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polymorphism of cyp2d6 gene encoding for debrisoquine hydroxylase was determined genotypically for 94 controls and 77 lung cancer patients using a polymerase chain reaction - based application . both of the point mutations that give rise to deficient alleles of the cyp2d6 gene are detectable by this method . out of the 94 healthy controls , 3 individuals ( 3.2% ) had poor metabolizer ( pm ) genotypes , whereas no pm genotypes were detected in the lung cancer patient group . we observed no difference in the allelic frequencies for either homozygous extensive metabolizers ( ems ) or heterozygous ems between the lung cancer patients and the healthy controls . however , the absence of the poor metabolizer genotype ( 0/77 ) in the lung cancer patients is compatible with the hypothesis that there is an increased risk of lung cancer for individuals who are extensive metabolizers of debrisoquine . another member of the cytochrome p450 gene superfamily that has attracted interest for its potential role in human pulmonary carcinogenesis is the cyp1a1 gene . in cyp1a1 gene studies , a polymorphic site assessable to mspi gives rise to two different hybridizable fragments in a southern blot analysis ( alleles c1 and c2 , respectively ) . the c2c2 genotype has previously been associated with an increased risk of lung cancer . so far 74 lung cancer patients , 30 patients with lung diseases other than cancer , and 118 healthy controls have been studied for cyp1a1 gene polymorphism . no association between the mspi restriction fragment length polymorphism in the cyp1a1 gene and lung cancer susceptibility has been found.imagesfigure 1 . afigure 1 . b

Images

pathogenic bacterial infections afflict countless numbers of individuals annually , with outcomes ranging from minor and treatable complications to death . food and waterassociated bacterial illnesses , estimated at 76 million illnesses per year , cost the us economy well in excess of $ 10 billion annually ( buzby and roberts , 1997 ; mead et al . , 1999 ) . development of accurate approaches for detecting and identifying diverse groups of pathogenic bacteria is essential for diagnosis and treatment of bacterial infections as well as assessing the toxicity potential of bacterial contamination . despite enormous advances in molecular and immunological ( dna and antibody ) detection technologies , pathogenic bacteria still escape detection . detection methods based on protein or dna structure specific to an organism often can not distinguish between live and dead bacteria or the potential of these organisms to cause disease ( mckillip et al . , 1998 ; sheridan et al . , 1998 ; nogva et al . , previously , a cellbased biosensor consisting of cultured primary chromatophore pigment cells , specifically erythrophores , from betta splendens ( siamese fightingfish ) , was described in terms of its functionbased detection of toxic agents such as herbicides , fungicides , genotoxins ( mojovic et al . , 2004 ) , cell receptor agonists and purified toxins ( dierksen et al . erythrophore cells are specialized pigmented nerve cells that can change appearance when physiologically stimulated ( elwing et al . , 1990 ; karlsson et al . , 1991 ; danosky and mcfadden , 1997 ) . erythrophore cell response to toxic agents can be easily detected by monitoring changes in optical density due to movement of pigment organelles ( chromatosomes ) within the erythrophore cell . pigment organelle movement can be dispersive ( pigment organelles move outwards toward the cell edge ) or aggregative ( movement towards the centre of the cell ) . the movement of pigment organelles along a radically arrayed microtubule network achieves visual changes in erythrophore cell morphology . pigment organelle movement is thought to be controlled through signal transduction pathways and other regulatory mechanisms in response to triggering events at various cell surface receptor sites , including gprotein coupled receptorbinding events ( danosky and mcfadden , 1997 ) . the movement of the pigment organelles within erythrophore cells can be measured by recording the change in pigment area occupied by the erythrophore , allowing realtime assessment of erythrophore cell response to toxic conditions ( dierksen et al . , 2004 ; little is known about the potential of erythrophore cells to detect biologically active foodassociated bacterial pathogens . bacillus cereus was selected as the initial model system because of its increasing implication in emetic and diarrheal forms of food poisoning ( turnbull , 1986 ; kramer and gilbert , 1989 ) . in addition to its link to gastrointestinal infections , b. cereus , an opportunistic pathogen , has been linked to numerous nongastrointestinal infections including , osteomyelitis , pulmonary and wound infections , bacteremia and septicemia ( kramer and gilbert , 1989 ; turnbull , 1986 ) . in this study , we present findings in support of the potential for erythrophore cells to be used to detect , as well as potentially assess toxicity for , the foodassociated pathogenic bacteria , b. cereus , clostridium perfringens , clostridium botulinum and salmonella enteritidis . a desirable feature of an erythrophore cellbased biosensor would be the ability to distinguish uninoculated bacterial culture medium ( representing the negative control ) from the cultured bacterial strains , thus erythrophore cell response to culture medium was included in this analysis . cultured in bhi ( brain heart infusion ) medium : bacillus subtilis 1a1 ( a laboratory reference strain ; yoshida et al . , 1995 ) , b. cereus atcc 49064 ( identified in a gastroenteritis outbreak and produces diarrheogenic enterotoxin ; midura et al . , 1970 ; taylor and gilbert , 1975 ; melling et al . , 1976 ) , b. cereus atcc 14579 ( frankland and frankland , 1887 ) and a b. cereus plcr deletion mutant strain that is a derivative of atcc 14579 ( salamitou et al . , 2000 ) . the negative control ( uninoculated bhi media ) caused slight dispersion of pigment organelles within the erythrophore cell ( fig . erythrophore cell response to b. subtilis 1a1 culture was similar in strength and rate to the response elicited by the negative control ( fig . 1b and d ) , and this response was found to be statistically insignificant ( p = 0.635 ) . 1c ) or atcc 14579 induced erythrophore pigment organelles to rapidly aggregate ; this response was detected within two to 5 min after addition of the bacterial culture ( fig . erythrophore cell response to b. cereus atcc 49064 or atcc 14579 was found to be statistically significant compared with the negative control ( p < 0.001 and p < 0.001 respectively ) . erythrophore cells appearance at time 0 ( left ) and time 20 min ( right ) upon exposure to : ( a ) bhi medium , ( b ) b. subtilis 1a1 , ( c ) b. cereus atcc 49064 . images are at 100 , the size bar represents a length of 100 m . ( d ) graphical display of erythrophore cell pigment area change in response to bacillus spp . a negative change in pigment area is indicative of pigment aggregation in erythrophore cells whereas a positive change in pigment area represents pigment dispersion . ( ) bhi medium ; ( ) b. subtilis 1a1 ; ( ) b. cereus atcc 49064 ; ( ) b. cereus atcc 14579 ; ( ) b. cereus plcr deletion mutant strain that is a derivative of atcc 14579 . data represent the mean values of three trials . to investigate the potential mechanism of erythrophore cell response to b. cereus , erythrophore cells were tested against a b. cereus atcc 14579 strain lacking the global gene regulator , plcr ( salamitou et al . , 2000 ) . the b. cereus plcr deletion mutant strain induced a similar response ( slightly dispersive rather than aggregative ) in erythrophore cells as the negative control ( bhi ) ( p = 0.0754 ; fig . to further evaluate erythrophore cell response , we investigated the response of erythrophore cells to additional foodassociated pathogenic bacteria including , the gramnegative bacterium , s. enteritidis atcc 4931 and the grampositive spore forming bacterium , c. perfringens sm101 ( zhao and melville , 1998 ) . erythrophore cells were exposed to s. enteritidis atcc 4931 grown in luria bertani broth ( lb ; fig . the difference between s. enteritidis and the negative control ( lb media ) was statistically significant ( p < 0.001 ) . clostridium perfringens sm101 was cultured in duncanstrong sporulation medium to allow production of the potent enterotoxin cpe ( elwing et al . clostridium perfringens induced aggregation of erythrophore pigment within 4 min followed by slow dispersion for the remainder of the assay ( fig . this response was very different from the dispersive response of the negative control ( duncanstrong sporulation medium ) and was statistically significant ( p < 0.004 ) . a negative change in pigment area is indicative of pigment aggregation whereas a positive change in pigment area represents pigment dispersion . a. ( ) lb medium ; ( ) s. enteritidis atcc 4931 . b. ( ) duncanstrong sporulation medium ; ( ) c. perfringens sm101 . clostridium botulinum was chosen as a second representative of a grampositive , spore forming , yet anaerobic bacterium associated with food contamination and potential use as a biological weapon . erythrophore cells were challenged with c. botulinum nctc 7272 ( type a ) or c. botulinum nctc 7273 ( type b ) cultured in bhi . erythrophore cell response was monitored for 1 h. very little response to c. botulinum was observed during this hour ; therefore monitoring time was extended to 6 h. two hours after exposure to c. botulinum nctc 7272 or nctc 7273 , pigment organelles began to slowly aggregate ( fig . 3a ) and aggregation of pigment organelles was complete at approximately 5 h. the erythrophore response to both c. botulinum cultures was statistically significant compared with the negative control ( bhi ) ( c. botulinum nctc 7272 p < 0.005 and c. botulinum nctc 7273 p < 0.006 respectively ) . the observed morphological changes of the erythrophore cells were unique from changes induced by other bacterial pathogens . extensive dendrite formation was observed in erythrophore cells treated with c. botulinum ( fig . 3c ) , while other bacterial pathogens induced pigment organelles to centrally localize within the erythrophore cell ( fig . ( a ) b. splendens erythrophore cell response after 6 h exposure to bacterial isolate . a negative change in pigment area is indicative of intracellular pigment aggregation whereas a positive change in pigment area represents pigment dispersion . ( ) c. botulinum nctc 7272 ; ( ) c. botulinum nctc 7273 ; ( ) bhi medium . ( b ) and ( c ) b. splendens erythrophore cells at 100 , the size bar represents a length of 100 m ; left , time = 0 h , right , time = 6 h , exposure to : ( b ) bhi medium , ( c ) c. botulinum nctc 7272 . this study demonstrates the potential of a novel bacterial biosensor populated with b. splendens erythrophore cells . when erythrophore cells are exposed to a bacterial pathogen , the response is easily observed and quantified . erythrophore cells respond to selected foodassociated bacteria , b. cereus , s. enteritis , c. perfringens and c. botulinum , and this response is different from the respective culture medium ( negative control ) . erythrophore cells were unable to distinguish the nonpathogenic bacterial strains , b. subtilis and the b. cereus plcr mutant strain from the negative control . the statistical and graphical evidence suggests that erythrophore cell response has potential for use as a biosensor in the detection or toxicity assessment for foodassociated pathogenic bacteria . what is it about a bacterial pathogen , such as b. cereus , that causes erythrophore cells to respond by relocating pigment organelles to the centre of the erythrophore cell ? the failure of erythrophore cells to respond to the nonpathogenic b. cereus plcr mutant strain provides insight to this intriguing question . plcr , a pleiotropic transcriptional regulator , controls the expression of numerous extracellular compounds , including several virulence factors ( agaisse et al . , 1999 ; the observation that b. cereus lacking plcr does not induce pigment aggregation in erythrophore cells suggests that failure to express one or several of the plcr regulated genes results in the failure of b. cereus to induce a response in erythrophore cells . lereclus and coworkers have shown that b. cereus pathogenicity is dependent on plcr ( lereclus et al . , 2000 ; salamitou et al . , 2000 ) , suggesting that erythrophore cells may interact with bacterial virulence traits or other gene products dependent on plcr . identification of b. cereus mutants that no longer induce a response in erythrophore cells is currently underway . identifying the mutant bacterial gene products will define the mechanism(s ) by which this bacterial pathogen induces a change in pigment organelle location within erythrophore cells . sm101 cause a slower rate of aggregation and less overall aggregation when compared with b. cereus atcc 49064 and atcc 14579 , suggesting that bacterial pathogens may interact with erythrophore cells through different mechanisms . additionally , when erythrophore cells are exposed to c. botulinum , the erythrophore cell morphology in response to this bacterium is different from reactions with other bacterial pathogens . the observation of specific erythrophore cell morphology in response to specific bacterial pathogens as well as differences in the kinetics of erythrophore cell response may provide another means to measure and characterize bacterial detection . reports of salmonella contaminated tomatoes , peanut butter and spinach and c. botulinum contaminated canned meats indicates the ease by which foodassociated bacterial pathogens evade detection , often resulting in massive recalls of popular food items . given the economic impact of massive recalls , as well as the lack of effective detection methods to enforce the usa 's and uk 's zero tolerance policy for certain foodassociated pathogenic bacteria , suggests a need for detection methods based on parameters different from those describing bacterial presence . although newer detection methods can identify single microbes , thus meeting zero tolerance requirement , many technical challenges remain for these methods . these challenges often include time intensive sampling and testing practices , long culture times to increase the number of bacteria to detectable levels , and costly shipment methods to move samples to a central laboratory for analysis ( cady et al . , 2005 ; batt , 2007 ) . furthermore , many of these methods rely on detecting a structure through the use of a dna or antibody probe , and thus are limited in the evaluation of toxicity and the potential of bacteria to cause disease . additionally , dna or antibodybased detection methods are limited in the detection of unidentified ( e.g. genetically rearranged ) or newly emerging ( e.g. new toxin producing variants ) pathogenic bacteria . while dna technology such as pcr may be able identify offending bacteria in the food , it can not assess if the detected bacteria are alive and exhibiting the pathogenic behaviour that make them a health risk . for example , the bacteria may be nonviable and not exhibiting pathogenic behaviour , but their dna may still be detectable by pcr technology , thus suggesting that information gathered from dna characterizing techniques , if taken alone , may be seriously misleading . the b. splendens erythrophore cell response described here represents a unique class of cellbased biosensors with potential to complement current detection methods as well as circumvent a number of limitations and challenges with the available technology . all bacillus spp . [ b. subtilis 1a1 ( yoshida et al . , 1995 ) , b. cereus atcc 14579 ( frankland and frankland , 1887 ) and a b. cereus plcr deletion mutant strain that is a derivative of atcc 14579 ( salamitou et al . , 2000 ) clostridium perfringens sm101 ( zhao and melville , 1998 ) was generously provided by dr mahfuzur r. sarker . briefly , a 0.1 ml aliquot of a frozen stock of c. perfringens sm101 was transferred into 6 ml of fluid thioglycollate ( ftg ) and then heat shocked for 20 min at 70c . the heatshocked culture was then incubated for 14 h at 37c , and 0.4 ml of this starter culture was transferred to a second 6 ml ftg before this culture was incubated for 9 h at 37c ( kokaikun et al . , 1994 ) . an aliquot ( 0.4 ml ) of this ftg culture was then added to 20 ml of duncan strong ( ds ) sporulation medium ( mcdonel et al . , 1988 ) and incubated for 8 h at 37c ( mcdonel et al . , 1988 ) . clostridium botulinum nctc 7272 and c. botulinum nctc 7273 were obtained from the national collection of type cultures , phls central public health laboratory . a 0.1 ml aliquot of a cooked meat medium stock of c. botulinum nctc 7272 and nctc 7273 was transferred to 1.0 ml of bhi medium . a 0.1 ml aliquot of the starter culture was transferred to a second 1 ml bhi tube before the culture was incubated for 24 h at 37c . all c. botulinum culturing was done in an anaerobic chamber using anaerogen gas packs ( fisher ) . all biosafety level 2 ( bsl2 ) bacterial agents were cultured in an approved bsl2 facility and performed under the requirements and regulations of and approved by oregon state university 's institutional biosafety committee . erythrophore cells were isolated from the tails and fins of b. splendens fish as described previously ( mojovic et al . , 2004 ) . briefly , tissue was washed in skinning solution [ 1 mm naedta , 5.6 mm glucose and penicillin streptomycin mixture 1:100 ( w / v ) in calcium and magnesiumfree phosphatebuffered saline ( pbs ) ] , digestion with an enzyme solution [ 2030 mg of collagenase type 1 ( worthington ) , and 13 mg of hyaluronidase ( worthington ) , in 7 ml of pbs ] . after several washes , the cell pellet was resupsended in a predetermined volume of leibovitz l15 medium+ ( l15 + , gibco # 21083027 , 500 ml of dyefree l15 was supplemented with 10 ml 1 m hepes ( gibcobrl 15630080 ) buffer and 5 ml antibioticantimycotic ( gibcobrl 15240062 ) to ensure the desired plate density in the centre of each well of a 24well microtitre plate or 48well microtitre plate . the l15 + medium was added in each well to a final volume of 1.5 ml ( 24well microtitre plate ) or 0.75 ml ( 48well microtitre plate ) and 5% fetal bovine serum ( hyclone # sh3007101 ) was added to each well . validation tests were performed for each preparation of erythrophore cells to ensure that isolated erythrophore cells were physiologically responsive . erythrophore cells were treated with a known dispersive agent , melanocyte stimulating hormone ( msh ) , and a known aggregative agent , clonidine , to verify physiological responsiveness ( dierksen et al . , 2004 ; mojovic et al . , 2004 ) . this effort was performed under the requirements and regulations of and approved by oregon state university 's institutional animal care and use committee , approval # 2979 and # 3513 . monitoring erythrophore cell response is based on observing and recording changes in the pigmented area of erythrophore cells exposed to bacterial agents and control agents as previously reported ( dierksen et al . a field of view containing approximately 100 erythrophore cells was monitored under a magnification of 100 . bacterial suspension or physiological response testers ( 400 nm clonidine , sigma c7897 ; 400 nm msh , sigma m4135 , respectively ) was added to each well to a final dilution of 1:10 in l15 medium ( viable cell counts were 10 for bacillus spp . and 10 cells salmonella and clostridium spp . respectively ) . digital images ( jpeg format ) at 300 300 resolution were taken using a spot insight 320 colour camera ( diagnostic instruments , sterling heights , mi , usa ) and spot software version 3.5.6.2 ( diagnostic instruments ) , through a leica ( leica , wetzlar , germany ) dmil inverted microscope ( bartels and stout ) . erythrophore response was recorded for 20 min ; images were captured at 0 , 0.25 , 0.5 , 1 , 2 , 4 , 10 and 20 min . for experiments lasting 6 h , images were captured at the same time interval as the 20 min experiments , then every 10 min up to 60 min . after 60 min , images were captured every 30 min until the experiment was completed . the images were opened in image pro plus 4.1 ( media cybernetics ) , exported and processed into excel . the total area in pixels ( digital area units ) for each captured image was determined and the percentage pigmented area change [ cell area change ( % ) = (a0 ax)/a0 100 , where a0 is the initial area and ax is the final area ] . an aggregative response would result in a negative per cent area change and a dispersive response would result in a positive per cent area change . the statistical analysis was carried out with s plus statistical software ( insightful technologies , seattle , wa ) . all bacterial challenges were completed in triplicate using three different erythrophore cell preparations ; reported values are the average of three trials . final time points were analyzed using a twosample ttest , all reported pvalues were declared significant at p < 0.05 . all bacillus spp . [ b. subtilis 1a1 ( yoshida et al . , 1995 ) , b. cereus atcc 14579 ( frankland and frankland , 1887 ) and a b. cereus plcr deletion mutant strain that is a derivative of atcc 14579 ( salamitou et al . , 2000 ) clostridium perfringens sm101 ( zhao and melville , 1998 ) was generously provided by dr mahfuzur r. sarker . briefly , a 0.1 ml aliquot of a frozen stock of c. perfringens sm101 was transferred into 6 ml of fluid thioglycollate ( ftg ) and then heat shocked for 20 min at 70c . the heatshocked culture was then incubated for 14 h at 37c , and 0.4 ml of this starter culture was transferred to a second 6 ml ftg before this culture was incubated for 9 h at 37c ( kokaikun et al . , 1994 ) . an aliquot ( 0.4 ml ) of this ftg culture was then added to 20 ml of duncan strong ( ds ) sporulation medium ( mcdonel et al . , 1988 ) and incubated for 8 h at 37c ( mcdonel et al . , 1988 ) . clostridium botulinum nctc 7272 and c. botulinum nctc 7273 were obtained from the national collection of type cultures , phls central public health laboratory . a 0.1 ml aliquot of a cooked meat medium stock of c. botulinum nctc 7272 and nctc 7273 was transferred to 1.0 ml of bhi medium . a 0.1 ml aliquot of the starter culture was transferred to a second 1 ml bhi tube before the culture was incubated for 24 h at 37c . all c. botulinum culturing was done in an anaerobic chamber using anaerogen gas packs ( fisher ) . all biosafety level 2 ( bsl2 ) bacterial agents were cultured in an approved bsl2 facility and performed under the requirements and regulations of and approved by oregon state university 's institutional biosafety committee . erythrophore cells were isolated from the tails and fins of b. splendens fish as described previously ( mojovic et al . , 2004 ) . briefly , tissue was washed in skinning solution [ 1 mm naedta , 5.6 mm glucose and penicillin streptomycin mixture 1:100 ( w / v ) in calcium and magnesiumfree phosphatebuffered saline ( pbs ) ] , digestion with an enzyme solution [ 2030 mg of collagenase type 1 ( worthington ) , and 13 mg of hyaluronidase ( worthington ) , in 7 ml of pbs ] . after several washes , the cell pellet was resupsended in a predetermined volume of leibovitz l15 medium+ ( l15 + , gibco # 21083027 , 500 ml of dyefree l15 was supplemented with 10 ml 1 m hepes ( gibcobrl 15630080 ) buffer and 5 ml antibioticantimycotic ( gibcobrl 15240062 ) to ensure the desired plate density in the centre of each well of a 24well microtitre plate or 48well microtitre plate . the l15 + medium was added in each well to a final volume of 1.5 ml ( 24well microtitre plate ) or 0.75 ml ( 48well microtitre plate ) and 5% fetal bovine serum ( hyclone # sh3007101 ) was added to each well . validation tests were performed for each preparation of erythrophore cells to ensure that isolated erythrophore cells were physiologically responsive . erythrophore cells were treated with a known dispersive agent , melanocyte stimulating hormone ( msh ) , and a known aggregative agent , clonidine , to verify physiological responsiveness ( dierksen et al . , 2004 ; mojovic et al . , 2004 ) . this effort was performed under the requirements and regulations of and approved by oregon state university 's institutional animal care and use committee , approval # 2979 and # 3513 . monitoring erythrophore cell response is based on observing and recording changes in the pigmented area of erythrophore cells exposed to bacterial agents and control agents as previously reported ( dierksen et al . a field of view containing approximately 100 erythrophore cells was monitored under a magnification of 100 . bacterial suspension or physiological response testers ( 400 nm clonidine , sigma c7897 ; 400 nm msh , sigma m4135 , respectively ) was added to each well to a final dilution of 1:10 in l15 medium ( viable cell counts were 10 for bacillus spp . and 10 cells salmonella and clostridium spp . respectively ) . digital images ( jpeg format ) at 300 300 resolution were taken using a spot insight 320 colour camera ( diagnostic instruments , sterling heights , mi , usa ) and spot software version 3.5.6.2 ( diagnostic instruments ) , through a leica ( leica , wetzlar , germany ) dmil inverted microscope ( bartels and stout ) . erythrophore response was recorded for 20 min ; images were captured at 0 , 0.25 , 0.5 , 1 , 2 , 4 , 10 and 20 min . for experiments lasting 6 h , images were captured at the same time interval as the 20 min experiments , then every 10 min up to 60 min . after 60 min , images were captured every 30 min until the experiment was completed . the images were opened in image pro plus 4.1 ( media cybernetics ) , exported and processed into excel . the total area in pixels ( digital area units ) for each captured image was determined and the percentage pigmented area change [ cell area change ( % ) = (a0 ax)/a0 100 , where a0 is the initial area and ax is the final area ] . an aggregative response would result in a negative per cent area change and a dispersive response would result in a positive per cent area change . the statistical analysis was carried out with s plus statistical software ( insightful technologies , seattle , wa ) . all bacterial challenges were completed in triplicate using three different erythrophore cell preparations ; reported values are the average of three trials . final time points were analyzed using a twosample ttest , all reported pvalues were declared significant at p < 0.05 .

summarycellbased biosensors have been proposed for use as functionbased detectors of toxic agents . we report the use of betta splendens chromatophore cells , specifically erythrophore cells , for detection of foodassociated pathogenic bacteria . evaluation of erythrophore cell response , using bacillus spp . , has revealed that this response can distinguish pathogenic bacillus cereus from a nonpathogenic b. cereus plcr deletion mutant and a nonpathogenic bacillus subtilis . erythrophore cells were exposed to salmonella enteritidis , clostridium perfringens and clostridium botulinum . each bacterial pathogen elicited a response from erythrophore cells that was distinguished from the corresponding bacterial growth medium , and this observed response was unique for each bacterial pathogen . these findings suggest that erythrophore cell response has potential for use as a biosensor in the detection and toxicity assessment for foodassociated pathogenic bacteria .

Introduction Results Discussion Experimental procedures Bacterial culture preparation Erythrophore cell preparation Erythrophore cell response and computer analysis Statistical analysis

it is the main independent risk factor for developing type 2 diabetes mellitus ( t2dm),1,2 leading to a condition known as diabesity.2,3 in patients with morbid obesity , the likelihood of developing t2 dm and glucose intolerance ( gi ) is further increased.1,4 bariatric surgery provides sustained weight loss and leads to well - documented remission of t2 dm in obese , diabetic patients.5,6 patients who undergo bariatric surgery have a decreased rate of long - term mortality compared to obese patients who do not receive bariatric surgery,7 with 136 lives saved per 10,000 surgical procedures performed.8 currently , bariatric surgery is the most effective choice of treatment for morbidly obese patients with t2dm.9 surgical procedures to treat morbid obesity are divided into two groups : gastric restrictive procedures and procedures that combine gastric restriction and malabsorption.10 the roux - en - y gastric bypass ( rygb ) procedure is well - established and is the most frequent bariatric surgery performed,7,11 whereas sleeve gastrectomy ( sg ) is an emerging restrictive procedure.12 sg can be performed as the first of a two - stage operation in patients at high risk of death13,14 or as a definitive surgical procedure.15 it has shown good results with regard to weight loss16 and glycemic control in various studies.6,14,16,17 the potential advantages of sg include a lower probability of vitamin and mineral deficiencies than rygb , access to the entire intestinal tract , a lack of need for a subcutaneous access port and a lower risk of intestinal obstruction . in addition , sg can be performed in patients who have inflammatory bowel disease or have undergone previous bowel surgery , and it can be easily converted into a rygb.12,15 both sg and rygb can be performed with or without the placement of a silastic ring.18,19 metabolic control can be achieved with gastric restrictive procedures such as vertical banded gastroplasty,5 adjustable gastric banding20 and , more recently , sg.17 however , previous studies have found that glucose homeostasis is affected by various intestinal mechanisms that are only altered by bariatric surgery procedures that include an intestinal element,21 such as rygb.2224 a systematic review showed resolution of t2 dm in 76.8% of patients undergoing rygb and improvement of glycemic control in 86% of patients.25 of the criteria used to diagnose the metabolic syndrome , fasting glucose levels26 are the first to return to normal in patients who have undergone silastic ring sleeve gastrectomy . the achievement of normoglycemia after bariatric procedures results from multiple changes that occur postoperatively,6,27,28 such as dietary control,20,21 decreased plasma ghrelin levels,29,30 weight loss , reduction of body fat,6 and the release of gastrointestinal hormones that interfere with the function of pancreatic cells ( incretins).23,24,31,32 the main purpose of this study was to assess weight loss in morbidly obese patients undergoing srsg as well as to evaluate the effects of the srsg procedure on glucose homeostasis . it was approved by the research ethics committee of the university hospital of the federal university of esprito santo , brazil ( protocol no . 049/06 ) . to obtain a homogenous sample , we adopted the following inclusion criteria : we included female patients 2060 years with a bmi of 4045 who agreed to provide written informed consent . the exclusion criteria included the following : secondary obesity , alcohol or drug use , presence of a severe psychiatric disorder , binge - eating disorders and previous stomach or bowel surgery . the patients had a mean age of 36.7 9.4 years , a mean bmi of 42.33 1.5 and a mean waist circumference of 118.7 5.98 cm . the mean preoperative fasting glucose level of the included patients was 108.5 43.76 mg / dl . the diagnoses of diabetes and gi were based on the criteria adopted by the brazilian diabetes society.33 t2 dm was found in 11 patients ( 33.3% ) , and gi was found in 4 patients ( 12.1% ) . therefore , 45.4% of the morbidly obese patients analyzed in the present study presented with elevated fasting glucose levels . most of the diabetic patients were treated with oral hypoglycemic agents . assessment was performed one year ( range : 12 to 14 months ) after surgery . the surgical procedures were performed between december 08 , 2006 and july 27 , 2007 at hospital at hospital universitrio cassiano antonio moraes da universidade federal do esprito santo ( hucam / ufes , cassiano antonio moraes university hospital , federal university of esprito santo ) . the procedures were performed by the same surgeon using similar anesthetic techniques ( epidural anesthesia combined with general anesthesia ) . silastic ring sleeve gastrectomy was performed as follows : the vessels of the greater curvature of the body and fundus of the stomach were ligated , and the fundus and part of the body of the stomach was resected using a linear stapler ( 80 mm , tyco ) . a 32-fr tube was then used to calibrate the diameter of the remaining stomach and a 6.2-cm silastic ring was placed around the stomach , 5.0 cm below the esophagogastric junction . the staple lines were then oversewn , and a methylene blue test was performed to verify that the staple line was secure . the patients were given a liquid diet on the first postoperative day and were discharged on the third postoperative day . they received dietary guidance and instructions regarding physical activities and were also prescribed vitamin and mineral supplements . the percentage of excess bmi lost was calculated using the following formula : excess bmi loss = ( preoperative bmi - current bmi ) ( preoperative bmi 25 ) 100.34 glucose homeostasis was assessed through the measurement of fasting plasma glucose levels and an oral glucose tolerance test , after hipoglicemic agents were discontinued . the results of the descriptive analyses were expressed as means , standard deviations , medians , frequency ( % ) , minimum values and maximum values . the wilcoxon matched pairs test was used to analyze the differences between pre - treatment and post - treatment plasma glucose levels . the mcnemar test was used to compare the rates of t2 dm and gi pre- and post - treatment . it was approved by the research ethics committee of the university hospital of the federal university of esprito santo , brazil ( protocol no . 049/06 ) . to obtain a homogenous sample , we adopted the following inclusion criteria : we included female patients 2060 years with a bmi of 4045 who agreed to provide written informed consent . the exclusion criteria included the following : secondary obesity , alcohol or drug use , presence of a severe psychiatric disorder , binge - eating disorders and previous stomach or bowel surgery . the patients had a mean age of 36.7 9.4 years , a mean bmi of 42.33 1.5 and a mean waist circumference of 118.7 5.98 cm . the mean preoperative fasting glucose level of the included patients was 108.5 43.76 mg / dl . the diagnoses of diabetes and gi were based on the criteria adopted by the brazilian diabetes society.33 t2 dm was found in 11 patients ( 33.3% ) , and gi was found in 4 patients ( 12.1% ) . therefore , 45.4% of the morbidly obese patients analyzed in the present study presented with elevated fasting glucose levels . assessment was performed one year ( range : 12 to 14 months ) after surgery . the surgical procedures were performed between december 08 , 2006 and july 27 , 2007 at hospital at hospital universitrio cassiano antonio moraes da universidade federal do esprito santo ( hucam / ufes , cassiano antonio moraes university hospital , federal university of esprito santo ) . the procedures were performed by the same surgeon using similar anesthetic techniques ( epidural anesthesia combined with general anesthesia ) . silastic ring sleeve gastrectomy was performed as follows : the vessels of the greater curvature of the body and fundus of the stomach were ligated , and the fundus and part of the body of the stomach was resected using a linear stapler ( 80 mm , tyco ) . a 32-fr tube was then used to calibrate the diameter of the remaining stomach and a 6.2-cm silastic ring was placed around the stomach , 5.0 cm below the esophagogastric junction . the staple lines were then oversewn , and a methylene blue test was performed to verify that the staple line was secure . the patients were given a liquid diet on the first postoperative day and were discharged on the third postoperative day . they received dietary guidance and instructions regarding physical activities and were also prescribed vitamin and mineral supplements . the percentage of excess bmi lost was calculated using the following formula : excess bmi loss = ( preoperative bmi - current bmi ) ( preoperative bmi 25 ) 100.34 glucose homeostasis was assessed through the measurement of fasting plasma glucose levels and an oral glucose tolerance test , after hipoglicemic agents were discontinued . the results of the descriptive analyses were expressed as means , standard deviations , medians , frequency ( % ) , minimum values and maximum values . the wilcoxon matched pairs test was used to analyze the differences between pre - treatment and post - treatment plasma glucose levels . the mcnemar test was used to compare the rates of t2 dm and gi pre- and post - treatment . although it was not the main focus of the present study , it was noteworthy that two patients ( 6% ) developed a fistula at the staple line at the level of the cardiac notch . one patient died , and the other underwent total gastrectomy , which led to the resolution of the fistula . these patients , neither of whom had t2 dm or gi , were excluded from the postoperative analysis . therefore , a total of 31 patients were included in the postoperative analysis . in these 31 patients , bmi decreased from 42.27 1.46 kg / m to 27.4 2.42 kg / m ( p < 0.001 ) , a reduction of 35.18% ( figure 1 ) . waist circumference decreased from 118.42 5.71 cm to 89.87 6.66 cm ( p < 0.001 ) ( figure 2 ) . the percentage of excess bmi loss was 86.51 14.2% ( 46.6 108.5% ) . the reduction in glucose levels was also significant ( p < 0.001 ) , with mean plasma glucose values decreasing from a preoperative mean of 109.77 44.19 to a postoperative mean of 80.94 6.3 mg / dl ( figure 3 ) . srsg proved to be effective in promoting the resolution of t2 dm and gi in affected patients ( p < 0.001 ) ( figure 4 ) . all patients were able to discontinue the use of oral hypoglycemic agents , insulin , or both , during the follow - up period ( table 1 ) . obesity can cause deleterious effects on many organic functions and impair health and quality of life.35 the sg procedure is used with increasing frequency in bariatric surgery.1217 however , there are few prospective clinical studies in the literature that compare this emerging procedure with the gold standard , rygb.12,17 in the present study , sg was performed , and a silastic ring was placed around the stomach . we thus created a small , functional stomach , much like that created during traditional vertical gastroplasty . we also removed the principal site of ghrelin production , which gave the srsg the characteristics of both bariatric and endocrine surgery.19 the removal of the principal site of ghrelin production led to a decrease in ghrelin levels , adding a hormonal component to srsg that other restrictive procedures lack , such as adjustable gastric banding.31 some authors have reported the use of added restriction in sg to increase the intensity and duration of weight loss.19,36,37 some researchers have reported that sg is less risky than rygb.1214 in our sample , however , serious complications and death occurred after sg . we observed significant weight loss , bmi reduction , waist circumference reduction and excess bmi loss in this study . these findings are in accordance with several studies12,15,17 but are in disagreement with others13,14 that regarded sg as simply the first stage of a definitive surgery . the promising results of the present study are most likely due ( at least in part ) to the judicious inclusion criteria we chose , which excluded patients with a bmi greater than 45 and patients who had undergone prior stomach or bowel surgery . in these patient populations , other factors that might have contributed to the promising results of the present study include the calibration of the remaining stomach using a 32-fr tube and the placement of a silastic ring . in other studies in which weight loss was less pronounced , tubes of a larger caliber were used39 , and a silastic ring was not placed.13,14 resolution of t2 dm has been well - documented in various types of bariatric surgery.5 in two studies of patients undergoing sg , control of t2 dm was achieved in 80% of patients.13,14 this remission rate was higher than the rate that is commonly reported for restrictive procedures like vertical banded gastroplasty5 and adjustable gastric banding.20 this rate , however , is lower than that obtained with rygb9,26 and biliopancreatic diversion procedures.21 in the present study , all of the patients with t2 dm or gi went into clinical remission , a surprising result also found in another study.17 because srsg is basically a restrictive procedure that does not affect incretin expression , it was expected that the results obtained from this procedure would be inferior to those obtained from procedures in which a duodenal switch is performed with regard to glucose homeostasis.23,24,27,28 in the present study , glucose levels might have decreased as a result of the marked weight loss observed in all patients , which led to increased insulin sensitivity and decreased leptin production , and thus to increased insulin secretion and remission of gi and t2 dm , as noted in previous studies.6,39 other hormones produced in adipose tissue might also have been involved in the notable rate of glycemic control observed in the present study . it is notable , however , that only one patient who underwent surgery in the present study used insulin preoperatively . the other 14 patients with gi or t2 dm were treated with oral hypoglycemic agents . according to previous studies , remission or dietary control of gi / t2 dm is more likely to be possible in these patients after bariatric surgery than patients who required insulin preoperatively.22,24 the greatest limitations of the present study were the short follow - up period and the lack of a control group for comparison . however , the study sample was comprised of patients with similar baseline characteristics , namely age , bmi , waist circumference and preoperative fasting glucose levels . the similar baselines among patients is an advantage for this study over previous studies in which the patient populations were not similar at baseline,12 and the patients often had bmis of < 4012,15,17 and/or > 50.1217 the surgical procedure in the present study , srsg , resulted in marked weight loss , bmi reduction , waist circumference reduction , excess bmi loss , improved glucose homeostasis and remission of gi and t2 dm in our study population . further studies need to evaluate srsg with a longer follow - up period , a control group and the inclusion of other variables , such as hormonal changes , to solidify srsg s standing as a bariatric surgery procedure .

objectiveto assess glucose homeostasis and weight loss in morbidly obese patients undergoing silastic ring sleeve gas-trectomy.methodsthis was a prospective clinical study . thirty - three female patients with a mean body mass index ( bmi ) of 42.33 1.50 kg / m2 ( range : 4045 kg / m2 ) , a mean age of 36.7 9.4 years and a mean waist circumference of 118.7 5.98 cm were included in this study . type 2 diabetes mellitus was observed in 11 patients ( 33.3% ) , and glucose intolerance was observed in 4 patients ( 12.1% ) . mean plasma fasting glucose levels were 109.77 44.19 mg / dl ( 75320 ) in the preoperative period . all silastic ring sleeve gastrectomy procedures were performed by the same surgical team using the same anesthetic technique . the patients were monitored for at least 12 months after surgery.resultsthe mean weight of the patients decreased from 107.69 6.57 kg to 70.52 9.36 kg ( p < 0.001 ) , the mean bmi decreased to 27.4 2.42 kg / m2 ( p < 0.001 ) , and the mean waist circumference decreased to 89.87 cm 6.66 ( p < 0.001 ) in the postoperative period . excess bmi loss was 86.5 14.2% . fasting glucose levels were reduced to 80.94 6.3 mg / dl ( p < 0.001 ) . remission of diabetes and glucose intolerance was observed in all patients.conclusionsilastic ring sleeve gastrectomy was effective in promoting weight loss , waist circumference reduction and control of glucose homeostasis in morbidly obese patients .

INTRODUCTION METHODS Study protocol Sample Surgical procedure Assessment Statistical analysis RESULTS DISCUSSION CONCLUSIONS

obesity rapidly becomes a worldwide epidemic disease with increased risk of cardiovascular diseases , type 2 diabetes mellitus , and metabolic syndrome . metabolic syndrome is characterized by increased visceral adiposity , hyperlipidemia , insulin resistance , and hypertension . the liver is the largest visceral organ for maintaining homeostasis in glucose , lipid , and protein . hepatic steatosis is characterized by massive fat accumulation in the liver and thus is strongly related to several features of metabolic syndrome , including hyperlipidemia and insulin resistance . indeed , reduction or loss of insulin action in the liver leads to abnormally increased hepatic gluconeogenesis , glucose production , and lipogenesis , as well as decreased insulin clearance , hepatic glucose uptake , and lipolysis , consequently resulting in dyslipidemia . age and sex are physiologic factors that have strong association with the prevalence and features of metabolic syndrome . the state of estrogen deficiency as seen in postmenopausal women and the state of androgen deficiency as seen in aging men predispose older population to the metabolic syndrome and associated diabetes and cardiovascular diseases , indicating that sex hormones play important roles in regulating energy metabolism [ 5 , 6 ] . nonalcoholic fatty liver disease ( nafld ) disproportionally affects people with obesity , diabetes with insulin resistance , and dyslipidemia [ 79 ] . the prevalence of nafld varies among ethnicities , with the highest prevalence in hispanics , correlated with the high prevalence of obesity and insulin resistance in this ethnic group , compared to whites and blacks . similar to the incidence of metabolic syndrome , the frequency of nafld varies between genders , with greater prevalence in men than in women among whites ( 42% in white men versus 24% in white women ) but not in other ethnicities . this is consistent with another epidemiology study showing that the rate of nafld is a little higher in men than in women with all ethnicities combined . interestingly , nafld is twice as common in postmenopausal women as in premenopausal women whose estrogen levels are higher than postmenopausal women [ 7 , 11 ] , which suggests the protective role of estrogens in nafld [ 12 , 13 ] . in general , androgens are considered as hormones of the male sex due to their masculinizing effects and their roles in regulating male sexual behavior , whereas estrogens are considered as hormones of the female sex due to their roles in regulating female reproductive physiology and behaviors , although all sex hormones are present in both males and females , albeit at different levels between these two sexes . the most important biologically relevant forms of estrogens and androgens in humans are estradiol ( e2 ) and testosterone , respectively . understanding of how estrogens and androgens regulate energy metabolism via their receptors may shed light on potential pharmaceutical applications . in the present review , we discuss the roles of estrogens and androgens in regulating liver glucose and lipid homeostasis in rodents and humans . we also deliberate the distinct , important effects of estrogen receptors ( ers ) and androgen receptors ( ars ) on the regulation of liver metabolism . in both males and females , e2 is derived from the aromatization of testosterone . in premenopausal women , e2 is mainly synthesized from cholesterol in the ovaries , with e2 concentration being approximately 5 times higher than that in men , while in postmenopausal women e2 is primarily converted from testosterone by aromatase in peripheral tissues , such as adipose tissue , adrenal glands , bones , vascular endothelium , and smooth muscle , with e2 concentration being similar compared with men ( http://www.hemingways.org/gidinfo/hrt_ref.htm ) . estrogens act on ers , including classic nuclear receptors er- and er- , and membrane - bound receptors , including g protein - coupled er ( gper , also known as gpr 30 ) and membrane - associated er- and er- variants . all these nuclear and membrane er subtypes are expressed in the livers of male and female humans and rodents , but at a lower level compared with reproductive organs such as uterus , prostate , testis , ovary , and breast [ 1618 ] . er- is less abundant in liver cells than er- [ 19 , 20 ] and gper ( unpublished observation ) . one study by lax et al . determines levels of ers in male and female rat livers and reports that the levels of nuclear ers are not sex dependent but are age dependent , as levels of ers are similar between male and female rats and vary with the course of life in a comparable manner in males and females . specifically , levels of ers in the liver of both male and female rats are the highest during the perinatal period , decline till the onset of puberty , and increase to reach postpubertal peak . additionally , levels of ers are maintained as a stable level across the estrous cycles of female rats . consistently , eisenfeld group has reported that er concentration in the rat liver increases evidently at puberty . ovariectomy ( ovx ) , a procedure that removes ovaries and thus majority of endogenous estrogens , is a suitable preclinical model to study postmenopausal diseases . liver er- expression does not change following ovx ; however , it significantly increases by e2 treatment at a superphysiological level in rats with ovx , higher than sham - operated rats with intact ovaries and normal levels of endogenous estrogens . these studies indicate that er- expression in the liver is similar between gonad intact males and females and remains stable in postmenopausal females but could increase following hormone replacement therapy or during puberty . there is no available literature showing changes of expression of er- and gper during menstrual period or postmenopausal stage , and these questions remain unknown . males also express ers in the liver , and aromatase metabolizes androgens to generate e2 and other estrogen metabolites locally in many target tissues . a growing body of evidence suggests that estrogens also have important metabolic functions in males . the aromatization of testosterone to e2 is beneficial for preventing intra - abdominal adiposity in men , demonstrated by a clinical study showing increased intra - abdominal fat in men by reduced estrogens due to aromatase inhibition . the effects of estrogens on male and female reproductive organs have been extensively studied , but the beneficial effects of estrogens in nonclassical endocrine targets including the liver are less appreciated . we will discuss how hepatic estrogen signaling via ers regulates metabolism in male and female animal and human models . upon estrogen binding , classic estrogen nuclear receptors er- and er- form homo- or heterodimers and bind to estrogen response element ( ere ) in target gene promoters or to other transcription factors , such as activator protein-1 ( ap-1 ) and stimulating protein-1 , to induce expression of target genes . the genomic action following e2-er binding varies as the level of sex hormone changes . specifically , the transcriptional activity of er- alters during the 4-day estrous cycle , demonstrated by using ere - luciferase reporter mice which have luciferase reporter controlled by activated ers . the peak of the transcriptional activity of er- in the liver occurs in proestrus , indicating dynamics of er- transcriptional activity that is possibly modulated by different concentration of estrogens . these findings suggest that liver er- could recognize the changes in circulating e2 levels and response to reproductive cues during transition of different stages of the estrous cycles and select appropriate genetic programs to adapt the hepatic metabolism to the energy requirements of each stage . there are many lines of evidence showing that the full length er- and truncated er- may exert actions via nongenomic signaling which is faster than the classic genomic signaling . such nongenomic signaling usually involves activation of intracellular second messenger systems , such as protein kinase a ( pka ) , protein kinase c , and mitogen - activated protein kinase ( mapk)/extracellular signal - regulated protein kinase ( erk ) [ 2830 ] . gper is structurally unrelated to er- and er- and is a seven - transmembrane domain g protein - coupled receptor located at the cell membrane and endoplasmic reticulum membrane . gper is reported to rapidly activate different nongenomic estrogen signaling pathways , including pka , mapk / erk , and phosphoinositide 3-kinase ( pi3k ) ( figure 1 ) . females , as compared with males , tend to store more energy in subcutaneous fat instead of in visceral fat . the liver is a key visceral organ for controlling energy storage , as the liver has high capacity for lipid transport , de novo lipogenesis , lipid oxidation , and lipolysis . liver steatosis , as seen in the nonalcoholic fatty liver disease ( nafld ) , is due to the excess of triglyceride ( tg ) accumulation within the hepatocytes . incidence of hepatic steatosis is frequently associated with low levels of high density lipoprotein cholesterol ( hdl - c ) and high levels of low density lipoprotein cholesterol ( ldl - c ) in the circulation . epidemiological studies have showed higher plasma level of ldl - c and lower plasma level of hdl - c in men and postmenopausal women compared with premenopausal women , suggesting that lower circulating estrogen levels may promote fat deposition in the liver . further evidence is supported by using ovx mouse model combined with pair - feeding between sham operation and ovx groups . removal of the ovaries and thus the majority of endogenous estrogens in female mice results in increased fat proportion in the liver even when they are pair - fed with the same amount of calories as females with intact ovaries , which indicates the direct role of estrogens in inhibiting lipogenesis in the liver , rather than the secondary effects to ovx induced overfeeding . in another e2-deficient aromatase knockout ( arko ) mouse model , spontaneous obesity and hepatic steatosis result from impaired fatty acid -oxidation and elevated fatty acid synthase ( fas ) in the liver in both female and male mice . these findings are further supported by previous studies demonstrating that e2 inhibits lipogenic gene expression and lipid uptake in the liver by decreasing lipoprotein lipase activity , as well as promoting lipolysis by increasing expression of hormone - sensitive lipase and adipose tg lipase in the liver [ 35 , 36 ] . er- is the predominant er subtype presented in both male and female hepatocytes [ 19 , 20 ] . estrogen signaling is important in both males and females in the regulation of lipogenesis , demonstrated by using animal models and human studies . specifically , estrogens regulate the activity and expression of lipogenic genes to directly inhibit lipogenesis in several animal species [ 37 , 38 ] . one study of genome - wide analyses demonstrated that the subtle oscillations of estrogens occurring during the estrous cycle are sufficient to influence liver gene expression , and that ers are involved in the pulsatile synthesis of fatty acids and cholesterol in the liver . thus this study demonstrated the importance of the maintenance of estrogen oscillation to limit fat deposition in the hepatic tissues in females . additionally , treatment of the specific er- agonist ppt decreases weight , fat mass , and tg in the liver in both wild - type mice and obese ob / ob mice [ 39 , 40 ] . thus , the metabolically protective effect of estrogen may be attributed to estrogen signaling via er- . this is further demonstrated by investigation of estrogen and estrogen signaling using knockout or transgenic animal models . male and female er- knockout mice exhibit hepatic steatosis by increasing gene expression of lipogenic transcription factors such as sterol regulatory element binding protein 1c ( srebp-1c ) and decreasing lipid transport genes [ 42 , 43 ] . mice with liver - specific er- knockout [ 44 , 45 ] or liver - specific gper knockout show increases in fat accumulation in the liver and develop disturbed insulin signaling under high - fat diet ( hfd ) feeding . thus , hepatic steatosis has been observed in both of the above genetic models , one with liver - specific er- knockout with functional gper and the other with liver - specific gper knockout with functional er-. thus , although it is widely recognized that estrogens regulate liver lipid metabolism and reduce triglyceride accumulation in the liver mainly via er- [ 47 , 48 ] , both er- and gper are required to be present in the liver to maintain lipid homeostasis . male but not female mice in which the aromatase gene has been deleted ( arko ) develop hepatic steatosis that can be normalized by estrogen treatment . thus , e2 treatment reduces fatty acid synthesis and lipid accumulation and prevented nafld in castrated male rats . hepatic tg and diacylglyceride increase in the livers of er- knockout male mice under hfd feeding , explained by dysregulation of insulin - stimulated acc phosphorylation and dgat1/2 protein levels . interestingly , a recent study using specific plasma membrane er- knockout has demonstrated that it is the membrane - localized er- , but not nuclear er- , that is responsible for protection from hyperlipidemia by decreasing expressions of many hepatic genes involved in lipid synthesis , at least in female mice with ovx . although er- is antilipogenic in the liver , the role of er- in the liver is not consistent in the literature . er- deficient mice have higher body weight but lower liver weight due to increased insulin sensitivity and decreased tg accumulation in the liver , indicating that er- might be lipogenic and diabetogenic in the liver . opposite finding has been reported where , different from treatment of e2 or er- agonists that decrease hepatic ppar expression , treatment of er- agonist 8-ve2 comparably elevates ppar expression to the same mrna level as non - drug treated group in the liver of hfd - fed female rats with ovx . interestingly , all treatments of e2 , er- agonist , or er- agonist are capable of reducing tg accumulation in the liver of hfd - fed rats with ovx . thus , the mechanism for reduced hepatic lipid accumulation in both suppressed er- signaling as seen in er- knockout mice and activated er- signaling as seen in er- agonist - treated rats is awaiting further elucidation . hepatic steatosis is also found in gper deficient female mice fed with hfd rather than male mice . although both 6-month - old female and male gper ko mice display increased body weight , only female mice had glucose intolerance , while male mice developed glucose intolerance at the age of 18 months . furthermore , gper agonist g-1 decreases fatty acid synthesis and tg accumulation in both human and rodent pancreatic cells , but the effect of g-1 treatment on lipid metabolism in the liver is not clear . however , it is possible that gper has greater impacts on male lipid regulation , whereas membrane - associated er- variant may have greater impacts on female lipid regulation , as female livers have markedly higher expression of all three membrane - associated er- variants compared with male livers . hepatic glucose homeostasis is determined by glucose uptake and glucose production . the major glucose transporter ( glut ) in the liver is glut2 that bidirectionally transports glucose across liver cell plasma membrane , efflux of glucose formed from gluconeogenesis or glycogenolysis out of liver cells , and uptake of circulating glucose into liver cells . since estrogen treatment has been shown to increase insulin synthesis and release , estrogens might indirectly increase glut2 expression in the liver , which has not been demonstrated yet . a recent study demonstrates that it is estriol , instead of e2 , that downregulates glut2 in pregnant women during late stages of pregnancy whose peak postprandial glucose levels are much lower than glucose levels of healthy nonpregnant women . several lines of evidence show that intravenous conjugated estrogen treatment or low dose of oral contraceptive does not significantly alter insulin sensitivity but slightly increases hepatic insulin clearance in postmenopausal women [ 60 , 61 ] . estrogens reduce gluconeogenesis and increase glycogen synthesis and storage in the liver , lowering circulating glucose level [ 43 , 62 ] . additional observations using rodents with ovx that lacks majority of endogenous estrogens support the notion that estrogens lower glucose levels [ 63 , 64 ] . a recent study reports increased glucagon signaling due to increased amount of glucagon receptor that accounts for enhanced glucose production , accompanied with increased gluconeogenic enzymes in rats with ovx . interestingly , such changes can not be prevented by e2 replacement , which indicates that disrupted liver glucose homeostasis following ovx is not merely caused by deficiency of endogenous e2 but could be caused by deficiency of other ovarian hormones such as progesterone . although classic nuclear progesterone receptor has not been found in the liver [ 22 , 66 ] , progestins can either bind to membrane - bound progesterone receptors or bind to ars in human liver and carry metabolic effects . on the other hand , estrogens are also found to facilitate epinephrine 's action via 2-adrenergic receptor in regulating glycogenolysis and gluconeogenesis in the rat liver to increase circulating glucose level . estrogen signaling is important in both males and females in the regulation of glucose homeostasis , improving glucose tolerance and insulin sensitivity , demonstrated by using animal models and human studies [ 6971 ] . additionally , although estrogens do not affect hepatic glucose metabolism in vivo , estrogens increase insulin receptor to enhance glucose metabolism in vitro [ 72 , 73 ] . er- deficient mice exhibit significantly impaired glucose tolerance and hepatic insulin resistance , while er- deficient mice exhibit normal glucose tolerance , suggesting that er- instead of er- plays an important role in the regulation of hepatic glucose homeostasis . the importance of er- in the regulation of hepatic glucose tolerance is further supported by inadequate suppression of hepatic glucose production during hyperinsulinemic clamp study in er- deficient mice . although impaired glucose tolerance is seen in gper1 knockout mice , glut2 and glucokinase are not affected , and glucose production in liver has not been measured yet . the rats treated with e2 or er- agonist have reduced ppar expression in the liver , whereas the rats treated with er- agonist maintain a similarly high mrna level of ppar as non - drug treated hfd - fed rats with ovx . the sustained hepatic ppar gene expression correlates with increased glucose uptake into the liver of rats with ovx . the liver is the principal organ for cholesterol de novo biosynthesis , which is catalyzed by the rate limiting enzyme 3-hydroxy-3-methyl - glutaryl - coa reductase ( hmgr ) . the srebp-1c is the master regulator of cholesterol by stimulating transcription of ldl and hmgr . a previous in vitro study points out that hmgr promoter is induced by estrogen treatment in the breast cancer cell line mcf-7 but not in any hepatic cell line , indicating differential regulation of hmgr by estrogens among different tissues . estrogen treatment does not increase cholesterol synthesis in liver cells in vitro . in an in vivo study using castrated male rats , dht , but not e2 , treatment increases phosphorylation of hmgr to decrease cholesterol synthesis in the liver . thus , at least in castrated male rats , androgen action is associated with downregulation of cholesterol biosynthesis in the liver . estrogens also decrease ldl level and increase hdl to promote cholesterol secretion into bile in postmenopausal women . total cholesterol and ldl are elevated in arko mice with e2 deficiency . increased hepatic hmgr activity and subsequently increased levels of cholesterol and ldl are seen in rats with ovx with reduced level of endogenous estrogens estrogen replacement in both arko mice and rats with ovx normalizes the levels of ldl and cholesterol . the above mentioned cell , animal , and human studies collectively indicate important roles of estrogens in reducing ldl and increasing hdl . er- is able to protect the liver from hypercholesterolemia [ 47 , 48 ] . to support this , lack of er- ( whole body ) is associated with increased expression of genes involved in lipid biosynthesis and lipid metabolism . a male patient without functional er- has been reported with dyslipidemia , supporting the importance of er- in regulating cholesterol homeostasis . consistently , the expressions of er- ( and ar ) and phosphorylated hmgr are significantly reduced in the human liver samples from male severe steatotic nafld patients compared with the liver samples from subnormal men . aromatase deficient mice without endogenous estrogen production exhibit obesity and dyslipidemia and mice with liver - specific er- knockout accumulate liver triglycerides and diacylglycerides [ 42 , 43 ] . in contrast , er- agonist ppt increases the expression of genes involved in lipid oxidation and metabolism . additionally , er- deficient mice and er- and er- double knockout mice display increased body fat and serum cholesterol level , but these changes are not found in er- deficient mice . in gper ko mice , ldl levels increase approximately by 200% , but hdl levels do not show any significant differences from wt , which indicates that gper mainly regulates the ldl metabolism instead of hdl . a recent study shows that human individuals with hypofunctional p16l genetic variant of gper have increased plasma ldl [ 84 , 85 ] . in contrast , gper activation upregulates ldl receptor expression in the liver via downregulation of proprotein convertase subtilisin kexin type 9 to enhance ldl metabolism . interestingly , animals with estrogen deficiency do not increase cholesterol synthesis ; instead they decrease cholesterol catabolism by reducing activity of 7-hydroxylase , the enzyme that catalyzes the initial step in cholesterol catabolism and bile acid synthesis in calcium supplementation - induced hypercholesterolemia . this study further demonstrates that estrogen treatment protects against increase in circulating level of cholesterol by activating of gper . the major circulating androgens include dehydroepiandrosterone , androstenedione , testosterone , and dihydrotestosterone ( dht ) , in descending order of circulating concentrations . only testosterone and dht bind to the ar whereas the rest are considered as proandrogens . within target cells , testosterone can be converted to active androgen dht via 5-reductase or converted to e2 by aromatase . ars are expressed in the liver of male and female humans and rodents , and ar expression in the liver is sex dependent . in adult rats , basal ar expression in the liver of male rats is about 20 times higher than that in the liver of female rats . ar expression is also age dependent in the liver of either sex , which is very low , almost undetectable , before puberty , increases in postpubertal life , and gradually declines during aging , reaching an almost nondetectable level after about 2224 months of age in rats . the sex- and age - dependent ar expression in the liver is programed by a regulatory element in the ar gene promoter . there are isoforms of ars which are ar - a with n - terminal truncated that resulted from proteolysis and ar - b with full length [ 90 , 91 ] ; among these two ar isoforms , the ar - b with full length is more potent than ar - a . it is not clear , however , which isoform of ar is dominant in the liver . . the genomic effect of androgens is achieved through activation of nuclear receptor , followed by binding to specific dna known as androgen response element ( are ) motifs in its target gene . ar can recruit other transcription factors such as ap-1 , nuclear factor-b , sex - determining region y , and the e26 transformation - specific family of transcription factors and bind to dna regions other than are , to participate in transcription activation of many other genes . the nonnuclear receptor of androgens function is independent of dna interaction and is more rapid by interacting with cytoplasmic signal transduction pathways , including pka and mapk / erk ( figure 1 ) . the ar knockout animals are well developed , but the membrane - only ar knockout animals are not established yet , and that is why the exact role of membrane ar in liver metabolism is unclear . many studies have shown that androgens and androgen signaling suppress the development of hepatic steatosis [ 96 , 97 ] . one population - based cross - sectional study has reported a close association between low serum testosterone level and hepatic steatosis in men . these mice upregulate expression for the genes involved in lipid storage and downregulate genes for fatty acid oxidation and accumulate lipid in their livers when they are fed with hfd . therefore , normal level of active androgen is critical to prevent liver steatosis . besides androgen level , ars are also critical in maintaining lipid metabolism in the liver . testicular feminized ( tfm ) mice with nonfunctional ar and very low serum testosterone levels greatly increase hfd feeding - induced hepatic lipid deposition compared with control male mice with functional ar and normal circulating levels of testosterone . replacement of testosterone reduces lipid deposition in the liver of tfm mice to a similar level to control males . moreover , kelly et al . found that the expressions of key regulatory enzymes for fatty acid synthesis , including acetyl - coa carboxylase ( acc ) and fas , are elevated in placebo - treated tfm mice comparing with placebo - treated wild - type littermates and tfm mice receiving testosterone treatment , indicating that the action of androgens on lipid deposition is independent of ar and at least partially via affecting key regulatory lipogenic enzymes to protect against hepatic steatosis . male but not female hepatic arko mice fed with a normal chow diet developed liver steatosis at 10 months with reduced fatty acid oxidation and increased de novo fatty acid synthesis . thus , males with either functional ar or normal circulating testosterone level would maintain normal level of fatty acid synthesis and avoid increased lipid deposition in the liver . although many studies have shown that androgens protect against nafld [ 50 , 103 ] , other studies have reported an opposite finding that androgens promote nafld development and progression [ 104 , 105 ] . the inconsistencies might be due to different animal models employed and different treatments utilized in various studies . the findings reported by mnzker et al . indicate that the testosterone / dht ratio is more important for nafld development and progression than concentrations of testosterone and/or dht . the total ar knockout mice develop liver steatosis and insulin resistance in both male and female mice . hepatic ar knockout mice with hfd feeding also show hepatic steatosis and insulin resistance , via upregulation of hepatic expression of srebp-1c , acc , and ppar to increase lipid synthesis and downregulation of ppar to decrease fatty acid oxidation ; interestingly , such effects are evident in males but absent in females [ 102 , 108 ] . thus , hepatic ar plays more critical roles in maintaining liver lipid metabolism in males than in females . testosterone is either converted to e2 binding to ers or converted to dht binding to ars . from the above studies , ars are vital in regulating liver lipid homeostasis in both males and females , although hepatic ars have greater impact in males than in females [ 102 , 108 ] . in order to test the role of androgen - ar signaling in female metabolic process , kanaya et al . replace dht in female mice with ovx and find that those mice accumulate greater amount of fat in the liver and develop other symptoms and signs of metabolic dysfunction when these mice are fed with either a standard chow diet or hfd . women have lower basal levels of androgens compared with males , and increased androgen level can affect metabolism in women . the role of androgens in females is not well established , but many lines of evidence indicate that hyperandrogenism in women with polycystic ovary syndrome ( pcos ) increases risk of developing nafld . nafld is frequently present in pcos women with excessive production of androgens by the ovaries and thus elevated circulating level of androgens , suggesting that abnormally high level of androgens in women may contribute to increased fat storage in the liver . it is noteworthy that the risk for nafld in women with pcos is independent of obesity or insulin resistance but is triggered directly by the hepatotoxic , destructive effect in the liver , indicated by elevated level of alanine aminotransferase . to summarize , normal level and signaling of androgens prevent hepatic lipid accumulation in males , while androgen deficiency in males is associated with fatty liver . androgen deprivation therapy for prostate cancer patients lowers their circulating testosterone level and increases their risk of diabetes [ 112 , 113 ] and not only increases circulating level of glucose but also diminishes pancreatic cell function . glut2 directionally transports glucose across liver cell plasma membrane to maintain glucose homeostasis , as mentioned above in section 2.3 . upregulation of glut2 plays a more critical role in regulating glucose export out of , rather than regulating glucose import into , the liver . it has been reported that blood glucose level , along with the mrna and protein levels of glut2 in the liver , significantly increases following castration in male rats with deficiency of endogenous androgens . supplementation of testosterone or a combination of testosterone with e2 normalizes glut2 mrna and protein levels in the livers of castrated rats , whereas treatment of e2 alone does not have any effect . these findings suggest that testosterone maintains glucose homeostasis by regulating hepatic glucose output , and testosterone deprivation due to castration increases hepatic glucose output , induces hyperglycemia , and develops symptoms seen in type 2 diabetes and metabolic syndrome . testosterone replacement restores glut2 mrna and protein levels suggesting that testosterone may have a direct effect on glut2 transcription and translation of mrna . although the presence of are has not been identified in the promoter region of glut2 , ar could function as a ligand - activated transcription factor by itself or bind to some other coactivators [ 118 , 119 ] to increase glut2 expression . in contrast , estrogens have little effect on hepatic glut4 and insulin receptor in male rats , but estrogens increase level of insulin receptor in hepg2 , a liver cancer cell line . interestingly , insulin receptor mrna level as well as insulin sensitivity is increased in a human liver cell line when being treated with testosterone . similarly , replacement of testosterone in castrated male mice also increases insulin receptor mrna and protein levels in the liver and normalizes castration - induced glucose metabolic impairment . treatment of testosterone induces glycogen synthesis in both intact and castrated male rats [ 108 , 121 ] . high testosterone level is associated with a low risk of diabetes in men , whereas it is associated with a high risk of diabetes in women [ 111 , 122124 ] . excess androgen in women with pcos impairs hepatic glucose metabolism by decreasing insulin - stimulated glucose uptake and glycogen synthesis and predisposes women with pcos to insulin resistance [ 125 , 126 ] . metformin , the most commonly used first - line drug to treat diabetes , is found to be effective to treat nafld and also suppresses the serum androgen concentration in pcos patients [ 127 , 128 ] . increased androgen activity in postmenopausal women correlates with impaired glucose tolerance [ 129 , 130 ] . to summarize , testosterone in males favors hepatic glucose metabolism , whereas testosterone in females impairs it . old men have increased risks of developing dyslipidemia with increased serum cholesterol and ldl levels , and decreased hdl level , and testosterone replacement reverses such dyslipidemia . hepatic scavenger receptor class b member 1 ( sr-1b ) is important in regulating cholesterol uptake from circulating hdl . dht treatment in castrated obese mice increases sr-1b compared with vehicle - treated castrated mice . at the same time , ldl secretion is decreased by dht treatment . cholesterol 7-hydroxylase , a key enzyme in bile formation and cholesterol removal , is also decreased after dht treatment . all these above results provide a comprehensive explanation for how chronic androgen replacement can decrease serum levels of cholesterol and ldl via enhancing liver cholesterol uptake and via suppressing cholesterol removal , which in turn increases liver cholesterol accumulation . a clinical study , however , shows that a single dose of testosterone treatment increases the serum cholesterol level after two days by increasing the expression of hmgr , the rate limiting enzyme for cholesterol de novo biosynthesis in the liver , but 15 days after the testosterone administration the cholesterol levels in the volunteers were back to baseline levels . the mechanisms for the androgen induced upregulation of hmgcr transcription as well as the physiological consequences have not been investigated and need to be further elucidated . the metabolic syndrome and its related diseases , such as obesity and diabetes , increase the health problems worldwide . the liver is the largest organ in the body that regulates lipid , glucose , and cholesterol homeostasis . several lines of epidemiological data have suggested that sex hormones are associated in fatty different types of receptors . estrogens seem to play protective roles against hepatic fat accumulation via suppressing lipogenesis and gluconeogenesis and promoting lipolysis and glycogen storage . interestingly , estrogens increase both cholesterol synthesis and secretion . er- and its membrane form are more important in regulating energy homeostasis than er-. gper and its roles in energy homeostasis are currently under intensive investigation ; however , there is less evidence about the role of gper in the liver compared with classic nuclear estrogen receptors . since the gper specific agonist and antagonist have been developed , further studies should apply these new chemicals to examine the role of gper in liver energy homeostasis , yet the underlying molecular mechanisms are still unclear and longing for further investigation . we review and discuss the roles played by estrogens , androgens , and their receptors in regulating liver energy homeostasis ( figure 2 ) . the action mechanisms of estrogens are complicated in the body , as they work through multiple different subtypes of estrogen receptors . estrogens promote liver glucose storage via increasing glucose transporters and glycogen synthesis and suppress liver glucose production via decreasing gluconeogenesis . estrogens also actively participate in maintaining lipid and cholesterol balance and play protective roles against hepatic lipid accumulation , via suppressing lipogenesis , lipid uptake , and cholesterol synthesis and promoting lipolysis and cholesterol removal . classic nuclear er- and its membrane form are more important in regulating energy homeostasis than er-. gper and its roles in energy homeostasis are currently under intensive investigation ; however , there is less evidence about the roles of gper in the liver compared with classic nuclear ers . since the gper specific agonist and antagonist have been developed , further studies should apply these new chemical compounds to examine the role of gper in liver energy homeostasis . androgens and nuclear ar have been shown to increase insulin receptor , decrease lipogenesis , and promote cholesterol storage in the liver . the membrane ar , however , is not well studied , which is also a potential research area to explore . it must be emphasized that the integration of nongenomic effects via membrane receptor signaling and genomic effects via nuclear receptor signaling of sex hormones is critical to produce the final sex hormone cellular outcomes . androgen deficiency , or excessive androgens as seen in women with pcos , the most common endocrine disorder and cause of infertility among women of reproductive age , is closely associated with disturbed lipid and glucose metabolism in the liver .

the liver is one of the most essential organs involved in the regulation of energy homeostasis . hepatic steatosis , a major manifestation of metabolic syndrome , is associated with imbalance between lipid formation and breakdown , glucose production and catabolism , and cholesterol synthesis and secretion . epidemiological studies show sex difference in the prevalence in fatty liver disease and suggest that sex hormones may play vital roles in regulating hepatic steatosis . in this review , we summarize current literature and discuss the role of estrogens and androgens and the mechanisms through which estrogen receptors and androgen receptors regulate lipid and glucose metabolism in the liver . in females , estradiol regulates liver metabolism via estrogen receptors by decreasing lipogenesis , gluconeogenesis , and fatty acid uptake , while enhancing lipolysis , cholesterol secretion , and glucose catabolism . in males , testosterone works via androgen receptors to increase insulin receptor expression and glycogen synthesis , decrease glucose uptake and lipogenesis , and promote cholesterol storage in the liver . these recent integrated concepts suggest that sex hormone receptors could be potential promising targets for the prevention of hepatic steatosis .

1. Introduction 2. The Role of Estrogens in Regulating Liver Energy Homeostasis 3. The Role of Androgens in Regulating Liver Energy Homeostasis 4. Summary and Future Directions

neurofibrillary tangles ( nft ) are the cardinal intracellular lesion of alzheimer disease ( ad ) , and are also found in normal aging , albeit to a lesser extent . highly phosphorylated tau protein is considered the predominant proteinaceous component of nft 1 , however , numerous other proteins have also been localized to these lesions including neurofilaments 2 , ubiquitin 3 , 4 , amyloid- 5 , and cell cycle markers 6 - 11 . notably , nft associated with normal aging are viewed as being quantitatively different , but qualitatively identical 12 . whether the mechanisms responsible for the genesis of nft in ad are similar or different from the genesis of nft in normal aging is unknown . brca1 is expressed in dividing neuronal cells during development , and is present in smaller amounts in fully differentiated cells 13 . brca1 is known to regulate transcription , regulate cell cycle progression , and may even have a role in maintaining telomere function and as such the presence of brca1 is indicative of cell cycle changes and dna damage , both of which are pathogenic changes in ad . nucleic acid damage is well - documented in ad , specifically within the pyramidal neurons , the population susceptible to neurodegeneration and death 14 - 18 . consequently , tumor suppressor proteins such as p21 , p27 , p53 are activated by brca1 , are indicative of dna damage 19 , and are activated in ad 6 , 20 . such tumor suppressors play a role in suppression of the cell cycle and cell survival instead of apoptosis and their presence may be a neuroprotective factor to prolong the life of the cell after re - entry into the cell cycle , protecting neurons from completion of apoptosis 21 . these proteins have come to the forefront as molecular candidates to be used in discrimination between normal aging and pathological diseases . neuroprotective factors have also been suggested as a possible target for drug design efforts with the goal of halting the progression of the cell cycle and delaying apoptosis . brca1 is also associated with a spectrum of functions related to the preservation of genomic stability 25 . for example , brca1 is involved in transcriptional activation and growth inhibition 26 - 28 , transcription coupled repair ( tcr ) of oxidative damage to dna and other dna repair 29 , 30 , and association with -tubulin , a central component of the microtubule organizing center and centrosomes , thus implying a regulatory role in g2/m progression 31 . there are also a host of putative functions assigned to brca1 based on its structure and associations . among these include association with bard1 , cyclin a and cyclin d kinases which phosphorylate brca1 32 . oxidative dna damage , as well as rna damage 15 , 16 , has been well documented in the aging brain , contributing to the development of ad 18 . further , even cases of mild cognitive impairment display the same abnormalities , prompting the search for increased dna repair mechanism in cases of neurodegeneration 33 . evidence of cell cycle dysfunction and the oxidative dna damage profile in ad caused speculation that brca1 may play a role in disease pathogenesis . there are clearly a number of striking parallels between ad and cancer , including age , and likely multiple etiologies and risk factors 34 . as for cancer , the notion of a two - hit hypothesis has also been proposed 35 , 36 . the latter may separate ad from normal aging . indeed , while cells , in this case neurons , have the capacity to maintain homeostatic balance and function under condition of stress , several hits may disrupt the cells ' regeneration capacity leading to neurodegeneration and death . possibilities for other hits include genetic mutations in apolipoprotein e , presenilins , or amyloid- protein precursor , hormonal dysregulation , environmental or education status , inflammatory responses , or perhaps even the induction of oncogenic - like pathways 37 . to this end , we found that the brca1 protein is strongly associated with nft in ad yet a feature of only about half of the cases of normal aging containing tau - positive nft . elevations in brca1 in neurons in ad may represent an attempt towards homeostasis by the cell , working with other factors to halt the cell cycle and mediate dna repair . interestingly , a much higher proportion of nft were labeled in ad cases than in control cases . these findings hint at differential mechanisms of nft genesis in ad and in normal aging and/or distinct cellular responses to these changes . hippocampal and cortical specimens were obtained postmortem from patients with histopathologically confirmed ad ( n = 33 , age 65 - 93 , mean 82.3 ) and control ( n = 28 , age = 47 - 89 , mean 73.5 ) . tissue was fixed either in 10% buffered formalin or in methacarn ( methanol : chloroform : acetic acid , 6:3:1 ) , and embedded in paraffin . 6m sections were deparaffinized in xylene and rehydrated in graded alcohol , the endogenous peroxidase activity eliminated by incubation in 3% hydrogen peroxide in methanol for 30 min , and finally to tris buffered saline ( tbs , 50 mm tris , 150 mm nacl , ph=7.6 ) . sections were blocked in 10% normal goat serum ( ngs ) for 30 min followed by overnight incubation with primary antibody in 1% ngs at 4c in a humidified chamber . staining was completed using the peroxidase - anti - peroxidase procedure with 3,3-diaminobenzidine ( dab ) as chromagen , and sections were dehydrated and mounted with permount . antibodies used included monoclonals recognizing brca1 amino acids 1 - 304 ( clone ms110 , oncogene research products ) , rabbit polyclonal against phosphorylated brca1 amino acids 1489 - 1500 ( upstate cell signaling solutions ) , and phosphorylated tau ( at8 , endogen ) to label nft . antibody specificity for brca11 - 304 was confirmed by performing an adsorption experiment with its corresponding antigen . diluted antibody was incubated overnight with 20g of brca1 peptide and applied to an adjacent section with antibody alone . additionally , cross - adsorption with purified tau protein was performed as well as omission of primary antibody . to further analyze the presence of brca1 in cases of control , mild cognitive impairment , as well as ad , formalin fixed blinded sections were analyzed for brca1 and phosphorylated tau . using images obtained with a zeiss axiocam and associated image analysis software , the number of nft immunostained in 3 fields ( 1 mm ) encompassing the ca1 and ca2 areas of the hippocampus were determined . brca1 is found to be specifically and intensely localized with intracellular nft in hippocampal neurons in ad ( figure 1a ) . in young control cases and those without any tau pathology , no cellular staining was seen ( figure 1b ) . the specificity of our findings was demonstrated in adjacent sections where brca1 immunoreactivity in nft ( figure 1c ) was completely abolished following adsorption with the specific brca1 peptide ( figure 1d ) . on the other hand , cross - adsorption with tau protein brca1 localization to nft was detected in all cases of ad , independent of fixation methods . hippocampal sections from 17 clinically normal cases containing pathological accumulations consistent with normal aging were specifically chosen and immunostained for brca1 and at8 . it was noted that in many of the control cases containing phosphorylated tau - positive nft , brca1 was absent . analysis of this series of cases shows that while all cases with ad exhibited brca1-positive nft , brca1 was present to a lesser extent and in smaller and more variable numbers in control cases with pathology across all age ranges ( figure 2 ) . to further assess the relationship between brca1 and ad , blinded sections were stained for brca1 and at8 in well characterized cases classified as control ( no neurological diagnosis ) , mild cognitive impairment ( mci ) and ad . the numbers of nft stained for each marker in three fields were quantified using a computer assisted image analysis ( figure 3 ) and expressed as the percentage of brca1 positive compared to at8 positive nft . in control cases ( n = 4 , age range 83 - 93 ) , an average of only 9% of nft contained brca1 . in cases with mci ( n = 3 , age range 78 - 96 ) , 18% of nft were brca1 were positive , and in cases of ad ( n = 3 , age range 69 - 91 ) , the number increased to 28% . the percentage of nft stained in mci cases is essentially midway between ad and control cases consistent with clinical findings that mci is a transition . nonetheless , as expected , by looking only at tau , there was a wide variety of pathology in each category . in the control cases , the number of tau - positive nft ranged from 5 to 260 , and in ad cases , from 39 to 321 nft . the colocalization of at8 and brca1 representative of the different disease states is shown in figure 4 . adjacent serial sections of ad showed that large numbers of nft are positive for brca1 ( figure 4a ) with significant overlap with at8 ( figure 4b ) . in a case of mci , while fewer at8-positive nft are present ( figure 4d ) , again there is significant overlap with brca1 ( figure 4c ) . as was seen in about half of the clinically normal cases with tau pathology , while even moderate numbers of at8-positive nft are present ( figure 4f ) , brca1 ( figure 4e ) is not present . qualitative analysis for the presence of phosphorylated brca1 ( pbrca1 ) was also performed . in some cases of ad , pbrca1 stained neuronal nuclei as well as a smaller population of nft ( figure 5b ) compared to non - phosphorylated brca1 ( figure 5a ) . in this study , we show that , controlled for age , there is a progression in the percentage of nft containing brca1 from cases with no dementia to mci to ad . the differences in brca1 and tau co - localization in control versus mci or ad cases may point to different etiologies and/or different cellular responses . indeed , mechanisms involved in the formation of nft in ad may be very different to the process during normal aging , i.e. , that the development of ad requires two or more hits . neurons can maintain normal function and combat assault from oxidative damage throughout aging , unless there is another hit , whether it be a genetic mutation or metabolic dysfunction , from which the cell can not overcome and maintain balance , resulting in neuronal death . as ad is a disease that can last ten years or more , rather than succumbing to apoptosis immediately , neurons may attempt to survive by initiating cell cycle progression , and attempting to control the deregulated cell cycle and concurrent apoptotic signaling . brca1 is a tumor suppressor protein , involved in dna repair , suspension of the cell cycle and probable temporary delay of apoptosis when problems are suspected . since the prevalence of brca1 increases as the disease progresses this is consistent with the hypothesis that cell cycle changes take place very early in the progression of the disease , long before the presence of other pathology . over time , dna and cell cycle changes may compound , and brca1 and other protein expression increases , eventually resulting in cell death . these findings raise the possibility that brca1 accumulates in neurons early in the disease and only in those cases in the early stages of ad and may or may not be independent of tau formation and the expression of other cell cycle markers . the association of brca1 with neurodegenerative pathology in ad implicates genomic instability and possibly a neuroprotective element in neurons in ad . the emerging evidence of genomic instability as a proximal feature in the pathogenesis of neurodegeneration in ad may possibly be a feature of cell cycle instability in neurons 38 . taken together with the association of brca1 , this phenotype bears many resemblances to a mitotic lesion or , at minimum , the presence of oncogenic signaling in ad , providing another driving force , or hit the presence of brca1 and other tumor suppressor proteins is also indicative of protective mechanisms against the formation of a cancer or unnecessary apoptosis . for instance , phosphorylation of specific residues dictate both localization and function 39 , which could be related to the varying nuclear accumulations seen in the brain in the present study . pbrca1 has also been implicated to play a role in maintaining genomic integrity in mitochondria and in the nucleus 40 . recent work has related these functions specifically to telomere maintenance . in brca1 -/- cells , telomere dysfunction evidenced by a loss of telomere repeats was found 41 , a distinctive feature of degenerating neurons in the ad brain 42 . while the mechanisms responsible for the localization of brca1 to nft remain to be determined , one intriguing hypothesis is that the presence of brca1 signifies a neurogenic / oncogenic stimulus that is found in ad and other neuropathology . in this regard , there are several examples showing cognitive improvements in dementia patients undergoing chemotherapy 43 . it would be interesting to investigate the therapeutic efficacy of combination or simply agent antimitotic therapy with vincristine , carmustine , melphalan , cyclophosphamide , or prednisone for ad 44 monoclonal antibody to brca1 recognizes intracellular nft in all cases of ad ( a ) , yet in many control cases , no structures are stained ( b ) . on adjacent serial sections , the specific localization of brca1 to nft ( c ) all ad cases and all clinically normal cases showing at8-positive nft consistent with normal aging were also analyzed for the presence of brca1-positive nft . all ad cases ( 100% ) at all age ranges exhibited brca1 positive nft . yet only about half of the control cases with nft displayed brca1 positivity . the number of nft stained for brca1 and at8 were counted in the ca1/ca2 regions of hippocampus in well characterized cases of ad ( n = 3 ) , mci ( n = 3 ) , and control ( n = 4 ) . in ad , an average 28% of at8-positive nft contained brca1 . that number was only 18% for cases of mci and 9% for the control cases . while all four control cases contained at8-positive nft , only two cases displayed brca1 , a finding similar to that observed with the aged controls examined in figure 2 . ad cases show high numbers of at8-positive nft ( b ) , with many overlapping with brca1 localization ( a ) . in cases of mci , while there are fewer at8-positive nft ( d ) , many overlap with brca1 ( c ) . yet in about half of the aged control cases , while there are moderate numbers of at8-positive nft ( f ) , brca1 is absent ( e ) . phosphorylated brca1 is localized in some cases of ad to both nuclei as well as some nft ( b ) . in adjacent serial sections stained for brca1 ( a ) , many of the cells containing nft ( arrows ) also contain pbrca1 ( b ) .

in alzheimer disease , neuronal degeneration and the presence of neurofibrillary tangles correlate with the severity of cognitive decline . neurofibrillary tangles contain the antigenic profile of many cell cycle markers , reflecting a re - entry into the cell cycle by affected neurons . however , while such a cell cycle re - entry phenotype is an early and consistent feature of alzheimer disease , the mechanisms responsible for neuronal cell cycle are unclear . in this regard , given that a dysregulated cell cycle is a characteristic of cancer , we speculated that alterations in oncogenic proteins may play a role in neurodegeneration . to this end , in this study , we examined brain tissue from cases of alzheimer disease for the presence of brca1 , a known regulator of cell cycle , and found intense and specific localization of brca1 to neurofibrillary tangles , a hallmark lesion of the disease . analysis of clinically normal aged brain tissue revealed systematically less brca1 , and surprisingly in many cases with apparent phosphorylated tau - positive neurofibrillary tangles , brca1 was absent , yet brca1 was present in all cases of alzheimer disease . these findings not only further define the cell cycle reentry phenotype in alzheimer disease but also indicate that the neurofibrillary tangles which define alzheimer disease may have a different genesis from the neurofibrillary tangles of normal aging .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion Figures and Tables

breast cancer is the most common form of cancer among women in developed countries ; and 12% of all breast cancers are diagnosed among women aged 20 - 34 . breast cancer has the second place after skin cancer in iran in both sexes in terms of prevalence . it is the most common malignancy in iranian women and is the fifth leading cause of death among malignancies . eight thousands ninety new cases of breast cancer occur annually and more than 1300 of breast cancer patients die every year . survival from breast cancer has improved significantly , and the potential late effects of treatment and its impact on the quality of life have become of paramount importance . an individual s psychological response to cancer is influenced by specific aspects of the cancer , and her ability to manage the cancer diagnosis and treatment which constantly changes over the course of the illness due to medical , psychological , and social factors . factors influencing the states of the disease include ( a ) the personal variables of socio demography , personality and coping style , beliefs , and prior adjustment ; ( b ) the variables associated with stage of illness , rehabilitation options , illness - related behaviours , and the positive feeling toward the treatment team ; ( c ) the availability of social support ( family , friends , community , and socio cultural influences ) ; and ( d ) concurrent stresses related to illness that add to the psychological burden , such as loss of a spouse . the diagnosis of breast cancer encompasses not only physical , but also social and psychological concerns due to the importance of breast in woman s body image , sexuality and motherhood . body image is a phrase used to describe how one feels about his / her body . it includes not only physical appearance , but also the psychological feelings one has about " breast fullness " , and can be considered as significant predictor of sexual activity . mastectomy as a treatment option can result in a sense of mutilation and diminished self - worth and loss of sense of feminity and sexual attractiveness . losing a breast or poor breast appearance would be more distressing to women as they are supposed to give women high expectations for physical beauty . data from studies among longer - term breast cancer survivors show that sexual problems occur with considerable frequency and often do not resolve over time , even among women who do not undergo mastectomy , or who have subsequent breast reconstruction . radiation can also lead to redness and soreness on the affected area , and chemotherapy often causes hair and weight loss . significant similar psychosexual and body image problems occur in patients treated for breast cancer with either mastectomy or breast conserving therapy ( bct ) . however , there is a growing acknowledgement that sexual problems are not being appropriately addressed by providers . these problems arise early in the course of the disease and therefore their detection and treatment should be addressed during the patients ' initial assessment and in the early phases of the treatment . there is a growing body of literature on disability for achieving healthy sexual function among breast cancer survivors . the ability to achieve healthy sexual function involves both psychological and physical factors that affect the sexual response cycle ( e.g. desire , arousal , orgasm and resolution ) . review of researches on breast cancer and sexuality from 1998 to 2010 , has documented a range of physical changes to woman 's sexuality following breast cancer , including disturbances to sexual functioning , as well as disruptions to sexual arousal , lubrication , orgasm , sexual desire , and sexual pleasure , resulting from chemotherapy , chemically induced menopause , tamoxifen , and breast cancer surgery . women 's intrapsychic experience of changes to sexuality includes a fear of loss of fertility , negative body image , feelings of sexual unattractiveness , loss of femininity , as well as alterations to a sense of sexual self . the impact of such changes can last for many years after successful treatment , and can be associated with serious physical and emotional side effects [ 7 , 9 - 20 ] . sexual functioning can be affected by illness , pain , anxiety , anger , stressful circumstances and medications . distress , depression , anxiety and other psychiatric morbidities [ 22 - 28 ] are common in breast cancer patients . there is a strong link between depression and sexual dysfunctions ; moreover sexual dysfunction is considered as an important underestimated adverse effect of certain antidepressant drugs . most of this disturbance probably results from the patients ' inability to cope psychologically with disease - related and treatment - related stresses and the thought of losing their health , role and life . sexual dysfunctions as side effects of antidepressant treatments are being reported more and more frequently and are one of the main reasons of dropout from therapy . numerous side effects associated with most antidepressants , include inability to get an erection , sexual dysfunction and reduction in sexual interest or function [ 31 , 32 ] . according to serrette and chiesa study , the introduction of psychiatric medications with relatively good safety profiles , such as selective serotonin reuptake inhibitors ( ssris ) and serotonin norepinephrine reuptake inhibitors , has led to increasing attention on side effects such as sexual dysfunction ( sd ) , which , although unrelated to risks of mortality , could undermine compliance with treatment regimens and impair quality of life . indeed , there is consistent evidence to suggest that a large number of psychiatric medications affect one or more of the three phases of normal sexual response : desire , arousal , and orgasm ( table 1 ) . sexual problems are a widespread concern among patients and survivors , and many patients do not receive the information they need from their oncology providers . there are large differences in sexual function between patients who do and do not ask providers about sexual problems . female sexual function has been largely ignored in treatment of breast cancer . despite the prevalence and documented persistence of sexuality and intimacy problems among large numbers of breast cancer survivors most have focused on helping women adjust to global changes in their lives or on accruing generic , illness - related coping skills . sexual assessment and counselling there is an urgent need to make oncologists aware of the importance of the sexual life of their patients ; furthermore , cancer patients need open communication on intimacy and sexuality . they should learn to investigate and treat sexual dysfunctions while assessing and treating the breast cancer . moreover , they must be aware of the sexual adverse effects of many commonly used antidepressant drugs , particularly ssris . since sexual activity has undeniable benefits , such as pain reducing effects decreasing muscular tension for several hours [ 34 - 37 ] , sleep enhancing effects and releasing oxytocin , [ 38 - 40 ] , diminishing the autism resembling aspects of behaviour , mood - enhancing and antidepressant effects , basic questions regarding sexual functioning should be part of any complete medical history and part of the treatment plan discussion , particularly if the prescribed treatment has the potential to alter sexual function . therefore , it is of great importance for all the oncology professionals to encourage open discussion in addition to making appropriate referrals . psychologists can be helpful in sharing information meaningfully and supportively with patients and their spouses . for bio psychosocial intervention addressing interruptions and weaknesses in woman 's sexual response cycle identified during the assessment , forms the basis of therapy . behaviour therapy plus sex therapy are two rehabilitative options for women suffering from sexual dysfunction . the multi factorial nature of female sexual concerns defies a quick fix ; therapeutic interventions should be tailored to address each source of distress ( psychological , interpersonal , socio - cultural , and physiologic ) and to attend to each affected functional domain ( desire , arousal , orgasm , pain ) . available modalities range from education , psychotherapy , and lifestyle interventions to mechanical devices , pelvic floor exercises , and medications . sexual response in women is based on desire , arousal , lubrication , plateau , orgasm and resolution . the first three components are interdependent and greatly responsible for reaching plateau , orgasm and resolution . there is growing evidence that women treated for breast cancer with surgery and chemotherapy commonly experience disturbances in sexual functioning . over the past years , studies have found that breast cancer , chemotherapy , endocrine treatment or psychiatric drugs have had a negative effect on the sex life of breast cancer survivors resulting in sexual dysfunction as a quite common aspect . thus , growing attention has been paid to a range of changes in women 's sexuality following breast cancer including : disturbances of body image and sexual functioning . such problems push these patients to lose schema of attractiveness ; and impact of such changes can last for many years after successful treatment and can be associated with serious physical and emotional side effects . these patients are then referred to psychiatrists and take psychiatric medications , not knowing this orientation may increase sexual dysfunction . despite the prevalence of sexuality and intimacy problems among large numbers of breast cancer survivors it is of crucial importance for all the oncology professionals to encourage open discussions in addition to making appropriate referrals . basic questions regarding sexual functioning should be part of any complete medical history and treatment plan discussion , particularly , if the prescribed treatment has the potential to alter sexual functioning . in recent years , we have focused on sexual functioning , its assessment and effects on quality of life . we have examined the role of sexual desire , sexual efficacy [ 43 , 45 ] , body image , sexual fantasy , and personality in sexual function and dysfunction . we have studied the etiology of female sexual dysfunction and the role of drugs in sexual behaviour . our findings confirmed that sexual relationship typically plays a powerful role in human bonding ; moreover , based on our culture , we examined psychological treatment in decreasing sexual dysfunction . along with these studies , our researches on cancer patients , particularly breast cancer patients , in the context of sexual behaviour have shown the role of body image depression and anxiety in breast cancer patient 's sexual behaviours . we have also examined the role of increasing awareness , hope , relaxation and four - factor psychotherapy in breast cancer patients mental health and quality of life . we strongly believe that the evaluation and management of sexual difficulties should be the standard parts of the clinical care of those women treated for breast cancer . we agree with anderson and elliot who stated that treatment of sexual dysfunction / disorders in women with cancer is a challenging process . first a psychological and medical assessment of realistic limitations created by the disease or treatment is required ; second , the clinician must facilitate creative problem solving in response to these realistic limitations . the ultimate goal of treatment is to support women with cancer in the reacquaintance with their bodies and to facilitate a positive sense of sensuality and sexuality . the general principles of sex therapy may offer women with cancer the opportunity to see themselves as capable sexual women . interventions are critical to the self esteem of women with cancer and may significantly contribute to their recovery . health care professionals must realize the impact of sexuality on the quality of life for these women and consequently address their sexual concerns . sexual problems of the cancer patients are more challenging and difficult to treat , but this does not deny the burden of responsibility to help them . professionals are bound to create an environment of concentrated effort , and should incorporate a broad repertoire of psychosexual interventions . we agree with anderson and elliot who stated that treatment of sexual dysfunction / disorders in women with cancer is a challenging process . first a psychological and medical assessment of realistic limitations created by the disease or treatment is required ; second , the clinician must facilitate creative problem solving in response to these realistic limitations . the ultimate goal of treatment is to support women with cancer in the reacquaintance with their bodies and to facilitate a positive sense of sensuality and sexuality . the general principles of sex therapy may offer women with cancer the opportunity to see themselves as capable sexual women . interventions are critical to the self esteem of women with cancer and may significantly contribute to their recovery . health care professionals must realize the impact of sexuality on the quality of life for these women and consequently address their sexual concerns . sexual problems of the cancer patients are more challenging and difficult to treat , but this does not deny the burden of responsibility to help them . professionals are bound to create an environment of concentrated effort , and should incorporate a broad repertoire of psychosexual interventions .

backgroundclinical experiences have revealed that patients with breast cancer experience various sexual problems following their treatment . breast cancer negatively impacts the sexual life of the afflicted couples , and as a traumatic event can influence women s psychosexual functioning and intimate relationship . this review focuses on sexuality after breast cancer and on a growing need for bio - psycho - social guidelines for breast cancer treatment . methodsthis study aims to review the literature on management , psychological outcomes and sexual dysfunction in patients with breast cancer . resultsalthough the benefits of the current treatment strategies are well established , many cancer survivors are at risk for developing psycho physiological symptoms including sexual dysfunction . cancer and treatment - related factors can influence sexual functioning . we review current treatment - related side effects on sexual functioning such as desire , arousal and orgasm in breast cancer patients . despite the impact of medical treatment on survival of patients with breast cancer , no satisfactory steps have been taken towards improving sexual functioning of these patients.conclusionbreast cancer affects many aspects of sexuality , including changes in physical functioning and in the perception of feminity . sexual dysfunction following breast cancer should be diagnosed and managed as a systematic approach with multidisciplinary inputs . healthcare professionals should assess the effects of medical and surgical treatment on the sexuality of breast cancer survivors .

