasmic hmgb1 is involvedin immune responses by increasing autophagy inhibitingapoptosis and regulating mitochondrial function atthe membrane hmgb1 promotes axonal sprouting andneurite growth activates platelets and induces cellmigration as a typical damage associated molecular pattern damp extracellular hmgb1 is involved in manyimmune responses by promoting immune cell maturation activation and cytokine production extracellular hmgb1 can also interact with chemokines such ascxcl11 to enhance immune responses as a multifunctional protein hmgb1 exerts different biologicaleffects under different stimuli the deregulation ofhmgb1 is associated with many diseases especiallyinflammatory disorders and cancerhmgb1 in inflammationhmgb1 plays a critical role in the regulation of bothinnate and adaptive immune responses to promoteimmune response to sterile or infectious stimulus insterile inflammation during ischemiareperfusion injuryiri hmgb1 becomes disulfidebonded to increasemacrophage production of proinflammation cytokinesin a tlr4 dependent manner indicating that disulfidebonded hmgb1 can be identified as a diagnosis and the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang and zhang of hematology oncology page of fig the multifunctions of hmgb1 hmgb1 in the nucleus regulates dna damage repair and genome stability as a nonhistone chromatinassociated protein and dna chaperon in the cytoplasm or mitochondria hmgb1 increases autophagy inhibits apoptosis and regulatesmitochondria functions at the membrane hmgb1 promotes axonal sprouting and neurite growth hmgb1 can be transferred to extracellularcontext by two ways active secretion from immunocompetent cells or passive release from apoptotic or necrotic cells participating inimmune responsesin contrasttreatment biomarker for iri when initially secreted byinnate immune cells like macrophages hmgb1 is proinflammatory during the early stages of sepsis howeverthe extracellular hmgb1 could also induce immune tolerance and immunosuppression when released by othersomatic cellsintracellular hmgb1 caninduce protective autophagy and contribute to cellsurvival by delivering lipopolysaccharide lps andpromoting endocytosis hmgb1 activates the noncanonical inflammasome pathway and induces pyroptosis pyroptotic macrophage death may accelerateundesirable immune hyperactivity and immunosuppression which is a potential mechanism associatedwith late mortality from sepsis in hepatic infectious disease the release and activity of hmgb1 as acytokine could be suppressed by glycyrrhizinic acidga by binding with tlr4 hmgb1 regulatesthe hepatitis virusesinduced immunological axis providing a new therapy strategy for the treatment ofacute viral hepatitis in the clinical practice canbesecretedin severe pulmonary inflammatory diseases including covid19 hmgb1inabundance by necrotic pulmonary epithelial cells andinnate immune cells disulfidehmgb1 triggers proinflammatory cytokine release and further exacerbatessevere inflammation therefore hmgb1 mightbeofinflammationtreatmentpotentialtargetaforthethe dual effects of hmgb1 in canceraccording to the alterations of the subcellular locationsreceptors and expression levels hmgb1 is associatedwith the hallmarks of cancer proposed by hanahan andweinberg hmgb1 appears to play paradoxicalroles during the development and therapy of cancer onthe one hand hmgb1 can contribute to tumorigenesisexcessive hmgb1 production caused by chronic inflammatory response seems to be associated with tumorigenesis for example by combining with rage hmgb1plays an important role in regulating oval cells activationand inflammationassociated liver carcinogenesis in mice in established cancers hmgb1 produced by tumorcells may exacerbate inflammationrelated immunosuppression for instance previous research indicated thatlps induced the release of proinflammatory cytokinessuch as il1 il6 and tnfα in a hmgb1dependentmanner to improve colon cancer progression however the underlying mechanism of hmgb1 in the transformation ofinflammation and cancer needs to befurther studied it has been reported that hmgb1 canbe released to extracellular context by necrotic cellsunder hypoxia in growing solid tumor extracellularhmgb1 promotes the release of cytokines such as il6and il8 by activating mapk and myd88dependentnfκb pathways which in turn stimulates tumor cellsproliferation angiogenesis emt invasion and metastasis nucleusand cytoplasmic hmgb1 promotes 0cwang and zhang of hematology oncology page of autophagy and inhibits apoptosis of tumor cells to induce chemotherapy resistance on the other handhmgb1 plays a protective role in the suppression oftumor and tumor chemoradiotherapy and immunotherapy nucleus hmgb1 assists in the regulation of telomere and maintenance of genome stability loss ofhmgb1 results in the instability of genome and leads totumorigenesis thus the roles of hmgb1 in regulationof dna damage repair and cancer etiology indicate thattargeting chromosomal architectural hmgb1 may provide a new perspective for cancer therapy hmgb1located in the cytosol or mitochondria may bind toautophagy associated genes like beclin to regulate cellautophagy and mitophagy absence of hmgb1 resultsin autophagy deficiency and increased