Introduction Breast Cancer and Sexual Dysfunction What Can Be Done Intervention Guideline Discussion Conclusion

hair loss is a common problem encountered either due to pathological causes or nutritional deficiencies . there are various tests available to investigate hair loss such as hair pull test , hair pluck test ( trichogram ) , phototrichogram , digital phototrichogram , unit area trichogram , hair diameter assessment , and trichotillometry . trichotillometer is a device that is used to determine the force required to pluck the hair . we devised a simple and inexpensive hand - held trichotillometer to assess the epilation force ( ef ) in normal individuals . a simple hand - held trichotillometer was assembled by modifying the laboratory spring balance . a laboratory spring balance ( 1 newton ( n)/100 grams ( g ) ) 16 cm long and 3 cm diameter was selected for the study . the loading hook of the spring balance was replaced with a paper clip of 2 cm length . the spring balance had a range of 1 n ( 100 g ) with a resolution of 2 g . two holes were drilled on diametrically opposite locations on the sides of the spring balance , at a distance of 4.5 cm from the top edge of the cylindrical barrel . two nylon cords were introduced through these holes and a hand - made paper cup was cemented to the end of these cords . this paper cup with a diameter matching the inner diameter of the barrel serves as a non - return marker . two slots were provided at the diametrically opposite points of the indicator cone to allow the nylon cord to pass through freely [ figure 1 ] . the trichotillometer was calibrated with analytical weights for accuracy before and after the modification . assembling a trichotillometer by modifying a spring balance thirty healthy volunteers who did not complain of hair loss volunteers were advised not to wash the scalp at least three days prior to the date of ef estimation . for estimating the ef , volunteers were made to sit on the stool and the clip at the lower end of the trichotillometer was attached to the distal end of a single hair shaft and the spring balance was pulled manually , upward gently at the top end with the help of the ring attached to its top till the hair snaps from the scalp [ figure 2 ] . force indicator slides down the inner wall of the spring balance and stops at the point when the hair is detached from the scalp ; the force in g was noted and taken as the ef [ figure 3 ] . the force indicator was pulled to the base line by the nylon thread and trichotillometer was set for assessing the ef of another hair . if the hair shaft broke during the process of pulling the spring balance , then another hair shaft was selected for the estimation of ef . the root of the plucked hair was examined under microscope to determine whether the follicle plucked was in the anagen or telogen phase . the ef required to pluck 5 anagen and 5 telogen hair from 4 regions ( frontal , vertex , occipital , and parietal ) on each volunteer was determined separately . subsequently , the mean force required to pluck the anagen hair and telogen hair from each area was calculated . volunteer position during the epilation force estimation and clamping of the hair shaft to the device estimation of epilation force a total of 30 volunteers ( 15 males and 15 females ) were included in the study . the mean ef in our study was 70.15 g. the mean ef required to pluck the anagen hair was 86.61 g and telogen hair was 53.69 g [ table 1 ] . in females , the mean ef for anagen hair was 88.55 g and mean ef for telogen hair was 53.55 g , and the mean ef for females was 71.05 g [ table 2 ] . in males , the mean ef for anagen hair was 84.67 g and mean ef for telogen hair was 53.83 gm , and the mean ef for males is 69.25 g [ table 3 ] . in frontal area , the mean ef required to pluck the anagen hair was 83.44 g ( sd : 10.46 ) and telogen hair was 49.04 g ( sd : 9.61 ) . in vertex area , the mean ef required to pluck the anagen hair was 83.46 g ( sd : 10.21 ) and telogen hair was 51.46 g ( sd : 11.67 ) . in parietal area , the mean ef required to pluck the anagen hair was 84.04 g ( sd : 11.14 ) and for telogen hair was 50.02 g ( sd : 10.91 ) . in occipital area , the mean ef required to pluck the anagen hair was 95.50 g ( sd : 4.70 ) and telogen hair was 64.24 g ( sd : 7.01 ) . mean ef for anagen and telogen hairs on various regions of scalp mean ef for anagen and telogen hairs on various regions of scalp in females mean ef for anagen and telogen hairs on various regions of scalp in males ef for the anagen hair was higher than that for the telogen hair on all the regions of scalp in our study group . the ef of anagen hair has been reported to be higher than the telogen hair in previous studies . mechanical resistance encountered during the plucking of anagen hair could be due to the deeper location of the anagen follicle , the firm attachment of the dermal papilla to the fibrous sheath , and the close apposition of inner root sheath to the hair shaft . force required to pluck the hair in normal healthy volunteers using a hand - held trichotillometer has been reported in few studies . reported that the average observed range among well - nourished patients , when plucking 10 individual hairs , was 36.0 12.4 g. wyness et al . reported that mean hair pluckability measurements for the 12 participants obtained by three observers were 39.5 g , 41.2 g , and 32.7 g , respectively . smelser et al . determined the usefulness of trichotillometry under field conditions by measuring hair ef of 69 subjects at a hospital in nigeria , the mean force was 36.5 9.5 g. el rifaie et al . calculated the shear strain of the follicle rather than the ef alone on different regions of the scalp and separately for men and women . the mean ef in our study group was 70.15 g , which is higher than the previous reports . in our study , the ef was calculated separately for the anagen and telogen hairs in various regions of scalp and for a larger sample size ; this could explain the variations in the findings . the force required to pluck hair is likely to vary throughout the different stages of growth , over the different regions of scalp , and in different races and these biological variations might contribute to the differences in ef in our study compared to the others . the mean ef of anagen and telogen hairs was higher in occipital region than other regions of the scalp . the mean ef of anagen and telogen hairs were comparable in other regions of the scalp . these different behaviors of occipital and frontal / parietal follicles may result from the embryological derivation of the dermis in these two regions . dermis of the frontal / parietal scalp is of neural crest origin , whereas the dermis of the occipital scalp is of mesodermal origin . it is a common observation that the occipital hairs are retained in hamilton- norwood scale owing to its androgen independent nature compared to the other regions on the scalp . el rifaie et al . reported that the shear strain of plucking the follicles is not different in anagen and telogen hairs on the various regions of the scalp and there was no difference between men and women . the variations in ef among the individual and the observers has been reported in previous studies . this variation could be due to mechanical error in the instruments . in order to obtain a reproducible and representative mean hair pluckability measurement , an adequate amount of individual hairs need to be plucked and a standard instrument needs to be designed . limitations of our study are patient discomfort during the repeated plucking of hairs ; other hair parameters such as the growth rate , density , hair diameter , and the shear strain during the follicular plucking were not estimated . even though our device was calibrated with analytical weights , the final reading may not be as accurate as a digital reading . our device was assembled by modifying a spring balance and the method described can be easily replicated . tricotillometer is a useful device to measure the ef in various physiological and pathological conditions and may have diagnostic , prognostic , and therapeutic value in various hair diseases . the tensile strength of the hair shaft can be estimated in various disorders using a trichotillometer .

aim : to assemble a simple , inexpensive hand - held trichotillometer and to determine the epilation force ( ef ) required to pluck the anagen hair and telogen hair from four regions of the scalp in healthy volunteers.materials and methods : a simple hand - held trichotillometer was assembled by modifying a laboratory spring balance , and the instrument was calibrated after attachments . ef was measured in 30 healthy individuals on four regions of the scalp.results:a total of 30 volunteers were included in the study , among them 15 were males and 15 were females . a total of 1200 hairs were examined . the mean ef in our study was 70.15 grams ( g ) . the mean ef required to pluck the anagen hair was 86.61 g and telogen hair was 53.69 g.conclusion:the assembled- hand held trichotillometer is a simple and reliable device . tricotillometer is a useful device to measure the ef in various physiological and pathological conditions and may have diagnostic , prognostic , and therapeutic value in various hair diseases .

Introduction Materials and Methods Results Discussion Conclusion None Financial support and sponsorship Conflicts of interest

nutrition ( that is , eating food ) is not only a prerequisite for survival ; it is also a frequently enjoyable and social function . as a consequence , when the ability to swallow is impaired ( dysphagia ) people with neuromuscular disease may develop acute respiratory failure and be admitted to critical care for mechanical ventilation . the article by macht and colleagues in a previous issue of critical care throws more light on this area of clinical uncertainty and begins to answer some questions and at the same time opens up further avenues for exploration . the presence of dysphagia is frequently under - recognized and suboptimally managed , especially in patients discharged from critical care back to a medical or surgical ward following extubation . in a retrospective study of patients with acute respiratory failure and pre - existing neuromuscular disease , macht and colleagues reported that 93% of those patients discharged from critical care following extubation and transferred to a surgical or medical ward had post - extubation dysphagia , which in 33% of cases was considered severe . of those with moderate or severe dysphagia , 66% were discharged from the hospital with dysphagia still present . in a similar cohort , without pre - existing neuromuscular disease , 84% had dysphagia , and only 17% were assessed as being severe . in both cohorts , those patients with dysphagia had a prolonged length of hospital stay and increased morbidity ; mortality was not increased in the recent study . as in many retrospective studies , only a minority of all the patients admitted and mechanically ventilated on critical care were included ; 9% in the neuromuscular group and 25% in the non - neurological group were included in the studies . this is a recurring problem with many studies in this area ; the numbers are small , raising questions about generalizability of results . what is the true incidence of post - extubation dysphagia ? could the number with severe dysphagia be higher than suggested , as many patients did not undergo a bedside swallow examination ( bse ) . how common is aspiration , silent or otherwise ? in a small cohort of 35 patients studied by tolep and colleagues , 16 had neuromuscular disorders ; of these 16 , 14 had an abnormal videofluoroscopy ( vf ) ( modified barium swallow ) ; and of these 14 , 4 were aspirating . leder , using fiberoptic endoscopic evaluation of swallowing ( fees ) , noted that 17 out of 52 patients aspirated , the majority silently . aspiration pneumonia is the most common form of hospital - acquired pneumonia and carries a mortality of 20% to 65% . the development of complications , including pneumonia , following aspiration is dependent on many factors , including the physiological condition of the patient , and by definition many of these patients are at the limits of their physiological reserve , the frequency of aspiration , the contents of the aspirate and the quality of mouth care . macht and colleagues are concerned that the bse will not detect silent aspiration , and this is true by definition , but does that matter ? we all aspirate , particularly at night when the frequency of swallowing is greatly reduced or ceases . many studies investigate preselected patients , who have been referred for assessment , whereas those studies investigating un - selected patients do not find any particular association with aspiration . silent aspiration is detected only by instrumentation ( vf / fees ) and , when detected in this way , appears to have no clinically apparent affect and this would not be logistically practical in all patients and should be reserved for those patients who are most at risk of infection or continually develop chest infections or desaturation . this cohort of patients with neuromuscular disease was a heterogeneous population , and how optimal was the management of the dysphagia ( as good clinical management can reduce pulmonary complications ) , or was mouth care very diligent to prevent aspiration pneumonia secondary to infected saliva ? the etiology of post - extubation dysphagia is likely to have multiple factors , including progression of the underlying neuromuscular disease critical care syndrome and trauma to the airway and muscle wasting / weakness . it is possible that this study by macht and colleagues missed significant variation in the occurrence of dysphagia because of the heterogeneity of the cohort or because the subgroups were too small for separate analysis . therefore , future studies in critical care populations should be undertaken prospectively and need to be adequately powered to detect clinically significant differences , and determine predefined sub groups must be identified for analysis . the data collected need to explore all possible etiology factors , including trauma due to a difficult intubation ( 1.5% to 8.5% of intubation are classed as difficult ) , and answer questions such as these : was pre - intubation dysphagia that had not been clinically apparent present , and what was the functional ability of the patient prior to intubation ? this study has begun to answer some of the questions around the complex problem of post - extubation dysphagia in neuromuscular disease . this should spur others on to further elucidate the relationship between extubation dysphagia , neuromuscular disease , and acute respiratory failure . bse : bedside swallow examination ; fees : fiberoptic endoscopic evaluation of swallowing ; vf : videofluoroscopy .

post - extubation dysphagia is a common and serious problem . the presence of neuromuscular disease at the time of intubation is likely to increase this . until recently , the prevalence and the association with length of intubation had not been clarified . results published in this journal suggest that 93% of extubated patients with neuromuscular disease had post - extubation dysphagia , which in 33% of cases was considered severe . the number of days ventilated was the single predictor of severe dysphagia and a consequent prolonged hospital stay . further work to build on these results to unravel the complex interplay between disease , trauma , and other unknown factors will be required .

Introduction Discussion Conclusions Abbreviations Competing interests

musculoskeletal disorders ( msds ) are a major problem for patients as well as for society and can lead to functional limitation and absence from work [ 1 , 2 ] . health care workers have physically and psychologically demanding work and are at high risk of developing long term msds and sickness absence [ 35 ] . research regarding work ability and prevention of sickness absence is a great challenge because of its complexity . the international classification of function ( icf ) provides a classification system for function and disability associated with health . the theoretical model of icf explains functioning as all body function , activity and participation as well as personal and environmental factors that interact with these concepts . hence , work ( dis)ability may be explained by physical , mental and social aspects of functioning , in addition to environmental and organizational demands of a person s work and personal factors that influence his or her capacity to meet these demands . socio - demographic factors such as age , gender and educational level are important predictors for work ability [ 711 ] . other factors associated with insufficient work ability are heavy physical work [ 9 , 10 , 12 , 13 ] , high pain intensity [ 12 , 14 ] , social and environmental workplace factors [ 15 , 16 ] , and psychological variables [ 8 , 11 , 17 ] . besides these factors , some studies have focused on the relation between deconditioning and poor work ability [ 11 , 1820 ] . deconditioning refers to a decrease of capacity over time expressed by weakened muscle strength , reduced aerobic fitness or altered coordination during activity . although it is argued that deconditioning may be a result of fear avoidance and altered behavioral performance , the evidence is inconclusive [ 19 , 2124 ] . there is also conflicting evidence concerning deconditioning among patients with chronic low back pain ( lbp ) [ 19 , 25 , 26 ] . although self - reported functioning and physical tests have been used to predict and evaluate work ability in several studies , only a few studies have compared the function of employees on sick leave and employees still working despite msds [ 17 , 2730 ] . it has been found that employees on sick leave have poorer health and more disability [ 17 , 28 ] , higher perceived workload , more fear - avoidance beliefs [ 27 , 29 ] , lower pain acceptance [ 17 , 27 , 28 ] and lower functional capacity compared to employees still working . more knowledge about the differences between employees on sick leave due to msds and employees staying at work despite msds can give us insight into what could be emphasized in work interventions and contribute to increase work participation . employment policies in scandinavian countries have focused on active approaches for employees with reduced work ability . partial sick leave has been used to give employees the possibility to combine work and sickness benefits . however , there is a lack of evidence regarding functional ability in workers on partial sick leave compared to those on full sick leave . the aim of this study was to describe self - reported and physically tested function in health care workers with msds and to examine how function was associated with work participation . by using the icf s model to understand the complexity of work ability , a wide range of bio - psycho - social and work - related factors were investigated . this study examines possible differences of functioning in ( a ) health care workers staying at work despite msds , ( b ) on partial sick leave , or ( c ) on full sick leave . this study was part of a larger study called function , activity and work of health care workers with msds in the municipality of bergen . the participants were recruited from the department of health and social service in the municipality of bergen , norway , from january 2012 to december 2013 . about 7,000 health care workers are employed in this department ; working in nursing homes , home care service and in special homes for disabled . through their managers and/or brochures we invited employees who were on sick leave or at risk of being sick - listed due to msds , to a functional evaluation . health care workers with msds took direct contact with the university of bergen and booked an appointment with a physiotherapist in the project . exclusion criteria were insufficient knowledge of the norwegian language and being on full sick leave for more than 4 months continuously . within 2 weeks after requesting an appointment , the participants met for an evaluation completed by a physiotherapist in the project . the evaluation ended with a verbal and written presentation of the self - reported and physical findings for all participants , except 56 who were recruited to an randomized controlled trial for participants with low back pain ( lbp ) . they practiced several times together before the start of the project and also examined the first 10 participants together . the participants performed the tests that required minimal effort first , in order to prevent fatigue and pain from having a significant impact on scores . age , gender , marital status , number of children , education , exercise , smoking , and duration of sick leave were registered . in addition , different questionnaires regarding pain , function , psychosocial health and work environment were filled in . for the logistic regression analysis we dichotomized educational level into secondary school / vocational education and university degree , episodes of sick leave into 01 and 2 , exercises into < 1/week and 1/week , and smoking to yes ( yes , daily and yes , sometimes ) and no . the nprs has shown better reliability and responsiveness than the visual analogue scale [ 33 , 34 ] . the participants marked on a pain drawing the area or areas that had been painful the last 14 days . subjective health complaints inventory ( shc ) consists of 29 items regarding subjective somatic or psychological complaints experienced during the last month . hopkins symptoms checklist ( hscl-25 ) has 25 items with 10 items for anxiety symptoms and 15 for depression symptoms . the hscl has been shown to have a satisfactory validity and reliability in psychiatric outpatients and in a normal population [ 38 , 39 ] . the tampa scale of kinesiophobia ( tsk ) consists in short form of 13 items concerning fear of movement / re - injury . the tsk has been validated in numerous studies including patients with neck pain , acute and chronic lbp and fibromyalgia [ 4143 ] . . the short form of the rebro musculoskeletal pain screening questionnaire has 10 items and is appropriate for clinical and research purposes since it is nearly as accurate as the longer version . norwegian function assessment scale ( nfas ) is an instrument for self - report of work related functioning with basis in the icf s classification system . test retest reliability has been tested in a normal population and found acceptable . to measure social and psycho - social characteristics of jobs the demand - control - support questionnaire ( dcsq ) , based on a shortened and modified version of the job - demand- social support model ( jcq ) [ 46 , 47 ] . the psychometric properties of dcsq have been demonstrated to be satisfactory [ 46 , 47 ] . the short form-12 ( sf-12 ) , a 12-item version of the sf-36 , was used to measure physical and mental health - related quality of life . the sf-12 has shown good internal consistency , validity , and responsiveness in patients with lbp . bis can refer to high internal consistency , adequate reliability and good convergent and discriminative validity . body mass index was calculated by dividing weight ( kg ) by the square of height ( m ) . the physical tests were chosen to get a general impression of physical function according to body functions or activities in the icf s model . a more detailed description of the tests is given in table 1.table 1description of physical testsphysical testscontentscoreicf - dimensionsglobal body examination ( gbe ) ( 51 , 52)six tests : truncal flexibility and ability to relax during passive movements : elbow - drop flexibility , lumbar - sacral flexibility , head rotation resistance and resistance to hip circumduction , hip - knee flexion and arm / shoulder flexioneach test : 07 . healthy ( 34 individuals ) : median = 5.5 , mean = 7.2body functionback performance scale ( bps ) ( 53 , 54)five tests reflecting mobility - related activities for trunk and lower extremities ( sock - test , pick - up test , roll - up test , fingertip - to - floor and a lift test where a box weighing 4 kg ( women ) or 5 kg ( men ) is lifted from floor to waist for 1 min).each test : 03 . normative data for people without back pain ( n = 150 ) : median = 0 , mean = 0.8activity / participationhigh lift testa high lift test was a modified lift test included in bps . the participants lift a box of 2 kg ( for women ) or 3 kg ( for men ) from waist to shoulder height and back again . the lifting technique was optional.number of lifts performed in 1 min is counted.activity/participationbieringsrensen test ( 5558)static endurance of the back . participants are positioned prone with the upper body extending beyond the edge of the plinth and the lower body is fixed to the bench with three straps.the length of time holding the upper body straight is recorded . max time 240 s. healthy ( 31 individuals ) : median = 138body functionabdominal endurance / strength ( 59 , 60)three levels of dynamic sit - up test with increased demand for each level . the participants are supine with the knees flexed and with feet supported on the plinth by the tester.the number of completed repetitions is counted ( 015).body functiontender points ( 61)18 defined fibromyalgia tender points with four kilos pressure are tested.painful points are countedbody function description of physical tests the global body examination ( gbe ) is used to assess bodily function in patients with long - lasting musculoskeletal pain and/or with psychosomatic complaints . discriminating ability between healthy and different patients groups has shown to be very good to excellent . good inter - tester reliability has been demonstrated in a former version of the gbe . back performance scale ( bps ) consists of five tests reflecting mobility - related daily activities for trunk and lower extremities . retest reliability and responsiveness to change have been demonstrated in patients with long - lasting lbp [ 53 , 54 ] . this is a modified lift test from the lifting test in the bps , but not described elsewhere . to assess static endurance of the back extensors we used the biering retest reliability has been reported as satisfactory , but variability has been high [ 5658 ] . for testing of abdominal endurance / strength we chose a three levels dynamic sit - up test with increasing demands for each level [ 59 , 60 ] . four kilos pressure of 18 defined fibromyalgia tender points were tested , and painful points counted . the study was accepted by the regional committee for medical and health research ethics , western - norway , and was performed according to the helsinki declaration . chicago , il , 2011 ) and matlab ( version 7.10 ; mathwork , 2010 ) . differences between groups ( full sick leave , partial sick leave , not sick leave / working ) were analyzed by chi square exact for categorical data and kruskal wallis and mann whitney u tests for continuous variables . a personal mean was given for missing data if < 30 % of a sub - scale was missing . to examine which factors were associated with being on sick leave , a logistic regression analysis was performed using sick leave groups as the dependent variable and several independent variables ( gender , age , self - reported physical and mental function , perception of work environment , physical tests ) . we estimated both an unadjusted model for each independent variable and a fully adjusted model containing all independent variables . from those models and a correlation analysis we selected a final model based on statistical significance and clinical relevance . work demands were reported in both back - ground data and in the dcsq and reflect similar aspect . we chose the dcsq in the logistic regression model because this is a standardized measurement tool . taking into account multiple effects , a bonferroni adjustment was too conservative , therefore we used p 0.01 as marginal level . the participants were recruited from the department of health and social service in the municipality of bergen , norway , from january 2012 to december 2013 . about 7,000 health care workers are employed in this department ; working in nursing homes , home care service and in special homes for disabled . through their managers and/or brochures we invited employees who were on sick leave or at risk of being sick - listed due to msds , to a functional evaluation . health care workers with msds took direct contact with the university of bergen and booked an appointment with a physiotherapist in the project . exclusion criteria were insufficient knowledge of the norwegian language and being on full sick leave for more than 4 months continuously . within 2 weeks after requesting an appointment , the participants met for an evaluation completed by a physiotherapist in the project . the evaluation ended with a verbal and written presentation of the self - reported and physical findings for all participants , except 56 who were recruited to an randomized controlled trial for participants with low back pain ( lbp ) . they practiced several times together before the start of the project and also examined the first 10 participants together . the participants performed the tests that required minimal effort first , in order to prevent fatigue and pain from having a significant impact on scores . age , gender , marital status , number of children , education , exercise , smoking , and duration of sick leave were registered . in addition , different questionnaires regarding pain , function , psychosocial health and work environment were filled in . for the logistic regression analysis we dichotomized educational level into secondary school / vocational education and university degree , episodes of sick leave into 01 and 2 , exercises into < 1/week and 1/week , and smoking to yes ( yes , daily and yes , sometimes ) and no . the nprs has shown better reliability and responsiveness than the visual analogue scale [ 33 , 34 ] . the participants marked on a pain drawing the area or areas that had been painful the last 14 days . subjective health complaints inventory ( shc ) consists of 29 items regarding subjective somatic or psychological complaints experienced during the last month . hopkins symptoms checklist ( hscl-25 ) has 25 items with 10 items for anxiety symptoms and 15 for depression symptoms . the hscl has been shown to have a satisfactory validity and reliability in psychiatric outpatients and in a normal population [ 38 , 39 ] . the tampa scale of kinesiophobia ( tsk ) consists in short form of 13 items concerning fear of movement / re - injury . the tsk has been validated in numerous studies including patients with neck pain , acute and chronic lbp and fibromyalgia [ 4143 ] . the short form of the rebro musculoskeletal pain screening questionnaire has 10 items and is appropriate for clinical and research purposes since it is nearly as accurate as the longer version . norwegian function assessment scale ( nfas ) is an instrument for self - report of work related functioning with basis in the icf s classification system . retest reliability has been tested in a normal population and found acceptable . to measure social and psycho - social characteristics of jobs the demand - control - support questionnaire ( dcsq ) , based on a shortened and modified version of the job - demand- social support model ( jcq ) [ 46 , 47 ] . the psychometric properties of dcsq have been demonstrated to be satisfactory [ 46 , 47 ] . the short form-12 ( sf-12 ) , a 12-item version of the sf-36 , was used to measure physical and mental health - related quality of life . the sf-12 has shown good internal consistency , validity , and responsiveness in patients with lbp . bis can refer to high internal consistency , adequate reliability and good convergent and discriminative validity . body mass index was calculated by dividing weight ( kg ) by the square of height ( m ) . the physical tests were chosen to get a general impression of physical function according to body functions or activities in the icf s model . a more detailed description of the tests is given in table 1.table 1description of physical testsphysical testscontentscoreicf - dimensionsglobal body examination ( gbe ) ( 51 , 52)six tests : truncal flexibility and ability to relax during passive movements : elbow - drop flexibility , lumbar - sacral flexibility , head rotation resistance and resistance to hip circumduction , hip - knee flexion and arm / shoulder flexioneach test : 07 . healthy ( 34 individuals ) : median = 5.5 , mean = 7.2body functionback performance scale ( bps ) ( 53 , 54)five tests reflecting mobility - related activities for trunk and lower extremities ( sock - test , pick - up test , roll - up test , fingertip - to - floor and a lift test where a box weighing 4 kg ( women ) or 5 kg ( men ) is lifted from floor to waist for 1 min).each test : 03 . normative data for people without back pain ( n = 150 ) : median = 0 , mean = 0.8activity / participationhigh lift testa high lift test was a modified lift test included in bps . the participants lift a box of 2 kg ( for women ) or 3 kg ( for men ) from waist to shoulder height and back again . the lifting technique was optional.number of lifts performed in 1 min is counted.activity/participationbieringsrensen test ( 5558)static endurance of the back . participants are positioned prone with the upper body extending beyond the edge of the plinth and the lower body is fixed to the bench with three straps.the length of time holding the upper body straight is recorded . max time 240 s. healthy ( 31 individuals ) : median = 138body functionabdominal endurance / strength ( 59 , 60)three levels of dynamic sit - up test with increased demand for each level . the participants are supine with the knees flexed and with feet supported on the plinth by the tester.the number of completed repetitions is counted ( 015).body functiontender points ( 61)18 defined fibromyalgia tender points with four kilos pressure are tested.painful points are countedbody function description of physical tests the global body examination ( gbe ) is used to assess bodily function in patients with long - lasting musculoskeletal pain and/or with psychosomatic complaints . discriminating ability between healthy and different patients groups has shown to be very good to excellent . good inter - tester reliability has been demonstrated in a former version of the gbe . back performance scale ( bps ) consists of five tests reflecting mobility - related daily activities for trunk and lower extremities retest reliability and responsiveness to change have been demonstrated in patients with long - lasting lbp [ 53 , 54 ] . this is a modified lift test from the lifting test in the bps , but not described elsewhere . to assess static endurance of the back extensors we used the biering retest reliability has been reported as satisfactory , but variability has been high [ 5658 ] . for testing of abdominal endurance / strength we chose a three levels dynamic sit - up test with increasing demands for each level [ 59 , 60 ] . four kilos pressure of 18 defined fibromyalgia tender points were tested , and painful points counted . the study was accepted by the regional committee for medical and health research ethics , western - norway , and was performed according to the helsinki declaration . age , gender , marital status , number of children , education , exercise , smoking , and duration of sick leave were registered . in addition , different questionnaires regarding pain , function , psychosocial health and work environment were filled in . for the logistic regression analysis we dichotomized educational level into secondary school / vocational education and university degree , episodes of sick leave into 01 and 2 , exercises into < 1/week and 1/week , and smoking to yes ( yes , daily and yes , sometimes ) and no . the nprs has shown better reliability and responsiveness than the visual analogue scale [ 33 , 34 ] . the participants marked on a pain drawing the area or areas that had been painful the last 14 days . subjective health complaints inventory ( shc ) consists of 29 items regarding subjective somatic or psychological complaints experienced during the last month . hopkins symptoms checklist ( hscl-25 ) has 25 items with 10 items for anxiety symptoms and 15 for depression symptoms . the hscl has been shown to have a satisfactory validity and reliability in psychiatric outpatients and in a normal population [ 38 , 39 ] . the tampa scale of kinesiophobia ( tsk ) consists in short form of 13 items concerning fear of movement / re - injury . the tsk has been validated in numerous studies including patients with neck pain , acute and chronic lbp and fibromyalgia [ 4143 ] . the short form of the rebro musculoskeletal pain screening questionnaire has 10 items and is appropriate for clinical and research purposes since it is nearly as accurate as the longer version . norwegian function assessment scale ( nfas ) is an instrument for self - report of work related functioning with basis in the icf s classification system . retest reliability has been tested in a normal population and found acceptable . to measure social and psycho - social characteristics of jobs the demand - control - support questionnaire ( dcsq ) , based on a shortened and modified version of the job - demand- social support model ( jcq ) [ 46 , 47 ] . the psychometric properties of dcsq have been demonstrated to be satisfactory [ 46 , 47 ] . the short form-12 ( sf-12 ) , a 12-item version of the sf-36 , was used to measure physical and mental health - related quality of life . the sf-12 has shown good internal consistency , validity , and responsiveness in patients with lbp . bis can refer to high internal consistency , adequate reliability and good convergent and discriminative validity . body mass index was calculated by dividing weight ( kg ) by the square of height ( m ) . the physical tests were chosen to get a general impression of physical function according to body functions or activities in the icf s model . a more detailed description of the tests is given in table 1.table 1description of physical testsphysical testscontentscoreicf - dimensionsglobal body examination ( gbe ) ( 51 , 52)six tests : truncal flexibility and ability to relax during passive movements : elbow - drop flexibility , lumbar - sacral flexibility , head rotation resistance and resistance to hip circumduction , hip - knee flexion and arm / shoulder flexioneach test : 07 . healthy ( 34 individuals ) : median = 5.5 , mean = 7.2body functionback performance scale ( bps ) ( 53 , 54)five tests reflecting mobility - related activities for trunk and lower extremities ( sock - test , pick - up test , roll - up test , fingertip - to - floor and a lift test where a box weighing 4 kg ( women ) or 5 kg ( men ) is lifted from floor to waist for 1 min).each test : 03 . normative data for people without back pain ( n = 150 ) : median = 0 , mean = 0.8activity / participationhigh lift testa high lift test was a modified lift test included in bps . the participants lift a box of 2 kg ( for women ) or 3 kg ( for men ) from waist to shoulder height and back again . the lifting technique was optional.number of lifts performed in 1 min is counted.activity/participationbieringsrensen test ( 5558)static endurance of the back . participants are positioned prone with the upper body extending beyond the edge of the plinth and the lower body is fixed to the bench with three straps.the length of time holding the upper body straight is recorded . max time 240 s. healthy ( 31 individuals ) : median = 138body functionabdominal endurance / strength ( 59 , 60)three levels of dynamic sit - up test with increased demand for each level . the participants are supine with the knees flexed and with feet supported on the plinth by the tester.the number of completed repetitions is counted ( 015).body functiontender points ( 61)18 defined fibromyalgia tender points with four kilos pressure are tested.painful points are countedbody function description of physical tests the global body examination ( gbe ) is used to assess bodily function in patients with long - lasting musculoskeletal pain and/or with psychosomatic complaints . discriminating ability between healthy and different patients groups has shown to be very good to excellent . good inter - tester reliability has been demonstrated in a former version of the gbe . back performance scale ( bps ) consists of five tests reflecting mobility - related daily activities for trunk and lower extremities retest reliability and responsiveness to change have been demonstrated in patients with long - lasting lbp [ 53 , 54 ] . this is a modified lift test from the lifting test in the bps , but not described elsewhere . to assess static endurance of the back extensors we used the biering retest reliability has been reported as satisfactory , but variability has been high [ 5658 ] . for testing of abdominal endurance / strength we chose a three levels dynamic sit - up test with increasing demands for each level [ 59 , 60 ] . four kilos pressure of 18 defined fibromyalgia tender points were tested , and painful points counted . the study was accepted by the regional committee for medical and health research ethics , western - norway , and was performed according to the helsinki declaration . chicago , il , 2011 ) and matlab ( version 7.10 ; mathwork , 2010 ) . differences between groups ( full sick leave , partial sick leave , not sick leave / working ) were analyzed by chi square exact for categorical data and kruskal wallis and mann whitney u tests for continuous variables . a personal mean was given for missing data if < 30 % of a sub - scale was missing . to examine which factors were associated with being on sick leave , a logistic regression analysis was performed using sick leave groups as the dependent variable and several independent variables ( gender , age , self - reported physical and mental function , perception of work environment , physical tests ) . we estimated both an unadjusted model for each independent variable and a fully adjusted model containing all independent variables . from those models and a correlation analysis we selected a final model based on statistical significance and clinical relevance . work demands were reported in both back - ground data and in the dcsq and reflect similar aspect . we chose the dcsq in the logistic regression model because this is a standardized measurement tool . taking into account multiple effects , a bonferroni adjustment was too conservative , therefore we used p 0.01 as marginal level . a total of 250 participants ( 92.4 % women ) were consecutively recruited to the functional evaluation study . self - reports showed that 83 % of the participants had experienced their present complaints for more than 8 weeks . about 50 % reported previous contact with health personal for treatment of their msds . the group not on sick leave ( working group ) included 168 participants and the groups on partial and full sick leave each included 41 participants . in table 2 , workers on partial sick leave had statistically significant longer duration of sick leave compared to workers on full sick leave . the group on full sick leave reported more heavy physical work compared to the working group . the differences in function , health and work related variables between the three groups are presented in table 3 . major differences in self - reported and physically tested function were observed between the group on full sick leave and the working group . participants on full sick leave had statistically significant poorer function and higher ( worse ) score on rebro questionnaire compared to those working . when comparing those on partial and full sick leave , the group on partial sick leave had statistically significant ( p < 0.05 ) better scores on nfas , the physical dimension of sf-12 , nprs , rebro questionnaire , bsi , gbe and high lift test , compared to the group on full sick leave.table 2demographic variablesvariablesgr.1 working n = 168 n ( % ) gr.2 partial sick leave n = 41 n ( % ) gr.3 full sick leave n = 41 n ( % ) p value sosiodemographic factors age 49 ( 2164)47 ( 2662)49 ( 2167).414 gender , women155 ( 85.4)41 ( 100)35 ( 95.8).052 education.273 secondary school11 ( 6.6)2 ( 4.9)5 ( 12.2 ) vocational education82 ( 49.1)23 ( 56.1)24 ( 58.5 ) university degree74 ( 44.3)16 ( 39.0)12 ( 29.3 ) work status full - time work110 ( 65.5)23 ( 56.1)24 ( 58.5).436 sick leave ( weeks ) 0 ( 0)9 ( 262)3 ( 010 ) < .001 sick leave episodes ( number).214 027 ( 17.1)8 ( 19.5)10 ( 24.4 ) 132 ( 20.3)13 ( 31.7)10 ( 24.4 ) 299 ( 62.7)20 ( 48.8)21 ( 51.2)type of work ( mainly ) .034 sedentary work / sitting13 ( 7.8)1 ( 2.5)0 ( 0 ) standing / walking97 ( 58.1)23 ( 57.5)17 ( 42.5 ) heavy physical work57 ( 34.1)16 ( 40.0)23 ( 57.5 ) health related factors main disorder.067 neck- and shoulder pain53 ( 32.3)14 ( 34.1)7 ( 17.1 ) low back pain61 ( 36.3)19 ( 46.3)19 ( 46.3 ) widespread pain3 ( 25.6)3 ( 7.3)10 ( 24.4 ) other10 ( 6.0)5 ( 12.2)5 ( 12.2)smoking.403 yes , daily31 ( 18.8)9 ( 22.0)13 ( 32.5 ) yes , sometimes18 ( 10.9)4 ( 9.8)5 ( 12.5 ) no116 ( 70.3)28 ( 68.3)22 ( 55.0)exercise.752 < 1/week30 ( 17.9)7 ( 17.0)9 ( 21.9 ) 12/week73 ( 43.5)17 ( 41.5)13 ( 31.7 ) 35/week65 ( 38.7)17 ( 41.5)19 ( 46.4)body mass index 24.9 ( 18.842.1)25.2 ( 17.639.6)25.4 ( 17.236.4).904 median ( min max ) kruskal wallis test chi square , exact test bold = significant at p < 0.05table 3differences in health , work characteristics and function between three groups : those working , on partial sick leave , or on full sick leavevariablesngr wallis testmedian ( min max)median ( min max)median ( min max ) p values pain pain intensity2506 ( 210)5 ( 310)7 ( 210 ) 0.005 pain drawing area25010 ( 170)9 ( 237)10 ( 140)0.72 health factors and function rebro questionnaire25044 ( 1484)46 ( 1770)56 ( 3280 ) 0.001 sf-12 mental23250.1 ( 26.761.1)48.6 ( 30.563.2)48.4 ( 2.961.2)0.412sf-12 physical23245.5 ( 12.859.9)42.2 ( 24.33.2)38.7 ( 24.648.4 ) < 0.001 nfas2501.2 ( 1.002.10)1.23 ( 1 ( 1.00 1.84)1.42 ( 1.0 2.38 ) < 0.001 hscl2441.44 ( 1.002.87)1.42 ( 1.002.58)1.45 ( 1.043.08)0.665shc ( n)17910 ( 315)9 ( 313.0)10 ( 315)0.245tsk24721.7 ( 13.046.0)21.0 ( 13.035.8)21.0 ( 13.043.0)0.952bis24416.5 ( 042)17.0 ( 036)24.0 ( 241)0.065 work characteristics dcsq social2470.78 ( 0.221.00)0.78 ( 0.331.00)0.72 ( 0.331.00)0.108dcsq demand2460.67 ( 0.001.00)0.67 ( 0.270.93)0.67 ( 0.271.00)0.214dcsq control2400.67 ( 0.220.94)0.64 ( 0.390.83)0.67 ( 0.390.83)0.186 physical assessment acr - tender points ( n)2507 ( 018)6 ( 018)7 ( 018)0.616gbe flexibility25016 ( 235)16 ( 530)19 ( 535 ) 0.038 high lift test ( n)25016 ( 029)15 ( 824)13 ( 325 ) < 0.001 abdominal strength ( n)24812.5 ( 015)9 ( 015)5 ( 015 ) < 0.001 back strength ( s)24870 ( 0240)33 ( 0220)36 ( 0240 ) 0.002 bps2503 ( 015)4 ( 011)6 ( 013 ) < 0.001 sf-12 quality of life , short form-12 , nfas norwegian function assessment scale , hscl hopkins symptoms checklist , shc subjective health complaints , tsk tampa scale of kinesiophobia , bis bergen insomnia scale , bmi body mass index , dcsq demand - control - support questionnaire , acr - tender points american criteria of rheumatology , gbe global body examination , bps back performance scale . bold = significant at p < 0.05 demographic variables chi square , exact test bold = significant at p < 0.05 differences in health , work characteristics and function between three groups : those working , on partial sick leave , or on full sick leave sf-12 quality of life , short form-12 , nfas norwegian function assessment scale , hscl hopkins symptoms checklist , shc subjective health complaints , tsk tampa scale of kinesiophobia , bis bergen insomnia scale , bmi body mass index , dcsq demand - control - support questionnaire , acr - tender points american criteria of rheumatology , gbe global body examination , bps back performance scale . bold = significant at p < 0.05 the results of the logistic regression analysis are presented in table 4 . the group on full sick leave and the group on partial sick leave were compared with the working group . complete data were available in 210 participants ( 142 working , 30 on full sick leave , 38 on partial sick leave ) . reduced level of the physical dimension of sf-12 and on high lift test were significantly related to full sick leave ( or 0.86 , p < 0.001 ) ( or 0.79 , p = 0.002 ) . there was also a tendency ( p < 0.05 ) that being on full sick leave was associated with gender , the mental dimension of sf-12 , the hscl-25 , the demand dimension of the dcsq , and the abdominal strength test . the physical dimension of sf-12 ( or 0.91 , p = 0.005 ) was the only variable that was associated to partial sick leave ( table 4 ) . table 4logistic regression comparing group on full sick leave and on partial sick leave with the working groupvariablesnunadjusted model , full sick leaveadjusted model , full sick leave n = 30nunadjusted model , partial sick leaveadjusted model , partial sick leave n = 38or95 % ci p valueor95 % ci p valueor95 % ci p valueor95 % ci p valuegender410.49(0.171.38)0.1760.10(0.020.67)0.01841 age411.00(0.971.04)0.8840.96(0.921.01)0.126410.98(0.951.01)0.2410.97(0.941.01)0.164smoking400.67(0.461.00)0.0490.84(0.471.51)0.567410.92(0.611.41)0.7111.16(0.691.96)0.580sf-12 physical370.86(0.820.91 ) < 0.001 0.86(0.790.94 ) < 0.001 400.91(0.870.96 ) 0.001 0.91(0.850.97 ) 0.005 sf-12 mental370.96(0.911.01)0.1100.89(0.801.00)0.043400.98(0.931.04)0.5090.95(0.871.03)0.210hscl401.99(0.844.72)0.1180.10(0.010.90)0.040401.16(0.452.98)0.7540.25(0.041.43)0.119dcs support410.12(0.020.80)0.0290.67(0.0218.82)0.816411.20(0.168.77)0.8611.96(0.1526.35)0.611dcs demand403.63(0.324.86)0.18937.07(1.73792.84 ) . 021414.75(0.6932.65)0.1136.65(0.5876.37)0.128dcs control380.18(0.012.88)0.2234.43(0.03578.40)0.549400.21(0.013.34)0.2720.16(0.014.88)0.295gbe flexibility411.07(1.021.12 ) 0.009 1.02(0.941.10)0.676411.00(0.951.05)0.9570.98(0.921.04)0.559high lift test410.82(0.750.90 ) < 0.001 0.79(0.680.91 ) 0.002 400.97(0.891.06)0.4781.04(0.921.17)0.567abdominal strength400.84(0.780.91 ) < 0.001 0.84(0.730.97)0.014410.94(0.871.00)0.0650.95(0.851.06)0.335back strength410.99(0.981.00)0.0131.00(0.001.01)0.385410.99(0.991.00)0.0390.99(0.991.00)0.305bps411.29(1.151.45 ) < 0.001 0.85(0.661.09)0.206411.15(1.031.29)0.0161.06(0.891.28)0.513 sf-12 quality of life , short form-12 , hscl hopkins symptoms checklist , dcsq demand - control - support questionnaire , gbe global body examination , bps back performance scale . bold = significant at p 0.01 only womentable 5logistic regression - full model . comparing full sick leave and partial sick leave with the working groupn = 189nunadjusted model , full sick leaveadjusted model , full sick leavenunadjusted model , partial sick leaveadjusted model , partial sick leaveor(95 % ci ) p valueor(95 % ci ) p valueor(95 % ci ) p valueor(95 % ci ) p valuegender410.49(0.171.38)0.1760.06(0.000.70)0.02541age411.00(0.971.04)0.8840.96(0.911.02)0.160410.98(0.951.01)0.2410.98(0.931.02)0.284education410.52(0.251.09)0.0830.67(0.192.39)0.541410.80(0.401.62)0.5410.49(0.171.38)0.177working ( full / partly)411.37(0.682.75)0.3811.93(0.527.17)0.324411.51(0.753.03)0.2451.16(0.403.40)0.781sick leave episodes410.63(0.311.25)0.1840.42(0.131.38)0.153410.57(0.281.13)0.1090.53(0.201.38)0.190smoking400.67(0.461.00)0.0490.72(0.361.44)0.351410.92(0.611.41)0.7111.25(0.672.32)0.486exercise410.77(0.331.79)0.5470.94(0.194.70)0.937411.06(0.432.61)0.9061.49(0.346.45)0.594pain categories410.71(0.471.08)0.1070.83(0.332.09)0.692410.79(0.521.19)0.2660.89(0.461.73)0.738pain drawing area410.99(0.961.02)0.5820.98(0.931.04)0.572410.98(0.941.02)0.2930.97(0.921.02)0.280nprs411.33(1.101.60 ) 0.003 0.95(0.641.41)0.809410.95(0.791.13)0.5460.77(0.561.06)0.112rebro questionnaire411.05(1.021.08 ) < 0.001 1.04(0.981.11)0.211411.01(0.991.04)0.3261.02(0.961.08)0.470nfas4120.89(5.6277.70 ) < 0.001 2.47(0.1348.00)0.550412.99(0.7212.47)0.1320.56(0.039.48)0.685sf-12 physical370.86(0.820.91 ) < 0.001 0.88(0.790.98)0.015400.91(0.870.96 ) 0.001 0.89(0.810.97 ) 0.006 sf-12 mental370.96(0.911.01)0.1100.91(0.801.03)0.131400.98(0.931.04)0.5090.94(0.841.05)0.246hscl401.99(0.844.72)0.1180.08(0.011.15)0.063401.16(0.452.98)0.7540.66(0.085.35)0.699tsk411.00(0.951.06)0.8630.86(0.760.98)0.019410.99(0.941.05)0.7800.95(0.861.05)0.333dcs social410.12(0.020.80)0.0290.34(0.0116.50)0.583411.20(0.168.77)0.8613.13(0.1373.77)0.479dcs demand403.63(0.5324.86)0.189153.40(2.778,508.64)0.014414.75(0.6932.65)0.1135.53(0.27112.31)0.266dcs control380.18(0.012.88)0.2231.11(0.00363.95)0.973400.21(0.013.34)0.2720.14(0.0011.13)0.379bis401.04(1.001.07 ) . 2230.87(0.741.03)0.097410.94(0.871.00)0.0650.94(0.831.07)0.343back strength410.99(0.981.00)0.0131.00(0.991.02)0.597410.99(0.991.00)0.0390.99(0.981.01)0.314bps411.29(1.151.45 ) < 0.001 0.87(0.631.21)0.415411.15(1.031.29)0.0161.10(0.871.38)0.432 nprs numeric pain rating scale , nfas norwegian function assessment scale , sf-12 quality of life , short form-12 , hscl hopkins symptoms checklist , shc subjective health complaints , tsk tampa scale of kinesiophobia , dcsq demand - control - support questionnaire , bis bergen insomnia scale , bmi body mass index , acr - tender points american criteria of rheumatology , gbe global body examination , bps back performance scale . bold = significant at p 0.01 logistic regression comparing group on full sick leave and on partial sick leave with the working group sf-12 quality of life , short form-12 , hscl hopkins symptoms checklist , dcsq demand - control - support questionnaire , gbe global body examination , bps back performance scale . bold = significant at p 0.01 logistic regression - full model . comparing full sick leave and partial sick leave with the working group nprs numeric pain rating scale , nfas norwegian function assessment scale , sf-12 quality of life , short form-12 , hscl hopkins symptoms checklist , shc subjective health complaints , tsk tampa scale of kinesiophobia , dcsq demand - control - support questionnaire , bis bergen insomnia scale , bmi body mass index , acr - tender points american criteria of rheumatology , gbe global body examination , bps back performance scale . in this study we found that workers on full sick leave had reduced self - reported and physically tested function compared to workers still working despite msds , as well as compared to those on partial sick leave . lower physical function measured by the physical function score on sf-12 and the high lifting test were strongest associated with being on full sick leave . being female , lower mental health score ( worse ) on sf-12 , in addition to lower scores ( better ) on the hscl-25 , increased self - reported work demands ( dcsq ) and lower abdominal strength showed a tendency to be associated with being on full sick leave . for the group on partial sick leave , only the physical function scale of sf-12 was associated with being on sick leave , those on sick leave having lower ( worse ) scores . our findings are supported by several studies , but there are also new and interesting findings . low self - reported physical health and disability have been found to be associated with being on sick leave in patients with chronic lbp . in a systematic review of factors that promote staying at work with msds , an association was found between low perceived physical disability and staying at work . however , only a few studies have compared measures of physical tests / capacity between workers on sick leave and workers who continue working despite pain . compared functional capacity between workers staying at work despite msds , workers on sick leave due to msds and a group of healthy workers . in accordance with our findings , they found that the two groups with msds had significantly lower functional capacity than the healthy group , with the lowest capacity observed in the group on sick leave . other studies have shown that physical tests can predict return to work after being on sick leave . cardiovascular fitness was identified as one of the strongest predictors for return to work in a norwegian study . in a systematic review , better results on physical tests , and especially the lifting test , appeared to be predictive of work participation for patients with msds . as our study was cross - sectional , prediction of work participation could not be estimated . low lifting capacity was , however , strongly associated with being on full sick leave . an explanation may be that lifting captures several components such as gripping , holding , bending and lowering . several explanations were considered in order to explain why the participants on full sick leave in the present study had lower scores on the physical tests compared to workers not on sick leave . a possible explanation could have been different level of exercise between groups . however , the three groups in the present study reported quite similar level of regular exercising , in accordance with earlier research [ 12 , 27 ] . increased fear avoidance has been observed in workers on sick leave with msds [ 19 , 27 , 29 ] . our findings did not support this association , as scores on the tsk were similar for those on sick leave versus those working . even if a state of deconditioning is present , the functional capacity could still be sufficient to meet actual work demands , especially if they are not too excessive . however , health care workers usually have physically demanding work , including lifting , transferring patients and working in uncomfortable positions . in accordance with several studies showing that perceived workloads are associated with being on sick leave [ 12 , 19 , 27 ] , the workers on full sick leave in this present study reported higher perceived work demands than the other two groups . the reason might be more demanding work tasks for this group , but decreased physical capacity might also influence an individual s perception of work demands . this highlights the need for research that takes into account work demands and work environment for specific occupational groups . high pain intensity has also been associated with being on sick leave [ 8 , 11 , 17 ] . our study showed a statistically significant difference of pain intensity between the groups , with the highest level in the group on full sick leave and the lowest in the group on partial sick leave . however , there was only one point in difference on the nprs between those on full sick and the working group . it is therefore not likely that the pain level was of great importance for the result regarding physical functioning in the present study . in previous years , much attention has been given to the role of psycho - social factors related to work ability [ 17 , 64 ] . there were only small differences in measures of the psychological variables between the groups in our study . reduced physical function was more strongly associated with being sick - listed than pscyho - social factors , also reported in previous research [ 12 , 27 ] . there was only a tendency that being on full sick leave was associated with mental health , and the results were conflicting . the group on full sick leave showed worse function at the mental health component of sf-12 , but surprisingly , better score on hscl-25 . the hscl-25 has a higher number of items related to mental health and may therefore provide a more precise picture than the less detailed generic questionnaire sf-12 . being on short time sick leave , as in our study , may to a lesser degree influence psycho - social factors . the authorities in norway , sweden and denmark have strongly promoted the use of partial sick leave as the primary choice , if sick leave is needed . it is assumed that partial sick leave has positive effects on health and well - being , compared to full - time absence , and it is believed to facilitate return to full - time work . to our knowledge , the present study is the first study comparing self - reported and physical tested function in workers with those on full or partial sick leave , due to msds . the group on partial sick leave had statistically significant better function on some of the functional questionnaires and physical tests compared to those on full sick leave . more women than men have been on partial sick leave according to register data from norway . further research is needed to get insight into factors affecting workers on partial and full sick leave , and the decisions around sick leave . the high number of participants in our study ( n = 250 ) gave us enough power to detect differences between workers on full , partial or not on sick leave , and to identify variables related to work status . in accordance with the icf- model a variety of demographic variables , questionnaires and physical tests were used to cover the different dimensions in the model when evaluating the participants functioning and working ability . we used well - known standardized questionnaires measuring pain , physical- and mental functioning and conditions at work . this is in line with wand et al . who argued that both self - reported and physically tested functioning need to be assessed to get a better understanding of msds and how they could be managed . the tests demonstrate good levels of reliability and validity , but two of the tests ( abdominal and high lift tests ) are still under evaluation . the physical tests were able to discriminate functioning between workers on sick leave and not , although most of the workers were not on long - term sick leave . this indicates that the test battery could be a useful assessment of function at an early stage of sick leave and a tool when giving advice about rehabilitation and work adaption . different batteries of physical tests are designed to evaluate work ability and daily functioning [ 18 , 66 , 67 ] . most of them are costly and time - consuming and are mainly used as assessment tools in the return to work process . in contrast to this , our test battery is cheap , quick to apply and require little equipment and therefore could also be a useful clinical tool in private practice for physiotherapists . workers were provided with information about the project by their leaders and through pamphlets and took direct contact to participate . although several workers were pushed by their employer to participate , we can not be sure that the least motivated and the workers with more complex health problems actually contacted us . our target population was workers on sick leave or at risk of becoming sick - listed due to msds . only 20 % of all that were examined had never been on sick leave due to msds before ; indicating that we have included the target group . interviews with managers in the midst of the total project supported that we had managed to get a representative sample of participants ( not yet published ) . although self - reported sick leave data has been evaluated as being less reliable than register recorded data , other studies [ 69 , 70 ] have demonstrated good agreement between self - reports and register data in cross - sectional design . the length of the last sick leave and number of sick leave episodes the last years are reported , but not the length of all absences . workers on sick leave could recently have returned to work , and workers on partial sick leave could have changed to full sick leave . our study did not record this , and it is quite surprising that the differences between the groups still were so significant . over 90 % of the participants in the present study were women working in the health- and social sector . being male and/or having a less demanding work may not affect work ability in the same way . the present study was cross - sectional and therefore causality can not be inferred , and only associations are reported . it was conducted in a single country with a highly established social insurance system , thereby reducing generalizability of the study to countries that have similar social and security system . more specific knowledge about occupational sub - groups is needed to catch groups at risk for prolonged sick leave , and further research in this field should emphasize longitudinal studies . the high number of participants in our study ( n = 250 ) gave us enough power to detect differences between workers on full , partial or not on sick leave , and to identify variables related to work status . in accordance with the icf- model a variety of demographic variables , questionnaires and physical tests were used to cover the different dimensions in the model when evaluating the participants functioning and working ability . we used well - known standardized questionnaires measuring pain , physical- and mental functioning and conditions at work . this is in line with wand et al . who argued that both self - reported and physically tested functioning need to be assessed to get a better understanding of msds and how they could be managed . the tests demonstrate good levels of reliability and validity , but two of the tests ( abdominal and high lift tests ) are still under evaluation . the physical tests were able to discriminate functioning between workers on sick leave and not , although most of the workers were not on long - term sick leave . this indicates that the test battery could be a useful assessment of function at an early stage of sick leave and a tool when giving advice about rehabilitation and work adaption . different batteries of physical tests are designed to evaluate work ability and daily functioning [ 18 , 66 , 67 ] . most of them are costly and time - consuming and are mainly used as assessment tools in the return to work process . in contrast to this , our test battery is cheap , quick to apply and require little equipment and therefore could also be a useful clinical tool in private practice for physiotherapists . workers were provided with information about the project by their leaders and through pamphlets and took direct contact to participate . although several workers were pushed by their employer to participate , we can not be sure that the least motivated and the workers with more complex health problems actually contacted us . our target population was workers on sick leave or at risk of becoming sick - listed due to msds . only 20 % of all that were examined had never been on sick leave due to msds before ; indicating that we have included the target group . interviews with managers in the midst of the total project supported that we had managed to get a representative sample of participants ( not yet published ) . although self - reported sick leave data has been evaluated as being less reliable than register recorded data , other studies [ 69 , 70 ] have demonstrated good agreement between self - reports and register data in cross - sectional design . the length of the last sick leave and number of sick leave episodes the last years are reported , but not the length of all absences . workers on sick leave could recently have returned to work , and workers on partial sick leave could have changed to full sick leave . our study did not record this , and it is quite surprising that the differences between the groups still were so significant . over 90 % of the participants in the present study being male and/or having a less demanding work may not affect work ability in the same way . the present study was cross - sectional and therefore causality can not be inferred , and only associations are reported . it was conducted in a single country with a highly established social insurance system , thereby reducing generalizability of the study to countries that have similar social and security system . more specific knowledge about occupational sub - groups is needed to catch groups at risk for prolonged sick leave , and further research in this field should emphasize longitudinal studies . health care workers on full sick leave due to msds have reduced function on self - reported and physically tested function , compared to those working despite msds , as well as compared to those on partial sick leave . lower physical function measured by the physical dimension on sf-12 and the high lift test were strongest associated with being on full sick leave , and only the physical dimension on sf-12 was associated with being on partial sick leave . the authors , tove ask , jan sture skouen , jrg assmus , and alice kvle have no conflict of interest .

purpose the aim of this study was to describe self - reported and physically tested function in health care workers with musculoskeletal disorders ( msds ) and to examine how function was associated with work participation.methodsa cross - sectional study was conducted . 250 health care workers attended an evaluation where self - reported and physical function were measured . differences between groups ( full sick leave , partial sick leave , not on sick leave / working ) were analyzed for categorical data ( chi square exact test ) and continuous variables ( kruskal wallis and mann whitney u tests ) . logistic regression analysis was performed to examine which factors were associated with being on sick leave.results participants on full sick leave had statistically significant poorer function compared to those working and the group on partial sick leave . logistic regression showed that a reduced level of the physical dimension of sf-12 and a high lift test were significantly related to full sick leave ( or 0.86 , p < 0.001 ) ( or 0.79 , p = 0.002 ) . the physical dimension of sf-12 was the only variable that was associated to partial sick leave ( or 0.91 , p = 0.005).conclusionhealth care workers on full sick leave due to msds have reduced function on self - reported and physically tested function , compared to those working despite msds , as well as when compared to those on partial sick leave . more knowledge about work ability in occupational sub - groups is needed .

Introduction Methods Participants Procedure Measures Self-Report Physical Tests Statistical Analyses Results Discussion Strengths and Limitations Conclusion Conflict of interest

breast cancer remains the leading cause of cancer related morbidity and mortality in women , worldwide . every tenth new cancer diagnosed is that of breast , and nearly a quarter of cancers in the women are breast cancer . about 1.4 million new breast cancer cases are diagnosed every year , and the burden is going to rise tremendously . the incidence of breast cancer rises with increasing age , about 80% of breast cancer occur in women above 50 years of age and nearly half in the age group range of 50 to 69 . the majority of this postmenopausal women with early breast cancer are hormone , that is , estrogen ( er ) and progesterone ( pr ) receptor positive hormone therapy , which essentially deprives the tumor cells of hormone , relies on the fact that the tumor cells from the breast retain the property of thriving in the presence of female sex hormones , and dispossession of which results in inhibition of cancer cell regrowth . hormonal therapy is confirmed to render at least a benefit of 47% risk reduction for recurrence and that of 26% for mortality . this benefit alone surpasses the combined gain obtained by all other interventions in the adjuvant setting . most noticeably , the hormonal therapy intervention benefits all the patients who are hormone positive , regardless of their tumor size and nodal status . tamoxifen has traditionally been considered the gold standard for adjuvant hormonal therapy in hormone receptor positive breast cancer . the newer third generation aromatase inhibitors have challenged this unparalleled status of tamoxifen in postmenopausal women , in whom cessation of ovarian function leaves only peripheral conversion of steroids as the source of production of estrogen and therefore making it an attractive target to block . data from randomized phase iii trials has now shown for most available ais to have better toxicity profile and at least equal , if not superior , efficacy than the traditionally used tamoxifen . the physicians who still prefer to use tamoxifen quote unique mechanism of action with possible intrinsic benefits and low cost as the reason behind the choice . while the advocates of the newer third generations ai cite greater safety and improved pfs including a possible overall survival benefit as strong reasons for shifting the gear in favor of ais as initial therapy . there is now in addition a data to support the switch strategy , in which one agent is alternated with the other , to have actually more beneficial effects than traditionally used continuous single agent for five years . this mix of emerging and existing data has understandably expanded the options for physician prescription ; besides , the many unanswered or half answered questions regarding whether to use ais , which ai , longer use and switching between ais and tamoxifen , has left the treating physician to use the best personal judgment in making a choice before solid clinical data are available . with this background we conducted a survey to on practicing oncology physicians worldwide as to see the trends of prescriptions of hormonal treatment for postmenopausal women in early adjuvant setting . with the propose of identifying what questions still need to be answered unequivocally from clinical trials that would set the standards from which this , the majority , of breast cancer patients would benefit the most . a web page was developed , which asked physicians five main questions , after collecting data on their profile and country of practice . the questions asked about the choice of physicians hormonal agent in early adjuvant setting for the postmenopausal women without having a significant co - morbid , such as a history of osteoporosis , stroke , ischemic heart disease , and hyperlipidemia . the questionnaire also asked duration of use of hormonal therapy , switching therapy , laboratory investigations done on followup , and the frequency of follow up . the webpage address was sent to contactable physicians whose emails were available in the oncology conference database . the survey was sent out to 522 physicians , a total of 182 physicians responded to the questionnaire , and the response percentage is 34.8% . most of participants that are 142 ( 78.0% ) were medical oncologists , 24 ( 13.2% ) clinical oncologists , while 14 ( 7.8% ) were radiation oncologists by profession . approximately one - third ( 38.5% ) of them were above 45 years of age while remaining participants were younger than 45 ; likewise one - third ( 33% ) of them were in clinical practice for less than 5 years while remaining two - thirds have clinical experience of more than 5 years . most of the responding physicians were working either as private physicians or in teaching hospitals ( 40.7% each ) . participants were from most parts of the world , including pakistan , india , saudi arabia , middle east , uk , europe , and usa . a detailed list of the participants is given in table 1 . in response to question what is your first choice of hormonal agent in early adjuvant setting for receptor positive postmenopausal breast cancer patients , without significant comorbid like osteoporosis , dvt , heart disease ? 36.3% chose anastrozole as their choice of therapy , 35.2% favored tamoxifen , and 22% preferred letrozole as their first choice . more than two - thirds ( 67% ) of the participants gave the reason of having a robust clinical data behind their choice of therapy , while one - third stated concerns regarding safety as their primary motivation behind the choice of the agent of their preference . 31% physicians stated they consider cost , and 34% consider ease of availability as an additional factor in making a choice for the hormonal agents . about 8% stated that a trustworthy pharmaceutical company was the major factor behind the decision regarding the drug of their choice . figure 2 summarizes the reason considered for prescription by different physicians . in answer to the question do you switch your patient from one agent to other during treatment , in a patient who does not have any progression of disease ? 63.7% responding physicians said yes while 33% replied in negative . amongst the physicians who switched to another agent 67.2% based their decisions on presence of data , and 24% had to switch the therapy due to problems encountered during the patient management . of 33% of physicians who do not switch their patients from one agent to other 80% of them based their decisions on lack of data favoring the benefit of switching the therapy according to them . medical oncologists switched therapy more often than a clinical or radiation oncologists ( 0.002 ) . when the participants were asked about the number of investigation do they undertake before starting the treatment of the patients in early adjuvant breast cancer treatment for postmenopausal women , without a major known comorbidity , 73.6% replied that they do some investigations . table 2 summarizes the laboratory and radiological investigations frequently requested by the physicians on followup . nearly two - thirds of physicians ( 61.5% ) followed their patients 36 monthly during the 1st year of followup while 21% follow them every other month , 10% every month , and 2% follow their patients every 6 monthly . most of physicians ( 73.6% ) keep their patients for 5 years on chosen hormonal therapy ; 8.8% want their patients to take hormonal therapy for 2 years , 6.6% for 7 years , and 4.4% for 10 years . insignificant differences were also noticed based on designation , type , country , age , and years of practice . the concept of breast cancer 's hormone sensitivity is more than a century old , when oophorectomies were shown to result in regression of advanced breast cancers . deriving benefits from the same concept , subsequent pharmacologic developments , and refining breast cancer subtypes , today hormonal therapies are the mainstay of hormone receptor positive breast cancers , which make up about 75% to 80% of the breast cancer population . the data looking at effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival by evaluation of all randomized trials from early breast cancer trialists group ( ebctg ) suggest a 40% reduction in the risk of recurrence and 30% reduction in the risk of death with intervention from hormonal therapy , in women with hormone positive breast cancer , making hormone therapy , the single most powerful intervention , in terms of patient benefit . tamoxifen an oral nonsteroidal , antiestrogenic compound with an intrinsic proestrogenic activity was first discovered in 1966 and first approved for clinical use in 1977 for metastatic breast cancer . tamoxifen was found , through series of studies , to render sufficient therapeutic benefits without significant toxicity to justify the title of being gold standard for antiestrogen treatment in breast cancer . the status of tamoxifen as a gold standard has been significantly challenged by the newer generation of aromatize inhibitors which in themselves are a robust group of antiestrogenic therapy . the mechanism of action of ais relies on the fact that most estrogen in the postmenopausal women comes from conversion of steroid hormones to estrogen . the conversion takes place primarily in the adrenals and at multiple other sites and is mediated by the aromatase enzyme . the third - generation aromatase inhibitors first made their impact when they were compared to megestrol acetate in metastatic breast cancer ( mbc ) setting and showed superior time to progression ( ttp ) . to date in at least five randomized phase iii studies done subsequently ais demonstrated mostly superior response rate and time to progression compared to tamoxifen , when used in metastatic breast cancer [ 14 , 15 ] . ais have been clinically tested in adjuvant setting where they have unequivocally proved , in at least four phase iii trials ; that early adjuvant therapy with third - generation aromatase inhibitor in postmenopausal women with hormone positive breast cancer provides better disease - free survival ( dfs ) compared to the traditionally used tamoxifen . all three available ais anastrozole , letrozole , and exemestane have been separately compared with tamoxifen . the intergroup exemestane study studied switching to exemestane after initial 2 - 3 years of tamoxifen therapy in more than 4500 patients and showed improved disease free and a modest improvement in overall survival in er positive women . the followup study results of long - term use letrozole versus placebo after 5 years of tamoxifen in the ncic ctg ma.17 trial analysis were recently published and suggest that extended adjuvant letrozole was superior to placebo in dfs and os . there are , however , serious considerations associated with the use of ais in the form of increased expense and adverse impact on quality of life with increased bone loss and joint pains . there have , thus , been calls for careful interpretation of complex scientific data available and to identify important subsets of population who are to actually derive the greatest benefit from the use of ais [ 19 , 20 ] . the existing and evolving knowledge and research on refinement in hormonal therapy for postmenopausal women generally is favoring the use of ais , a longer duration of therapy and possibly for using the agents alternatively . however , the existing data appears not to have reached a threshold of generating distinct guidelines in favor of their use in this particular patient population , except for suggestions for the same , neither has been the duration of therapy , and the switch strategy reached a point where recommendation can be made unequivocally . controversies in setting up the end points , complexity of the data and crossover of the patients from one arm to the other are some of the factors responsible for the ambiguity . our survey clearly demonstrates the discord that is seen in general amongst the treating physicians . although our survey has limitations in the form of number of participants and geographical variance to claim global representation ; we , nevertheless , find that tamoxifen is still prescribed as frequently in postmenopausal women with early stage breast cancer in adjuvant setting as are ais . switch therapy has been found to be sufficiently practiced amongst our survey population where close to 64% physicians actually do mention to switch the patients to exemestane based on the data . there were individual differences in the frequency of followups and laboratory or radiological investigations requested by physicians . it seems that in general there is a lack of clear guidelines available to physicians on how to follow the patients after they are over with the active adjuvant treatments that involve chemotherapy and radiation therapy . physicians vary over choices on the preferred agents of hormonal therapy in postmenopausal women in whom there are no significant comorbid . they also differ quite significantly among the duration , switching the therapy , frequency of followup , and necessary laboratory and radiological investigations in such setting . in order to benefit the patients the most from what is believed to be corner stone of the therapy , further optimization of hormonal therapy with answers from clinical trials designed to identify the subgroups , benefitting the most from a specific hormonal therapy , shall perhaps set the standards .

the choice of adjuvant hormonal therapy in postmenopausal women with hormone receptor positive breast cancer has remained a matter of controversy and debate . the variety of agents is available , with each claiming to be superior . this clinical survey was undertaken to get an impression of the physician 's first choice of therapy in an attempt to find out what questions still need to be answered in the making of standard of care . a web - based clinical survey was sent to the cancer physicians around the world , and 182 physicians responded to the survey . most were medical oncologists in a tertiary care hospital . 36.3% preferred anastrozole , 35.2% tamoxifen , and 22.2% letrozole as their first choice . data support ( 67.8% ) and safety concerns ( 30% ) were given as the main reasons for the choice , 63.7% switched their therapy , and 24% had to switch because of side effects . 73.6% used 5 years of adjuvant hormonal therapy , 6.6% for 7 years , and 4.4% for 10 years . 61.5% follow their patients 3 times monthly , and 73.2% used laboratory and radiological assessment at each followup . conclusion . physicians show disagreement over the choice and duration of hormonal therapy in this patient population . clinical trials leading to firm recommendations to set standards from which patients benefit the most are needed .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

acetyl - coa carboxylase ( ec6.4.1.2 ) ( acc ) is a biotin carboxylase that catalyzes the atp - dependent condensation of acetyl - coa and carbonate to form malonyl - coa . it is an essential and rate - limiting substrate for de novo lipogenesis ( dnl ) , and it acts as an allosteric inhibitor of the enzyme carnitine - palmitoyl transferase i ( cpt-1 ) . cpt-1 is responsible for the transport of long - chain fatty acyl - coas across the mitochondrial membrane into the mitochondria where they become available for fatty acid oxidation . thus , acc is positioned as a key physiologic switch regulating the transition from oxidative to lipogenic metabolism . metabolic perturbations , including suppressed fatty acid oxidation and increased hepatic dnl , have been hypothesized to contribute to ectopic accumulation of lipid species in muscle and liver , which in turn have been hypothesized to play a causative role in the molecular pathogenesis of insulin resistance . inhibition of malonyl - coa production by acc is expected to simultaneously inhibit dnl and increase flux through cpt-1 , leading to increased -oxidation of long - chain fatty acids , and thus may lead to reduced ectopic lipid accumulation and improved insulin sensitivity . acc inhibition is therefore an attractive biological target for the treatment of metabolic diseases such as t2 dm and nonalcoholic fatty liver disease . consistent with this hypothesis , antisense oligonucleotide inhibition of acc significantly reduced diet - induced hepatic steatosis and hepatic insulin resistance . the two closely related isoforms , acc1 and acc2 , are encoded by separate gene products that differ in tissue and subcellular distribution . acc1 is primarily located in liver and adipose tissue , while acc2 is the dominant isoform in skeletal and heart muscle . acc1 is also expressed in multiple human cancers , making it an attractive oncology target . we sought balanced inhibitors of acc1 and acc2 to gain benefit from inhibition of the enzyme in both liver and muscle . several acc inhibitors have been disclosed in recent years , with much consideration directed toward understanding whether selective or balanced inhibition of acc1/acc2 is preferable . published results to date abbott described an acc2-selective thiazole ether ( r = ome ) that elicited dose - dependent reductions in muscle malonyl - coa levels . however , significant neurological and cardiovascular safety events were observed and attributed to the alkyne - containing structure of the specific compound . using a related acc2-selective compound from the abbott disclosures ( r = me ) , boehringer ingelheim observed reductions in malonyl - coa , stimulation of fatty acid oxidation , improvements in glucose tolerance , and hba1c reductions following chronic treatment of db / db mice . a phenyl ether from sanofi - aventis , with unselective activity against acc1/acc2 , increased lipid oxidation but failed to decrease hepatic triglycerides or body weight in diet - induced obese ( dio ) mice or in zucker diabetic fatty rats after chronic administration . takeda described a spiro - pyrazolidinedione with balanced acc1/acc2 activity that showed dose - dependent changes in respiratory quotient in rats , providing evidence of increased fatty acid oxidation . amgen s piperazine oxadiazole with dual acc1/acc2 inhibition decreased malonyl - coa levels but unexpectedly increased plasma glucose and impaired glucose tolerance in dio mice treated for 28 days . the natural product soraphen a , also an inhibitor of both acc1/acc2 , decreased weight gain and body fat content in mice and improved insulin sensitivity , although a narrow safety window may have confounded the results . nimbus disclosed favorable impact on weight gain , triglycerides , cholesterol , and insulin sensitivity in dio rats with a compound whose specific structure was not reported . pfizer described a spiroketone ( 1 ) that decreased malonyl - coa in liver and muscle ; the subject of this paper is follow - up to that disclosure , along with preclinical and human biology data for a lead compound . the n2-alkyl pyrazole ketones with substitution at the -position to the ketone and the n1-alkyl pyrazole ketones described in this work were synthesized by the general methods shown in schemes 1 and 2 . the mono- and dimethyl substituted ketone cores in scheme 1 were synthesized by -alkylation of the respective precursor ketones . although the desired enolates could be formed at low temperature by treatment of the ketone with lithium diisopropylamide ( lda ) or lithium hexamethyldisilazide ( lhmds ) , the rate of alkylation ( for example , with methyl iodide ) was slow relative to the rate of ring - opening -elimination of the oxy - pyrazole anion . notably , addition of 1,3-dimethyl-3,4,5,6-tetrahydro-2(1h)-pyrimidinone ( dmpu ) achieved both increased efficiency of enolate formation and increased reactivity of the enolate to the desired alkylation . the pyrazoloketones in scheme 2 were prepared by regioselective addition of alkyl hydrazines to a cyclohexenone intermediate , followed by functionalization of the olefin . compound 1 was previously disclosed as a candidate for in vivo efficacy and toxicology evaluation , demonstrating good potency against human acc1 and acc2 ( ic50 = 111 and 9.8 nm , respectively ) and low human liver microsomal clearance ( < 4.9 ml key observations during the preparation for and analysis of those in vivo efficacy and safety studies guided the search for improved properties in subsequent compounds . first , during synthetic scale - up , a byproduct arising from ring - opening elimination of oxy - pyrazole was observed , presumably via a retro - michael reaction analogous to that shown in scheme 1 , an obstacle that was overcome but which increased concern about potential instability of the -pyrazoloxy - ketone functionality . second , crystalline compound 1 exhibited low solubility , requiring a spray - dried dispersion formulation to achieve adequate compound exposure in toxicology studies . third , the ketone functional group in 1 was reduced in vivo to provide substantial circulating levels of the alcohol metabolite , 1 m . the alcohol metabolite was formed upon incubation of 1 with rat , dog , and human liver microsomes , with reduction rates highest in dog and lowest in rat . pharmacokinetic ( pk ) studies in dog showed the alcohol metabolite 1 m had nine - times higher exposure relative to parent compound 1 ( see figure 2 ) . the metabolite had substantially reduced acc1 and acc2 potency ( ic50 = 8.7 and 1.0 m , respectively ; see supporting information , table s1 ) relative to parent , but the high level of ketone reduction was a concern for two main reasons : ( a ) decreased confidence in human pk predictions with ketone reduction as a primary metabolic pathway and ( b ) increased off - target pharmacology risk due to high circulating levels of metabolite , with our attention focused primarily on herg inhibition because ketone 1 and alcohol 1 m had similar potencies versus this ion channel ( ic50 = 34 and 57 m , respectively ) . plasma concentrations of 1 and its alcohol metabolite 1 m in beagle dogs following oral administration of 1 at 10 mg / kg . metabolite 1 m had higher exposure ( auc0 = 27800 ngh / ml ) than parent 1 ( auc0 = 3030 ngh / ml ) . as a result of these observations from the study of compound 1 , a decreased rate of ketone reduction became the primary objective in the design of new compounds . the initial hypothesis for decreasing ketone reduction was to increase steric hindrance around the ketone , leading to two approaches for substitution proximal to the ketone : adding alkyl substituents on the methylene adjacent to the ketone and altering the pyrazole to the regioisomeric n1-alkyl ( rather than n2-alkyl ) derivative . consideration of the crystal structure of 1 bound in acc provided support for exploring the -substitution strategy . as described previously , the acc ct - domain is an obligate homodimer , with the active site composed of residues from each monomer ( denoted a and b ) . an x - ray cocrystal structure of 1 bound in a humanized yeast chimeric ct - domain of acc demonstrated binding to the putative biotin binding site consistent with previous structures ( figure 3 ) . compound 1 bound in a generally hydrophobic cleft , with all of its polar atoms satisfied and sequestered from competing solvent interactions . the ketone and amide carbonyls made key hydrogen - bond contacts to the protein , orienting the rigid spirocycle in a low - energy conformation that allowed for ideal placement of polar and hydrophobic interactions with a minimal loss of conformational entropy . there appeared to be sufficient space adjacent to the ketone to accommodate substitution with alkyl groups , though specific interactions with the protein were not expected with small substituents . a crystallographic water was hydrogen - bonded to n1 , but the water was calculated using hydrosite to be low energy and easily displaced by a potential n1-substituent . however , there was also potential that the increased steric congestion would negatively impact the hydrogen - bond between the ketone and the backbone nh . co - crystal structure of 1 bound in the ct - domain binding domain of acc . compound 1 was oriented in the channel by hydrogen bonds between the ketone and nh - gly - b1958 and between the amide carbonyl and nh - glu - b2026 . the pyrazolopyranone group was sandwiched in a hydrophobic cleft lined with side chains of leu - a1762 , val - a1765 , leu - a1766 , ala - b1920 , val - b1923 , and phe - b1925 . the hydrogen bonding potential of the pyranone ether oxygen was unsatisfied , but this region of the active site was capped by the side chain of arg - b1954 . the distance between the ether oxygen and the guanidinium group of the arginine ( 3.8 ) was consistent with a weak electrostatic interaction , which likely alleviated the thermodynamic penalty associated with burying an unsatisfied hydrogen bonding group . the results of introducing substituents on the carbon adjacent to the ketone are shown in table 1 . for routine screening , potencies were assessed versus human acc1 and acc2 using recombinant enzymes in a transcreener format . consistent with the strategy employed in the identification of compound 1 , lipophilic efficiency ( lipe ) was a key parameter that was used to evaluate the quality of new compounds . unfortunately , the monomethyl stereoisomers , 2 and 3 , and the dimethyl derivative , 4 , demonstrated insufficient potency as acc inhibitors . the additional lipophilicity relative to parent compound 1 also increased intrinsic clearance in human liver microsomes ( hclint , table 1 ) . on the specific property of ketone reduction , the qualitative rate of ketone reduction as assessed by rate of alcohol formation in human liver microsomes was 1 > 2 3 > 4 ( data not shown ) . evaluation of the dimethyl derivative 4 in a dog pk study ( figure 4 ) showed a marked decrease in the amount of alcohol metabolite 4 m relative to parent ketone ( 23-fold greater exposure for 4 ) . although the -alkylation approach did not seem likely to lead directly to a candidate , these adme data provided strong support for the hypothesis that increased steric hindrance was a viable strategy for decreasing the rate of reduction . human acc1 and acc2 potencies are the geometric mean of at least three replicates ( 95% confidence interval ) . hclint = intrinsic clearance in human liver microsomes ; herg = inhibition of the herg channel in a patch - clamp assay ; nd = not determined . plasma concentrations of 4 and its alcohol metabolite 4 m in beagle dogs following oral administration of 4 at 1 mg / kg . metabolite 4 m had lower exposure ( auc0 = 14 ngh / ml ) than parent 4 ( auc0- = 321 ngh / ml ) . the regioisomeric n1-alkyl pyrazole derivatives were the alternative structural change designed to increase steric hindrance around the ketone . the synthetic route developed for these compounds also provided the opportunity to replace the cyclic ether in compound 1 with a carbocycle , thus obviating the potential for retro - michael ring - opening . the potency of the corresponding cyclic ether and carbocyclic analogues was similar for both the n2- and n1-alkyl series across a range of analogues ( data not shown ) . although there was some concern that removing the electron - donating ether might increase electrophilicity of the pyrazoloketone and the associated rate of ketone reduction , we believed that the steric effects of the n1-alkyl substituent would dominate any change in electronics . compounds 5 , 6 , and 7 with varying steric bulk of the n1-substituent showed an encouraging profile . importantly , the structural changes from 1 to 5/6 retained both acc potency and selectivity versus herg . the relatively high lipe of compound 6 , which was reflected in the desirable balance of acc potency and human liver microsomal ( hlm ) stability , led to selection of r = isopropyl as the preferred pyrazole n1-substituent . having made structural changes to the core of the molecule that increased steric hindrance around the ketone and removed the potentially labile cyclic ether linkage , we focused on the piperidine amide group to improve solubility . within the scope of 5,6-bicycloheteroaromatic groups ( a topology found to be important for potency in the discovery of compound 1 ) , weakly basic rings were targeted to achieve the most desirable balance between solubility and herg potency . pyrrolopyridine 8 and benzimidazole 9 were two of the most promising derivatives , with the benzimidazole 9 favorably differentiating on both hlm stability and herg activity . furthermore , the solubility at ph 1.2 of crystalline compounds 8 ( 2320 g / ml ) and 9 ( 1980 g / ml ) increased substantially as compared to crystalline compound 1 ( 20 g / ml ) . to assess the impact of pyrazole n1-substitution on the rate of ketone reduction , the benzimidazole 9 was tested in a dog pk study ; the data depicted in figure 5 demonstrated a 24-fold lower exposure to alcohol metabolite 9 m as compared to parent compound 9 . human acc1 and acc2 potencies are the geometric mean of at least three replicates ( 95% confidence interval ) . plasma concentrations of 9 and its alcohol metabolite 9 m in beagle dogs following oral administration of 9 at 3 mg / kg . plasma levels of 9 m were below the lower level of quantitation beyond the 4 h time point . metabolite 9 m had lower exposure ( auc0 = 330 ngh / ml ) than parent 9 ( auc0 = 7930 ngh / ml ) . comparison of the x - ray cocrystal structures of 1 and 6 bound in the ct - domain of acc showed that both inhibitors maintained hydrogen bonds between the two carbonyls and protein backbone nhs ( figure 6 ) . as predicted , the n1 substituent of 6 displaced the crystallographic water that was hydrogen - bonded to the ketone and to the pyrazole n1 in the bound structure of 1 . notably , however , the pyrazoloketone group of compound 6 shifted by 17 relative to 1 such that the n1-ipr group bound in a similar position to the n2-tbu group . the concomitant shift in the ketone position resulted in a less favorable geometry but a nearly identical hydrogen bond length for 6 as compared to 1 , suggesting comparable binding energies for the two interactions . the piperidine and indazole groups were essentially undisturbed by the n1 substitution and made similar interactions with the protein as in the structure of 1 . co - crystal structure of 6 ( magenta ) bound in the ct - domain of acc , overlaid with the bound conformation of 1 ( orange ) . compound 9 met our initial project objectives with its attractive overall profile of acc inhibitory potency , relatively low extent of ketone reduction , increased solubility , diminished herg activity , and good hlm stability . compound 9 exhibited low protein binding in both rat and dog plasma but approximately 10-fold higher binding in both monkey and human plasma . the in vitro metabolism of 9 was evaluated in microsomes from rat , dog , and human hepatocytes . compound 9 was not metabolized ( as assessed by disappearance of parent ) in rat , dog , or human microsomes . compound 9 was also stable in human hepatocyte incubations ( data not shown ) , but was minimally metabolized by recombinant human cyp3a4 and cyp3a5 , suggesting it is a substrate for cyp3a4 and cyp3a5 . clh microsomes estimated from well - stirred model , including blood : plasma ratio and plasma and microsomal binding . fed , vehicle : 20% sbecd . fed , vehicle : 40% 2-pyrrolidinone/60% ph 4.5 citrate buffer , solution , sterile filtered , ph = 5.41 . fed , vehicle : 10% dmso/20% 2-pyrrolidinone/70% of 20% sbecd in water . in vivo , the plasma clearance of 9 was low following intravenous ( iv ) administration ( 1 mg / kg ) to rats , dogs , and monkeys ( table 4 ) . oral ( po ) administration ( 3 mg / kg ) to rats and dogs showed bioavailability of 40% and 54% , respectively , consistent with the low microsomal clearance and good solubility at low ph . the bioavailability following a 50 mg / kg oral dose in rats was 106% , suggesting saturation of clearance . in vitro potencies are the geometric mean of at least 3 replicates the standard error of the mean ( sem ) . a 96-well radioenzymatic assay using both recombinant human and purified rat acc isozymes was used to confirm in vitro inhibitory potency for the determination of pharmacokinetic / pharmacodynamic ( pk / pd ) relationships . this assay format was also used with recombinant human acc2 to determine the mode and reversibility of inhibition . compound 9 was found to be uncompetitive with atp and noncompetitive with carbonate and acetyl - coa ( see lineweaver burk plots in the supporting information ) , consistent with previously reported inhibitors binding in this pocket of the protein . dose response experiments were performed at saturating concentrations of atp and acetyl - coa where the inhibitory concentration ( ic50 ) approximated the equilibrium dissociation constant for an enzyme ( ki ) for uncompetitive inhibitors . compound 9 was shown to inhibit both rat and human acc1 and acc2 in vitro with similar potencies ( table 4 and supporting information ) . the human acc1 and acc2 potencies in the radiometric assays were 3- and 2-fold more potent , respectively , than values determined in the transcreener assays . ex vivo , the effect of 9 on the acc / malonyl - coa axis was assessed in primary rat hepatocytes ( figure 7 ) . compound 9 inhibited formation of malonyl - coa in a concentration - dependent manner with a potency ( ec50 = 30 nm ) in rat hepatocytes consistent with its potency against rat acc1 ( 24 nm ) . cryopreserved rat hepatocytes were plated on collagen - coated 96-well plates and overlaid with matrigel . the cultured hepatocytes were incubated in the presence and absence of a range of concentrations of 9 . following incubation , malonyl - coa levels in lysates from the treated and untreated hepatocytes were analyzed using high - throughput mass spectrometry . on the basis of the robust enzyme and cellular activity of 9 , the effects of the compound on modulation of acc activity in vivo was assessed first as changes in levels of malonyl - coa , and subsequently as the downstream impact on dnl and fuel substrate utilization . the low plasma clearance and high exposure following oral administration in rats ( see table 3 ) enabled the ready examination of in vivo pharmacology . formation of the direct product of acc , malonyl - coa , in the skeletal muscle ( quadriceps ) and liver of lean sprague dawley ( sd ) rats was assessed 1 h following an acute oral dose of 9 , showing concentration - dependent reductions in both skeletal muscle and liver malonyl - coa ( figure 8) . at the nadir , quadriceps and liver malonyl - coa levels were reduced by 76% and 89% , respectively . the ec50s for inhibition of quadriceps and liver malonyl - coa were 870 and 540 nm , respectively , determined from unbound plasma concentrations of 9 . effect relationship of orally dosed 9 and malonyl - coa tissue levels in liver and quadriceps from male sd rats . rats were dosed by oral gavage with vehicle or a range of doses of 9 . one hour postdose , rats were euthanized , plasma was collected for compound exposure measurements , and liver and skeletal muscle ( quadriceps ) were rapidly removed and freeze - clamped in liquid nitrogen . malonyl - coa content of tissue extracts was determined by liquid chromatography mass spectrometry ( lc - ms ) . tissue concentrations of malonyl - coa for each dose group were plotted against the corresponding free plasma concentrations of 9 measured from the same animals . to confirm that the observed malonyl - coa biomarker reduction led to the anticipated changes in lipid metabolism , we examined the impact of 9 on dnl and respiratory exchange ratio ( rer ) , the latter being an indication of whole - body fuel substrate utilization . acute oral administration of 9 inhibited hepatic dnl in rats in an unbound plasma drug concentration - dependent manner . compound 9 inhibited up to 82% of the incorporation of [ c]acetate into [ c]lipids with an ec50 of 326 nm ( figure 9 ) . lean , male sd rats fed ad libitum with standard chow were dosed by oral gavage with vehicle or a range of doses of 9 . one hour later , animals were euthanized and liver punch samples and plasma were collected . the [ c]lipid generated from the [ c]acetate through dnl was isolated and measured from the liver samples by organic extraction with ethanol and petroleum ether and quantified by scintillation counting . consistent with the role of acc in modulating fatty acid oxidation , oral treatment with 9 also produced acute reductions in whole - body rer . to drive the basal fuel substrate utilization toward increased dependence on carbohydrate usage ( increased rer ) , rats were fasted for 24 h and then re - fed a high sucrose diet for 48 h prior to the experiment . following baseline rer measurement , the rats were dosed by oral gavage with a range of doses of 9 or vehicle . immediately following dosing , animals were returned to the metabolic chambers and rer was monitored for an additional 105 min . following the last rer measurement , animals were euthanized and skeletal muscle and plasma were collected for malonyl - coa and drug exposure measurements , respectively . within 15 min of oral administration of 9 , dose - dependent reductions in rer indicated a shift in fuel substrate utilization toward increased net whole - body dependence on fatty acid utilization ( figure 10a ) . at the nadir , rer values in the highest two doses of 9 dropped 0.19 units and at 120 min postdose the ec50 was 119 nm ( figure 10b ) . further , the change in rer from predose was found to be proportional to the quadriceps malonyl - coa levels measured from the same animals ( figure 10c ) . respiratory exchange ratio ( rer ) in male sd rats dosed orally with 9 . rats were treated with a single oral dose of 9 at 100 mg / kg ( red circle ) , 40 mg / kg ( blue square ) , 20 mg / kg ( green inverted triangle ) , 10 mg / kg ( light - blue triangle ) , 5 mg / kg ( orange diamond ) , 2 mg / kg ( green star ) , 1 mg / kg ( purple square ) , or vehicle ( black open circle ) at time zero , immediately following the baseline measurement period shown . quadriceps were collected and freeze - clamped for quantification of malonyl - coa levels ; plasma was collected for drug exposure measurements . ( b ) unbound plasma concentration - change in rer relationship at steady state following treatment . ( c ) relationship between quadriceps malonyl - coa levels and change in rer measured in the same animal . overall , the mechanistic studies with compound 9 in rats demonstrated that decreases in liver and skeletal muscle malonyl - coa lead to downstream decreases in dnl and increases in the use of fatty acids as fuel substrate . secondary in vitro pharmacology studies did not identify any significant off - target activity for 9 . in vivo safety pharmacology studies of 9 were conducted in rat and dog ; doses with no observed adverse effects were identified in both species . the effects described above for liver and muscle malonyl - coa , dnl , and rer in rats were the foundation for a rat pk / pd model that was then applied to predict human dosing for 9 . quadriceps muscle malonyl - coa level ( qmcoa ) in rat was chosen as the primary mechanistic biomarker . on the basis of the rapid formation and elimination ( seconds to min ) of malonyl - coa in muscle tissue , a direct response model was selected as a reasonable approximation to describe the pk / pd relationship . using the resulting rat qmcoa pk / pd model and the predicted human pk of 9 , gastroplus was used to project the steady - state dose necessary to achieve 50% inhibition of human quadriceps acc2 at the steady - state average concentration ( cave ) . these projections , after correction for species differences in acc2 enzyme potency ( radiometric assay ) and plasma / tissue protein binding , were based on assumptions of : ( a ) a 1:1 translation in qmcoa response between rats and humans , and ( b ) as observed in rat , a 1:1 relationship between human free muscle and free plasma drug concentrations . on the basis of these assumptions and models , the clinical plasma concentration of 9 to achieve 50% inhibition of human quadriceps acc2 was predicted to be 14800 ng / ml ( 473 nm free ) , which would be achieved by a dose of 125 mg once - daily . in the first - in - human study , the safety , tolerability , and pharmacokinetics of single doses of 9 were tested according to a randomized , placebo - controlled , double - blind , parallel - group design . seven sequential cohorts of healthy volunteers were randomized to receive either 9 or placebo ( six and three subjects per cohort , respectively ) . compound 9 was administered at escalating doses ranging from 10 to 800 mg ; the 800 mg dose was provided as split doses of 400 mg in the a.m. and p.m. overall , single ascending doses of 9 were safe and well - tolerated , exhibiting a benign adverse event profile similar to placebo - treated subjects . five adverse events were considered to be potentially treatment related , including nightmare in two subjects [ one treated with placebo and one with 9 ( 800-mg split dose ) ] , gastroesophageal reflux disease in one subject treated with 9 ( 300 mg ) , dyspepsia in one subject treated with 9 ( 600 mg ) , and dry mouth in one subject treated with 9 ( 300 mg ) . all potentially treatment - related adverse events were mild in intensity . in the fasted state , rapid absorption was noted with median peak concentrations observed 12 h postdose ( figure 11 ) . the pharmacokinetic profiles exhibited low - to - moderate interindividual variability in auc and cmax . auc was approximately proportional to dose over the 10800 mg range , while cmax was observed to be slightly less than dose - proportional . dosing in the presence of food ( 300 mg dose ) resulted in a delayed tmax ( 3.5 h ) , with marginal differences in peak and total exposure . terminal half - life values were consistent across doses and were independent of dosing in the presence of food , with mean values of 1013 h ( data not shown ) . time - course of plasma concentration of 9 in healthy human volunteers at a range of doses . compound 9 was administered orally at 10 ( blue circles ) , 30 ( red squares ) , 100 ( green triangles ) , 300 ( purple triangles ) , 600 ( orange circles ) , and 800 ( as 2 400 , pink circles ) mg doses . the pharmacodynamic effects of 9 on metabolic parameters were evaluated at the top ( nonsplit ) dose in a double - blinded , placebo - controlled crossover study in healthy volunteers in each of two study periods . hepatic dnl was assessed by measuring the incorporation of c - labeled acetate into very low density lipoprotein triglyceride ( vldl - tg ) , quantified using mass isotopomer distribution analysis ( mida ) . whole - body fuel substrate utilization was assessed by measuring rer using indirect calorimetry . subjects were randomized to receive a single oral dose of 9 ( 600 mg ) or placebo in the first period and were crossed over to receive the other treatment at least 1 week apart . oral fructose loading was used during the 10 h duration of dnl and rer assessments to provide reproducible fractional contribution of dnl to vldl - tg as well as elevated and reproducible rer measures from one assessment period to the other . oral fructose administration increased the fractional contribution of dnl over fasting values to a peak fractional dnl contribution of approximately 27% ( figure 12a ) . in subjects treated with compound 9 , fructose stimulated dnl was inhibited over this time course ; peak fructose - stimulated fractional dnl was reduced by 63.6% ( 90% ci = 75.152.0% ) relative to placebo treatment . all subjects responded to treatment with similar reductions in peak fractional dnl ( figure 12b ) . ( a ) fructose - stimulated fractional dnl in subjects treated with placebo ( pbo , black circles ) or a single dose of compound 9 ( 600 mg , blue squares ) using a crossover design . ( b ) treated subjects had significantly lower peak fractional dnl ( 10.5% ; 90% ci = 7.613.5% ) compared with placebo ( 26.5% ; 90% ci = 20.932.1% ) . all data were corrected for baseline dnl . the effect of 9 on rer was assessed by indirect calorimetry in the same subjects concurrent with the above dnl determinations . over the course of the study , the auc0.510h for rer values in subjects treated with 9 were significantly lower than for subjects treated with placebo ( ratio = 0.86 , 90% ; ci 0.810.92 ) , indicating an increase in net whole - body fatty acid utilization in the 9 subjects compared with placebo subjects ( figure 13 ) . baseline corrected respiratory exchange ratio ( rer ) over time in subjects treated by crossover design with placebo or a single dose of compound 9 ( 600 mg ) . the design , synthesis , and biological characterization of a dual acc1/acc2 inhibitor with low clearance and high off - target selectivity have been described . metabolic ketone reduction was greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl . phase i clinical studies demonstrated dose - proportional increases in exposure , a pharmacokinetic profile suitable for once - daily dosing , single - dose inhibition of dnl , and increased net whole - body fatty acid utilization that support its further clinical evaluation in type 2 diabetes patients . all chemicals , reagents , and solvents were purchased from commercial sources and used without further purification . data for h nmr spectra are reported as follows : chemical shift ( ppm ) , multiplicity , coupling constant ( hz ) , and integration . the multiplicities are denoted as follows : s , singlet ; d , doublet ; t , triplet ; q , quartet ; sept , septet ; m , multiplet ; ( v ) br s , ( very ) broad singlet ; app , apparent . silica gel chromatography was performed using a medium pressure biotage or isco system and columns prepackaged by various commercial vendors including biotage and isco . whatman precoated silica gel plates ( 250 m ) were used for analytical thin - layer chromatography ( tlc ) . the terms concentrated and evaporated refer to the removal of solvent at reduced pressure on a rotary evaporator with a water bath temperature not exceeding 60 c . purity of final compounds was assessed by reversed - phase hplc with uv detection at 215 nm ; all tested compounds were > 95% purity , unless otherwise noted . sodium borohydride ( 0.37 g , 10 mmol , 2.5 equiv ) was added to a suspension of 2-(tert - butyl)-1-(1h - indazole-5-carbonyl)-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazol]-7(6h)-one ( 1.67 g , 4.10 mmol , 1 equiv ) in methanol ( 24 ml ) . after 1 h , the mixture was partitioned between water and ethyl acetate ( 2 ) . the combined organic layers were washed sequentially with water and with brine , and the organics were dried over sodium sulfate , filtered , and concentrated to afford a foam that was triturated with ether to afford an off - white solid that was dried under vacuum ( 1.0 g , 67% ) . h nmr ( 400 mhz , dmso - d6 ) 13.19 ( s , 1h ) , 8.10 ( br s , 1h ) , 7.82 ( s , 1h ) , 7.54 ( d , j = 8.6 hz , 1h ) , 7.36 ( s , 1h ) , 7.36 ( dd , j = 8.6 , 1.2 hz , 1h ) , 5.19 ( d , j = 5.4 hz , 1h ) , 4.66 ( app q , j = 5.5 hz , 1h ) , 4.08 ( v br s , 1h ) , 3.51 ( br s , 1h ) , 3.23 ( br s , 2h ) , 1.98 ( dd , j = 14.1 , 5.9 hz , 1h ) , 1.91 ( br s , 1h ) , 1.81 ( dd , j = 14.1 , 6.1 hz , 1h ) , 1.70 ( m , 3h ) , 1.42 ( s , 9h ) . + esi ms ( m + h ) 410.2 . a solution of hydrogen chloride in dioxane ( 4 m , 3 ml , 12 mmol , 12 equiv ) was added to a solution of tert - butyl 2-(tert - butyl)-6-methyl-7-oxo-6,7-dihydro-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazole]-1-carboxylate ( 400 mg , 1.1 mmol , 1 equiv ) in dioxane ( 4 ml ) . after 2 h , the mixture was concentrated and the residue was triturated with ether . the resulting solid was partitioned between aqueous sodium hydroxide solution ( 3 ml ) and ethyl acetate ( 2 3 ml ) . the combined organics were dried over magnesium sulfate , filtered , and concentrated to afford the amine as an oil ( 215 mg , 65% ) , which was used without further purification . a solution of 1h - indazole-5-carboxylic acid ( 111 mg , 0.69 mmol , 1.0 equiv ) , 2-chloro-4,6-dimethoxy-1,3,5-triazine ( 120 mg , 0.69 mol , 1.0 equiv ) , and n - methylmorpholine ( 0.15 ml , 1.4 mmol , 2.0 equiv ) in dmf ( 2 ml ) was stirred for 1 h , then a solution of 2-(tert - butyl)-6-methyl-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazol]-7(6h)-one ( 215 mg , 0.69 mmol , 1 equiv ) in dmf ( 3 ml ) was added and the resulting solution was stirred for another 2 h. the product mixture was partitioned between saturated aqueous ammonium chloride solution ( 25 ml ) and ethyl acetate ( 2 15 ml ) . the combined organics were concentrated , and the resulting residue was purified by column chromatography twice , first using 4% methanol in dichloromethane as eluent , then using 50100% ethyl acetate in heptanes , to afford the racemic product ( 144 mg , 50% ) . the enantiomers were separated by sfc ( chiralpak ad - h , 65:35 co2:methanol ) to afford compounds 2 ( retention time 3.61 min , 51 mg , 18% , chiral purity > 99% ) and 3 ( retention time 5.63 min , 40 mg , 14% , chiral purity > 99% ) . 2 : h nmr ( 400 mhz , dmso - d6 ) 13.21 ( s , 1h ) , 8.12 ( s , 1h ) , 7.84 ( s , 1h ) , 7.77 ( s , 1h ) , 7.55 ( d , j = 8.5 hz , 1h ) , 7.38 ( d , j = 8.5 hz , 1h ) , 4.30 ( v br s , 1h ) , 3.59 ( v br s , 1h ) , 3.19 ( v br s , 2h ) , 2.61 ( q , j = 7.0 hz , 1h ) , 1.90 ( br s , 2h ) , 1.70 ( m , 2h ) , 1.49 ( s , 9h ) , 1.06 ( d , j = 7.0 hz , 3h ) ; + esi ms ( m + h ) 422.4 . 3 : h nmr ( 400 mhz , dmso - d6 ) 13.21 ( s , 1h ) , 8.12 ( s , 1h ) , 7.84 ( s , 1h ) , 7.77 ( s , 1h ) , 7.55 ( d , j = 8.5 hz , 1h ) , 7.38 ( d , j = 8.5 hz , 1h ) , 4.29 ( v br s , 1h ) , 3.60 ( v br s , 1h ) , 3.19 ( v br s , 2h ) , 2.61 ( q , j = 7.0 hz , 1h ) , 1.89 ( br s , 2h ) , 1.71 ( m , 2h ) , 1.49 ( s , 9h ) , 1.06 ( d , j = 7.0 hz , 3h ) ; + esi ms ( m + h ) 422.2 . a solution of hydrogen chloride in dioxane ( 4 m , 0.86 ml , 3.5 mmol , 15 equiv ) was added to a solution of tert - butyl 2-(tert - butyl)-6,6-dimethyl-7-oxo-6,7-dihydro-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazole]-1-carboxylate ( 90 mg , 0.23 mmol , 1 equiv ) in dioxane ( 2.5 ml ) . after 18 h , the mixture was concentrated and the resulting residue was concentrated from heptanes . the resulting solid ( 81 mg ) was used without further purification . a solution of 1h - indazole-5-carboxylic acid ( 40 mg , 0.25 mmol , 1.0 equiv ) , 2-chloro-4,6-dimethoxy-1,3,5-triazine ( 43 mg , 0.25 mol , 1.0 equiv ) , and n - methylmorpholine ( 82 l , 0.74 mmol , 3.0 equiv ) in dmf ( 3 ml ) was stirred for 1 h , then a solution of 2-(tert - butyl)-6,6-dimethyl-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazol]-7(6h)-one hydrochloride ( 81 mg , 0.25 mmol , 1 equiv ) in dmf ( 3 ml ) and n - methylmorpholine ( 54 l , 0.50 mmol , 2.0 equiv ) were added sequentially and the resulting solution was stirred for another 1.5 h. the product mixture was diluted with saturated aqueous ammonium chloride solution ( 1 ml ) , and the resulting mixture was partitioned between water ( 5 ml ) and ethyl acetate ( 30 ml + 20 ml ) . purification of the residue by column chromatography ( 05% methanol in dichloromethane ) afforded the product as a solid ( 74 mg , 69% ) . h nmr ( 400 mhz , dmso - d6 ) 13.19 ( s , 1h ) , 8.10 ( br s , 1h ) , 7.84 ( s , 1h ) , 7.76 ( s , 1h ) , 7.54 ( app d , j = 8.6 hz , 1h ) , 7.38 ( dd , j = 8.1 , 1.6 hz , 1h ) , 4.32 ( v br s , 1h ) , 3.84 ( br s , 1h ) , 3.12 ( v br s , 2h ) , 1.84 ( m , 2h ) , 1.73 ( m , 2h ) , 1.48 ( s , 9h ) , 1.07 ( s , 6h ) . + esi ms ( m + h ) 436.6 . sodium borohydride ( 11 mg , 0.28 mmol , 1.1 equiv ) was added to tert - butyl 2-(tert - butyl)-6,6-dimethyl-7-oxo-6,7-dihydro-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazole]-1-carboxylate ( 100 mg , 0.26 mmol , 1 equiv ) in methanol ( 2 ml ) at 0 c . after 1 h at ambient temperature , the mixture was again cooled to 0 c and an additional portion of sodium borohydride was added ( 10 mg , 0.26 mmol , 1.0 equiv ) . after an additional 2 h at ambient temperature , the mixture was cooled to 0 c and water ( 1 ml ) was added . the mixture was concentrated , and the resulting residue was partitioned between water ( 5 ml ) and ethyl acetate ( 2 30 ml ) . the combined organic layers were dried over sodium sulfate , filtered , and concentrated to afford a solid ( 98 mg , 98% ) , which was used without further purification . a solution of hydrogen chloride in dioxane ( 4 m , 1.8 ml , 7.2 mmol , 30 equiv ) was added to a solution of tert - butyl 2-(tert - butyl)-7-hydroxy-6,6-dimethyl-6,7-dihydro-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazole]-1-carboxylate ( 95 mg , 0.24 mmol , 1 equiv ) in dioxane ( 2 ml ) . after 6 h , the mixture was concentrated to afford an oil which was used without further purification . a solution of 1h - indazole-5-carboxylic acid ( 39 mg , 0.24 mmol , 1.0 equiv ) , 2-chloro-4,6-dimethoxy-1,3,5-triazine ( 43 mg , 0.24 mol , 1.0 equiv ) , and n - methylmorpholine ( 80 l , 0.73 mmol , 3.0 equiv ) in dmf ( 5 ml ) was stirred for 1 h , then a solution of 2-(tert - butyl)-6,6-dimethyl-6,7-dihydro-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazol]-7-ol hydrochloride ( 80 mg , 0.24 mmol , 1 equiv ) in dmf ( 3 ml ) and n - methylmorpholine ( 52 l , 0.50 mmol , 2.0 equiv ) were added sequentially and the resulting solution was stirred for another 18 h. the product mixture was diluted with saturated aqueous ammonium chloride solution ( 3 ml ) , and the resulting mixture was partitioned between water ( 10 ml ) and ethyl acetate ( 2 50 ml ) . the combined organics were dried over magnesium sulfate , filtered , and concentrated . purification of the residue by column chromatography ( 08% methanol in dichloromethane ) afforded the product as a solid ( 67 mg , 63% ) . h nmr ( 400 mhz , dmso - d6 ) 13.19 ( s , 1h ) , 8.10 ( br s , 1h ) , 7.82 ( s , 1h ) , 7.53 ( d , j = 8.6 hz , 1h ) , 7.36 ( dd , j = 8.6 , 1.6 hz , 1h ) , 7.35 ( s , 1h ) , 5.25 ( d , j = 6.3 hz , 1h ) , 4.30 ( v br s , 1h ) , 4.19 ( br s , 1h ) , 3.60 ( v br s , 1h ) , 3.09 ( v br s , 2h ) , 1.99 ( br s , 1h ) , 1.68 ( m , 3h ) , 1.42 ( s , 9h ) , 0.89 ( br s , 6h ) . + esi ms ( m + h ) 438.5 . a suspension of 1-(tert - butyl)-1,4-dihydrospiro[indazole-5,4-piperidin]-7(6h)-one hydrochloride ( 158 mg , 0.53 mmol , 1 equiv ) , 1h - indazole-5-carboxylic acid ( 86 mg , 0.53 mmol , 1.0 equiv ) , hatu ( 207 mg , 0.53 mmol , 1.0 equiv ) , and triethylamine ( 0.15 ml , 1.1 mmol , 2.0 equiv ) were combined in dmf ( 7 ml ) and stirred at room temperature for 16 h. the mixture was partitioned between water ( 5 ml ) and ethyl acetate ( 3 15 ml ) . the combined organics were washed sequentially with saturated aqueous sodium bicarbonate solution ( 5 ml ) and brine ( 5 ml ) . after concentration , the resulting residue was partitioned between dichloromethane ( 20 ml ) and 10% aqueous citric acid solution ( 5 ml ) to remove residual triethylamine hydrochloride . the resulting residue was purified by column chromatography ( 38% methanol in dichloromethane ) to afford the title compound as a solid ( 69 mg , 32% ) . h nmr ( 400 mhz , cdcl3 ) 10.23 ( br s , 1h ) , 8.10 ( s , 1h ) , 7.82 ( br s , 1h ) , 7.49 ( d , j = 8.6 hz , 1h ) , 7.43 ( dd , j = 8.6 , 1.4 hz , 1h ) , 7.29 ( s , 1h ) , 3.46 ( m , 4h ) , 2.81 ( s , 2h ) , 2.61 ( s , 2h ) , 1.64 ( s , 9h ) , 1.57 ( m , 4h ) . 1-isopropyl-1,4-dihydrospiro[indazole-5,4-piperidin]-7(6h)-one hydrochloride ( 30.3 g , 94.6 mmol , 1 equiv ) and 1h - indazole-5-carboxylic acid ( 16.96 g , 104.6 mmol , 1.1 equiv ) were suspended in n , n - dimethylacetamide ( 430 ml ) and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride ( 22.3 g , 115 mmol , 1.2 equiv ) was added , followed by the dropwise addition of triethylamine ( 65 ml , 475 mmol , 5.0 equiv ) . 1-hydroxybenzotriazole hydrate ( 16.2 g , 106 mmol , 1.1 equiv ) was then added , and the reaction mixture was stirred at 60 c for 2 h. the reaction was poured into half - saturated aqueous ammonium chloride solution ( 500 ml ) and extracted with ethyl acetate ( 1 1 l , 2 500 ml ) . the combined organic layers were washed with aqueous sodium bicarbonate solution ( 2 500 ml ) , water ( 3 500 ml ) , and aqueous saturated sodium chloride solution ( 500 ml ) . the organic layer was dried over sodium sulfate , filtered , and concentrated under reduced pressure to an oil . the oil was purified via column chromatography ( 16% methanol in dichloromethane ) to afford the desired product ( 27.1 g ) . a small amount was crystallized from ethyl acetate / heptanes , which was then used to seed the following crystallization . the product was dissolved in ethyl acetate ( 100 ml ) and heated to reflux until the solution turned hazy . a small amount of seed crystal was added . the mixture was cooled to room temperature , and a precipitate formed and was stirred for 80 h. the precipitate was collected by filtration and washed with cold ethyl acetate ( 2 30 ml ) . the material was air - dried and then further dried under high vacuum to afford an off - white solid ( 23 g , 62% ) . h nmr ( 400 mhz , dmso - d6 ) 13.19 ( s , 1h ) , 8.10 ( m , 1h ) , 7.79 ( m , 1h ) , 7.53 ( m , 1h ) , 7.43 ( s , 1h ) , 7.34 ( m , 1h ) , 5.24 ( sept , j = 6.6 hz , 1h ) , 3.45 ( v br s , 4h ) , 2.78 ( s , 2h ) , 2.59 ( s , 2h ) , 1.48 ( br s , 4h ) , 1.32 ( d , j = 6.6 hz , 6h ) . + esi ms ( m + h ) 392.5 . a mixture of benzyl 1-ethyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4-piperidine]-1-carboxylate ( 358 mg , 0.97 mmol , 1 equiv ) and palladium on carbon ( 50% wet , 60 mg ) in ethanol ( 20 ml ) was treated with 50 psi hydrogen gas for 4 h. the mixture was filtered through celite , rinsing with ethanol ( 50 ml ) . the filtrate was concentrated , then the resulting residue was slurried in heptanes ( 20 ml ) and concentrated . the resulting solid was dissolved in dichloromethane ( 5 ml ) and was treated with triethylamine ( 0.40 ml , 2.9 mmol , 3.0 equiv ) and 1h - indazole-5-carbonyl chloride ( 211 mg , 0.97 mmol , 1.0 equiv ) for 18 h at ambient temperature . the mixture was partitioned between water ( 50 ml ) and ethyl acetate ( 2 50 ml ) . purification by column chromatography ( 210% methanol in dichloromethane ) afforded the title compound as a solid ( 172 mg , 47% ) . h nmr ( 400 mhz , cdcl3 ) 8.13 ( s , 1h ) , 7.85 ( s , 1h ) , 7.51 ( d , j = 8.6 hz , 1h ) , 7.46 ( d , j = 8.6 hz , 1h ) , 7.37 ( s , 1h ) , 4.52 ( q , j = 7.2 hz , 2h ) , 3.64 ( v br s , 4 h ) , 2.82 ( s , 2h ) , 2.61 ( s , 2h ) , 1.63 ( br s , 4h ) , 1.40 ( t , j = 7.2 hz , 3h ) . + esi ms ( m + h ) 378.2 . a suspension of 1-isopropyl-1,4-dihydrospiro[indazole-5,4-piperidin]-7(6h)-one hydrochloride ( 100 mg , 0.35 mmol , 1 equiv ) , 1h - pyrrolo[3,2-b]pyridine-6-carboxylic acid ( 57 mg , 0.35 mmol , 1.0 equiv ) , hatu ( 83 mg , 0.35 mmol , 1.0 equiv ) , and triethylamine ( 0.10 ml , 0.70 mmol , 2.0 equiv ) were combined in dmf ( 3 ml ) and stirred at room temperature for 16 h. the mixture was partitioned between saturated aqueous sodium bicarbonate solution ( 10 ml ) and ethyl acetate ( 2 5 ml ) . the combined organics were washed sequentially with water ( 5 ml ) and brine ( 5 ml ) . after concentration , the resulting residue was purified by column chromatography ( 5% methanol in ethyl acetate ) to afford the title compound as a solid ( 65 mg , 47% ) . h nmr ( 400 mhz , dmso - d6 ) 11.76 ( br s , 1h ) , 8.43 ( d , j = 1.8 hz , 1h ) , 7.95 ( br s , 1h ) , 7.86 ( t , j = 2.9 hz , 1h ) , 7.46 ( s , 1h ) , 6.65 ( m , 1h ) , 5.27 ( sept , j = 6.6 hz , 1h ) , 3.56 ( m , 4h ) , 2.82 ( s , 2h ) , 2.63 ( s , 2h ) , 1.53 ( m , 4h ) , 1.36 ( d , j = 6.6 hz , 6h ) . + apci ms ( m + h ) 392.2 . n , n - dimethylformamide ( 0.33 ml , 4.3 mmol , 0.05 equiv ) and oxalyl chloride ( 22.1 ml , 257 mmol , 3.0 equiv ) were added to a solution of 2-methyl-1h - benzimidazole-5-carboxylic acid ( 15 g , 85 mmol , 1 equiv ) in tetrahydrofuran ( 500 ml ) . the reaction solution was stirred at ambient temperature for 16 h. the solution was concentrated , and the resulting residue was twice taken up in dichloromethane and concentrated under reduced pressure . to the resulting acid chloride was added tetrahydrofuran ( 500 ml ) , 1-isopropyl-1,4-dihydrospiro[indazole-5,4-piperidin]-7(6h)-one hydrochloride ( 25.9 g , 91 mmol , 1.1 equiv ) , and triethylamine ( 71.2 ml , 510 mmol , 6.0 equiv ) . the solution was stirred at room temperature for 16 h , then saturated aqueous sodium bicarbonate solution ( 250 ml ) was added and the resulting mixture was stirred for 5 min . the layers were separated , and the aqueous layer was extracted with 1:1 ethyl acetate tetrahydrofuran . the organic layers were combined , diluted with ethyl acetate ( 1 l ) , and washed sequentially with saturated aqueous sodium bicarbonate solution ( 200 ml ) and saturated aqueous sodium chloride solution . the organic layer was dried over sodium sulfate , filtered , and concentrated to afford a light - yellow solid . the solid was dissolved in hot methanol ( 300 ml ) and then heated to reflux . to the solution was added 350 ml of ethyl acetate , and 300 ml of solvent was then removed by distillation . additional ethyl acetate was added dropwise until an internal temperature of 70 c was reached . the solution was cooled to ambient temperature over 3 h. the resulting solids were collected by filtration and dried in a vacuum oven ( 40 c ) for 16 h to afford a white solid ( 20.5 g , 59% ) . h nmr ( 400 mhz , dmso - d6 ) 12.29 ( br s , 1h ) , 7.43 ( m , 3h ) , 7.11 ( m , 1h ) , 5.24 ( sept , j = 6.5 hz , 1h ) , 3.45 ( m , 4h ) , 2.77 ( s , 2h ) , 2.57 ( s , 2h ) , 2.45 ( s , 3h ) , 1.46 ( m , 4h ) , 1.32 ( d , j = 6.5 hz , 6h ) . + esi ms ( m + h ) 406.5 . sodium borohydride ( 27 mg , 0.76 mmol , 2.2 equiv ) was added to 1-isopropyl-1-(2-methyl-1h - benzo[d]imidazole-6-carbonyl)-1,4-dihydrospiro[indazole-5,4-piperidin]-7(6h)-one ( 139 mg , 0.34 mmol , 1 equiv ) in methanol . after 1 h , water ( 1 ml ) was added dropwise and the resulting mixture was partitioned between ethyl acetate ( 20 ml ) and water . the organic layer was washed with brine , dried over sodium sulfate , and concentrated . the resulting solid was purified by column chromatography ( 220% methanol in dichloromethane ) to afford a white solid ( 75 mg , 54% ) . h nmr ( 400 mhz , dmso - d6 ) 12.32 ( br s , 1h ) , 7.43 ( m , 2h ) , 7.13 ( s , 1h ) , 7.11 ( dd , j = 8.2 , 1.6 hz , 1h ) , 5.19 ( d , j = 6.6 hz , 1h ) , 4.71 ( m , 2h ) , 3.47 ( v br s , 4h ) , 2.46 ( s , 3h ) , 2.41 ( d , j = 15.3 hz , 1h ) , 2.32 ( d , j = 15.3 hz , 1h ) , 1.96 ( dd , j = 13.4 , 5.8 hz , 1h ) , 1.66 ( dd , j = 13.4 , 6.8 hz , 1h ) , 1.56 ( br s , 1h ) , 1.43 ( m , 3h ) , 1.34 ( d , j = 6.4 hz , 3h ) , 1.28 ( d , j = 6.6 hz , 3h ) . + esi ms the preparation and crystal structure analysis of humanized acc ct domain has been described previously . compounds 1 or 6 were soaked for 24 h into humanized acc ct domain crystals at a concentration of 3 mm , then flash cooled in liquid propane prior to data collection . data for compound 1 was collected at beamline x29a at the national synchrotron light source ( brookhaven national laboratory , upton , ny , usa ) . data for compound 6 was collected at sector 17bm of the industrial macromolecular crystallography association collaborative access team ( imca - cat ) ( advanced photon source , argonne , il ) . the structures of acc - ct were determined by rigid body refinement of a reference structure against the isomorphous data . compound 1 was refined using the program autobuster ; compound 6 was refined using refmac . the intensity of reflection hkl , and ihkl is the average intensity of multiple observations . rwork = |fo fc|/fo , where fo and fc are the observed and calculated structure factor amplitudes , respectively . rfree is the r - factor for a randomly selected 5% of reflections which were not used in the refinement . % ( preferred ) + % ( allowed ) recombinant human acc1 ( rhacc1 ) was utilized in enzyme inhibition assays as previously described . recombinant human acc2 ( rhacc2 ) was prepared , purified , and utilized in enzyme inhibition assays as previously described . rat acc1 and acc2 were purified from rat liver and muscle , respectively , as previously described . cryopreserved male wistar rat hepatocytes from xenotech were thawed and isolated using a percol rat hepatocyte isolation kit . hepatocytes were plated in seeding media containing high glucose dmem , 5% fbs , 1 m dexamethasone , 4 g / ml insulin , 1% pen / strep , and 1% glutamine . hepatocytes were plated in 48-well collagen coated type i plates using a viable seeding density of 125000 cells / well in seeding media to result in 8090% confluency at 5% co2 and 37 c . media was aspirated 46 h later and fresh hepatocyte modified eagle medium , dr . fresh cold mcm media containing a 1:40 dilution of matrigel , 250 ng / ml , 200 l / well was added 24 h post seeding . the assay was run 72 h post seeding with a mcm media change the night before the experiment . on the day of the study , media was aspirated and cells were treated with fresh mcm media containing dmso vehicle or varying concentrations of 9 as indicated . an aliquot of this solution was added to the fresh mcm in each well , further diluting the compound by 1:10 . this dilution progression ensured that all wells had a final dmso concentration of 0.1% . after 5 h at 37 c , incubation media was removed and the experiment was terminated by washing the cells with ice - cold pbs . cells were lysed with the addition of 70 l of 10% trichloroacetic acid ( tca ) . plates were mixed for 20 min at 4 c and centrifuged at 2200 g for 5 min . supernatant ( 48 l ) was transferred to a screenmates clear 384 well v bottom polypropylene microplates and frozen . tca , ammonium acetate , methanol , acetone , acetonitrile , and water were used to make the necessary mobile phases . then 2 l of malonyl - c3-coa internal standard ( 0.4 pmol/l final ) and 2 mm final atp in 10% tca were added to the reaction and mixed . the samples were analyzed on a rapidfire platform coupled to a sciex api4000 triple quadrupole mass spectrometer ( rf - ms ) . malonyl - coa and malonyl - c3-coa were monitored in positive ion mode following mrm transitions at 854.1/347.1 and 857.1/350.1 , respectively . sample ( 10 l ) was aspirated directly from assay plates quenched with tca ( 10% final ) . the aspirated sample was loaded onto the rf ms microscale solid - phase hypercarb extraction cartridge ( d ) and washed with hplc grade h2o in a 3 s wash cycle to remove the nonvolatile assay components . the product and internal standard were coeluted to the mass spectrometer in 3 s with 50% h2o , 25% mecn , 25% acetone , and 5 mm ammonium acetate . peak area ratios ( malonyl - coa / malonyl - c3-coa ) were compared against a standard curve prepared in 10% aqueous tca to determine sample concentrations of malonyl - coa . malonyl - coa values were expressed as pmol/l and compared against a standard curve . dose response curves of malonyl - coa inhibition by acc inhibitor compound 9 were generated by plotting percent of control . male sd rats were weighed and randomized by body weight into treatment groups consisting of vehicle , 0.25 , 0.5 , 1 , 2 , 4 , 8 , 15 , 25 , 50 , and 100 mg / kg of 9 . immediately prior to initiation of the study , the dosing solutions were prepared in dosing vehicle ( 0.5% methyl cellulose:0.1% polysorbate 80 ) , giving a final dosing volume of 5 ml / kg . animals were orally dosed 2 h into the light cycle with their respective treatments and fed ad libitum . one hour postdose , the animals were sacrificed via co2 asphyxiation followed by cervical dislocation . blood for plasma exposure of compound 9 was collected via cardiac puncture , transferred to bd microtainers tubes with k2edta , centrifuged at 4 c , and the plasma transferred to a 96-well microtiter plate and stored at 20 c . liver and quadriceps were rapidly removed , freeze - clamped in a wollenberg clamp ( precooled in liquid nitrogen ) , and subsequently stored at 80 c . approximately 200 mg of pulverized tissue were added to the lysing matrix tube ( mp bio , lysing matrix tube a for quadriceps and lysing matrix tube d for liver ) containing ice - cold 10% tca , giving a final ratio of 1:5 , tissue to acid . the extracted samples were then centrifuged at 4 c and 20000 g for 15 min ( liver ) and 30 min ( muscle ) . the supernatants containing malonyl - coa were frozen at 80 c until analysis was completed via lc - ms ms using an abi sciex api-3000 triple quadrupole mass spectrometer with turbo ion spray . tissue concentrations for malonyl - coa were calculated from a standard curve prepared in a 10% solution of tca in water and ranged from 0.01 to 1 pmol/l . malonyl - c3-coa ( final concentration of 0.4 pmol/l ) was added to each standard curve component and served as an internal standard , and the resulting chromatograms were integrated using analyst software ( applied biosystems ) . this procedure was derived from a published method for the examination of rat liver de novo lipid synthesis . on the day of the study , male sd rats , previously randomized into groups of seven based on body weight , were administered a single dose of compound 9 or vehicle via oral gavage 2 h into the light cycle ( 8 a.m. ) . [ 2-c]-acetic acid ( amersham ) was diluted to 64 ci / ml with saline prior to being administered intraperitoneally at 2.5 ml / kg 1 h postdose of compd / vehicle . then 1 h following c - acetic acid administration , the animals were sacrificed ( co2 euthanasia ) and liver samples collected ( 400 mg , bifurcated median lobe ) using disposable tissue biopsy punches . briefly , tissues were saponified in aqueous naoh ( 1.5 ml of 2.5 m ) . following complete degradation , absolute ethanol ( 2.5 ml ) was added to each sample and vigorously mixed . following centrifugation to separate the organic and aqueous phases , the upper organic phase was removed and discarded . concentrated hcl ( 0.6 ml of 12 m ) was added to the remaining aqueous phase and vortexed vigorously . the acidified aqueous phase was subsequently extracted with petroleum ether ( 4.8 ml ) and then centrifuged to separate the organic and aqueous phases . the remaining aqueous phase was extracted with petroleum ether as described above , and the organic phase was combined with the previous extract . the combined organic phases were evaporated to dryness under gentle flow of nitrogen at room temperature . compatible scintillation fluid was added to each vial , and the level of c in the extraction was determined . the data were analyzed with microsoft excel and plotted using graphpad prism 5 . statistical analysis ( one - way anova , dunnett s posttest ) male sd rats were fasted for 24 h and then re - fed a diet high in sucrose ( d10001 , research diets ) for 2 days prior to study initiation to elevate baseline respiratory exchange ratio ( rer ) . on the day of experiment , rats were removed from their home cage , weighed , and individually placed into the calibrated indirect calorimetery chambers ( oxymax , columbus instruments , columbus , oh ) with free access to water and diet ( d10001 ) . baseline oxygen consumption and carbon dioxide production rates were measured every 15 min for 75 min before treatment . after collecting baseline calorimetery data , rats were dosed orally with either vehicle control ( 0.5% methylcellulose/0.1% tween 80 ) or 9 ( 1 , 2 , 5 , 20 , 40 , or 100 mg / kg ) and then returned to the calorimetry chambers . oxygen consumption and carbon dioxide production were measured for an additional 2.25 h after being placed back in the calorimetry chamber . immediately following completion of the calorimetry measurement period , plasma for determining plasma exposure of compound 9 was collected via cardiac puncture , and quadriceps muscles were rapidly removed , freeze - clamped as described above , and stored at 80 c . studies involving human subjects were conducted in compliance with the ethical principles originating in or derived from the declaration of helsinki and in compliance with all international conference on harmonisation ( ich ) good clinical practice ( gcp ) guidelines . in addition , all local regulatory requirements were followed , in particular , those affording greater protection to the safety of trial participants . final study protocols and informed consent document were reviewed and approved by the investigational centers participating in the studies and by an independent institutional review board ( irb ) . the investigator was required to inform the irb of the study s progress and occurrence of any serious and/or unexpected adverse events . a signed and dated informed consent was required before any screening procedures were initiated . the investigator or his / her delegate explained the nature , purpose , and risks of the study to each subject . each subject was informed that he / she could withdraw from the study at any time and for any reason . each subject was given sufficient time to consider the implications of the study before deciding whether to participate . safety , tolerability , and pharmacokinetics were evaluated in the first - in - human study . a total of 63 healthy volunteers ( all male ; mean age 32.6 years [ range 2045 years ] ; mean body mass index ( bmi ) 27.0 kg / m ; [ range 21.335.1 kg / m ] ) participated in the study . seven sequential cohorts of volunteers were randomized to receive either 9 or placebo ( six and three per cohort , respectively ) . compound 9 was administered at escalating doses ranging from 10 to 800 mg ; the 800 mg dose was provided as split doses of 400 mg in the a.m. and p.m. due to the prediction that cmax might exceed the exposure limits . compound 9 was administered after 10 h of fasting in a 10 , 50 , and 100 mg powder - in - capsule formulation . the effect of food on the pharmacokinetics of 9 was evaluated at the 300 mg dose . safety and tolerability were assessed by adverse event monitoring , laboratory values , and cardiovascular parameters including blood pressure , heart rate , and electrocardiogram . the effects of 9 on inhibition of dnl and on whole - body fuel substrate utilization was assessed in a randomized , double - blinded , placebo - controlled crossover study in healthy volunteers ( mean age 35.4 years [ range 2050 years ] ; mean body mass index ( bmi ) 29.9 kg / m ; [ range 25.835.5 kg / m ] ) . substrate utilization , assessed by rer , was measured in each of two study periods . subjects were randomized to receive a single oral dose of 9 ( 600 mg ) or placebo in the first period . in the second period , subjects were crossed over to receive the other treatment ( placebo or 9 ) at least 1 week apart . oral fructose loading was used during the dnl and rer assessments to provide reproducible fractional contribution of dnl to vldl - tg from one assessment period to the other . a continuous infusion of c - acetate ( 99.5 mg c - acetic acid sodium salt per min via an infusion pump ) was started at approximately 10:00 a.m. on the evening prior to each treatment period and continued until approximately 6:30 p.m. on the study day . blood samples for assessment of the fractional contribution of dnl to vldl - tg were collected and rer measures made hourly for 10 h. subjects received study medication ( 9 or matching placebo ) at approximately 08:00 h ( plus or minus 2 h ) . subjects received a bolus of 0.25 g fructose / kg body weight every 30 min starting at approximately 08:30 h for approximately 9.5 h ( total of 20 fructose administrations ) during each of the two study periods . urine samples for purpose of urinary nitrogen determination were collected during each study period . subjects refrained from eating and drinking beverages other than water and the fructose drinks administered as part of study procedures . dnl was measured by c - incorporation ( derived from intravenous c - acetate infusion ) into vldl palmitate using mass isotopomer distribution analysis ( mida ) . rer values reported are nonprotein rer as no changes in urinary nitrogen were observed during or between the study periods .

acetyl - coa carboxylase ( acc ) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus ( t2 dm ) , hepatic steatosis , and cancer . however , the identification of tool compounds suitable to test the hypothesis in human trials has been challenging . an advanced series of spirocyclic ketone - containing acc inhibitors recently reported by pfizer were metabolized in vivo by ketone reduction , which complicated human pharmacology projections . we disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl . incorporation of weakly basic functionality improved solubility and led to the identification of 9 as a clinical candidate for the treatment of t2 dm . phase i clinical studies demonstrated dose - proportional increases in exposure , single - dose inhibition of de novo lipogenesis ( dnl ) , and changes in indirect calorimetry consistent with increased whole - body fatty acid oxidation . this demonstration of target engagement validates the use of compound 9 to evaluate the role of dnl in human disease .

Introduction Results and Discussion Conclusion Experimental Section

hypnic headache ( hh ) is a primary headache disorder characterized by attacks that occur only during sleep . it was first described by raskin in 1988 . since then , about 100 similar cases have been reported in the literature . however , recently two cases of probable hh are reported in the children less than 10 years [ 3 , 4 ] . we report a case of relapsing remitting variety of hh in an adolescent who showed complete response to indomethacin . a 19-year - old male presented with a 4-year history of relapsing remitting type of nocturnal headache , which awakened him during sleep . in each relapse , he had headache almost every night at a consistent time ( about 12 a.m. ) , 34 h after he fell asleep . the periods of relapse and remission were variable , few weeks to 6 months . the patient described the headache as non - throbbing and moderate to severe in the intensity . however , it used to become bilateral in about 10% of the attacks ( especially with severe attacks ) . the usual duration of attacks was about 60 min ( range 30 min to 5 h ) . he denied nausea , vomiting , photophobia , phonophobia , any autonomic features , and restlessness during the attacks . however , rarely , after falling back to sleep he was awakened with another similar headache , usually occurring 34 h after the first attack . the patient noticed that at least 3 h sleep was essential to get an attack . because of the fear of an attack , he used to wake ( spontaneously or with the help of an alarm clock ) within 3 h of the onset of sleep . he would have an attack , immediately or within an hour , if he again goes to bed in less than 60 min after the awakening . waking for the 12 h a nap of more than 34 h even during the daytime was occasionally associated with the similar type of headache . the patient consulted in our out patient clinic in the recent relapse of the headache , that was going on for the last 2 months . the patient was alcoholic , but did not recall any relation of alcohol intake to the onset , frequency , duration , or severity of headache . he had a trial of sodium valproate ( 500 mg bid ) , amitryptiline ( 75 mg od ) , duloxetine ( 60 mg bid ) , naproxen ( 500 mg bid ) , ibuprofen ( 600 mg tds ) , and propranolol ( 60 mg bid ) . he could not recall any effect of these drugs in the frequency or severity of headache . treatment with oral sumatriptan ( 100 mg ) , oral rizatriptan ( 5 mg ) , and oxygen inhalation during an attack provided no relief . it was ineffective with 25 mg dose ( bedtime ) . gradually increasing the dose to 75 mg at the bedtime ( over 4 days ) produced complete relief . drug was restarted with the same dose and response was noted on the same day . as patient had relapsing remitting course in the past , slow tapering ( over 7 days ) was tried on two occasions in the next 2 months . the second trial was successful and patient did not have any type of headache in the next 3 months . however , his symptoms recurred after 3 months . this patient presented with a type of headache fulfilling the international headache society ( ihs ) criteria of hh . hh is incorporated into group 4 of the ihs classification in the subheading of other primary headaches . the differential diagnosis of headache with an exclusive onset during sleep includes secondary headache and primary headaches such as migraine , trigeminal autonomic cephalalgias and hh . exclusion of intracranial disorders and differentiation from one of the trigeminal autonomic cephalalgias is recommended in the patient of hh in the ihs diagnostic criteria . the features against the diagnosis of cluster headache ( ch ) were the absence of autonomic features and restlessness , no response to triptans and oxygen inhalation during the attacks , and response to indomethacin as a preventive therapy . the absence of autonomic features , duration up to 5 h , only one attack per day , and bilateral involvement in few attacks were against the diagnosis of paroxysmal hemicrania . the mean age at onset of headache is 63 years.the onset after the age of 50 years is one of the features in the ihs diagnostic criteria of hh ( although not essential ) . to the best of our knowledge , he is the youngest person reported who fulfills the diagnostic criteria for hh . there are two case reports of probable hh in the literature younger than 15 years [ 3 , 4 ] . besides the age , these two patients did not fulfill the criteria regarding the frequency of attacks per month . the frequency of more than 15 attacks per month is essential in a person less than 50 years of age . reported a 9-year - old girl with hypnic - like headache with frequency of 23 attacks per week . recently , scagni et al . described an 8-year - old girl with a 5-year history of hypnic - like headache with frequency of one attack per month , and suggested that lower frequency might be observed in children with hh . these two cases and our observation suggest that the age range at onset may be broader than initially claimed . the intensity of pain is moderate to severe in more than 95% patients in the literature . the usual time of onset of the headache attacks in the course of sleep is 24 h after falling asleep . three hours sleep was usually required to get an attack of headache in our patient . the average duration of attacks is about 1 h. however , the range may be 15360 min . the maximum duration of an attack in our patient was about 5 h. the average frequency of headaches is about 1.2 . however , the patients may have six attacks per night to one in a week . the episodic form is further subdivided into two types , episodic with no recurrence , and relapsing and remitting variety ( and relapse is not because of withdrawal of the effective drug ) . episodic form without recurrence may show spontaneous resolution or sustained remission even after withdrawal of the effective drug [ 5 , 6].our case was relapsing remitting type . there are only six cases of relapsing remitting type in the literature ( and 5 of them were reported recently ) [ 5 , 9 ] . lithium ( 300600 mg at the bedtime ) is the most effective drug for the patients with hh . the efficacy similar to lithium could be obtained with caffeine , flunarizine , and indomethacin . the response to indomethacin in our patient confirms this notion ( as our patient had predominantly unilateral headache ) . peters et al . reported indomethacin responsive hh in patients with bilateral and holocephalic pain . the effective dose of indomethacin for the patients of hh is 25150 mg at bedtime ( mean 75 mg ) . there was temporal relation in the disappearance of headache and the administration of indomethacin on four occasions in the patient . however , we can not rule out the possibility of coincidence ( disappearance of headache and the administration of indomethacin ) in our patient , as he had many episodes of spontaneous relapse and remission in the past . the reason as to why few primary headache ( including hh ) disorders are indomethacin sensitive is unknown . the effects of many other drugs ( such as lithium , melatonin , caffeine , flunarizine , etc . ) in the patients of hh suggest that pathophysiology may be heterogeneous . the adverse effects of indomethacin in the patients of hh are largely unknown as long - term follow up is lacking in the literature . although indomethacin is used as a single dose ( bedtime ) in hh ( bid or tds in other headache disorders ) , it is difficult to predict the incidence of side effects as most of the patients are elderly . daytime headaches were one of the unique side effects of indomethacin in the patients of hh ( not reported with other types of headache ) . moreover , this case suggests that frequent tapering of the effective drug should be done to look for the spontaneous resolution or relapsing remitting variety of hh .

hypnic headache ( hh ) is a rare sleep - associated primary headache disorder that usually begins after the age of 60 years . here we report a 19-year - old male with 4-year history of predominantly left sided hh . he is the youngest person reported who fulfills the ihs diagnostic criteria for hh . the patient had history of relapsing remitting course . the headache occurred every night at a constant time in each relapse . it was non - throbbing , moderate to severe , for 30 min to 5 h , and usually after 3 h of sleep . the patient showed complete response to indomethacin ( 75 mg at bedtime ) . frequent tapering of indomethacin was required to look for the remission phase .

Introduction Case report Discussion

social media , web - based applications , and internet has revolutionized communication process over the last 2 decades . similarly , learning and teaching process in the field of medicine had transformed slowly and progressively to suit the changes according to the geographical and economic constraints . we in this study , aimed to determine what the thoughts were among the current undergraduate students and the teachers in the irish republic on introduction of technology and web - based applications in current medical curriculum . we also have questioned if this is necessary or not to bring forth any change from learning and teaching perspective . in the near future , one should not be surprised to come across an advertisement on the web stating complete medicine from home in 4 years from a virtual university offering distance education . we hope this does not happen , compromising the traditional art of learning and teaching medicine in medical schools across the globe . recently , a news in the media stated that indian government has announced a major program in partnership with a british company to produce a tablet computer that will be made available to students for only 26 . we certainly believe that the addition of the gadgets and inclusion of web - based technology in medical school will be certainly being beneficial . recent reports also have revealed that the republic of ireland has been lagging behind in information technology based learning . this is another reason why we felt this study may be beneficial in highlighting the potential this study is based on the assumption that a 4 year medical student would reasonably be competent in the basics of information technology particularly using the internet and using web - based resources for learning . the teachers and medical professionals were also included to make this study more complete getting bilateral views than being biased towards the students . to determine if modern information technology and web - based applications can be incorporated in the current undergraduate medical curriculum in the republic of ireland.to determine how receptive the medical students are in incorporating the recommended views / changes in this study.to determine if the teachers and medical professionals agree or disagree to the same proposed . to determine if modern information technology and web - based applications can be incorporated in the current undergraduate medical curriculum in the republic of ireland . to determine how receptive the medical students are in incorporating the recommended views / changes in this study . to determine if the teachers and medical professionals agree or disagree to the same proposed a cross - sectional study involving 202 participants of which 152 of them were 4 year medical students and 50 of the medical professionals ( teachers & hospital doctors ) within the republic of ireland . this involved three different medical universities namely university college dublin ( ucd , national university of ireland , galway ( nuig ) and university college cork ( ucc ) representing the country . a 15-point questionnaire was framed incorporating various fields of technology where changes in teaching and learning in medical education can be introduced [ table 1 ] . participants were made to answer the questions or comments based on likert psychometric five - point scale ( strongly agree-1 ; agree-2 ; do nt know-3 ; disagree-4 ; and strongly disagree-5 ) . the participants in the ucc had the questionnaire distributed by the authors in person and the other group involving ucd and nuig had an online version of the same questionnaire using survey monkey . the data from the survey was analyzed using statistical package for social sciences ( spss ) software to determine statistical significance . the overall scores obtained from the survey shows that both the medical students and the teachers have a positive attitude towards the introduction of more up - to - date technology in medical education . no statistically significant difference was noted between the two methods utilized for data collection utilized in this study . does a more modern classroom ( 3d projectors and laptops ) likely to make you more interested in going to class compared to the conventional classroom ? few statements among the 15 had a mean of about 2.5 out of 5 , which indicated that participants were unsure about the benefits these ideas could bring to the current medical curriculum . we subsequently proceeded to analyze these statements in detail and identified that two of the statements that were not be favorable to the participants . the first statement was regarding the introduction of medical e - books ( online books ) and the idea that they may be better than actual textbooks . we also noted that 50% of the participants actually did indeed favor the introduction of medical e - books . however , it was also very obvious that there were some participants completely against the idea of medical e - books . table 3 summarizes the overall individual responses of the participants to the 15 questions . the introduction of medical e - books ( online books ) is better than actually buying the medical text books the second statement that got mixed reviews was on inclusion of the rest of the questions clearly seemed favorable to both the groups ( students and teachers ) . table 4 depicts the statistical comparison with the mean difference to the rest of the values . most of the statements that we adopted to incorporate in the questionnaire were well - accepted and in fact some of the statements that received the best responses included the utility of blackboard and dropbox application . medical students seem to favor online methodology of managing their work , as it proved easier and handier than the conventional method . utility of software like skype and vodoo was also well accepted as an alternate methodology of attending lectures from different universities . podcast of medical lectures seemed to be appreciated well as students had the luxury of listening to their lectures of choice without limitations to the number of times they wanted to view them to get their concepts clear . these ideas may have logistical setbacks to make its way into the current curriculum , however these ideas seemed to have been received well by the students and teachers . we are hoping that this study may potentially initiate a change in the system if feasible . a plethora of literature supporting this already exists and we have attempted to review a few that we felt may be appropriate . in the united states in 1997 , an association of american medical colleges ( aamc ) medical school graduation questionnaire revealed that use of computers had increased from 37 to 71% , over a 9 year period . a decade later ; a finnish study looked at the opportunities new technology may provide for medical education . they concluded that in both universities involved , it appeared that medical teachers and students had a very positive attitude toward the advances in modern technology . teachers , however , used information technology more in their research work than in teaching . looking beyond the web - based applications there are studies to prove that inclusion of modern gadgets may prove beneficial in the field of medical education . recently , a study was carried out looking into incoporation of ipads into a preclinical curriculum revealing a mixed attitude towards the idea . focus group data indicate students appreciate certain aspects of ipad use in the curriculum , including improved curriculum interactivity , but the majority believes it can not replace printed handouts at this time . a separate study carried out in the united kingdom on the use of smartphone and medical related apps among medical students and junior doctors also showed promising results . the study concluded that there is a high level of smartphone ownership and usage among medical students and junior doctors . both groups endorse the development of more apps to support their education and clinical practice . another interesting research was carried out in brazil to see the impact of cybertutor in dermatological teaching . according to this study , multimedia programs may be used for undergraduate education in dermatology as a complementary educational tool . it is generally accepted that information technology ( it ) is a highly desirable and a very necessary ingredient of modern healthcare . a survey carried out by nebraska national air guardon the combined use of skype and the storz cmac video laryngoscope in field intubation training also highlighted the fact that web - based applications are being considered not only by medical students , but also by the other sectors to aid in medical education using skype and voip . studies have also proven student preference for computer - assisted or digital technologies ; a study was developed to determine whether it was more successful than a conventional radiology textbook in assisting dental students with the learning of radiographic anatomy . results showed that whilst traditional textbooks are still valued in the dental curriculum , it is evident that the preference for computer - assisted learning of oral radiographic anatomy enhances the learning experience by enabling students to interact and better engage with the course material . the above examples show the diverse nature of how technology can be adopted for medical education . our study had taken a fraction of the omnipotent technology into medicine where some of the advancements can be successfully utilized for teaching and learning in irish medical schools . small number of participants involved in the study . only three medical universities were involved including only 4 year medical students . as our main focus was on the students , more numbers of students were chosen in comparison to the teachers ( 1:3 ratio ) . this is another limitation of the study . perhaps involving the management of the medical universities can be included to see the logistics of bringing forth the changes eventually or not . with our preliminary results obtained there is an obvious need for computer - based learning and teaching in medical education . modern technology and digital learning environments aid different kinds of learning processes pertaining to different group of students and teachers around the world . medical teachers may be able to meet these challenges and adapt appropriately to the evolving trends utilizing methods that may enhance their own and medical students ways of learning . the survey was well received by the participants with 91% of them having positive attitudes to the idea being proposed . a 10 of the participants seemed doubtful of the need for change and our ideas , but nevertheless the vast majority is hoping for a revamp of the education methodology in the irish republic . this study is an initiative to look at the thoughts of students and teachers and we acknowledge there may be significant logistical limitations to bringing forth changes and few may disagree to our ideas . a large study is recommended involving more participants across the medical schools in ireland and may throw more light on this topic . we also hope that the concept utilized may become reality in the near future if the educational facilitators feel that this is appropriate and also to meet the challenges medical education may face in the future in ireland .

background / aim : significant change has been happening in the introduction of technology in medical teaching all over the world . we aim to determine if the undergraduate medical students and teachers are open to incorporating changes in the current medical curriculum or if there is a need for the same in the republic of ireland.materials and methods : a cross - sectional study involving 202 participants of whom 152 were medical students and 50 medical professionals ( teachers and hospital doctors ) were carried out involving three different medical universities namely ; university college cork ( ucc ) , university college dublin ( ucd ) , and national university of ireland in galway ( nuig ) . participants were requested to answer a series of 15 questions designed incorporating various fields of technology necessary for the study . the data was collected and analyzed using statistical package for social sciences ( spss ) software to determine statistical significance.results:the participants overall had a positive attitude toward the utility of modern technology and web - based applications in current medical curriculum . ninety - one percent of the participants preferred the introduction of modern technology into medical education and 7% were against the idea and a further 2% of them remained undecided.conclusion:there seems to be a technology gap in the current undergraduate medical curriculum in ireland . a large - scale study involving more participants from all the medical schools in ireland is recommended . we believe , changes can be brought into the current medical teaching and learning to make the process more fruitful and successful .

INTRODUCTION OBJECTIVES MATERIALS AND METHODS RESULTS DISCUSSION LIMITATIONS OF THE STUDY CONCLUSION

control of the trunk of the body is a necessary condition for humans to maintain their balance against gravity1 . however , patients with stroke caused by brain damage experience a diminished ability to balance because of the weakened muscle strength of the trunk2 . training to control the trunk and maintain balance is essential for functional improvement in chronic stroke patients3 . improved trunk control enhances the ability to balance , which is a necessary condition for the prevention of falling . hence , the probability of falling is decreased because the ability to maintain balance is enhanced4 . proprioceptive neuromuscular facilitation ( pnf ) improves the functions of the muscles and tendons by stimulating the proprioceptive sense , which enhances muscle strength , flexibility , and balance5 . neck pattern exercises are known to increase the stability of the head and neck6 . the resistance against the neck exercise causes irradiation in the body - trunk muscle exercise7 . however , few studies have investigated the improvement in trunk control in chronic stroke patients as a result of using the pnf neck pattern . hence , the purpose of this study was to examine the effects of the pnf neck pattern exercise on the ability to control the trunk and balance in chronic stroke patients . the subjects of this study were patients who were diagnosed with stroke through ct or mri at hospital f located in daegu , korea , between january and july 2015 ( table 1table 1.general characteristics of the subjects ( meansd)experiment group ( n=15)control group ( n=15)age ( years)59.49.155.99.8after onset ( months)11.23.610.93.5gender ( m / f)5 /107 /8side ( r / l)7 /89 /6 ) . the subjects had a duration of illness of six months or longer , a mmse - k score over 24 , manual muscle testing results for neck flexion and extension graded as better than fair , and the ability to sit and stand independently . those who were incapable of training , including patients with high - risk heart disease or musculoskeletal disease , were excluded from the sample . prior to the experiment , the institutional review board ( irb ) at daegu fatima hospital approved the study . this research was conducted after signed agreements to participate in the study were received from the patients and their guardians . after 30 research subjects were selected , they were divided randomly and evenly into an experimental group in which the pnf neck pattern was applied and a control group in which traditional rehabilitation therapy was applied . traditional rehabilitation therapy was applied daily for 30 minutes to both groups five times a week for six weeks . the experimental group received additional application of the pnf neck pattern , while the control group received additional application of 30 minutes more traditional rehabilitation therapy per day , three times a week for six weeks . the patients sat on a mat that was knee height with their feet placed shoulder width apart and their hands on their knees . for the neck pattern , both the flexion pattern and extension pattern were performed . the experimenter stood behind the patient on the right side and put the tip of his right finger below the chin of the patient . the experimenter then put his left hand slightly left of the top of the patient s head in a diagonal direction . the experimenter pulled the patient s chin so that it was lifted and the neck was extended . hence , in the preparation position , the patient s head was tilted and rotated towards the right side . the preliminary exercise was performed with sufficient explanation so that the patient could accurately recognize the exercise direction . hence , the patient s head , neck , and upper thoracic spine had sufficient extension , left rotation , and left lateral flexion . the experimenter provided resistance against left rotation , flexion , and lateral flexion by providing traction to the patient s chin . the identical method was applied on the opposite side . for the neck extension pattern , he put his right thumb on the right side of the patient s chin and placed his left hand slightly right of the top of the patient s head in a diagonal direction . the patient assumed the preparation position in which the chin was pulled , the neck was flexed , and the head was rotated and tilted to the left . lift your head to look above . hence , the patient s head , neck , and upper thoracic spine had complete extension , right rotation , and right lateral flexion . resistance against right rotation , extension , and lateral flexion was provided during the exercise in order to induce strong muscle contractions . in this study , the trunk impairment scale ( tis ) was used to evaluate the ability to control the trunk . the tis consists of 17 subitems in three categories : static sitting balance , dynamic sitting balance , and coordination . because the tis has high reliability and validity , it is used to evaluate the degree of motor damage to the trunk in stroke patients8 . in the tis , static sitting balance has a minimum score of zero and a maximum score of seven , dynamic sitting balance has a minimum score of zero and a maximum score of 10 , and coordination has a minimum score of zero and a maximum score of 6 ; the maximum tis score is 23 . the berg balance scale ( bbs ) , which has proven reliability and validity , was used to examine the ability of patients to balance before and after the exercise9 . the bbs consists of 14 items , each of which has a minimum score of zero and a maximum score of four ; the maximum score is 56 . the nonparametric wilcoxon signed - rank test was performed to compare the tis and bbs scores before and after the intervention . statistically significant changes in all items of the tis , tis total score , and bbs were observed in both the experimental group and the control group ( p<0.05 ) ( table 2table 2.comparison between before and after the interventionexperiment group ( n=15)control group ( n=15)beforeafterbeforeafterbbs41.84.247.63.342.04.344.64.3tisstatic sitting6.21.06.80.36.30.76.60.6dynamic sitting4.31.86.51.96.41.75.31.8coordination3.51.35.11.63.21.23.81.4total14.03.418.53.314.03.015.73.1*p<0.05 . table 3table 3.comparison of effects between the groupsexperiment group ( n=15)control group ( n=15)bbs5.82.32.61.6tis static sitting0.60.90.20.4dynamic sitting2.21.40.90.8coordination1.60.70.50.5total4.41.81.71.0*p<0.05 . bbs : berg balance scale ; tis : trunk impairment scale shows the comparison results according to the therapy method . a significant between - group difference was found in all items among the subitems of the tis except the static sitting balance ( p<0.05 ) . * selective control of the trunk affects breathing , speaking , balancing , walking , and the functions of the hands and the upper limbs10 . improvement in the ability to control the trunk is necessary to enhance the ability to balance in chronic stroke patients11 . hence , this study applied the pnf neck pattern to chronic stroke patients in order to investigate the effects on their ability to control the trunk and to maintain balance . mobility and ability of the trunk to maintain balance are important factors in the functional independence of stroke patients12 . moreover , improved motor control of the trunk is known to assist independent performance of daily tasks in stroke patients13 . both the experimental group and the control group showed a significant difference in the bbs and tis after the intervention . this implies that both traditional rehabilitation therapy and pnf neck pattern exercises are effective in enhancing the ability of chronic stroke patients to control the trunk and to maintain balance . using the tis , park predicted falling among patients with subacute stroke , reporting that a tis score between 14.5 and 15.5 corresponded to the range of risk for falling14 . in the present study , both groups showed average tis scores of 14.0 before the intervention , which was within the range of risk for falling . after the intervention , the average improved to 18.5 in the experimental group and 15.7 in the control group . it is possible that the pnf neck pattern exercise contributed to the ability to control the trunk and maintain balance more than the traditional rehabilitation therapy did , eventually decreasing the risk of falling . moreover , park observed that better tis dynamic sitting balance was connected to a lower risk of falling . in the present study , significant results were found for all items except tis static sitting in the between - group comparison . this indicates that the pnf neck pattern exercise resulted in stronger improvement in tis dynamic sitting balance , tis coordination , and bbs scores compared with the traditional rehabilitation therapy . in particular , the improvement in dynamic sitting balancing was expected to contribute to the prevention of falling14 overall , the pnf neck pattern exercise was shown to have a positive effect on increasing the ability to control the trunk and maintain balance in the chronic stroke patients in this study . however , because of the small number of subjects , the results of the study can not be generalized . thus , in future research , studies should be conducted to apply the pnf neck pattern exercise to a large sample of chronic stroke patients .

[ purpose ] the aim of this study was to investigate the effects of proprioceptive neuromuscular facilitation neck pattern exercise on the ability to control the trunk and balance in chronic stroke patients . [ subjects and methods ] a total of 30 study subjects were selected and randomly divided into an experimental group of 15 subjects , who received the proprioceptive neuromuscular facilitation neck pattern exercise , and a control group of 15 subjects , who received a traditional rehabilitation treatment . [ results ] statistically significant changes in all the items of the trunk impairment scale , the trunk impairment scale total score , and the berg balance scale were observed in both the experimental group and the control group . significant between - group differences were found in all items among the subitems of the trunk impairment scale except the static sitting balance . [ conclusion ] proprioceptive neuromuscular facilitation neck pattern exercise was shown to have a positive effect on increasing the ability to control the trunk and maintain balance in chronic stroke patients .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

forty years ago , given the evidence available , it was appropriate to label ldl ( low - density lipoprotein ) , bp ( blood pressure ) and smoking as risk factors rather than causes of vascular disease . all the links in the evidentiary chain of causality temporality , strength , dose response , specificity , consistency , biological plausibility and experimental confirmation are in place . to pretend there is doubt is to disregard the masses of interlocking biological , pathophysiological , epidemiological and clinical trial results . but if ldl , bp and smoking cause vascular disease , why are they such weak risk factors for the likelihood of clinical events ? wald and law , in particular , have enunciated and emphasized this paradox [ 13 ] and it is a fact that , except at the extremes , the level of ldl , however it is estimated , the level of bp and the extent of smoking only marginally influence the estimates of risk . indeed , wald and law argue that , although ldl and bp cause vascular disease , they are of no practical value to identify those who would benefit from preventive therapy . that is the core of their argument that the polypill should be given to all those over 55 years of age . in the conventional sense , this is correct : the causes of vascular disease are weak risk factors for vascular disease . however , it does not follow that we should not identify and treat the causes of vascular disease within individuals . on the contrary , we will try to demonstrate that the reason that the causes of vascular disease are weak risk factors for clinical events is primarily a function of how we treat age as a determinant of vascular disease . age is , by far , the dominant risk factor in any risk factor model for cardiovascular events . from 25 years of age indeed , once age and gender are taken into account , all of the accepted modifiable risk factors add only marginally to the predictive power of the risk factor engines such as framingham . but does that mean that age causes vascular disease ? or does it point to the fact that the causes of vascular disease act progressively over time ? the most important difference between the risk factor and causal exposure models is how they treat age . in the risk factor model , age is regarded as the simple , purely chronological , variable that it is thought to be in every day life , a variable , which is known to be non - modifiable and which , operationally , is presumed to be independent of all the other risk factors . however , age is , in reality , a complex variable , pointing to , on the one hand , all the non - modifiable biological changes that occur within our arteries over time and , on the other , all the modifiable consequences of the cumulative injuries to our arteries over time due to ldl , bp , smoking and diabetes . put simply , the injuries to arteries owing to age are due to exposure and to disintegration . in the risk factor model of cardiovascular events : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{eqnarray * } & & { \mathrm{risk = { \it f}\ ; [ age\,\times\,apo\ ; ( apolipoprotein)\ ; b}}\nonumber\\ & & \qquad\quad{\mathrm{\times\ ; smoking}{\rm \times\ , bp\,\times\ , diabetes ] } } \end{eqnarray * } \end{document } but actually age represents the duration of exposure of arteries to apob , smoking and diabetes . therefore in the causal exposure model of vascular disease : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{eqnarray * } { \rm disease = ( level\ ; of\ ; apob\ ; \times\ ; duration\ ; of\ ; time\ ; the\ ; arterial\;}\\[-6pt ] { \rm wall\ ; is\ ; exposed\ ; to\ ; the\ ; apob\ ; lipoproteins)\ ; + \ ; ( level\ ; of\ ; bp } \\ { \rm \times\ ; duration\ ; of\ ; time\ ; the\ ; arterial\ ; wall\ ; is\ ; exposed\ ; to\ ; bp)}\\ { \rm + \ ; ( intensity\ ; of\ ; smoking\ ; \times\ ; duration\ ; of\ ; smoking)}\\[-5pt ] { \rm + \ ; duration\ ; of\ ; diabetes\ ; + } \\ { \rm non\hbox{-}modifiable\ ; changes\ ; due\ ; to\ ; disintegration } \end{eqnarray * } \end{document } the strengths of the risk factor model are that it is well known , well accepted and that it works , that is it identifies risk within broad , but reasonable , limits for the next decade . to be sure , there are , in fact , multiple risk factor models : framingham , score , qrisk and reynolds . nevertheless , their predictions are quite similar because age is the principal determinant of risk in all . events early in life are much less common than events later in life . that simple reality is what makes age such an overwhelmingly powerful predictor . even numerically early events account for an important portion of the total toll of disease and early death is particularly tragic and costly . thus , in framingham , one in six events in men and one in ten in women occur before 55 years of age . indeed , the majority of those at low short - term risk are at high long - term risk : in the framingham study , those in the lowest tertile of 10-year risk at 50 years of age face a lifetime risk of a coronary heart event almost as high as those in the highest tertile . most importantly , a low likelihood of a clinical event in the next decade does not equal a low likelihood that disease is not developing within our arterial walls during that decade . on the contrary , if the causes of vascular disease are present , there is a high likelihood that it is . the consequence is that prevention is too often delayed until disease is advanced and the potential for benefit from therapy correspondingly reduced . moreover , there is a numbers mismatch at the core of the risk factor model which aims to identify the minority who are at greatest risk within the next decade , whereas the reality is the majority , two out of three in the case of the u.s.a . on the basis of ten large cohort studies in western populations , wald and law calculated that a reduction in ldl - cholesterol of 1.0 mmol / l at 50 , 60 and 70 years of age would reduce vascular events by 56 , 41 and 31% respectively , demonstrating that the same degree of ldl - lowering will produce much greater benefit earlier , rather than later , in life . in the bupa ( british united provident association ) study , even though the absolute number of deaths due to ischaemic heart disease over a 12-year period was four times greater in those 5564 years of age at entry compared with those 3544 years of age at entry , reducing cholesterol by 0.6 mmol / l will prevent almost a two and a half times greater percentage of fatal events in the younger group compared with the older one . this evidence supports the pathophysiological argument that the absolute gain from treating the younger rather than older subjects will be greater , arguing for earlier compared with later strategies of prevention . lloyd - jones and his co - workers have shown a strikingly low lifetime incidence of vascular disease in those who do not manifest the major risk factors by 50 years of age . that the duration of the time arteries are exposed to apob matters is illustrated by mutations in pcsk9 ( proprotein convertase subtilisin kexin 9 ) . gain - of - function mutations in pcsk9 result in reduced numbers of ldl - receptors and higher plasma ldl levels , whereas loss - of - function mutations result in more ldl - receptor numbers and life - long lower plasma ldl levels . strikingly , a gain - of - function mutation in pcsk9 that results in a reduction in ldl - cholesterol levels by 30% over a lifetime reduces clinical events by 88% . this decrease in events per mg of ldl - cholesterol is substantially more pronounced than the decrease in events per mg with ldl - lowering therapy . this finding of augmented benefit from lifelong lower levels has been confirmed in other studies of mutations of this gene . finally , on the basis of the results of the amoris ( apolipoprotein - related mortality risk ) study , reducing the apob / apoa - i ratio in all of those above the 80th percentile to the level of those in the 20th percentile would decrease deaths from acute myocardial infarction by 80% . that a very high proportion of the population , approximately 75% , is at a high total lifetime risk of a significant cardiovascular event should not be surprising , since this percentage corresponds reasonably closely to the known total incidence of cardiovascular events in the population . indeed , this is the core of the polypill argument to treat all over 55 years of age with a combination of fixed low - dose pharmacological agents . but pharmacological therapy will produce clinical benefit only in those in whom that specific cause is present . moreover , the degree of benefit will relate to the degree of abnormality , that is the absolute risk and the absolute benefit from pharmacological therapy depend on the absolute levels of the cause(s ) of vascular disease . thus patients with normal ldl levels but high bp do not require ldl - lowering therapy , whereas those with very high ldl levels require more intensive therapy than those with only moderately elevated ldl levels . targeted therapy would be more cost - effective than population - based single dose therapy precisely because it is limited to those who need it most . for example , if the atpiii ( adult treatment panel iii ) guidelines were applied to the u.s.a . population , substituting non - hdl ( high - density lipoprotein)-cholesterol as the target of therapy for ldl - cholesterol would result in 300000 more events prevented over a 10-year period . however , if apob were substituted for ldl - cholesterol , 800000 more events would be prevented over a 10-year period . in the causal exposure model , therapeutic intervention would be based on detection of the causes of vascular disease , not the calculation of short - term risk . age is the primary driver of decisions in both the risk factor and the polypill models . the causal exposure model would expand the numbers selected for early medical prevention of cardiovascular disease beyond the risk factor model and the polypill model . treating more people for longer periods of time would necessarily change the usual estimates of cost and benefit . the patent periods of most of the major statins and antihypertensive agents have expired or are close to expiring . the price of generics , particularly in markets such as the u.s.a . indeed , treatment of patients at intermediate risk for cardiovascular events has become cost - effective . nevertheless , long - term safety can not be taken for granted and , indeed , only recently has evidence emerged that the incidence of diabetes may be slightly increased with long - term ingestion of statins . naturally , this has been assumed to be a negative effect of statins , although it seems possible that this might represent a hazard of survival free of clinical events . in any event , the balance of risk and benefit still strongly favour statin therapy , even when the impact of a potential increase in the risk of diabetes is taken into account . that said , true assurance of long - term safety will only come with large numbers undergoing long - term therapy . the reality remains that cardiovascular disease is the major cause of death worldwide notwithstanding all the diagnostic tests and therapies that exist . prevention is , therefore , the best strategy both from an individual and a societal perspective . ideally , the causal exposure model hypothesis should be put to the test in a randomized clinical trial . but this will never occur for many reasons , not least the several decades such a trial would require . however , it is possible to model the impact of different strategies to prevent vascular disease , and this evidence can be very helpful . nevertheless , in the absence of unequivocal proof , guideline groups may find it impossible to recommend the causal exposure model and this alone may make widespread implementation impossible . equally important , many patients are reluctant to take medication and would not accept the equation of potential long - term gain in exchange for early medical therapy . our view is that patients should be aware of the risks that they face and the options available to avoid them . the choice is for the patient to make , a choice , which can be reviewed and revised as knowledge is gained . moreover , all the barriers that apply to early implementation of therapy to lower ldl also apply to hypertension and , in the case of hypertension , at least so far as the guideline groups are concerned and much of medical practice , they have been overcome . that is to say , virtually anyone with markedly increased bp will be offered the option of therapy even if their short- and medium - term risks of cardiovascular events are low . in this instance , the knowledge of the long - term hazards has overcome the barriers to therapy . the difference , we would contend , is that hypertension is recognized as a disease , whereas ldl , at least at present , is only a risk factor for disease . definition of when ldl - lowering therapy should be initiated needs to be defined for ldl . early treatment would not be accepted if only modest deviations from the norm are present . the challenge is to find the appropriate level at the appropriate age at which pharmacological therapy should be considered and this is where studies that systematically model outcome would be most helpful . with regard to ldl , statins will be most effective in those who are most abnormal . the younger the individual , the higher the threshold level for intervention that will be required . for those above 40 years of age , we suspect that treatment of those above the 70th percentile should be cost - effective , but this must be confirmed in quantitative modelling studies . it might also be helpful to couple the approach we suggest with appropriate non - invasive tests of vascular function . the difficulty is the lack of standardization and physiological validation of some of the most popular of these tests . the risk factor model for cardiovascular events is broadly known and broadly accepted by professionals and governments , and this facilitates the implementation of public health policy based on this model . the concept of appropriately weighting and integrating all of the relevant information is intuitively attractive and the method appears authoritative and precise . however , age , which is conventionally viewed as a non - modifiable risk factor , is the principal determinant of whether prevention will be initiated in the individual patient , not the modifiable causes of the disease process in the artery . once we appreciate that age describes the length of time over which exposure to the causal factors of disease occurs , age becomes a modifiable risk factor for vascular disease . once we couple this understanding with the recognition that the ability of therapy to prevent clinical events depends on the extent of disease within the arterial wall at the time therapy is initiated , the advantages of the causal exposure model for vascular disease over the risk factor model for cardiovascular events become apparent . after all , if disease in the wall is prevented , there will be no events to predict .

primary prevention of cardiovascular disease is governed at present by the risk factor model for cardiovascular events , a model which is widely accepted by physicians and professional associations , but which has important limitations : most critically , that effective treatment to reduce arterial damage is often delayed until the age at which cardiovascular events become common . this delay means that many of the early victims of vascular disease will not be identified in time . this delay also allows atherosclerosis to develop and progress unchecked within the arterial tree with the result that the absolute effectiveness of preventive therapy is limited by the time it is eventually initiated . the causal exposure model of vascular disease is an alternative to the risk factor model for cardiovascular events . whereas the risk factor model aims to identify and treat those at markedly increased risk of vascular events within the next decade , the causal exposure model of vascular disease aims to prevent events by treating the causes of the disease when they are identified . in the risk factor model , age is an independent non - modifiable risk factor and the predictive power of age far outweighs that of the other risk factors . in the causal exposure model , age is the duration of time the arterial wall is exposed to the causes of atherosclerosis : apob ( apolipoprotein b ) lipoproteins , hypertension , diabetes and smoking . preventing the development of advanced atherosclerotic lesions by treating the causes of vascular disease is the simplest , surest and most effective way to prevent clinical events .