apoptosis leadingto tumorigenesis intracellular hmgb1 functions as atumor suppressor by binding tumor suppressor proteinslike rb but it remains to be studied whether hmgb1interacts with other tumor suppressors or oncoproteinsextracellular hmgb1 enhances chemotherapy efficacyby transforming tumor cells from apoptosis to senescence in addition hmgb1 can mediate immunogenic cell death during chemoradiotherapy and enhanceantitumor immunity in response to chemotherapy likeanthracycline or radiotherapy hmgb1 can be rapidlyreleased from dead cells as an alarming molecule uponrelease from necrotic cells or secreted by activated macrophages hmgb1 can recruit inflammatory cells andmediate interactions between nk cells dendritic cellsdcs and macrophages activated nk cells provide anadditional source of hmgb1 which is released into theimmunological synapse between nk cells and immaturedcs promoting the maturation of dcs and the induction of th1 response in addition hmgb1 produced from nsclc cells induced by docetaxel canstimulate t cells for antitumor immune response andimprove immunotherapy effects like cart cells therefore modulating hmgb1 may provide a potentialcombination strategy for cancer chemoradiotherapy andimmunotherapyconclusionhmgb1 is a multifunctional molecule that plays a majorrole in homeostasis as damp molecule hmgb1 playsthe complex roles in various biological processes and isinvolved in the development of many diseases such asautoimmune diseases and cancers hmgb1targetedagents including antibodies and inhibitors have shownbeneficial results in preclinicalinflammatory modelssuch as sepsis these agents await clinical developmentabbreviationshmgb1 high mobility group box rage the receptor for advancedglycation end products tlr tolllike receptors damp damage associatedmolecular pattern iri ischemiareperfusion injury lps lipopolysaccharidega glycyrrhizinic acid covid19 coronavirus disease19 mapk mitogenactivated protein kinase myd88 myeloid differential protein88 nfκb nuclear factor kappalightchainenhancer of activated b cellsemt epithelialmesenchymal transition dcs dendritic cellsacknowledgementsnot applicableauthors’ contributionsyz designed directed and revised the manuscript sw drafted themanuscript both of the authors read and approved the final manuscriptfundingthis work was supported by the national key research and developmentprogram of china 2016yfc1303500availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1biotherapy center cancer center the first affiliated hospital ofzhengzhou university zhengzhou china 2state key laboratory ofesophageal cancer prevention treatment zhengzhou universityzhengzhou china 3henan key laboratory for tumor immunologyand biotherapy zhengzhou chinareceived july accepted august referencesgoodwin gh sanders c johns ew a new group of chromatinassociatedproteins with a high content of acidic and basic amino acids[j] febs j–paudel yn angelopoulou e piperi c balasubramaniam vrmt othman ishaikh mf enlightening the role of high mobility group box hmgb1 ininflammation updates on receptor signalling[j] eur j pharmacol huebener p gwak gy pradere jp et al highmobility group box isdispensable for autophagy mitochondrial quality control and anfunction in vivo[j] cell metab –rivera vargas t apetoh l danger signals chemotherapy enhancers[j]immunol rev –gao q wang sm chen xf et al cancercellsecreted cxcl11 promotedcd8 t cells infiltration through docetaxelinducedrelease of hmgb1 innsclc[j] for immunotherapy of cancer andersson u tracey kj hmgb1 is a therapeutic target for sterileinflammation and infection[j] annu rev immunol –kim hm kim ym hmgb1 lps delivery vehicle for caspase11mediatedpyroptosis[j] immunity –deng m tang y li w et al the endotoxin delivery protein hmgb1mediates caspase11dependent lethality in sepsis[j] immunity –753e747shi xd yu lj zhang yl et al glycyrrhetinic acid alleviates hepaticinflammation injury in viral hepatitis disease via a hmgb1tlr4 signalingpathway[j] int immunopharmacol saha b tornai d kodys k et al biomarkers of macrophage activation andimmune danger signals predict clinical outcomes in alcoholic hepatitis[j]hepatology baltimore md – andersson u ottestad w tracey kj extracellular hmgb1 a therapeutictarget in severe pulmonary inflammation including covid19[j] molecularmedicine cambridge mass 0cwang and zhang of hematology oncology page of hanahan d weinberg ra hallmarks of cancer the next generation[j] cell– pusterla t nèmeth j stein i et al receptor for advanced glycationendproducts rage is a key regulator of oval cell activation andinflammationassociated liver carcinogenesis in mice[j] hepatologybaltimore md – yang y yang l jiang s et al hmgb1 mediates lipopolysaccharideinducedinflammation via interacting with gpx4 in colon cancer cells[j] cancer cellint yuan s liu z xu z et al high mobility group box hmgb1 a pivotalregulator of hematopoietic malignancies[j] j hematol oncol mukherjee a vasquez km targeting chromosomal architectural hmgbproteins could be the next frontier in cancer therapy[j] cancer res liu y dong y kong l et al abscopal effect of radiotherapy combined withimmune checkpoint inhibitors[j] j hematol oncol publisher’s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
Colon_Cancer