INTRODUCTION AGE THE DOMINANT RISK FACTOR STRENGTHS AND LIMITATIONS OF THE RISK FACTOR MODEL FOR CARDIOVASCULAR EVENTS STRENGTHS OF THE CAUSAL EXPOSURE MODEL: EARLY AND LATE PREVENTION WHO SHOULD BE TREATED? BARRIERS TO IMPLEMENTATION OF THE CAUSAL EXPOSURE MODEL SUMMARY

corneal neovascularization is defined as the occurrence of neovessels developed from preexisting vascular structures in the limbus , invading areas of the cornea initially not vascularized . the avascular character results from a balance between angiogenic and antiangiogenic factors . in pathological conditions such as inflammatory , infectious , degenerative , or traumatic ones , this homeostasis can be broken and can be responsible for the occurrence of corneal neovessels . several therapeutic modalities have been proposed : steroids , physical therapy by superficial keratectomy or argon laser , cauterization , and local and subconjunctival application of anti - vegf ( bevacizumab , ranibizumab ) ; but there is so far no consensus in the therapeutic management of corneal neovessels . the aim of our study is to report the efficiency and safety of bevacizumab in intrastromal injection in the treatment of corneal neovascularization . this is a prospective , noncomparative study , conducted at the university hospital mohammed vi of marrakech , between september 2013 and december 2015 , and it included 25 eyes of 23 patients with deep predescemetic corneal neovessels who received intrastromal injections of bevacizumab . all patients underwent an eye examination with a measurement of best corrected visual acuity , examination at the slit lamp with photographs of the cornea , intraocular pressure measurement , and examination of the eye fundus with an ocular ultrasound in the absence of a refractive medium of the eye . the severity was assessed using a clinical grading scale ; this method aims to analyze directly photographs of the cornea ( table 1 ) . patients had to have a minimum setback of six months to be included in the study . all patients received initially a topical corticosteroid to reduce their diameter and act on their effects ( edema and inflammation ) . the bevacizumab at 25 mg / ml was prepared sterilely into a syringe with a needle of 30 gauge . an injection of about 0.01 ml was performed under topical anesthesia with instillation of oxybuprocaine hydrochloride 0.4% . the product was administered in the corneal stroma next to neovessels ; the exact location depends on the location of the neovessels compared to the limbus , their number , and their extension according to the quadrants . the intrastromal injections were spaced by a month and were continued until the stagnation of the regression of neovascularization . patients who received intrastromal injections of bevacizumab were monitored at day 7 , day 30 , and day 120 . patients received blood pressure test , visual acuity , intraocular pressure measurement , and the slit lamp exam with photographs . the photographs on photographic slit lamp were taken in the inclusion visit then monthly until the end of the treatment . the primary evaluation criterion was the corneal surface covered with neovessels before and after treatment . the analysis was performed by clinical grading scale and photoshop software ( adobe systems , inc . the second criterion for efficacy was the variation in visual acuity before and after treatment . twenty - five eyes of twenty - three patients ( 15 men and 8 women ) with nine months of setback after the beginning of bevacizumab therapy were included in the study . the average age of patients at the time of treatment with bevacizumab was 31 years ( between 16 and 44 years ) . the etiologies of corneal neovascularization were scars of herpetic keratitis in 10 patients ( 43.4% ) , ocular trauma in 6 patients ( 26.1% ) , postinfectious keratitis in 3 patients ( 13% ) , chemical burns in 2 patients ( 8.7% ) , and toxic epidermal necrolysis in 2 patients ( 8.7% ) . 10 patients received 3 intrastromal injections of bevacizumab , 9 received four injections , and 4 had 5 injections . the evolution was marked by the total regress in 15 patients ( figures 1 , 2 , and 3 ) , partial in 5 . overall , the percentage of corneal neovascularization is reduced by 43 19% ( between 14 and 70% ) at 18 16% ( between 0 and 39% ) in day 120 ( p < 0.001 ) . later on , intraoperatively , 6 patients received subconjunctival corticoids injections , and 2 were injected with subconjunctival bevacizumab . postoperatively , there was recurrence of neovessels at the collar of the receiver in 3 patients , which was well controlled under topical corticosteroids , and a backlash in 3 patients . there was also a recurrence of neovascularization in 2 patients after an average of five months . our study seems to suggest the efficacy of bevacizumab in the treatment of deep corneal neovascularization . the development of corneal neovessels occurs in several phases : a perivascular latency phase , a second active neovascularization , and finally a maturation phase of new vessels . the first two phases will be a potential target for angioregressive therapy by blocking angiogenic factors . classical treatment modalities include corticoids , nonsteroidal anti - inflammatory drugs , laser photocoagulation , and reconstruction of the ocular surface . however , these treatments have demonstrated limited therapeutic effect with considerable complications [ 710 ] . the development of inhibitors of vegf , a humanized monoclonal antibody bound to isoforms of vegf - a , introduced a new perspective in the treatment of various ophthalmic disorders , including damage to the ocular surface such as corneal neovascularization . the efficiency of bevacizumab in intrastromal injection has been reported ; the dose of 25 mg / ml appears to be effective and well tolerated in several studies [ 1215 ] ( table 2 ) . several hypotheses can be made to explain this variability in response to bevacizumab as the different etiologies of neovascularization are involved : the extension of the neovascularization , the state of limbo , and the delay between the appearance of neovessels and the beginning of treatment . indeed , in these patients , the decline was not only due to corneal neovascularization , but also due to corneal scars . compared to other forms of administration , the intrastromal injection allows greater exposure to bevacizumab and delivery of a known concentration of the drug . the penetration of the topical form of this drug may be limited by an intact epithelium because of the high molecular weight of bevacizumab . the intrastromal administration also ensures less risk of failure due to lack of patient compliance . patients may sometimes forget to install the medication at home , when using a topical preparation . the subconjunctival injection of bevacizumab allows better diffusion compared to topical instillation ; several studies have shown its effectiveness in the treatment of superficial and average depth neovessels [ 17 , 18 ] , and others have proved that subconjunctival injection of bevacizumab allows only partial reduction of deep neovessels [ 1921 ] . a recent study showed that the subconjunctival injection has no effect on mature neovessels ; it is quite effective in the active phase of the process of angiogenesis . used a ranibizumab having a lower molecular weight than subconjunctival topical instillation and found satisfactory results . the intrastromal injection of bevacizumab increases the concentration and duration of exposure ; in addition , it is also very effective in mature neovessels [ 13 , 14 ] . the intrastromal injection of bevacizumab might be a useful option in the management of corneal neovascularization . . however , further studies are needed to determine the optimum dosage and to define the indications , the frequency , and risk factors for developing possible side effects .

introduction . corneal neovessels are a major risk factor for corneal graft rejection , due to the loss of the immune privilege . the purpose of this study is to evaluate the effectiveness of intrastromal injection of bevacizumab in the treatment of corneal neovascularization . material and methods . this is a prospective study that included 25 eyes of 22 patients with deep corneal neovessels , treated with intrastromal injections of bevacizumab . results . the average age of patients was 31 years ranging from 16 to 44 years . the causes of neovascularization were dominated by herpetic keratitis ( 10 cases ) . the evolution was marked by complete regress of neovessels in 16 patients , partial regress in 6 cases , and reduced opacity and improved visual acuity in 5 patients . no side effects were noted . discussion . short - term results demonstrated the effectiveness of intrastromal injection of bevacizumab in the treatment of corneal neovessels . it may be an option or a complement to other useful treatments in stabilizing or improving vision . conclusion . bevacizumab is an effective additional treatment for the improvement of corneal transplants prognosis with preoperative corneal neovascularization .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusion

short - term memory ( stm ) persists for several hours at most , while long - term memory ( ltm ) can last for up to a lifetime . a clear biochemical feature of memory consolidation at the cellular level , which occurs immediately after learning ( an episode ) , is the induction of gene expression . this gene expression is considered to induce plastic changes in neurons , thereby allowing the long - term retention of memory . for example , experiments in rodents using a pavlovian fear conditioning task and other tasks showed that when gene expression in the brain was inhibited immediately after conditioning ( learning ) , stm ( up to 2 - 4 h ) was intact , but ltm ( approximately 24 h ) was disrupted [ 2 , 3 ] . in addition , long - term potentiation ( ltp ) is considered to be a cellular model that reflects one aspect of memory formation . field recording analyses showed that long - lasting ltp ( l - ltp ) induced by tetanic stimulation of hippocampal ca1 neurons also requires gene expression ; the inhibition of gene expression impairs l - ltp without affecting the induction or early phase of ltp [ 1 , 3 ] . creb was cloned in 1988 as a transcription factor that binds to the camp - responsive element ( cre ) . subsequent studies showed that creb belongs to the creb / atf family together with atf-1 , camp - responsive element modulator ( crem ) , atf2 and atf3 and atf4 , which are highly homologous to creb at the primary structure level . the n - terminal region of creb contains two glutamine - rich regions ( q - rich domains ) and a region with serine residues ( kid ; kinase induceble domain ) , the latter of which is the target of various kinases and functions as a transcriptional regulatory domain ( fig . , the c - terminal region harbors a b - zip domain with a leucine zipper following a basic region and contributes to the formation of homo- and hetero - dimers and binding to cre ( fig . the activation of transcription by creb depends on its phosphorylation at serine 133 ( s133 ) [ 1 , 5 - 8 ] , and creb is activated when s133 is phosphorylated mainly by protein kinase a ( pka ) or ca / calmodulin - dependent kinase iv ( camkiv ) . creb can interact with creb - binding protein ( cbp ) , a transcriptional co - activator , only when s133 is phosphorylated , thereby inducing transcription . in this way , the activation of transcription by creb is strictly controlled by the phosphorylation of s133 ; therefore , s133 phosphorylation is widely used as a marker of transcriptional activation by creb . to clarify the roles of creb in learning and memory formation , importantly , previous studies using mutant mice demonstrated that the genetic loss of creb function impaired the formation of ltm without affecting stm [ 2 , 9 , 10 ] . furthermore , mutant mice with inhibited creb activity also showed deficits in hippocampal l - ltp . these findings indicate that creb is required for memory consolidation and ltp , suggesting that creb plays a central role in these processes . creb target genes include c - fos , activity - regulated cytoskeleton - associated protein ( arc ) , and brain - derived neurotrophic factor ( bdnf ) [ 11 - 13 ] ; creb is believed to control memory consolidation and ltp by regulating the expression of these genes . importantly , previous studies have shown that creb plays critical roles in memory formation not only in rodents but also in aplysia and drosophila [ 1 , 14 , 15 ] the finding that the loss of creb function blocks memory consolidation and ltp suggests that it functions as a positive regulator of these processes . to examine this hypothesis , three types of transgenic mice displaying gain of creb function have been generated and investigated . vp16 , a virus - derived protein , contains a transcription activation domain displaying significantly high transcription activity and has been used in abundant transcription studies as a model of transcription activation domain in eukaryotes . to investigate the gain - of - function of creb , creb - vp16 , a chimeric transcription factor , transgenic mice expressing creb - vp16 in the forebrain region were generated using the tetracycline system . these mutant mice showed that creb - vp16 is highly expressed in the hippocampal ca1 and dentate gyrus areas . however , even though these mice were expected to have improved memory formation , behavioral studies showed that they displayed abnormalities in spatial memory formation . these observations are contradictory compared to the results of behavioral experiments using creb - y134f and creb - diedml mice ( see below ) . these findings are discussed in the next session . previous studies using field recordings have shown that ltp was induced in the hippocampal ca1 neurons of wild - type ( wt ) mice when 1 train of 100-hz tetanic stimulation was applied for 1 s , although this ltp disappeared after approximately 2 h. on the other hand , when this tetanic stimulation was applied 4 times at 5-min intervals , l - ltp that lasted for over 3 h was induced . importantly , similarly with memory consolidation , l - ltp requires the induction of gene expression . interestingly , electrophysiological analyses showed that one train of tetanic stimulation is sufficient to induce l - ltp - like ltp in hippocampal ca1 neurons of creb - vp16 mice . further studies indicated that the threshold of l - ltp induction was lower in creb - vp16 mice than in wt mice . as a next step , the mechanism underlying the reduction in the threshold of l - ltp induction observed in creb - vp16 mice was investigated . in wt hippocampal slices , even one train of tetanic stimulation was sufficient to induce l - ltp once l - ltp was induced in the other synapses of the same neuron by four trains of tetanic stimulation . this observation is thought to reflect the fact that the application of tetanic stimulation four times induces creb - mediated gene expression ; then , the resulting gene products are transported to the synapses of the stimulated neuron , even to those in which l - ltp was not induced . therefore , since the expression of creb target genes is significantly enhanced in creb - vp16 mice , one train of tetanic stimulation , as with four trains of tetanic stimulation , is thought to induce l - ltp . this hypothesis was supported by detailed analyses showing that the ltp induced in wt mice by one train of tetanic stimulation following four trains of tetanic stimulation has similar characteristics to that induced in creb - vp16 mice by one train of tetanic stimulation . furthermore , the increased expression of bdnf in creb - vp16 mice under the basal condition , i.e. , without any tetanic stimulation , has been shown to contbibute to the lowered threshold of l - ltp induction . consistently , four trains of tetanic stimulation failed to enhance ltp induced by one train of tetanic stimulation in creb - vp16 mice . these observations suggested that synaptic capture of bdnf is sufficient for induction of l - ltp by a single tetanic stimulation and provided potential molecular mechanisms of synaptic tagging [ 19 - 21 ] . creb - y134f ( y134f ) contains a mutation in which tyrosine is changed to phenylalanine at position 134 . this mutant protein displays increased affinity to pka ( a creb kinase ) , thereby leading to the lowering of the threshold for creb activation ; this mutation makes it easier for the protein to be activated . on the other hand , for creb - diedml ( diedml ) , six amino acids ( rrpsyr , which include s133 ) are replaced with diedml ( fig . 1 ) , the cbp - binding motif of sterol - responsive element binding protein ( srebp ) . therefore , diedml interacts constitutively with cbp without phosphorylation by creb kinases . transgenic mice were generated in which y134f or diedml is expressed specifically in the forebrain region under the control of the camkii promoter ( y134f and diedml mice ) . these mutant mice display higher expression levels of c - fos , a creb - target gene , than wt mice , indicating that the expression of these dominant active mutants leads to the enhanced activation of creb - mediated transcription . similarly with the results from the creb - vp16 mice , electrophysiological analysis using field recordings in a transgenic line with a high level of y134f expression ( line c ) confirmed enhanced l - ltp in hippocampal ca1 neurons . further analysis using the patch clamp method showed enhanced spike - timing ltp in the hippocampal ca1 neurons of y134f mice ( line c ) . taken together with the observations from the creb - vp16 mice , these results strongly suggest that creb functions as a positive regulator of ltp , even though these studies were not performed under similar conditions to those used for the creb - vp16 mice . in contrast to the results from the creb - v16 mice , behavioral studies showed that all of the transgenic y134 and diedml lines displayed improved ltm at 24 h in social recognition and fear conditioning memory tasks . furthermore , improved ltm was also observed in a spatial memory task using the morris water maze and a passive avoidance memory task . in a contextual fear conditioning task , wt mice display impaired contextual discrimination at one month after conditioning . these mice display fear ( freezing ) responses when they are exposed to a box that is novel , but similar to the original box in which they had received an electrical foot shock . however , they did not display such a high level of freezing responses in the novel box compared to the original box at one day after conditioning . in contrast , y134f mice ( line c ) , which highly express the creb mutant , could discriminate between the novel and familiar boxes , even at one month after conditioning ; mutant mice displayed significantly higher fear responses in the familiar context in which they received a foot shock compared to the novel context . these observations indicated that y134f mice formed more accurate ( stronger ) memory than wt mice . thus , the behavioral experiments using y134f and diedml mice showed that , in contrast to the results from creb - vp16 mice , the activation of creb significantly improved memory consolidation , indicating that creb functions as a positive regulator of this process ( fig . i compare the results of behavioral analyses among creb - vp16 , y134f , and diedml mice . in y134f and diedml mice , creb was activated only a few fold higher than in wt mice , whereas in creb - vp16 mice , since vp16 contains a strong transcriptional activation domain , the activation level was 20- to 30-fold higher than in wt mice . importantly , the dominant active creb mutants were expressed simply under the control of the camkii promoter in the y134f and diedml mice . in contrast , in the creb - vp16 mice , the expression of creb - vp16 is amplified using the tetracycline system . therefore , these comparisons suggest that the activation of creb - mediated transcription was unusually high in the creb - vp16 mice compared to the y134f and diedml mice ; y134f and diedml mice display enhanced creb - mediated transcription at a physiologically moderate level , while creb - vp16 mice do not . thus , although enhanced ltp was observed in the creb - vp16 and y134f /diedml mice , the levels of creb activation seemed to exert a strong influence on memory ; only y134f /diedml mice , which show moderate creb activation , display enhanced memory consolidation . interestingly , y134f and diedml mice demonstrated improved stm from 30 min to 2 h as well as ltm . especially , diedml mice , with higher creb activation levels than y134f mice , displayed improved stm at 30 min . these observations strongly suggest that stm is improved in a dose - dependent manner by creb activity . importantly , as stm is thought to be formed independently of new gene expression , it is suggested that creb plays a regulatory role in stm , but this enhanced stm is not mediated by the transcriptional activation of creb target genes immediately after training . expression analysis showed that y134f and diedml transgenic lines with enhanced stm display increased levels of bdnf in the hippocampus ; transgenic lines expressing higher levels of bdnf also exhibit enhanced stm . importantly , a micro - infusion of bdnf or a bdnf inhibitor into the hippocampus of wt mice generated enhanced or impaired stm , respectively . additionally , the infusion of a higher dose of the bdnf inhibitor into the hippocampus was required for the impairment of stm in diedml mice than in wt mice . these observations suggest that an increase in the expression levels of bdnf improves stm in y134f and diedml mice . from these findings , creb is thought to play a regulatory role in stm through the regulation of bdnf expression ( fig . 2 ) . on the basis of the analysis of y134f and diedml mice described above , it is suggested that creb is a positive regulator of memory consolidation . in addition , it is suggested that creb indirectly controls stm by regulating the expression levels of bdnf . vp16 , a virus - derived protein , contains a transcription activation domain displaying significantly high transcription activity and has been used in abundant transcription studies as a model of transcription activation domain in eukaryotes . to investigate the gain - of - function of creb , creb - vp16 , a chimeric transcription factor , transgenic mice expressing creb - vp16 in the forebrain region were generated using the tetracycline system . these mutant mice showed that creb - vp16 is highly expressed in the hippocampal ca1 and dentate gyrus areas . however , even though these mice were expected to have improved memory formation , behavioral studies showed that they displayed abnormalities in spatial memory formation . these observations are contradictory compared to the results of behavioral experiments using creb - y134f and creb - diedml mice ( see below ) . these findings are discussed in the next session . previous studies using field recordings have shown that ltp was induced in the hippocampal ca1 neurons of wild - type ( wt ) mice when 1 train of 100-hz tetanic stimulation was applied for 1 s , although this ltp disappeared after approximately 2 h. on the other hand , when this tetanic stimulation was applied 4 times at 5-min intervals , l - ltp that lasted for over 3 h was induced . importantly , similarly with memory consolidation , l - ltp requires the induction of gene expression . interestingly , electrophysiological analyses showed that one train of tetanic stimulation is sufficient to induce l - ltp - like ltp in hippocampal ca1 neurons of creb - vp16 mice . further studies indicated that the threshold of l - ltp induction was lower in creb - vp16 mice than in wt mice . as a next step , the mechanism underlying the reduction in the threshold of l - ltp induction observed in creb - vp16 mice was investigated . in wt hippocampal slices , even one train of tetanic stimulation was sufficient to induce l - ltp once l - ltp was induced in the other synapses of the same neuron by four trains of tetanic stimulation . this observation is thought to reflect the fact that the application of tetanic stimulation four times induces creb - mediated gene expression ; then , the resulting gene products are transported to the synapses of the stimulated neuron , even to those in which l - ltp was not induced . therefore , since the expression of creb target genes is significantly enhanced in creb - vp16 mice , one train of tetanic stimulation , as with four trains of tetanic stimulation , is thought to induce l - ltp . this hypothesis was supported by detailed analyses showing that the ltp induced in wt mice by one train of tetanic stimulation following four trains of tetanic stimulation has similar characteristics to that induced in creb - vp16 mice by one train of tetanic stimulation . furthermore , the increased expression of bdnf in creb - vp16 mice under the basal condition , i.e. , without any tetanic stimulation , has been shown to contbibute to the lowered threshold of l - ltp induction . consistently , four trains of tetanic stimulation failed to enhance ltp induced by one train of tetanic stimulation in creb - vp16 mice . these observations suggested that synaptic capture of bdnf is sufficient for induction of l - ltp by a single tetanic stimulation and provided potential molecular mechanisms of synaptic tagging [ 19 - 21 ] . creb - y134f ( y134f ) contains a mutation in which tyrosine is changed to phenylalanine at position 134 . this mutant protein displays increased affinity to pka ( a creb kinase ) , thereby leading to the lowering of the threshold for creb activation ; this mutation makes it easier for the protein to be activated . on the other hand , for creb - diedml ( diedml ) , six amino acids ( rrpsyr , which include s133 ) are replaced with diedml ( fig . 1 ) , the cbp - binding motif of sterol - responsive element binding protein ( srebp ) . transgenic mice were generated in which y134f or diedml is expressed specifically in the forebrain region under the control of the camkii promoter ( y134f and diedml mice ) . these mutant mice display higher expression levels of c - fos , a creb - target gene , than wt mice , indicating that the expression of these dominant active mutants leads to the enhanced activation of creb - mediated transcription . similarly with the results from the creb - vp16 mice , electrophysiological analysis using field recordings in a transgenic line with a high level of y134f expression ( line c ) confirmed enhanced l - ltp in hippocampal ca1 neurons . further analysis using the patch clamp method showed enhanced spike - timing ltp in the hippocampal ca1 neurons of y134f mice ( line c ) . taken together with the observations from the creb - vp16 mice , these results strongly suggest that creb functions as a positive regulator of ltp , even though these studies were not performed under similar conditions to those used for the creb - vp16 mice . in contrast to the results from the creb - v16 mice , behavioral studies showed that all of the transgenic y134 and diedml lines displayed improved ltm at 24 h in social recognition and fear conditioning memory tasks . furthermore , improved ltm was also observed in a spatial memory task using the morris water maze and a passive avoidance memory task . in a contextual fear conditioning task , wt mice display impaired contextual discrimination at one month after conditioning . these mice display fear ( freezing ) responses when they are exposed to a box that is novel , but similar to the original box in which they had received an electrical foot shock . however , they did not display such a high level of freezing responses in the novel box compared to the original box at one day after conditioning . in contrast , y134f mice ( line c ) , which highly express the creb mutant , could discriminate between the novel and familiar boxes , even at one month after conditioning ; mutant mice displayed significantly higher fear responses in the familiar context in which they received a foot shock compared to the novel context . these observations indicated that y134f mice formed more accurate ( stronger ) memory than wt mice . thus , the behavioral experiments using y134f and diedml mice showed that , in contrast to the results from creb - vp16 mice , the activation of creb significantly improved memory consolidation , indicating that creb functions as a positive regulator of this process ( fig . i compare the results of behavioral analyses among creb - vp16 , y134f , and diedml mice . in y134f and diedml mice , creb was activated only a few fold higher than in wt mice , whereas in creb - vp16 mice , since vp16 contains a strong transcriptional activation domain , the activation level was 20- to 30-fold higher than in wt mice . importantly , the dominant active creb mutants were expressed simply under the control of the camkii promoter in the y134f and diedml mice . in contrast , in the creb - vp16 mice , the expression of creb - vp16 is amplified using the tetracycline system . therefore , these comparisons suggest that the activation of creb - mediated transcription was unusually high in the creb - vp16 mice compared to the y134f and diedml mice ; y134f and diedml mice display enhanced creb - mediated transcription at a physiologically moderate level , while creb - vp16 mice do not . thus , although enhanced ltp was observed in the creb - vp16 and y134f /diedml mice , the levels of creb activation seemed to exert a strong influence on memory ; only y134f /diedml mice , which show moderate creb activation , display enhanced memory consolidation . interestingly , y134f and diedml mice demonstrated improved stm from 30 min to 2 h as well as ltm . especially , diedml mice , with higher creb activation levels than y134f mice , displayed improved stm at 30 min . these observations strongly suggest that stm is improved in a dose - dependent manner by creb activity . importantly , as stm is thought to be formed independently of new gene expression , it is suggested that creb plays a regulatory role in stm , but this enhanced stm is not mediated by the transcriptional activation of creb target genes immediately after training . expression analysis showed that y134f and diedml transgenic lines with enhanced stm display increased levels of bdnf in the hippocampus ; transgenic lines expressing higher levels of bdnf also exhibit enhanced stm . importantly , a micro - infusion of bdnf or a bdnf inhibitor into the hippocampus of wt mice generated enhanced or impaired stm , respectively . additionally , the infusion of a higher dose of the bdnf inhibitor into the hippocampus was required for the impairment of stm in diedml mice than in wt mice . these observations suggest that an increase in the expression levels of bdnf improves stm in y134f and diedml mice . from these findings , creb is thought to play a regulatory role in stm through the regulation of bdnf expression ( fig . 2 ) . on the basis of the analysis of y134f and diedml mice described above in addition , it is suggested that creb indirectly controls stm by regulating the expression levels of bdnf . previous studies have shown that the loss of creb function impairs memory consolidation and ltp . conversely , recent studies using mouse genetics indicated that the gain of creb function improves memory and ltp . taken together , these findings clearly indicate that creb positively regulates memory consolidation and ltp . furthermore , creb is suggested to play a regulatory role in stm though the activation of target gene expression such as bdnf .

camp response element - binding protein ( creb ) , a transcription factor , has been shown to play a central role in memory formation , and its involvement in this process has been investigated using a wide range of animal models , from nematodes to higher animals . various creb mutant mice have been developed and investigated . several types of mutant mice with loss of creb function have impaired memory formation and long - term potentiation ( ltp ) , suggesting that creb plays essential roles in these processes . to characterize the roles of creb in memory formation and ltp further , mutant mice displaying gain of creb function have been generated and analyzed . importantly , creb - diedml mice and creb - y134f mice showed enhanced memory formation , whereas creb - vp16 mice displayed a lowered threshold of long - lasting ltp ( l - ltp ) induction , strongly suggesting that creb functions as a positive regulator of memory formation and ltp . in this review , i focus on the effects of the genetic activation of creb in ltp and memory formation and summarize previous findings .

ROLES OF GENE EXPRESSION IN MEMORY FORMATION AND LONG-TERM POTENTIATION CREB, MEMORY FORMATION, AND LTP EFFECTS OF THE GENETIC ACTIVATION OF CREB ON LTP AND MEMORY FORMATION CREB-VP16 mice Y134F mice and DIEDML mice SUMMARY

although new bedford harbor ( nbh ) , ma , is one of the largest polychlorinated biphenyl ( pcb ) superfund sites in the united states , it has not been studied as an important source of airborne pcbs . in 1983 , nbh was placed on the national priorities list of superfund cleanup sites because of the extremely high levels of pcbs in the sediments . aroclors 1242 and 1016 were discharged into the harbor for more than 30 years ( 1940s1970s ) . pcb concentrations of 1 g g in flounder and 10000 g g ( dry weight ) in sediments have been reported . these high pcb levels motivated release of seafood consumption advisories starting in 1979 and sediment dredging since 1994 . concentrations of pcbs in air have been measured and reported by the u.s . environmental protection agency ( u.s . epa ) since 1999 , and although concentrations are elevated , the magnitude of the harbor as an emission source is unclear and the potential risk by inhalation caused by emissions is unknown . while remediation is driven by pcb levels in the sediments , pcbs are mobilized from sediment to overlying water and air , contributing to human exposure via inhalation . it is important to understand the specific contribution of nbh to local levels of airborne pcbs as part of the risk - based decision making regarding remediation . we recently developed a strategy for predicting concentrations of airborne pcbs as a function of emissions from contaminated water . we measured pcbs in water , calculated the emissions as gross volatilization flux , and predicted dispersion into the surrounding region . in northwest indiana , we found that pcbs released from the indiana harbor and ship canal ( ihsc ) accounted for 15% of the observed pcb concentrations in the adjoining neighborhoods . the community surrounding the ihsc is one of the most industrially dense regions in the united states and has a long history of environmental contamination . using this two - pronged strategy of calculated emissions and atmospheric dispersion modeling , we concluded that there were many sources of airborne pcbs in this region in addition to ihsc . pcb concentrations in nbh water are at least 10 times higher than in ihsc , and its area is 3 times larger than that of ihsc , which could dramatically increase the contribution of airborne gas - phase pcbs to the local atmosphere . therefore , we hypothesized that pcb emissions from nbh explain ambient air concentrations of pcbs in the surrounding communities . to address our hypothesis , in response to and in collaboration with community and environmental organizations , we launched a field effort in 2015 to evaluate the effect of emissions from nbh on airborne pcbs . we calculated congener - specific pcb emissions from nbh as a function of reported water concentrations , chemical properties , and local meteorology . we used an atmospheric dispersion model to predict gas - phase pcb concentrations in the region surrounding nbh . in addition , we compared our predictions to measured values from our own samplers and those measured by the u.s . lastly , we examined the long - term trends in pcb emissions and air concentrations for 20062015 using historical data reported by the u.s . epa . airborne pcbs were measured using polyurethane foam passive air samplers ( puf - pas ) as previously described . the puf - pas collects both gas and particle phases , but because pcbs are mostly in the gas phase ( 90% ) , the values reported here are assumed to be the gas phase . samplers were placed at the same 18 locations for three consecutive periods , from july to november of 2015 in new bedford , fairhaven , dartmouth , and acushnet , ma ( figure s1 ) , except for one location where we sampled two rounds . the sampling locations were selected by community members following discussions about study objectives and the need for the spatial distribution of pcb monitors . all but two selected locations are near residential homes . prior to placement , puf disks were cleaned in a soxhlet apparatus for 24 h with hexane , followed by 24 h with acetone , and finally with a 1:1 ( v / v ) hexane / acetone solution for 24 h. pufs were dried for 1 h in a ventilated fume hood , wrapped in combusted aluminum foil within ziploc bags , and stored at 4 c . hourly sampling rates ( r ) specific to each sampler and pcb congener were modeled from local meteorology , ranging from 2 to 3 m day . these sampling rates were used to calculate effective sampling volumes ( veff ) for each sampler and pcb congener , ranging from 25 to 110 m. we used these volumes to calculate the concentration of airborne pcb from the mass collected in the passive samplers . additional details , including the qa / qc for the airborne pcb measurements , airborne pcb emission calculations , air dispersion model aermod , and meteorological data utilized in this investigation , are provided in the supporting information . pcb concentrations in water and from high - volume air sampling were reported elsewhere , and details are also provided in the supporting information . concentrations of airborne pcb ranged from 0.4 to 38 ng m , with a geometric mean of 3.1 3.8 ng m. the values of > 10 ng m ( n = 9 ) are the highest values reported for outdoor pcb puf - pas samples by our laboratory ( chicago , cleveland , and east chicago ) and also by others ( toronto , on ) . epa large - volume air sampler ( hi - vol ) gas - phase measurements for the same months of 2015 ( mann whitney ; p = 0.32 ) ( figure 1 ) . u.s . epa high - volume ( hi - vols ) and puf - pas measurements for comparison of the gas phase . no significant difference was found between the methods ( mann whitney ; p = 0.32 ) . epa hi - vol samplers used in this comparison are given in figure 3 . a clear and large spatial variability was found , with the highest values located closest to the water ( figure 2 ) . indeed , no statistical difference was found among the three sampling period average concentrations ( kruskal wallis ; p = 0.83 ) . samples from the same location varied by a factor of <3 , whereas puf - pas samples in chicago varied in average > 6-fold for the same location . in addition , the highest value showed the lowest variability ( 10% ) , suggesting a single and constant pcb source . spatial and temporal distributions of airborne pcb ( nanograms per cubic meter ) in new bedford harbor . sampling round 1 ( green ) from july 9 , 2015 , to august 20 , 2015 , sampling round 2 ( blue ) from august 20 , 2015 , to october 1 , 2015 , and sampling round 3 ( yellow ) from october 1 , 2015 , to november 12 , 2015 . the inset shows the box - and - whisker plot for the concentration distribution of the three sampling periods . map source : office of geographic information ( massgis ) , commonwealth of massachusetts , massit . very similar pcb congener profiles were found in all the samples , dominated by low and middle chlorinated congeners ( < pentachlorobiphenyls ) . the majority of the congeners found in the samples are present in aroclor mixtures ( figure s4 ) . the samples have strong aroclor 1242 and 1016 profiles , consistent with numerous reports of sediment contamination with these two commercial mixtures and a consistent congener profile ( sample average cos = 0.97 ) . samples collected farther from nbh exhibit a larger relative contribution of non - aroclor pcb11 ( figure s5 ) . the pcb11 fraction in the samples exhibits one of the highest variabilities ( standard error ) of the 171 pcb congeners measured , suggesting that nbh is not the major source of this congener . these results suggest a single and continuous source of airborne pcbs , consistent with our hypothesis that the water of nbh is a large source of pcbs to the local atmosphere . pcb gross volatilization fluxes ranged from 160 to 1200 g m day for the 2015 samples . these fluxes result in pcb emissions from the upper and lower harbor areas ranging from 90 to 140 kg year , with an average of 110 kg year . the only previous pcb emission calculations for nbh were published by garton et al . using their data from 1983 , we were able to predict a gross volatilization flux of 70 g m day , which is on the same order of magnitude as the lower - end estimates reported here ( 2015 water data ) . nbh fluxes are higher than those of other well - known pcb - contaminated water systems in the united states . for example , gross volatilization fluxes from green bay in 1989 ranged from 0.2 to 5.3 g m day . the values for the hudson river estuary in 19992001 ranged from 0.05 to 0.9 g m day . the values for the delaware river in 20012003 ranged from 0.2 to 2.5 g m day . although it is very difficult to compare pcb fluxes between different studies , all these fluxes are at least 2 orders of magnitude lower than the 2015 nbh values . nbh fluxes are a result of the notably high concentrations of dissolved pcbs measured in nbh , which in some cases were in the hundreds of nanograms per liter ( table s1 ) . indeed , some values are only a factor of 100 below the water solubility limit . we excluded from consideration any water samples that were collected while there was active dredging , as indicated in the u.s . although some values are extremely high , a clear decrease in emission over time was observed ( figure s6 ) . this reduction is consistent with the observed decline in air and sediment pcb concentrations over time . aermod is an epa model for predicting the dispersion of airborne pollutants from a source , in this case nbh . we applied aermod to compare the measured pcb concentrations to those predicted as a function of our calculated emissions . the aermod predictions exhibit a pcb spatial distribution consistent with our field measurements and validate our hypothesis that nbh waters are the source of pcb in the nearby surrounding air ( figure 3 ) . aermod prediction map for mean pcb concentrations ( nanograms per cubic meter ) from july to november 2015 . circles represent our puf - pas samplers , and squares represent high - vol samples placed by the u.s . pink circles and squares represent the samplers used for comparison of methods ( figure 1 ) . both puf - pas and hi - vol samplers were active between july and november 2015 , although the puf - pas samplers were continuously collecting while the hi - vol samplers were sampling in 24 h periods . aermod predicted pcb within a factor of 2 of our puf - pas measurements 50% of the time ( 9 of 18 samples ) ( figure 4 ) . interestingly , all the locations where the prediction exceeded the measurements by a factor of > 3 were located close to the water and east of nbh . in general , the predictions exceeded our field measurements by an average factor of 2.6 , ranging from 0.5 to 11 . epa has been monitoring airborne pcbs in nbh since 1999 using hi - vol samplers . their hi - vol measurements for the same period of time were somewhat closer to the predicted concentrations with an average factor of 2.1 , ranging from 0.7 to 5 , with only one location with an average factor of > 4.0 . given that the range of pcb concentrations is large and a factor of 100 , we conclude that the modeling approach we used is appropriate and accurately predicts the effect of nbh emissions on ambient pcbs in the air of the surrounding communities . circles represent mean puf - pas measurements ( n = 3 ) , and triangles represent geometric means of hi - vol measurements from the u.s . error bars represent one standard deviation for the puf - pas samplers and one geometric standard deviation for the hi - vol samplers . the black line represents the 1:1 line , and the red lines represent the 1:2 and 2:1 lines ( i.e. , factor of 2 ) . the results of this study support our working hypothesis : pcb emissions from nbh explain nearby air concentrations . first , we note the large spatial variation in measured airborne pcbs , with much higher concentrations close to the shoreline . second , we found that the profiles of pcb congeners in the air samples are remarkably similar , and also similar to those of the commercial mixtures aroclor 1016 and aroclor 1242 . third , we found that our predicted and measured air concentrations exhibit similar ranges of values and similar spatial distributions , both decreasing in magnitude with distance from the water . to the best of our knowledge , this is the first study to show that a pcb - contaminated waterway is responsible for the nearby measured pcbs . it is likely that pcbs have been emitted from nbh water for many years . using the same modeling approach , we predicted airborne pcb concentrations from u.s . our calculations illustrate a decrease in pcb emissions as well as airborne pcb concentrations ( figure 5 ) . our findings indicate that nbh is one of the largest ongoing sources of airborne pcbs in the united states and the cause of the highest concentrations in the neighborhoods surrounding the harbor . it is likely that nbh has been an important source of airborne pcbs in the new bedford area since it was contaminated with aroclors . asterisks indicate a significant difference between 2006 predicted concentrations and 2011 and 2014 ( mann whitney ; p = 0.004 ) .

qualitatively and quantitatively , we have demonstrated that airborne polychlorinated biphenyl ( pcb ) concentrations in the air surrounding new bedford harbor ( nbh ) are caused by its water pcb emissions . we measured airborne pcbs at 18 homes and businesses near nbh in 2015 , with values ranging from 0.4 to 38 ng m3 , with a very strong aroclor 1242/1016 signal that is most pronounced closest to the harbor and reproducible over three sampling rounds . using u.s . environmental protection agency ( u.s . epa ) water pcb data from 2015 and local meteorology , we predicted gas - phase fluxes of pcbs from 160 to 1200 g m2 day1 . fluxes were used as emissions for aermod , a widely applied u.s . epa atmospheric dispersion model , to predict airborne pcb concentrations . the aermod predictions were within a factor of 2 of the field measurements . pcb emission from nbh ( 110 kg year1 , average 2015 ) is the largest reported source of airborne pcbs from natural waters in north america , and the source of high ambient air pcb concentrations in locations close to nbh . it is likely that nbh has been an important source of airborne pcbs since it was contaminated with aroclors more than 60 years ago .

Introduction Materials and Methods Results and Discussion

endobronchial fibroepithelial polyps are a rare neoplasm with only a few case reports in the english literature ( 1 - 4 ) . histologically , these lesions are composed of collagen fibers in a stroma covered by normal respiratory epithelium ( 3 ) . we report a case of a fibroepithelial polyp of the bronchus , of which a lobulating contour is well visualized at computed tomography ( ct ) . however , a non - contrast chest ct scan ( brilliance 6 , philips healthcare , cleveland , oh , usa ) revealed a 1 cm , endobronchial polypoid nodule abutting the anterior wall of the left main bronchus , which had a soft - tissue attenuation of 40 hu . the endobronchial nodule showed a conspicuous lobulating contour that resembled an appearance of a blackberry ( fig . at bronchoscopic examination , a lobulated endoluminal nodule with a glossy surface was arising from the anterior wall of the left main bronchus , resulting in significant luminal narrowing . the polypoid nodule was removed using an electrosurgical snare during rigid bronchoscopy under general anesthesia . at gross examination of the polypectomy specimen , the tumor measured 1.2 1.0 cm in diameter with a lobulating contour and a glossy surface ( fig . histopathologically , the nodule showed marked papillary projections , which resulted in the gross morphology of a lobulating contour of the polyp ( fig it consisted of fibrovascular stroma covered by normal respiratory epithelium , enabling the histological diagnosis of a fibroepithelial polyp ( fig . a fibroepithelial polyp is a common type of tumor in the skin or the genitourinary tract . especially , it is the most common benign tumors of the ureters ( 5 - 8 ) . however , fibroepithelial polyps of the bronchus are rare with only a few case reports in the english literature ( 1 - 4 ) . according to these case reports , it manifests as an endobronchial polypoid nodule that is covered with normal respiratory mucosa , consisting of fibrovascular stroma with or without few inflammatory cells and adipocytic components ( 1 - 4 ) . some reports have classified fibroepithelial polyps as inflammatory , while others have argued that the inflammation is a result of the intermittent respiratory tract obstruction caused by its mass effect ( 4 ) . histologically , fibroepithelial polyps of ureters are known to be predominantly composed of urothelial epithelium and fibrovascular stroma , showing a finger - like or polypoid growth pattern ( 8) . such a papillary growth pattern on histology may result in a lobulating contour of the nodule on gross morphology , which can be readily identifiable on radiological images . the gross appearances of fibroepithelial polyps arising in the tracheobronchial tree and the ureter described in the previous case reports are summarized in table 1 . the majority of appearances of the polyps suggested their lobulating contours , and many of those figures actually shown in reports revealed quite a similar appearance of the nodules to that of our case . although these appearances had not been emphasized in those reports , the lobulating contour of the polyp was clearly identified on ct images in our case , and this feature had been derived from a typical papillary growth pattern of the fibroepithelial polyp . it has been known that an endobronchial nodule can develop in a variety of neoplasms including squamous cell carcinoma , adenoid cystic carcinoma , mucoepidermoid carcinoma , neurogenic tumor , leiomyoma , hamartoma and lipoma , and so on . therefore , we think that an awareness of the implication of such a lobulating contour of an endobronchial tumor might be important in the differential diagnoses of various tracheobronchial polypoid nodules . in summary , we report a case of a fibroepithelial polyp of the bronchus in which a lobulating contour of the endophytic nodule was well identified at ct scan , and we believe that its lobulating shape represents a typical papillary growth pattern of the tumor on histopathology . although endobronchial fibroepithelial polyps are a rare neoplasm , such a characteristic lobulating contour of the polyps might be helpful in the differential diagnosis from other various endobronchial polypoid nodules .

a fibroepithelial polyp of the bronchus is a rare , benign , and endobronchial tumor , histologically consisting of fibrovascular stroma covered by normal respiratory epithelium . we report a case of a fibroepithelial polyp arising from the left main bronchus . on ct , a characteristic lobulating contour of the endobronchial nodule was well visualized , which histopathologically represented a typical papillary growth pattern of the nodule . such a lobulating contour of the nodule might help make a correct diagnosis of this rare disease among other various endobronchial neoplasms .

INTRODUCTION CASE REPORT DISCUSSION

multiple sclerosis ( ms ) is an immune - mediated disease of the central nervous system with a relapsing - remitting course in the majority of the early stages of the disease . as for other multifactorial diseases this limits the opportunities provided by the advancements in genetics , immunology , and neurobiology since it is difficult to contextualize each single discovery . the uncertainties in the interpretation of genome - wide association studies ( gwas ) reflect , to some extent , this problem . these studies carried the expectation to define the heritable component in multifactorial diseases and , through this , also sketch the nonheritable ( environmental ) contribution to the phenotype . missing heritability in multifactorial diseases , also this powerful approach appears to be in need of interpretative keys as neither genes nor the environment seem to harbour factors that , alone or jointly , are strong enough to explain the disease etiology [ 2 , 3 ] . likewise situations are rather common in the physics of nonlinear systems ; here the observed large variations are explained through the effects induced by small random perturbations [ 46 ] . an example is the theory of the earth 's climate : the cooperative effect of a small stochastic perturbation and periodic forcing ( variation of astronomical parameters ) provides an amplification of the climate response known as stochastic resonance that leads to the transition from a temperate climate to an ice - covered earth state and vice versa [ 57 ] . such variation can arise from random fluctuations in gene expression [ 8 , 9 ] , and in fact the term gene expression noise is typically used in broad reference to variations among seemingly identical cells experiencing the same environment . noise in genetic circuits can have profound implications also in multicellular organisms enabling physiological regulation mechanisms , differentiation strategies , and facilitating adaptation and evolution , often with a qualitatively different outcome than a deterministic one . as such , these discrete and random fluctuations in the expression of individual genes may also participate in the transition between health and disease by exposing hidden genetic and environmental risk . we show that , in ms , transitions between remissions and relapses ( i.e. , states of relative health and disease , resp . ) indeed occur randomly suggesting that stochastic events may contribute to disease pathogenesis . this is described in a model that accommodates the erratic course of the disease and concedes that soft heritable and nonheritable perturbations can be amplified over time . the model is based on the equation for a double - well forced by a random perturbation ( noise ) . we assume that the patient has two states , disease ( relapse ) and health ( remission ) , and that the barrier to overcome for going from one state to the other is determined by his genetic background and environmental exposures . the small random perturbation is thought to be the inherent variability in gene expression that triggers the erratic sequence of relapses . in the likely hypothesis that the mechanisms leading to the transition between remission and relapse recapitulate those that are responsible for the transition between health and disease at onset , time and noise appear as crucial variables that can amplify gene - environment interactions and effects until , at one time or the other , the transition towards the disease state occurs in any susceptible patient , we used a dataset of relapses and remissions of 70 patients ( 28 males and 42 females ) with definite ms [ 14 , 15 ] who were monitored prospectively along the years at the ms clinic of sapienza university of rome ( italy ) by four experienced ms neurologists . all patients were free of any disease - modifying therapy ( all data are antecedent 1993 ) and had received short courses of corticosteroids if deemed necessary during relapses . times to disability end - points were not different between this cohort and published data on the natural history of the disease . the period of interest starts with the first relapse at onset and ends with the last one before the shift to a secondary progressive form . a relapse was defined as the occurrence of new symptoms , the reappearance of former ones , or the worsening of current symptoms of at least 24 hours duration but less than 6 month . its duration was calculated as the interval between onset of the first symptom or sign and maximum improvement of the last symptom or sign . as part of the standard procedures of specialized ms clinics , which require to firmly establish the presence or absence of disease activity and progression in order to ensure the most appropriate therapeutic interventions , being this a analysis of anonymous clinical data ( collected until 1993 , before the institution of ethics committee in italy in 1998 ) , stored for both clinical and research purposes in the database of the university hospital , ethics committee approval and written informed consent of patients are not needed ( http://www.garanteprivacy.it/garante/doc.jsp?id=1884019 ) . each subject has been represented by a sequence of plus one ( + 1 ) and minus one ( 1 ) corresponding to the states of relapses and remissions , respectively . for clarity , events are reported on a weekly scale ( shorter exacerbations have been rounded up to one week ) . first , the dataset is analyzed to study the time evolution of relapses , their time duration , and their statistical distribution . secondly , a simple model consisting of a mechanistic component and a stochastic perturbation is introduced to represent the main characteristics observed in the clinical histories of patients . the mechanistic component provides the deterministic ( predictable ) component of the model , while the stochastic perturbation describes an external stimulus whose behaviour is intrinsically non - deterministic ( random ) and that can be analyzed only in probabilistic terms . the latter is usually called stochastic forcing since it acts as a force that perturbs the deterministic component , making the model solution unpredictable . the underlying hypothesis of the model here proposed is that the health state of a patient in time behaves likewise the motion of a particle of unit mass in a double - well energy potential as schematically illustrated in figure 1 . now , given any initial position of the particle and if no perturbation is applied on it ( i.e. , under deterministic conditions ) , the particle will fall in one of the two potential wells and will remain there for infinitely long time , independent of whether the well is deep or flat ( figure 1(a ) ) . if , instead , there is a strong stochastic perturbation acting on the particle , it will randomly move forth and back between the two wells , independently of their depth . the most interesting situation for us occurs when there is a small / moderate stochastic perturbation acting on the particle : the particle will typically stay for some time in the well it occupies , until the random diffusion drives it over the potential barrier into the other well ( figure 1(b ) ) . it is intuitively clear that the exit from a flat well happens faster than the exit from a deep well . in the phenomena of diffusion , like the one just described , the random perturbation is represented by the wiener process , which consists of a normally distributed white noise ( i.e. , the amplitude of the noise is normally distributed with given mean and standard deviation , and all frequencies are involved with no time periodicity ) . we note that the noise in physical systems was considered for a long time just as a nuisance that degrades the signal . later on it was found that , in complex ( nonlinear ) processes , as the two - state process here considered , it may introduce a longitudinal dependence corresponding to the erratic switching between the steady states that , otherwise , would be persistent forever . this property of noise should be distinguished from the mechanism of stochastic resonance introduced to explain the cyclic recurrence of ice ages and that is based on the combined effect of noise and a periodic external forcing . in that case , useful applications have been found in physical , technological , and biomedical contexts ( [ 1720 ] and references therein ) . coming back to the metaphor particle - patient , the noise may represent small biological variations of random character experienced by the patient at each time . the bottom of the two wells represents the health and no health states , while the height of the wells is the barrier to be overcome for going from one state to the other , here supposed to be determined by the genetic background and environmental exposures of the patient . only the effect of noise over time can let the patient change his state of health into disease and be subjected to random relapses . the time series suggest the unpredictable nature of relapses ( + 1 states ) followed by periods of relative quiet remission with no new signs of disease activity ( 1 states ) . also , the time in a health state seems much longer than the one spent in a disease state . it is worth noting that the transition between the two states , by construction , is here represented by a step function ( abrupt change of state ) . such a choice can be reductive or unrealistic since patients usually report a slow or subacute onset of the relapses . as shown in figure 3 , the duration of the relapsing - remitting phase ( i.e. , period of analysis ) varies from one patient to the other , from a minimum of 40 weeks to a maximum of 1311 weeks ( about 27 years ) . as illustrated by figure 3 , for 25 of 70 patients ( about 36% ) the relapsing - remitting phase lasts for about 200 weeks ( about 4 years ) , and moving towards longer periods the percentage of interested patients quickly decreases to zero ( exponential decaying distribution ) . similarly , the distributions of the time duration ( or exit time ) of relapses and remissions follow exponential decaying behaviours ( figures 4(a ) and 4(b ) ) . the mean duration of disease attacks is about 4.3 weeks , with a minimum of 1 week to a maximum of about 24 weeks ( rare events ) . the mean time spent by patients in the health state is instead about 100 weeks , with a minimum time interval between two consecutive disease events of a few weeks up to about 1000 weeks in exceptional cases . it is worth noting that the absence of any peak in the distributions at some specific time corroborates what has been noted before for sample patients ( figure 2 ) : disease events occur randomly in time . moreover , the mean duration of disease events is much shorter than that of remission . in the following , we will show how these features can be taken into account in a simple mechanistic model forced by random perturbations . the aim is to contribute to the understanding of the mechanisms that underlie the disease , highlighting the role of noise in a system ( patient ) that is primarily governed by mechanistic laws . the model to simulate the random transitions between the health and no health states ( 1 and + 1 , resp . let x be the health state of a patient ( the position of the particle in our metaphor ) . for a two - state process , as the one we are interested in , the time evolution of x is given by the following equation : ( 1)dx = x(1x2)dt , where t is the time , is a control parameter , and dx and dt are the infinitesimal variation of the health state and of time , respectively . note that in case = 1 , we have x1 = 1 and x2 = 1 . now , in agreement with the convention used for clinical data , let x1 be the state of health and x2 the state of no health . the potential v , associated with ( 1 ) can be easily computed as , ( 2)v(x)=x(1x2)dx=12x2+14x4 . it can be noted that v(x ) is a symmetric function with respect to a change of sign of the x variable ( it is the same for x greater than zero or x less than zero ) , describing a double - well . for clarity , we show in figure 5 the potential v as a function of x for two values of the control parameter : = 1 and = 0.7 . it should be noted that the difference of the potential in x1 ( x2 ) and x0 ( denoted by v in the figure ) is the barrier that must be overcome to jump from one state to the other ; it increases when is decreased with respect to the reference value = 1 ( the opposite occurs when is greater than 1 ) . in the context of ms , such a barrier is though to be set by the combination of heritable and nonheritable risk . thus , according to ( 1 ) and ( 2 ) , we haveif , by any chance , at initial time the patient is nearby x1 , he will be there forever;if , by any chance , at initial time the patient is nearby x2 , he will be there forever;if , by any chance , at initial time the patient is nearby x0 , he will have 50% chance to be forever nearby x1 or x2.now , as shown by clinical data ( figure 2 ) , the patient ( x ) stays a longer time in the health state compared to the no health state . this feature suggests that the depth of the two wells differs and , in particular , the one associated to the health state is deeper than the other , so that it is more difficult to exit from the health condition than from the relapse . in our mathematical model , this implies the introduction of an asymmetry in v(x ) , as ( 3)v(x)=[x(1x2)]dx=12x2+14x4+x , where the parameter determines the magnitude and shape of the asymmetry . if , by any chance , at initial time the patient is nearby x1 , he will be there forever ; if , by any chance , at initial time the patient is nearby x2 , he will be there forever ; if , by any chance , at initial time the patient is nearby x0 , he will have 50% chance to be forever nearby x1 or x2 . figure 6 illustrates an example of asymmetric double - well ( = 0.08 ) for = 1 and = 0.7 . in figure 6 , the barriers are denoted as v1 and v2 and are the difference in the potential between the states x1 and x0 , and x2 and x0 , respectively . in all the three circumstances above , such barriers separate the states x1 and x2 , preventing the switching between the wells . now the question is : can some external random stimuli provide enough energy for diffusing x across the barriers , regardless the initial conditions ? unless new risk factors are discovered , those identified so far in ms appear unsuitable as deterministic triggers of continuous and random transitions between health and disease , inducing very different disease courses not only in different patients but also in the same patient at different time points . as noise is a source of variability in biological systems , we tried to model it as a driving force . let us suppose that on average the noise has zero effect ( i.e. , its time mean is zero ) , has a given variance , and it is time decorrelated ( i.e. , at a given time the value of the stimulus depends only on the previous time ) : this is the zero order approximation of the statistics of noise ( i.e. , the wiener process previously discussed ) . by introducing such external random perturbation into ( 1 ) , we have ( 4)dxchange of the healthstate of a patient=[x(1x2)]dtmechanistic component(double - well ) + 1/2dw , external randomstimuli where dw is the noise of variance . because of the external random stimuli , this equation has important deviations from its deterministic version described by ( 1 ) : now the patient can change randomly his state from health to no health and vice versa . averaged over time it is impossible that x crosses the barrier since the noise , by construction , has zero mean . however , given the statistical property of the stimulus , in due time , we expect that a sequence of small driving occurrences may be equally signed so that x may overcome the barrier and switch from one state to the other . in other words for the sake of clarity , the relationship among disease characteristics and model parameters are summarized in table 1 . as we shall see , this model fits nicely the statistics of the exit times of disease events and explains a few paradoxes encountered in understanding the disease course . to better understand the model , let us look at the solutions of ( 4 ) for different values of the parameters . setting = 1 , the solution of ( 4 ) as a function of time is shown in figure 7 for the symmetric and asymmetric double - well cases ( = 0 and = 0.08 , resp . ) . according to our metaphor , the patient alternates periods of wellness and disease randomly and , as expected , when a small asymmetry is taken into account , the patient spends more time in the health state than in the no health state . this means that there is a finite probability that for any initial condition nearby x1 ( or x2 ) the patient will jump across x0 at a finite time ( i.e. , time duration in a given state ) , switching from one state to the other . as seen from figure 4 , the mean value of in the health state ( x1 ) can be estimated and is given by ( 5)x1e2v1/ while in the no health state ( x2 ) is ( 6)x2e2v2/ , where e is the nepero number . it is interesting to note that , knowing the mean values of duration times in the health and no health states , it is possible to estimate the ratio between v1 and v2 , say the asymmetry of the double - well . for the dataset discussed in the previous section , we have x1 100 weeks and x2 4.3 weeks . on the other hand , taking the logarithm of ( 5 ) and ( 6 ) , and performing their ratio thus , the ratio of the logarithms of duration times in the states x1 and x2 provides an estimate of the ratio between the barriers that must be overcome to move from one state to the other . by considering the duration times x1 and x2 averaged over all the cases of 70 patients it is found v1/v2 3.1 . this implies that the well in x1 is about three times deeper than that in x2 . we point out that the ratio between the depths of the wells can be estimated for each patient , provided that the mean duration times of the two states are known from the clinical history . as an example , we have estimated such a ratio for the three sample patients considered in figure 2 . we found x1 117.7 weeks , x2 1.5 weeks , v1/v2 11.8 , for the patient no . 23 ; x1 54.0 weeks , x2 2.1 weeks , v1/v2 5.3 , for the patient no . 32 ; x1 47.0 weeks , x2 4.3 weeks , v1/v2 2.7 , for the patient no . this stage , the potential v associated with each patient can be estimated by fixing = 1 and changing so that the two wells are asymmetric in the predetermined ratio . plots of the three potentials are shown in figure 8 . by assuming that random stimuli of the same variance are applied to the three patients , the shapes of the potentials reflect the differences in the duration times of health and disease states observed in figure 1 . the first potential has a very deep well associated with the health state and a very shallow well associated with the no health state : this configuration lets the patient be affected by a few disease attacks of short duration . given the same observation period , the patient no . 32 is expected to have more attacks compared to the previous patient due to the difference in the depth of the wells . 32 should experience disease events lasting more time because of the deeper well associated with the no health state . x1 117.7 weeks , x2 1.5 weeks , v1/v2 11.8 , for the patient no . 23 ; x1 54.0 weeks , x2 2.1 weeks , v1/v2 5.3 , for the patient no . 32 ; x1 47.0 weeks , x2 4.3 weeks , v1/v2 2.7 , for the patient no . 53 . the contribution of the heritable or non - heritable factors that drive ms onset and course appear uniformly too small to explain its etiology , variable severity , and erratic manifestations . we present a mechanistic , stochastic model that describes how the effects of the above forces may be randomly amplified over time . given a time of observation t ( e.g. , the average lifespan of a person ) , patients may experience a transition between the two states with a mean time . thus , the probability to observe a transition requires that t is much longer than . however , since is randomly distributed , by mere chance , the transition may or may not occur within the observation time t. within this approach , time is conceived not only as a random variable characterizing disease events but also as the observation time needed to detect the onset of the disease in any susceptible patient . stochastic noise in gene expression , through its pervasive effects on virtually all biological processes , may be the factor that amplifies and reshapes the deterministic effects of genetic and environmental risk factors . the model is in accord with a variety of data in ms and may be general enough to explain features of other complex traits . first , time is a key variable in this model but also in experimental autoimmunity where there is evidence of a multistep process made of subtle alterations resulting from quantitative trait loci variations that may accumulate with time and end up in susceptibility when a threshold for the occurrence of the disease is passed [ 21 , 22 ] . furthermore , defects of the immune response that originate from predisposing genetic variants are present before any onset of autoimmunity . these relatively recent observations are in accord with classical epidemiological data suggesting that fairly long time intervals are a necessary factor for the development of ms [ 24 , 25 ] . it is therefore not surprising that , if time is a crucial factor to understand the nature of the disease in diagnostic terms , it may also be instrumental for the pathogenetic understanding of the process . second , our model links the degree of genetic variation with the probability that the transition between health and disease states occurs over time . in fact , ms risk alleles influence age of onset and correlation of age of onset in ms relative pairs is proportional to genetic sharing . interestingly , the model applies also when individuals share genetic protection as witnessed by our observation of an underrepresentation of ms among italian twins , possibly due to protective factors shared by twins in a mediterranean area . furthermore , as expected , a link between genetic variation and transition between the two states over time seems to be maintained during the disease course : with one exception , the vast majority of the studies on the natural history of ms has shown that , while the occurrence of relapses is unpredictable , their frequency and severity tend to be conserved as a longer first interattack interval is associated with a better prognosis [ 3134 ] . while this is the first time that a model combining deterministic factors and stochastic forcing is applied to the interpretation of a multifactorial disease , determinism and stochasticity have been already shown to fit basic biological mechanisms ( [ 812 ] and references therein ) . here , stochasticity is beneficial to the individual , the colony , or the species in that it may exert a physiological regulatory role introduced to explain earth 's glaciations and sometimes erroneously associated with the random transitions illustrated above , the model solution is driven by the cooperative effect of the stochastic perturbation and the small periodic forcing , leading to a flip from one state to the other with the frequency proper of the external periodic forcing ; the small stochastic perturbation provides the energy necessary for the transition , while the periodic forcing sets the periodicity of the transitions . the stochastic resonance occurs for low frequency of the periodic forcing , say for long time periods . for climate changes that induce glaciation cycles , such a period is set to 100000 years that describes the long - term variation of the energy input prescribed by astronomical theory . in the case of relapsing - remitting ms , as clinical data suggest , there is not a typical periodicity of relapses induced by some external factors . however , we can imagine to exploit a small external cyclic component in order to stabilize the solution around a given state ( the health state ) deamplifying it with some short periodicity , contrary to what happens in the stochastic resonance , in order to prevent relapses . a nonmutually exclusive approach may be to decrease the variance of the noise ( possibly acting on fluctuations of gene expression ) . this study offers a new interpretative key of the events that characterize the clinical course of ms . by inference , it also suggests that time and noise amplify and adjust the deterministic influence of genes and environment on the disease . it will not be easy to obtain direct experimental evidence for this model at the disease level . however , basic science data increasingly supporting the notion that noise provides critical functions at the cell level suggest that this effort will probably be a rewarding one . thus far the model may contribute to the understanding of phenotypic variation in health and disease states . despite the advent of genomewide association studies ( gwas ; an approach to look for associations between hundreds of thousands genetic variations and diseases ) , many questions remain about the causative mechanisms of multifactorial diseases . in particular , the effect size of the genetic variants identified through these studies explains relatively little of the heritability ( the proportion of total variance in a population for a particular measurement , taken at a particular time or age , that is attributable to variation in genetic values ) of most complex traits . multiple sclerosis is one of the diseases that best exemplify this key problem . by observing the course of this disease in its relapsing - remitting phase we found that a mechanistic model with a random forcing describes the main features of the disease course . if the model is applied to the events that precede the disease ( i.e. , those that derive from the effects of the genetic and environmental risk factors ) , it may explain how relatively small effects may be amplified by comparably small random perturbations . as such , the model may inform the design of future studies on gene - environment interactions in multifactorial diseases .

heritable and nonheritable factors play a role in multiple sclerosis , but their effect size appears too small , explaining relatively little about disease etiology . assuming that the factors that trigger the onset of the disease are , to some extent , also those that generate its remissions and relapses , we attempted to model the erratic behaviour of the disease course as observed on a dataset containing the time series of relapses and remissions of 70 patients free of disease - modifying therapies . we show that relapses and remissions follow exponential decaying distributions , excluding periodic recurrences and confirming that relapses manifest randomly in time . it is found that a mechanistic model with a random forcing describes in a satisfactory manner the occurrence of relapses and remissions , and the differences in the length of time spent in each one of the two states . this model may describe how interactions between soft etiologic factors occasionally reach the disease threshold thanks to comparably small external random perturbations . the model offers a new context to rethink key problems such as missing heritability and hidden environmental structure in the etiology of complex traits .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

polycystic ovary syndrome ( pcos ) is a heterogeneous disorder characterized by chronic anovulation and the cutaneous effects of hyperandrogenism [ 13 ] . there is a high prevalence of pcos in women with type 2 diabetes mellitus , and there is evidence that insulin resistance and associated compensatory hyperinsulinaemia play a central role in the pathogenesis of pcos in some women [ 47 ] . the metabolic aspects of this syndrome include insulin resistance , obesity , lipid abnormalities , and an increased risk for impaired glucose tolerance and type 2 diabetes mellitus [ 79 ] . hyperinsulinaemia and insulin resistance are more common among women with pcos than women without the condition regardless of whether they are obese or not [ 1 , 8 ] . high insulin levels can increase the amount of androgens produced by ovarian theca cells in synergism with lh . it has been suggested that hyperandrogenism and insulin resistance occur early on in life and might even originate during foetal life and detected clinically before puberty [ 9 , 10 ] . a potential mechanism for insulin resistance in pcos women increased serine phosphorylation of the insulin receptors . serine phosphorylation of irs-1 ( insulin receptor substrate-1 ) has been suggested as a mechanism for tnf--mediated insulin resistance and modulated the activity of the key regulatory enzyme of androgen biosynthesis , p450c17 in the adrenal and ovarian steroidogenic tissues [ 4 , 6 , 8 ] . the high prevalence of pcos observed in certain ethnic groups also seems to be identified with a high risk of insulin resistance , and certain drugs used to improve insulin sensitivity have been included in the treatment for these women [ 6 , 7 , 11 ] . these drugs include metformin and the glitazones , which have had good results in controlling insulin resistance in patients with the syndrome [ 1216 ] . new studies would be of utility for research into new drugs to increase another therapeutical possibilities . diamel is a well - known product as a vitamin supplement used to treat type 2 diabetes mellitus . it can improve insulin sensitivity and the -cell function in people suffering from this disease . it could be an alternative therapy for women with pcos if it can be proved that it is capable of decreasing insulin resistance in these patients and improve the performance of insulin produced by pancreatic cells . we decided to conduct research on women diagnosed with pcos and evaluate the possible effect that this supplement has on the insulin sensitivity index , and to find out how the gonadotropins and testosterone levels in these patients react to the treatment . the studies carried out on the experimental and preclinical models showed that this vitamin supplement improves glucose tolerance , and it does not contain any toxic elements [ 17 , 18 ] . the product is approved by the national institute of nutrition and food safety and although earlier tests with diamel have not been carried out on patients with pcos , favourable results in diabetics with insulin resistance have been obtained in which a decrease in the triglyceride levels was observed along with an increase in hdl - cholesterol and an improvement of homa - ir and homa - b indexes in a 6-month period . this implies that it could have a favourable response in women with pcos and insulin resistance . diamel contains trace elements , amino acids , vitamins , lettuce extract , and cranberry extract , which are activated by means of a molecular magnetization process . the laboratory researchers who produce it state that it is designed to stimulate the pancreatic metabolism with its natural activated components that work like biocatalysts . the secret to its effectiveness lies in molecular activation process of the natural ingredients that it contains . moreover , if we take into account the fact that the stability of human organic systems is progressively deteriorated due to the harmful chemical reactions that cause this cellular oxidative stress , antioxidants that it contains could prevent or delay such reactions . after the molecular activation process developed by the catalysis laboratories , a biophysical analysis was carried out on the electric conductivity and the thermal stability of different products , including diamel , whose molecules and components were seen to be powerful reagents [ 17 , 18 ] . it is worthwhile mentioning the fact that even though no previous studies have been published about the diamel and pcos , but some gynaecologists from kuwait have used this supplement on patients with pcos , and they have observed an improvement in their menstrual disorders with ovulation recovery ( personal data ) . therefore , this paper would be the first documented study to provide results to actually consider using the supplement . we aimed at identifying the response of women with the pcos in treatment with diamel for to evaluate insulin sensitivity and sexual hormones results . vaginal ultrasound was performed in all subjects , and polycystic ovaries were diagnosed if ovaries were enlarged with more than 1012 small antral follicles of 28 mm diameter with the typical cortical arrangement . one hundred four women with pcos were recruited consecutively during two years , and those that presented homa - ir index greater than 2.6 were included in the investigation.a phase iii triple - blind , placebo - controlled , random clinical trial was carried out with placebo that had similar characteristics as those of diamel . 60 boxes of vials were handed out , numbered 1 to 60 , according to the random number chart . one separate vial was assigned to each patient ( 30 of which correspond to diamel and 30 to the placebo ) . neither the researchers nor the patients knew whether the a or b vials contained the diamel or the placebo capsules . at the end of the 6-month follow up and so as to statistically process the results , the first sealed envelope was opened . it contained information about the random distribution of the corresponding vial numbers for group a and group b. the identification of these vials was kept secret in another second - sealed envelope that was kept by an intermediary who was not involved in the research programme . this was handed over once the study with the statistical process had been completed to identify the vials . the envelopes and the administration of the diamel or placebo were monitored by a nurse who was not directly involved in the study . this person was in charge of giving the researchers the corresponding envelope for each patient included in the trial . until all the statistics had been processed , the link between the groups a and b and the placebo and diamel were not revealed . diagnostic criteria . including criteria are the following : women in the reproductive stage ( between 18 and 35 years old ) who have been diagnosed with pcos according to rotterdam criteria , homa - ir greater than 2.6 , and women who have not been treated by pcos beforehand . excluding criteria are the following : patients with type 2 diabetes mellitus , patients with cushing 's syndrome , congenital adrenal hyperplasia due to a 21-hydroxylase deficiency , virilizing ovarian tumor , ovarian cysts that are bigger than 10 mm . they are the follows : serious adverse effects related to the supplement , patients who refused to carry on with the study and decided to leave , development of some of the processes or entities that are established in the exclusion criteria , and pregnancy ( this will be recorded as being a favourable response to the supplement ) . members of both groups were trained about the change of their life styles on their diet , and an increase in physical activity through walks or supervised exercises at least 5 times a week was recommended . the maximum maintenance dose of diamel was 3960 mg ( 6 capsules ) divided out into 2 capsules before breakfast , midmorning snack , and lunch were prescribed , based on the previous experience of the recommended therapeutic dosage . before the study began , to each patient was made a transvaginal ultrasound in order to need anatomical characteristics the ovaries . fasting blood samples were drawn during the morning in follicular phase ( cycle days 35 ) ; patients with cycle length > 3 months had the blood samples drawn on a random cycle day . all the laboratory measurements and indexes were recorded at the beginning , at 3 months and at 6 months of the follow up , as follows fasting blood glucose levels , fasting insulin levels , testosterone , lipids ( cholesterol , triglycerides , hdl - cholesterol and ldl - cholesterol ) , postinhibition of basal cortisol with 1 mg of dexamethasone . at start study ( point 0 ) , at 3 months and at 6 months , blood glucose and insulin levels were taken in the morning , then after approximately 1012 hours of fasting , three samples were taken every 5 minutes to measure the homa - ir , established by wallace and colleagues [ 20 , 21 ] . moreover , raynaud 's index was used to determine insulin sensitivity [ 22 , 23 ] . homa - ir : insulin resistance index : ( 1)homa - ir = fasting insulinemia ( u / ml)fasting glucose ( mmol / l)22.5 . raynaud : insulin sensitivity index : ( 2)raynaud=40fasting insulinemia ( u / ml ) . normal sensitivity was considered to be at the cut - off point equal or less than 0.33 . the results were shown as means sd , and two - tailed fisher 's exact test was used to determine differences between groups . for menstrual pattern data and homa - ir and raynaud indexes results comparisons within and between groups , nonparametric test ( kruskal - wallis h. test and the mann - whitney u test ) were used . nonparametric tests were also used to examine treatment effects at start , 3 months and 6 months ( within and between groups ) . this form outlines the objectives of clinical trial and details of such . by signing the form , the subject expresses their willingness and agrees to take part in this research programme . figure 1 is a data - flow diagram on screening and subsequent follow up of the patients ; we took two years to find 104 women with pcos . two patients from group a ( diamel ) and another 3 from group b ( placebo ) left the study by different causes . figure 1 shows the patient screening process over two years in which 36.5% of the 104 patients with pcos were found to meet the criteria established on the existence of insulin resistance ; 16 patients in each group completed the follow up stage up to the ending of study . obesity is predominant in both groups , and there are not any significant differences in the features assessed . table 3 contains the baseline values of the lipid and hormone measurements taken at the start of study . no significant differences were observed . table 4 contains the patients ' physical evolution shown in weight and body mass index ( bmi ) . although there was a slight gradual decrease in weight in both groups , there were no significant differences . on studying the evolution of the homa - ir index ( table 5 ) , a significant drop from the initial value to the value given at 6 months is observed in the group that is administered diamel . meanwhile the decrease in this index in the group administered placebo is much smaller , even insignificant . table 6 contains data on the evolution of the insulin sensitivity by raynaud index , which increases considerably especially in the group given diamel . more patients in the group with diamel start having normal periods again , compared to those in the control group , and two of the patients from the former group even manage to conceive : one of them obviously leaves the study for this reason at 3 months and the other patient conceives two weeks after the study has finished . table 8 shows the evolution of hormone levels . there is a significant decrease in testosterone at 3 months in the group treated with diamel compared to the group that is given placebo . the lh also drops in the former group from the start at 6-month point ; although it was smaller in this group than the used placebo , these differences did not reach statistical signification . the use of drugs such as metformin , which improves insulin sensitivity and decreases resistance to this hormone , has had good results in some studies with the decrease of high insulin levels and improvement of hyperandrogenism [ 14 , 16 ] . some studies have suggested a lower miscarriage rates if metformin treatment is continued in the first trimester , but this remains unresolved . there is less risk of miscarriage in women using this drug [ 24 , 25 ] . however , there are not many other alternative drug treatments to reduce or prevent the signs and symptoms caused by pcos ; for this reason , we decided to study the use of the food supplement called diamel . it has already proved to be effective in patients with type 2 diabetes mellitus ; it does not have any adverse effects [ 17 , 18 ] , and it could be another alternative for women suffering from the syndrome if it proves to be capable of reducing insulin resistance . the components in diamel are activated by means of electromagnetic procedures that can enhance the metabolic changes in patients , and the lettuce extract reduces the amount of glucose absorbed during the digestion process . the cranberry extract contains anthocyanosides and tannins which improve the microcirculation and the secretion of insulin . cranberries and blueberries have proved to be beneficial for both men and women as it enhances insulin sensitivity and reduces resistance to this hormone . our research work involved two years of screening patients with pcos and insulin resistance which was identified by the homa - ir index , as shown in the follow up algorithm ( figure 1 ) . even though the proposed figure of 60 women with pcos , and a homa - ir index > 2.6 was not reached , these circumstances limit the reach and the results of our investigation , despite we considered of utility in present to these data , since it is a first study that analyzes the possible influence of diamel in a sample of women with pcos and we were able to make an acceptable evaluation with the 37 patients who did satisfy aforesaid criteria . none of the patients left the study during the follow up stage due to adverse effects caused by the food supplement . indeed insulin sensitivity progressed favourably , and there was a significant decrease in the resistance to the hormone in the group being treated with diamel . we found diminution of the insulin resistance in the group with the supplement , when compared the beginning with month 6 although statistical meaning is not appraised when it is compared with the placebo group ; the decrease in the insulin resistance was not significance in this control group , and this is thought to have been due to a slight weight loss and drop in the bmi . changing the life style is a proven first - line treatment for the majority of overweight women with pcos . it improves the psychological results , the reproductive conditions ( menstrual cycle , ovulation , and fertility ) , and the metabolic conditions , insulin resistance , cardiovascular risk factors and those of diabetes mellitus . evidence proves that changing the patients ' life style by setting tiny goals gets clinically beneficial results even when the woman in question is still overweight or obese [ 7 , 14 , 27 ] . practically all our patients had oligomenorrhoea or amenorrhoea at the beginning of the study , and it was remarkable to see that 6 of 16 patients in the diamel treatment group were able to recover menstrual periods again , two from this group even managed to conceive , although one miscarried at 12 weeks . these results tie in with the evolution of the hormone biochemistry , as the lh is seen to decrease significantly in the group treated with diamel ; the testosterone gradually decreases too although when comparing the two groups it is only significant at the 3-month point in the treatment group . insulin resistance leads to hyperinsulinaemia , it reduces the shbg , and it increases the free circulating testosterone , and together hyperandrogenism and hyperinsulinaemia alter the development of the follicles in the ovaries [ 2 , 4 , 8 ] . a favourable tendency is observed in our results with the diamel since it seems that it helps reduce insulin resistance , lh , and testosterone levels . the possible utility of the diamel would be in those women with pcos that they suffer insulin resistance although other investigations with a greater reach as far as the study of sexual hormones would be necessary to justify their indication like therapeutic line in these patients . this syndrome is a chronic disease with symptoms that are suffered throughout a woman 's life . it is a serious and common problem for public health that affects both the individual and the economy . a large percentage of women with pcos also suffer from hyperandrogenism and insulin resistance , especially if they are overweight or if they are prone to the risk of metabolic syndrome , prediabetes , and type 2 diabetes . tackling the problem must include providing support , education , and managing psychological factors , along with highlighting the importance of having a healthy life style and medical treatment . for a large majority of patients , the treatment involves a major change in their life style which is based on a multidisciplinary effort to prevent long - term complications . there must also be other alternative medicine available that do not have any adverse effects , and it would be completely acceptable to use diamel in other investigations considering the results obtained . in conclusion the food supplement diamel reduces insulin resistance and improves insulin sensitivity in a small sample of women with pcos and shows promise to be a nonmedical alternative if our findings are confirmed in larger randomized control trials .

for to determine the effect of diamel on the insulin resistance , insulin sensitivity , and sexual hormones results in women with polycystic ovary syndrome ( pcos ) . a study was carried out on 37 patients with this disorder . a triple - blind clinical trial was designed in which the diamel food supplement was compared with a placebo . the women with reproductive ages were randomly distributed in two groups , with 18 and 19 women respectively , and they took diamel or placebo and were followed up during 6 months with clinical and biochemical evaluation . a significant decrease in the homa - ir from the initial value at six months was observed in the group with diamel . the insulin sensitivity improved considerably in this group . the rate of menstrual recovery was higher in the group with diamel , and two patients from this group obtained pregnancy . the hormone levels shows a significant decrease in testosterone at 3 months in the group with diamel compared with the control group . the lh also decreases in the same group when comparing the start with 6 months.we concluded that the diamel decreases insulin resistance and improves sensitivity to this hormone in women with pcos , with improvement in the levels of lh and testosterone .

1. Introduction 2. Patients and Methods 3. Results 4. Discussion

sexual instinct which is the cause of numerous changes in an individual s life could be influenced by different factors such as menopause and ageing . participants were 174 menopausal women who referred to medical health centers of isfahan , iran . data were collected through self constructed sexual dysfunctions questionnaire in relation to their sexual activities before and after menopause . the reliability and validity of this questionnaire was determined by content validity and cronbach s alpha coefficient . findings showed that the relative frequency of sexual dysfunctions was 38% in the productive period and 72.4% in the menopause period . there was a significant association between sexual dysfunctions before and after the menopause period ( p < 0.001 ) . sexual dysfunctions in these women in the productive and menopause period were 49.2% and 62.6% in desire disorder , 34.3% and 34.9% in dyspareunia , 26.8% and 75.3% in arousal disorder , 23.5% and 56.3% in orgasmic disorder and 16.4% and 15.6% in vaginismus , respectively . a considerable percentage of women experienced sexual dysfunctions in productive and menopause periods , and menopause could be a factor to maintain or intensify sexual dysfunctions . sexual desire is a natural instinct which needs attention and it is impossible to neglect because it is presented since the birth of a child and it changes and flourishes according to his or her growth . the world health organization ( who ) has defined sexual health as a kind of coordination between mind , senses ( feelings ) and body that can affect the social and intellectual aspects of human personal growth . in other words , sexual desire is an important and integral aspect of the human personality however , sexual desire can be disrupted because of factors such as illnesses , drug usage , social problems , aging , menopause , and etcetera , which can lead to psychological disorders . castelo - branco et al . reported that the prevalence of these dysfunctions in middle aged women was 50% . moreover , he reported that the prevalence of these dysfunctions will increase as a result of age . although , there are some physiological and pathological changes in the menopause period that result in these problems , the attitude of the women toward menopause has an important role in creating or eliminating these problems . in this regard , it has been seen that a large number of these women consider the menopause period as a period of freedom because of the culmination of the productive period the decrease of the responsibilities over the children , and no fear of pregnancy . therefore , they feel comfortable and more active ( regarding sexual interactions ) compare to the pre - menopause period . however , for others this period may cause anxiety and be a sign of aging and the end of being pretty and attractive . it seems that if they were sexually inactive or had undesirable psychological sexual condition during the productive period ; the menopause period may decrease their sexual ability and any kind of sexual feeling in them . therefore , menopause can be a factor in intensifying or eliminating sexual problems in this period . these problems result in high incidence of sexual dysfunctions and the declining of psychological health in families . this problem has caused some negative attitudes and wrong social customs in relation to sexual relationships . therefore , considering this problem and the limitation of such studies in our country , the present research was designed to compare women s sexual dysfunctions before and after menopause . the population under study included 174 menopausal women ( living with their spouses ) , which referred to the medical health centers , after one year of being menopausal , to receive medical services . subjects in this study were randomly selected from 10 medical health centers , 4 of which were affiliated to medical health center number1 ( because 40% of families in the city of isfahan are under the coverage of this center ) and 6 were affiliated to medical health center number 2 ( because 60% of families in the city of isfahan are under the coverage of this center ) . the exclusion criteria of the study were as follows : illnesses in the women or their spouses which affect sexual interaction including vasculitis , thyroids , illnesses of adrenal cortex , diabetes , blood pressure , lung or heart problems , central nervous system damages , infectious diseases and venereal system operations.using drugs which interfere with sexual interactions such as blockers of h2 receiver , psychoactive drugs , and heart and antihypertensive drugs , anti - convulsion drugs , narcotics , anti - cholinergic and anti-histamines.experiencing some stresses like the spouse cheating , fatal diseases or imprisonment of immediate relatives in the previous year.menopausal women who lived apart from their spouses at the time of the interview.women whose spouses had premature ejaculation or sexual disabilities . illnesses in the women or their spouses which affect sexual interaction including vasculitis , thyroids , illnesses of adrenal cortex , diabetes , blood pressure , lung or heart problems , central nervous system damages , infectious diseases and venereal system operations . using drugs which interfere with sexual interactions such as blockers of h2 receiver , psychoactive drugs , and heart and antihypertensive drugs , anti - convulsion drugs , narcotics , anti - cholinergic and anti - histamines . experiencing some stresses like the spouse cheating , fatal diseases or imprisonment of immediate relatives in the previous year . the nominated women were invited to the health center and the eligible candidates were interviewed and they completed the questionnaire after being ensured of the confidentiality of the questions . it should be mentioned that the participants were interviewed by midwifes s in the health centers and interviewers received explanations during a 2 hour session . these interviewers were asked to have in mind that different participants may have different conceptions of the questions , and thus to make sure each question is understood correctly . finally interviewers asked the participants to give information about their sexual interaction during the menopause period and before it . in this research , the pre - menopause ( productive ) period was considered to be between the ages of 15 to 50 . due to the fact that the pre - menopause period is so long and women have different sexual experiences in each stage of this period , the researchers and interviewers classified this period into several categories such as the first years of marriage , the delivery intervals , the school years of children and etcetera . this classification can help the participants to better remember their sexual interactions , make a careful conclusion from these interactions , and to state if their sexual interaction in these periods was satisfactory or disordered sexual interaction ( in one or more stages ) . the tools for gathering information included self constructed sexual dysfunctions questionnaire based on diagnostic measures of psychiatric disorders of usa ( dsm iv- tr ) . this questionnaire which had 18 closed multiple choice ( 5-choices ) questions , was designed to measure the dysfunctions of sexual desires phases , arousal , orgasm and sexual dysfunctions resulting from pain ( dyspareunia vaginismus ) in the periods of before and after menopause . in order to answer each question based on likert spectrum , the choices of never to always ( never , seldom , often , and always ) were considered for assessing the intensity of sexual dysfunctions . if the participants mentioned dysfunctions in one or more phases , they had sexual dysfunctions ; otherwise , they had no problems in their sexual functioning . in order to evaluate the validity of this questionnaire , content validity was used and in order to investigate its validity , retake exam and cronbach s alpha index ( = 0.875 ) were used . moreover , in order to determine the distribution frequency of sexual dysfunctions in these women in productive and menopause periods , the mcnemar test was used . participants were between the age of 40 to 74 ( mean = 54.67 6.3 years ) , the pregnancy age range was from 0 to 16 ( mean = 6.8 3.14 years ) , the age range of menopause was from 30 to 56 years ( mean = 47.6 4.5 years ) , and the weight range was from 38 to 95 kilograms ( mean = 66.12 10.13 years ) . findings showed that the relative frequency of women s sexual dysfunctions before and after menopause were 38.5% and 72.4% , respectively . the frequency distributions of sexual dysfunctions in the productive period based on the intensity of disorder were as follows : dysfunctions in the desire phase ( 49.2% ) , dyspareunia ( 34.6% ) arousal disorder ( 26.8% ) , orgasmic disorder ( 23.8% ) and vaginismus ( 16.4% ) . furthermore , the frequency distributions of these disorders during the menopause period were as follows : arousal disorder ( 75.3% ) , sexual desire disorder ( 62.6% ) , orgasmic disorder ( 56.3% ) , dyspareunia ( 34.9% ) and vaginismus ( 15.6% ) . according to the results , 41.1% of the units did not mention sexual dysfunctions , and 56.9% mentioned sexual dysfunctions in the menopause period in the group which did not have any sexual dysfunction before menopause , and in the group which had sexual dysfunctions before the menopause period , 6% of the units reported the absence of these dysfunctions in menopause period and 94% reported these dysfunctions during the menopause period . a significant correlation was seen between sexual dysfunctions before and after menopause ( p < 0.001 ) ( table 1 ) . the frequency distribution of sexual dysfunctions before and after menopause periods in units under study based on the results of this study , the most common sexual dysfunctions in the productive period were the desire phase and the dyspareunia dysfunctions , and in the menopause period , the highest amount of dysfunction was seen in the arousal and the sexual desire phases . these problems in the menopause period can be due to the attitudes of these women toward the menopause phenomenon and sexual interaction , or some physiological changes resulting from decreasing of the hormone levels especially estrogen , and in productive period , they can be due to cultural and psychological problems . as it was mentioned in this research , relative frequency of sexual dysfunctions before menopause was estimated as 38.5% , but abdo et al . kabudi reported 70% sexual response disorders in the two phases of desire and arousal in 141 menopause women . olaolorun and lawoyin reported the incidence of these dysfunctions 40.4% , and valadares et al . reported 35% . the considerable difference between the obtained results in our country and other countries can be due to racial , religious , cultural and attitude differences . about the relation of sexual dysfunctions before and after menopause , results showed that more than half of the women who did not have any sexual dysfunctions before the menopause period reported these dysfunctions during the menopause period , and in the group which had these dysfunctions before the menopause period , most women also reported sexual dysfunctions during menopause . , it seems that sexual status before menopause is associated with sexual interaction status during menopause they recognized that the study of their sexual history ( background ) in the productive period is the first step in diagnosing and curing the sexual dysfunctions of menopausal women . for example , olaolorun and lawoyin studied this phenomenon in urban populations of nigerian women , they reported that sexual problems will be influenced by aging ; so that , the women who were older had the highest problems premenopausal and postmenopausal . reported that the sexual dysfunction of middle aged women will be influenced by the two factors of age and the post menopause status . these studies were in conformity with our findings . while , gott and hinchliff reported that aging and long lasting interactions are two affirmative factors increasing the sexual desire in the spouses . therefore , people can have a suitable sexual interaction during their middle ages and elderly priods . in conclusion , the present research showed the relation of sexual dysfunctions before and after menopause periods and the high prevalence of these dysfunctions during menopause period . therefore , according to the results , the staff of medical health center in the counseling and educational sessions regarding sexual interactions , recommend to these women who are in their menopause or productive period , to pay careful attention to the physiological changes of the menopause period and find ways to overcome these dysfunctions by referring to psychologists and counselors . in fact , through their instructions the staff should prepare women to accept the menopause period and remove any misconceptions they may have in this field .

background : sexual instinct which is the cause of numerous changes in an individual s life could be influenced by different factors such as menopause and ageing . this study was designed to compare sexual dysfunction before and after menopause.materials and methods : this was a cross - sectional study . participants were 174 menopausal women who referred to medical health centers of isfahan , iran . data were collected through self constructed sexual dysfunctions questionnaire in relation to their sexual activities before and after menopause . the reliability and validity of this questionnaire was determined by content validity and cronbach s alpha coefficient.findings:findings showed that the relative frequency of sexual dysfunctions was 38% in the productive period and 72.4% in the menopause period . there was a significant association between sexual dysfunctions before and after the menopause period ( p < 0.001 ) . sexual dysfunctions in these women in the productive and menopause period were 49.2% and 62.6% in desire disorder , 34.3% and 34.9% in dyspareunia , 26.8% and 75.3% in arousal disorder , 23.5% and 56.3% in orgasmic disorder and 16.4% and 15.6% in vaginismus , respectively.conclusions:a considerable percentage of women experienced sexual dysfunctions in productive and menopause periods , and menopause could be a factor to maintain or intensify sexual dysfunctions .

Background: Materials and Methods: Findings: Conclusions: I M F D

thoracic aortic aneurysms have altered cellular composition and degeneration of the extracellular matrix in the aortic wall . there are several different etiologies of thoracic aortic aneurysm such as monogenic syndromes ( marfan and loeys - dietz syndromes ) , aneurysm associated with bicuspid aortic valves , and idiopathic aneurysms . the pathogenesis of aneurysm formation in the monogenic syndromes has been extensively studied , whereas the molecular and cellular mechanisms of the other forms , which constitute the majority of thoracic aortic aneurysms , remain largely unknown . most studies of the cell and molecular biology of thoracic aortic aneurysm have used entire wall specimens [ 26 ] . consequently , the biology of the different cell types in the aneurysmal wall remains largely unknown . single studies comparing smooth muscle cells ( smcs ) from aneurysm patients with the cells from patients with acute aortic dissection revealed differences in expression of some smc specific genes . in addition , smcs from aneurysms were demonstrated to have significantly shorter telomeres , reduced metabolic activity , and impaired proliferation and migration rates . comparison of smc derived from either bicuspid aortic valve ( bav ) or normal tricuspid aortic valve ( tav ) in thoracic aortic aneurysms showed expression difference in several markers including osteopontin and tissue inhibitor of metalloproteinase 3 which may reflect different etiologies of tav- and bav - associated aneurysms [ 510 ] . however all above - mentioned studies did not address the functional properties of endothelial cells . as a consequence , a possible role of the endothelium in the developing of aneurysms is largely unknown , although a recent study suggests that aneurysms at least from bav patients may be associated with endothelial dysfunction . recent studies show that endothelial cells could directly influence smooth muscle cell phenotype [ 13 , 14 ] . the major role of mature differentiated vascular smooth muscle cells is to maintain blood vessel tone and to regulate blood pressure through constriction or relaxation . this is achieved through the expression of a complement of regulatory and contractile genes that provide the machinery for this response . the differentiated contractile phenotype is largely characterized by expression of coordinately regulated smooth muscle - specific markers that include smooth muscle ( sm ) -actin ( acta2 ) , and sm22 ( tagln ) and some other proteins . we sought to investigate if smcs from aortic tissue of the patients with thoracic aneurysm undergo phenotypic and functional change such as growth , apoptosis , and extracellular matrix synthesis and whether this change is also accompanied by endothelial cell changes in the cells of the patient tissues compared to the cells from healthy tissue . our data demonstrate downregulation of smooth muscle as well as endothelial cell specific markers in the patient cells and also changes in functional state of both smc and endothelial cells . the clinical research protocol was approved by the local ethics committee of the almazov federal medical research center and was in accordance with the principle of the declaration of helsinki . samples of the aneurysmal wall of the thoracic aorta were harvested during aortic surgery at the almazov federal medical research center . thirty specimens were sampled from patients with bav ( n = 17 ) or tav ( n = 13 ) ( table 1 ) . control aortic specimens were obtained from organ transplant donors ( n = 11 ) and all had tav . all tissues were sampled from the outer curvature of the thoracic aorta . to obtain smc cultures human aortic endothelial cells ( haec ) were isolated from tissue fragments of patients after surgery for aneurysm corrections . under sterile conditions tissue fragments , the tissue fragments were first incubated for 30 min at 37c in 0.1% collagenase solution ( collagenase , type 3 , worthington biochemical corporation , usa ) . then endothelial layer was removed mechanically by scraper , and endothelial cells were washed twice and plated onto fresh 3 cm culture dish covered with 0.1% gelatin ( sigma ) in egm2 medium ( promocell ) and incubated at 37c . the next day endothelial cells were washed by pbs and culture medium smcs were grown to confluence on 6-well plates ; after the cells formed a monolayer , the medium was exchanged for serum free medium containing 10 mm hydroxyurea and 10 ng / ml pdgf_bb growth factor to inhibit proliferation and to stimulate migration and the cell monolayer was scraped with a 200p pipette tip to create a cell - free zone . the number of cells which migrated into the wounded area was counted after six and 24 hours . experiments were performed in duplicate and then repeated three times . for estimation of apoptosis smcs were seeded at a density of 10 10 cells / cm , and 10 10 m hydrogen peroxide ( h2o2 ) was added to the culture medium 48 hours later . after two hours the cells were removed and labeled with fitc - conjugated annexin v ( sigma ) . the number of annexin v labelled cells was estimated by flow cytometry using calibur ii ( bd ) . total rna was extracted from smcs or endothelial cells using trizol reagent ( invitrogen ) according to the instructions of the manufacturer . real - time pcr was performed in the lightcycler system with sybr green detection ( fermentas ) using specific primers . changes in target genes expression levels were calculated as fold differences using the comparative ct method . specimens were homogenized in a lysis buffer ( 50 mm tris ( ph 8) , 150 mm nacl , 1% triton x-100 , 1% sodium deoxycholate , and 5 mm edta ) , containing protease inhibitors ( roche ) . extracts were separated by 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) . primary antibodies used are sm22 ( ab14106 , abcam ) , sma , vimentin ( m072529 , dako ) , beta - actin ( ab 6276 , abcam ) , collagen i , fibrillin , and elastin . positive bands were quantified by densitometry using a gel documentation system fusion fix ( vilber lourmat ) and fusion - capt software . primary antibodies used are sma ( sc-32251 , santa cruz ) , sm22alpha ( ab14106 , abcam ) , vimentin ( sc-6260 , santa cruz ) , ve - cadherin ( mab938 , randd ) , von willebrand factor ( ab20435 , abcam ) , and calponin ( ab700 , abacam ) . microphotographs were taken using axioobserver microscope ( zeiss ) at 20 magnification with axiovision software . smcs from aneurysms of the thoracic aorta and from control aortas were analyzed regarding the expression of smc markers like -smooth muscle actin ( sma ) , vimentin , and sm22. figure 1 shows typical immunofluorescent staining of smc from control aortas and from aneurysms in patients with bav and tav . both tav- and bav - derived smcs appeared to have decreased level of sma , vimentin , and sm22 . however , there were no visible differences between smc from patients with bav and tav . at both mrna and protein level expression of sma and vimentin was reduced both in the bav- and tav - derived smc and in the aortic media ( figure 2 ) . however , although sma was lower in aortic media of aneurysms from patients with bav than in controls , it was still higher than in patients with tav . sm22 expression was decreased only in smc and aortic media from patients with tav . in aneurysms from patients with bav the expression of sma was higher than in tav patients both in smc and in aortic media ( figure 2 ) . figure 3 demonstrates primary cultures of endothelial cells and icc staining of the cells including staining for sma to confirm that the cultures were not contaminated with smc . endothelial markers appeared to be reduced in endothelium in aneurysms from patients with both tav and bav ( figure 3 ) . we compared also the mrna level of sma , cd31/pecam , ve - cadherin , and vwf in endothelial cells derived from control aortas and from aneurysms . the expression of sma mrna was elevated in both tav- and bav - derived endothelial cells ( figure 4 ) . typical sma microfilament staining was not observed in our endothelial cultures ( figure 3 ) ; thus the elevation of sma mrna level was not due to contamination with smc . expression of the endothelial markers vwf and cd31/pecam was substantially decreased in endothelial cells from aneurysms ( figure 4 ) ; the level of ve - cadherin mrna was not changed . specific degenerative processes associated with reduced cellularity are observed in the aneurysm wall . to evaluate the possible contribution of smc and endothelial cells to these changes , we compared cell proliferation and migration in healthy donors and patients with aneurysms of the thoracic aorta . smc proliferation rate ( figure 5(a ) ) in both bav and tav aneurysm was lower compared to healthy donors , but without any difference between the two types of aneurysms . endothelial cells from aneurysm patients had also lower proliferation than controls , but endothelium from patients with bav had lower proliferation rate than endothelium from aneurysms of patients with tav ( figure 5(b ) ) . smc migration rate was higher in aneurysm patients with tav , but not in bav patients compared to controls ( figures 5(a ) and 5(b ) ) . reduced cell number has been shown in aortic tissue of thoracic aortic aneurysm patients and may reflect increased apoptotic level in the vessel wall cells . indeed , the number of cells that are positive for dna double strand breaks ( an apoptotic marker ) is increased in the media of the wall of thoracic aneurysms . therefore we studied apoptosis in smc cultures from aneurysm patients and controls ( figure 6(a ) ) . the number of annexin v positive cells was significantly higher in smc cultures from patients with both bav and tav . oxidative injury might be a cause of increased wall weakness in both abdominal and thoracic aortic aneurysm . the ability of oxidative stress to cause apoptosis might be altered in smc from aneurysms . to test this hypothesis h2o2 we measured apoptosis as a residual between the percentage of annexin v positive cells after h2o2 treatment and the amount of annexin v positive cells in normal cultures ( baseline ) . h2o2-induced apoptosis was reduced in smc from aneurysms compared to smc from nonaneurysm aortic walls ( figure 6(b ) ) . to evaluate smc contribution to extracellular matrix protein synthesis ( elastin , fibrillin , and collagen i ) in the aortic wall we estimated the protein content in aortic media specimens and in smc protein extracts ( figure 7 ) . the elastin and fibrillin content was reduced in aortic media from aneurysms in both bav and tav patients ( figure 7(a ) ) . collagen i content was higher in aortic media from both types of aneurysm patients but was not significantly changed in smc from aneurysm patients . however , the amount of collagen i was higher in smc of aneurysms from patients with tav only ( figure 7(a ) ) . the culture media from endothelial cells were analyzed for elastin , collagen i , and fibrillin content ( figure 7(b ) ) . our data show that aortic endothelial cells are capable of synthesizing these proteins and synthesizing fragmented collagen i. mmp expression may be increased in thoracic aneurysms [ 2 , 22 ] , but their role in the etiology of aneurysms is not clarified . the exact type of cells in the aortic wall that synthesizes mmps is still unknown . we did not detect increase in mmp2 or mmp9 activity in total aneurysm medial tissue samples ( figure 8) , whereas mmp-9 activity was significantly increased in smcs and smc culture media from aneurysm patients with both bav and tav . however the ultimate target of any therapy approach is a cell , whose healthy properties are changed in the pathology . that is why , it is important to know not only a set of genes changed at a definite pathology , but also cellular properties that are the cause of a pathology and thus to find a possible tool for a correction . this study aimed to characterize two major aortic cellular populations , smc and ec , from taa patients and healthy donors . in the present study we demonstrated that both smc and endothelial cells from thoracic aortic aneurysms have impaired functional properties in terms of proliferation , contractile and extracellular protein expression , and apoptosis with significant difference between bav- and tav - associated aneurysms . of most importance our findings are in agreement with a recent publication describing endothelial dysfunction in bav patients . smc from thoracic aneurysms demonstrated decreased expression of key smc proteins such as sma and sm22 both at mrna and at protein level . endothelial cells from thoracic aneurysms also demonstrated downregulation of specific markers and impairment in growth . our data suggests that the cells from the aneurysm aortic wall are in less differentiated state comparing to normal aortic wall . this observation is well in line with the findings that endothelial cells influence differentiation and functions of underlying smc [ 13 , 14 ] . thus , endothelial changes may contribute to impairment of the aortic wall structure built by smc . cultured smc from aneurysm patients demonstrated surprisingly high amounts of annexin v positive cells suggesting a high level of apoptosis in the aortic wall of aneurysm patients . this may also be partly a consequence of the cell culture , but nevertheless there were differences between the different groups . philippi and coauthors showed that smc from aneurysms in bav patients had the poorest resistance to oxidative stress , but they did not show a baseline level of apoptosis in their smc cultures but counted the viability of the cells under oxidative stress . we suggest that both decreased proliferation rate and increased apoptosis contribute to the loss of cells in the aortic wall in aneurysms . the mechanisms of apoptosis resistance / susceptibility in aortic smc population may be important in the development of aneurysms . we compared the content of some extracellular matrix protein in aortic tissue samples , smc and supernatants from smc and endothelial cells . the observed results suggest that smc are not the only cells that synthesize extracellular matrix proteins in the aortic wall . also endothelial cells are capable of synthesizing extracellular matrix proteins in aneurysms and the extracellular matrix composition is different between aneurysms from patients with tav and bav taa . finally the integrity of the extracellular matrix in the wall of aneurysms is influenced by changes in the ratio between different proteins . fibrillin content was different between aneurysms from tav and bav patients . both endothelial cells and smc from aneurysm patients had synthesis of fragmented collagen i. the complex nature of biosynthesis of extracellular matrix proteins in the aneurysm wall is in agreement with other recent studies [ 22 , 23 ] the role of different mmp in thoracic aneurysm pathogenesis has been discussed [ 22 , 23 ] . there are a number of papers describing mainly the elevated gene expression levels for some mmps in the aneurysm wall [ 2 , 2224 ] . in our study the activity for mmp2 and mmp9 was elevated in smc and in supernatants from smc cultures . this is in accordance with previously reported elevated gene expression of mmp2 and mmp9 in the wall of thoracic aneurysms [ 22 , 23 ] . the study is heterogeneous because from every tissue sample it is not possible to get smc and endothelial cell cultures ; the cells are in culture for limited amount of passages that limits the possibilities to study them . for the endothelial cells it is difficult to get cultured cells and it is not possible to get enough cells from many patients for different kind of analysis . another limitation is that we have studied only two types of cells whereas the aorta consists of more types of cells including stem cells , macrophages , and fibroblasts . a very important limitation is that gene expression and cell behavior in dilatative aortopathy can be affected by hemodynamics as well as by physical and biochemical environment , other than by their interaction with the genetic background and these factors are absent in cultured cells . further research of the cell alterations in the wall of thoracic aneurysms might lead to a deeper understanding of pathogenesis and pathology and thus to find a potential therapeutic tool .

thoracic aortic aneurysm develops as a result of complex series of events that alter the cellular structure and the composition of the extracellular matrix of the aortic wall . the purpose of the present work was to study the cellular functions of endothelial and smooth muscle cells from the patients with aneurysms of the thoracic aorta . we studied endothelial and smooth muscle cells from aneurysms in patients with bicuspid aortic valve and with tricuspid aortic valve . the expression of key markers of endothelial ( cd31 , vwf , and ve - cadherin ) and smooth muscle ( sma , sm22 , calponin , and vimentin ) cells as well extracellular matrix and mmp activity was studied as well as and apoptosis and cell proliferation . expression of functional markers of endothelial and smooth muscle cells was reduced in patient cells . cellular proliferation , migration , and synthesis of extracellular matrix proteins are attenuated in the cells of the patients . we show for the first time that aortic endothelial cell phenotype is changed in the thoracic aortic aneurysms compared to normal aortic wall . in conclusion both endothelial and smooth muscle cells from aneurysms of the ascending aorta have downregulated specific cellular markers and altered functional properties , such as growth rate , apoptosis induction , and extracellular matrix synthesis .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

implant restoration is a common practice and considered the first choice for replacing maxillary anterior teeth . one of the challenges in restoring anterior space with implant restoration is maintaining natural looking for the restoration . to reach patient satisfaction , optimal esthetic result has to be achieved for the teeth and the soft tissue to create a natural emergence profile . one method of preserving soft and hard tissue is through immediate implant placement and placement of a provisional restoration . however , this is not always possible with implant treatment . with delay implant placement cases , bone resorption and soft tissue collapse occur following tooth extraction , results in flat anatomical contour . restoring natural emergence profile of the restoration provisional restoration for implant - supported restoration can help in predicting and achieving the esthetic result . one of the important advantages of provisional restorations is modeling of soft tissue during the healing process . several techniques have been proposed to restore gingival contour and create emergence profile for an esthetic restoration during provisional restoration . neale and chee , chee and donovan advised performing gingivoplasty procedures to recontour the tissues before making provisional restorations . hinds described the fabrication of a custom impression coping for the replication of the healed tissue around the implant . bain and weisgold and ormianer et al . recorded the soft tissue contour by inserting autopolymerizing acrylic resin directly into the sulcus during impression making . however , use of acrylic resin monomer intraorally may cause thermal and chemical irritation of the soft tissue . attard and barzilay presented a technique using a provisional restoration - level impression to register the soft tissue configuration and shape , as well as the implant position . the aim of this clinical article is to report a simple , indirect and nonsurgical clinical procedure to create a soft tissue emergence profile for anterior maxillary teeth . this clinical method helps to avoid soft tissue collapse that may occur during final impression , and minimize peri - implant soft tissue irritation . a 49-year - old nonsmoking male presented to the dental office complaining of missing maxillary right lateral incisor [ figure 1 ] . the tooth was extracted 2 months before presenting to the clinic due to root fracture . previous dental history revealed that the tooth had received a root canal treatment and had a postretained crown . a removable partial denture was fabricated for him by his previous dentist immediately after extraction to restore the space of the extracted maxillary lateral incisor . pre - treatment clinical view , missing of maxillary right lateral incisor radiographic examination revealed a socket of recently extracted maxillary lateral incisor with bone level 23 mm below the cementoenamel junction ( cej ) of the adjacent teeth [ figure 2a ] . intra - oral examination revealed a fair oral hygiene with 12 mm recession of the gingival margin below the cej . the space available of the area related to maxillary lateral incisor was 6 mm mesio - distally and 7 mm interocclusal distance . upon presentation of the various options of treatments , the patient opted to restore the space with implant treatment . the decision was made to proceed with a single implant to restore the space of the maxillary right lateral incisor and to maintain and to create natural soft tissue contour and emergence profile around the tooth to improve esthetic . periapiacl rediograph : ( a ) two months after extraction , ( b ) immediately before impalnt placement maxillary and mandibular study models with a face bow transfer were obtained , and a diagnostic waxing of the maxillary lateral incisor was performed . a surgical guide was then fabricated to insure proper position of the implant during placement procedure . the patient had decided to concentrate only on replacing the missing lateral incisor , declining esthetic enhancement of the surrounding dentition . before surgery , new periapiacl rediograph was taken [ figure 2b ] , and surgical placement of the implant completed using surgical splint . single implant with 3.5 mm diameter and 13 mm height ( replace select tiu np , nobel biocare , switzerland ) was placed and covered with 3 mm height healing abutment [ figure 3 ] . one week later , the healing abutment was removed and replaced with an immediate temporary abutment with a plastic coping ( nobel biocare , switzerland ) . the temporary abutment was screwed to the implant , and a provisional crown was fabricated using temporization material ( pro temp , 3 m espe , mn , usa ) directly on the plastic coping . area related to the gingival tissue ( transmucosal area ) was built up by adding composite resin ( filtek z100 , 3 m espe , mn , usa ) . the composite resin has been added buccally and interproximally to gently push the soft tissue and create a concavity on the ridge area until the optimum esthetic result of the emergence profile achieved . finishing and polishing were completed using finishing polishing discs ( sof - lex , 3 m espe , mn , usa ) , and the occlusal was adjusted to keep the provisional nonfunctional . finally , the provisional crown was cemented using temporary cement ( temp bond , kerr , bioggio , switzerland ) , and it served as a guide for soft tissue healing [ figure 4 ] . cliniucal view , immediately after placement of the implant and healing abutment cliniucal view for the provisional restoration two months later , the provisional restoration was removed , and the implant was examined for osseointegration . soft tissue healing was completed and stable with the required gingival contour to keep the emergence profile created [ figure 5 ] . one of the objectives during the final impression was to transfer the created emergence profile to the permanent restoration accurately . clinical view for the peri - implant soft tissue , two months after placement of the provisional restoration the temporary abutment was removed , and the provisional restoration was cemented properly on the abutment . a silicon putty impression material was mixed and placed in a plastic cup . while the putty material was still soft , the temporary abutment with the restoration fitted on an implant replica ( np , nobel biocare , switzerland ) , and placed directly in the soft mix . the implant replica and the gingival third of the crown were immersed in the putty material as shown in figure 6a . after complete setting of the putty material , the temporary abutment with the restoration was removed [ figure 6b ] , and impression coping was screwed to the implant replica [ figure 6c ] . a resin material powder and liquid ( duralay , reliance dental mfg , worth , il ) was mixed and placed around the impression coping to fill the area of the gingival contour [ figure 6d ] . after complete setting , the impression coping transferred to patient mouth and fitted accurately [ figure 6e ] . final impression was taken using vinyl polysiloxane impression material ( virtual , ivoclar vivadent , italy ) [ figure 6f ] . the impression was sent to the laboratory , and screw - retained porcelain - fused to metal crown was fabricated . cliniucal steps for taking final impression : ( a ) provisional restoration with implant replica immerssed in putty impression . ( b ) the provisional restoration removed from the immediate temporary abutment ( c ) impression coping screwed to the implant replica.(d ) acrylic resin ( duralay ) added to the impression coping . this case report describes a clinical method that helps to control gingival contour around implant crown to create an emergence profile for the crown . it is relatively easy , precise and predictable method for accurate duplication of soft tissue profile . achievement of the good result is dependent on the amount of keratinized mucosa , height and thickness of bone and shape and material of the transmucosal implant prosthetic components . the technique presented in this article differs from other techniques in that it reduces gingival trauma by eliminating the intra - oral use of resin monomer that avoid chemical or thermal insult to the tissues . it also minimizes surgical procedure by remodeling the soft tissue during the healing process to create the proper contour needed . in addition to that , the important advantage is avoiding the possibility of soft tissue collapsing that may occur during the impression procedure , which gives accurate of the peri - implant soft tissue contours . the emergence profile recorded with this technique from the contour of the provisional restoration not the soft tissue contour . this technique can be used for one ( or more ) single implant - supported restoration ( s ) in anterior areas , where an optimal esthetic result is required . in this clinical case , composite resin , rather than acrylic resin , has been used in the transmucosal area to reduce soft tissue irritation . using screw - retained provisional restoration however , screw - retained will simplify corrections / modifications required for the provisional restoration , and will eliminate the needed for cementation and possible soft tissue irritation , especially in subgingival areas . this clinical report describes an alternative indirect impression procedure that accurately captures the emergence profile and soft tissue contours around implants after a provisional restoration has been placed . the definitive restoration is shaped exactly like the provisional , and excellent esthetics results can be achieved .

one of the challenges in restoring anterior space with implant restoration is maintaining the natural looking of peri - implant area . this case report presents a clinical procedure to create the soft tissue emergence profile for anterior maxillary teeth . a 49-year - old male presented with missing right maxillary lateral incisor . a provisional restoration was inserted 1 week after implant placement . area of the provisional restoration related to the gingival tissue ( transmucosal area ) was adjusted to create an optimum emergence profile . two months later , an indirect method was used to accurately transfer the soft peri - implant tissues to the master cast . this clinical technique minimizes surgical procedure and avoids the possibility of soft tissue collapsing that may occur during the impression procedure .

Introduction Clinical Case Discussion Conclusion

it has a dual structure , with the upper part of the joint being ligamentous and the lower part of the joint being a true synovial joint . the sij moves in several planes , with the largest plane of motion being sagittal . in this plane , the sij has been reported to have 24 degrees of flexion extension ( nutation counternutation ) with less motion in lateral bending and internal / external rotation.1 the synovial part of the joint can undergo osteoarthritic changes , including joint space narrowing , osteophyte formation , subchondral sclerosis , and cyst formation.2 inadequate functioning of the sij and its associated musculature , as well as deterioration of the joint capsule and surrounding ligaments , results in increased stresses , pathologic motion , and altered biomechanics , causing chronic pain of the buttocks , lower back , as well as thigh and legs.3 as early as the 1800s , the sij was thought to explain a significant proportion of all low back pain.4 the sij has been demonstrated to have both mechanoreceptors5 and nociceptive receptors.6 it has a rather complex innervation , with contribution from lateral branches of multiple lumbosacral nerve roots . pressurization of the sij in healthy volunteers can elicit pain7 and anesthetics applied to the exiting dorsal sacral nerve roots block sensation outside of the joint but not pain elicited by joint pressurization.8 the sij is thought to explain 15%23% of all chronic low back pain.9,10 however , the exact prevalence is unknown , at least in part due to the lack of a universally agreed - upon diagnostic standard . the sij may explain an even larger proportion of pain in patients who have had prior lumbar spine fusion.11 being immediately below the lumbosacral junction , it falls into the spectrum of adjacent segment disease after prior lumbar arthrodesis . radiographic findings of degeneration in the sij are common,2,12 both on ct and mri , and are not necessarily predictive of the presence of sij pain . this is similar to the presence of radiographic degenerative disk disease in both cervical and lumbar spines in asymptomatic volunteers.13 moreover , potentially due to the nonaxial compressive forces through the sij , chronic sij dysfunction can occur in patients with ligament and/or capsular failure . mri and ct scan may not show classic articular cartilage deteriorations and/or degenerative patterns seen in other joints . acute sij pain is fairly common and frequently transient , with most patients requiring either observation alone or simple measures such as physical therapy , nonsteroidal anti - inflammatory drugs , sacroiliac belts , exercise , chiropractic treatment , and sacroiliac blocks . however , there is very little evidence to support the effectiveness of nonsurgical interventions for long - term treatment of chronic established debilitating sij pain . two blinded , controlled trials of radiofrequency ablation of lateral branches of sacral nerve roots have shown only short - term improvement in pain;14,15 a 12-month follow - up study showed a modest long - term response rate following this treatment.16 no high - quality study reporting long - term outcomes has been published . sij fusion ( sijf ) was first described in the 1920s.17 a variety of approaches have been reported , including anterior , posterior , and lateral transiliac . sijf thus preceded the first reports of lumbar discectomy for disk herniations by about a decade.18 the original reports of sijf included a number of patients with infection - related sij pain ( including tuberculosis ) and subsequent reports have included patients with arthritic conditions as well . several single - center retrospective reports have suggested that open sijf may be effective for the treatment of pain in this patient population.1924 regardless of the approach , open fusions of the sij were quite invasive and associated with long hospital stays and recovery times , high nonunion rates ( 9%41% ) , 21,25,26 poor long - term results , and low levels of satisfaction.27 they also required prolonged periods of immobilization to achieve solid arthrodesis , mostly due to lack of adequate internal fixation techniques . in the past decade , there has been a resurgence of interest in the sij as the pain generator in a substantial number of patients requiring surgical interventions . several device systems are now commercially available for minimally invasive sijf , and the minimally invasive approach is now used in 90% of cases.28 most of the reported literature describe patients treated with triangular titanium implants ( ifuse implant system ; si - bone , inc . the current surgical literature for this system includes single - center retrospective cohorts,2935 a combined multicenter analysis,36 and three comparative studies of open and minimally invasive approaches.3739 a prospective randomized trial of minimally invasive sijf vs nonsurgical management showed improved 12-month outcomes after sijf compared with those after nonsurgical management,40 and a single - arm multicenter trial showed similar 24-month outcomes.41 herein , we report intermediate- to long - term ( 3 + years ) outcomes after sijf . we report a retrospective cohort study with a prospective evaluation component conducted at seven centers ( each with one surgeon ) in the us . the study was sponsored by the device manufacturer ( si - bone , inc . ) . the study includes patients at one center ( d sachs ) , which has been previously reported.36 all centers obtained institutional review board approval prior to participation ( from a commercial irb for 6 authors and the local hospital for 1 author ) , and all participants signed a study - specific consent form . eligible patients were adults ( at least of age 21 years ) who underwent sijf using the ifuse implant system prior to december 2012 , whose charts documented preoperative pain scores , and who provided consent to complete questionnaires . unified criteria to diagnose sij dysfunction were not used , as this study was retrospective in nature . however , diagnosis at all sites was made on the basis of history ( buttocks pain with optional radiation into the groin or upper leg ) , typical pain reproduced on at least three physical examination maneuvers , and a confirmatory diagnostic anesthetic block of the sij producing acute pain relief . physical examination signs are predictive of a positive sij block,42 and diagnostic block is recommended by multiple us specialty societies to diagnose sij pain.4347 sijf for all patients was performed through a transiliac , muscle - sparing approach , as described previously ( figure 1).29 the triangular shape of the implant is designed to minimize rotation and maximize surface area . patients had to be willing to complete questionnaires and sign a consent form allowing review of medical records by study personnel . chart review included abstraction of demographic details , preoperative sij pain score , medical history focused on the sij and lower back , and procedure details ( procedure date , side treated , and adverse events ) . charts were also reviewed for postoperative follow - up , including dates of visits , numeric pain scale ratings assessed during the visit , global assessments of health status , the occurrence of sij complications and revisions , and the occurrence of new conditions related to the spine and/or hip . details about revision surgery were not collected . as per the study protocol , patients completed questionnaires in clinic , over phone or through email . questionnaires included sij pain rating using a numeric rating scale score ( 010 scale ) , oswestry disability index ( odi , version 2.0 ) , satisfaction with surgery , and a customized survey consisting of questions related to ability to perform various activities compared with that prior to surgery . there was no interventional aspect to this study , and no imaging was reviewed or analyzed . continuous variables were analyzed using mean and standard deviation and compared using student s t - test . ordinal and nominal values were tabulated and compared with chi - square test or fisher s test . one hundred seven patients at seven centers had undergone sijf prior to the cutoff date and completed surveys . patient age varied widely ( 18.687.0 years ) and a history of perisacral trauma ( mostly falls , less commonly motor vehicle accidents ) resulting in sij pain was common ( 35 patients , 32.7% ) . concomitant spine and hip disease were common , and a large proportion of patients had undergone prior lumbar spine surgical procedures ( 36.4% had prior lumbar fusion ) . lumbar stenosis was more common in older patients ; otherwise age was not related to preoperative historical factors . patients were highly debilitated by sij pain , as indicated by high baseline pain ratings ( mean 7.5 ) . the duration of pain prior to enrollment averaged 5.9 years ( range 0.346.0 years ) . over half of the patients had undergone prior physical therapy ( although it could not be determined whether physical therapy was focused on the sij ) ; 1.9% had undergone sij steroid injections ; and 2.8% had undergone rf ablation of the sij nerve root branches . one patient underwent concomitant removal of a subcutaneous gluteus lesion in the right buttock ( pathology report showed calcinosis with focal ossification ) . adverse events related to sijf were uncommon : one patient had mild ileus postoperatively , one had suture material extending from the wound at a second postoperative visit , and one had an adhesive tape allergic reaction . five patients had sijf revision surgery : one patient had early postoperative neuropathic pain related to implant malposition and underwent revision surgery at day 41 . second patient had initial improvement in sij pain followed by pain recurrence at month 18 ; ct scan showed no evidence of bridging bone across the sij , possible loosening of the uppermost implant and inadequate placement of the second implant . third patient had recurrent pain at month 6 ; ct showed posterior placement of the third implant . the patient underwent a revision surgery through an open approach with placement of bone graft . fourth patient had little postoperative improvement and ct scan showed inadequate placement of the caudal - most implant . the patient underwent revision surgery ~3.3 years after index surgery and had also undergone l5s1 lumbar decompression with interbody fusion and pedicle screw instrumentation for lumbar pain . fifth patient was injured in a motor vehicle accident ( t - bone mechanism ) ~9 months postoperatively ; this patient underwent contralateral sijf ( during which further implants were placed on the original side ) as well as a t9 laminotomy and placement of a spinal cord stimulator . prospective follow - up assessments were done in the clinic in 64 cases , by phone in 13 cases and through email in 30 cases . mean sij numeric rating pain score at follow - up was 2.6 , with a mean change of 4.8 points from baseline ( p<0.0001 , table 2 ) . eighty - six patients ( 80.4% ) had improvement in sij pain from baseline of at least 2 points . the proportion of patients who would undergo the procedure again was 83.2% ( 69.2% would definitely and 14.0% would probably undergo the procedure again , table 3 ) . patients reported improvement in ability to perform various activities related to sij pain ( eg , sitting , standing , walking , ascending , and descending stairs ) . improvements in pain scores and final odi level as a function of baseline potential predictors were evaluated using analysis of variance . no statistically significant relationship was seen between improvement in sij pain and a history of prior lumbar fusion , piriformis syndrome , lumbar stenosis , degenerative disk disease , spondylolisthesis , hip osteoarthritis , and workers compensation status . smaller changes in mean improvement in sij pain ( ~1.4 points ) were seen in smokers vs nonsmokers ( p=0.0346 ) . similarly , odi at final follow - up wa10 points higher in smokers vs nonsmokers ( p=0.0427 ) . improvements in sij pain at last follow - up were highly correlated with satisfaction levels , walking compared with prior to surgery , ability to sit for long periods , ability to work , pain medication use , getting in and out of chair , going up and down stairs and getting in and out of a car ( all p<0.0001 ) , desirability of having surgery again ( p=0.0021 ) , and ability to sleep ( p=0.0004 ) . final odi was also closely related to patient responses to specific sij - related questions ( figure 2 ) . twenty - seven ( 25.2% ) patients had non - sij lumbar spine or hip - related surgeries during follow - up ( eg , lumbar spine fusion , kyphoplasty , and hip replacement ) and 26 ( 24.3% ) had nonsurgical procedures related to the spine or hip ( eg , rhizotomies , repeat sij injection , facet or bursa injections ) . patients who underwent subsequent spine or hip surgeries had somewhat smaller improvement in sij pain ( 4.3 vs 5.0 , p=0.2710 ) and somewhat higher final odi score ( 32.9 vs 26.6 , p=0.1582 ) . it is associated with conditions commonly treated surgically48 and was diagnosed at all sites on the basis of typical history , physical examination findings , and a confirmatory diagnostic anesthetic block of the sij producing acute pain relief . this diagnostic method is commonly practiced in the us , is recommended by specialty societies,4347 and was used in prospective clinical trials.40,41,49 joint fusion is a commonly performed procedure in modern spine surgery , and evidence to support minimally invasive sijf using titanium implants is increasing , with a large number of published retrospective case series,2939,50,51 including some with 4 - 35 and 5-year follow - up,34 comparative case series,3739 and prospective multicenter trials.40,41,49 revision rates after sijf are low and have decreased over time.52 moreover , in - trial health care utilization data from the landmark insite ( investigation of sacroiliac fusion treatment ) study showed that minimally invasive sijf is cost - effective compared with nonsurgical treatment.53 ignoring the sij during workup of chronic low back pain may increase health expenditures due to misdiagnosis and potentially failed lumbar fusion surgeries.54 several short - term outcome studies , including a description of operative parameters , have been published . but for two retrospective cohorts,34,35 little information is available regarding long - term outcomes after sijf . in our study , with a minimum follow - up of 3 years , outcomes after sijf using titanium implants were excellent , with large improvements in sij pain and only moderate residual follow - up disability ( oswestry ) scores . mean odi at follow - up ( 28.2 ) in the current study was similar to 12-month values observed in a prospective randomized trial40 ( mean 28.1 ) and 24-month values in a prospective multicenter single - arm study41 ( mean 30.9 ) . although , like in most trials of spine surgery procedures / devices , odi is not restored to 0 ( ie , complete absence of disability related to back pain ) , our results suggest stability of improved function over time . self - rated improvement in various activities of daily living associated with chronic sij dysfunction was high . satisfaction with the surgical procedure was high , and most patients stated they would have the procedure again . improvements in sij pain and final odi values were highly correlated with responses to specific questions about activities of daily living commonly impaired in sij dysfunction . smokers appeared to have somewhat smaller improvements , a finding common in orthopedic trials.55 this finding might be in sync with similar findings from other fusion procedures . the reoperation rate was low and mostly related to two factors : symptomatic implant malposition and recurrence of symptoms due to suboptimal implant placement and/or nonunion . combined with the results from prospective trials , our study indicates that minimally invasive transiliac sijf with triangular titanium implants is an excellent treatment option for patients with sij dysfunction who have failed nonsurgical treatments . patients in our study ( and in our practices in general ) were complex , and many patients were subsequently diagnosed with other conditions of the spine or hip and underwent either surgical ( 25.2% ) or nonsurgical ( 24.3% ) procedures for such conditions . it is likely that the same underlying pathology that causes spine disease ( eg , trauma or osteoarthritic degeneration ) also causes sij dysfunction . although we postulated that final odi and satisfaction rates would be related to the need to undergo subsequent non - sij surgery for these associated conditions , we found only a modest relationship between these factors . however , we do note that the co - occurrence of other spine / hip conditions may result in poorer pain scores , ie , patients with other conditions might experience pain ( eg , at fortin s point ) that they attribute to the sij , which would result in higher disability scores . importantly , no evidence suggests that these other hip / spine conditions are caused by sijf . moreover , finite element analysis has suggested only minimal increases in adjacent segment motion after sijf.56 ( because the sij moves only minimally during normal daily life , stabilization and long - term fusion are not expected to increase adjacent stresses . ) taken together , these data suggest that sij dysfunction can be identified in clinical practice , and sijf can be an effective treatment in the long term . however , many patients are complex , with multiple pain sources , and treatment of other lumbar spine conditions remains challenging . advantages of our study include its combined retrospective and prospective multicenter design , enrollment of patients in different geographic areas , and various practice types ( private , teaching , hybrid , etc ) . our study was retrospective by design and could be subject to biases inherent in this design . some patients did not participate because of inability to make contact or patient refusal . although this could have contributed a bias to our results , the directionality of such bias is not known . methods to diagnose sij pain may have varied across sites and time ; however , the diagnostic algorithm is considered standard , and typically included history , findings on at least three physical examination tests that stress the sij , and a confirmatory diagnostic sij anesthetic block . physical therapy is often provided to patients with chronic low back pain , but not all patients in our cohort underwent such treatment . however , there is no high - quality evidence that physical therapy is effective in chronic sij pain . baseline odi scores were not available in most patients , limiting our ability to determine per patient improvements in this commonly reported parameter . however , follow - up odi scores were similar to those reported in two prospective multicenter us trials at 12 months.40,57 residual odi scores were higher than those reported in studies of degenerative lumbar spondylolisthesis58 or lumbar stenosis,59 but whether this reflects patient complexity or effectiveness of sijf is not known . we did not perform standardized long - term imaging of the sij . in the absence of clinical signs suggestive of implant loosening ( eg , failure of sij pain to improve after sijf or pain recurrence ) , routine cross - sectional imaging of the sij may have little , if any , clinical value . one study of the same sijf procedure showed a high rate of growth across the sij at 5 years.34 mean follow - up in our cohort was 3.7 years , which represents one of the longest postoperative experiences for this procedure reported to date . however , continued follow - up of such patients may help to define even longer term ( 5 years ) outcomes . many patients in our cohort had concomitant spine disease at baseline and a substantial fraction underwent other spine surgeries or interventional spine or hip procedures . such interventions may have limited improvements in odi or affected patients abilities to perform activities of daily living . our data do not allow us to discern whether the rate of subsequent non - sij surgeries was high or low ; rather these data reflect the complexity of the patient population . many patients had multiple pain generators . whether some patients underwent sijf when the underlying diagnosis was different however , responses at 3 + years appeared positive and consistent with improvements seen in prospective clinical trials of sijf . patients participating in this study represent the earliest use of the ifuse device ; it is possible that with increased experience with the device , both patient selection and technical aspects related to the procedure may have improved . prospective trials have shown decreases in opioid use after minimally invasive sijf.40,41,57 our study collected outcomes of patients treated through a transiliac lateral approach , with implants designed to resist rotation after implantation ( due to the triangular shape ) and for biological fixation in bone . other devices are available to perform sijf ; however , because these devices differ from those we used , it is unknown whether our results are applicable to such devices . moreover , our results may not apply to other surgical approaches to sijf , such as direct posterior or combined approaches , allograft - only fusions , distraction arthrodesis using cannulated or hollow screws , or other procedures . intermediate- to long - term follow - up after lateral , transiliac sijf using titanium implants shows durable , clinically important improvements in pain and disability , with high satisfaction rates .

backgroundsacroiliac joint ( sij ) fusion ( sijf ) , first performed 95 years ago , has become an increasingly accepted surgical option for chronic sij dysfunction . few studies have reported intermediate- or long - term outcomes after sijf.objectivethe objective of this study is to determine patient - based outcomes after sijf for chronic sij dysfunction due to degenerative sacroiliitis or sij disruption at 3 years of follow-up.methodsconsecutive patients who underwent sijf prior to december 2012 were contacted over phone or through email . participants completed questionnaires in clinic , over phone or by email , regarding sij pain , activities related to sij dysfunction , and the oswestry disability index . charts were reviewed to extract baseline parameters and the clinical course of follow-up.resultsone hundred seven patients were eligible and participated in this study . mean ( standard deviation ) preoperative sij pain score was 7.5 ( 1.7 ) . at mean follow - up of 3.7 years , the mean sij pain score was 2.6 ( representing a 4.8-point improvement from baseline , p<0.0001 ) and the mean oswestry disability index was 28.2 . the ability to perform activities commonly impaired by sij dysfunction showed positive improvements in most patients . sij revision surgery was uncommon ( five patients , 4.7% ) . fourteen patients ( 13.1% ) underwent contralateral sijf during follow - up , 25.2% of patients had additional non - sij - related lumbar spine or hip surgeries during follow-up.conclusionin intermediate- to long - term follow - up , minimally invasive transiliac sijf was associated with improved pain , low disability scores , and improved ability to perform activities of daily living .

Background Methods Results Discussion Conclusion

biological nitrification and denitrification are key processes to remove nitrogen from wastewater and have become more important due to stringent discharge regulations . research has shown that nitrifier 's population ( ammonia oxidizing bacteria ( aob ) + nitrite oxidizing bacteria ( nob ) ) should be more than 58% of the biomass for good nitrification . however , nitrifiers have slow growth rates and they are also believed to be sensitive to environmental changes such as toxic shocks , ph , and temperature changes . due to these characteristics , it is difficult to obtain and maintain sufficient nitrifiers in wastewater treatment plants ( wwtps ) , if solids retention time ( srt ) gets shorter . and as a consequence , it is difficult to maintain nitrification in municipal and industrial wastewater treatment plants ( wwtps ) . in conventional activated sludge processes , the long srt increases the concentration of mixed liquor suspended solids ( mlss ) which requires large tanks and clarifiers to accommodate the accumulation of solids inventory . in wastewater treatment plants a significant source of ammonia is generated within the plant in anaerobic digesters . dewatering operations on anaerobic digester sludge and supernatant produces a stream of filtrate and centrate called reject water which contains up to 15~25% of the total nitrogen load to the plant [ 6 , 7 ] . a number of side - stream reject water treatment processes have emerged in recent years , such as oxygen - limited autotrophic nitrification / denitrification ( oland ) process and anaerobic ammonium oxidation ( anammox ) process . in addition to reducing the impact of recycle nitrogen loadings , some processes promote the potential for cultivating a stable source of nitrifying microorganisms to bioaugment the main stream , such as mainstream autotrophic recycle enabling enhanced n removal ( maureen process ) , bioaugmentation batch enhanced technology ( babe ) . the amount of nitrifiers needed to be grown in the main treatment stream is reduced by the amount of new nitrifiers supplied from the reject water side - stream treatment system . since a smaller amount of nitrifiers need to be grown in the mainstream treatment plant , the nitrification section ( oxic section ) can be smaller and the mlss concentration decreases since higher srt is not required . considering the nitrifier 's diversity and kinetic differences , even what might seem to be a small environmental difference seemingly can impact nitrifier 's activity . the addition of enriched nitrifiers from reject water to municipal wastewater with its unique microbial communities may have limited success if the added nitrifiers are not adaptive to the conditions inherent to a sewage treatment system , which can lead to the reduction of nitrifier 's activity or even to a decay of nitrifiers , or nitrifiers washout with effluent head et al . . therefore , the community survivability and functional stability of the seed source are common problems in bioaugmentation applications , which may determine the results of bioaugmentation . so , it is important to investigate the enriched nitrifier 's population diversity , fate , and functional variation after bioaugmentation to the main stream . berends et al . have pointed out that nitrosomonas and nitrobacter were the dominant aob and nob in the seed source , respectively . so far , many studies have measured structural diversity of aob and stability of ammonia oxidization in main stream activated sludge systems bioaugmented from side - stream reject water treatment systems [ 5 , 15 ] . smith and oerther have also theorized that increased population diversity from input of microorganisms possessing desirable properties from side - stream processes to a mainstream process creates a more robust system that is less susceptible to inhibition or upsets . however , there is a lack of information for the variation of nitrifier 's community structure and function during bioaugmentation . furthermore , there is also a lack of information about the diversity of nob in order to determine which species are successfully bioaugmented and to understand the mechanisms that enable them to be incorporated into main - stream biomass . this information will provide useful insights into the second step of the nitrification process since the nitrite oxidation step may be less stable than the ammonia oxidation step for side - stream reject water treatment systems . this study focuses on investigating functional stability ( ammonia uptake rate ( aur ) and nitrite uptake rate ( nur ) ) of the inoculated nitrifiers after being bioaugmented from side - stream reject water treatment systems by measuring the shifts in nitrification ability in the main stream . furthermore , the fate of the seed source was analyzed by comparing the shifts in the population and structure of nitrifiers in the activated sludge and that in the effluent of the main - stream reactor by using fluorescent in situ hybridization ( fish ) . side - stream reactor , a 5-l sequencing batch reactor ( sbr ) , was operated with a hydraulic retention time ( hrt ) of 2.5 d and srt of 7 d at 20c . feeding consisted of adding 500 ml of sludge reject water four times per day , every 6 h ( feed , 1 min ; anoxic , 59 min ; oxic , 240 min ; settle , 50 min ; decant , 1 min ; idle , 9 min ) . the ph was controlled at 7~8 by addition of sodium bicarbonate ( nahco3 ) to the sludge reject water . oxygen concentration was monitored automatically and maintained approximately 2.0 mg / l in the oxic phase . the sludge reject water used in the experiment was the supernatant of the anaerobic digestion tank of the dangjiacun waste water treatment plant in xi'an , china . it was delivered to the laboratory once per week and stored in a closed container at 4c . sludge processing includes cothickening of primary sludge and waste activated sludge and anaerobic digestion for about 14~17 d at ambient temperature , followed by rethickening and dewatering by centrifuge . the sludge reject water contained 1949 562 mg / l total chemical oxygen demand ( tcod ) , 455 131 mg / l soluble chemical oxygen demand ( scod ) , 812 135 mg / l total kjeldahl nitrogen ( tkn ) , and 3035 395 mg / l alkalinity ( caco3 ) ( mean value of fresh sludge reject water during the experiment ) . main - stream reactor , a 4-l sbr , was operated to treat a synthetic domestic wastewater , containing 300 mg / l glucose - cod , 50 mg / l nh4-n , and 5 mg / l phosphorous , 50 10 mg / l edta , 5 10 mg / l znso47h2o , 5.06 10 mg / l mncl24h2o , 4.99 10 mg / l feso47h2o , 1.1 10 mg / l ( nh4)6mo7o244h2o , and 1.57 10 mg / l cuso45h2o . the ph was controlled at 7~8 by addition of sodium bicarbonate ( nahco3 ) to the synthetic domestic water . oxygen concentration was monitored automatically and maintained approximately 2.0 mg / l in the oxic phase . the reactor was operated with a hydraulic retention time ( hrt ) of 8 h and sludge retention time ( srt ) of 5 d at 20c , and the nitrogen load was 150 mg n / l / d , about half of the side stream ( 812 mg n / l/2.5 d = 324.8 mg the reactor was fed 2 l of synthetic wastewater six times daily , every 4 h ( feed , 1 min ; anoxic , 89 min ; oxic , 90 min ; settle , 50 min ; decant , 1 min ; idle , 8 min ) . the main - stream reactor was run for about three months before it was seeded once daily for 50 d with 50 ml seed source . the volume of the seed source for bioaugmentation was calculated as follows : for the side stream reactor , the nitrogen load was 812 mg n / d , and the wasted sludge ( enriched nitrifiers ) was equivalent to the amount of mlvss in 700 ml mixed liquor per day ( the srt was about 7 days ) , so the yield of enriched nitrifiers was 700 ml/1624 mg n = 0.43 ml / mg n. for the main - stream reactor , the nitrogen load was 50 mg n / l 12 l / d = 600 mg n / d , and accordingly the nitrogen load was about 20% of the main stream , so the volume of seed source matched with main - stream reactor should be 600 mg n 20% 0.43 ml / mg n = 50 ml . to evaluate the activity of nitrifiers , nitrification rates were measured in batch scale ( 400 ml ) outside of the reactors . the 400 ml biomass for these tests originated from the reactors and was maintained at 20c . / l . considering the nitrifier 's kinetic difference between the side stream and main stream , the initial ammonia and nitrite concentration used in the test was about the maximum concentration in the reactors . l for the side stream and 50 mg / l for the main - stream reactor , and the initial nitrite concentration was 50 mg / l for the side stream and 20 mg / 8 samples were taken over time , the aur or nur ( linear correlation coefficient r > 0.98 ) was determined by measuring the consumption of nh4-n and no2-n . mixed liquor volatile suspended solids ( mlvss ) , nh4-n , no2-n , no3-n , and chemical oxygen demand ( cod ) were analyzed in accordance with standard methods . dissolved oxygen ( do ) was measured with hanna hi9143 dissolved oxygen meters ; oxidation - reduction potential ( orp ) and ph were measured with hanna hi9025 orp / ph meter . samples ( 2 ml ) were taken at days 30th ( the day before bioaugmentation ) , 38th , 57th , and 77th and fixed in 4% paraformaldehyde . direct ultrasonification ( 20 khz , five minutes ) was applied to break up large flocs prior to hybridization . the fish analysis was performed according to the protocol previously described by amann et al . with fluorescently labeled probes listed in table 1 . a 3 l sample was applied to each well of the slide and then dried at 46c . the samples were then dehydrated in 50% , 80% , and 98% for 3 minutes and dried at 46c . 8 l of hybridization buffer ( 0.9 m nacl , 20 mm tris - hcl , 0.01% ( w / v ) sds ) with x% ( v / v ) deionized formamide ( x is listed in table 1 ) and 1 l of fluorescently labeled probe ( 50 ng/l ) were added to each well . the slide then washed in 50 ml prewarmed washing buffer ( 0.3 m nacl , 20 mm tris - hcl , 0.01% ( w / v ) sds ) . slides were then stained with 10 l of 1 g / ml dapi for 5 minutes . the slides were rinsed again by serial washing in deionized water and allowed to natural air drying in dark . the software image pro - plus 7.0 was used for counting the targeted population in relation to the total microbial population in the sample . the cell counts were performed in triplicate using independently prepared fluorescence in situ hybridization ( fish ) slides for each probe . the side - stream reactor was operated about two years before the bioaugmentation experiment , the reactor performance was stable , and full nitrification was achieved during the experiment . g / l , the effluent nh4-n concentration was lower than 10 mg / l and the conversion efficiency was greater than 98% , and the effluent no2-n concentration was lower than 1 mg / l ( figure 1 ) . the maximum ammonia uptake rate ( aur ) was 66.9 mg nh4-n / lh , and the maximum nitrite uptake rate ( nur ) was 34.4 mg no2-n / lh of the activated sludge in the side - stream reactor ( seed source or inoculums ) . fish analysis showed that the aob / dapi was about 18.8% 2.7% in the seed source , aob consisted of 83% nitrosococcus mobilis lineage , and no nitrosospira spp . the nob / dapi was about 14.4% 2.8% , nob consisted of 65% nitrobacter , 19% nitrospina gracilis , 10% nitrospira , and 6% nitrococcus mobilis ( mean values for four samples , table 2 ) . and the kinetics characterization of the enriched nitrifiers was shown in yu et al . the main - stream reactor was operated with an apparent srt near design srtmin in engineering ( 5 days ) as demonstrated by high nh4-n concentration and low nox - n concentration in the effluent for about three months and achieved a stable condition before the start of bioaugmentation . during the stable stage before bioaugmentation , the mlvss was 1.75 0.08 g / l ( mean value ) , and the nh4-n , no2-n and no3-n concentration in the effluent was 40~42 mg / l , 0.4~0.8 mg / l , and 1~2 mg / l , respectively . it showed that the nh4-n concentration in the effluent decreased gradually after bioaugmentation , and at day 79 , the nh4-n can not be detected in the effluent , while the concentration of no2-n , no3-n in the effluent increased gradually . at the same time , the sum of total inorganic nitrogen concentration ( nh4-n + no2-n + no3-n ) in the effluent of main - stream reactor decreased gradually after bioaugmentation , and about half of the removed ammonia is not retrieved in the total oxidized metabolites ( sum of nitrite and nitrate ) . because the fill ratio of the reactor is 50% , and the reactor went to an anoxic phase after fill , then about 50% of the nitrite and nitrate in a cycle will be denitrified in next cycle , and the denitrification ability increased gradually along with the increase of nitrification ability . the shifts in nitrification performance had a strong relationship with the chemical analysis of nitrogen parameters ( figure 3 ) . the aur increased from 2.88 to 13.36 mg nh4-n / lh and the nur increased from 0.76 to 4.34 mg no2-n / lh for the main - stream reactor . the aur increased much more than the nur , corresponding to the evident nitrite accumulation in the main - stream reactor after bioaugmentation ( figure 2 ) . the shifts in aob and nob composition in the main - stream reactor during bioaugmentation were shown in table 3 . before bioaugmentation , the aob / dapi and nob / dapi were 3.7 1.2% and 0.11 0.1% , respectively ( day 30 , samples pre - bioaugmentation ) . once bioaugmentation started , the percentage of aob / dapi and nob / dapi increased quickly at the beginning of bioaugmentation ( days 0~8 ) , the aob / dapi and nob / dapi increased to 7.0 2.2% and 3.4 1.1% , respectively , while during days 57~77 , the fractions of aob and nob relative to the total microbial population in the study did not exhibit an evident difference . table 3 also showed the percentage of nitrifiers in the ( suspended solids ) ss flow out with the effluent after bioaugmentation . both the percentage of aob and nob tend to decrease with the bioaugmentation time . in the initial time of bioaugmentation ( day 38 ) , the percentage of aob / dapi in the effluent was about 2.5 times of that in activated sludge , but the ratio was close at day 57 and day 77 . it was surprising that the percentage of nob / dapi in the effluent was lower than that in the activated sludge at day 57 and day 77 . figure 4 showed the community structure shifts in aob and nob in the main - stream reactor during bioaugmentation according to the detection of nitrifiers in the activated sludge by fish techniques . aob were dominated by members related to nitrosococcus mobilis lineage ( probe nmv ) , which made up about 60~80% of all detected aob ( probe nso1225 ) . no ammonia - oxidizing bacteria stainable with nsv443 ( nitrosospira spp . ) were presented . the nob in the main - stream reactor belonged to the genus nitrospira ( probe ntspa662 ) , and no nitrobacter ( probe nit3 ) , nitrococcus mobilis ( probe ntcoc206 ) , and nitrospina gracilis ( probe ntspn693 ) appeared before bioaugmentation . however , after bioaugmentation , the percentage of nitrobacter in nob increased and that of nitrospira decreased gradually , the dominant nob transferred gradually from nitrospira to nitrobacter ( the dominant nob in the side - stream reactor ) , and the nitrospina gracilis and nitrococcus mobilis were not detected . the low concentration of ammonia and nitrite detected in the effluent of the side - stream reactor ( figure 1 ) reflected that stable and full nitrification was achieved . and the biomass in the side - stream reactor achieved high nitrification rates ( 66.9 mg nh4-n / lh of aur and 34.4 mg no2-n / lh of nur ) , wett et al . also reported a high nitrification rate of 50~58.3 mg nh4-n / lh in an sbr treating sludge reject water at 20~25c . fish analysis showed that aob / dapi and nob / dapi in the seed source averaged 18.8% and 14.4% , respectively ( table 2 ) , and the average ratio of nitrifiers to total bacteria ( dapi ) was about 33.2% . sludge reject water ( 631 47 mg nh4-n / l , lower than the ammonia concentration in the research ) . these data were much higher than data in the activated sludge of the normal wwtp with full nitrification in which nitrifiers account for approximately 5~8% . the big difference of nitrifier 's content in the activated sludge between the main - stream reactor and the side - stream reactor is due to the difference of c / n ratio in the feedings . high ammonium concentration and low c / n ratio stimulates a high fraction of nitrifiers content in the sludge . . the significant increases of the aur and the nur in the main stream suggests that bioaugmentation with nitrifiers enriched by sludge reject water is a promising approach to enhance the nitrification performance in sewage treatment . some problems have to be pointed out , such as nitrite accumulation in the main - stream and the decrease of nitrification ability of the seed source . for the main stream reactor , nitrite concentration accumulated gradually after bioaugmentation and reached 10 mg no2-n / l at the end of experiment . the nitrite concentration at the end of the experiment was due to the unequal and asynchronous increase of the aur and nur . the increase in the aur was 10.48 mg nh4-n / lh and the increase in the nur was just 3.58 mg no2-n / lh . bioaugmentation indeed enhances nitrification in the main - stream reactor , but can not function fully . that is to say , not all nitrifiers added to the main stream work well and part of their nitrification capability is lost during bioaugmentation . for aob , 50 ml per day of the seed source was added to 4 l mainstream bioreactor volume / day , that is , an 80-fold dilution . srt was 5 d. the aur due to the seed source was calculated to be 0.67 mg nh4-n / lh per day ( ( 1 1/5 ) 66.9/80 = 0.67 ) , but the slope of the aur curve ( figure 4 ) was about 0.28 mg nh4-n / lh . the nur due to the seed source should have been 0.34 mg no2-n / lh per day ( ( 1 1/5 ) 34.4/80 = 0.34 ) , but the slope of the nur curve ( figure 3 ) was about 0.07 mg no2-n / lh . this difference indicated that most of the nitrification ability ( 58% of aur and 80% of nur ) introduced by the seed source was lost in the main - stream reactor . therefore , the nitrite oxidation step may be less stable than ammonia oxidation for main - stream systems after bioaugmentation . compared to side stream , the anoxic / aerobic phase ratio in the main - stream reactor was much higher and relativly lower srt leading to a lower nitrifier growth . the percentage of nitrifiers to total biomass increased quickly and reached a stable level at day 38 ( 8 days after bioaugmentation ) , and the aob+nob / dapi was approximate 14.0 5.2% , which was much higher than the pre - bioaugmentation value of 3.8 1.3% . the ratio of aob / nob was 1.72.1 in the main - stream reactor after bioaugmentation due to the longer generation time of nob . however , aobi/nobi ( aobi = aobi aob0 , nobi = nobi nob0 , i , the day number after bioaugmentation . aob0 , nob0 , the content of aob or nob in the activated sludge before bioaugmentation ) in the main - stream reactor was approximate 1.0 , lower than the ratio of aob / nob in the seed source (= 1.3 ) . these results indicate that much more of the population of aob in the seed source was lost than the population of the nob . in addition , the nur decrease was much more than the aur decrease of the seed source after bioaugmentation . apparently , the nitrifier 's loss with the decant liquor could not be accounted for by the nitrite accumulation . the aob / dapi in the effluent was about 2.5 times of that in the activated sludge in the initial stage of bioaugmentation , while it approached to the aob / dapi in the activated sludge during the course of the experiment . it is interesting that the percentage of nob / dapi was much lower than the percentage of aob / dapi in the effluent , and it also was lower than the nob / dapi in the activated sludge at day 57 and day 77 . these results also indicated that washout with effluent should not be the main reason for nitrification loss of the seeded nitrifiers . the adhesion characteristics of nitrifiers in activated sludge also contributed to the nitrifier 's behavior . larsen et al . discovered that nitrifiers can form relatively dense and strong microcolonies in activated sludge and remain almost intact even under extreme physical and chemical conditions so that nob are much more difficult to deflocculate than aob . comparing the microbial structure in two reactors revealed that the community structure of aob in the side - stream reactor is similar with that in the main - stream reactor , while a significant difference is observed for the nob community . nitrobacter was the dominating nob in the seed source , which exhibits a low affinity to the substrate and a high maximum growth rate [ 2931 ] . however , after being added to the main - stream reactor , in which the nitrite concentration was relativly low and the nitrospira was the dominating nob , the nitrobacter can not surpass the nitrospira in competing for nitrite . therefore , nob structure mismatch resulting from substrate concentration may be one of the main reasons for more nur loss than aur loss during the course of bioaugmentation . in conclusion , bioaugmentation exerted a strong influence on the microbial community and activity of the nitrifiers.after bioaugmentation , the ammonia uptake rate ( aur ) increased from 2.88 to 13.36 mg nh3-n / lh , and the nitrite uptake rate ( nur ) increased from 0.76 to 4.34 mg no2-n / lh for main - stream reactor.fish analysis showed that nitrosococcus mobilis lineage was the dominant aob which matches with the main - stream reactor , while the nob in the seed source was nitrobacter , and mismatches the main - stream reactor in which the dominant nob was nitrospira spp . before bioaugmentation ; however , it gradually transferred to nitrobacter spp . ( the dominant nob in the seed source ) after bioaugmentation.although aob was inclined to wash out with effluent compared with nob , the nur of the seed source lost more than that of aur due to the mismatch of nob structure . after bioaugmentation , the ammonia uptake rate ( aur ) increased from 2.88 to 13.36 mg nh3-n / lh , and the nitrite uptake rate ( nur ) increased from 0.76 to 4.34 mg no2-n / lh for main - stream reactor . fish analysis showed that nitrosococcus mobilis lineage was the dominant aob which matches with the main - stream reactor , while the nob in the seed source was nitrobacter , and mismatches the main - stream reactor in which the dominant nob was nitrospira spp . before bioaugmentation ; however , it gradually transferred to nitrobacter spp . although aob was inclined to wash out with effluent compared with nob , the nur of the seed source lost more than that of aur due to the mismatch of nob structure .

this study used two laboratory - scale sequencing batch reactors ( sbrs ) to evaluate the shifts in nitrification kinetics and microbial communities of an activated sludge sewage treatment system ( main stream ) during bioaugmentation with nitrifiers cultivated on real sludge reject water ( side stream ) . although bioaugmentation exerted a strong influence on the microbial community and the nitrification kinetics in the main stream , there was 58% of maximum ammonia uptake rate ( aur ) and 80% of maximum nitrite uptake rate ( nur ) loss of the seed source after bioaugmentation . in addition , nitrite accumulation occurred during bioaugmentation due to the unequal and asynchronous increase of the aur ( from 2.88 to 13.36 mg n / lh ) and nur ( from 0.76 to 4.34 mg n / lh ) . fish results showed that ammonia oxidizing bacteria ( aob ) was inclined to be washed out with effluent in contrast to nitrite oxidizing bacteria ( nob ) , and nitrosococcus mobilis lineage was the dominant aob , while the dominant nob in the main stream gradually transferred from nitrospira to nitrobacter . nitrospina and nitrococcus which existed in the seed source could not be detected in the main stream . it can be inferred that nitrite accumulation occurred due to the mismatch of nob structure but washed out with effluent .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

because traditional methods only provide a static picture of the chromatin landscape , it is not surprising that steve henikoff ( fred hutchinson cancer research center , seattle ) , who gave the first talk , chose to summarize efforts from his lab to develop newer methodologies that allow the study of a highly dynamic epigenome at single base pair resolution . he started by explaining that by combining the use of standard mnase digestion and paired - end sequencing to map the budding yeast epigenome , his group was able to characterize dna - binding features of more than a hundred transcription factors , identifying protected and exposed regions around the binding site of each of them . method for the study of the drosophila genome , they showed that the rate of nucleosome turnover is faster at sites bound by trithorax group proteins than at sites bound by polycomb - group proteins ; a difference that is likely to be important for epigenome maintenance and gene regulation . these studies provided an extraordinarily detailed genome - wide view of rnap ii pausing and nucleosome turnover . finally , he described a method that employs in vivo biotin labeling of nuclear envelope proteins present in individual cell types that allowed the purification of nuclei from different cell types in arabidopsis for the analysis of cell - specific gene expression patterns and chromatin features . recently , the same method was used to isolate nuclei from muscle and non - muscle cells of adult c. elegans . henikoff and colleagues were able to identify genes that are specifically expressed in muscle tissues and found that these genes are depleted of nucleosomes at promoters and gene bodies in muscle cells but not in other tissues . this method could potentially be applied to compare epigenetic profiles among different cell types or tissues within any organism . also interested in developing assays that facilitate the study of a dynamic epigenome , peter jones s lab ( university of southern california , los angeles ) used a single - molecule , high - resolution nucleosome positioning assay , called nucleosome occupancy methylome - sequencing ( nome - seq ) , to demonstrate that active ( but not inactive ) non - cpg island promoters display a nucleosome - depleted region ( ndr ) , something previously studied only at cpg island promoters . jones explained that the strength of the non - cpg ndr correlates with the expression level of the corresponding gene and suggested that epigenetic status of non - cpg island promoters should therefore be also taken into consideration in cancer studies . jones explained that nucleosome occupancy precedes dna methylation and that de novo dna methylation does not occur in the absence of nucleosomes . studying the process on individual dna molecules , jones and colleagues showed that the unmethylated oct4 distal enhancer has a ndr that is maintained by binding of oct4 , which is itself required for oct4 expression . interestingly , de novo methylation follows the loss of the ndr , stabilizing a silenced configuration of the gene . in a different example , jones described his myod1 studies . the myod1 gene is repressed by polycomb - group proteins and is autoregulated ( having binding sites for the protein it codes for in its enhancer and promoter ) . exogenous myod1 activates its own transcription by binding first to the myod enhancer , which leads to a ndr at the promoter . interestingly , cells that normally express myod1 ( human rhabdomyosarcoma cell line ) and cells in which myod1 is repressed ( normal human fibroblast cell line ) respond differently to forced expression of myod1 than cells in which myod1 is silenced ( rko colorectal cancer cell line ) . jones found that the myod1 enhancers in both active and repressed myod1 cell lines ( but not in rko cells ) are enriched in the h3k4me1 modification , which appears to mark a permissive state that is receptive and could potentially be activated by the binding of master regulatory factors . in genome - wide studies , jones observed that a high percentage of polycomb target genes have enhancers that acquire a permissive state , which could indicate a general mechanism for the regulation of cell - fate reprogramming . moving the focus of attention now to heterochromatin function and formation , hiten madhani ( university of california , san francisco ) discussed studies from his lab investigating how noncoding rnas ( ncrnas ) trigger heterochromatin formation . he presented data that identifies a conserved sequence - specific rna binding protein that recognizes ncrnas to promote silencing . this essential protein is physically recruited to centromeric repeat - derived ncrnas in fission yeast and represents the molecular component linking ncrnas and the histone h3 methyltransferase clr4 in an rnai - independent pathway . he discussed the implications of these findings as h3k9 methylation is generally triggered in fungi and animals , where it is clear that rnai is not a universal trigger of heterochromatin formation . heterochromatin was also the focus of thomas jenuwein ( max - plank institute , freiburg , germany ) , who presented work from his lab on the identification of transcription factor binding sites within major satellite repeats . specifically , his lab showed that pax3 binds to satellite repeats and represses their transcription . in imef cells mutant for pax3 , he observed loss of heterochromatic histone methyl marks . in addition , double mutant cells in which pax9 is also mutated , display massive deregulation of heterochromatic transcripts , indicating that pax3 and pax9 may perform redundant functions . pax3 and pax9 bind in the vicinity of the transcription start sites within major satellite repeats . jenuwein proposed an interesting model in which the distinction between euchromatin and heterochromatin would be given by the way transcription factor binding sites are organized . in this model , heterochromatin is crowded with transcription factor binding sites , but these sites are not organized in the same way in which they appear in euchromatin ( i.e. , mainly in promoters and enhancers ) . he argued that the same transcription factors work in heterochromatin and euchromatin but , because in heterochromatin the binding sites lack the typical euchromatic organization , transcription factor binding is uncoordinated , defining that region to become heterochromatic ( e.g. , by triggering the recruitment of specific enzymes that modify chromatin ) . identifying the epigenetic status of all genomic loci in each cell type at every given time is the goal of many laboratories working in the field . the potential application of this knowledge is enormous , including the design of diagnostic tools that could detect disease at earlier stages , just by analyzing the epigenome . a pioneer in this type of work , bing ren ( university of california , san diego ) described efforts from his lab toward the identification of tissue - specific gene expression programs by genome - wide prediction of enhancers , promoters and insulators . a few years ago , his lab identified specific chromatin signatures present at different cis - regulatory regions . now , they have begun to systematically map these signatures in a genome - wide scale . ren and colleagues performed the first survey of this kind using a chip - seq approach on 20 mouse tissues . ren reported the identification of almost 300,000 regulatory sequences in the genome , the majority of which were confirmed by reporter assay arrays . these newly identified cis - elements provide annotation for 11% of the mouse genome , which represent a significantly larger portion than that occupied by protein - coding sequences . they could also associate putative enhancers with tissue - specific chromatin modifications and with the enrichment for lineage - specific transcription - binding motifs . interestingly , out of 206 de novo motifs , 126 appear to match a known transcription binding motif and , of these , 75% have been shown to play a role in gene regulation in a particular tissue . ren s lab is also trying to understand how these cis - regulatory elements are organized along the genome and work together in order to achieve gene regulation . using hi - c technology , which allows genome - wide analysis of higher order chromatin structure this topological view indicates that the genome is composed of megabase sized topological domain structures that determine specific chromosomal territories , which confirm previous computational models . raul mostoslavsky ( massachusetts general hospital , harvard medical school , ma ) and katrin chua ( stanford university , ca ) moved the discussion toward sirtuins . mostoslavsky and chua talked about sirt6 , one of the seven mammalian sirtuins that shares homology with the yeast sir2 . sirt6 is a chromatin - associated protein that promotes resistance to dna damage and suppresses genomic instability in mouse cells . loss of sirt6 leads to an aging - like phenotype in mice that is likely to arise from its involvement in aging , metabolism and cancer . in the absence of sirt6 , the uptake of glucose increases significantly . in sirt6 knockout cells , lactate production increases and oxygen consumption decreases . sirt6 regulates h3k9 acetylation at the promoter of glycolytic genes , causing an increase in their transcription . sirt6 therefore functions as a critical regulator of glucose uptake and glycolysis . in his talk , mostoslavsky discussed some new results indicating a role for sirt6 in the modulation of transcription beyond its involvement in chromatin compaction . chua discussed ongoing work from her lab attempting to identify new cellular functions and contexts for sirt6 , in order to understand its role in physiology and disease . she explained that sirt6 knockout mice die in approximately 4 weeks due to a hypoglycemic crisis but , when provided with a diet rich in fat , mice are able to survive past this crisis period . these longer - lived sirt6 mice show behavioral defects that are reminiscent of mouse models of alzheimer or autism , for example . paolo sassone - corsi ( uci ) discussed how the cellular metabolism and the epigenome might communicate with each other in previously unsuspected ways . specifically , he suggested that changes in the levels of cellular metabolites ( which are partly controlled by the circadian clock ) could influence the epigenome . sassone - corsi explained that because histone - modifying enzymes could sense the cellular metabolism , it is possible that they interpret the metabolic state of a cell at a given time by modifying chromatin in a specific cyclic manner . as an example , the oscillatory nature of nad+ dictates oscillatory acetylation of sirt1 targets , even though sirt1 levels do not oscillate . an important fraction of the genome is transcriptionally controlled in a circadian manner ; it is intriguing to now start to understand how the circadian clock could act through the epigenome to exert its regulatory function . the interpretation of this epigenetic signal is mediated by the so - called readers , which are specialized proteins that are able to interpret the epigenetic language . or gozani s lab ( stanford university ) investigates the role of lysine methylation in disease . at this symposium gozani and colleagues identified the bah domain in orc1 ( the largest subunit of the origin recognition complex and previously implicated in primordial dwarfism ) as the link between h4k20me2 , orc1 and primordial dwarfism . importantly , mutation in the bah domain of orc1 is implicated in the etiology of the meier - gorlin syndrome ( a primordial dwarfism syndrome ) , due to the impairment of h4k20me2 recognition . there is therefore a potential role for h4k20me2 in determining organism size in mice . remarkably , h4k20me2 depletion in zebrafish results in dwarfism . axel imhof ( ludwig - maimilians universitt , mnchen , germany ) discussed the role that rna molecules play in chromatin . he explained that the binding of rna to in vitro assembled chromatin appears to open chromatin , and the removal of rna from chromatin leads to its compaction . this may be due to the removal of many ( rna - dependent ) factors from chromatin , which he analyzed using an in vitro chromatin assembly system prepared from drosophila embryos . in his talk , imhof suggested that the formation of a chromatin associated rnp network may be responsible for maintaining an accessible chromatin structure . the role of cohesin in the regulation of gene expression was the topic of kyoko yokomori s ( uci ) talk . in studies of the -globin locus , she showed that cohesin , and the cohesin loading factor nipbl , bind to the locus control region at the ctcf insulator region and distal enhancer , upon differentiation . cohesin binding is critical for long - range chromatin interactions between the enhancer and the promoter and is important for -globin gene expression in human cells . nipbl haploinsufficiency affects cohesin binding , altering chromatin interactions and affecting gene expression . continuing the discussion on the mechanism by which the epigenome regulates transcriptional activation , jean marc egly ( igbmc , inserm , strasbourg , france ) talked about his studies of the intriguing role of nucleosome excision repair ( ner ) factors in transcription . egly explained that , upon gene activation , the rnap ii transcription machinery associates with ner factors at the promoter . egly and colleagues observed that , in patients with silenced ner factors , the changes necessary for transcription to initiate ( such as , h3k4me , h2k9me , h3k9/k14ac and dna demethylation ) do not occur . deficiencies in ner factors impede the recruitment of other necessary remodeling factors that modify chromatin . specifically , xpg and xpf appear to have a role in the formation of the necessary chromatin loop between the promoter and terminator . joseph ecker ( hhmi and the salk institute for biological studies , san diego ) talked about his arabidopsis studies of phenotypic diversity . his lab analyzed spontaneous changes in dna methylation , which are able to produce stable epialleles that can modify the phenotype . through examination of plants propagated by single - seed descent across 30 generations , ecker and colleagues identified single methylation polymorphisms and cg differentially methylated regions that provide evidence for an epigenetic mechanism of phenotypic diversity . these new epialleles are sequence - independent , can alter transcription and can be transmitted to the offspring , providing evidence for an epigenetic mechanism of phenotypic diversity that can occur in the absence of genetic mutation . ecker ended his talk with the interesting question of whether these events could be regulated by the environment . jean antoine girault ( inserm , paris , france ) discussed the chromatin changes involved in the response to behavioral modifications . he explained that the dopamine signaling pathway is involved in several neurological and psychiatric disorders and in drug addiction . dopamine regulates a nucleosomal response through a kinase / phosphatase cascade that mediates the nuclear accumulation of darpp-32 , a potent inhibitor of protein phosphatase-1 . mutations in darpp-32 alter the effect of drugs of abuse , which underlines the importance of darpp-32 in this response . girault and colleagues are currently measuring transcriptional and epigenetic changes mediated by darpp-32 using a series of clever tools that allow the isolation of polysomes or nuclei from specific cells of interest . in preliminary results , they observed that cocaine exerts different effects on histone posttranslational modifications in nuclei from dopamine receptor 1 ( d1r)-expressing cells or in nuclei from cells expressing the dopamine receptor 2 ( d2r ) . emiliana borrelli ( uci ) discussed the exciting possibility that neurological epigenetic effects could be induced by dysfunctional neurotransmitter 's control of brain functions . she presented results from studies of dopamine receptor d2r conditional knockout mice in which d2r expression is lost specifically in presynaptic neurons . these animals appear to be excellent models of psychosis and schizophrenia , as suggested by their specific behaviors , which mimic the symptoms of these human neurological disorders . analysis of rna extracted from the frontal cortex , an area shown to be involved in schizophrenia , showed that almost 2,000 ( out of a total of 25,000 ) rnas were downregulated in d2r presynaptic mutant mice . further study of this region showed that specific repressive histone marks are significantly enriched in these samples , compared with samples from wild type animals , suggesting that dopamine might mediate its effects in the frontal cortex via epigenetic events . as an example , borrelli mentioned that the akt1 gene , which encodes one of the downregulated rnas in d2r presynaptic mutants , shows significant enrichment of repressive histone marks at its promoter . it is appealing to speculate that disorders such as schizophrenia could be regulated by epigenetic events . but , particularly at this meeting , their enthusiasm and vitality was refreshing and contagious . the arrangement of speakers gathered at this first symposium on epigenetic control and cellular plasticity and the content of their presentations warrant success for many more international symposiums of this kind .

with the goal of discussing how epigenetic control and chromatin remodeling contribute to the various processes that lead to cellular plasticity and disease , this symposium marks the collaboration between the institut national de la sant et de la recherche mdicale ( inserm ) in france and the university of california , irvine ( uci ) . organized by paolo sassone - corsi ( uci ) and held at the beckman center of the national academy of sciences at the uci campus december 1516 , 2011 , this was the first of a series of international conferences on epigenetics dedicated to the scientific community in southern california . the meeting also served as the official kick off for the newly formed center for epigenetics and metabolism at the school of medicine , uci ( http://cem.igb.uci.edu ) .

A Dynamic Epigenome When the Epigenome Says Silence Going (epi)genome-wide Sirtuins make an appearance Signaling To and From Chromatin Epigenetic Phenotypic Diversity Epigenetics of the Brain

millions of medical devices are used each year , and despite many advances in biomaterials , a proportion of each type of device becomes colonized by bacteria.1 implanted devices may be colonized by bacteria at the time of surgery or via a hematogenous route from a distant source . the most significant factor in the development of device - related infections appears to be the skill of the surgical team ; prosthetic hips have been reported to become infected in < 0.2% of cases , but in as many as 4% of cases in less proficient facilities . generally , large and complex medical devices that require long and complicated surgery for their placement are at high risk of bacterial infection.2 when infection occurs , it can be life threatening . device - related infections may occur almost immediately postsurgery or may be very slow to develop , with overt symptoms occurring months , or even years , after the device is implanted . this strategy targets free - floating planktonic bacteria and fails to target the biofilm - forming bacteria that are more sessile . the threat of antibiotic resistance is also real because of the increasing occurrence of antibiotic - resistant strains of bacteria , especially multi drug resistant staphylococcus aureus ( mrsa ) . nanotechnology is a promising alternative to overcome the problems faced in the traditional administration of proteins , peptides , and new drugs found in the discovery pipeline . many of these drugs that are used to fight infections have been shown to be poorly soluble in aqueous and organic media and show erratic absorption and poor bioavailability . nanoparticles can be used to increase efficacy and decrease toxicity by controlling biodistribution , improving intracellular penetration , facilitating absorption through the mucosa , and improving protection against degradation . implant materials that prevent initial bacterial adhesion are an important tool for fighting infections.3 bacterial growth and adhesion can be controlled by altering various surface properties such as porosity , roughness , hydrophobicity , hydrophilicity , the use of a functional group , etc . the biocompatibility of titanium is closely related to the properties of the surface oxide layer in terms of its structure , morphology , and composition.4 the titanium oxide is a naturally occurring surface property of titanium . various physical and chemical treatments of the ti surface have been proposed with the aim of enhancing osseointegration and improving initial stability . the approaches that have been found to be beneficial to the biological performance of the implants include increasing the surface roughness and oxidation to form thicker or otherwise modified titanium dioxide ( tio2 ) layers on the surface.5 several antibiotics ( such as vancomycin ) have been incorporated into coatings on ti surfaces , but issues of optimal incorporation and controlled release have yet to be dealt with.6 one major restriction on the incorporation of antibiotics in coatings is the use of high temperature processes such as plasma spraying that denatures the coating.79 furthermore , the loading capacity and the release kinetics of antibiotics are restricted by their physical adsorption onto substrates and their use could trigger antibiotic resistance , which is a problem yet to be solved.10,11 coatings that reduce bacterial attachment are possible by modifying the implant surface to have structures that alter the surface energy and also provide mechanical cues that disrupt the bacterial membrane.12 many studies have indicated that surface roughness and hydrophobicity are primarily responsible for controlling bacterial attachment , and since nanostructures give us the ability to do so , nanostructured implant surfaces are far better for reducing orthopedic infections than conventionally structured implants . thus , by incorporating nanofeatures onto implant surfaces , the orthopedic infections can be reduced by up to five times as compared to plasma - sprayed implants , which create conventional , micron - structured features.1315 there are three primary crystalline phases of tio2 ( anatase , rutile , and brookite ) with different sizes of crystal cells in each case.1 the photolytic abilities of tio2 have been utilized widely for the preparation of different types of nanomaterials , including nanoparticles , nanorods , nanowires , nanotubes , and mesoporous and nanoporous materials.2 the photolytic abilities of ti are maintained regardless of the scale . in addition , nanoscale tio2 has a surface reactivity that fosters interactions with biological molecules , such as phosphorylated proteins and peptides,3 as well as some nonspecific binding with dna.4 compared to other advanced shaping techniques , electro - phoretic deposition ( epd ) is a very versatile plasma - based coating process since it can be modified easily for a specific application . for example , the titanium oxide texturing can be made on flat , cylindrical , or any other shaped substrate with only minor changes in electrode design and positioning . in particular , despite being a wet process , epd offers easy control over the thickness and morphology of the surface topography through the simple adjustment of the coating time , solution ph , and applied potential.16 the following study used epd to treat ti-6al-4v to modify the surface of tio2 to create a nanotopography to prevent bacterial adhesion and proliferation while either maintaining or accelerating osteoblast growth . using ethanol as the electrophoretic medium , the epd cell was established using titanium as the anode and the titanium sample as the cathode . a potential difference of 4080 v for a minute per sample the treated samples ( the surface modified titanium samples ) were dried overnight and sintered in a furnace to finish the process . the processing parameters were adjusted to produce the two distinct surface nanotopographies for ti-120 and ti-160 . after the nanotreatment , the samples were characterized under scanning electron microscopy to visualize the nanoscale topography.17 the nanosurfaces were also characterized by atomic force microscopy ( afm ) to provide scale and quantitative topography measurements . following material characterization , the samples were cleaned by washing with 70% ethanol for 5 minutes and then were sterilized under ultraviolet light overnight . they were washed thrice with phosphate - buffered saline ( pbs ) prior to seeding the cells on the surface . bacterial assays were conducted using the following three strains of bacteria : staphylococcus aureus ( atcc 29740 ) , pseudomonas aeruginosa ( atcc 39324 ) , and an ampicillin - resistant strain of escherichia coli ( bio - rad strain hb101 k-12 # 166 - 0408 and pglo plasmid # 166 - 0405 ) . agar ( sigma - aldrich , cat # a1296 ) and 0.03% tryptic soy broth ( tsb ; sigma - aldrich , st louis , mo , usa ; cat # 22092 ) were used as the media . a small amount of bacteria was taken from the stock culture , streaked onto an agar plate , and then used as the stock plate for further experiments . one colony from the tsb agar plate was added to 5 ml of 3% tsb and incubated at 37c in humidified conditions under a 5% carbon dioxide atmosphere for 16 hours . a total of 10 cells / ml were seeded onto the material surface and incubated for 16 hours at 37c . the supernatant was removed , and the samples were rinsed twice with pbs followed by sonication for 5 minutes in pbs . the supernantant was once more switched with fresh pbs , and the samples were sonicated for an additional 10 minutes . the final bacterial suspension was diluted to create subsequent dilutions ( 10 and 10 ) . following this , 0.1 ml of each of the 10 and 10 dilutions was plated and incubated for 16 hours . the number of bacterial colonies formed on each sample was counted , and using these values , the number of bacteria per milliliter was determined . osteoblasts from atcc ( cat # c12720 , population numbers 13 ; american type culture collection , manassas , va , usa ) were grown in dulbecco s modified eagle s medium supplemented with 10% fetal bovine serum and 1% penicillin ( hyclone ; thermo fisher scientific , waltham , ma , usa ) . the samples were sterilized with 70% ethanol for 20 minutes and then rinsed thrice with pbs . an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium , inner salt ( mts ) assay was used to determine cell adhesion and proliferation after 1 , 3 , and 5 days . the cells were seeded at 5,000 cells / cm for the adhesion and proliferation assays , and the media were changed every other day . the mts ( celltiter 96 aqueous one solution cell proliferation assay , g3581 promega ) reagent ( 1:5 ratio with cell culture media ) was added to each well and incubated for 3 hours on the day of the measurement . absorbance from each well was measured by a spectramax m3 ( mt05412 ) at 490 nm , and a color change from pink to dark brown was seen . each experiment was completed in triplicate with new bacteria , osteoblasts , media , and samples . a p - value of < 0.01 was deemed to be statistically significant . using ethanol as the electrophoretic medium , the epd cell was established using titanium as the anode and the titanium sample as the cathode . a potential difference of 4080 v for a minute per sample the treated samples ( the surface modified titanium samples ) were dried overnight and sintered in a furnace to finish the process . the processing parameters were adjusted to produce the two distinct surface nanotopographies for ti-120 and ti-160 . after the nanotreatment , the samples were characterized under scanning electron microscopy to visualize the nanoscale topography.17 the nanosurfaces were also characterized by atomic force microscopy ( afm ) to provide scale and quantitative topography measurements . following material characterization , the samples were cleaned by washing with 70% ethanol for 5 minutes and then were sterilized under ultraviolet light overnight . they were washed thrice with phosphate - buffered saline ( pbs ) prior to seeding the cells on the surface . bacterial assays were conducted using the following three strains of bacteria : staphylococcus aureus ( atcc 29740 ) , pseudomonas aeruginosa ( atcc 39324 ) , and an ampicillin - resistant strain of escherichia coli ( bio - rad strain hb101 k-12 # 166 - 0408 and pglo plasmid # 166 - 0405 ) . agar ( sigma - aldrich , cat # a1296 ) and 0.03% tryptic soy broth ( tsb ; sigma - aldrich , st louis , mo , usa ; cat # 22092 ) were used as the media . a small amount of bacteria was taken from the stock culture , streaked onto an agar plate , and then used as the stock plate for further experiments . one colony from the tsb agar plate was added to 5 ml of 3% tsb and incubated at 37c in humidified conditions under a 5% carbon dioxide atmosphere for 16 hours . a total of 10 cells / ml were seeded onto the material surface and incubated for 16 hours at 37c . the supernatant was removed , and the samples were rinsed twice with pbs followed by sonication for 5 minutes in pbs . the supernantant was once more switched with fresh pbs , and the samples were sonicated for an additional 10 minutes . the final bacterial suspension was diluted to create subsequent dilutions ( 10 and 10 ) . following this , 0.1 ml of each of the 10 and 10 dilutions was plated and incubated for 16 hours . the number of bacterial colonies formed on each sample was counted , and using these values , the number of bacteria per milliliter was determined . osteoblasts from atcc ( cat # c12720 , population numbers 13 ; american type culture collection , manassas , va , usa ) were grown in dulbecco s modified eagle s medium supplemented with 10% fetal bovine serum and 1% penicillin ( hyclone ; thermo fisher scientific , waltham , ma , usa ) . the samples were sterilized with 70% ethanol for 20 minutes and then rinsed thrice with pbs . an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium , inner salt ( mts ) assay was used to determine cell adhesion and proliferation after 1 , 3 , and 5 days . the cells were seeded at 5,000 cells / cm for the adhesion and proliferation assays , and the media were changed every other day . the mts ( celltiter 96 aqueous one solution cell proliferation assay , g3581 promega ) reagent ( 1:5 ratio with cell culture media ) was added to each well and incubated for 3 hours on the day of the measurement . absorbance from each well was measured by a spectramax m3 ( mt05412 ) at 490 nm , and a color change from pink to dark brown was seen . each experiment was completed in triplicate with new bacteria , osteoblasts , media , and samples . the surface of the epd - coated samples showed enhanced nanoscale features as depicted in figure 1 , which is a scanning electron microscopic image of the ti-120- and ti-160-treated and -untreated samples . the number following ti in the nomenclature represents the afm root - mean - square ( rms ) roughness value in nanometers . the untreated ti alloy control was much smoother at the nanoscale , possessing rms values of 40 nm for this 22 m afm scan . the bacterial assays conducted demonstrated that there was a significant decrease in bacterial adhesion across all three strains of bacteria on the nanotextured ti . there was a 95.6% decrease ( a 1 log reduction ) for s. aureus , a 90.2% decrease ( a 1 log reduction ) for p. aeruginosa , and an 81.1% decrease ( close to a 1 log reduction ) for ampicillin - resistant e. coli for the ti-160 surfaces . for ti-120 , there was an 86.8% reduction in s. aureus , an 82.1% reduction in p. aeruginosa , and a 48.6% reduction in ampicillin - resistant e. coli . graphs were plotted to show the number of bacteria versus the rms roughness of the treated and control samples , and it was seen that there was an almost linear relationship with r values of 0.93855 , 0.93797 , and 0.99574 for s. aureus , p. aeruginosa , and ampicillin - resistant e. coli , respectively ( figure 5 ) . this demonstrates that the nanoscale roughness created was the primary factor , resulting in a decrease in bacteria density on the nanotextured samples . figure 6 illustrates the improved osteoblast proliferation achieved on the samples treated with ti-120 versus untreated ti . combined with the results mentioned earlier , the results indicated that the ti-120 surface topography led to decreased bacterial colonization with increased osteoblast proliferation . importantly , such results were achieved by only changing the raw surface roughness values at the nanoscale and not using antibiotics or growth factors . the surface of the epd - coated samples showed enhanced nanoscale features as depicted in figure 1 , which is a scanning electron microscopic image of the ti-120- and ti-160-treated and -untreated samples . the number following ti in the nomenclature represents the afm root - mean - square ( rms ) roughness value in nanometers . the untreated ti alloy control was much smoother at the nanoscale , possessing rms values of 40 nm for this 22 m afm scan . the bacterial assays conducted demonstrated that there was a significant decrease in bacterial adhesion across all three strains of bacteria on the nanotextured ti . there was a 95.6% decrease ( a 1 log reduction ) for s. aureus , a 90.2% decrease ( a 1 log reduction ) for p. aeruginosa , and an 81.1% decrease ( close to a 1 log reduction ) for ampicillin - resistant e. coli for the ti-160 surfaces . for ti-120 , there was an 86.8% reduction in s. aureus , an 82.1% reduction in p. aeruginosa , and a 48.6% reduction in ampicillin - resistant e. coli . graphs were plotted to show the number of bacteria versus the rms roughness of the treated and control samples , and it was seen that there was an almost linear relationship with r values of 0.93855 , 0.93797 , and 0.99574 for s. aureus , p. aeruginosa , and ampicillin - resistant e. coli , respectively ( figure 5 ) . this demonstrates that the nanoscale roughness created was the primary factor , resulting in a decrease in bacteria density on the nanotextured samples . figure 6 illustrates the improved osteoblast proliferation achieved on the samples treated with ti-120 versus untreated ti . combined with the results mentioned earlier , the results indicated that the ti-120 surface topography led to decreased bacterial colonization with increased osteoblast proliferation . importantly , such results were achieved by only changing the raw surface roughness values at the nanoscale and not using antibiotics or growth factors . since the natural hierarchy of the body shows that most interactions take place at the nanoscale , the effect of nanotopographical cues on cell adhesion and proliferation , inflammation , and infection has been widely investigated.1821 nanoscale features on the implant surface offer increased cell adhesion and protein adsorption due to changes in surface energy.22,23 the interaction of microscale cells with their environment occurs through a number of nanotopographical and biochemical cues . thus , material surfaces with biochemical2427 or topographical2830 modifications similar to that of the natural in vivo environment have been shown to elicit cell - specific functionality , enabled through biomimetic cues . various biomaterial surfaces such as nanoparticles , nanofibers,35,36 nanopores , nanowires , nanostructured hydrogels , and nanotube arrays have been fabricated and extensively studied . the goal of these nanostructures is to provide nanoscale cues for a variety of cell types.37 different approaches are being used in an effort to obtain the desired bone implant interface . the ideal implant should present a surface conducive to or that will induce osseointegration , regardless of implantation site , bone quantity , bone quality , etc . as kasemo and lausmaa,38 among others , have described , biological tissues interact mainly with the outermost atomic layers of an implant . although secondary and other by - product reactions will occur , the primary interaction zone is generally ~0.11 nm . consequently , much effort is being devoted to methods of modifying surfaces of existing biomaterials to achieve desirable biological responses . materials such as zno , tio2 , polymers , and carbon nanotubes have been good examples of this . tio2 is a naturally occurring oxide formed on titanium surfaces.38 nanotechnology serves as a promising tool in tissue engineering as it allows us to generate surfaces that mimic the constituent properties of natural tissues . by altering the surface energy of implants , one can subsequently change protein adsorption , alter protein bioactivity , and promote cell function while at the same time reducing bacterial adhesion and proliferation . this is now a popular concept in the industry , and nanoparticles are being incorporated into paints , dyes , etc . lysosomal fusion in mammalian cells causes the disruption of these free radicals and reduces the damage caused by free radicals . finally , while it is anticipated that these surfaces will reduce immediate bacterial colonization and at the same time promote osteoblast functions , thus providing an immediate positive interaction in the body , it is unclear over what sustained time period such properties will last ; this will require further investigation . biofilm formation and bacterial infection of implants is a complex issue in which many variables are involved . bacteria that are present throughout the body can be treated by the host immune system and traditional antibiotic treatment . however , once the bacteria have colonized and form a biofilm , the course of treatment becomes more challenging since biofilms are not very responsive to traditional treatments . s. aureus is the most common bacteria present in clinical infections and biofilms on medical devices,38 where preventing the adhesion and colonization of bacteria can be a tremendous benefit to reduce infections and to facilitate the treatment of free - floating planktonic bacteria not adherent to implant surfaces . technology that can create nanoscale surface roughness , similar to that presented here , can reduce bacterial colonization and , thus , may have a synergistic effect in treating infections . these promising results show that just by changing the surface topography of the implant surface by creating ti-120 and ti-160 nanotextured tio2 surfaces , one can significantly reduce bacterial adhesion and growth for both gram - positive and gram - negative bacteria . a reduction in bacteria adhesion and growth was also observed for an antibiotic - resistant bacterium , as well as inducing upregulation of osteoblast activity for ti-160 . finally , moving forward from these results , with the aid of a biological , mechanical , and mechanistic understanding of the coatings , optimal coatings can be developed that offer improved osteoblast adhesion and proliferation , while reducing bacterial colonization , all without the use of the antibiotics .

backgroundthe attachment and initial growth of bacteria on an implant surface dictates the progression of infection . treatment often requires aggressive antibiotic use , which does not always work . to overcome the difficulties faced in systemic and local antibiotic delivery , scientists have forayed into using alternative techniques , which includes implant surface modifications that prevent initial bacterial adhesion , foreign body formation , and may offer a controlled inflammatory response.objectivethe current study focused on using electrophoretic deposition to treat titanium with a nanophase titanium dioxide surface texture to reduce bacterial adhesion and growth . two distinct nanotopographies were analyzed , ti-160 , an antimicrobial surface designed to greatly reduce bacterial colonization , and ti-120 , an antimicrobial surface with a topography that upregulates osteoblast activity while reducing bacterial colonization ; the number following ti in the nomenclature represents the atomic force microscopy root - mean - square roughness value in nanometers.resultsthere was a 95.6% reduction in staphylococcus aureus ( gram - positive bacteria ) for the ti-160-treated surfaces compared to the untreated titanium alloy controls . there was a 90.2% reduction in pseudomonas aeruginosa ( gram - negative bacteria ) on ti-160-treated surfaces compared to controls . for ampicillin - resistant escherichia coli , there was an 81.1% reduction on the ti-160-treated surfaces compared to controls . similarly for surfaces treated with ti-120 , there was an 86.8% reduction in s. aureus , an 82.1% reduction in p. aeruginosa , and a 48.6% reduction in ampicillin - resistant e. coli . the ti-120 also displayed a 120.7% increase at day 3 and a 168.7% increase at day 5 of osteoblast proliferation over standard titanium alloy control surfaces.conclusioncompared to untreated surfaces , ti-160-treated titanium surfaces demonstrated a statistically significant 1 log reduction in s. aureus and p. aeruginosa , whereas ti-120 provided an additional increase in osteoblast proliferation for up to 5 days , criteria , which should be further studied for a wide range of orthopedic applications .

Introduction Materials and methods Nanotreatment electrophoretic processing, EPD Surface characterization Bacterial assays Cell culture Statistical analysis Results Surface characterization Bacterial adhesion and growth Osteoblast proliferation Discussion Conclusion

dendrimer - encapsulated nanoparticles ( dens ) are well - defined nanoparticles having sizes ranging from just a few atoms to perhaps 300 atoms . this is the most scientifically interesting range of metal particle sizes because the addition of just a few atoms can drastically change their optical , electrical , mechanical , and catalytic properties . for fundamental studies of catalytic properties , dens are particularly useful for two reasons . first , it is possible to control their size , composition , and structure over a fairly broad parameter space , which is important for comparing theoretical calculations with experimental data . second , the presence of the dendrimer protects the particles from agglomeration without poisoning the metal surface . for both of these reasons , dens are one of the best model materials available for studying the fundamental properties of electrocatalytic reactions on metal particles in the 12 nm size range . pt is one of the most important catalytic metals , and hence pt dens have been studied as catalysts for homogeneous , heterogeneous , and electrocatalytic reactions . however , we and others have previously pointed out that correlations between theory and experiment with dens are complicated by incomplete reduction of the pt salt used as the nanoparticle precursor . this situation is unique to pt dens and is a consequence of the method used to prepare them . first , the poly(amidoamine ) ( pamam ) dendrimer and precursor metal salt are mixed together , and this results in encapsulation of the precursor within the dendrimer interior . second , a strong reducing agent like bh4 is added to the resulting solution . this leads to reduction of the precursor and subsequent intradendrimer agglomeration of the resulting atoms to yield the final nanoparticle . for most metals , the addition of bh4 results in complete reduction of the precursor metal salt . pt is unusual , however , in that the synthesis leads to a bimodal distribution of fully reduced dens and fully unreduced , pt - containing dendrimers . we explained this observation by invoking a nucleation and growth mechanism for pt dens . within this framework , zerovalent pt seeds form in some dendrimers but not in others . in the presence of seeds , additional reduction of pt within that dendrimer however , if no seed forms , then the metal salt is kinetically trapped in its oxidized form . at this point we do not know with certainty why seeds form in some dendrimers and not in others , but the problem has been studied by others . for example , borodko et al . reported that multidentate binding of pt to amine groups within the dendrimer hinders the reduction of the precursor complex to zerovalent particles , presumably by shifting the redox potential of pt to more negative potentials . subsequently , this same group showed that uv irradiation of the precursor can yield linear pt chains containing 28 atoms , and that these seeds lead to the formation of nanocrystals . we have shown that small ( 1.5 nm ) , fully reduced pt dens can be prepared using the method of galvanic exchange . these materials are prepared by synthesizing cu dens using the usual bh4 reduction method , and then a pt salt , such as ptcl4 , is added to the solution . because of their relative reduction potentials , the cu dens are oxidized to cu , and the pt salt is reduced to zerovalent pt . although this method is highly effective , the largest pt dens that can be formed in sixth - generation pamam dendrimers by galvanic exchange contain just 64 atoms . this is a consequence of the fact that the maximum number of cu ions that can be sequestered within the dendrimer is 64 and that the cu : pt galvanic exchange stoichiometry is 1:1 . clearly , it would be advantageous to use this same approach to prepare larger pt dens so that a broader size range of materials could be reliably synthesized . in the present report , we use more advanced characterization tools , primarily scanning transmission electron microscopy ( stem ) and x - ray absorption near edge structure ( xanes ) spectroscopy , to confirm that the standard bh4-reduction method leads to a bimodal distribution of reduced and unreduced pt - dendrimer composite materials . more importantly , however , we describe a galvanic exchange - based synthetic procedure that leads to larger , fully reduced pt dens . first , cu dens containing more than 64 atoms are prepared by carrying out multiple sequential complexation / reduction steps . second , galvanic exchange of these larger cu dens with pt results in pt dens containing up to at least 225 atoms . vis spectroscopy , x - ray photoelectron spectroscopy ( xps ) , and extended x - ray absorption fine structure ( exafs ) spectroscopy . this advance in the synthesis of model pt dens will allow for more accurate comparison of experimental properties to theory , which is the goal of our research in this field . sixth - generation , hydroxyl - terminated ( g6-oh ) poly(amidoamine ) ( pamam ) dendrimers in methanol were purchased from dendritech ( midland , mi ) . before use , the dendrimer solution was dried under vacuum and reconstituted in water at a concentration of 100.0 m . isopropyl alcohol and nabh4 were purchased from sigma - aldrich , k2ptcl4 was from acros organics , cuso4 and naoh from fisher scientific , high - purity hclo4 from j.t . all solutions were made using deionized water having a resistivity of 18.2 mcm ( milli - q gradient system , millipore ) . the pt - dendrimer precursor complexes ( g6-oh(pt)n , n = 55 , 140 , or 225 ) were prepared by allowing the appropriate amount ( 55 , 140 , or 225 equiv , respectively ) of k2ptcl4 to complex with the interior of g6-oh ( 2.0 m ) for 72 h. the g6-oh(pt)n precursor was reduced by adding 50 equiv of nabh4 , and then tightly sealing the container for 24 h. to avoid high h2 pressures , 15 ml samples of dens were reduced in 20 ml vials . note that in the past we have typically used a nabh4:pt ratio of 10:1 , but it was increased here to facilitate maximum pt reduction . the first step of the pt den galvanic exchange synthesis is preparation of 55-atom cu dens using a previously reported procedure . briefly , a 2.0 m solution of g6-oh pamam dendrimer was prepared from a 100.0 m stock solution . to this aliquots of 0.30 m naoh were added to adjust the ph to 7.5 , and this solution was stirred for 15 min to allow for cu complexation to the dendrimer . at this point , the solution was purged with n2 , and it was kept under n2 for the remainder of the synthetic procedure . next , an equivalent ( molar ) amount of nabh4 was added from a freshly made 0.10 m stock solution . once reduction was complete , excess unreacted bh4 was oxidized by adding a 4-fold excess ( relative to the equiv of bh4 ) of 0.1 m hclo4 and allowing it to react for 5 min ( the ph was maintained above 5 with naoh throughout the synthesis ) . the ph of the solution was then raised to 6.5 so that additional equiv of cu could be complexed with the dendrimer . this cycle ( adjust ph to > 6.5 , add cu , wait for complexation , reduce , oxidize excess bh4 ) was repeated as many times as necessary to synthesize cu dens of the desired size . at this stage , the ph was lowered to 3.2 and galvanic exchange for pt was carried out by adding sufficient pt ( from a freshly prepared 0.10 m stock solution of ptcl4 ) to the cu dens solution so that the pt : cu ratio was 1 . the resulting pt dens solution was immediately immobilized on vulcan carbon using the procedures described in the next two sections . the g6-oh(pt)n precursor , pt dens synthesized by chemical reduction , and pt dens synthesized by galvanic exchange ( collectively referred to as pt - dendrimer composites ) were immobilized on vulcan carbon by diluting a 2.0 m solution of the pt - dendrimer composite to 200 nm and then adding 2.0 mg of vulcan carbon per ml of diluted pt - dendrimer composite . this ratio of dendrimers : carbon ensured sufficient separation ( for imaging purposes ) between individual pt - dendrimer composites on the surface of the vulcan carbon . isopropyl alcohol ( final concentration : 20 vol % ) was added to each solution to assist with dispersion of the carbon . the resulting ink was sonicated for 1 min , then 2.0 l of this solution was pipetted onto a lacey - carbon - over - ni transmission electron microscopy ( tem ) grid ( electron microscopy sciences , hatfield , pa ) , and finally the grid was dried in air . a jeol jem - arm200f stem with spherical aberration ( cs ) correction and a high - angle annular dark - field ( haadf ) detector was used for sample analysis . as for the stem analysis , the pt - dendrimer composites were also immobilized on vulcan carbon for xas . the xas analysis requires a higher surface concentration of the pt - dendrimer composites , however , so 2.0 mg of vulcan carbon per ml of the undiluted ( 2.0 m ) composite was used . the carbon - supported pt - dendrimer composites were then filtered using an advantec ptfe membrane filter ( 0.5 m pore size ) . the filtrate was rinsed with water and then isopropyl alcohol , and allowed to dry in air overnight . the dried powder was pressed into a pellet at 1 ton of pressure for xas analysis . xas analysis was performed at the national synchrotron light source at brookhaven national lab using beamline x18b . a pt foil was fit to obtain the amplitude reduction factor ( s0 = 0.87 for the pt l3 edge was used ) . the first shell was fit in r - space using a k - weight of 2 for the fourier transforms . a portion of the same pellet used for the xas analysis was used for xps . the pellet fragment was dissolved in water , and an aliquot was pipetted onto a chip of glassy carbon . xps was performed using a kratos axis ultra spectrometer ( chestnut ridge , ny ) having an al k source . individual elemental spectra were collected with a 0.1 ev step size and a band - pass energy of 20 ev . casaxps ( v 2.3.15 , casa software , teignmouth , uk ) was used for peak fitting , assuming a mixed gaussian / lorentzian model . before describing our new method for complete reduction of pt dens , we discuss the original bh4-reduction method , which leads to only partial reduction , for comparison . the uv vis spectra of the g6-oh(pt)n ( n = 55 , 140 , and 225 ) precursor before and after reduction with bh4 are provided in figure 1 . these spectra show that even after reduction with 50-fold excess bh4 , a fraction of the ligand - to - metal charge - transfer ( lmct ) band of g6-oh(pt)n ( max = 250 nm ) and the absorbance bands arising from the unreduced pt salt ( max = 215 and 230 nm ) are present . uv vis spectra of pt - dendrimer complexes , g6-oh(pt)n , and pt dens synthesized by bh4 reduction , g6-oh(ptn ) , where n = 55 , 140 , and 225 . the spectra were acquired using a 2.0 mm quartz cuvette and blanked with 2.0 m g6-oh . as discussed in the experimental section , pt dens prepared by galvanic exchange are synthesized by sequentially complexing and reducing aliquots of cu in the presence of g6-oh , and then reacting the resulting cu dens with pt . to prepare cu dens containing more than 64 atoms , this is because , as we have previously shown , the maximum number of cu ions that can be complexed with the interior tertiary amines of g6-oh is 64 . accordingly , the cu den synthesis begins by complexing a slightly substoichiometric amount of cu ( 55 equiv ) with g6-oh . as shown in figure 2a , this results in a well - defined lmct band at max = 300 nm corresponding to the g6-oh(cu)55 precursor . following reduction with bh4 , this lmct band disappears and the characteristic broad absorbance of 55-atom cu dens is observed in figure 2b . after removal of excess bh4 by addition of hclo4 , 55 additional equiv of cu are added to the solution and the lmct band is observed again ( figure 2a ) , but now it is superimposed on the spectrum of the reduced g6-oh(cu55 ) dens . importantly , the appearance of the lmct band confirms that no active bh4 is present in solution , because the added cu is still in its oxidized form and able to complex to the dendrimer ( at ph > 6.5 ) . after the second aliquot of cu is sequestered inside the dendrimer , additional bh4 is added to yield reduced dens containing an average of 110 atoms : g6-oh(cu110 ) . as shown in figure 2b this results in an increase in the featureless absorbance spanning the indicated wavelength range . this process is then continued until cu dens of the desired size are formed . in this case , we stopped the process at 140 total equiv of cu in order to make a direct comparison to bh4-reduced pt dens of the same nominal size ( 140 is a complete - shell magic number for a truncated octahedron nanoparticle ) . ( a ) uv vis spectra for each sequential complexation step of the synthesis of g6-oh(cu140 ) dens . ( b ) uv vis spectra for each sequential reduction step in the synthesis of g6-oh(cu140 ) dens . ( c ) comparison of the uv vis spectra of g6-oh(cu140 ) prepared by direct reduction with bh4 ( blue ) , and g6-oh(pt140 ) prepared by galvanic exchange at ph 3.2 ( black ) and at ph > 6.5 ( green ) . the concentration of the dendrimers in these solutions was 2.0 m , and the data were obtained using a 2.00 mm quartz cuvette and blanked with water . the cu dens are converted into pt dens by adding the same number of equiv of ptcl4 as were used to prepare the cu dens . as previously reported for the synthesis of g6-oh(pt55 ) , this galvanic exchange reaction is carried out at ph 3 , with the value here being 3.2 . figure 2c compares uv vis spectra of the 140-atom cu dens at ph > 6.5 and the pt dens immediately after galvanic exchange ( ph 3.2 ) . consistent with previous reports , the absorbance of the g6-oh(pt140 ) dens is significantly higher than that of the corresponding cu dens . additionally , the cu - lmct band ( max = 300 nm ) is absent after galvanic exchange at ph 3.2 , because the interior tertiary amines of the dendrimer are protonated and hence not available for complexation with cu . for direct comparison with the g6-oh(cu140 ) spectrum , the ph of the g6-oh(pt140 ) solution was then raised above 6.5 . in this case , the ph is high enough that the interior tertiary amines can complex free cu , and a cu - lmct band is again apparent . this confirms that galvanic exchange has occurred and that cu is present in the solution . pt dens larger than n = 140 can also be made by galvanic exchange . for example , figure s1 in the supporting information compares g6-oh(pt140 ) and g6-oh(pt225 ) dens . the absorbance of g6-oh(pt225 ) is higher at all wavelengths , qualitatively indicating the presence of larger nanoparticles . following synthesis , the pt dens were immobilized on vulcan carbon , which as we have shown previously is a good support for performing electrocatalytic experiments using dens . immobilization was carried out at ph 3.2 by the addition of vulcan carbon and subsequent filtration as described in the experimental section . tem images and size - distribution histograms of g6-oh(pt140 ) and g6-oh(pt225 ) immobilized on vulcan , prepared using the aforementioned procedure , are shown in supporting information figure s2 . consistent with expectations , these data indicate that the dens have diameters of 1.7 0.2 nm for g6-oh(pt140 ) ( calculated diameter = 1.6 nm ) and 1.9 0.2 nm for g6-oh(pt225 ) ( calculated diameter = 1.9 nm ) . the conclusion that a bimodal distribution of fully reduced and unreduced dens results from the standard bh4reduction method relies in part on results from a previous tem study . in that earlier analysis , micrographs showed that direct reduction resulted in pt dens having the expected size distribution despite other analytical methods indicating incomplete reduction . at that time , however , we did not have access to electron microscopy with sufficient resolution and contrast to image the g6-oh(pt)n precursor . now , by utilizing high - resolution aberration - corrected stem , individual pt atoms / ions are visible , and hence it is possible to distinguish between the g6-oh(pt)n precursor and g6-oh(ptn ) dens . the stem data discussed next confirm that bh4 reduction results in a bimodal distribution of reduced and unreduced dens . figure 3a is a stem micrograph of the g6-oh(pt)55 precursor that has not been exposed to bh4 . this image shows clusters of individual pt atoms / ions on the vulcan carbon surface . to highlight the nature of these groupings , red circles having diameters of 7 nm ( the approximate diameter of g6 pamam dendrimers ) have been overlaid onto the image . although highly qualitative , it is not difficult to imagine that these clusters of atoms / ions are contained within individual dendrimers . the micrograph in figure 3b was obtained after direct bh4 reduction of the g6-oh(pt)55 precursor . in this case , both ordered nanoparticles ( 1.3 nm , indicated by red arrows ) and a grouping of atoms ( red circle ) are visible on the vulcan carbon support . specifically , the approximate spread of the disordered atoms is maintained at 7 nm , suggesting that some of the complexes are unaffected by the chemical reduction process . representative stem images of ( a ) the g6-oh(pt)55 precursor complex , ( b ) g6-oh(pt55 ) synthesized by direct reduction with bh4 , ( c ) g6-oh(pt55 ) synthesized by galvanic exchange , ( d ) the g6-oh(pt)140 precursor complex , ( e ) g6-oh(pt140 ) synthesized by direct reduction with bh4 , ( f ) g6-oh(pt140 ) synthesized by galvanic exchange , ( g ) the g6-oh(pt)225 precursor complex , and ( h ) g6-oh(pt225 ) synthesized by direct reduction with bh4 . the red circles ( 7 nm in diameter ) represent the approximate diameter of a g6 pamam dendrimer , and they highlight the groupings of ions initially present in the precursor complex or after incomplete reduction of the precursor complex . figure 3c shows that g6-oh(pt55 ) dens synthesized by galvanic exchange of cu for pt reveal no sign of the unreduced g6-oh(pt)55 complex ( that is , no evidence of individual atoms were apparent despite extensive analysis of the grid ) . this same trend is observed for g6-oh(pt)140 , g6-oh(pt140 ) prepared by bh4 reduction , and g6-oh(pt140 ) prepared by galvanic exchange ( figure 3d , e , f , respectively ) and for g6-oh(pt)225 and g6-oh(pt225 ) prepared by bh4 reduction ( figure 3 g and h , respectively ) . in summary , the representative micrographs shown in figure 3 confirm , qualitatively , our earlier bimodal - distribution model , wherein a fraction of the g6-oh(pt)n species remain unreduced when exposed to bh4 , while the remainder are reduced to yield g6-oh(ptn ) dens . we wish to emphasize , however , that a much larger statistical analysis would be required to confirm these conclusions if they were solely based on electron microscopy . as discussed in the next three sections , although it is difficult to obtain quantitative information about the extent of pt den reduction from tem studies , xps is very well suited for this purpose . accordingly , we used xps to compare the extent of reduction using the bh4 and galvanic exchange approaches . as shown in figure 4a , the pt 4f7/2 peaks for g6-oh(pt)n ( n = 55 , 140 , and 225 ) are present at 72.9 , 73.3 , and 73.5 ev , respectively . these values can be compared with that of the ptcl4 starting material : 73.4 ev ( black vertical line ) . the slight shift to lower binding energy as the pt : dendrimer ratio decreases may result from the increased availability of dendrimer binding sites at lower pt concentrations and the corresponding increase in multidentate binding . high - resolution xps spectra of ( a ) g6-oh(pt)n , ( b ) g6-oh(ptn ) synthesized by direct reduction with bh4 , and ( c ) g6-oh(ptn ) synthesized by galvanic exchange ( ge ) , where n = 55 , 140 , and 225 . the vertical black lines at 73.4 and 71.3 ev represent literature values for the binding energies of ptcl4 and fully reduced pt dens , respectively . the spectra of the bh4-reduced dens ( g6-oh(ptn ) , n = 55 , 140 , and 225 ) exhibit multiple pairs of peaks , which is consistent with partial reduction and two populations of pt oxidation states . focusing on the pt 4f7/2 region , the peaks at 71.8 0.2 ev correspond to zerovalent dens . the black vertical line at 71.3 ev marks the value previously reported for the 4f7/2 peak for pt dens . in addition to these zerovalent pt 4f peaks , a second set of 4f peaks , corresponding to the g6-oh(pt)n precursor , are also present . these results are consistent with our previous finding that the bh4 method leads to only partial reduction of the precursor . the xps peaks in figure 4b were fit to quantify the extent of reduction for each sample . the fits are shown in supporting information figure s3 and quantitative results are provided in table 1 . in all cases , the total xps spectra are well represented by deconvolution into peaks corresponding to the pt and pt binding energies . the extent of reduction determined from these fits is 59% , 40% , and 43% for g6-oh(ptn ) ( n = 55 , 140 , and 225 ) dens , respectively . these values are somewhat different than those we have reported previously for n = 55 , 147 , and 240 : 14% , 44% , and 64% , respectively . we attribute the large difference in the percent reduction of g6-oh(pt55 ) to the larger excess of bh4 used for reduction in the present set of experiments ( 50-fold versus 10-fold ) . figure 4c shows xps spectra of the g6-oh(ptn ) dens prepared by galvanic exchange . in contrast to the spectra of the bh4-reduced dens in figure 4b , these spectra exhibit just one 4f7/2 peak corresponding to fully reduced pt . these results indicate that complete galvanic exchange has occurred with the cu dens and that little or no unreduced pt salt or complex is present in the sample . xanes linear combination analysis ( lca ) was used to further quantify the extent of pt reduction and to verify the xps results . figure 5a shows that the white line intensity of the g6-oh(pt55 ) dens prepared by direct bh4 reduction is between that of the corresponding g6-oh(pt)55 precursor and g6-oh(pt55 ) dens synthesized by galvanic exchange . the spectrum of g6-oh(pt55 ) prepared by direct reduction and the fit obtained from a linear combination of the g6-oh(pt)55 precursor and g6-oh(pt55 ) prepared by galvanic exchange are shown in figure 5b . the overlap is nearly exact , and it indicates a percentage reduction of the precursor of 59% , which compares favorably with the value of 54% determined by xps ( table 1 ) . xanes data ( left ) and lca fits ( right ) for ( a , b ) g6-oh(pt55 ) , ( c , d ) g6-oh(pt140 ) , and ( e , f ) g6-oh(pt225 ) . for the right panel , the data corresponding to the dens synthesized by bh4 reduction are shown in black and the lca best fit is in red . xanes spectra for the n = 140 and 225 dens and den precursors are shown in figure 5c and e , respectively . the corresponding lca fitting for the g6-oh(pt140 ) and g6-oh(pt225 ) dens ( direct bh4 reduction ) in figure 5d and f , respectively , provide good matches . the resulting percentage reductions of the precursors are within 7% of those determined by xps ( table 1 ) . exafs spectra were acquired to obtain the coordination number for the fully reduced g6-oh(ptn ) dens synthesized by galvanic exchange . the fits yield coordination numbers of 8.9 1.2 for g6-oh(pt140 ) and 9.2 1.2 for g6-oh(pt225 ) . these values can be compared to those calculated for 140-atom and 225-atom truncated octahedra of 9.09 and 9.49 , respectively . these values are substantially higher than those reported previously for pt dens reduced with bh4 : 3.99 0.61 for g6-oh(pt147 ) and 6.09 0.42 for g6-oh(pt240 ) . the latter very low coordination numbers indicate the presence of a substantial number of unreduced pt species , which is consistent with the previously discussed xanes and xps results . an important outcome of the exafs analysis is that in the future it will be possible to use in situ electrochemical exafs to study electrocatalysis at fully reduced pt dens . exafs data ( black ) and r - space fits ( red ) ( k - weight of 2 ) for pt dens prepared by galvanic exchange . the synthesis is carried out by first preparing cu dens of the desired size , and then using the process of galvanic exchange to convert these into pt dens containing the same number of atoms . we also re - examined the originally reported method for preparing pt dens by direct reduction using bh4 , and confirmed that this approach leads to a bimodal distribution of fully reduced dens and fully unreduced precursors . the present research focus of our group is reconciling the experimentally measured and theoretically calculated electrocatalytic properties of dens . due to their high activity for many catalytic reactions , pt dens are among the most important materials for developing this type of structure activity relationship . the types of future studies we envision require , to the maximum extent possible , homogeneity in catalyst size and structure , and the results reported here move us a step closer to this goal .

here we outline a new method for synthesizing fully reduced pt dendrimer - encapsulated nanoparticles ( dens ) . this is achieved by first synthesizing cu dens of the appropriate size through sequential dendrimer loading and reduction steps , and then galvanically exchanging the zerovalent cu dens for pt . the properties of pt dens having an average of 55 , 140 , and 225 atoms prepared by direct chemical reduction and by galvanic exchange are compared . data obtained by uv vis spectroscopy , x - ray absorption spectroscopy , x - ray photoelectron spectroscopy , and high - resolution electron microscopy confirm only the presence of fully reduced pt dens when synthesized by galvanic exchange , while chemical reduction leads to a mixture of reduced dens and unreduced precursor . these results are significant because pt dens are good models for developing a better understanding of the effects of finite size on catalytic reactions . until now , however , the results of such studies have been complicated by a heterogeneous mixture of pt catalysts .

Introduction Experimental Section Results and Discussion Summary and Conclusions

the accord trial was a double 2 2 factorial trial designed to test the effect of 1 ) intensive glucose control versus standard control , 2 ) intensive blood pressure control versus standard control , and 3 ) lipid treatment strategy that used fenofibrate plus a statin compared with statin monotherapy on the composite outcome of myocardial infarction , stroke , or cardiovascular death . details of the design of the accord lipid trial have already been published ( 1416 ) . all participants received simvastatin to assure good ldl control , and participants were started on masked medication ( 160 mg / day fenofibrate or placebo ) at the month 1 visit . all 77 participating clinics obtained approval from their local institutional review board prior to participating . this accord renal ancillary study was conducted among participants of a subset of 40 clinics with a total enrollment of 1,081 participants ( mean per clinic enrollment was 27 , range 291 ) . participants read and signed an additional informed consent before enrollment in this study . during the accord lipid trial , participants whose gfr fell below 50 ml / min/1.73 m for two consecutive visits had their fenofibrate dose reduced to 54 mg / day . participants whose gfr fell below 30 ml / min/1.73 m on two consecutive visits had their fenofibrate discontinued . to maintain masking , placebo arm participants who experienced similar decreases in gfr were provided with equivalent appearing , reduced dose placebo tablets or had their placebo pills discontinued . all gfr estimates for safety reporting used the modification of diet in renal disease method ( 17 ) . this study used a retrospectively defined , nested , three group , on - drug / off - drug study design . eligible participants within participating clinics were identified by the coordinating center approximately 3 months prior to their accord lipid trial close - out visit and reported to clinics while maintaining masking to participant randomization status . fenofibrate arm case subjects ( cases ) were defined as active participants in the fenofibrate arm who had experienced 20% increase in serum creatinine from trial baseline to month 4 and remained on study medication at close out ( either full or reduced dose ) . since the study drug was started 1-month postrandomization , study month 4 was equivalent to 3 months of therapy . fenofibrate arm control subjects ( controls ) were defined as active in the fenofibrate arm who experienced 2% increase in creatinine over the same period . placebo arm control subjects ( placebos ) were defined as randomized to placebo but without restriction on their change in serum creatinine . individuals with baseline renal disease ( creatinine > 1.5 mg / dl ) were excluded from accord . all eligible cases and controls that consented to participate were enrolled in the study , while eligible placebos were enrolled until maximum enrollment was achieved . the accord lipid trial close - out visit served as the baseline visit for the accord renal ancillary study . a follow - up visit for ancillary study participants was held 68 weeks after the trial close - out visit , referred to here as the postclose - out visit . at both visits , all trial participants were provided with a 3-month supply of simvastatin , as well as their current diabetes medications . no fenofibrate was provided to any trial participant at close out , and participants in the ancillary study were given a letter asking their physician to refrain from starting open label fenofibrate or other medications that could affect serum creatinine or creatinine excretion until after the postclose - out visit . this protocol was approved by the institutional review board at each clinic , at the coordinating center , and at the accord renal study center . serum creatinine was measured during the accord lipid trial at the baseline visit , every 4 months throughout the trial , and at the close - out visit . an additional blood sample was drawn at the close - out and postclose - out visits to assay serum creatinine and cystatin c. the same accord central laboratory performed all assays . serum creatinine was determined using the roche creatinine plus enzymatic assay with spectrometric analysis on a roche double modular p analytics analyzer ( roche diagnostics , indianapolis , in ) . the assay sensitivity was 0.03 mg / dl , and intra - assay coefficients of variation based on analysis of low- and high - quality control samples were 0.8% and 0.7% , respectively , while interassay coefficients of variation were 1.6% and 2.5% . the interassay precision is consistently < 1.4% for the high - quality and < 2.2% for the low - quality control samples . serum cystatin c concentrations were determined using the siemens diagnostics reagent on a roche hitachi p - module analyzer ( siemens diagnostics , deerfield , il ) . the interassay precision for the high- and low - quality control samples was 2.5 and 2.6% , respectively . all serum samples were analyzed for creatinine on the day of sample receipt ; cystatin c assays were performed in a single batch from plasma previously stored at 80c . estimated gfr ( egfr ) in this study was computed by the chronic kidney disease epidemiology collaboration ( ckd - epi ) method ( 18 ) . serum cystatin c estimated glomerular filtration rate ( cgfr ) was computed by the method of stevens et al . pairwise differences between groups were compared using two - sample t tests for continuous factors ( with satterthwaite correction for unequal variances where appropriate ) and tests for categorical factors . between - group comparisons of renal function measures ( serum creatinine , egfr , serum cystatin c , cystatin c - egfr ) were performed using linear analysis of covariance models . all analyses were performed using sas version 9.2 software ( sas institute , cary , nc ) . the study power was estimated for the change in creatinine levels after the trial postclose - out visit based on two - group t tests of differences in the log - transformed serum creatinine . the estimates used satterthwaite approximation for unequal variances at a bonferroni - adjusted global significance of 0.05 . based on projected variability estimates prior to study close - out , the power to detect a difference of 1.1 versus 1.0 mg / dl in serum creatinine ( i.e. , a difference of 0.095 between log means ) was 91% for fenofibrate cases versus fenofibrate controls ; 99% for fenofibrate cases versus placebo controls ; and 94% for fenofibrate controls versus placebo controls . observed variability was less than projected , yielding post hoc power estimates of 99% for all three comparisons . this study used a retrospectively defined , nested , three group , on - drug / off - drug study design . eligible participants within participating clinics were identified by the coordinating center approximately 3 months prior to their accord lipid trial close - out visit and reported to clinics while maintaining masking to participant randomization status . fenofibrate arm case subjects ( cases ) were defined as active participants in the fenofibrate arm who had experienced 20% increase in serum creatinine from trial baseline to month 4 and remained on study medication at close out ( either full or reduced dose ) . since the study drug was started 1-month postrandomization , study month 4 was equivalent to 3 months of therapy . fenofibrate arm control subjects ( controls ) were defined as active in the fenofibrate arm who experienced 2% increase in creatinine over the same period . placebo arm control subjects ( placebos ) were defined as randomized to placebo but without restriction on their change in serum creatinine . individuals with baseline renal disease ( creatinine > 1.5 mg / dl ) were excluded from accord . all eligible cases and controls that consented to participate were enrolled in the study , while eligible placebos were enrolled until maximum enrollment was achieved . the accord lipid trial close - out visit served as the baseline visit for the accord renal ancillary study . a follow - up visit for ancillary study participants was held 68 weeks after the trial close - out visit , referred to here as the postclose - out visit . at both visits , all trial participants were provided with a 3-month supply of simvastatin , as well as their current diabetes medications . no fenofibrate was provided to any trial participant at close out , and participants in the ancillary study were given a letter asking their physician to refrain from starting open label fenofibrate or other medications that could affect serum creatinine or creatinine excretion until after the postclose - out visit . this protocol was approved by the institutional review board at each clinic , at the coordinating center , and at the accord renal study center . serum creatinine was measured during the accord lipid trial at the baseline visit , every 4 months throughout the trial , and at the close - out visit . an additional blood sample was drawn at the close - out and postclose - out visits to assay serum creatinine and cystatin c. the same accord central laboratory performed all assays . serum creatinine was determined using the roche creatinine plus enzymatic assay with spectrometric analysis on a roche double modular p analytics analyzer ( roche diagnostics , indianapolis , in ) . the assay sensitivity was 0.03 mg / dl , and intra - assay coefficients of variation based on analysis of low- and high - quality control samples were 0.8% and 0.7% , respectively , while interassay coefficients of variation were 1.6% and 2.5% . the interassay precision is consistently < 1.4% for the high - quality and < 2.2% for the low - quality control samples . serum cystatin c concentrations were determined using the siemens diagnostics reagent on a roche hitachi p - module analyzer ( siemens diagnostics , deerfield , il ) . the interassay precision for the high- and low - quality control samples was 2.5 and 2.6% , respectively . all serum samples were analyzed for creatinine on the day of sample receipt ; cystatin c assays were performed in a single batch from plasma previously stored at 80c . estimated gfr ( egfr ) in this study was computed by the chronic kidney disease epidemiology collaboration ( ckd - epi ) method ( 18 ) . serum cystatin c estimated glomerular filtration rate ( cgfr ) was computed by the method of stevens et al . pairwise differences between groups were compared using two - sample t tests for continuous factors ( with satterthwaite correction for unequal variances where appropriate ) and tests for categorical factors . between - group comparisons of renal function measures ( serum creatinine , egfr , serum cystatin c , cystatin c - egfr ) were performed using linear analysis of covariance models . all analyses were performed using sas version 9.2 software ( sas institute , cary , nc ) . the study power was estimated for the change in creatinine levels after the trial postclose - out visit based on two - group t tests of differences in the log - transformed serum creatinine . the estimates used satterthwaite approximation for unequal variances at a bonferroni - adjusted global significance of 0.05 . based on projected variability estimates prior to study close - out , the power to detect a difference of 1.1 versus 1.0 mg / dl in serum creatinine ( i.e. , a difference of 0.095 between log means ) was 91% for fenofibrate cases versus fenofibrate controls ; 99% for fenofibrate cases versus placebo controls ; and 94% for fenofibrate controls versus placebo controls . observed variability was less than projected , yielding post hoc power estimates of 99% for all three comparisons . we recruited 321 active fenofibrate arm cases ( 30.2% ) , 175 active fenofibrate arm controls ( 16.5% ) , and 565 active placebo controls ( 53.3% ) plus 20 others ( 18 ineligible and 2 eligible but missing required study data ) for a total accord renal ancillary study enrollment of 1,081 participants . forty - nine individuals ( 4.6% ) were lost to follow - up between the trial close - out visit ( the first renal study on - drug visit ) and postclose - out ( off - drug ) visit . two of the forty - nine were deaths with causes not attributed to this study . the clinical characteristics for the three recruited study groups are listed in table 1 . as expected , the change in serum creatinine between the trial baseline and month 4 visits was significantly different between cases and controls with a mean ( sd ) increase of + 0.31 0.16 mg / dl in cases , decrease of 0.05 0.08 mg / dl in controls , and was also significantly different between controls and placebos , with placebos showing no change ( 0.00 0.13 mg / dl ) . other differences between the three groups included fewer control participants in the intensive arm of the accord glycemia trial ( 37% ) than cases ( 55% ) or placebos ( 49% ) ; differences among all three groups in the mean follow - up time during the main trial from randomization to close - out visit ( 5.0 1.0 vs. 5.6 1.6 vs. 5.2 1.2 years ) ; more asian participants among controls ( 17% ) than in cases ( 10% ) or placebo ( 10% ) ; a lower triglyceride level at trial close - out among cases ( 136 14 mg / dl ) than either controls ( 160 227 mg / dl ) or placebos ( 163 93 mg / dl ) ; greater use of insulin by cases at trial close - out ( 62% ) than among controls ( 49% ) ; and more cases ( 58% ) with a creatinine > 1.0 mg / dl at close - out than among either controls ( 38% ) or placebos ( 36% ) . there were no significant differences in mean time interval between close - out and postclose - out visits for the three groups ( table 2 ) . creatinine levels in cases remained higher than controls at study close - out after a mean of 5.2 years of follow - up , although the creatinine levels in cases declined from the month 4 visit to close - out visit , while that of the controls and placebos increased , narrowing the difference in the means between the groups . clinical characteristics of the nested fenofibrate case , fenofibrate control , and placebo control study groups time course of primary renal outcomes for the 3 study groups in accord lipid trial and renal ancillary study table 2 shows the adjusted mean creatinine and egfr for the three groups at four time points during the trial and the postclose - out visit . the mean values in table 2 were adjusted for glycemia treatment arm assignment , age , diabetes duration , sex , nonwhite race , insulin use , and systolic and diastolic blood pressure ; the change in values were additionally adjusted for the time interval between visits ( trial baseline to month 4 , or trial close - out to postclose - out visit ) . all subsequent results in the main text refer to these adjusted values ( unadjusted values are included for comparison in supplementary table 1 ) . the mean serum creatinine levels in the three study groups were not significantly different at baseline or month 4 from the other participants in the main lipid trial who would have satisfied the retrospective percent change in serum creatinine inclusion criterion and were thus representative of the entire eligible accord lipid trial cohort at month 4 . time course values of adjusted serum creatinine and cystatin c by accord renal study group . a : the changes in serum creatinine in units of mg / dl . the study group trends are shown as : black circles , fenofibrate cases ; red triangles , fenofibrate controls ; blue crosses , placebo controls . base , baseline visit ; m4 , month 4 visit ; close , close - out visit ; post , postclose - out visit . ( a high - quality color representation of this figure is available in the online issue . ) a : the changes in egfr in units of ml / min/1.73 m. b : changes in cgfr in units of ml / min/1.73 m. the horizontal trial time point axis is not shown to scale . the study group trends are shown as : black circles , fenofibrate cases ; red triangles , fenofibrate controls ; blue crosses , placebo controls . base , baseline visit ; m4 , month 4 visit ; close , close - out visit ; post , postclose - out visit . ( a high - quality color representation of this figure is available in the online issue . ) in the retrospectively defined nested groups , the mean ( se ) serum creatinine at the month 4 visit was greater in cases ( 1.16 0.01 mg / dl ) than in controls ( 0.90 0.01 mg / dl ) or placebos ( 0.90 0.01 mg / dl ) . no difference was seen between controls and placebos ( p = 0.9 ) . at the trial close - out visit after a mean trial follow - up period of 5.2 years ( range 5.05.6 years in the three groups ) , serum creatinine was still greater in cases than controls or placebos ( 1.11 0.02 mg / dl versus 1.01 0.02 mg / dl and 0.98 0.01 mg / dl , respectively ) although the difference in the means of the three groups decreased . at the postclose - out visit after discontinuation of the fenofibrate study drug for 51 days , the mean creatinine had decreased markedly in both cases ( average decrease 0.13 0.01 mg / dl ) and controls ( average decrease 0.09 0.01 mg / dl ) , but showed a slight increase in placebos ( average increase + 0.02 0.01 mg / dl ) . the absolute magnitude of reduction in serum creatinine was significantly greater in cases than in controls ( p = 0.002 ) , and also different in both cases and controls compared with a slight rise in placebos ( p < 0.0001 for both comparisons ) . after discontinuation of the study drug for 68 weeks , cases had a mean serum creatinine of 0.12 mg / dl above trial baseline mean , while controls were 0.04 mg / dl below their baseline mean , although identical to their mean value at trial month 4 . similar between - group trends were observed in egfr . at postclose - out , the mean egfr of cases was 1.1 ml / min/1.73 m greater than placebos but not significant ( p = 0.4 ) , while controls were 5.2 ml / min/1.73 m greater than placebos ( p < 0.0001 ) . these results did not change substantially after removing participants who stopped taking nonfenofibrate , renal function altering medications between close - out and postclose - out visits ( further details in the supplementary data online ) . the supplementary data also includes analyses that suggest that the reduction in serum creatinine was essentially complete after 51 days . changes in adjusted serum cystatin c levels between close - out and postclose - out visits recapitulated those of serum creatinine , table 2 , and figs . , cystatin c dropped from a mean ( se ) of 1.03 0.02 to 0.96 0.02 mg / dl and was not significantly different from placebos at postclose - out ( p = 0.9 ) . the mean change in cases and controls was not significantly different ( p = 0.4 ) . trends in adjusted cgfr were similar to egfr with mean cgfr in controls at postclose - out ( 91.4 1.9 ml / min/1.73 m ) greater than either cases ( 83.1 1.4 ) or placebos ( 83.1 1.0 ) , which were not significantly different ( p = 0.9 ) . the mean recovery of cgfr was also greater in cases and controls on drug cessation compared with placebos . we investigated the potential reversibility of the rapid serum creatinine increase observed on starting fenofibrate therapy . the fenofibrate cases ( participants who experienced a 20% or more increase in serum creatinine after 3 months of fenofibrate ; 47.4% of all participants randomized to fenofibrate in the accord lipid trial ) had a mean serum creatinine decrease on cessation of fenofibrate to a value that was no different than the mean creatinine of the placebo reference group , suggesting no residual loss of gfr after 5 years of therapy . by contrast , for the control subgroup of fenofibrate participants ( participants who experienced < 2% change in creatinine ; 24.6% of all participants randomized to fenofibrate ) , the mean serum creatinine was lower than the placebo reference group after 51 days off therapy , suggesting a net preservation of gfr and renal function . fenofibrate was also found to have a protective effect on the albuminuria levels in accord lipid trial participants . fewer participants randomized to fenofibrate progressed to frank microalbuminuria or proteinuria postrandomization than placebos ( 16 ) . this result is consistent with the reduced progression found in the dais and field trials ( 5,7 ) , and the greater reduction in mean urinary albumin / creatinine ratio in the fenofibrate arm compared with placebo over 5 years of follow - up in field ( 7 ) . previous studies support our key finding about the reversibility of serum creatinine levels postfenofibrate therapy . early comparative studies in patients who had undergone renal transplant and were on fenofibrate therapy demonstrated a reversible increase in creatinine if no chronic renal failure was present at baseline ( 1,2 ) . a limitation of these early reports is that they were all retrospective case studies in a limited number of patients with pre - existing renal disease or transplant . the field trial reported results from a large study of reversibility of renal function in patients with diabetes on fenofibrate therapy . after a 6-week prerandomization run - in period of all field study participants on fenofibrate therapy , mean levels of serum creatinine increased , but returned to baseline in those subjects randomized to placebo within 4 months of cessation of therapy . furthermore , a posttrial fenofibrate wash - out substudy at the conclusion of the 5-year trial demonstrated an acute decline in creatinine values during the wash - out period of 52 days resulting in a net protective effect of fenofibrate ( 7,8 ) . similar to accord , the field trial examined a broad population of type 2 diabetic participants who had normal renal function at study entry . the first is the previously studied rise in serum creatinine levels shortly after starting therapy . based on the accord results , this can be expected to occur in 47% of type 2 diabetic patients with a similar cardiovascular and renal function profile to the participants in the accord lipid trial . the second effect is a preservation of gfr in patients who experience little or no rise in serum creatinine immediately after initiating therapy , which is expected in about 25% of patients with a similar profile to accord lipid . a similar protective effect on gfr was also seen in fenofibrate participants more generally at the conclusion of the field trial ( 7 ) . whether this preservation is maintained or whether it reduces the long - term macro- or microvascular disease risk after the initial 5 years of therapy is unknown . the accordion ( accord follow - up ) study , which is currently underway , will help to answer the second question . accordion is a posttrial , prospective , observational study of 8,000 accord participants over 3.5 years to elucidate the long - term effects of the accord treatment strategy and will include additional measurements of serum creatinine , urinary creatinine , and albumin . potential mechanisms include increased muscular production of creatinine , decreased secretion from renal tubules , and a change in the glomerular filtration through altered hemodynamics . the investigators performed a crossover study of 15 patients with renal insufficiency ( average creatinine clearance 69 ml / min ) that had experienced at least a 10% increase in serum creatinine with fenofibrate therapy . serum creatinine increased with fenofibrate therapy , but indices of both gfr ( creatinine clearance , inulin clearance ) and renal blood flow ( p - aminohippuric acid clearance ) were unaffected . a similar , small , cross - over study in healthy individuals found that while creatinine clearance was reduced by fenofibrate , neither gfr as measured by inulin clearance nor the rate of creatinine secretion changed ( 10 ) . fifty - five patients treated with ciprofibrate showed similar results but no significant change was observed in 15 patients taking gemfibrozil . similar results were observed by westphal et al . ( 11 ) in a cross - over study of 22 men with hypertriglyceridemia and normal renal function randomized to either micronized fenofibrate 200 mg / day or gemfibrozil 900 mg / day . the cystatin c results in the accord renal study are consistent with the field helsinki substudy , which showed that an increase in cystatin c levels accompanied the initial increase in serum creatinine with fenofibrate ( 12 ) . the results suggest that the physiological mechanism for the rapid increase in serum creatinine on initiation of fenofibrate therapy ( and the reversion on cessation ) is not specific for creatinine and rule out increased creatinine production or secretion as a plausible mechanism . an unexpected benefit of this design was that it highlighted the differences in changes in serum creatinine and cystatin c in major subgroups of trial participants once these subgroups ceased on - trial therapy . these different responses would typically not have been identified in an on - drug / off - drug study that recruited without regard to the magnitude of the initial serum creatinine increase and analyzed the mean of the single active fenofibrate group , such as in the field trial ( 7,8 ) . the selection of the case and control subgroups in this study also had its limitations , however . interpretation of the serum marker trends in these groups during the trial and posttrial is complicated by the effect of single point - in - time selection of the extremes of percent change of serum creatinine and statistical regression to the mean . this study did not analyze the changes in the 28% of patients with intermediate response in percent serum creatinine after 3 months ( 220% change in serum creatinine ) . furthermore , the choice of 20% and 2% as thresholds for change in serum creatinine was arbitrary , albeit that they are clinically meaningful levels of change / no change . finally , participants who had significant worsening of renal function and required discontinuation of study medication are not included in this substudy . despite these limitations , these results can assist clinical decision making for type 2 diabetic patients with comparable cardiovascular and renal profile according to their percent serum creatinine change in response to initiation of fenofibrate therapy . this study found that about 50% of accord type 2 diabetic participants experienced an increase of 20% or more in serum creatinine levels immediately upon starting fenofibrate therapy , while approximately 25% had no increase . for those participants with a creatinine increase of 20% or more , the increase was reversible and not associated with an adverse effect compared with placebo on renal function over 5 years . those with no increase in creatinine upon starting fenofibrate appeared to have less renal function loss compared with placebo over 5 years of therapy . more work needs to be done to test these findings over longer durations of therapy , to determine if this apparent renal protection is also seen among the low hdl / high triglyceride subgroup that had apparent cardiovascular benefit in the main accord lipid study , and to determine the duration of the apparent renal protective effects .

objectiveto assess the reversibility of the elevation of serum creatinine levels in patients with diabetes after 5 years of continuous on - trial fenofibrate therapy.research design and methodsan on - drug / off - drug ancillary study to the action to control cardiovascular risk in diabetes ( accord ) lipid trial to investigate posttrial changes in serum creatinine and cystatin c. eligible participants were recruited into a prospective , nested , three - group study based on retrospective on - trial serum creatinine levels : fenofibrate case subjects ( n = 321 , 20% increase after 3 months of therapy ) ; fenofibrate control subjects ( n = 175 , 2% increase ) ; and placebo control subjects ( n = 565 ) . serum creatinine and cystatin c were measured at trial end and 68 weeks after discontinuation of trial therapy.resultsat trial end , case subjects had the highest adjusted serum creatinine ( se ) mg / dl ( 1.11 0.02 ) and the lowest adjusted estimated glomerular filtration rate ( egfr ) ( se ) ml / min/1.73 m2 ( 68.4 1.0 ) versus control subjects ( 1.01 0.02 ; 74.8 1.3 ) and placebo subjects ( 0.98 0.01 ; 77.8 0.7 ) . after 51 days off - drug , serum creatinine in case subjects was still higher ( 0.97 0.02 ) and egfr still lower ( 77.8 1.0 ) than control subjects ( 0.90 0.02 ; 81.8 1.3 ) but not different from placebo subjects ( 0.99 0.01 ; 76.6 0.7 ) . changes in serum cystatin c recapitulated the serum creatinine changes.conclusionsparticipants with significant initial on - trial increases in serum creatinine ( 20% ) returned to the same level of renal function as participants receiving placebo while participants who had 2% increase in serum creatinine had net preservation of renal function compared with the same unselected placebo reference group . the fenofibrate - associated on - trial increases in serum creatinine were reversible , and the reversal was complete after 51 days off - drug . the similarity of the cystatin c results suggests that the mechanism of this change is not specific for serum creatinine .

RESEARCH DESIGN AND METHODS Study population and design Renal measures Statistical analysis Study power RESULTS CONCLUSIONS

after the first characterization of thiol - specific antioxidant ( tsa ) in the yeast saccharomyces cerevisiae at the end of the 1980s ( kim et al . , 1988 ) , it turned out that this enzyme was a member of a major and highly expressed superfamily of thiol - dependent peroxidases able to reduce hydrogen peroxide , alkyl hydroperoxides and peroxynitrite in bacteria , archaea and eukaryotes ( perkins et al . , 2015 ; these peroxidases , named peroxiredoxins ( chae et al . , 1994 ) , are selenium- and heme - free peroxidases carrying a conserved peroxidatic cysteine ( cp ) located in the n - terminal domain of all members of this superfamily . a classification has been proposed based on sequence homology and structural data that finally classify peroxiredoxins of all kingdoms of life into six subfamilies ( hofmann et al . , 2002 ; knoops et al . , 2007 ; nelson et al . , 2011 ) . in a relatively simple but unexpectedly efficient mechanism , the cp of peroxiredoxins attacks the o - o bond of the peroxide during the peroxidase reaction and is subsequently oxidized to a cys sulfenic acid ( hall et al . , 2010 ; the cys sulfenic acid is then reduced back during the resolution step either by an external thiol in peroxiredoxins of the so - called 1-cys subgroup , either by a resolving cys ( cr ) of a second molecule resulting in the formation of an intermolecular disulfide bond in the typical 2-cys subgroup , or by a cr located within the same molecule resulting in the formation of an intra - molecular disulfide bond in the atypical 2-cys subgroup ( seo et al . , 2000 ) . the disulfide bond can be then reduced by thiol - dependent reductants such as thioredoxins ( perkins et al . , 2015 ) . although peroxiredoxins have been initially thought to be much less efficient peroxide reductases than catalases and glutathione peroxidases , it was shown that they are able to reduce hydrogen peroxide , alkyl hydroperoxides and peroxynitrite with rate constants as high as 1010 ms ( ferrer - sueta et al , 2011 ) . moreover , it has been proposed , based on models of redox kinetics , that peroxiredoxins reduce more than 90% of cellular hydrogen peroxide ( adimora et al . , 2010 ; cox et al , 2010 ) . thus , their central role as peroxide and peroxynitrite scavenging enzymes among the cellular arsenal of antioxidant enzymes , such as well - characterized catalase and glutathione peroxidases , has been probably underestimated until recently ( perkins et al . , 2015 ) . functionally , it was shown that in prokaryotic but also in eukaryotic cells , peroxiredoxins may act as cytoprotective antioxidant enzymes , protecting cells against deleterious oxidation of dna , proteins and lipids , but also other macromolecules , caused by physiological or pathophysiological production of intracellular as well as extracellular reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) ( perkins et al . , 2015 ) . this cytoprotective antioxidant activity could be considered as an ancestral function , endowed by oxidative stress - inducible adaptative peroxiredoxin expression in microorganisms but also by constitutively expressed peroxiredoxins ( with functions of house - keeping enzymes ) in metazoa including mammals in which peroxiredoxin expression is poorly inducible by hydrogen peroxide ( desaint et al . , 2004 ) . more interestingly , in the early 2000s , the role of hydrogen peroxide as mediator in cell signaling processes emerged ( rhee et al . , given that several eukaryotic peroxiredoxins were also shown to be susceptible to regulation by reversible overoxidative inactivation or other post - translational modifications , it was proposed that these peroxidases may act as modulators of peroxide signaling ( rhee et al . recently , several reports have been supporting these hypotheses and mammalian peroxiredoxins are now increasingly considered as regulators of peroxide signaling as local , at subcellular level , peroxide scavengers but also as redox relays ( rhee and woo , 2011 ; sobotta et al . , 2015 ) . in mammals , the six peroxiredoxin isoforms ( prdx1 - 6 ) , encoded by six different genes , are ubiquitous and multifunctional peroxidases addressed to different subcellular compartments and also found in the extracellular milieu ( hanschmann et al . , 2013 ; leyens et al . , 2003 ) . mammalian peroxiredoxins are constitutively expressed , although at different levels , in virtually all tissues and cell types ( hanschmann et al . , 2013 ; leyens et al . , 2003 ; perkins et al . , 2015 however , it was reported in the literature that peroxiredoxin expression is significantly increased upon acute inflammation in the lung ( kinnula et al . , 2002 ; knoops et al . , 1999 ) but also in other tissues ( wang et al . , 2002 ; yun et al . , 2015 ) . moreover , in macrophages and microglial cells , several peroxiredoxins are highly upregulated upon stimulation by interferon gamma ( ifn- ) and lipopolysaccharide ( lps ) ( abbas et al . , 2009 ; diet et al . , 2007 ; sun et al . , 2010 ) suggesting that peroxiredoxins may actually play key roles as cytoprotective antioxidant enzymes in cells that generate high levels of ros / rns upon pro - inflammatory stimulation but also as redox signaling modulators in innate immunity and inflammation . as emphasized by nathan and cunningham - bussel ( 2013 ) , in the immune system ros , but also rns , are neither unique products of one cell type , nor they have a unique effect that would be to kill microbes . it becomes clear that ros / rns have also physiological roles in signaling , that could probably extend to every cell type in immunology . however , as with any signaling system , ros / rns can trigger toxic mechanisms for the cells if the signal is too strong , if it lasts for too long or if it arises at the wrong time and place . in this context , peroxiredoxins might have unanticipated very specific and important roles . against this background , we tentatively report in this minireview published results of the notably increasing literature that suggests that peroxiredoxins are major players in inflammatory processes as : ( 1 ) cytoprotective enzymes for the host against elevated concentrations of ros / rns generated during inflammation , ( 2 ) modulators of redox signaling to control essential functions of inflammatory cells such as synthesis and release of inflammatory mediators , and ( 3 ) extracellular pathogen - associated molecular patterns ( pamps ) or damage - associated molecular patterns ( damps ) to regulate inflammation via pattern recognition receptors ( prrs ) . here , we would like also to apologize for any omissions of pertinent original references owing to space constraints of this minireview and to note that more extensive reviews on the roles of peroxiredoxins in innate immunity and inflammation have been published previously ( ishii , 2015 ; ishii et al . , 2012 ) . the protective role of peroxiredoxins in inflammation was revealed by the higher sensitivity of peroxiredoxin knockout ( ko ) mice to pro - inflammatory lipopolysaccharide ( lps ) challenge . indeed , it was shown that intratracheal instillation of lps triggered higher ros levels in macrophages and more oxidative damages to dna and proteins in prdx3 ko mice compared to control animals ( li et al . , 2007 ) . accordingly , prdx2 ko mice are also more sensitive to lps - induced endotoxic shock and can be rescued by intravenous injection of adenovirus encoding prdx2 or administration of catalase ( yang et al . , 2007 ) . of note , prdx1 is also upregulated upon lps exposure in microglia of the central nervous system and reduction of lps - mediated prdx1 upregulation was shown to sensitize the microglia to h2o2-mediated cell death suggesting that in this pro - inflammatory situation , prdx1 is indeed able to protect cells against h2o2-mediated oxidative attacks ( kim et al . , 2008 ) . in the same context , prdx1 ko mice are highly susceptible to bleomycin - induced pulmonary inflammation and fibrosis implicating oxidative damages ( kikuchi et al . , 2011 ) . it was also shown that in transgenic mice overexpressing human prdx4 in a model of type 1 diabetes mellitus , -cells in the islets are significantly protected against inflammatory insults and apoptosis ( ding et al . , 2010 ) . in the same transgenic mice , prdx4 was shown to protect against nonalcoholic steatohepatitis , type 2 diabetes mellitus and the metabolic syndrome by reducing oxidative stress - induced injuries ( nabeshima et al . , 2013 ) . ros / rns produced by inflammatory cells are increasingly recognized as key signaling molecules and no longer considered only as harmful molecules released to eradicate pathogens in host defense mechanisms of innate immunity ( mittal et al . , 2014 ; nathan and cunningham - bussel , 2013 ) . as noted previously , the role of peroxiredoxins as modulators of redox signaling is now widely accepted ( rhee et al . , 2005 ; sies , 2014 ; woo et al . , 2010 ) . so , although peroxiredoxins are constitutively expressed by virtually all cell types in mammals , and considering that peroxidase activity of many mammalian peroxiredoxins can be modulated by post - translational modifications including overoxidation , several reports have been focused on inducible peroxiredoxins in inflammatory cells and their potential roles as regulators in redox signaling during inflammatory processes . notably , drapier and collaborators have shown that expression of prdx1 , prdx5 and prdx6 is dramatically increased in mouse bone marrow - derived macrophages ( bbms ) upon stimulation with ifn- and lps ( abbas et al . , 2009 ; diet et al . , 2007 ) . ( 2010 ) also reported increased expression of prdx1 , prdx2 , prdx4 , prdx5 and prdx6 in murine bmms stimulated with lps and ifn-. it was speculated that the increased expression of several peroxiredoxins in such inflammatory situation could represent a negative feed - back loop to protect macrophages against oxidative insults but could also contribute to the control of redox - sensitive effectors ( diet et al . , 2007 ) . interestingly , prdx5 expression was shown to be upregulated via lps / tlr4 and the th1 cytokine ifn- pathways ( abbas et al . , 2009 ) . it was demonstrated that tlr4-dependent increase of prdx5 expression in mouse macrophages was mediated by a trif - dependent / ifn--independent pathway , and that ifn- increased prdx5 gene expression via a myd88- and tnf - dependent pathway ( abbas et al . , 2009 ) . mapks were shown to be a point of convergence downstream of ifn- and lps signaling pathways leading to prdx5 induction ( abbas et al . , 2009 ) . 2010 ) showed that prdx5 expression is also highly sensitive to lps stimulation in microglia . induced prdx5 expression was demonstrated to be under the control of cooperative action of ros , rns and the jnk signaling cascade . interestingly , knockdown of prdx5 increased microglial activation with augmented ros generation and jnk - dependent no production suggesting that prdx5 might indeed modulate a ros - dependent signaling cascade ( sun et al . , 2010 ) . moreover , choi et al . ( 2013 ) proposed that pdrx5 would be implicated in the regulation of lps / tlr4-induced il-6 production in mouse macrophages , by modulating the jak - stat signaling pathway . in the proposed mechanism , prdx5 interacts with jak2 via its cp which leads to the inhibition of the sequential phosphorylation of jak2 and stat5 and consequently reduces il-6 production . prdx1 was also involved in the control of ros / rns - dependent microglial activation ( kim et al . , indeed , it was shown that prdx1 is upregulated upon exposure of microglia to lps ( but not to h2o2 or paraquat ) in a ros / p38 mapk - dependent manner ( kim et al . , 2013 ) . under this pro - inflammatory situation , it was proposed that prdx1 can serve as a regulator of microglial activation by inhibiting no production via the suppression of a ros / nf-b / inos signaling pathway ( kim et al . , prdx6 was also shown to play an unexpected role in nadph oxidase-2 ( nox2 ) activation ( chatterjee et al . , 2011 ) . indeed , it was shown that in stimulated macrophages , prdx6 is phosphorylated , translocated to the membrane and the activation of its phospholipase a2 activity facilitates assembly and activation of the nox2 complex ( chatterjee et al . , 2011 ) . inflammation is triggered when innate immune cells , such as macrophages detect infection or tissue injury . at the molecular level , prrs , including toll - like receptors ( tlrs ) , on the cell surface or in the cytoplasm , respond to pamps or host - derived damps to trigger expression of genes coding for chemokines , cytokines , adhesion molecules and regulators of the extracellular matrix that finally promote the recruitment and activation of leukocytes to eliminate microbes and host debris ( newton and dixit , 2012 ) . several peroxiredoxins have been proposed to act as pamps or damps ( robinson et al . , 2010a ; 2010b ) . as ubiquitous enzymes involved in the defense against ros / rns , peroxiredoxins have been studied for their role in parasite survival and virulence that require efficient defenses against ros / rns produced by the host immune system ( gretes et al . , 2012 ) . interestingly , it was also shown that prdxs secreted by parasitic helminths ( parasitic round- and flatworms ) may act as pamps , triggering the recruitment of macrophages and a th2 response with the production of high levels of il-10 and prostaglandin e2 ( donnelly et al . , 2005 ; 2008 ) . moreover , a typical 2-cys malarial prdx from plasmodium berghei was reported to act as a pamp by binding to tlr4 receptor on macrophages and triggering a pro - inflammatory response ( furuta et al . , 2008 ) . it must be noted here that prior to its characterization as a peroxidase , mammalian prdx1 was initially described as a soluble protein released from lysed cells and exhibiting immune modulating function able to enhance the cytotoxic activity of natural killer cells ( shau et al . , 1993 ) . since these seminal works , it was shown that extracellular prdx1 is able to act as a damp to trigger tlr4-dependent secretion of tnf- and il-6 , and dendritic cell maturation when incubated with murine macrophages or immature bone marrow - derived dendritic cells ( riddell et al . , 2010 ) . it was also reported that prdx1 interaction with tlr4 is independent of its peroxidase activity but dependent on its ability to form decamers ( riddell et al . , 2010 ) . moreover , very recently , exosomal release by different cells upon exposure to lps and tnf- of oxidized dimeric forms of prdx1 and prdx2 , as well as glutathionylated prdx2 triggering the production of inflammatory cytokines by macrophages , has also been described ( mullen et al . , 2015 ; salzano et al . , 2014 ) . the role of prdx1 , prdx2 , prdx5 and prdx6 as damps was emphasized in post - ischemic inflammation in the brain ( shichita et al . , 2012 ) . ( 2012 ) showed that following ischemic injuries , peroxiredoxins that are released from necrotic brain cells can induce expression of inflammatory cytokines in macrophages through activation of tlr2 and tlr4 , leading to neural cell death . innate immunity and inflammation are ancient evolutionary mechanisms which have been partly conserved from invertebrates to mammals ( royet et al . , 2005 ) . interestingly , it has been reported that several prdxs are significantly regulated in immune cells upon virus , bacteria or parasite infection in non - mammalian vertebrates such as fishes ( reviewed in valero et al . , 2015 ) or in invertebrates such as insects ( ahn et al . , 2012 ; chen et al . , 2014 ; radyuk et al . , 2010 ; zhang and lu , 2015 ) and molluscs ( genard et al . , 2013 ) to cite only a few reports . in fishes , large yellow croaker ( pseudosciaena crocea ) prdx4 was shown to be upregulated upon bacterial challenge in spleen cells ( yu et al . , 2010 ) . moreover , prdx4 gene knockdown resulted in increased nf-b activity , increased expression of tnf- and cc chemokine , as well as downregulation of il-10 expression in the spleen ( yu et al . , 2010 ) 2014 ) showed that miiuy croaker ( miichthys miiuy ) prdx3 and prdx5 are highly upregulated in spleen and kidney , two lymphoid organs in fish , after bacterial infection suggesting that these peroxiredoxins may also play an important role in fish immune response . in insects , it was shown that drosophila prdx5 confers resistance to oxidative stress and extend fly life span by up to 30% ( radyuk et al . , 2009 ) . prdx5 ko flies are also more susceptible to oxidative stress and have a higher incidence of apoptosis as well as a shortened life span ( radyuk et al . , 2009 ) . furthermore , in the silkworm ( bombyx mori ) prdx5 was shown to be upregulated by hydrogen peroxide injection and bacterial infection in haemocytes indicating that prdx5 could also play an important role in immune response and oxidative stress in insects ( zhang and lu , 2015 ) . accordingly , prdx5 expression is increased by gut - specific dual oxidase ( duox ) activation in gut epithelia upon oral bacterial infection in drosophila and this prdx5 upregulation was shown to be mediated by the jnk - foxo signaling pathway ( ahn et al . , 2012 ) . ( 2010 ) proposed also that prdx5 plays an important modulatory role in drosophila immune response via the jnk signaling pathway albeit as a negative regulator according to prdx5 ko fly model . peroxiredoxins are now recognized as a major superfamily of peroxidases conserved throughout evolution in bacteria , archaea and eukaryotes ( perkins et al . , 2015 ) . in mammals , peroxiredoxins are constitutively expressed in virtually all tissues and cell types where they can act as peroxide and peroxynitrite scavenging enzymes in collaboration with catalase and glutathione peroxidases . functionally , peroxiredoxins have been shown to protect cells against deleterious oxidation of macromolecules triggered by physiological or pathophysiological production of ros and rns . this cytoprotective function of peroxiredoxins could be considered as a more ancestral function endowed initially by bacterial and archaeal peroxiredoxins . however , as the role of peroxides , and notably hydrogen peroxide , as signaling molecules is emerging , and because peroxiredoxin activity is regulated by post - translational modifications , it is now recognized that peroxiredoxins may modulate peroxide signaling implicated in essential cell functions . accordingly , during the last decade , several lines of evidence pointed that peroxiredoxins may be major players in immunity including innate immunity and inflammation . indeed , it was demonstrated that peroxiredoxins may act as cytoprotective enzymes against high levels of ros / rns produced during inflammatory processes , as modulators of redox signaling involved in the control of inflammatory cell functions , and as extracellular pamps or damps . in this context , and considering the multiple potential roles of these peroxidases , challenges for the coming years will be to determine where ( in which inflammatory cells , in which subcellular compartment or extracellularly ) , when ( at which step of the inflammatory process ) and how ( as peroxide reductase , as redox relay or as ligand for membrane or intracellular receptors ) peroxiredoxins act in innate immunity and inflammation .

inflammation is a pathophysiological response to infection or tissue damage during which high levels of reactive oxygen and nitrogen species are produced by phagocytes to kill microorganisms . reactive oxygen and nitrogen species serve also in the complex regulation of inflammatory processes . recently , it has been proposed that peroxiredoxins may play key roles in innate immunity and inflammation . indeed , peroxiredoxins are evolutionarily conserved peroxidases able to reduce , with high rate constants , hydrogen peroxide , alkyl hydroperoxides and peroxynitrite which are generated during inflammation . in this minireview , we point out different possible roles of peroxiredoxins during inflammatory processes such as cytoprotective enzymes against oxidative stress , modulators of redox signaling , and extracellular pathogen- or damage - associated molecular patterns . a better understanding of peroxiredoxin functions in inflammation could lead to the discovery of new therapeutic targets .

INTRODUCTION FUNCTIONS OF PEROXIREDOXINS MAMMALIAN PEROXIREDOXINS AND IMMUNITY PEROXIREDOXINS AS CYTOPROTECTIVE ANTIOXIDANT ENZYMES IN INFLAMMATION PEROXIREDOXINS AS MODULATORS OF REDOX SIGNALING IN INFLAMMATION PEROXIREDOXINS AS PAMPS AND DAMPS WHAT CAN WE LEARN FROM NON-MAMMALIAN VERTEBRATES AND INVERTEBRATES? CONCLUSION

annually , more than 7 million patients with chest pain present to emergency departments in the usa . however , of these patients , 75% do not have cardiac - related chest pain.1 evaluation and treatment of these patients imposes a cost of more than us$10 billion on the us health system.2 in iran , a frequent number of beds in coronary care units ( ccus ) are occupied by low - risk patients with suspected diagnosis of acute coronary syndromes ( acss ) . in 2003 , azizi et al found that approximately 70% of these patients did not have deterioration of their symptoms during their hospitalization and were discharged after 23 days.3 at the same time that these ccu beds were occupied by low - risk patients , 2%8% of patients with chest pain who actually had acs were discharged.4 in a study of 903 patients with acute chest pain , it was found that patients stayed an average of 3.01 days in ccu and that this hospitalization duration was associated with : the diagnosis of acute myocardial infarction and unstable angina , severity of complications , the use of invasive and noninvasive diagnostic methods , and primary triage for referring patients to ccus.5 factors such as time of admission , insurance coverage , cardiac monitoring , exercise tests , and echocardiography had effects on increasing the length of the patients stay period.5 the results of another study have shown that the probability of occurrence of serious complications in low - risk patients is much less than high - risk patients.6 gatein et al have shown that patients with a normal electrocardiogram without chest pain can be observed without cardiac monitoring and there is no indication for the admission of such patients to ccu.7 the current study was done in 2008 , the data were collected this year , to determine the rate of exploitation of resources and cardiovascular outcomes in low - risk patients with chest pain in ccus of hospitals affiliated to a major medical university in tehran , iran . in this descriptive study , 550 patients with chest pain who were hospitalized in the ccu of six affiliated general hospitals of shahid beheshti university of medical sciences , tehran , iran , were recruited by census method during a 4-month period . the statistics of the surveyed hospitals in 2008 are presented in table 1 . using the thrombolysis in myocardial infarction ( timi ) risk score , 95 patients were identified as low risk ( 17.27% ) , 110 ( 21.5% ) as moderate risk , and 336 ( 61% ) as high risk . the low - risk group was evaluated with respect to demographics , bed - occupancy rate ( bor ) , mean hospitalization period , expenses during admission to ccu , and cardiovascular outcomes in the 30-day period postdischarge . for gathering the required information , inclusion criteria , based on the timi score provided by the american college of cardiology for the categorizing of patients with chest pain , and patient medical records were used simultaneously . the inclusion criteria consisted of disease information , admission to duration of stay in ccu , the expenses of hospitalization , and cardiovascular outcomes . the timi score uses seven independent risk factors : presence of three or more coronary artery disease ( cad ) risk factors established cad on angiography use of aspirin for at least 1 week occurrence of angina at least twice in a recent 24-hour period st segment deviation of 0.5 mm or more increase in cardiac enzymes . patients are categorized according to their scores into low risk ( timi score < 3 ) , moderate risk ( timi score 35 ) , and high risk ( timi score > 5 ) . for determination of the validity of timi scores and information gathered according to the inclusion criteria , the content validity method was applied . for reliable testing of timi , the interobserver reliability method was used ; risk rate was determined in this way and , after ten observations , an acceptable rate of 0.80 was achieved . since this study was based on medical records of the surveyed hospitals and no diagnostic or therapeutic intervention was performed on patients , there was no medical ethics approval and no need to obtain informed consent , either written or verbal , from any patient . since this study was based on medical records of the surveyed hospitals and no diagnostic or therapeutic intervention was performed on patients , there was no medical ethics approval and no need to obtain informed consent , either written or verbal , from any patient . mean ( standard deviation ) hospitalization duration in the low - risk group was 3.04 ( 0.71 ) days ( range , 14 days ) ( table 2 ) . using analysis of variance ( anova ) , no significant difference was seen between the six hospitals regarding hospitalization duration ( p = 0.602 ) . the highest bor by low - risk patients was seen in taleghani and shohada tajrish hospitals and the lowest was in modarres hospital ( table 2 ) . mean total hospitalization expenses was us$205 ( range , 129240 ) ( table 3 ) . in patients who had insurance , 90% of hospitalization expenses using anova , no significant difference was seen between the six surveyed hospitals ( p = 0.699 ) . in terms of cardiovascular outcomes , the majority of patients ( 89.5% ) at 1-month follow - up did not have any cardiac complications and six cases ( 6.3% ) presented to emergency departments due to chest pain and were discharged after receiving outpatient treatment and four cases ( 4.2% ) were readmitted to ccu . at the 1-month follow - up of low - risk patients , in the six surveyed hospitals , of 550 patients who were hospitalized in ccus with the diagnosis of acute coronary syndromes , 95 cases ( 17.5% ) were low risk and their mean hospitalization duration was 3.04 days . in udvarhelyi et al s study , mean hospitalization period was reported as 3.01 days , which is in agreement with the present study.5 one of the reasons that bor was the lowest at modarres hospital is that the hospital is a specialty hospital for patients with cardiac disorders . mean treatment expenses of the patients studied was us$205 , whereas in udvarhelyi et al s study , hospitalization expenses were reported as us$2375.5 in the sirois and pimental study , the mean triage expenses of patients in emergency departments was us$189.6 the high cost of unnecessary hospitalization of low - risk patients and increased cost to insurance systems can be reduced by the prevention of hospitalization of such patients . in the current study , at 1-month follow - up no deaths occurred . similarly , in solinas et al s study , at 1-month follow - up after discharge from emergency unit , acs was seen in 5.2% ( three acute myocardial infarction cases ) but no deaths were reported.8 in the present study , the fact that there was no cardiovascular complications in 89.5% of patients at 1-month follow - up shows that the studied patients were not high risk and were not indicated for admission to ccus . additionally , they did not have any special guidelines for triage of patients with chest pain . as a result of this ineffective approach , a considerable number of low - risk patients were admitted to the limited ccu facilities , which should be reserved for the treatment of high - risk patients . since special care is expensive and requires expert personnel , patients should be categorized according to their treatment needs and those in critical condition should be prioritized for admission to ccus .

backgroundmost patients who present to medical centers due to chest pain do not suffer from acute coronary syndromes and do not need to be hospitalized in coronary care units ( ccus ) . this study was done to determine exploitation of resources and cardiovascular outcomes in low - risk patients with chest pain hospitalized in ccus of educational hospitals affiliated with a major medical university.methodsover a 4-month period , 550 patients with chest pain who were hospitalized in the ccus belonging to six hospitals affiliated to the authors medical university were recruited by census method . using thrombolysis in myocardial infarction risk score , 95 patients ( 17.27% ) were categorized as low - risk patients . this group was evaluated with respect to demographics , bed occupancy rate , mean hospitalization period , expenses during admission , and cardiovascular outcomes in the 30-day period postdischarge.resultsmean ( standard deviation ) hospitalization duration was 3.04 ( 0.71 ) days . no significant difference was seen between the six surveyed hospitals regarding hospitalization duration ( p = 0.602 ) . the highest bed occupancy rate was seen in taleghani and shohada tajrish hospitals and the lowest was in modarres hospital . the mean paid treatment expenses by low - risk patients was irr 2,050,000 ( us$205 ) . mean total hospitalization expenses was us$205 . no significant difference was seen between the six surveyed hospitals ( p = 0.699 ) . of the patients studied , 89.5% did not show any cardiovascular complications in 1 month and no deaths occurred.conclusiongiven the high bed - occupancy rate by low - risk patients , associated high hospitalization costs , and the lack of cardiovascular complications in patients observed at 1-month follow - up after discharge , it is recommended that appropriate evaluations be performed in emergency units to prevent unnecessary admissions .

Introduction Methods and materials Ethics statement Results Discussion Conclusion

in our current study , we found that early exponential growth phase cultures of p. aeruginosa elicited the most robust net release and presence of a functional flagellum was essential for this process . forced contact of immotile p. aeruginosa with pmns restored their ability to trigger maximal net extrusion . p. aeruginosa flagellin alone was unable to induce net release . in a genetic complementation study we found that both , motab and motcd loci of p. aeruginosa thus , we identified flagellar swimming motility as a novel microbial factor crucial to pmn activation and net formation . although it is undocumented whether nets are present at early stages of cf lung disease , bacterial motility - fueled net formation likely occurs at this initial phase because early cf clinical isolates of p. aeruginosa typically express flagellum and pmns are also present ( fig . 1 ) . the interaction of pmns with early forms of p. aeruginosa must be critical to determine later progression of cf lung disease . important questions to be answered are why nets released at this early stage would be incapable of clearing p. aeruginosa infection and instead drive bacterial adaption toward an aflagellated , biofilm - forming phenotype . over the course of cf lung disease , p. aeruginosa down - regulates its flagellum expression . in chronic cf airways , p. aeruginosa mainly exist in 3-dimensional , suspension biofilms also called non - attached aggregates ( fig . 1 ) . these suspension biofilms surrounded by pmns represent the characteristic clinical picture in chronic cf airways . biofilms are dynamic structures , and motile , flagellated bacteria likely break free from biofilms in chronic cf , possibly interacting with pmns ( fig . 1 ) . this is supported by recent data showing that p. aeruginosa flagellin is detected in sputa of chronic cf patients . a minor population of flagellated p. aeruginosa in chronic cf airways could also have been marginalized so far because this topic has not been intensely investigated yet , and this population is hard to study and could have been overlooked in the presence of much more abundant biofilm - bound bacteria accumulating well - characterized mutations ( muca , lasr ) . conclusions with respect to the population structure of p. aeruginosa in cf have largely been generalized based on results obtained on single bacterial isolates , although high levels of phenotypic diversity among p. aeruginosa isolates within individual cf patients have already been noted . a small population of flagellated p. aeruginosa could be found in cf airways while most p. aeruginosa are present in form of alginate - producing , elastase - negative bacteria . muca mutations drive the mucoid , biofilm - forming phenotype , lasr mutations contribute to pmn recruitment while outbreaks of flagellated bacteria from biofilms could be mainly responsible for pmn activation and net release . pmns quickly and easily recognize motile , flagellated forms of p. aeruginosa and launch their robust effector mechanisms including net release in response to them . on the other hand , bacterial biofilms therefore , the way motile p. aeruginosa interacts with pmns , the cell type representing most of cells found in chronic cf airways , is likely an important factor in influencing the progression of cf lung disease despite the fact that planktonic forms of p. aeruginosa are outnumbered by those found in biofilms ( fig . 1 ) . overall , we speculate that p. aeruginosa motility - driven pmn activation has clinical relevance not only at initial but also later stages of cf airway disease .

abstractneutrophil extracellular trap ( net ) formation represents a unique effector function of neutrophils ( pmn ) . the mechanism of net release in response to bacteria is largely unknown . we studied the process by which pseudomonas aeruginosa , an opportunistic pathogen , interacts with primary pmns , and found that flagellar swimming motility of the bacterium is essential for inducing net extrusion . cystic fibrosis ( cf ) lung disease is associated with p. aeruginosa infection and pmn - dominated inflammation . although nets are abundant in cf airways , the main factors triggering net release in cf remain unclear . our study implicates that motile p. aeruginosa is a strong net - inducer in cf . in early stages of cf lung disease flagellated , motile isolates of p. aeruginosa are characteristic and their interactions with pmns could lead to net formation . in chronic cf , p. aeruginosa down - regulates its flagellum expression to avoid recognition by the immune system and forms biofilms . flagellated bacteria , however , are released from biofilms and could interact with pmns to form nets . although flagellated forms likely represent only a small fraction of the total p. aeruginosa load in chronic cf , net release induced by them could have a significant impact on inflammation and lung function since flagellated forms trigger the most robust response of the immune system including pmns.overall , we speculate that net formation driven by motile p. aeruginosa could be a novel , significant contributor to pathogenesis at both , early and late stages of cf lung disease .

Flagellar motility drives Flagellated Disclosure of potential conflicts of interest

the majority of breast cancer patients are diagnosed with luminal tumors that are characterized by the expression of estrogen receptors ( er ) and progesterone receptors ( pr ) and the absence or only weak amplification of her2 ( this latter parameter depends on the subclass , whether luminal a or luminal b ) [ 1 , 2 ] . although er - expressing and pr - expressing patients typically experience a favorable outcome and a relatively good prognosis , eventually many of them become unresponsive to endocrine therapies and develop metastases at remote organs [ 13 ] . to date , the mechanisms that contribute to tumor progression and more importantly to metastasis formation in these patients are poorly understood . tumor cell dissemination to remote organs is a multifactorial process that is linked to upregulation of extracellular matrix ( ecm ) and adhesion receptors , to increased spreading and migration , and to epithelial - to - mesenchymal transition ( emt ) [ 410 ] . moreover , strong induction of metastatic traits is endowed on the tumor cells by elements of the tumor microenvironment that promote many different metastasis - related functions including tumor cell spreading and emt [ 1113 ] . the tumor milieu is an extremely complex and dynamic contexture comprised of many cell types , ecm components , and secreted factors . recently , intensive research indicates that there is an intimate link between inflammation and cancer , where inflammatory cells and cytokines promote processes of tumor growth and progression . in this respect , much emphasis has been attributed to the inflammatory cytokine tumor necrosis factor ( tnf ) . tnf was shown to induce antitumor effects when administered in high concentrations directly into tumors . thus , tnf was considered for quite some time as a potential therapeutic modality , whose introduction to patients would limit disease course . however , recent investigations challenged this view and indicated that chronic and consistent presence of tnf in tumors leads to procancerous consequences in many malignant diseases [ 1417 ] . specifically in breast cancer , studies in animal model systems have shown that tnf exerted causative procancerous activities through a diverse set of mechanisms [ 1821 ] . along these lines , we and others have shown that tnf was highly expressed in breast tumors [ 2225 ] , that the incidence of tnf expression was significantly increased in advanced stages of breast cancer ( detected in approximately 90% of the patients with recurrent disease ) , and that tnf induced emt and invasion of breast tumor cells [ 22 , 26 , 27 ] . moreover , by virtue of its inflammatory actions as inducer of inflammatory chemokines , tnf indirectly led to high presence of protumoral leukocyte subpopulations in tumors . the opposing roles of tnf in cancer may be due to interactions that the cytokine has with other procancerous elements that reside at the tumor milieu . in luminal breast tumors , such interactions could be taking place mainly with two arms of the tumor microenvironment : hormones that are key regulators of the malignant process and growth - supporting factors that promote tumor cell proliferation . indeed , the hormone estrogen is a key player in luminal breast tumors , where it enhances the proliferation of breast tumor cells , induces emt , and consequently increases the migratory and invasive abilities of these cells [ 2932 ] . although the lack of er is usually associated with worse prognosis [ 32 , 33 ] , the hormone by itself has definite potent tumor - promoting functions and thus is a major therapeutic target in breast cancer treatment . in parallel , growth - supporting factors like epidermal growth factor ( egf ) are of large relevance . luminal breast cancer cells usually do not exhibit amplification of the egf - signaling her2 receptor or show only low over - expression of this receptor ; nevertheless , they bind egf and respond to its tumor - promoting stimuli [ 3437 ] . egf enhances tumor cell proliferation , migration , invasion , and emt [ 36 , 3840 ] , and thus it should be taken into account when we consider joint activities of microenvironmental factors on breast cancer metastasis . in view of the multi - factorial nature of the tumor microenvironment , in this study we determined the combined impact of the three arms inflammatory ( tnf ) + hormonal ( estrogen ) + growth - supporting ( egf)on malignancy - promoting characteristics and functions of luminal breast tumor cells . combined stimulation by tnf + estrogen + egf provides a more relevant representation of the multifaceted nature of the tumor microenvironment in luminal breast tumors than the reductionist approach of testing the activity of each element alone . approach is supported by published findings demonstrating coregulatory intracellular interactions existing between tnf- , estrogen- , and/or egf - mediated pathways in breast cancer and in other malignancies [ 34 , 41 , 42 ] . accordingly , in this study we determined the impact of the tnf + estrogen + egf stimulus and compared it to the effect of each factor on its own . using the joint powers of the tnf + estrogen + egf stimulation , we found that mcf-7 luminal breast tumor cells have acquired very high metastasis - related functions . already at the initial phases of the study we found that the combined stimulation had a much higher influence than tnf alone , estrogen alone , or egf alone on the tumor - promoting aspects that were studied . therefore , in more advanced stages of the research we focused on the effects of the joint stimulation by tnf + estrogen + egf on functional tumor - promoting readouts , including tumor growth and metastasis formation . overall , our findings indicate that tnf induces many metastasis - related functions in luminal breast tumor cells and that its activities are largely amplified by cooperativity with estrogen and egf . the tnf + estrogen + egf stimulation has endowed the cancer cells with high spreading and emt characteristics and with tumor- and metastasis - promoting functions . moreover , although tnf was cytotoxic to some of the tumor cells , its cooperativity with estrogen and egf has led to selection of tumors cells that have gained high metastasizing abilities in vivo , in an animal model system . these observations suggest that a paradigm shift is required in the treatment of luminal breast cancer patients , in which therapies against tnf should be introduced to the clinical regimen rather than the use of tnf as a cytotoxic agent . inhibitors of tnf are already in clinical use for other indications ( autoimmune diseases ) , and our findings suggest that they should be combined with antihormonal approaches and modalities targeting the egf - her2 pathway . we propose that such integrative therapies targeting multiple tumor - promoting factors may achieve a high therapeutic impact in luminal breast cancer patients . mcf-7 cells are luminal breast tumor cells that express high levels of er and pr and show low levels of expression of her2 and of egf receptors ( egfr ) [ 4345 ] . these cells were found to provide the unique setup of luminal breast tumor cells which is required for this study by ( 1 ) responding to tnf [ 22 , 46 , 47 ] ; ( 2 ) expressing estrogen receptor ( er ) and responding to estrogen [ 34 , 48 , 49 ] ; ( 3 ) responding to egf despite relatively low expression of her2 and egfr [ 3437 ] . the cells were kindly provided by professor kaye ( weizmann institute of science , rehovot , israel ) . in line with published mcf-7 characteristics [ 4345 ] , the cells were authenticated on the basis of expression and activity of er ; in vitro estrogen responsiveness ; tumor formation requiring estrogen and matrigel ; and low expression of her2 . the cells were maintained in growth media containing dmem supplemented by 10% fetal calf serum ( fcs ) , 2 mm l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 250 ng / ml amphotericin ( all from biological industries , beit haemek , israel ) . mcf-7 cells were plated over - night in complete media , washed in pbs , and stimulated for three days with tnf , estrogen , and/or egf . the concentrations of the three stimulants were selected based on extensive titration and kinetics analyses ( data not shown ) , and they agree with the conventional dose range used in other research systems : tnf at 50 ng / ml ( cat . 300 - 01a ; peprotech , rocky hill , nj , usa ) , estrogen at 10 m ( cat . e8875 ; sigma , saint louis , mo , usa ) and egf at 30 ng / ml ( cat . 236-eg ; r&d systems , minneapolis , mn , usa ) . in all procedures , control non - stimulated cells were grown in the presence of the diluents of the above stimulators . media , including the stimulators , were changed daily . when indicated , the pharmacological inhibitor of src , pp2 ( cat . 529573 ; calbiochem , emd millipore , san diego , ca , usa ) was used in a conventional concentration of 2.55 m . the inhibitor was added to cell cultures simultaneously with the stimulation of the cells by tnf + estrogen + egf or to control non - stimulated cells and was present in culture throughout the duration of stimulation ( three days ) . control cells were treated with the solubilizer of the drug at similar dilutions ( dmso ; sigma ) . stimulated and non - stimulated mcf-7 cells were fixed with 8% paraformaldehyde ( pfa ; cat . 1.04005 ; merck kgaa , darmstadt , germany ) , permeabilized by 0.2% triton ( cat . sc-558 ; santa cruz biotechnology , santa cruz , ca , usa ) and mouse igg1 against paxillin ( cat . no . 624001 then , the cells were incubated with the secondary abs : dylight-549-conjugated against rabbit igg ( cat . no . 111 - 505 - 144 ; jackson immunoresearch laboratories , west grove , pa , usa ) or alexa-647-conjugated against mouse igg ( cat . e18 - 18 ; golden bridge international , mukilteo , wa , usa ) and read by zeiss lsm 510-meta confocal microscope ( carl zeiss , jena , germany ) at 63 magnification . expression levels of cell surface molecules were determined by flow cytometry ( facs ) in stimulated and non - stimulated mcf-7 cells , using a becton dickinson facsort ( mountain view , ca , usa ) . the following abs were used : pe - conjugated mouse igg1 against integrin 1 ( cd29 ; cat . 303004 ; biolegend ) , alexa 488 conjugated - rat igg2a against integrin 6 ( cd49f ; cat . the abs against e - cadherin were followed by fitc - conjugated abs against mouse igg ( cat . total rna was isolated from stimulated and non - stimulated mcf-7 cells using the ez - rna kit ( cat . rna samples were used for generation of first - strand complementary dna synthesis using the m - mlv reverse transcriptase ( cat . quantification of cdna targets by quantitative real - time polymerase chain reaction ( qpcr ) was performed on rotor gene 6000 ( corbett life science , concorde , nsw , australia ) , using rotor gene 6000 series software . ab-4163/a ; thermo fisher scientific , waltham , ma , usa ) according to the manufacturer 's instructions . in each reaction , two pairs of specific primers were used , designed for different exons . the sequences of the primers as follows : for zeb1-forward 5-tgcagctgactgtgaaggtgt-3 , reverse 5-cttgcccttcctttctgtcatc-3 ; for snail - forward 5-ctaatccagagtttaccttccagca-3 , reverse 5-agtcccagatgagcattggc-3 ; for slug - forward 5-cctggtcaagaagcatttcaa-3 , reverse 5-caggcatggagtaactctca-3 ; for the normalizing gene rs9-forward 5-ttacatcctgggcctgaagat-3 and reverse 5-gggatgttcaccacctgctt-3. pcr amplification of the genes rs9 and slug was performed over 40 cycles ( 95c for 15 sec , 59c for 20 sec , 72c for 15 sec ) , while amplification of zeb1 was performed over 40 cycles ( 95c for 15 sec , 59c for 20 sec , 80c for 15 sec ) , and of snail over 45 cycles ( 95c for 15 sec , 59c for 20 sec , 84.5c for 15 sec ) . dissociation curves for each primer set indicated a single product , and no - template controls were negative after 40/45 cycles . quantification was performed by standard curves , on the linear range of quantification . stimulated and non - stimulated mcf-7 cells were recultured in 96-well plates in growth medium containing the stimulants . after 8 hr , media were removed and the cells were exposed to combined stimulation by tnf + estrogen + egf , in the absence or presence of 1 m doxorubicin ( teva pharmaceutical , netanya , israel ; kindly provided by professor peer , tel aviv university ) . after additional three days , media were removed , cells were washed , and xtt reagent ( cat . 20 - 300 - 1000 ; biological industries ) was added to the wells according to the manufacturer 's instructions for 2 hr . for each group ( non - stimulated and stimulated cells ) the percentage of cell survival was calculated compared to cells that were not exposed to doxorubicin . in other cases , viable cells were counted using a hemocytometer , and total cell number was calculated . stimulated and non - stimulated mcf-7 cells were grown as described above . conditioned medium ( cm ) was removed from the last 24 hr of cultures , and cxcl8 and ccl2 levels were determined by elisa using standard curves with rhcxcl8 or rhccl2 ( cat . the following abs were used ( all from peprotech ) : for cxcl8 : coating polyclonal abs ( cat . 016 - 030 - 084 ; jackson immunoresearch laboratories ) , the substrate tmb / e solution ( cat . the reaction was stopped by the addition of 0.18 m h2so4 and was measured at 450 nm . in parallel , cells were removed by trypsinization and counted by trypan blue exclusion ( see above ) , and the results were normalized to cell numbers . the growth medium was removed and cells were stimulated for three days with tnf + estrogen + egf as indicated above . cm of the last 24 hr were collected and separated on 7.5% sds - polyacrylamide gels containing 0.1% gelatin substrate . after electrophoresis , gels were washed three times in 50 mm tris / hcl ph 7.5 , containing 2.5% triton x-100 . the gels were then washed three times in 50 mm tris / hcl ph 7.4 buffer , followed by incubation in buffer containing 50 mm tris / hcl ph 7.4 , 0.02% nan3 , and 10 mm cacl2 for 48 hr at 37c . following three washes in double distilled h2o , the gels were stained with 0.1% coomassie blue and distained in 20% methanol and 10% glacial acetic acid until clear bands of protein degradation were visualized . in parallel , cells that were removed by trypsinization were counted by trypan blue exclusion ( see above ) . the obtained bands were subjected to densitometry performed using scion image software , and their density was normalized to cell number . 6-well plates were incubated overnight on a rocker with 1.2% poly(2-hydroxyethyl methacrylate ) ( cat . mcf-7 cells were plated in phenol - red free dmem / f12 medium supplemented with 2 mm l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , 250 ng / ml amphotericin ( all from biological industries ) , 0.4% bsa ( cat . 17504 ; gibco , life technologies , grand island , ny , usa ) , 20 ng / ml basic fgf ( cat . no . 100 - 18b ; peprotech ) , 20 ng / ml egf ( cat . 236-eg ; r&d systems ) , and 5 g / ml insulin ( cat . , tumor - spheroids were formed , and cells were stimulated with tnf + estrogen + egf in the above - indicated concentrations or with the diluents of the above stimulators , for additional 2496 hr . cytometry analyses that followed tumor - spheroid formation , cells were passed through a 40 m nylon mesh cell strainer , in order to separate single cells from tumor - spheroids . tumor - spheroids were later dissociated by trypsinization and the cells that were obtained by this procedure were compared to single cells that migrated out of tumor - spheroids formed by stimulated cells . cell viability of all groups was determined by trypan blue exclusion ( see above ) and cells were stained using abs against e - cadherin ( see above ) . mcf-7 cells were infected to stably express mcherry ( by pqcxi - mcherry retroviral vector ) . the cells were either non - stimulated or stimulated by tnf + estrogen + egf at the above - mentioned concentrations for three days . then , the cells were washed and 4 10 cells / mouse were inoculated to the mammary fat pad of female athymic nude mice ( harlan laboratories , jerusalem , israel ) . prior to injection to mice , the cells were mixed 1 : 1 with matrigel ( cat . no . one week prior to tumor cell inoculation , all mice were implanted subcutaneously with slow - release estrogen pellets ( 1.7 mg / pellet , 60 days release , cat . se-121 ; innovative research of america , sarasota , fl , usa ) which are essential for the growth of mcf-7 cells in mice . the cri maestro noninvasive intravital imaging system was used to monitor intact mice , at four different time points along the time course of up to 37 days ( depending on the experiment ) . the maestro device has provided two readouts : ( 1 ) size of primary tumors along the growth process of tumors in the intact mice ; ( 2 ) absence or presence of metastases in the intact mice . when the experiments were terminated , organs were excised and metastasis formation was compared to the readouts obtained by the maestro device . this analysis has indicated that in intact mice , the maestro device detected macro - metastases in a reliable manner , but could not detect micrometastases that may have been formed . accordingly , the data retrieved by the maestro device at the different time points in intact mice actually provided information on the formation of macrometastases in different organs . the regulations of tel aviv university animal care committee did not allow continuation of the experiments to the stage of survival analysis . all procedures involving experimental animals were performed in compliance with local animal welfare laws , guidelines , and policies . values of p < 0.05 were considered statistically significant , and all in vitro data were presented as mean standard deviation ( sd ) . in the in vivo studies of primary tumors , data are presented as mean standard error of mean ( sem ) , and statistical analyses of tumor sizes were done using student 's t - tests , where values of p < 0.05 were considered statistically significant . tnf , estrogen , and egf were each shown to have the potential to promote metastasis - related properties in breast tumor cells , as described above ; however , different research systems were used for the study of each of these factors . in our study , we have compared side by side the ability of tnf , estrogen , and/or egf to affect spreading and emt properties , using the mcf-7 luminal breast tumor cells . these cells express receptors for all the three above - mentioned factors ( references provided above ) , and represent a nonadvanced stage of breast malignancy that can be pushed forward towards a more aggressive / invasive phenotype in terms of acquisition of emt properties [ 22 , 26 , 27 ] . first , we determined the effects of tnf , estrogen , egf , or all three factors together on tumor cell morphology , spreading and expression of adhesion molecules which promote tumor cell invasion and metastasis [ 5053 ] . stimulating the tumor cells for three days by tnf has induced the formation of actin - rich cellular protrusions , accompanied by definite concentration of actin fibers at the cell cortex ( figure 1(a2a ) ) . in contrast , estrogen alone had no effect on tumor cell morphology ( figure 1(a2b ) ) , and egf induced cell spreading but to lower extent than tnf ( figure 1(a2c ) ) . however , the most robust change in cell morphology , exemplified by extensive spreading and reorganization of stress fibers , was noted when estrogen and egf were added to tnf ( figure 1(b ) ) . the cells that were exposed to the combined stimulation by tnf + estrogen + egf have formed definite and large cellular protrusions , with actin stress fibers clearly apparent , which were minimally visible previously in the control non - stimulated cells ( figure 1(b ) versus figure 1(a1 ) ) . additional analyses indicated also that the triple stimulation of tnf + estrogen + egf was more effective in inducing spreading and cell remodeling than dual stimulations by estrogen + tnf or estrogen + egf ( figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2013/720536 ) ( the dual stimulations focused on combinations including estrogen because it is the most relevant factor to the luminal tumor cells we were using , characterized by er expression ) . together , these results provide evidence to strong impact of the combined stimulation by tnf + estrogen + egf over other combinations , indicating that the joint activity of all three arms of the tumor microenvironment together is advantageous in inducing spreading and adhesion - related functions in luminal breast tumor cells . additional analyses indicated that the morphological changes induced by tnf + estrogen + egf in the tumor cells were accompanied by redistribution of focal adhesion kinase ( fak ) and paxillin , two key regulators of cell adhesion and spreading [ 5053 ] ( figure 2 ) . moreover , we noticed that the cancer cells that were exposed to the combined stimulation by tnf + estrogen + egf have detached from each other , and have formed connecting tubes ( figures 2(a2b ) and 2(b2b ) ) . based on published reports [ 54 , 55 ] , such tubes may support exchange of intracellular components between the cancer cells . the activation of fak and paxillin and their contribution to formation of cellular protrusions were found to be src - mediated processes . this was indicated by potent inhibition of cell spreading and fak / paxillin localization at cellular extremities by the specific src inhibitor pp2 ( figure 3 ) . the powerful spreading induced by tnf + estrogen + egf has led us to monitor the expression of the 1 integrin , known to be strongly involved in processes of tumor cell adhesion , spreading , and metastasis formation [ 5660 ] . as shown in figure 4 , of the three factors mainly tnf induced detectable upregulation in 1 expression although to a very limited extent ; however , when tnf activities were joined by estrogen and egf , the resulting tnf + estrogen + egf stimulation has led to much more substantial integrin 1 upregulation , in an extent that was stronger than the minimal effects induced by each of the factors alone ( figures 4(a1)4(a3 ) versus figure 4(b ) ) . the 1 integrin has been shown in many studies to stand in the basis of increased adhesion and invasion of tumor cells , including of breast origin [ 5660 ] . since integrins are acting as heterodimers , we searched for the chain counterpart that would accompany the increased expression of 1 . a thorough search through many different subunits has identified increases in 6 in response to tnf + estrogen + egf stimulation ( figure 4(c ) ) . accordingly , the combined stimulation has led to increase in a subpopulation of tumor cells expressing high levels of 61 . the 61 heterodimer , otherwise known as vla6 , is a laminin receptor that has been identified in the past as invasion - supporting complex that promotes breast cancer progression [ 61 , 62 ] . in parallel , we found that the combined stimulation by tnf + estrogen + egf has induced strong upregulation in another adhesion molecule that is highly implicated in breast metastasis , cd44 ( figure 5 ) [ 6 , 7 , 63 , 64 ] . as previously demonstrated for all other functions , the impact of the combined stimulation on cd44 elevation was definitely more powerful than each of the stimulators tnf , estrogen , or egf alone ( figure 5 ) . of interest was the fact that due to stimulation by tnf + estrogen + egf , over 50% of the tumor cells acquired high expression levels of both 1 and cd44 together ( figure 5(c ) ) . overall , the above results indicate that of all three factors tnf was the strongest inducer , of spreading and expression of metastasis - related adhesion molecules by the luminal mcf-7 breast tumor cells and that its activities were strongly amplified by the cooperativity with the other two representatives of the tumor microenvironment , estrogen ( hormonal ) and egf ( growth - supporting ) . to follow on the above findings , we determined the abilities of tnf , estrogen , and egf each alone or together to induce emt properties in the tumor cells . in cells undergoing emt , reduced expression of e - cadherin facilitates detachment of cancer cells from each other [ 9 , 10 ] . accordingly , following three days of stimulation by tnf , egf , and estrogen , each separately , downregulation of cell surface expression of e - cadherin was noted to some extent , with tnf inducing the most prominent effects of all three factors ; however , very clearly , the most potent emt phenotype was obtained upon joint stimulation by all three factors together , given in the form of tnf + estrogen + egf ( figure 6 ) . also , in response to the combined stimulation , the cells have gained typical morphology of cells undergoing emt , detaching from each other and expressing definite cellular protrusions ( figure 7(a ) ) . further supporting the ability of tnf + estrogen + egf stimulation to induce emt was the prominent increase in the expression of the known emt activators zeb1 , snail , and slug [ 6570 ] in the tumor cells ( figure 7(b ) above , we have shown that the combined stimulation by tnf + estrogen + egf has strongly induced spreading and emt properties in luminal breast tumor cells . to follow on the above findings , we determined the impact of the combined stimulation on tumor cell functions that are involved in increased tumor growth and progression . because of its high clinical relevance to tumor progression , first we asked what is the effect of the combined stimulation on resistance of tumor cells to doxorubicin ( adriamycin ) , which is a chemotherapy commonly used in the treatment of breast cancer patients [ 71 , 72 ] . when doing this analysis , we were aware of the fact that mcf-7 cells are sensitive to tnf cytotoxicity [ 7375 ] , and accordingly our routine procedure of tnf + estrogen + egf stimulation for three days has led to death of approximately 40% of the tumor cells ( figure s2 ) . nevertheless , despite their apparent sensitivity to tnf-induced cytotoxicity , tumor cells that were exposed to the combined stimulation were endowed with higher resistance to doxorubicin ( figure 8(a ) ) . these results indicate that those tumor cells that have survived the tnf-induced cytotoxicity were selected for high resistance to chemotherapy - induced death . in parallel to the above , we determined the effects of the combined stimulation by tnf + estrogen + egf on the ability of the tumor cells to acquire additional promalignancy functions . doing a per cell analysis , we found that the stimulation of the tumor cells by tnf + estrogen + egf has given rise to potent elevation in the release of the inflammatory chemokines cxcl8 ( figure 8(b1 ) ) and ccl2 ( figure 8(b2 ) ) , which have been well characterized as strong tumor - promoting factors by virtue of their potent angiogenic activities and recruitment of tumor - supporting leukocytes to the tumors [ 7681 ] . in addition , in response to the combined stimulation by tnf + estrogen + egf , the tumor cells have acquired the ability to produce high levels of functional matrix metalloproteinase 9 ( mmp9 ; figure 8(c ) ) , a key enzyme in degradation of the extracellular matrix ( ecm ) during local invasion end extravasation of the tumor cells . moreover , to follow on the cell - remodeling , emt , and metastatic / invasive properties acquired by tumor cells that were exposed to the combined stimulation by tnf + estrogen + egf , we determined the migratory functions of the cells . we took advantage of the high ability of mcf-7 cells to form tumor - spheroids and analyzed the ability of cancer cells to detach from the spheroids and move away from them . to this end , we have formed tumor - spheroids and then introduced the combined stimulation for additional 2496 hr . these tests have shown that control , non - stimulated cells , kept the organized spherical structure throughout the 96 hr time course ( figure 9(a ) ) . in contrast , cancer cells that were exposed to the combined stimulation have migrated out of the tumor - spheroids already after 48 hr of stimulation ( figure 9(b ) ) . at the 96 hr time point , extensive outward migration was observed in the tnf + estrogen + egf - stimulated cells , and a large proportion of single cells was detected ( cell viability tests indicated that these single cells were alive ) ( figure 9(b ) ) . here , it is interesting to note that the single cells that migrated out of tumor - spheroids formed in the presence of tnf + estrogen + egf stimulation expressed lower levels of e - cadherin compared to the cells that remained in the spheroids ( table 1 and figure s3 ) . these results provide a direct connection between the processes of elevated emt and migratory events that were induced by the combined stimulation of tnf + estrogen + egf . the results presented so far in this study indicate that tumor cells exposed to the combined stimulation have acquired properties that may contribute to tumor growth and metastasis . these results have motivated us to determine the effects of the tnf + estrogen + egf stimulation on formation of primary tumors at the mammary fat pad and on dissemination of metastasis . to enable detection of the tumor cells in intact animals , mcf-7 cells were infected to express the fluorescent protein mcherry . the tumor cells were stimulated for three days by tnf + estrogen + egf in vitro then washed to remove the stimulators and inoculated to the mammary fat pad of mice . because mcf-7 cells are sensitive to tnf-induced cytotoxicity ( figure s2 ) [ 7375 ] , following the three days of stimulation by tnf + estrogen + egf , we assured that equal numbers of live stimulated and non - stimulated cells were inoculated to the mice . following tumor cell inoculation , the maestro device has provided data on the size of primary tumors and appearance of macro - metastases in intact mice , in analyses that were performed at four time points along the course of the experiments ( up to 37 days ) . the findings of figure 10(a1 ) show that cells exposed to the combined stimulation of tnf + estrogen + egf have given rise to smaller tumors than control non - stimulated cells , due to possible reasons described further on ( section 4 ) . taking into account the fact that mcf-7 cells are well - characterized as nonmetastatic cells [ 45 , 83 ] , it was exciting to see that the combined stimulation by tnf + estrogen + egf for three days in culture has given rise to cells with high metastasizing ability in vivo . as expected , the control cells did not form macro - metastases at all , but in contrast the tumor cells that have been exposed to tnf + estrogen + egf stimulation have given rise to macro - metastases in 38% of the animals ( figures 10(a2 ) , 10(b ) , and 10(c ) ) , as determined in 2 independent experimental repeats showing similar results . macro - metastases were also detected in 2/3 mice in another experiment in which non - stimulated cells were not included . the macro - metastases formed by tnf + estrogen + egf - stimulated cells were detected in the liver , colon , and abdomen ( figure 10(d ) shows metastases in the liver and in the colon ) . actually , the impact of the combined stimulation on the metastatic potential of the mcf-7 was dramatic : the tumor cells were exposed to this stimulus for only three days in culture , and based on our in vitro results only a subpopulation ( based on figure 5 , up to ~50% of the cells ) has gained tumor and metastasizing abilities in culture ( figures 46 ) . also , in other in vivo studies that we have performed with oncogene - expressing mcf-7 cells that were stimulated by tnf ( in which mice were also injected twice - weekly with cm of such cells ) suggest that the metastatic load of cells stimulated by tnf + estrogen + egf is higher than the one induced by tnf ( data not shown ) . taken together , the influence of the tnf + estrogen + egf stimulation on the metastasizing capabilities of these cells in vivo is of major importance and of high clinical relevance . in this study , we demonstrated that the combination between promalignancy factors has a dramatic impact on the ability of luminal tumor cells to acquire metastasis - related properties and to disseminate to remote organs . when used singly , tnf was more effective than the other two representatives of the tumor microenvironment estrogen and egf and its activities were potently increased by cooperating with these two factors . thus , it was the joint activities of all three arms together inflammatory , hormonal , and growth - supporting that led in a prominent efficacy to the devastating processes of tumor cell spreading , emt , and metastasis . our findings have shown that as a result of the combined stimulation by tnf + estrogen + egf , luminal breast tumor cells have gained an extensive spreading phenotype in which src activation has given rise to tumor cell spreading and to localization of fak and paxillin in tumor cell protrusions . in parallel , the cancer cells have detached from each other and underwent the metastasis - relevant process of emt and migration . as a result of tnf + estrogen + egf stimulation , new cell subtypes have dominated the tumor cell population , expressing high levels of vla6 and of the metastasis - related adhesion molecules cd44 and 1 , accompanied by high levels of cxcl8 , ccl2 , and mmps that were released by the cells . based on published findings [ 8487 ] , the elevation in 1 , cd44 , and cxcl8 may very much stand in the basis of the high resistance to doxorubicin gained by the tnf + estrogen + egf - stimulated cells . the above characteristics that were gained by the tumor cells following exposure to tnf + estrogen + egf have led to an intriguing in vivo phenotype , in which the stimulated cells have produced smaller local tumors but expressed very high metastatic phenotype compared to control non - stimulated cells . based on the in vitro results described previously , two nonexclusive mechanisms could lead to such results : ( 1 ) out of the three stimulators of the tumor cells , tnf is the only one that is cytotoxic while estrogen and egf are known to stimulate tumor cell growth . the tumor cells used in this study ( mcf-7 cells ) are known to be sensitive to tnf-induced cytotoxicity [ 7375 ] ; accordingly , approximately 40% of the tumor cells were killed in vitro by their exposure to the combined stimulation of tnf + estrogen + egf ( figure s2 ) . although after this stimulation only live cells were injected ( in equal numbers to control cells ) to the mice and the stimulus was removed beforehand , it is possible that some of the tumor cells were destined to die later on , after they have been introduced into the mouse . these cytotoxic effects of tnf may have given rise to reduced growth of primary tumors . ( 2 ) the high spreading , emt , and migration phenotypes endowed on the cancer cells by tnf + estrogen + egf stimulation may have led to migration of tumor cells out of the primary focus soon after their inoculation to the mammary fat pad ( as has been illustrated in vitro in figure 9 ) ; thus , the cell inoculum from which the tumor developed was smaller after stimulation and gave rise to a smaller primary tumor than control non - stimulated cells . such a mechanism is in good agreement with the high metastatic yield of the tnf + estrogen + egf - stimulated cells ( figure 10 ) . obviously , such mechanisms suggest that it would be interesting to determine the emt properties in primary tumors established by non - stimulated control cells compared to cells stimulated by tnf + estrogen + egf . overall , our results suggest that some of the cancer cells that were stimulated by tnf + estrogen + egf partly succumbed to the cytotoxic effects of tnf and others migrated out of the initial tumor inoculum , giving rise to smaller primary tumors than those generated by control cells . but at the same time , these cells have gained many metastasis - promoting properties and became aggressive in vivo . therefore , the small tumor growth endowed following tnf + estrogen + egf stimulation provided a false benefit , because it has led to selection of cells expressing a higher metastasizing potential . here , it is important to note that in the heterogeneous population of tumor cells , only some have acquired the high spreading - emt - metastasis "- related functions , and this can explain why metastases were not formed in all mice . nevertheless , we would like to emphasize that the acquisition of a metastatic ability by mcf-7 cells is by itself extremely unique and important , even if not observed in all mice . mcf-7 cells are considered completely nonmetastatic , and even following over - expression of powerful proto - oncogenes such as h - ras , they did not acquire the ability to form metastases in vivo , despite increased invasiveness in vitro . therefore , our findings indicate that under certain conditions endowed by combined stimulation by three arms of the tumor microenvironment mcf-7 cells became metastatic . in our in vitro analyses , tnf was the most effective of all three elements , but its activities were potentiated by estrogen and egf . based on these studies , we propose that tnf is the factor that dominated the high protumoral phenotypes and responses , leading to its most extreme impact on tumor cell spreading to remote organs . overall , while tnf had the ability to exert cytotoxic effects that may reduce tumor growth , it cooperated with the two other arms of the tumor microenvironment and eventually turned into a metastasis - promoting entity . here , it is important to note that all three factors tnf , estrogen , and egf are often expressed in luminal breast tumors in breast cancer patients . past findings from our laboratory indicated that tnf is expressed in approximately 90% of patients with recurrent disease , and many of these patients also express er , and are therefore estrogen - responsive . taken together , these observations suggest that a relatively high subpopulation of luminal breast cancer patients may experience coexposure to tnf + estrogen + egf and may thus acquire increased metastatic rate . moreover , based on our results with doxorubicin resistance , the joint powers of all three factors together may further increase the resistance to chemotherapy in breast cancer patients , demonstrating another level at which the combined exposure to tnf + estrogen + egf may be devastating to the patients . the findings presented in this study have very high clinical relevance . until a decade ago , many researchers suggested introducing tnf as a therapeutic agent in cancer because of its cytotoxic activities . however , an increasing body of evidence puts tnf on the stake as a key tumor - promoting factor that has harmful impacts on the malignancy cascade . our findings pinpoint the devastating tnf activities to be the life - threatening stage of metastasis formation , and these findings have a profound importance for breast cancer therapy . tnf inhibitors , such as infliximab and etanercept have been fda - approved and are being successfully used in the clinic for treatment of several autoimmune disorders [ 8992 ] . therefore , we suggest considering the addition of these established tnf inhibitors to the treatment protocols of luminal breast cancer patients . specifically , we suggest taking the results of this study one step further , towards personalized cancer therapy . knowing that antihormone therapies and inhibitors of egfr / her2 are already used for therapy of breast cancer [ 93 , 94 ] , we propose that patients diagnosed with high tnf , estrogen , and egf levels would benefit from targeting all three arms simultaneously and that clinicians should consider the possibility of treating such patients with a cocktail of all three modalities : tnf inhibitors + antihormonal therapies + inhibitors of egfr / her2 . obviously , extensive research is needed in order to assess the impact of tnf inhibitors on breast tumor cells both in vitro and in vivo , and to design the proper clinical administration mode . however , we believe that the paradigm shift presented in this study on the roles of tnf in metastasis may have a strong impact on therapeutic choices in the future . the feasibility of blocking several arms of the tumor microenvironment together should not be ignored , and reducing the cancer - related inflammation might also attenuate the tumor - promoting effects imposed by the other arms of the tumor microenvironment and thus inhibit tumor cells migration and invasion and their devastating outcome , metastasis formation .

breast cancer progression is strongly linked to inflammatory processes , aggravating disease course . the impacts of the inflammatory cytokine tnf on breast malignancy are not fully substantiated , and they may be affected by cooperativity between tnf and other protumoral mediators . here , we show that together with representatives of other important arms of the tumor microenvironment , estrogen ( hormonal ) and egf ( growth - supporting ) , tnf potently induced metastasis - related properties and functions in luminal breast tumor cells , representing the most common type of breast cancer . jointly , tnf + estrogen + egf had a stronger effect on breast cancer cells than each element alone , leading to the following : ( 1 ) extensive cell spreading and formation of fak / paxillin - enriched cellular protrusions ; ( 2 ) elevated proportion of tumor cells coexpressing high levels of cd44 and 1 and vla6 ; ( 3 ) emt and cell migration ; ( 4 ) resistance to chemotherapy ; ( 5 ) release of protumoral factors ( cxcl8 , ccl2 , mmps ) . importantly , the tumor cells used in this study are known to be nonmetastatic under all conditions ; nevertheless , they have acquired high metastasizing abilities in vivo in mice , following a brief stimulation by tnf + estrogen + egf . these dramatic findings indicate that tnf can turn into a strong prometastatic factor , suggesting a paradigm shift in which clinically approved inhibitors of tnf would be applied in breast cancer therapy .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

the identification of genomic regulatory elements is an important but unsolved problem in genome annotation ( 1 ) . of the 5% of mammalian genome that is estimated to be under evolutionary selection pressure , less than a third is coding ( 2,3 ) . the remaining portion is believed to be composed of untranslated regions , non - coding genes , chromosomal structural elements and regulatory elements that control a variety of biological processes including gene expression , translation , chromosomal replication and condensation . whereas the number of coding genes in many of the sequenced organisms can now be reasonably estimated , there is no clear estimate of the number of functional regulatory elements in these genomes , especially in the metazoa . in eukaryotes ranging from the nematodes to flies to the mammals , the number of coding genes is similar ( 2,46 ) , and it is now thought that organismal complexity may be attributed to phenomena such as alternative splicing , dna rearrangement and increased number of transcriptional regulatory elements as well as transcription factors ( tfs ) which regulate gene expression ( 7 ) . the identification of cis - regulatory elements controlling gene expression , and characterization of their interaction with the respective tfs , thus lie not only at the very heart of understanding of the network of gene interactions but also of explaining the origins of organismal complexity and development . as such these representations are general and can be used to describe any class of short dna sequence elements . however the theories for weight matrix model , which is a common way to represent a collection of dna elements , are based on the biophysical considerations of protein dna interactions ( 811 ) ( as described in the following section ) . therefore , here we primarily discuss the transcriptional regulatory elements , more specifically the dna sites that are bound by the tfs . it is estimated that there are 2000 tfs in the mammalian genomes ( 2,5 ) and 1000 in the flies and worms ( 7 ) . however , only for a minority of the tfs ( 900 in human , 700 in mouse , 200 in drosophila and 100 in caenorhabditis elegans ) is there currently any known information on binding sites or interacting protein partners ( 12 ) . dna - binding site models are available for 500 vertebrate tfs , and < 5000 genomic sites are known in all vertebrates in fewer than 3000 genes ( 12 ) . based on the information available from a few of the well - studied genes ( 13 ) , it appears that the total number of such sites in the multicellular genomes could be at least an order of magnitude higher than the number of coding genes , i.e. in the order of hundreds of thousands or more . thus , our knowledge of the tfs , and especially their binding sites and regulated genes , is severely limited at this present time . computational methods for modeling and identification of dna regulatory elements have been developed over the past two and a half decades ( 1420 ) . orthogonal information from comparative genomics or co - regulation at the transcriptional level have also been integrated into these methods to identify cis - regulatory sites ( 2125 ) . more recently , methods have been developed ( 2632 ) to analyze composite regulatory elements , i.e. modules consisting of multiple dna sites bound by the regulatory factors ( 33 ) . all of these methods have been valuable in expanding our limited knowledge of regulatory elements in the genome . here we discuss the progress made in the computational identification of genomic regulatory elements , recent advances , the utility of orthogonal data , existing challenges in the field and some selected examples where these methods have been successfully applied to discover novel functional elements . rather than exhaustively covering the literature , we focus on the key concepts . significant progress has also been made in the experimental characterization of regulatory sequences , a discussion of which is beyond the scope of this article , and the reader is referred elsewhere for further information ( 3443 ) . recurrent motifs in a collection of dna sites are most commonly modeled by sequence patterns ( also called regular expressions ) , or by position weight matrices ( pwms , also called profiles and position - specific scoring matrices , pssms ) . the sequence patterns are simply strings over the 4-letter alphabets [ a , c , g , t ] that form the dna . in order to capture variation in a specific position , the degenerate iupac nucleic acid codes ( 44 ) ( , nomenclature for incompletely specified bases in nucleic acid sequences ) are used ( figure 1 ) . an l - long sequence motif can also be represented by a 4 l matrix with weights giving the frequency of the four dna bases ( or the logarithm , see below ) in each of the l positions ( 18,45,46 ) ( figure 2 ) . it is worth noting here that some dna - binding site motifs are bipartite , i.e. have two halves that are sometimes separated by a spacer element in between . such bipartite motifs can often be palindromic , e.g. 5-cggnnnnnnnnnnnccg-3 , the binding site for yeast tf , gal4 ( 47 ) . the pattern and pwm representations serve as complementary approaches and have been used widely since the 1980s . the dna sequence patterns are simpler in representation , and advantageous in terms of exact enumeration of their significance in the genome using statistical methods ( explained in detail later ) . the pwms are able to capture information on the variability of a collection of dna sites in a quantitative manner , which is not possible with the dna patterns . however when detecting significant pwms in dna sequences , heuristic methods have to be used instead of exhaustive enumeration . perhaps the most important practical utility of these models is their application in scanning dna sequences for new regulatory element candidates . for this , one needs the appropriate motif models representing the regulatory elements , a statistical framework to score sites and determine their significance , and suitable thresholds to minimize false positives and false negatives . currently there are two comprehensive and curated databases containing information on tfs binding site profiles ( 12,48 ) . jaspar ( 48 ) contains a smaller set that is non - redundant ( i.e. each tf has only one profile ) , while transfac ( 12 ) contains multiple profile models for some tfs . in addition to the above generic databases , other organism - specific databases exist that host transcriptional regulation data ( 47,4951 ) ( ) . therefore if a large enough sampling of sites is not available , they may not capture all possible variations of the functional sites . this tends to produce false negative calls on new sites that do not match well with the previously characterized ones . the binding sites for structurally related tfs are often similar , and in such cases building familial binding profiles instead of profiles for each tf may be useful ( 52 ) . in both patterns as well as pwm representations , the significance of a particular motif is given by a measure of statistical surprise ( or likelihood ) for the motif given the data . in the case of patterns , significance can be calculated given the distribution of all occurrences of patterns using standard statistical procedures ( 20,53 ) . this has been used in several software packages that discover over - represented patterns from input sequences ( 5458 ) . in the methods using weight matrix models , the measure of significance is commonly given by the information content ( ic , also called relative entropy ) ( 8,9,11 ) : 1i(p)=j=1li = atfi , j logfi , jpi , where i(p ) is the ic for the pwm representing a pattern p , l is the pattern length , i is the index of a base { range a through t } at position j of the pwm , fi , j is the frequency of base i at position j of the pwm , and pi is the probability of observing that base in the data . based on biophysical models of protein dna binding , it has been shown that the contributions of the individual positions of a site to the total binding free energy of the tf may be given by log(fi , j / pi ) ( 8,11,46 ) , which are often used as the weights ( or log - odds scores ) in a pwm ( figure 2c ) . the ic is therefore the weighted average of the binding energies from each of the sites represented in the matrix , and lower ic indicates higher variability ( consequently lower specificity ) in the sites . statistical methods for computing the p - value of ic have been defined ( 59,60 ) . several algorithms use the ic measure to identify optimal motifs from input sequences ( 59,6164 ) . given a dna motif , searching sequences for candidate sites is straightforward ; but distinguishing real sites from artifactual ones is difficult . the problem of distinguishing true versus false sites arises from the fact that tf dna - binding sites are usually degenerate and many subsequences may match a given motif . the situation is illustrated with the collection of eight known sites for a saccharomyces cerevisiae tf , rox1 [ taken from the scpd database ( 47 ) ] ( figure 2 ) . even though there is a preferred base at 11 of the 12 positions , at 8 of those more than one base is tolerated ( in a larger collection of sites , even these 4 conserved positions may show variations ) . only two of the known binding sites correspond to the consensus pattern , ( t / c)ccattgttctc ( conserved positions are shown in boldface ) , so a search of the genome with this consensus pattern would miss many functional sites . on the other hand , the iupac representation of the sites , yyhmtkgttbdb , captures the variation of all the sites but is also likely to find a large number of false positives and non - functional sites in a genome - wide search . given a sequence and a pwm , one can score any subsequence in that input . suppose the length of the pwm is l and the weight of base i at position j is wi , j , the score of a subsequence ( s ) , when aligned to the pwm , is then given by the following equation : 2scores=j=1li = atwi , jsi , j , where xi , j = 1 if base i occurs at position j of subsequence s and 0 otherwise . this simple scheme is commonly used ( 59,65,66 ) , and assumes an additive contribution from each position towards the score . methods have been described to calculate the p - values of these scores based on their distribution ( 59,65,67 ) . similar to dna patterns , site predictions with the pwms can suffer from high false positive rates if motifs are degenerate . if base composition of a genome is assumed to be random ( which is not an accurate assumption , but helps us to discuss the following point in an approximate but simplified way ) , the rox1 pwm ( figure 2c ) , which has an ic of 11.2 bits , would be expected to have a site every 2500 nt [ for details see ( 9 ) ] , or 4700 total sites in the yeast genome . there are motifs for which the ic is even lower , leading to a much higher number of possible sites . for example , a collection of 13 known binding sites for uash ( 47 ) has an ic of only 8.5 bits , giving 32 000 potential sites in the yeast genome ( or 1 every 370 nt ) . thus , the problem of distinguishing real from false ( biologically non - functional ) sites is an important but challenging one . in addition to the motif degeneracy , there are other biological issues relating to the predictions of functional tf - binding sites in the genome , which are discussed below . although there are limitations , the pwm models work fairly well in representing the specificity of dna - binding sites and predicting tf - binding probability to a given site ( 11,18 ) . some tfs are by nature moderately or poorly specific in their dna binding and achieve higher specificity only in the context of other binding partners . one also has to remember that the chromatin structure and dna methylation play important roles in gene regulation ( 6870 ) . large portions of the chromosomal dna are sequestered by histones forming part of the nucleuosomal structure , and are therefore not accessible for binding by the tfs . dna methylation can inhibit interaction of the regulatory proteins with cognate dna sites and also influence the chromatin structure . while doing genome - wide searches for putative binding sites using a motif model , one typically does not know the chromosomal regions that are open for the regulatory proteins to bind , or ( with a few exceptions ) the binding partners for a given tf . a blind search of the entire genome without such information usually returns a large number of sites , many of which would probably bind to the tf ( strongly or weakly , depending on the match of the sites to the model and the specificity of the model ) if the dna sequences were open for binding , but are biologically non - functional in vivo . genomic sequences that play an active role in transcriptional regulation , such as the promoters , may often be outside of the nucleosome structure , or at least available a part of the time due to chromatin remodeling ( 71 ) . so the rate of non - functional site predictions in these sequences is likely to be lower than other parts of the genome . however , without the information on dna availability , methylation status or other binding partners , the binding site predictions with individual models are unlikely to achieve the same level of specificity that are achieved in vivo by the tfs . despite the fact that for individual pwm models determining thresholds for distinguishing real versus non - functional sites is difficult a sample - size adjusted ic may be defined as the true ic ( equation 1 ) minus the ic expected from an arbitrary alignment of an equal number of random sites . the patser program ( 59 ) ( ) uses a default cutoff score for which the loge(probability ) ( i.e. the probability of observing a score greater or equal to the cutoff ) equals the sample - size adjusted ic . another approach is based on prediction rates on sequences that contain known sites and on sequences that are likely not to contain sites ( 66,72 ) . false negative rates are computed from predictions made on the experimentally characterized tf - binding sites , and false positive rates may be computed , for example , from predictions in exons , where the number of regulatory elements is expected to be low ( 66 ) . given these prediction rates , the selection of the appropriate cut - off score depends largely on the user 's objectives . a third approach has been to use a distribution of scores for a pwm and use one or two standard deviations below the mean score as a threshold for filtering out low - scoring sites ( 73 ) , but retaining most of the true positive ones . recently , djordjevic et al . ( 10 ) have described a support vector machine ( svm ) method for representation of dna motifs based on thermodynamic principles of protein dna binding . the biophysical treatment and optimization of the svm automatically provides a threshold to distinguish the binding energies of the real sites versus those that are likely to be false . this natural and objective thresholding is one of the strengths of their approach and it provides a way to avoid the use of the more subjective or user - defined thresholds that are frequently employed . since many of the tfs bind dna in the context of other tfs , where information is available about the organization of multiple dna - binding sites or the binding partners for a given tf , it can be utilized to reduce the rate of false positive predictions . even when individual pwms tend to be fairly non - specific , searching for co - occurrence of binding sites that form regulatory modules has been shown to be an effective approach to increasing prediction specificity without losing sensitivity ( 74 ) . finally , we discuss one limitation of the additive pwm models in representing dna sites , and recent developments which address this issue . as seen from equations 1 and 2 , the simple pwm approach assumes independent contributions from each position within a dna site towards the binding free energy of the tf ( mononucleotide model ) . this has been shown not to hold true in several situations ( 37,75,76 ) where sufficient binding site data are available for detailed analysis . in such cases , higher - order models , i.e. those that consider interdependence between positions within a site , are better representations and give more accurate predictions when searching for candidate sites ( 10,7679 ) . most of the higher - order models ( 10,7581 ) use di - nucleotide interactions , which is a reasonable compromise between the mononucleotide pwms and the more complete models with interactions between multiple ( more than two ) positions . this is because ( i ) the large number of characterized binding sites that is needed to build complex models ( with increased number of parameters ) is rarely available for any particular tf ( 12 ) and ( ii ) in a few cases where sufficient experimental data are available ( 37,75 ) , interdependencies between adjacent nucleotide positions appear to be the most significant of the within - site interactions . from the dna - binding site data available for a small number of tfs , it has been estimated that 25% of sites may show significant within - site positional correlations ( 76 ) . even in cases where intra - site interactions exist , the simpler additive model has been suggested to be a good approximation ( 80 ) . as more experimental data become available with the application of high - throughput methods for characterizing tf - binding sites , it remains to be seen if significant interdependence between positions of dna - binding sites is prevalent in tf dna interactions , and whether the more complex sequence models provide a considerable enhancement in modeling accuracy over the additive pwms in many cases . since the structural contexts of both dna and protein can contribute towards specific dna binding by tfs ( 82 ) , some studies have investigated the use of structural information of protein the structure - based approaches are promising as they can predict binding sites for tfs where no previous sites have been characterized before ( 85 ) , and can provide improved predictive power over the simple sequence profile models ( 83 ) . however , they have been limited in their general use so far , since derivation of the quantitative structural parameters is dependent on the small number of solved protein dna complex structures . a more thorough discussion of the structural aspects of protein dna recognition is beyond the scope of this review and the readers are referred to the articles above ( and references therein ) for further information on this topic . with larger datasets , the issues with modeling complex dna protein interactions can be investigated more thoroughly than what has been done so far , and the building of models ( with or without structural parameters ) with higher accuracy is likely to become more feasible . discovery of motifs in sequence data was an early problem to be addressed in computational biology . the dna motif discovery algorithms that have been developed can be divided into two categories , namely pattern driven methods ( those that identify dna patterns ) and sequence driven or alignment driven methods ( those that identify profile models ) ( 16,20 ) . the concepts behind these algorithms , their advantages and limitations , and a few example methods are discussed below . in the pattern driven methods , given a set of dna sequences and a length l of pattern that we wish to find , the challenge is to identify the most significant patterns of that length . the solution to the problem can be obtained by generating all possible patterns of length l , searching for the number of occurrences of each pattern , and then reporting the ones with highest frequency as being the most significant in the given data ( 5258,8991 ) . this enumerative approach is exact and guaranteed to find optimal solutions in the restricted search space . approximate sequence patterns , or patterns that contain degeneracy at one or more positions , can also be identified from the sequence . the similarity between any two patterns may be given by the hamming distance ( the number of positions in which they differ ) or the levinstein distance ( the number of substitutions , insertions or deletions needed to transform one string into another ) ( 20 ) . when multiple patterns are close in terms of their distance , they can be merged into one approximate pattern ( 89,92 ) . although the exact enumeration is an advantage of these methods , one limitation is that searching for long patterns is computationally expensive , and an exhaustive search through the sequence space of 4 words often becomes impractical for l > 10 ( 93 ) . two general strategies have been taken to address this limitation : ( i ) the use of efficient methods [ e.g. pattern graphs ( 93 ) or projections ( 94 ) ] for pre - processing the data so that the search space is reduced and actual pattern search becomes less expensive , and ( ii ) combining the shorter overlapping patterns found from the data to yield longer or more complex patterns ( 89,92,95,96 ) . in addition to the exact enumerative methods , efficient data structures like the suffix trees ( 97 ) have also been applied to the dna pattern discovery problem ( 98100 ) . although not exact algorithms , the advantage of the suffix trees is that they allow one to search for patterns of longer lengths since the search time is not exponential in the length of the patterns , but exponential in the number of mutations to be tolerated in the sites ( 98,100 ) . in the sequence driven methods the challenge is to find the location of the sites and the representative pwm using only the sequence data , without any assumptions on the statistical distributions of patterns in the sequences . if the locations of sites are known , building a dna profile for them is trivial . however in the ab initio motif discovery problems , this information is not known ( missing information ) and has to be learned from the input data . such problems with missing information can be solved by employing machine learning algorithms . several machine - learning approaches have been applied to the problem of motif finding ( 59,6264,76,101107 ) . unlike some pattern driven methods where the most significant motifs can be identified by exact enumeration , obtaining the globally optimal results motifs of arbitrary lengths can be searched , since the search time does not depend significantly on the length of sites . the first amongst the sequence driven methods was the greedy algorithm ( 59,61,62 ) . given a set of n sequences , and a motif length l to be searched , this algorithm progressively builds matrices by including the sites which maximize the ic ( equation 1 ) . the algorithm first builds a set of significant matrices by comparing all pairs of sequences . in subsequent iterations , sites that increase the ic of the alignment another method that has been commonly used is the expectation maximization ( em ) algorithm ( 63,102,103 ) . the em algorithm simplifies the analysis of problems with missing information by iteratively substituting the locations of sites by expected locations . the algorithm starts with a guess pwm , which can be random or based on some prior knowledge about the binding sites [ see e.g. ( 52,94 ) ] . using the pwm , the probability of each subsequence being a binding site is estimated , and the pwm is updated based on those probabilities . a stochastic variant of the em algorithm that is now very widely used in sequence motif recognition is the gibbs sampling method ( 64,106 ) . gibbs sampling , which is a type of markov chain monte carlo ( mcmc ) algorithm , tends to provide a more robust optimization of the pwms as the probabilistic sampling of sites helps the avoidance of local minima . variations of the gibbs sampling technique have been implemented in many motif finding software that are used in the bioinformatics community ( 76,104,105,108,109 ) . for stochastic algorithms like the gibbs sampling , multiple searches have to be performed with the input dataset in order to confirm that the same motifs are discovered starting from different random points in the sequence space . controlling for background sequence is an important issue in dna motif discovery that has been effectively addressed in recent years . variation in local base composition can adversely affect sequence alignment and discovery of relevant motifs . because such variations can be complex , and since binding motifs are often at- or gc - rich , these adverse effects can be difficult to control using existing masking algorithms . in addition , some low complexity sequence motifs [ like ploy(a ) or poly(gc ) ] are widespread in genomes of certain organisms . as a consequence , often the strongest motifs that are discovered from any input data are these common motifs that are prevalent in the genome but do not represent the specific regulatory elements that are being sought . it is therefore important to detect those motifs that are significantly more frequent in the positive sequence set ( the input set in which we want to discover motifs ) relative to the background ( 91,92 ) . the available programs which identify such motifs ( often called discriminative motifs ) do so by modeling the background with a markov chain ( 105,109,110 ) , or by accounting for the motifs that are frequent in a given background set ( 26,92,108 ) through sampling or enumeration . recently , tompa et al . ( 111 ) reported the most comprehensive comparative study yet performed for different ab initio motif - finding algorithms . assessment was done for 13 commonly used dna motif discovery tools that do not use any auxiliary information , such as comparative sequence analysis , mrna expression levels or chromatin immunoprecipitation ( chip chip ) data . predictions were compared with known binding sites , using various statistics to assess their accuracy . one important , but not unexpected , observation from this work was that a few different tools tend to complement each other 's performance . for example , motifsampler 's ( 109 ) predictions complement well the predictions of meme ( 101,102 ) , oligo / dyad - analysis ( 56,90 ) , ann - spec ( 108 ) and ymf ( 110 ) . the authors rightly suggest that biologists would be well advised to use a few complementary tools in combination rather than relying on a single one , and to pursue the top few predicted motifs of each rather than the single most significant motif ( 111 ) . there are currently some examples where ab initio motif finding algorithms have been employed to identify novel regulatory elements that have been validated by follow - up experimental studies . a few of these are described below with the objective of illustrating the types of input data that can be used and the approaches that were taken in the studies . using the program alignace on the upstream promoter region of 248 distinct groups of genes in yeast s.cerevisiae , hughes et al . ( 104 ) , generated a list of 3311 putative regulatory motifs . by applying stringent thresholds and selecting motifs which had highest specificity for certain groups of genes , this large set was reduced to a small set of 54 motifs that were then grouped to 25 motif clusters based on the similarity of multiple motifs . of the 25 motif clusters , one of the previously unidentified motifs , representing the binding sites for the tf rpn4 , was verified using mrna expression analysis of the rpn4 protein knockout and overexpressing strains . ( 112 ) have described the identification of one novel regulatory element in the nematode c.elegans heat - shock response starting from a set of microarray experiments in which genes were robustly upregulated on heat - shock treatments at both early and late time points . using a set of 28 heat - shock upregulated genes they used the ab initio motif discovery algorithms , ann - spec ( 108 ) and consensus ( 59,108 ) , to identify two strong motifs that are over - represented in the promoters of these genes . one of those motifs was the previously characterized heat - shock element that is broadly conserved in eukaryotes and known to bind to the tf called heat - shock factor . the second motif , which was novel , was shown to be biologically functional in vivo through transgenic and mutational studies . in fact , the two elements were shown to function in a cooperative manner ; the contribution to heat - shock mediated expression by either one was weak , but when placed together in close proximity they strongly regulated expression . studies similar to the one described above was done to identify several new muscle regulatory elements in c.elegans , which were shown to contribute to muscle expression in a cooperative fashion ( 113 ) . the identified elements were also highly predictive of additional muscle - specific genes in that organism ( 114 ) . these studies show that ab initio prediction methods can be valuable in elucidating unknown regulatory elements not only in unicellular organisms , but also the more complex metazoan genomes . although much progress has been made in the methods for ab initio detection of dna regulatory elements , the problem still remains a difficult one , especially where the input sequences are long and motifs are weak . therefore , the incorporation of auxiliary information into such methods can be of significant benefit . since the availability of many complete genomes , the application of comparative genomics in the identification of dna regulatory elements has become an important area of study and it is covered in detail in the next section . here , as an example , we discuss a different approach which leverages mrna expression data in dna motif discovery . ( 24 ) described recently a method to discover regulatory elements that uses correlation of dna patterns with the expression levels of genes in a single profiling experiment . they use a simple linear regression model to fit the logarithm of the expression of each gene to the sum of contributions from a set of dna patterns in the upstream promoter region . all the genes are simultaneously fit , and statistically significant patterns that best fit the expression data are selected . using yeast expression datasets , they reconfirm most of the motifs originally found by clustering of expression data and then running motif finding algorithms on clustered gene sets ( 115,116 ) . since there are frequently cooperative ( non - additive ) interactions between tfs regulating a gene , the modeling is simplistic . however , the advantage of this method is the fact that limited expression data are required for analysis and discovery of the dna motifs . in addition to the above methods which integrate motif discovery with expression data , several tools and studies have been described that identify dna - binding site motifs for tfs from a set of sequences defined by mrna profiling ( 112,118,119 ) or chip chip data ( 120,121 ) . in multicellular organisms the sequence space in which regulatory elements can be present in the genome is vast . in addition to the core promoter region , auxiliary transcription regulatory elements like enhancers , silencers and insulators can be present in the distant 5 upstream region , 3 downstream region and the introns ( 122 ) . in drosophila such dna elements can be spread over a region of 10 kb around the genes whereas the average transcribed dna is 23 kb , and in mammals these elements can be scattered over distances of hundreds of kb ( 123 ) . approaches that help to limit this search space are hence of significant value to the analysis of regulatory elements . also , it is intriguing to study those regulatory mechanisms and elements that are likely to be of fundamental importance in maintaining certain cellular functions and therefore conserved in evolution . consequently , phylogenetic footprinting ( 124,125 ) , which is based on the premise that functional elements are likely to be under selection pressure and thereby evolve at a rate that is slower than the surrounding non - functional sequence , has been widely applied in recent years to the identification of regulatory sequences ( 21,123,126129 ) . purely for the purpose of illustration , a simple example of the application of phylogenetic footprinting for the identification of putatively conserved tf - binding sites is given in figure 3 . enrichment of regulatory elements has been clearly demonstrated in non - coding dna in the human mouse conserved regions ( 21,127,130 ) . for example , wasserman et al . ( 21 ) found that 98% of the known muscle regulatory elements are located within 19% of the sequence that is most conserved between human and mouse . non - coding sequences that can be aligned across two or more organisms have been shown to have functional regulatory roles ( 123,129 ) . analysis of motif frequencies and correspondence in conserved regions across multiple species has been used to identify known and novel regulatory elements in organisms ranging from yeast ( 25,131133 ) to the mammals ( 134,135 ) . rodent sequence alignments , and known sets of regulatory sequences and ancient repeats in those genomes , methods have been developed to distinguish conserved regulatory regions from neutral sequences ( 136,137 ) . in addition to its application in eukaryotic genomes , comparative genomic approaches have helped in elucidation of regulatory elements in prokaryotes and archaea ( 22,138141 ) . therefore , significant developments have been made over the past several years in utilizing the sequences from multiple species to identify functional regulatory elements in all phyla . there are now numerous methods that are available for alignment of genomic sequences from two or more species ( 142151 ) . some of these have been integrated with motif finding and visualization programs to provide practical tools for analysis of regulatory elements within cross - species conserved regions ( 152154 ) . multiple sequence alignment methods can be advantageous in comparative genomics since they utilize more information relative to pairwise sequence alignments ; they can also be more powered to identify regulatory motifs ( 135,140,155 ) . prakash and tompa ( 135 ) recently compared the performance of six global and local multiple alignment tools with respect to their potential of identifying highly conserved short ( 10mers ) dna patterns from the immediate upstream promoter sequences of orthologous genes from multiple vertebrate species ( human , chimp , mouse , rat , chicken ) . two of the methods tested , namely mlagan ( 147 ) and tba ( 156 ) , appeared to perform better than several others for this purpose ( 135 ) . more advanced methods have now been developed that directly take into consideration the phylogenetic distances between the organisms that are being aligned in order to identify conserved dna motifs ( 107,126,157159 ) . in addition to phylogenetic footprinting , comparative genomics is now being utilized in other sophisticated and intriguing ways to identify regulatory elements of potentially conserved function . some methods have not only utilized sequence conservation but also gene network conservation ( based on the hypothesis that multiple sets orthogonal genes may be regulated by common tfs ) to identify sets of regulatory motifs ( 133,160 ) . although developed in yeast , one of these studies show that such methods can be sufficiently powered to detect regulatory elements in much longer sequences in the multicellular organisms , including mammalian genomes ( 133 ) . there are now many examples where comparative genome sequence analysis has been used successfully to elucidate novel regulatory elements which would have been difficult to identify without that information ; three are illustrated below . mccue et al . ( 140 ) used an extended gibbs sampling algorithm to identify probable transcription regulatory sites upstream of escherichia coli genes by cross - species comparison . a set of 184 genes with orthologs from two or more other gamma proteobacterial organisms were analyzed . of their predictions 81% corresponded with the documented sites known to regulate these genes , whereas 67% corresponded when data from only one other species were available , suggesting that addition of one more species aids in sensitivity of the binding site detection . one of the novel predictions , a dna site bound by the tf yijc , was verified by experiments . in an elegant study , loots et al . ( 123 ) demonstrated the utility of phylogenetic footprinting by identifying a regulatory region conserved between human and mouse for il-5 that is located 120 kb away from the gene itself . in another study comparing genomic sequences from multiple primate species , boffelli et al . ( 129 ) , identified regulatory elements for apoa , a recently evolved primate gene . a region of high conservation adjacent to the transcription start site was shown to interact with one or more dna - binding proteins using electrophoretic mobility shift assays with nuclear extracts from liver cells . identification of such primate - specific functional elements would be unattainable through the comparison of species that are evolutionarily more distant . as evident from the discussions above , approaches that utilize conservation across species are very useful in elucidating functional dna regulatory elements . significant advances have been made in this area over the past 6 years . despite its utility and widespread use , there are limitations in phylogenetic footprinting approaches with respect to their use in the identification of regulatory elements . the short regulatory elements may be missed if the genomic sequences that are being aligned come from distant organisms ( 112,161,162 ) . if the organisms are too close , however , the alignments are extensive and therefore unable to distinguish the conserved functional elements from the non - functional ones ( 161,162 ) . it is unclear at this time whether cross - species alignments would be equally useful in finding regulatory elements in all phylogenetic clades ( 163,164 ) . in a recent comparison of two drosophila species , the known regulatory elements were found to be only modestly enriched in the conserved regions ( 164 ) , although the amount of conservation in the non - coding regions of these drosophila species was roughly the same as in human mouse . in another study , individual binding sites appear to be conserved across two nematode species , but they were not located in sequences that were aligned by the software for phylogenetic footprinting ( 112 ) . therefore , whether cross - species sequence alignment is likely to be an effective approach in all organisms in elucidating most of their functional regulatory elements , as well as the issues such as optimal phylogenetic distances , and the number of organisms needed to detect these elements , are still matters of debate and investigation ( 163 ) . in eukaryotes , tfs rarely act alone in regulating the expression of a given gene . in most cases multiple factors bind dna , often in close proximity with each other , forming regulatory modules ( 13,33,165,166 ) . by utilizing combinatorial interactions between multiple factors these cis - regulatory modules ( crms ) confer specific spatial and temporal patterns of transcription . therefore identification of composite modules and higher order regulatory structures is currently an active area of research in computational analysis of regulatory sequences . the problem is difficult however since the combinatorial interactions between the regulating factors can be very complex ( e.g. see the array of regulatory interactions in the immediate upstream region of endo16 in sea urchin ( 13 ) ] . there have been some developments in the past 6 years in addressing this problem in dna sequence analysis . the current approaches can be classified as follows : methods that identify modules given a set of dna motifs representing sites for tfs that are known to act together in regulating transcription ( 27,2931,74,167171).methods for ab initio identification of multiple dna motifs representing the sites for a crm ( 26,93,98,105,172174 ) . methods that identify modules given a set of dna motifs representing sites for tfs that are known to act together in regulating transcription ( 27,2931,74,167171 ) . methods for ab initio identification of multiple dna motifs representing the sites for a crm ( 26,93,98,105,172174 ) . softwares that identify crms given a set of known dna motifs fall into two categories , those that use hidden markov models to represent the crms ( 27,169 ) , and those that rely on observation of frequent joint occurrence of sites within a certain window , modeling the frequency of multiple sites with an appropriate statistical ( e.g. poisson ) distribution ( 30,31,74,167,169,170,175,176 ) . because the dna - binding site models for the majority of tfs are currently unknown and the information on tfs that bind dna together in crms is very limited ( 166 ) , several ab initio methods have been developed to identify composite dna elements given just a set of input sequences ( 26,28,98,105,172174 ) . a few of these methods use efficient suffix - tree structures to identify multiple or dyad patterns ( 28,98 ) , and others employ gibbs sampling or monte carlo strategy to identify multiple pwms that may represent binding sites in regulatory modules ( 26,105,172174 ) . by combining information from multiple sites , these methods have the potential to identify motifs that are too weak ( i.e. information poor ) to be identified individually ( 26 ) . two examples of the application of computational methods for identification of novel crms are discussed . the first describes the utility of an ab initio motif finder in identifying a novel composite motif regulating cell - cycle genes in yeast , and the second describes how the information on tfs that are known to bind dna jointly can be leveraged to make genome - wide predictions of regulatory modules involving sites for those factors . with the application of an ab initio composite motif discovery program , ( 177 ) identified a crm involving the binding sites for yox1 and mcm1 from the promoters of a set of 28 genes upregulated in the yeast yox1 knockout strain . yox1 , which represses the expression of genes in the m / g1 interval of yeast cell - cycle , binds dna in conjunction with the generic mads family tf , mcm1 . the binding sites for both tfs were jointly identified using the software and subsequently validated through mutational and gel mobility shift studies . the second example is of the transcriptional program in drosophila embryo . by using known dna binding specificity data for five tfs , berman et al . ( 170 ) identified genomic regions containing unusually high concentrations of predicted binding sites for these factors . a significant fraction of these binding site clusters overlap known crms . in addition , many of the remaining clusters were adjacent to genes that were expressed in a pattern characteristic of those regulated by these factors . the authors tested one of the newly identified clusters , mapping upstream of the gap gene giant ( gt ) , and showed that it acted as an enhancer that recapitulates the posterior expression pattern of gt . although the above approaches show potential , the developments in computational identification of crms are recent and there is significant room for further investigations and improvement , since the arrangements of sites in the modules are complex . the number of datasets containing collections of known composite regulatory elements that exist today is very limited [ a few examples are ( 74,164,167 ) ] , which poses an obstacle in the training and testing of general computational methods in this realm . understanding the mechanisms of transcriptional regulation has been an object of extended and difficult quest in biological disciplines . clearly , significant advances have been made over the past two and half decades not only in the representation and modeling of the dna regulatory elements , but also methods for their identification in genomic dna . however , our knowledge of the transcriptional regulatory elements in the genome and their contribution to gene expression in different spatial and temporal contexts is still limited . given the complex pattern of regulatory interactions , the challenges involved in the complete elucidation of these elements in the genome are substantial . one of the major challenges is to associate the computationally identified regulatory elements with their cognate tfs . genome - wide analyses often identify a host of putative regulatory elements ( 25,132135 ) . in order to get an understanding of the regulatory processes it is essential to associate the regulatory proteins with these elements . there have been some investigations into this problem ( 119,178,179 ) in prokaryotes and yeast , but further developments are required . an important utility of characterizing the cis - regulatory elements is their use in the computational reconstruction of gene regulatory networks . several studies have applied the information on cis - regulatory elements , either in isolation or in combination with other orthogonal sources of information ( e.g. microarray expression data ) , to construct regulatory networks ( 180183 ) . these studies demonstrate how the information on transcriptional regulatory elements can be integrated to create gene networks . however , there are significant opportunities for investigations in this area . the ab initio motif discovery tools and comparative genomics approaches have made it possible to detect regulatory elements in many genomes . in addition , information that can guide the search for regulatory elements to the most relevant regions of the genome is becoming available , e.g. the accurate location of transcriptional start sites ( 184 ) , dna - ase hypersensitive sequences within nuclear chromatin that represent regulatory regions ( including promoters , enhancers , silencers , locus - control regions ) ( 43 ) , and tf binding locations from the chip chip experiments ( 38,40,41 ) . individually , there are methodological or practical limitations in the computational and experimental approaches in elucidating the location and function of the full set of regulatory elements . for example , the computational dna motif finding algorithms have limitations in terms of the sensitivity and specificity of signals they can detect , whereas the high - throughput tf - binding site location technologies ( e.g. chip chip ) are currently limited in the extent of intergenic sequences they can explore ( 185,186 ) . therefore it appears that the most efficient path to elucidating the novel regulatory elements and mechanisms will lie in the judicious integration of the various methods and data ( 182 ) . despite inherent challenges in the field , rapid progress has been made over the past few years in the computational identification of regulatory elements . successes of the computational methods have been demonstrated through experimental validations , and efficient methods for comparative genomics and analysis of crms are being fruitfully utilized to elucidate complex regulatory elements in organisms ranging from the unicellular bacteria to the mammals . the iupac ( international union of pure and applied chemistry ) code for representing degenerate nucleotide sequence patterns . ( a ) the collection of eight known rox1-binding sites taken from scpd ( 47 ) . the cells represent the number of times a base i is observed at position j in the alignment of sites . the frequencies , fi , j , of base i at position j of the binding sites can be obtained by dividing the values in the cells of the alignment matrix by the total number of sites , e.g. fc,1 = ft,1 = 4/8 = 0.5 . each weight is given by log2(fi , j / pi ) ( see text ) , where pi is the probability of observing the base i in the data ; here we have taken pa = pt = 0.32 , and pc = pg = 0.18 ( corresponding to the s.cerevisiae genome ) . this matrix was used to score the sites in a. as an example , the score of the site in red ( sequence ccaattgttttg , score 13.87 ) is given by the summation of the scores that are circled in red . note that the scores of the two consensus sequences , cccattgttctc and tccattgttctc are different because pc pt . ( d ) sequence logo representation ( 187 ) of the alignments , visually showing the ic and conservation at each of the alignment positions . the ic of this matrix is 11.3 bits or 7.83 nats ( equation 1 ) . the genomic regions , along with 5 kb upstream and 2 kb downstream , of the ckm gene were extracted from the human and mouse genomes and aligned using the blastz software ( 151 ) . the blastz alignments were then fed into the rvista program ( 153 ) through the website . binding sites for several tfs that are known to regulate gene expression in the muscle tissue were then predicted on the human sequence using the pwm models available from the transfac database ( 12 ) . the predicted sites can be dynamically viewed and clustered through the above website . for the purpose of this current figure , we required that at least two binding sites belonging to different tfs be present within a window of 100 nt . a cluster of sites was observed in the immediate 5 upstream region of this gene ( boxed ) . percent conservation between the two sequences is shown ; regions with 75% conservation are colored .

identification and annotation of all the functional elements in the genome , including genes and the regulatory sequences , is a fundamental challenge in genomics and computational biology . since regulatory elements are frequently short and variable , their identification and discovery using computational algorithms is difficult . however , significant advances have been made in the computational methods for modeling and detection of dna regulatory elements . the availability of complete genome sequence from multiple organisms , as well as mrna profiling and high - throughput experimental methods for mapping protein - binding sites in dna , have contributed to the development of methods that utilize these auxiliary data to inform the detection of transcriptional regulatory elements . progress is also being made in the identification of cis - regulatory modules and higher order structures of the regulatory sequences , which is essential to the understanding of transcription regulation in the metazoan genomes . this article reviews the computational approaches for modeling and identification of genomic regulatory elements , with an emphasis on the recent developments , and current challenges .

INTRODUCTION REPRESENTATION AND SEARCH OF DNA REGULATORY ELEMENTS DISCOVERY OF DNA MOTIFS COMPARATIVE GENOMICS IN THE SEARCH FOR REGULATORY ELEMENTS COMPOSITE MOTIFS AND CONCLUDING REMARKS Figures and Tables

amyloidosis is particularly difficult to diagnose because the signs and symptoms are subtle . additionally , there are no specific imaging or laboratory tests , except histopathology . although it is considered to be a systemic disorder , a small portion of cases may be localized . a 54-year - old man presented with nonspecific symptoms ( jaundice and back pruritus ) . biochemical tests showed a high level of bilirubin and elevated serum tumor markers ( ca199 and ca125 ) . amyloidoses are a group of disorders that primarily consist of 3 forms , namely primary amyloidosis , secondary amyloidosis , and familial amyloidosis . amyloidosis is characterized by the deposition of abnormal proteins undergoing a conformational change to form insoluble -sheet protofilaments . the difficulty associated with diagnosing amyloidosis relates to the lack of specific imaging or laboratory tests , which poses a great challenge for clinicians . cases of amyloidosis that are primarily localized in the liver rarely have been reported . here , we report a case involving a patient with primary hepatic amyloidosis who presented with liver dysfunction . a 54-year - old man was admitted to our hospital for gradual jaundice and weight loss over 2 months . his vital signs were stable and afebrile . on physical examination , his skin and sclera were mildly jaundiced , with scratch marks on his arms and back . biochemical tests showed a high level of both total and direct bilirubin ( 97.8 mol / l and 82.6 mol / l , respectively ) . serum alkaline phosphatase ( alp ) ( 374 /l ) and gamma glutamyl transpeptidase levels ( -gtt ) ( 319 /l ) were also elevated ( listed in table 1 ) . computed tomography ( ct ) and magnetic resonance ( mr ) imaging showed hepatomegaly with no suspicious nodules ( fig . 1 ) . to determine whether there were blockages in the bile ducts , magnetic resonance cholangiopancreatography ( mrcp ) was used and showed hepatomegaly with cholecystitis . he then was treated with ursodeoxycholic acid capsules and compound ammonium glycyrrhetate single s and ademetionine for a week . after consent was obtained from the patient , ultrasonography - guided liver biopsy was utilized . the liver biopsy demonstrated a massive amount of amyloid deposition along the sinusoids ( fig . 2c ( a ) a biopsy specimen from the skin , photomicrograph ( h&e stain ) showed collagen - type extracellular material , ( b ) characteristic apple - green birefringence on polarized light microscopy , which is consistent with amyloidosis , ( c ) biopsy specimen from the liver tissue , photomicrograph ( h&e stain ) showed collagen - type extracellular material , ( d ) congo red stain demonstrated characteristic staining , ( e ) methyl violet stain revealed strong affinity for the material , ( f ) immunostaining with antibodies to kappa light chains was negative , ( g ) immunostaining with antibodies to lambda light chains was positive , ( h ) the biopsy specimen from the skin , immunofluorescence with antibodies to lambda light chains was positive . since the patient was found to have amyloid deposition in his liver , we had to conduct other tests to determine whether it was localized or systemic . echocardiographic and renal function tests , bone marrow aspiration , serum - free light chain test , and skin biopsy ( from multiple sites ) were performed . histopathological evaluation showed positive staining with congo red and characteristic apple - green birefringence on polarized microscopy . all these results indicated primary hepatic amyloidosis , and the skin biopsy was positive for lambda light chains ( fig . serum bilirubin and amino transaminases levels measured at admission and afterward are listed in table 2 . the patient refused to take melphalan or undergo stem cell transplant ; therefore , we provided him with supportive therapies . subsequently , he was discharged from the hospital and continued to take oral chemotherapy ( thalidomide 100 mg / d and prednisone 20 mg / w ) . immunoglobulin light chain amyloidosis is a clonal plasma cell disorder in which amyloid fibrils that are derived from immunoglobulin light chains are deposited in organs and tissues . the prognosis is often poor if fibril deposits build up in vital organs such as cardiac muscles . with no treatment , the median survival time is 2 years . as reported , a patient with primary amyloidosis presented symptoms that were similar to those of crohn 's disease , delaying accurate diagnosis . since the presenting symptoms tend to mimic those of other diseases , physicians should suspect amyloidosis in any patient with generalized fatigue , weight loss , skin lesions , paresthesias , nondiabetic nephrotic syndrome , and hepatomegaly . amyloidosis is usually considered to be systemic , but 10% to 20% of cases can be localized . here , we reported a case in which a patient presented with jaundice and altered liver function tests . at first , the elevated serum tumor markers and enlarged liver suggested liver cancer or carcinoma of the gall bladder . however , imaging results did not support cancer . since the patient was allergic to iodine , contrast - enhanced ct was forbidden . the diagnosis of amyloidosis requires 2 components : a generic and a type - specific diagnosis . invasive organ biopsy is not always necessary , as fat pad aspiration , in particular , is even more sensitive ( 72% ) than bone marrow biopsy in diagnosing amyloidosis . however , if a fat pad aspiration fails to demonstrate amyloidosis , then biopsies of involved organs should be considered . currently , mass spectrometry - based proteomics have emerged as a new technique to classify systemic amyloidosis and analyze serum transthyretin in patients with potentially amyloidogenic mutations . serum - free light chains , protein immunofixation , mr elastographic , shear - wave elastography , and other examinations support the diagnosis of amyloidosis , but they are not sufficient to define it . a proposed strategy for evaluating a patient with suspected amyloidosis treatment for amyloidosis depends on the type of amyloidogenic protein . for amyloid light - chain ( al ) it has been reported that melphalan and prednisone are superior to colchicine in 2 randomized phase iii studies . melphalan in combination with dexamethasone is highly effective in treating al amyloidosis with minimal toxicity . autologous stem cell transplantation is a reasonable primary option , but only 20% of patients meet the requirements . researchers have explored new drugs ( thalidomide , lenalidomide , pomalidomide , and bortezomib ) to treat al amyloidosis in combination with dexamethasone or melphalan and dexamethasone or dexamethasone and cyclophosphamide . melphalan combined with dexamethasone is still considered to be the standard treatment for nontransplant candidates . however , more data are required on treatment options in association with the pathophysiology of the amyloid formation that is generated . amyloidosis is uncommon , and its prognosis is poor , especially when irreversible organ damage occurs . thus , the most urgent requirement is an early and accurate diagnosis of the disease . clinicians should be aware of all the possible signs and initiate an effective treatment as early as possible .

abstractbackground : amyloidosis is particularly difficult to diagnose because the signs and symptoms are subtle . additionally , there are no specific imaging or laboratory tests , except histopathology . although it is considered to be a systemic disorder , a small portion of cases may be localized.introduction of the case : a 54-year - old man presented with nonspecific symptoms ( jaundice and back pruritus ) . biochemical tests showed a high level of bilirubin and elevated serum tumor markers ( ca199 and ca125 ) . routine imaging showed hepatomegaly without heterogeneous enhancement . liver biopsy confirmed the diagnosis of hepatic amyloidosis . no cardiac or renal involvement was found . the patient accepted treatment involving oral chemotherapy.conclusion:a rare and unique presentation of hepatic amyloidosis was highlighted in this case .

Background: Introduction of the case: Conclusion: Introduction Case report Discussion

relapsing polychondritis ( rp ) is a rare autoimmune disease of unknown etiology characterized by recurrent inflammatory episodes primarily affecting the auricular , nasal or laryngotracheal cartilage , as well as the ocular , audiovestibular and cardiovascular systems . the myelodysplastic syndromes ( mds ) are clonal disorders of hematopoiesis characterized by peripheral cytopenias and a dysplastic bone marrow that are usually hypercellular . mdss characteristically exhibit a high number ( between 5% and 50% ) of erythroid precursors in the marrow . however , a minority have merely a small amount ( less than 5% ) of recognizable erythroid cells in the bone marrow . within this group , most of the patients with erythroid hypoplasia have the so - called mds with erythroid hypoplasia / aplasia whose pathogenic mechanism remains unclear . a review of the literature reveals only 16 well - documented cases of mds with erythroid hypoplasia / aplasia . there are several reports of a simultaneous or temporally close occurrence of rp and myelodysplastic / myeloproliferative conditions . here , we report a 79-year - old patient with rp who developed mds subtype refractory anemia ( ra ) with erythroid hypoplasia / aplasia . to our knowledge , this is the first case of rp associated with mds with erythroid hypoplasia / aplasia . a 79-year - old male was admitted to our hospital in october 2002 because of a five - day history of fever , chills , and productive cough . he was diagnosed with rp in 1997 due to recurrent swelling and redness of both ears ( figure 1 ) , conjunctivitis , general ache and myalgia , which was confirmed by an ear biopsy ( figure 2 ) . since then manifestations of rp were cleared under treatment with low - dose glucocorticoid over 5 years . on admission the blood count showed macrocytic anemia : hemoglobin 6.8 g / dl , hematocrit 20.6% , mcv 107 fl , and mch 35.2 pg . slight leukocytosis ( wbc count=10,400/l ) and moderate thrombocytopenia ( platelet count=99,000/l ) were noted . esr was 43 mm / h and crp was positive ( 36.9 mg / dl ) . biochemical tests were all within normal ranges except for slightly elevated ldh ( 499 iu / l ) and decreased protein / albumin levels ( 6.0/2.7 g / dl ) . serum iron level and saturation of transferrin were increased ( 164 g / dl and 91% , respectively ) . there was no vitamin b12 deficiency ( serum level : 963 pg / ml ; normal 200950 pg / ml ) or folic acid deficiency ( serum level : 14.5 ng / ml ; normal 2.520 ng / ml ) . tests for serum anti - nuclear antibodies , anti - neutrophil cytoplasmic antibodies and rheumatoid factor were all negative . chest radiographs showed air - space consolidations in the upper and lower right lobes with a small amount of right pleural effusion . however , transfusion - dependent anemia ( hemoglobin=7.8 g / dl ) and thrombocytopenia ( platelet count=24,000/l ) remained . a myeloid to erythroid ratio was markedly increased ( 21:1 ) due to the markedly decreased erythroid series . he was treated with prednisolone 5 mg daily until discharge , and received packed red cell transfusion once a week . with low dose prednisolone therapy and intermittent transfusion , the patient has remained in a stable clinical and hematologic condition . at a follow up three months after diagnosis , his blood count showed wbc count 2,800/l , hemoglobin 6.4 g / dl and platelets 119,000/l . rp is a rare , episodic inflammatory condition affecting cartilaginous tissues , especially the ear , nose and tracheobronchial cartilage . the diagnostic criteria were described by mcadam et al . our patient fulfilled these criteria , having both recurrent auricular chondritis and ocular inflammation . autoantibodies to type 2 collagen , association with other autoimmune disorders and response to steroids by reduction of the inflammatory response all support this view . in our patient , the manifestations of rp were cleared under treatment with low - dose glucocorticoid over 5 years . mdss are a group of acquired neoplastic disorders of bone marrow , characterized by quantitative and functional abnormalities of all three hematopoietic lineages caused by a defect at the stem cell stage . however , a minority have merely a small amount ( less than 5% ) of recognizable erythroid cells in the bone marrow . within this group , most of the patients with erythroid hypoplasia have the so - called mds with erythroid hypoplasia / aplasia . our patient showed 4% of erythroid precursors and markedly increased myeloid to erythroid ratio ( 21:1 ) in the marrow . van besien et al . reported a case of rp associated with mds subtype refractory anemia with excess blasts in transformation ( raeb - t ) . mongin et al . reported 12 cases of rp associated with mds , in which mds preceded rp , occurred simultaneously , or developed subsequently . in 1997 , our patient was diagnosed with relapsing polychondritis . five years later , a diagnosis of mds was made by a bone marrow aspiration . the association between mds and rp certainly does not appear fortuitous , but the mechanism of this association continues to remain obscure . however , the fact that there was anemia when he was diagnosed with rp and that rp preceded the bone marrow changes by five years add currency to the notion that rp may represent a paraneoplastic phenomenon of an underlying mds , or alternatively , that mds and rp may be caused by a single immunological abnormality leading to an autoimmune condition . it showed a dearth of erythroid series , and he was diagnosed with mds with erythroid hypoplasia / aplasia , a very rare subtype of mds . our patient is the first case reported in the literature with rp associated with mds with erythroid hypoplasia / aplasia . cook reported a case of polymyalgia rheumatica concurrent with erythroid hypoplasia / aplasia associated with myeloproliferative and myelodysplastic syndrome . in his case , the patient initially responded well to prednisolone , but relapsed after tapering off the steroid . although mds with erythroid hypoplasia / aplasia is usually highly resistant to therapy , the efficacy of steroids for erythroid hypoplasia / aplasia occuring in patients with mds is also reported in several cases . williamson et al . reported six cases of erythroid hypoplasia / aplasia occuring in patients with mds . three patients died within a short time of developing erythroid hypoplasia / aplasia associated with an increased blast percentage in the marrow . the other three patients showed at least a partial recovery of erythropoiesis after being treated with prednisolone . williamson et al . suggested that the response to steroid supports an autoimmune etiology superimposed on mds rather than an intrinsic stem cell defect . this supports the view that those patients who have erythroid hypoplasia / aplasia and no excess of blasts may have a relatively good prognosis and should be considered for immunosuppressive treatment . mds with erythroid hypoplasia / aplasia is a rare form of myelodysplasia , with only 16 well - documented cases reported in the literature . patients with this disorder were predominantly elderly males at presentation , all requiring regular blood transfusions , and with an unfavorable prognosis because of a high risk of blastic transformation . the pathogenic mechanism of mds with erythroid hypoplasia / aplasia in rp patients remains uncertain . although , a number of autoimmune diseases including crohn s disease , ulcerative colitis , behcet s disease , pernicious anemia , hypothyroidism , polymyalgia rheumatica , inflammatory arthritis and rp - have been suggested to occur rather frequently in mds , garcia - suarez et al . reported that most patients ( 15/16 ) with mds with hypoplasia / aplasia did not have immunologic disorders known to be associated with autoimmune erythroid hypoplasia / aplasia . , we add another immunologic disorder associated with the very rare mds with erythroid hypoplasia / aplasia . we believe that mds with erythroid hypoplasia / aplasia may also be related to autoimmune diseases , a feature not different from other mds subtypes .

relapsing polychondritis ( rp ) is a rare multisystem disorder . myelodysplastic syndrome ( mds ) with erythroid hypoplasia / aplasia is a rare form of myelodysplasia . several cases of rp associated with mds have recently been described . however , rp associated with mds with erythroid hypoplasia / aplasia has never been reported . there was only one case report of polymyalgia rheumatica associated with mds with erythroid hypoplasia / aplasia . in this study , we report a 79-year - old patient with rp , who developed mds subtype refractory anemia ( ra ) with erythroid hypoplasia / aplasia , a very characteristic subtype of mds .

INTRODUCTION CASE REPORT DISCUSSION

about 80% of strokes are ischaemic , resulting from an obstruction of blood flow , while about 15% are due to a primary intracerebral hemorrhage . stroke is one of the leading causes of chronic adult disability and death in western industrialized countries . neurological deficits reflect the location of the tissue damage and , in particular , the extent of the neuronal loss . neurons deprived of their normal metabolic substrates cease to function in seconds and show signs of structural damage after only 2 minutes . as energy - dependent processes fail , neurons are unable to maintain their normal transmembrane ionic gradients , resulting in ion and water imbalance that triggers apoptotic and necrotic cell death cascades and , ultimately , leads to focal neurological signs and symptoms . according to the who 's international classification of function , disability , and health ( icf , who 2001 ) , the impairment of brain functions may originate different activity limitations ( disability ) and participation restriction ( handicap ) . motor impairments , including hemiparesis , incoordination , and spasticity , are the most common deficits after stroke . however , functional recovery frequently occurs following stroke , although its extent is highly variable . some patients with initial severe hemiparesis may eventually achieve full recovery , while others have little or no improvement and remain permanently disabled . there are many reasons for the variable degrees of recovery , including the age of the patient , the location and extent of the lesion , and individual variations in anatomical and functional connections . the neural bases for poststroke recovery rely on the concept of plasticity , namely , the ability of central nervous system ( cns ) cells to modify their structure and function in response to a variety of external stimuli ( experience ) . the plastic / reparative properties of the brain are determined by the balance between cell - intrinsic mechanisms and extrinsic regulatory molecules , which is regulated by activity - dependent processes and different kinds of interaction with the external world [ 4 , 5 ] . molecules in the adult cns milieu , such as myelin - associated proteins ( e.g. , nogo , mag , and omgp ) , factors secreted by astrocytes near the stroke site ( e.g. , chondroitin sulfate proteoglycans ) , and repulsive axonal guidance cues ( e.g. , semaphorins , netrins , and members of the ephrin family ) , constrain axonal sprouting and hamper the formation of new connections . in preclinical stroke models it has been shown that pharmacological blockade of nogo , nogo receptor antagonism , or digestion of chondroitin sulfate proteoglycans by chondroitinase induce axonal sprouting and promote functional recovery [ 79 ] . blocking the semaphorin pathway other growth inhibitors , such as epha4 and ephrin - a5 , have also recently been identified , which limit functional recovery and are promising targets for repair after stroke [ 11 , 12 ] . interestingly , inhibition of rock , a downstream target of several growth inhibitors , greatly improves outcome after ischemic stroke . several studies have also uncovered pharmacological targets that promote a neuronal growth state in the adult cns . for example , inosine triggers a serine / threonine kinase ( mst3b ) , enhancing axonal sprouting [ 13 , 14 ] . the therapeutic potential of replacement strategies in laboratory models of stroke is also under investigation . transplantation of neural progenitor cells , bone marrow - derived mesenchymal stem cells or human - induced pluripotent stem cells into the ischemically lesioned brain have been proved to be a safe and efficient approach to promote significant functional recovery in experimental animals [ 1517 ] . nonetheless , the mechanisms underlying the beneficial effects of cell transplantation in the ischemic cns remain uncertain , and , most importantly , to date there is no clear evidence that donor cells may directly contribute to the structural repair of neuronal circuits . in addition to pharmacological or replacement therapies , clinical and preclinical studies are currently focusing on noninvasive strategies for post - stroke rehabilitation . motor rehabilitation after hemiparetic stroke typically involves combinatory approaches , including neurofacilitation techniques , task - specific training , and task - oriented training [ 19 , 20 ] . furthermore , stroke units , in which patients have access to daily skill training therapies in highly stimulating environments , such as during physical , occupational , or language therapy , result in decreased deficits , increased performance on self - care tasks , lower 1-year mortality , and lower probability to be in a nursing home at followup . finally , in recent years brain stimulation , mirror therapy , action observation , or mental practice with motor imagery is emerging as interesting options as add - on interventions to standard physical therapies . here , we will provide an overview of recent noninvasive strategies employed to enhance functional recovery in patients after stroke and discuss the current knowledge of rehabilitative strategies and the associated neural plastic events in preclinical models of stroke . stroke rehabilitation aims to guarantee that stroke survivors reach the maximum physical , functional , and psychosocial recovery possible within the limits of their impairment . in order to help stroke patients to fully participate in life , the final goal of rehabilitation should be to maximize performance of activities of daily living and independence . through learning - dependent processes recovery of stroke patients is extremely heterogeneous and determined by a combination of processes including functional restoring of damaged nervous tissue , relearning of lost skills through reorganization of spared pathways ( plasticity ) , adaptation , and compensation for deficits . compensation reflects the use of alternate behavioral strategies in order to solve a specific task . most recovery of specific neurological focal deficits occurs during the first 3 to 6 months after stroke , but it is largely accepted that improvements can continue for years after stroke . general principles of stroke rehabilitation include the start of intensive rehabilitation programs carried out in a stroke unit within the first few days after stroke [ 24 , 25 ] . evidence demonstrates that comprehensive intensive rehabilitation , as well as the presence of a structured multidisciplinary team , may be more effective than less intense programs . in agreement with the learning nature of the rehabilitative process , involvement , engagement , and motivation of patients , families and caregivers most recent neurorehabilitative approaches are based on a task - oriented model of motor learning , whose main feature is an intensive training with specific tasks in an environmental context ( task - specific and context - specific trainings ; [ 2730 ] ) . in this context , a number of new rehabilitative techniques potentially capable of stimulating cerebral plasticity have been proposed and tested in the last years . among these techniques , large interest is devoted to treatment approaches aimed to improve motor functions , including constraint - induced movement therapy , mental practice , mirror therapy , virtual reality , robotics , and brain stimulation techniques . constraint - induced movement therapy ( cimt ) involves the restriction of usage of the unaffected limb , forcing the use of the paretic one , and aiming to contrast the maladaptive learned nonuse of the paretic limb ( the subject learns to ignore the damaged limb because of its lack of functionality and learns to use exclusively the healthy limb ) . a number of studies including randomized controlled trials and a cochrane review have shown that cimt is effective in improving motor performance in human patients after stroke [ 31 , 32 ] with a large effect size and robust effects especially on arm function . in particular , the ecxite trial demonstrated that daily intensive cimt training for upper limb paresis was superior to the control treatment 3 to 9 months after stroke , and that a modest improvement in motor function persisted in the cimt group after 2 years . important limitations to the routine use of cimt training derive from the fact that it is labor intensive and suitable only for patients with some conservation of motor functions ( in particular wrist and finger ) , thus its use is recommended only for selected patients . mental practice with motor imagery is considered a promising additional treatment to improve motor functions of severely affected upper limb , although its clinical effectiveness is not yet clearly proven . this approach grounds on the statement that imaging a movement requires activation of brain circuits involved in the preparation and execution of the same movement and consists in a repetitive cognitive training during which the patient imagines performing a task or body movement without actually physically performing it . it has been demonstrated that mental practice may modulate cerebral perfusion and neural activity in brain regions similar to those activated during actual movements [ 34 , 35 ] . following few systematic reviews [ 36 , 37 ] suggesting that mental practice may be beneficial for post - stroke disabilities in addition to conventional treatments , a recent cochrane review concluded that there is only limited evidence that mental practice may increase the effectiveness of usual physiotherapy and occupational therapy . another approach based on multisensory stimulation is represented by the mirror therapy . in this technique a mirror is placed at 90 in the patient midsagittal plane , so that the paretic limb is hidden behind the mirror and the patient watches the image on the mirror of the unaffected arm as if it was the affected arm . in a certain sense , the patient receives the impression that the affected limb is functioning . it has been demonstrated that viewing the image of one 's moving hand reflected by the mirror increases the excitability of neurons in the ipsilateral primary motor cortex more than directly viewing the inactive hand . mirror therapy effects ( as well as those related to mental practice ) may be related to the activity of the so - called mirror neurons , which discharge both following performance of motor acts and simply observing the same action done by another individual [ 40 , 41 ] . in fact , by means of fmri it has been demonstrated that prolonged and repetitive observation of an action may enhance the activity in the ventral premotor cortex , the supplementary motor area , and the superior temporal gyrus . a recent systematic review including 14 studies and a total of 567 patients treated with mirror therapy concluded that , when compared to other rehabilitative approaches , this treatment has a significant effect on motor function even though this result is strongly influenced by the type of intervention used as control . thus , it remains unclear if mirror therapy should replace other treatments for motor rehabilitation after stroke , while its role as additional intervention is confirmed . moreover , mirror therapy improves activities of daily living , but this statement is limited by the small number of studies ( four ) examining this effect . the virtual reality idea is based on the possibility that a computer can generate a three - dimensional graphical environment from numerical data , so that , by using visual , aural , or haptic devices , the operator can experience the environment as if it were a part of the world . a key feature of all virtual reality applications is interaction : virtual environments are created to allow the user to interact also with virtual objects within the environment . in some systems , the interaction may be achieved via a mouse or a joystick button , while in others , a representation of the user 's hand may be generated within the environment with movement of the virtual hand reflecting the user 's hand , thus allowing a more natural interaction with objects . therefore , virtual reality represents a unique instrument to achieve several requirements for effective rehabilitation , such as repetitive practice , feedback about performance , and motivation to endure practice [ 45 , 46 ] . specifically , by using virtual reality it is possible to drive and control exercises for patient rehabilitation within a functional , purposeful , and motivating context . different virtual reality approaches have been used , in particular , for upper limb motor rehabilitation . a cochrane review published two years ago , analyzing 19 randomised and quasi - randomised trials of virtual reality that involved 565 participants , concluded that there is a limited evidence that virtual reality and interactive video games may be beneficial in improving arm function and activity of daily living function when compared with the conventional treatments . another , contemporary meta - analysis , including 12 studies ( 5 randomized controlled trials and 7 observational studies ) for a total of 195 patients , showed that in the large majority ( 11 over 12 ) of these studies virtual reality added a significant benefit on arm motor recovery after stroke . however , to gain convincing evidence of virtual reality effectiveness in poststroke rehabilitation , further research is needed based on good randomized controlled trials . in the last years a growing interest has been addressed to robot - assisted rehabilitative treatments after stroke . in theory several studies have demonstrated a significant result in motor recovery of the upper limb of patients who trained with robotic devices but no significant effect on functional ability . however , the conclusion of a randomized controlled trial ( ul - robot ) and a cochrane meta - analysis limited the significance of these results . in the ul - robot trial two groups of patients receiving 36 therapy sessions over 12 weeks of robot - assisted therapy or intensive conventional physical therapy , respectively , were compared with patients receiving usual ( not intensive ) care . the study failed to demonstrate a superiority of the intensive robot therapy when compared to intensive conventional physical therapy , but both techniques were superior to usual care , suggesting that intensity of training may be a crucial factor for motor recovery . the cochrane review , including 19 trials and 666 patients , concluded that electromechanical and robot - assisted arm training after stroke may improve generic activities of daily living as well as paretic arm function , but not arm muscle strength . a phase iii randomized and controlled trial ( leaps- ) , designed to test the efficacy of a popular technique that utilizes partial body - weight support with treadmill training , was concluded in 2011 . the leaps trial included 408 patients randomly assigned to three groups : two groups were subjected to a locomotor training with treadmill and body - weight support ( one group initiating treatment 2 months after stroke and the second 6 months after stroke ) , the third group received a home exercise program . the results were someway surprising : no significant difference was found between the three groups concerning the improvement in walk speed , motor recovery , balance , functional status and quality of life . thus , locomotor training with body - weight support and treadmill can not be considered superior to a structured , progressive , and intensive at home treatment . also , in this trial all intensive interventions were more effective when compared to non - intensive and structured care . a promising robotic interface has been recently developed by courtine 's group to evaluate , enable , and train pattern generation and balance during walking in rats . the devise continuously and independently assists or perturbs propulsion and balance along four degrees of freedom , while rats are progressing overground within a large workspace . in a model of stroke the use of noninvasive techniques of brain stimulation to stimulate adaptive plasticity is very appealing , and the results obtained are exciting . two main techniques are available to obtain both cortical enhancement and inhibition : repetitive transcranial magnetic stimulation ( rtms ) and transcranial direct current stimulation ( tdcs ) . rtms , using a coil placed on the scalp , generates a focal magnetic field , which induces ( transiently , focally , and reversibly ) an electric current in the underlying cortex . low frequency stimulation ( in the range of 1 hz ) reduces cortical excitability , while higher stimulation frequencies increase the cortical excitability . in tdcs , weak direct currents are delivered to the cortex through two electrodes that polarize the underlying tissue . electrode position is crucial to modulate the distribution and direction of the current flow : anodal stimulation has an excitatory effect by cortical neuron depolarization , while cathodal tdcs hyperpolarizes neurons by decreasing cortical excitability . in general , two different approaches can be described using noninvasive brain stimulation : one addressed to increase excitability of ipsilateral damaged hemisphere ( e.g. , by stimulating primary motor cortex ) , and the other one directed to reduce the activity of intact surrounding or contralateral area that can produce intra- or interhemispheric inhibition . the purpose of these applications is to restore the unbalance between intact and lesioned hemisphere according to the interhemispheric competition model . moreover , bestmann and coworkers suggested an unexpected role of the contralesional dorsal premotor cortex , with an elegant demonstration by means of rtms which showed the supporting activity of contralesional dorsal premotor cortex to ipsilesional sensorimotor regions in particular for greater clinical and neurophysiological impaired patients . the application of these approaches have produced very promising results , in both acute and chronic stroke patients , recently reviewed by corti et al . . that review suggests that rtms applied to the affected hemisphere is safe and could be considered effective for modulating brain function and contributing to motor recovery after stroke . however , the authors stressed the need of double - blinded , sham - controlled phase ii and phase iii clinical trials involving larger sample sizes to validate this treatment . in a meta - analysis of 18 randomized controlled trials dedicated to the effects of rtms on upper limb motor impairment , hsu et al . found a significant effect size ( 0.5595% ci , 0.370.72 ) for motor outcome function , with more clear effects for subcortical stroke and low - frequency rtms applied to the unaffected hemisphere . questioned about the real duration and anticipated size of the treatment effects in chronic stroke patients . in such patients they showed with a small semirandomized clinical trial that rtms application does not augment the gains from a late rehabilitation program . the need for randomized controlled trial is even more evident to validate efficacy of tdcs , considering that its use in stroke patients is quite new [ 59 , 60 ] . recently , khedr et al . provide an interesting evidence that both anodal and cathodal tdcs are superior to sham stimulation in enhancing the effect of rehabilitation training to improve motor recovery after subacute stroke in a pilot randomized controlled trial . however , it must be stressed that our knowledge about mechanisms underlying brain stimulation are largely incomplete . rehabilitative conditions in stroke units , such as physical therapy and various kinds of stimulating activities , can be partially mimicked in animal studies by housing the animals in an enriched environment ( ee ) . ee is a widely employed paradigm to study the influence of external stimuli on brain plasticity in animal models both in physiological conditions and after damage . environmental enrichment refers to housing conditions that facilitate enhanced sensory , social , cognitive stimulation , and motor activity . home cages used for enrichment are larger than standard cages to allow room for several objects , which generally vary in composition , shape , size , texture , smell , and colour . enrichment may also involve access to running wheels for enhanced voluntary exercise ( figure 1 ) . key aspects appear to be the provision of environmental complexity , with objects that offer a range of opportunities for visual , somatosensory and olfactory stimulation , and environmental novelty , obtained by changing the objects and their position in the cage , which might provide additional cognitive stimulation . increased complexity and novelty social interactions are also favored by housing rather large groups of animals of both sexes together ( see for review ) . several studies show that in experimental models of stroke , ee strongly promotes recovery of motor functions , such as skilled limb function [ 6468 ] and gait . compensatory mechanisms have been shown to substantially contribute to functional improvement after stroke [ 70 , 71 ] . compensation reflects the use of alternate behavioral strategies in order to solve a specific task [ 70 , 72 ] . to what extent ee enhances functional outcome after stroke due to compensation for lost functions rather than their restoration is not entirely clear . witte and coworkers addressed this question by focusing on the time course of functional recovery versus motor compensation in skilled forelimb movements after cerebral ischemia in rats . the skilled reaching task allows the distinction between recovery and compensation by quantitative ( reaching success ) and qualitative ( movement pattern ) analysis . it has been shown that ee facilitates effective compensation in skilled reaching , while it does not promote restitution of function . namely , rotating movements of the forelimb during reaching are permanently impaired and require functional compensation through intensified use of the upper body . interestingly , in one of the first studies on the effects of ee on stroke animals , ohlsson and johansson addressed whether preoperative and postoperative environments can differently influence functional outcome after focal brain ischemia . rats were subjected to ligation of the right middle cerebral artery ( mca ) then transferred from a non enriched to an enriched environment or reared in an ee already before the operation . rats kept in an ee before and after the mca ligation improved sooner and to a slightly higher degree than those placed in the ee only after the ischemia . the beneficial effects of ee in the animals enriched before mca ligation suggest that complex experiences during healthy conditions may provide a brain reserve against late brain damage , according to previous findings [ 7378 ] . among the ee - induced changes in physiological conditions , the development of new synapses [ 79 , 80 ] and dendritic spines [ 8183 ] has been demonstrated . in addition , there is evidence that exposure to ee reduces the expression of growth - inhibitory molecules in the intact cns tissue [ 84 , 85 ] . therefore , it is conceivable that reduced inhibitory mechanisms together with a reserve of synapses in enriched animals may provide neuroprotection and facilitate functional compensation after stroke . a bulk of evidence highlights the functional benefits induced by motor training after focal ischemic injury in humans . a useful method of training for chronic and acute individuals after a stroke is treadmill training ( figure 1 ) . when applied to ischemic rats starting 24 h after ischemia , it leads to a significant reduction of infarct volume and improves neurological function . interestingly , functional recovery after stroke ( such as forelimb foot placing , parallel bar crossing , and rope or ladder climbing ) can be further improved by complex motor training ( which can be obtained by using rotarod ) rather than simple repetitive exercise , such as treadmill training . this suggests that repeated complex movements involving motor balance and coordination are more effective for functional recovery after stroke than either simple activity or inactivity . in line with this view , specific behavioral experience , such as skilled - reaching training ( figure 1 ) , after focal experimental infarct , provides substantial behavioral recovery of skilled hand function in monkeys . in experimental animals , skilled reaching training consists of daily practice of the impaired forelimb to retrieve food pellets . this kind of rehabilitation provides positive reinforcement ( i.e. , food reward ) associated with use of the impaired limb , thereby encouraging animals to practice spared motor function or promoting development of compensatory motor strategies , resulting in lessened functional deficiency . interestingly , by combining both enriched living conditions and daily skilled - reaching training , biernaskie and corbett obtained dramatic long - term improvement both in skilled use of the impaired forelimb and digits and in limb placement in stroke rats . these findings reinforced the idea that skilled learning therapy coupled with enriched surroundings may facilitate neurologic recovery in humans . it should be noted , however , that the effect of forced exercise on functional recovery after stroke is controversial . forced exercise , such as treadmill running or constraint - induced movement therapy , has been shown to enhance the functional recovery of motor skills after experimental ischemic stroke [ 90 , 91 ] . other studies , however , demonstrate that treadmill running produced negative physiological adaptations induced by stress , and a constraint - induced movement study did not show improved functional outcome after brain ischemia . patients with high levels of social support or large social networks exhibit more rapid and extensive functional recovery after stroke than socially isolated individuals [ 94 , 95 ] . the importance of social influences on stroke outcome have been also highlighted in experimental animals by johansson and ohlsson ( figure 1 ) . these authors assessed the relative importance of postoperation physical activity and social interaction for functional outcome . rats were housed together in a large cage with no equipment or housed individually in cages with free access to a running wheel and compared to rats kept in an ee . interestingly , rats housed together in a large cage with no activity - stimulating facilities improve more than rats housed in individual cages with access to a running wheel . however , rats housed in an ee improve significantly more than the other two groups , suggesting that , although increased physical or social activity alone might result in some of the beneficial effects observed with enrichment , they do not fully account for the broader behavioural improvements observed following exposure to complex stimuli . to study social influences on experimental stroke outcome , devries ' group addressed the effects of social isolation versus pair housing on stroke - induced infarct size and functional recovery in mice . they observed that pair housing decreased infarct size and improved functional outcome of stroke mice when compared to socially isolated mice . social interaction influences locomotor activity and introduces auditory , olfactory , and visual stimuli , which in turn may influence pathophysiological mechanisms and recovery . further , the same authors asked whether one aspect of social interaction , namely , physical contact , may mediate the effects of social interaction . to control for the element of physical contact during pair housing , the experiment included the use of standard cages fitted with a grid partition that allowed the experimental mouse to see , hear , and smell its partner but not engage in physical contact . interestingly , only paired animals that were in unobstructed physical contact showed smaller infarct volumes and exhibited recovery of locomotor activity following mca occlusion , indicating that physical contact during social interactions influences stroke outcome . further clinical research is , therefore , needed to determine the influence of physical contact on patient recovery . another potential noninvasive treatment that might have a significant impact upon recovery of skilled motor behaviors after stroke is tactile stimulation ( figure 1 ) . when stroke rats are given tactile stimulation , which involves petting animals individually with a baby hairbrush or a paintbrush , they show dramatic improvement in the single pellet reaching task relative to untreated lesioned animals . these data suggest that massage therapy might be beneficial in resolving motor deficits in human stroke patients . interestingly , intermittent single whisker stimulation , if initiated within 2 h of permanent mca occlusion in the rat , induces complete protection from ischemic stroke by 24 h after injury , preventing the expected damage and deficits . an initial absent or severely disrupted whisker functional representation is followed by gradual recovery to baseline responses over the treatment period . evoked subthreshold activity and spiking and blood flow levels , which are severely decreased immediately after occlusion , return gradually to preocclusion levels . blood flow data suggest that the protection induced by early stimulation is due to reorganized blood flow via collateral vessels ( interarterial connections ) . in contrast , animals that do not receive treatment until 3 h post - mca occlusion show compromised function and large infarcts [ 101 , 102 ] . these studies raise hope for the development of stimulation - based strategies to mitigate stroke pathology in humans . recent studies employed animal models to investigate the positive effects of tdcs and define the optimal time window of its application after stroke ( figure 1 ) . both early ( 1 day after ischemia ) and late ( 1 week after ischemia ) anodal tdcs treatments exert beneficial effects on cognition , behavioral function ( i.e. , improved barnes maze performance and motor behavioral index scores ) , and neural plasticity , without exacerbating ischemic volume and metabolic alteration . however , only the rats receiving late tdcs treatment showed improvement in the beam balance test . accordingly , in the study by jiang et al . anodal and cathodal tdcs applications from day 1 to day 3 after cerebral infarction do not improve the beam walking test scores of rats on day 3 , but significant amelioration of motor function is observed if the animals receive continuous application of tdcs till day 7 or 14 . these findings suggest that late application of tdcs may result in stronger motor function improvement than earlier intervention after stroke . accordingly , one study , in which anodal tdcs was applied during five daily sessions to the ipsilesional primary motor cortex in acute stroke patients starting on the 2nd day , did not reveal any significant difference in motor function between the tdcs and sham groups , indicating that tdcs application from day 2 to day 5 after stroke does not promote functional recovery . ltp and ltd may be candidates processes to explain the cellular correlates for tdcs - induced effects [ 106 , 107 ] . despite the observed beneficial effects in humans ( see for review ) , the cellular / molecular mechanisms underlying rtms action are far from clear . it is likely that rtms induces ltp or ltd , which , in turn , produce enduring changes on neocortical excitability and synaptic connections [ 109111 ] . in humans , an increase in motor - evoked potential amplitude [ 110 , 112 ] , regional cerebral blood flow , glucose metabolism , and eeg response amplitude has been reported . studies in animal models ( figure 1 ) have shown that rtms effects depend on changes in nmda receptor activity . provided the first evidence that rtms induces changes in bdnf - trkb signaling in the rat brain , which are reflected in lymphocytes . transcription of glial fibrillary acidic protein ( gfap ) is increased in astrocytes of the mouse dentate gyrus ( the magnitude of this response depends on the number of stimulus trains ) , suggesting that rtms induces the first stage of a reactive response that is similar to what occurs following nervous tissue injury . however , the consequences of rtms on experimental animals after stroke have been poorly investigated . report a significant recovery of neurological severity score in stroke rats treated with tms , which is accompanied by increased expression of c - fos and bdnf in the cerebral cortex surrounding the infarction areas . after clinical stroke , the initiation of physical rehabilitation programs varies from days to several weeks after the insult . determining whether there is a period during which the poststroke brain is most sensitive to physical rehabilitation is essential to maximize the functional gains from such therapy . hypothesized that implementing rehabilitative treatment early after the stroke would enhance functional outcome . to characterize a potential critical period for successful rehabilitation after stroke , animals received enriched rehabilitative training at 5 d , 14 d , or 30 d after mca occlusion . early initiation of enriched rehabilitation ( 5 d after stroke ) provides enhanced functional outcome relative to ischemic animals receiving delayed rehabilitation , suggesting that the poststroke brain is in a state of heightened sensitivity to behavioral experience . in line with those findings , barbay et al . demonstrate a time - dependent , rehabilitation - induced map reorganization after ischemic injury in primates . similarly , early treadmill training ( started 24 h post - mca occlusion ) was found to have significant effects in reducing brain infarct volume and in improving neurologic function , when compared with late training ( started 1 week post - mca occlusion , ) . nevertheless , some evidences suggest that early training after focal brain ischemia in rats exacerbates brain damage and worsens the general outcome after excessive use of the impaired limb . namely , when the intact forelimb is constrained immediately after the surgical procedure , thus forcing the animal to overuse the impaired forelimb for postural support and movements , functional improvement is reduced [ 120 , 121 ] . the intensity of training may contribute to early exclusive use - dependent exaggeration of injury . for example , in the study by yang et al . , the intensity of treadmill training for 30 min / day seems to be mild compared to forced use by casting procedures . excessive sensorimotor activation too early after the insult may exacerbate injury through a use dependent , nmda - mediated process , possibly stimulating an excitotoxic cascade . this process may dissipate over days , explaining why rehabilitative experience beginning 35 d after insult does not worsen injury size or behavioral outcome [ 89 , 123 ] . in addition , during the first week after injury , the tissue surrounding the infarct is reported to show decreased phasic inhibition and thus become hyperexcitable . however , carmicheal 's group show that while phasic gaba signaling is reduced in the first weeks after stroke , tonic gaba signaling is potentiated in peri - infarct motor neurons . behavioral and electrophysiological studies in mice suggest that the overall effect in terms of motor cortex circuitry is a diminished neuronal excitability , which when reversed leads to recovery . therefore , the precise signaling systems in brain excitability that are deleterious in the early phases , become beneficial in later phases of recovery ( see for a comprehensive review on brain excitability in stroke ) . rehabilitation may act by affecting this delicate balance between hypo- and hyperexcitability of neuronal circuits in peri - infarct cortex . interestingly , immediate exposure to ee improves functional outcome , despite exacerbation of ischemic injury [ 67 , 126 ] , perhaps as a consequence of removal of functionally abnormal neurons . nonetheless , early ee combined with training enhances recovery when compared with conditions in which rehabilitation is started later and is not accompanied by any exacerbation of injury . in addition , a window of opportunity extends also to neurovascular changes , which can facilitate full protection . in summary , the efficacy of rehabilitative therapy after stroke is influenced by the time of its initiation , with mild intensity physical training provided early after brain injury being beneficial for functional improvement . delaying the beginning of rehabilitation may instead reduce the efficacy of treatment and , as a consequence , more intense or longer duration therapies are required to achieve the same functional gains . much of the recovery after stroke is likely due to brain plasticity , with some areas of the brain taking over the functions previously performed by the damaged regions . proposed mechanisms include : ( i ) redundancy of brain circuitry with alternative pathways taking over when another one has been damaged ; ( ii ) unmasking of previously existing but functionally inactive networks ; ( iii ) sprouting of fibers from surviving neurons with formation of new synapses [ 127 , 128 ] . when damage to a functional system is partial , within - system recovery is possible , whereas after complete destruction , substitution by a functionally related system may be the only alternative . in stroke patients , improved arm and hand movement and clinical scores have been found in correlation with an enlargement of the hand region in the ipsilesional cortex [ 130135 ] . activity changes in specific cortical areas may result from a reduction in inhibition from horizontal or callosal connections . alternatively , new connections may form due to lesion - induced sprouting at the cortical or subcortical level [ 137 , 138 ] . reorganization of neuronal connectivity around the lesion site and also in the undamaged contralateral cortex has been detected [ 139141 ] . interestingly , following an ischemic subtotal lesion of the rat forelimb motor cortex , spontaneous recovery of forelimb function is correlated with hindlimb corticospinal neurons forming new connections with cervical , forelimb - related , spinal cord neurons . for example , while an ischemic lesion confined to a small portion of the representation of one hand results in a further loss of hand territory in the adjacent , undamaged cortex , early rehabilitative training prevents the loss of hand territory adjacent to the infarct . in some instances , the hand representation expands into regions formerly occupied by representations of the elbow and shoulder . functional reorganization in the undamaged motor cortex is accompanied by behavioral recovery of skilled hand function . moreover , stroke rats housed in an ee or receiving tactile stimulation have significantly increased dendritic branching and spine density on pyramidal cortical neurons than control stroke rats , suggestive of increased sprouting of intracortical connections in the enriched / stimulated group . indeed , ee can influence a number of factors , such as functional enforcement of existing neuronal circuits , sprouting , formation of new connections , and angiogenesis [ 63 , 144 ] . ee may also modulate ischemia - induced glutamate excitotoxicity , thus leading to attenuated oxidative damage and neurodegeneration . one candidate mechanism underlying the beneficial effects of ee on functional recovery after stroke involves upregulation of neurotrophic factors [ 146 , 147 ] , which may stimulate neuritic remodeling and synaptogenesis . cortical neurons that sprout a new connection after stroke activate a neuronal growth program that consists of transcription factors , cell adhesion , axonal guidance , and cytoskeletal modifying molecules . it is known that ee modulates the expression of several genes in the infarcted cortex . namely , postischemic ee or social interaction modulate the expression of substances associated with neuronal plasticity , such as nerve growth factor - induced gene a ( ngfi - a ) and ngfi - b . ngfi - a ( also known as egr1 , krox24 , zif/268 , and tis8 ) , a transcription factor belonging to the early growth response family , is associated with stabilisation of ltp and learning [ 151 , 152 ] . ngfi - a target genes are synapsin - i and -ii , which are involved in synaptic vesicle trafficking and release as well as synaptogenesis [ 153155 ] . synapsin - i and ii are increased in the ipsilateral cortex of stroke rats following skilled training . in addition , ngfi - a is a master switch for the initiation of inflammatory gene expression under ischemic stress . ngfi - b ( also known as nur77 , n10 , tis1 , or tr3 ) , a member of the steroid / thyroid receptor family without any known ligand , has also been associated with ltp . at one month following mca occlusion the mrna expression of ngfi - a and ngfi - b is increased after ee in the cerebral cortex and the hippocampus . however , other reports show a decreased expression of ngfi - a in both cortices of ee rats [ 161163 ] , likely reflecting the suppression of postischemic inflammation in the brain . differences in the intensity and the duration of exercise administered to the rats may account for the different results obtained . postlesional plasticity in the adult brain is not restricted to structural modifications at the level of axons , dendrites , and synapses but also comprises the generation , differentiation , and maturation of new neurons in circumscribed brain regions ( reviewed by ) . numerous studies utilizing different experimental models have shown that an ischemic cns lesion leads to a substantial increase in proliferation of neural stem cells and subsequently increased generation of new neurons in the subgranular zone of the dentate gyrus and in the subventricular zone ( svz ) ( see for review ) . dentate neurogenesis is stimulated by focal ischemic infarcts even when the site of the injury is located in remote cortical brain areas [ 165 , 166 ] . newborn neurons in the svz are recruited to infarcted areas and may start to express region - specific mature neuronal markers [ 167172 ] . however , newborn cells expressing mature and region - appropriate neuronal markers have only been observed in the ischemic striatum but not in the cerebral cortex , with low fractions of newly generated cells surviving into maturity [ 167 , 168 , 173 ] . possible reasons for the reduced incidence of neuronal replacement in the ischemic striatum and cortex could be low cell survival or hampered neuronal phenotypic maturation due to detrimental factors in the perilesional environment , lack of neurotrophic support and of necessary developmental cues . notably , ablation of doublecortin - positive neuronal precursors from the rostral svz and dentate gyrus abolishes neurogenesis and associated neuronal migration induced by focal cerebral ischemia . this results in increased infarct size and worsened neurologic deficits , indicating that neurogenesis contributes to neuroprotection and short - term functional outcome after experimental stroke in mice . those beneficial effects may depend on the release of chemical mediators ( e.g. , growth factors ) by immature neurons . studies on the effects of ee and exercise on the adult germinal niches in intact animals have shown that both these paradigms lead to increased neurogenesis in the hippocampus and the svz . for example , postischemic ee enhances cell proliferation in the svz , with stronger effects in the chronic poststroke phase [ 171 , 176 ] , while wheel - running exercise after neocortical infarction attenuates the early poststroke activation of the svz germinal niche . interestingly , no effect of ee or exercise on hippocampal progenitor cell proliferation is reported after transient global ischemia in rats , suggesting that common pathways of regulation by lesion and environmental interventions may exist [ 178180 ] . in contrast , specific rehabilitative training of the impaired forelimb ( skilled reaching training ) is able to increase dentate neurogenesis relative to nontrained stroke rats , although at lower levels when compared with sham - operated animals . moreover , increased levels of newborn granule cells generated in the dentate gyrus correlate with better functional outcomes [ 181 , 182 ] . interestingly , postischemic ee combined with spatial learning ( which simulates occupational therapy in human rehabilitation and activate hippocampus and prefrontal cortex ) restores the perturbed dentate gyrus neuroblast production resulting from focal ischemic insult and increases neuroprotection in the ischemic penumbra . the lack or inadequacy of endogenous neuronal replacement after brain lesions encouraged investigations on the role of glial cells in poststroke recovery process . increasing evidence indicate that glial cells crucially contribute to the degenerative and regenerative processes following ischemic brain lesions [ 184 , 185 ] . also , some of the beneficial effects of ee on the postischemic brain might be mediated by a dynamic modulation of different glial populations . it is well known that astrocytes are essential for optimal neuronal function and take an active part in synaptic generation and plasticity as well as in maintenance of neuronal and synaptic homeostasis [ 186188 ] . recently , it has been revealed that astroglia may represent neural stem cells in the adult brain and may also direct neuronal differentiation of adult neural stem cells [ 189191 ] . they immediately proliferate in response to the lesion , increase their expression of gfap , and contribute to the formation of the glial scar [ 192 , 193 ] . reactive astroglia might provide a protective environment in the perilesional zone by shielding neurons from oxidative stress [ 194 , 195 ] or producing antiapoptotic and trophic factors . accordingly , they might promote neuronal survival , synaptic remodelling , and neurite outgrowth [ 184 , 196199 ] . postischemic ee or daily training of the impaired forelimb enhances astrogliosis in the perilesional area [ 171 , 176 , 193 ] . reactive astroglia , although representing an impediment for axon growth , may fulfill important protective and reparative functions after ischemic injuries and rehabilitation [ 199203 ] . immediately after the ischemic insult , resting microglia change their morphology from a ramified to an activated hyperramified phenotype and express the cd68 antigen . the activated microglia migrate towards the lesion , remove the necrotic tissue by phagocytosis , and thereby become macrophages [ 205 , 206 ] . some macrophages derive from monocytes that cross the blood - brain barrier after the ischemic lesion [ 207 , 208 ] . besides the degradation of necrotic cells , activated microglia and macrophages release growth factors and scavenge - free radicals [ 209 , 210 ] . however , activated microglia could also harm the injured brain with the synthesis of potentially toxic substances like nitric oxide and reactive oxygen radicals or the release of glutamate and proinflammatory cytokines [ 209 , 211216 ] . indeed , recent studies show that suppression of activated microglia and macrophages significantly improve functional recovery after focal ischemic infarcts [ 217 , 218 ] . in stroke animals exposed to ee or training a reduction of proliferating microglia and macrophages finally , proliferation and survival of immature and mature oligodendrocytes are only slightly influenced by ee . it has been shown that ee increases the number of ng2-positive glia , in intact ipsi - and contralateral cortical regions remote from the infarct . ng2-positive cells possess some characteristics of multipotent progenitor cells , may support neuronal function , and can turn into myelin - forming oligodendrocytes [ 220223 ] . however , the role of this cell population in the injured brain is still obscure . novel noninvasive interventions for stroke patients , such as mental practice , mirror therapy , virtual reality , robotics , and brain stimulation techniques , are emerging as potentially efficient strategies to promote functional recovery , but in most cases only when provided in combination with physical rehabilitation [ 43 , 224 ] . the expansion of rehabilitative programs with a wide range of possible interventions is more likely the key to obtain optimal results , by stimulating different reparative and adaptive brain processes . particularly , the use of noninvasive techniques of brain stimulation to promote adaptive plasticity , such as tdcs and rtms , is very appealing , and the results obtained in preclinical and clinical models of stroke are exciting . in this context , however , randomized controlled trials are needed to validate the efficacy of these techniques . this knowledge may allow the identification of biological markers suitable to monitor plastic processes in human patients undergoing specific rehabilitative programs , predict the outcome of the treatments , and optimise existing procedures . in conclusion , in the last few years there has been an enormous progress in the field of rehabilitative trials after stroke , for example , in terms of standardized interventions and tools for assessment of function and patient selection ( e.g. , recruitment of homogeneous groups of patients ) . crucial issues , however , remain to be addressed in future studies , including the sample wideness , repeatability of the results , and effective outcome measurements .

stroke is a common and disabling global health - care problem , which is the third most common cause of death and one of the main causes of acquired adult disability in many countries . rehabilitation interventions are a major component of patient care . in the last few years , brain stimulation , mirror therapy , action observation , or mental practice with motor imagery has emerged as interesting options as add - on interventions to standard physical therapies . the neural bases for poststroke recovery rely on the concept of plasticity , namely , the ability of central nervous system cells to modify their structure and function in response to external stimuli . in this review , we will discuss recent noninvasive strategies employed to enhance functional recovery in stroke patients and we will provide an overview of neural plastic events associated with rehabilitation in preclinical models of stroke .

1. Introduction 2. Novel Noninvasive Strategies for Patients Rehabilitation 3. Noninvasive Therapies in Animal Models of Stroke 4. Is There a Critical Period for Successful Rehabilitation? 5. Cellular and Molecular Correlates of Rehabilitation-Induced Plasticity 6. Conclusions

preoperative complete blood count showed hemoglobin at 8.9 g / dl and hematocrit at 26.5% . the electrocardiography , chest x - ray , and pulmonary function test showed no abnormal findings . after arriving at the or , the patient monitoring system ( polymount philips , usa ) was used to monitor the blood pressure , heart rate , pulse rate , and oxygen saturation by pulse oximeter . she appeared stable , with a heart rate of 85 bpm , blood pressure 134/80 mmhg , and respiratory rate 14 bpm , but oxygen saturation by pulse oximeter was 85% on both fingers but normal on the anesthesiologist 's finger . oxygen 6 l / min was administered by mask , and no abnormal breathing sounds were detected . in arterial blood gas analysis , however , the conjunctiva were pale ; her lips and fingernails were cyanosed ; and the oxygen saturation by pulse oximeter did not rise above 90% . the patient 's guardian indicated that she had a 10-year history of grayish lips and fingernails . although we considered delaying the surgery , the guardian wanted the surgery to proceed and the patient had the advanced gastric cancer accompanied by hemorrhage and perforation . before the anesthesia , a 20 g catheter was placed in the radial artery for continuous hemodynamic monitoring and arterial blood gas analysis . after preoxygenation with oxygen at 6 l / min , lidocaine 20 mg , propofol 120 mg , and vecuronium 10 mg were administered , and manual ventilation was performed with oxygen at 6 l / min . however , the patient experienced severe cyanosis , and the oxygen saturation by pulse oximeter decreased to 79% . after the tracheal intubation and controlled ventilation with oxygen at 3 l / min , the pulse oximeter increased to 83 - 85% . in arterial blood gas analysis after induction , the sampled blood was dark blackish brown , oxygen partial pressure was 224.7 mmhg , and oxygen saturation was 96.6% ( table 1 ) . the difference between oxygen saturation by arterial blood gas analysis and oxygen saturation by pulse oximeter was presumably due to dyshemoglobin . for confirmation , arterial blood gas analysis and hemoglobin analysis using a co - oximeter ( abl520 , radiometer , cofenhagen , denmark ) was ordered for the department of laboratory medicine . arterial blood gas analysis showed normal findings and hemoglobin analysis showed methemoglobin at 18.3% and oxyhemoglobin at 81.5% . ascorbic acid 600 mg mixed in fluid was administered , and methylene blue 1 mg / kg was intravenously injected slowly over 3 minutes . the methemoglobin level reduced to 11.2% after the administration of ascorbic acid and reduced to 4.3% and then 2.3% after methylene administration . anesthesia was sustained with oxygen at 3 l / min and enflurane 1.5 - 2.0 vol% . after the surgery , the patient had a normal recovery of consciousness and spontaneous respiration , but was transferred to the intensive care unit ( icu ) . in the icu , one day after the surgery , we found that the patient had taken daspone 100 mg / day for 30 years for hansen 's disease without her family ever knowing . after the surgery , methemoglobin in the blood went from 8.5% to 11.0% , but was untreated . from postoperative day 3 , it was treated under the diagnosis of adrenal apoplexy due to hypotension . methemoglobin forms when hemoglobin 's fe ion oxides from ferrous ( fe ) to ferric ( fe ) . in healthy adults , a small amount of hemoglobin oxides to methemoglobin and then it is rapidly converted back to hemoglobin such that methemoglobin levels remain below 1% . this balance is maintained by the intracellular enzyme systems , nadh cytochrome - b5 methemoglobin reductase and nadph methemoglobin reductase . methemoglobin can not reversibly bind with oxygen or carbon monoxide because of its additional cation , so it reduces the oxygen - carrying capacity in arterial blood . it also causes a leftward shift of the oxygen dissociation curve , increasing the affinity of the hemoglobin for oxygen and reducing tissue oxygen supply . symptoms of methemoglobinemia are primarily related to the level of methemoglobin but are also influenced by other factors , such as the total hemoglobin level and the cardiovascular status and the respiratory function . usually , at methemoglobin levels below 15% , patients have no clinical symptoms if adequate hemoglobin levels and preserved cardiac output is sufficient . however , when methemoglobin levels are 15 - 20% , blackbrown blood and cyanosis occur . higher levels ( 20 - 45% ) cause headaches , somnolence , tachycardia , lethargy , and dizziness . when it is 45% or above , respiratory difficulty , acidemia , arrhythmia , heart failure , and seizures occur . when above 70% , there is a high risk of death . methemoglobinemia can be caused congenitally , by certain foods , or by other unknown causes , but usually occurs when the patient is exposed to drugs or chemical substances that disturb the conversion of methemoglobin to hemoglobin and quicken the development of methemoglobin . when discovered in the perioperative period , it is usually due to the exposure of drugs or chemical substances such as : amyl nitrite , ethyl nitrite , sodium nitrite , silver nitrate , bismuth subnitrate , nitroglycerin , quinones , sulfonamide , sulfapyridine , sulfathiazole , aniline dye , acetanilid , aminobenzenes , aminophenol , benzocaine , prilocaine , and phenacetin . the drugs used in anesthetic practice that cause methemoglobinemia are prilocaine , benzocaine , amyl nitrite , nitroglycerine , phenacetin , and sulfonamide . dapsone is a sulfone antibiotic used for the treatment of hansen 's disease and other dermatologic diseases , as well as in immunosuppressed patients to prevent pneumocystitis carinii pneumonia or organ transplant patients , especially those intolerant to sulfonamide . methemoglobinemia is a known side - effect of dapsone [ 4 - 6 ] and the patients of most cases had a history of dapsone - intake . the patient here had a 30-year history of dapsone - intake , although this information was not obtained initially . the plasma half - life varies from 10 to 80 hours and is dose - dependent . the pulse oximeter uses 2 wavelengths , 660 nm and 990 nm , to calculate oxygen saturation because the absorbance difference between oxyhemoglobin and deoxyhemoglobin is maximized . methemoglobin has the same absorbability as oxyhemoglobin and deoxygemoglobin so can affect oxygen saturation measurements by pulse oximeter . low methemoglobin levels slight decrease oxygen saturation measurements to a limit of 85% , even with fatal methemoglobin levels . arterial blood gas analysis calculates oxygen saturation based on arterial oxygen tension and the temperature and may not reflect actual oxygen saturation . methemoglobinemia diagnosis includes cyanosis ; low arterial oxygen saturation that can not be explained by a problem in the respiratory system or cardiovascular system ; and when the normal oxygen partial pressure in the arterial blood gas analysis does not align with the oxygen saturation by the pulse oximeter . oxygen carrying status is most accurately measured by analysis of all hemoglobin types with a co - oximeter . congenital , chronic methemoglobinemia involves a family history or exposure to drugs and toxins , which could be captured in an accurate history . here , before anesthesia the patient showed cyanosis and low oxygen saturation on the pulse oximeter , which did not align with the arterial blood gas analysis . however , the diagnosis of dyshemoglobins was delayed because of the patient refusal to share her medical history . methemoglobin levels were 18.3% initially , and the patient had asymptomatic cyanosis and was hemodynamically stable , but she was given immediate treatment . despite adequate manual ventilation with oxygen at 6 l / min during anesthetic induction , the patient developed worsened cyanosis and showed decreased oxygen saturation by pulse oximeter to 79% . the eldely patient had diabetes , hypertension , and anemia , potentially indicating ischemic heart disease or impairment in the oxygen - carrying capacity . ascorbic acid and methylene blue treatment reduced methemoglobin levels to 11.2% , 4.3% , followed by maintenance of hemoglobin level with 2 u of packed red cells . cyanosis and 85% oxygen saturation on pulse oximeter before anesthesia could indicate high methemoglobin levels . despite adequate preoxygenation and manual ventilation with oxygen at 6 l / min anesthesia reduced oxygen saturation by pulse oximeter to 79% and the cyanosis worsened . the anesthetics propofol , vecuronium , and enflurane are not oxidants that develop methemoglobin , although local anesthetics like prilocaine and benzocaine do , and it is uncertain that lidocaine is a oxidant . after tracheal intubation and manual ventilation with oxygen at 6 l / min , the pulse oximeter went up to 84 - 85% and cyanosis decreased . therefore , reduced oxygen saturation during anesthetic induction resulted from normal respiratory depression and not anesthetic drugs . in healthy patients , the slight respiratory depression and reduced arterial oxygen partial pressure caused respiratory depressants may be ignored , but in methemoglobinemia , they may cause seriously low oxygen saturation . methemoglobinemia treatment is immediate intravenous administration of methylene blue ( 1 - 2 mg / kg ) and the removal of toxins or drugs that could cause it . methylene blue acts as a cofactor of methemoglobin reductase and accelerates the enzymatic breakdown of methemoglobin . higher levels ( 4 - 15 mg / kg / day ) can directly oxidize hemoglobin to methemoglobin and cause hemolytic anemia in glucose-6-phosphate dehydrogenase deficient patients . methemoglobin levels above 30% should be treated , but methemoglobin levels below 15% may seem clinically insignificant . however even when the methemoglobin level is low , treatment is recommended for patients with symptoms or underlying diseases with decreased oxygen - carrying capacity such cardiac disease , pulmonary disease , carbon monoxide intoxication , anemia , or acidosis . life - threatening methemoglobinemia unresponsive to methylene blue may occasionally respond to ascorbic acid as well , although ascorbic acid works slowly . other reported treatments are homogeneous transfusion , exchange transfusion , and hyperbaric oxygen treatment . in conclusion the anesthesiologist should perform pulse oximeter and arterial blood gas analysis on patients with cyanosis . when the two results do not align , or when the oxygen saturation by pulse oximeter does not align with the clinical manifestations , methemoglobinemia should be suspected . the presence of dyshemoglobin should be confirmed by performing a hemoglobin analysis with a co - oximeter . methemoglobinemia should be treated with methylene blue when necessary according to methemoglobin level , symptom , and underlying diseases .

methemoglobinemia is an uncommon but potentially fatal disorder . most cases have no adverse clinical consequence and require no treatment , but methemoglobinemia is often overlooked as a cause of low oxygen saturation , and often mistaken for the more common causes of hypoxia by anesthesiologists despite simple bedside tests that indicate the presence of this treatable abnormality . we present a 68-year - old female patient who underwent gastrectomy for advanced gastric cancer with bleeding . in the preoperative period , the patient showed cyanosis and oxygen saturation was 85% by pulse oximeter , but oxygen saturation by arterial blood gas analysis was 100% . after tracheal intubation , the methemoglobin level was 18.3% . ascorbic acid and methylene blue were administered . during preanesthetic evaluation , the patient had not informed the anesthesiologist that she had been taking dapsone .

Case Report Discussion

leprosy , also known as hansen s disease , is a worldwide health problem , varying from tuberculoid to lepromatous leprosy ( ie , paucibacillary to multibacillary disease ) according to the host immune response . it is caused by mycobacterium leprae , and affects the skin , eyes , and nerves , leading to skin lesions , eye pain , loss of vision , weakness , and numbness.1 the final diagnosis is based on a combination of findings on skin biopsy , smear , and physical examination . the treatment options differ according to the clinical manifestations.1 leprosy can be associated with type 1 ( reversal ) and type 2 ( erythema nodosum leprosum ) immunologic reactions that may occur at any time before , during , or after the start of treatment.1 during this process , this disease can worsen dramatically , and antibacterial therapy should continue during these periods.1,2 since 1947 , dapsone ( 4,4-diaminodiphenyl sulfone ) has been known to be a useful drug in the treatment of leprosy.2 its mechanism of action is via competitive inhibition of m. leprae dihydropteroate synthase , thus inhibiting the synthesis of folic acid.3,4 dapsone ( figure 1 ) is classified as a class ii drug according to the biopharmaceutics classification system , and has high permeability and low solubility in water ( log p = 0.97).5 thus , despite its therapeutic potential , the low solubility of dapsone in water results in low bioavailability and microbial resistance.5 a viable alternative for dose reduction and therefore limitation of side effects , never described before in the literature for dapsone , is the use of an intestinal permeation enhancer system capable of maintaining the drug in a soluble state after administration . nanoemulsions are homogenous systems with high solubilization capacity , thermodynamic stability over a wide range of ph and ionic environments , low viscosity , and can be formed spontaneously by simple mixture of various components.6,7 a nanoemulsion formulation of dapsone could improve its bioavailability and stability , and reduce the dosage necessary and the side effects of this drug , thereby representing a novel alternative in the treatment of leprosy . the aim of this study was to develop and evaluate the potential for better bioavailability of dapsone when the drug is incorporated into a nanoemulsion system using in vitro and in silico approaches . dapsone from g amphray laboratories ( mumbai , india ) was used in the production of all formulations , and dapsone usp standard lot 02/572 a ( rockville , md ) was used in all quantitative analyses . the isopropyl myristate , propylene glycol , ethanol , polyoxyethylene sorbitan monooleate - tween 80 , 40 and 20 , and sorbitan monooleate - span 80 and 20 ( sigma chemical company , st louis , mo ) used in preparation of the formulation were all of pharmaceutical grade . methanol and hydrochloric acid 37% were of chromatography grade ( tedia company inc , fairfield , oh ) . potassium phosphate monobasic , sodium phosphate dihydrate , and sodium hydroxide ( vetec chemicals , rio de janeiro , brazil ) , and fluorescein ( lgc biotechnology , sao paolo , brazil ) of analytical grade were also used . glucose , sodium bicarbonate , fetal bovine serum , nonessential amino acids , penicillin , streptomycin , and l - glutamine were purchased from sigma chemical company . water was obtained from a milli - q gradient a-10 water purification system ( millipore , bedford , ma ) . oral nanoemulsion systems containing dapsone were prepared using isopropyl myristate as oil phase and propylene glycol and ethanol as cosurfactant , as well as the surfactants span 80 or 20 , combined with tween 80 , 40 or 20 , and , the formulation was prepared according to the method reported by nandi et al.8 a pseudoternary phase diagram was constructed to determine the exact proportion of each component needed in the formulation to obtain the best parameters for the ideal nanoemulsion.9 a tween 80 and span 80 surfactant mixture was used at a fixed ratio of 1:1 in the first vertex . the other vertex represented the proportion of oil and cosolvent in a 8:1 mass ratio of isopropyl myristate and propylene glycol , respectively , while the third vertex represented water added in 10 to 10 l using an automatic micropipette in the titration process.8 this process was carried out to evaluate the maximum amount of water that could be incorporated into the system containing the mixture of surfactants , oil , and cosolvent . a pseudoternary phase diagram was then developed using the following proportions of oil phase + cosolvent and surfactants : 90:10 , 80:20 , 70:30 , 60:40 , 50:50 , 40:60 , 30:70 , 20:80 , and 10:90 . water was titrated in each of these proportions and , finally , the proportions were recalculated . we then evaluated the solubility of dapsone in the finished formulation and in the oil phase of the nanoemulsion . dapsone concentrations of 1.5 , 2.0 , 2.5 , 3.0 , 3.5 , and 4.0% w / w were tested . the dapsone solubility studies were performed with vigorous stirring for 24 hours followed by filtration through a 0.45 m filter . determination of the total amount of solubilized dapsone was done by ultraviolet spectroscopy at 295 nm . the oral dapsone nanoemulsion was characterized by droplet size distribution , refractive index , conductivity , and drug content . determination of the droplet size was performed by dynamic light scattering using a horiba lb-550 dls analyzer ( kyoto , japan ) , with a detection angle of 90 , 100 scans over two minutes for each sample , a refractive index adjusted to 1.330 , and a temperature of 22c.10 the refractive index was determined using an abbe refractometer ( model ar-001 , afab enterprises , eustis , fl ) , calibrated with distilled water at 25c . this parameter is important for assessment of the stability of nanoemulsions , because it is related to the optical clarity of these systems and helps to determine the type of nanoemulsion as w / o or o / w.11 the conductivity of the oral dapsone nanoemulsion was measured using a fe30 fiveeasy mettler toledo conductivity meter ( bedfordshire , uk ) calibrated with a nacl solution at 5.0 mg / l . a stress stability study of the oral dapsone nanoemulsion was conducted in a climatic chamber ( nova tica 420/ cldts 300 model , so paulo , brazil ) at 40c 2c and 75% 5% relative humidity over 3 months.12 samples were collected on days 15 , 30 , 60 , and 90 . at each interval , the formulation was characterized for droplet size distribution , refractive index , drug content , and conductivity . a sample of each formulation was stored under shelf conditions , ie , 25c 2c and 75% 5% humidity , for comparison with samples stored under stress temperature and humidity conditions . quantification of the amount of dapsone in the formulations was carried out using high - performance liquid chromatography ( hplc ) coupled with a photodiode array detector ( elite labchrom model , merck - hitachi , darmstadt , germany ) , according to a method described elsewhere,13 with some modifications . the dapsone assay was carried out on a kromasil c18 column ( 150 mm 4.6 mm , 5 m ) . the mobile phase consisted of a mixture of methanol and 30 mm phosphate buffer ph 5.9 ( 30:70 , v / v ) , and was isocratically delivered at 1.0 ml per minute . quantification of dapsone in the samples was based on the ultraviolet absorption at 295 nm using a six - level standard curve prepared from the stock solution in simulated gastric fluid and simulated intestinal fluid , with diluents at concentrations of 2.5 , 5 , 10 , 15 , 20 , and 25 g / ml for both conditions . stock dapsone solutions were prepared by transferring 25 mg of dapsone usp standard to a 100 ml volumetric flask . the required volume was completed using methanol , reaching a final concentration of 250 g / ml . dissolution tests were carried out with the nanoemulsion in capsules in order to standardize the dose and facilitate the assay . the nanoemulsion was placed in hard gelatin capsules ( number 00 ) , corresponding to a total volume of 0.95 ml , thereby simulating a commercial formulation . the maximum quantity of dapsone nanoemulsion incorporated in this capsule was 800 mg , equivalent to 16 mg of the drug considering the formulation containing 2.0% ( w / w ) . the capsules were weighed on an analytical balance before and after being filled with the nanoemulsion . the dissolution tests were performed in usp apparatus i using 900 ml of simulated gastric fluid and simulated intestinal fluid at 100 rpm.14 during the study , the temperature was maintained at 37c 0.5c . was added to each vessel and aliquots of 5 ml were collected at intervals of 15 , 30 , 45 , 60 , and 120 minutes , with replacement of the medium on each occasion . in order to evaluate the effect of the surfactant on the solubility and release of the active pharmaceutical ingredient , dapsone was encapsulated at the same concentration as in formulation ii with addition of tween 80 and span 80 to the dissolution medium . the dissolution test was performed following the same conditions described for the nanoemulsion , and aliquots were collected at intervals of 15 and 120 minutes . the data obtained in the dissolution test were fitted to four different equations to describe the drug release kinetics , ie , zero order , first order , higuchi , and hixon - crowell.14,15 the mathematical model that best expressed the kinetic release profile was selected based on the highest coefficient of determination ( r ) , obtained from linear regression analysis using the statistica 7 software package ( statsoft company , tulsa , ok).15 a caco-2 human intestinal cell line ( htb-37 ; american type culture collection , manassas , va ) was obtained from the cell bank at the universidade federal do rio de janeiro . the cells were routinely grown in 75 cm culture flasks with complete dulbecco s modified eagle s medium ( containing 4.5 g / l glucose , 3.7 g / l nahco3 , supplemented with 10% fetal bovine serum , 1% nonessential amino acids , penicillin 100 u / ml , streptomycin 100 g / ml , and glutamine 4 mm ) . cultures were maintained at 37c in a humidified atmosphere of 5% co2 . , the cells were washed with phosphate - buffered saline , without ca and mg , and detached from the flasks by trypsin buffer ( 0.25% in phosphate - buffered saline containing 0.2% ethylenediamine tetra - acetic acid ) . the cells were then resuspended in complete dulbecco s modified eagle s medium , and 5 10 cells were seeded on culture inserts fitted into 24-well culture plates ( transwell , costar , cambridge , ma ) . after 1418 days in culture , the monolayers were used for the permeability assays.16 the integrity of the monolayers was determined using an fei morgagni microscope operating at 80 kv ( hillsboro , or ) , as well as by measuring transepithelial electrical resistance and fluorescein passage . transepithelial electrical resistance values , expressed in . cm , were obtained using millicell ers-2 apparatus ( millipore ) . during the growth and differentiation period , the cell monolayer was considered to be fully formed when stable values were 300 cm . during the permeability experiments , transepithelial electrical resistance was measured at different time points , including at the start and end of the assays . fluorescein permeation was also used to assess the integrity of the monolayer . at the end of the permeability experiments , 200 l of fluorescein 1 mg / ml in phosphate - buffered saline was added to each apical chamber and 400 l of phosphate - buffered saline was added to each basolateral chamber.1719 after one hour of incubation at 37c , the fluorescein content in the basolateral medium was determined by hplc , as previously described . all transport studies were performed at 37c in an atmosphere of 5% co2 in phosphate - buffered saline ( ph 7.2 ) as the transport medium . before the permeability assay , the cell monolayers were washed using the transport medium and preincubated for 30 minutes . dapsone nanoemulsion or phosphate - buffered saline solution ( 200 l , donor solution ) at final concentrations of 14 transport was monitored for a period of 240 minutes , after which time 100150 l samples were removed from the basolateral chamber for hplc analysis and replaced with an equal volume of phosphate - buffered saline.17,20 to calculate the permeability of the dapsone nanoemulsion in caco-2 cells , the apparent permeability coefficient ( papp ) was obtained from the following equation : papp = ( v / a c ) ( dc / dt ) , where dc / dt is the permeate rate ( the amount of drug transported per unit time ) , v is the volume in the receptor chamber , a is the surface area of the filter , and c is the initial drug concentration in the donor chamber . the gastroplus version 7.0 ( simulations plus inc , lancaster , ca ) was used to simulate the effective permeability ( peff ) of dapsone and output pharmacokinetic parameters in the human body . the program has three tabs used for data input ; the first is a compound tab , followed by a physiology tab , and finally a pharmacokinetic tab . the compound tab inputs the basic physicochemical data for dapsone , such as solubility , pka , and dose , as derived from the literature . the input parameter required for peff simulation was experimentally determined based on permeability studies using the caco-2 cell line.21 in the physiology tab , the default values for transit time were selected for each compartment , with the fasted option checked . pharmacokinetic parameters , such as clearance and distribution volume , were used in the pharmacokinetic tab for simulation by gastroplus . these values were taken from the literature.22,23 statistical analysis was performed with the one - way analysis of variance test ( tukey s multiple comparisons ) using graph- pad prism 5.0 software ( la jolla , ca ) . dapsone from g amphray laboratories ( mumbai , india ) was used in the production of all formulations , and dapsone usp standard lot 02/572 a ( rockville , md ) was used in all quantitative analyses . the isopropyl myristate , propylene glycol , ethanol , polyoxyethylene sorbitan monooleate - tween 80 , 40 and 20 , and sorbitan monooleate - span 80 and 20 ( sigma chemical company , st louis , mo ) used in preparation of the formulation were all of pharmaceutical grade . methanol and hydrochloric acid 37% were of chromatography grade ( tedia company inc , fairfield , oh ) . potassium phosphate monobasic , sodium phosphate dihydrate , and sodium hydroxide ( vetec chemicals , rio de janeiro , brazil ) , and fluorescein ( lgc biotechnology , sao paolo , brazil ) of analytical grade were also used . glucose , sodium bicarbonate , fetal bovine serum , nonessential amino acids , penicillin , streptomycin , and l - glutamine were purchased from sigma chemical company . water was obtained from a milli - q gradient a-10 water purification system ( millipore , bedford , ma ) . oral nanoemulsion systems containing dapsone were prepared using isopropyl myristate as oil phase and propylene glycol and ethanol as cosurfactant , as well as the surfactants span 80 or 20 , combined with tween 80 , 40 or 20 , and , the formulation was prepared according to the method reported by nandi et al.8 a pseudoternary phase diagram was constructed to determine the exact proportion of each component needed in the formulation to obtain the best parameters for the ideal nanoemulsion.9 a tween 80 and span 80 surfactant mixture was used at a fixed ratio of 1:1 in the first vertex . the other vertex represented the proportion of oil and cosolvent in a 8:1 mass ratio of isopropyl myristate and propylene glycol , respectively , while the third vertex represented water added in 10 to 10 l using an automatic micropipette in the titration process.8 this process was carried out to evaluate the maximum amount of water that could be incorporated into the system containing the mixture of surfactants , oil , and cosolvent . a pseudoternary phase diagram was then developed using the following proportions of oil phase + cosolvent and surfactants : 90:10 , 80:20 , 70:30 , 60:40 , 50:50 , 40:60 , 30:70 , 20:80 , and 10:90 . water was titrated in each of these proportions and , finally , the proportions were recalculated . we then evaluated the solubility of dapsone in the finished formulation and in the oil phase of the nanoemulsion . dapsone concentrations of 1.5 , 2.0 , 2.5 , 3.0 , 3.5 , and 4.0% w / w were tested . the dapsone solubility studies were performed with vigorous stirring for 24 hours followed by filtration through a 0.45 m filter . determination of the total amount of solubilized dapsone was done by ultraviolet spectroscopy at 295 nm . the oral dapsone nanoemulsion was characterized by droplet size distribution , refractive index , conductivity , and drug content . determination of the droplet size was performed by dynamic light scattering using a horiba lb-550 dls analyzer ( kyoto , japan ) , with a detection angle of 90 , 100 scans over two minutes for each sample , a refractive index adjusted to 1.330 , and a temperature of 22c.10 the refractive index was determined using an abbe refractometer ( model ar-001 , afab enterprises , eustis , fl ) , calibrated with distilled water at 25c . this parameter is important for assessment of the stability of nanoemulsions , because it is related to the optical clarity of these systems and helps to determine the type of nanoemulsion as w / o or o / w.11 the conductivity of the oral dapsone nanoemulsion was measured using a fe30 fiveeasy mettler toledo conductivity meter ( bedfordshire , uk ) calibrated with a nacl solution at 5.0 mg / l . a stress stability study of the oral dapsone nanoemulsion was conducted in a climatic chamber ( nova tica 420/ cldts 300 model , so paulo , brazil ) at 40c 2c and 75% 5% relative humidity over 3 months.12 samples were collected on days 15 , 30 , 60 , and 90 . at each interval , the formulation was characterized for droplet size distribution , refractive index , drug content , and conductivity . a sample of each formulation was stored under shelf conditions , ie , 25c 2c and 75% 5% humidity , for comparison with samples stored under stress temperature and humidity conditions . quantification of the amount of dapsone in the formulations was carried out using high - performance liquid chromatography ( hplc ) coupled with a photodiode array detector ( elite labchrom model , merck - hitachi , darmstadt , germany ) , according to a method described elsewhere,13 with some modifications . the dapsone assay was carried out on a kromasil c18 column ( 150 mm 4.6 mm , 5 m ) . the mobile phase consisted of a mixture of methanol and 30 mm phosphate buffer ph 5.9 ( 30:70 , v / v ) , and was isocratically delivered at 1.0 ml per minute . quantification of dapsone in the samples was based on the ultraviolet absorption at 295 nm using a six - level standard curve prepared from the stock solution in simulated gastric fluid and simulated intestinal fluid , with diluents at concentrations of 2.5 , 5 , 10 , 15 , 20 , and 25 g / ml for both conditions . stock dapsone solutions were prepared by transferring 25 mg of dapsone usp standard to a 100 ml volumetric flask . the required volume was completed using methanol , reaching a final concentration of 250 g / ml . dissolution tests were carried out with the nanoemulsion in capsules in order to standardize the dose and facilitate the assay . the nanoemulsion was placed in hard gelatin capsules ( number 00 ) , corresponding to a total volume of 0.95 ml , thereby simulating a commercial formulation . the maximum quantity of dapsone nanoemulsion incorporated in this capsule was 800 mg , equivalent to 16 mg of the drug considering the formulation containing 2.0% ( w / w ) . the capsules were weighed on an analytical balance before and after being filled with the nanoemulsion . the dissolution tests were performed in usp apparatus i using 900 ml of simulated gastric fluid and simulated intestinal fluid at 100 rpm.14 during the study , the temperature was maintained at 37c 0.5c . a capsule containing dapsone nanoemulsion was added to each vessel and aliquots of 5 ml were collected at intervals of 15 , 30 , 45 , 60 , and 120 minutes , with replacement of the medium on each occasion . in order to evaluate the effect of the surfactant on the solubility and release of the active pharmaceutical ingredient , dapsone was encapsulated at the same concentration as in formulation ii with addition of tween 80 and span 80 to the dissolution medium . the dissolution test was performed following the same conditions described for the nanoemulsion , and aliquots were collected at intervals of 15 and 120 minutes . the data obtained in the dissolution test were fitted to four different equations to describe the drug release kinetics , ie , zero order , first order , higuchi , and hixon - crowell.14,15 the mathematical model that best expressed the kinetic release profile was selected based on the highest coefficient of determination ( r ) , obtained from linear regression analysis using the statistica 7 software package ( statsoft company , tulsa , ok).15 a caco-2 human intestinal cell line ( htb-37 ; american type culture collection , manassas , va ) was obtained from the cell bank at the universidade federal do rio de janeiro . the cells were routinely grown in 75 cm culture flasks with complete dulbecco s modified eagle s medium ( containing 4.5 g / l glucose , 3.7 g / l nahco3 , supplemented with 10% fetal bovine serum , 1% nonessential amino acids , penicillin 100 u / ml , streptomycin 100 g / ml , and glutamine 4 mm ) . , the cells were washed with phosphate - buffered saline , without ca and mg , and detached from the flasks by trypsin buffer ( 0.25% in phosphate - buffered saline containing 0.2% ethylenediamine tetra - acetic acid ) . the cells were then resuspended in complete dulbecco s modified eagle s medium , and 5 10 cells were seeded on culture inserts fitted into 24-well culture plates ( transwell , costar , cambridge , ma ) . after 1418 days in culture , the monolayers were used for the permeability assays.16 the integrity of the monolayers was determined using an fei morgagni microscope operating at 80 kv ( hillsboro , or ) , as well as by measuring transepithelial electrical resistance and fluorescein passage . transepithelial electrical resistance values , expressed in . cm , were obtained using millicell ers-2 apparatus ( millipore ) . during the growth and differentiation period , the cell monolayer was considered to be fully formed when stable values were 300 cm . during the permeability experiments , transepithelial electrical resistance was measured at different time points , including at the start and end of the assays . fluorescein permeation was also used to assess the integrity of the monolayer . at the end of the permeability experiments , 200 l of fluorescein 1 mg / ml in phosphate - buffered saline was added to each apical chamber and 400 l of phosphate - buffered saline was added to each basolateral chamber.1719 after one hour of incubation at 37c , the fluorescein content in the basolateral medium was determined by hplc , as previously described . all transport studies were performed at 37c in an atmosphere of 5% co2 in phosphate - buffered saline ( ph 7.2 ) as the transport medium . before the permeability assay , the cell monolayers were washed using the transport medium and preincubated for 30 minutes . dapsone nanoemulsion or phosphate - buffered saline solution ( 200 l , donor solution ) at final concentrations of 14 g / ml in phosphate - buffered saline were added to the apical chamber . transport was monitored for a period of 240 minutes , after which time 100150 l samples were removed from the basolateral chamber for hplc analysis and replaced with an equal volume of phosphate - buffered saline.17,20 to calculate the permeability of the dapsone nanoemulsion in caco-2 cells , the apparent permeability coefficient ( papp ) was obtained from the following equation : papp = ( v / a c ) ( dc / dt ) , where dc / dt is the permeate rate ( the amount of drug transported per unit time ) , v is the volume in the receptor chamber , a is the surface area of the filter , and c is the initial drug concentration in the donor chamber . the gastroplus version 7.0 ( simulations plus inc , lancaster , ca ) was used to simulate the effective permeability ( peff ) of dapsone and output pharmacokinetic parameters in the human body . the program has three tabs used for data input ; the first is a compound tab , followed by a physiology tab , and finally a pharmacokinetic tab . the compound tab inputs the basic physicochemical data for dapsone , such as solubility , pka , and dose , as derived from the literature . the input parameter required for peff simulation was experimentally determined based on permeability studies using the caco-2 cell line.21 in the physiology tab , the default values for transit time were selected for each compartment , with the fasted option checked . pharmacokinetic parameters , such as clearance and distribution volume , were used in the pharmacokinetic tab for simulation by gastroplus . statistical analysis was performed with the one - way analysis of variance test ( tukey s multiple comparisons ) using graph- pad prism 5.0 software ( la jolla , ca ) . different combinations of surfactants and cosurfactants were evaluated to identify a stable w / o nanoemulsion . these nanoemulsions can improve the biodisponibility of dapsone and the in vivo stability of this drug by in vivo phase inversion to an o / w nanoemulsion of dapsone due to the aqueous environment of the gastrointestinal tract.24,25 it was not possible to obtain stable nanoemulsions when constructing the pseudoternary phase diagrams using combinations of tween 40/span 20 and tween 20/span 20 as surfactants , ethanol or propylene glycol as the cosurfactant , or isopropyl myristate as oil phase . however , it was possible to establish several stable combinations when span 80 and tween 80 were used with propylene glycol as the cosurfactant and isopropyl myristate as oil phase ( figure 2 ) . use of propylene glycol as cosurfactant has been shown to be promising in oral pharmaceutical formulations . yang et al used this excipient in the preparation of a paclitaxel nanoemulsion and sharma et al used it in oral formulations of insulin.26,27 thus , based on the pseudoternary phase diagram ( figure 2 ) , a stable formulation able to incorporate more water was selected for the characterization tests ( table 1).9 dapsone was efficiently incorporated in the nanoemulsion formulation selected ( table 1 ) in two ways , ie , dissolved in oil phase ( i and iii ) and in the finished formulation ( ii and iv ) at concentrations of 2.0% ( i and ii ) and 2.5% ( iii and iv ) w / w ( table 2 ) . table 3 characterizes the nanoemulsions at time zero and after 90 days of storage under stress conditions . the parameters evaluated were droplet size , polydispersity index , refractive index , conductivity , and dapsone content ( table 3 ) . the nanometer scale droplets in these formulations were found to have a low polydispersity index . conductivity was 0 s / cm for all the formulations studied at time zero and after storage , indicating that all the nanoemulsions produced were of the w / o type . the droplet size below 100 nm and surfactant concentration used in the nanoemulsions produced allowed prediction of in vivo phase inversion and formation of a stable o / w nanoemulsion.24 the results of the stress stability studies showed that the nanoscale dimensions were maintained with uniform emulsion droplets and a narrow size distribution in all the study formulations ( i iv ) during the 3 months of testing . formulations iii and iv showed an increase in droplet size , and formulation i showed a slight reduction . moreover , formulations i to iv in the stress stability studies were considered to be stable , and formulations i and ii did not show drug precipitation during the shelf - life studies . like dapsone , paclitaxel is a chemotherapeutic agent with high lipophilicity and low water solubility , and has a high potential for bioavailability problems.28 paclitaxel nanoemulsions are described in the scientific literature as being systems capable of increasing the solubility of the active drug.29,30 although an o / w type of paclitaxel nanoemulsion is more common , researchers have developed a paclitaxel nanoemulsion of the w / o type , which has been shown to have increased clinical efficacy.30 this is probably due to in vivo phase inversion , as expected for the i and ii dapsone nanoemulsions . this hypothesis reinforces the use of a nanoemulsion system to modulate the bioavailability of dapsone . formulations i and ii showed the best results in the stability studies . however , formulation ii was chosen for the permeability and kinetic release studies because of its capacity to incorporate a larger volume of water ( figure 2 ) , thereby avoiding precipitation of dapsone and in vivo formation of an o / w nanoemulsion.24 the drug release profile from formulation ii was studied in the different media . using simulated gastric fluid , more than 90% of drug release occurred within the first 15 minutes , with 91.5% of the drug similarly released in 15 minutes using simulated intestinal fluid . dapsone appeared to be maintained in a solubilized form in the dissolution media when the nanoemulsion was tested , and was confirmed by a mean droplet size of 84.5 12 nm , a mean maximum size of 98.0 26 nm measured with phase inversion , and by conductivity measurement ( 1.12 s / cm ) . in order to evaluate the enhancement of dissolution of dapsone in nanoemulsion by the surfactant effect , a control study based on a dissolution test of the dapsone contained in the capsules was performed . the total amount dissolved was only 76.0% in simulated gastric fluid medium and 78.8% in simulated intestinal fluid at 120 minutes . at 15 minutes , the total amount dissolved was 27.9% in simulated gastric fluid and 5.6% in simulated intestinal fluid . an r of 0.95441 for simulated gastric fluid and 0.96026 for simulated intestinal fluid was found for release of the active pharmaceutical ingredient from the dapsone capsules based on the dissolution data fitted to the higuchi model . this indicates slow diffusion of dapsone and low solubility of the powdered active pharmaceutical ingredient in the dissolution media used in our study.24 our results for permeation across human caco-2 cells showed that formulation ii had higher permeation than dapsone in phosphate - buffered solution with or without addition of surfactant ( figure 3 ) . these data indicate that the nanoemulsion was able to enhance the bioavailability of dapsone , and the apparent permeability coefficient ( papp ) of the nanoemulsion was increased compared with dapsone solution alone ( table 4 ) . thus , dapsone , a class ii drug , when incorporated in a nanoemulsion , behaves as a class i drug according to the biopharmaceutics classification system , due to its higher solubility and higher permeability , with a papp of 11.04 10 cm / sec.31,32 moreover , dapsone formulated as a nanoemulsion showed more mass permeated by area over time than dapsone dissolved in phosphate - buffered saline only , indicating that the presence of pharmaceutical adjuvants helped in the process of permeation . the small droplet size contained in the nanoemulsion enables better adherence to the membrane during transport of the drug and optimizes intestinal absorption and permeation.33 therefore , both the dose required and the ensuing adverse effects can be reduced , with improved absorption and permeability parameters . drugs incorporated into nanoemulsion systems show reproducible bioavailability and a constant plasma concentration profile , which is clinically important for drugs known to have severe adverse effects.33,34 all these characteristics and benefits contributed to a high coefficient of permeability for the dapsone nanoemulsion . the surfactants , span 80 and tween 80 confer stability to the nanoemulsion , decreasing the surface tension of the two immiscible liquids ( water and oil ) . moreover , these surfactants are classified as promoters of permeation because they form pores in the cell membrane ( enterocytes in this model ) , which increase drug permeation across the intestinal epithelium after oral administration.35 the dapsone solution containing the tween 80 and span 80 surfactants did not produce significantly different permeability results . the pharmacokinetic parameters simulated by gastroplus indicate that dapsone nanoemulsion increases the drug absorption rate ( time taken to reach peak concentration decreased from 3.0 to 2.3 hours ) , with a slight increase in peak dapsone concentration ( 1.12 m / ml for solution and 1.21 m / ml for nanoemulsion ) . the efficiency of dapsone nanoemulsion in promoting drug absorption can be attributed to this drug delivery system ( table 4 ) . the peak plasma dapsone concentration values reported by mirochnick et al36 using in vivo assays in health volunteers were 1.94 0.31 m / ml for a propylene glycol solution and 1.86 0.47 m / ml for a tablet formulation . both formulations contained 100 mg dapsone and the times taken to reach peak concentration ( 1.01 and 1.13 hours ) were not significantly different . these findings confirm that gastroplus is a useful tool for prediction of bioavailability in silico and the effect of nanoemulsion systems in increasing the dapsone absorption rate , as well as the robustness of the experimental procedure established in this work.36 the nanoemulsion contains droplets of a nanoscale size , and its refractive index and conductivity characterizes it as a w / o system with probable in vivo phase inversion . formulation ii enhanced the drug dissolution rate compared with a powdered dapsone dispersion , with kinetic release according to the higuchi model . therefore , the dapsone nanoemulsion system could modulate absorption , bioavailability , and stability parameters , and be an alternative to the class ii antibacterial agents structurally related to dapsone , with feasible reduction of adverse effects while delivering a pharmacologically active dose .

backgrounddapsone is described as being active against mycobacterium leprae , hence its role in the treatment of leprosy and related pathologies . despite its therapeutic potential , the low solubility of dapsone in water results in low bioavailability and high microbial resistance . nanoemulsions are pharmaceutical delivery systems derived from micellar solutions with a good capacity for improving absorption . the aim of this work was to develop and compare the permeability of a series of dapsone nanoemulsions in caco-2 cell culture against that of effective permeability in the human body simulated using gastroplus software.methods and resultsthe release profiles of the dapsone nanoemulsions using different combinations of surfactants and cosolvent showed a higher dissolution rate in simulated gastric and enteric fluid than did the dispersed dapsone powder . the drug release kinetics were consistent with a higuchi model.conclusionthis comparison of dapsone permeability in caco-2 cells with effective permeability in the human body simulated by gastroplus showed a good correlation and indicates potential improvement in the biodisponibility of dapsone using this new system .

Introduction Materials and methods Materials Nanoemulsion preparation Characterization of dapsone nanoemulsion High-performance liquid chromatography analysis Dissolution testing of dapsone capsules Evaluation of release kinetics Permeability testing conditions Permeability study of dapsone nanoemulsion In silico studies Statistical analysis Results and discussion Conclusion

melanogenesis is inherently cytotoxic and uniquely occurs in melanocytic cells ; thus , tyrosine analogs that are tyrosinase substrates are good candidates for melanoma - specific targeting and therapy . n - propionyl and n - acetyl derivatives of 4-s - cysteaminylphenol ( npr- and naccap ) were synthesized and found to possess both cytostatic and cytocidal effects on in vivo and in vitro melanomas through the oxidative stress resulting from production of cytotoxic free radicals [ 26 ] . we now provide evidence that the unique melanogenesis cascade can be exploited for developing a chemo - thermo - immunologic approach ( cti therapy ) targeted to melanoma by conjugating nprcap with magnetite nanoparticles ( nprcap / m ) . magnetite nanoparticles have been employed for thermotherapy in a number of cancer treatments including human prostate cancers [ 79 ] . they consist of 10100 nm sized iron oxide ( fe3o4 ) with a surrounding polymer coating and become magnetized when placed in an external alternating magnetic field ( amf ) . in hyperthermia treatment , the expression of heat shock proteins ( hsps ) plays an important role in immune reactions [ 1119 ] . accumulating evidence from our group [ 2023 ] and from others implicates hsp expression induced by hyperthermia in tumor immunity and opens the door to cancer therapy based on hyperthermia treatment ( thermo - immunotherapy ) . in such a strategy , a tumor - specific hyperthermia system that can induce necrotic cell death via hsp expression without damaging noncancerous tissues would be highly desirable . an intracellular hyperthermia system using tumor - targeted magnetite nanoparticles facilitates tumor - specific hyperthermia ; this can induce necrotic cell death via hsp expression , which in turn induces antitumor immunity . we synthesized , in our initial study , magnetite cationic liposomes ( mcls ) loaded with 4-s - cysteaminylphenol ( cap ) . there was , however , a risk of nonspecific electrostatic interaction between mcls and various nontarget cells . a promising technique is the use of tumor - targeted magnetite nanoparticles , and this approach is extended by synthesizing another type of magnetite nanoparticles , nprcap / m , on which nprcap is superficially and directly bound on the surface of magnetite nanoparticles . nprcap / m is chemically stable and can be produced in large quantities and employed to effect melanoma - targeted chemotherapy ( by nprcap ) and thermoimmunotherapy ( by magnetite with hsp ) . in this study , we evaluated their thermo - therapeutic effect on distant metastatic melanomas , using the mouse b16 melanoma system . specifically we assessed the in vivo growth inhibition of a subsequently transplanted melanoma growth ( re - challenge melanoma ) after treating the initial melanoma transplant . we also investigated the possible association of hsp production with growth inhibition of the re - challenge melanoma . the details of the preparation of nprcap / m are described elsewhere . briefly , magnetite nanoparticles ( fe3o4 ; average particle size , 10 nm ) were coated with aminosilane and conjugated with nprcap via maleimide cross - linkers . all of the animal experiments were conducted by an approval of animal experiment ethics committee of sapporo medical university . all the surgical , transplantation and drug administration procedures were carried out after anesthesia by diethyl ether . mouse b16f1 and b16f10 and b16 ova melanoma cells ( 3.0 10 ) in 0.1 ml of phosphate - buffered saline ( pbs ) were s.c . transplanted into the right flanks of 4-week - old female c57bl/6 mice ( weighing approximately 10.0 g and purchased from hokudo laboratory , sapporo , japan ) . b16ova is a b16f1 melanoma cell line stably transfected with chicken ovalbumin ( ova ) cdna and was kindly provided by dr . y. nishimura , kumamoto university , kumamoto , japan . on day 5 after transplant mice with primary melanoma transplantation the b16 melanoma - bearing mice were injected with 0.1 ml of nprcap / m ( 40.0 mg / ml solution ) directly into the tumor site in a single - dose administration ( approximately 0.5 l / mm tumor volume ) . treated tumors on the right flank in mice were excised on day 13 after the first s.c . transplantation . on day 40 after the surgical excision ( on day 53 after primary transplantation ) , mice were re - challenged with 1.0 10 b16 cells which were injected into the left flank . the total number of melanoma cells at the re - challenge experiments was 1/3 of melanoma cells as that of the primary transplants because there was no nprcap / m administration which might cause some tissue destruction . the control group , naive mice of the same age and sex , received transplantation of melanoma cells into the right flank as with the treated groups . on day 14 after the secondary transplantation ( day 67 after primary transplantation ) , tumor diameters were measured in millimeters with calipers , and tumor volumes were calculated by the formula : longaxis ( shortaxis ) 0.5 . the mice in the treated groups were judged to be tumor free ( rejection ) if the tumor was less than about 2 mm in diameter by palpation on day 60 after the secondary transplantation ( day 113 after the primary transplantation ) . an alternating magnetic field ( amf ) was generated using a horizontal coil ( inner diameter : 7 cm ; length : 7 cm ) with a transistor inverter ( ltg-100 - 05 ; dai - ich high - frequency co. , tokyo , japan ) . the magnetic field frequency and intensity were 118 khz and 30.6 ka / m ( 384 oe ) , respectively . surface ( peripheral ) and core ( central ) temperatures of the tumor were continuously monitored and measured using two optical fiber probes ( fx-9020 ; anritsu meter , tokyo , japan ) ; that is , one inserted into the tumor core and another fixed on the tumor surface . measurement time points were 0 , 1 , 2 , 3 , 4 , 5 , 10 , 15 , 20 , 25 , 30 , 31 , 32 , 33 , 34 , and 35 minutes for 30 minutes thermotherapy . the therapeutic temperatures at 41c , 43c or 46c were monitored by measuring the surface temperature and adjusting the transistor inverter during exposure to amf ( figures 1(a ) , 1(b ) , and 1(c ) ) . each experimental group of protocols number 1 through number 4 consisted of six to eleven mice . all the treatment protocols were again approved by the animal experimental ethics committee of sapporo medical university . the experimental conditions for melanoma transplantation and the methods of nprcap / m administration were identical in all four protocols and all the animal experiments including drug administration were carried out after anesthesia by diethyl ether . on day 5 after the s.c . transplantation of b16 melanoma cells , mice were divided into four groups . in groups i and administration of 0.1 ml of 40.0 mg / ml aminosilane - coated magnetite ( m ) once a day every other day for a total of three days ( days 6 , 8 , and 10 ) with amf ( group ii ) or without amf ( group i ) . in groups iii and iv mice received s.c . administration of 0.1 ml of nprcap / m ( 4.0 mg magnetite equivalent ) once a day every other day for a total of three days ( days 6 , 8 , and 10 ) with ( group iv ) or without ( group iii ) amf . the temperature at the tumor surface was maintained at 43c during exposure for 30 minutes by controlling amf intensity . transplantation of melanoma cells into the right flank , as with the treated groups . on day 13 after the primary transplantation all mice underwent total resection of melanoma nodules . on day 53 after the first transplantation ( postoperative day 40 ) , surviving mice in each group received a second transplantation of b16 melanoma cells into the opposite flank . tumor volumes were calculated at day 14 after the second transplantation of b16 melanoma cells . they were exposed to amf once on day 6 ( group i ) , twice ( on days 6 and 8) ( group ii ) , twice ( on days 6 and 10 ) ( group iii ) , three times ( on days 6 , 8 , and 10 ) ( group iv ) , three times ( on days 6 , 7 , and 8) ( group v ) , and five times ( on days 6 through 10 ) ( group vi ) . mice of groups i and ii were exposed to the amf to maintain the surface temperature at 41c once a day for two days ( days 6 and 10 ) and for three days ( days 6 , 8 , and 10 ) , respectively . using the same day schedule mice of groups iii and iv were exposed to the amf at 43c and mice of groups v and vi at 46c . temperatures at the surface of the tumors in groups i and ii were maintained at 43c and 46c , respectively , during amf exposure for 15 minutes . the surface temperatures in groups iii and iv were maintained at 43c and 46c , respectively , during therapy for 30 minutes . after thermotherapy , the amount of hsp70 in the primary tumor was measured using an hsp70 eia kit ( stress gen biotechnologies , british columbia , canada ) according to the manufacturer 's instructions . the total protein content of the tumor homogenates was determined using the bca protein assay kit ( pierce biothechnology , inc . the control group was composed of mice without nprcap / m administration or amf exposure . administration of nprcap / m directly at the tumor site once a day without amf exposure . mice of groups ii and iii received thermotherapy at 41c for 15 minutes and 30 minutes , groups iv and v at 43c for 15 minutes and 30 minutes , and groups vi and vii at 46c for 15 minutes and 30 minutes , respectively . then , 24 hours later , all tumors were removed , and their lysates were processed for the hsp70 assay . in separate groups , tumors were excised at 24 , 48 , and 72 hours after the 43c thermotherapy for 30 minutes , and amounts of hsp70 were measured . after thermotherapy in the primarily transplanted melanoma at 43c for 30 minutes once a day for three days ( figure 4(a ) , group iv ) , melanomas in re - challenge mice were excised on the 18th day after second transplantation of b16f1 cells . the frozen tumor tissues were stained with antimouse cd4 ( santa cruz biotechnology inc . , ca , usa ) or cd8 ( chemicon international inc . , data were analyzed by one- or two - way analysis of variance ( anova ) , and then differences in experimental results for tumor growth and expression of hsp were assessed by sheffe 's test to compare all the experimental groups , or by dunnett 's test , which compared the experimental versus the control groups . differences in survival rates were analyzed by the kaplan - meier method and log - rank test with bonferroni correction for multiple comparisons . in the search for successful cancer treatment it is self - evident that the exploitation of a specific biological property is one of the best approaches for developing the targeted therapy [ 27 , 28 ] . we have previously shown that the melanogenesis substrate , nprcap , is a good candidate for developing melanoma chemotherapy because melanogenesis is uniquely expressed in melanocytic cells and is inherently cytotoxic from the action of tyrosinase on tyrosine with formation of highly reactive free radicals [ 1 , 4 ] . nanoparticles may also provide a good platform to coadminister anticancer therapeutics directed at different targets . specifically hyperthermia with the use of magnetite nanoparticles has been shown to possess great potential to develop thermo - immunotherapy [ 23 , 29 ] . in this study the conjugate of nprcap and magnetite nanoparticles , nprcap / m was synthesized with the hope of developing a chemotherapeutic as well as a thermo - immunotherapeutic effect . we employed two cell lines of b16 melanoma , that is , b16f1 and b16f10 , and b16ova cells and compared the thermo - therapeutic protocols in detail by evaluating the growth of the re - challenge melanoma as well as the duration and rates of survival of melanoma - bearing mice . in the previous intratumor mcl hyperthermia for b16 melanoma the skin surface temperature above the subcutaneous tumor rose to 43 or 46c . to obtain a rapid and steady temperature increase at the core and the surface of the b16 melanoma , nprcap / m was injected into the center of the tumor nodules , and internal and surface tumor temperatures were measured during amf exposure . both temperatures increased within one minute to the target of 41c , 43c , or 46c ( figures 1(a ) , 1(b ) , and 1(c ) ) , indicating that nprcap / m injection followed by amf exposure could immediately and steadily heat the subcutaneously transplanted melanoma nodules . the temperature at the tumor center was approximately 2c higher than that at the tumor surface . nprcap / m without heat inhibited the growth of primary transplants to the same degree as did nprcap / m with heat , indicating that nprcap / m alone has a chemotherapeutic effect . the critical temperature for thermotherapy was documented to be 43c for various cell lines [ 7 , 8 ] . using two melanoma cell lines , b16f1 , and b16f10 and b16ova , we examined melanoma growth inhibition by intra - tumor administration of nprcap / m into primary tumors on days 6 , 8 , and 10 after transplantation with exposure to amf at 43c for 30 minutes ( figures 2(a ) and 2(c ) ) under the experimental conditions of protocol number 1 ( figure 3(a ) ) . both nprcap / m with and without amf exposure resulted in a significant and equal reduction of melanoma tumor volume in both b16f1 and f10 cells at the site of primary transplantation ( p < .01 by two - way repeated measure anova , figures 2(a ) and 2(c ) ) compared to tumor volume of naive control mice . control studies comparing magnetite alone and magnetite plus nprcap ( nprcap / m ) without amf exposure showed that magnetite alone does not have any melanoma growth inhibiting effect whereas nprcap / m without amf significantly inhibited melanoma - growth ( p < .01 by two - way repeated measure anova , figure 2(b ) ) . since we obtained basically same growth inhibition results for both the primary and secondary transplants from the two cell lines , the majority of subsequent studies listed in protocols number 1 through number 4 were conducted on b16f1 cells . we then evaluated whether nprcap / m treatment with or without heat in the local primary tumor could inhibit the growth of distant tumors which were not given an intra - tumor injection of nprcap / m . there was a significant difference in the melanoma growth inhibition of re - challenge transplants between the groups of nprcap / m with and without heat . nprcap / m with amf exposure showed the most significant growth inhibition in re - challenge melanoma and increased life span of the host animals , that is , 30%50% complete growth inhibition ( rejection ) of re - challenge melanoma growth , indicating that nprcap / m with heat possesses a thermo - immunotherapeutic effect . for this , we treated the primary b16f1 and f10 melanoma cells by nprcap / m and then measured the volume of the secondary melanoma after the second transplantation at a different site to the first transplant . we also evaluated the survival periods and rates of host melanoma - bearing mice . these secondary melanomas were not directly exposed to nprcap / m ; hence we could evaluate the thermo - immunotherapeutic effect of nprcap / m treatment . first , we compared the therapeutic effects among groups i ( intratumor injection of magnetite nanoparticles alone without amf exposure ) , ii ( magnetite injection and heat at 43c for 30 minutes by amf ) , iii ( nprcap / m injection without amf ) , and iv ( nprcap / m injection and heat at 43c for 30 minutes by amf ) of protocol number 1 ( figure 3(a ) ) . as shown in figure 3(b ) , nprcap / m - mediated hyperthermia at 43c showed the most significant growth inhibition of secondary b16f1 melanoma in re - challenged mice . both magnetite nanoparticles with heat at 43c and nprcap / m without heat also inhibited the growth of secondary melanomas , though statistically not significant ( figure 3(b ) and ( c ) ) . most importantly , nprcap / m alone without heat caused equal growth inhibition of secondary melanomas to that induced by magnetite with amf exposure , suggesting some immunotherapeutic effect of nprcap / m . a similar growth inhibition of secondary transplanted melanoma cells was obtained in b16f10 ( figure 2 ) . the survival of mice in group iv was prolonged , compared with that of the control group ( p = .0003 ) and group i ( p = .0003 ) . three of the ten mice in group iv ( 30% ) were protected completely from re - challenge with b16f1 cells ( figure 3(c ) ) . magnetite alone with amf exposure at 43c ( group ii ) and nprcap / m alone without heat ( group iii ) failed to show any statistically significant prolongation of the host animal survival . to evaluate the effect of the number of treatments for the primary tumor on the re - challenge tumor they consisted of hyperthermia once on day 6 ( group i ) , twice on days 6 and 8 ( group ii ) or days 6 and 10 ( group iii ) , three times on days 6 , 8 , and 10 ( group iv ) or 6 , 7 , and 8 ( group v ) , and a total of five times on days 6 through 10 ( group vi ) ( figure 4(a ) ) . melanoma tumor volumes in re - challenged mice were smallest in groups iii and iv , while the longest survival periods and rates were obtained in group iv with complete growth inhibition ( rejection ) of second re - challenge being 33% ( n = 9 ) on day 60 ( figures 4(b ) and 4(c ) ) . the consecutive irradiation on days 6 , 7 , and 8 ( group v ) or days 6 through 10 resulted in larger volumes of secondary tumors and poorer survival periods and rates compared to group iii or iv ( figures 4(b ) and 4(c ) ) . these findings suggested that repeated hyperthermia , once a day every other day for a total of three days , could induce effective degradation of b16 melanoma cells , which then most likely induced host immunity against melanoma . our study indicated that the most effective thermo - immunotherapy for re - challenge b16 melanoma can be obtained at a temperature of 43c for 30 minutes with the treatment repeated three times on every other day intervals without complete degradation of the primary melanoma . we compared growth inhibition of secondly transplanted melanomas at therapeutic temperatures of 41c , 43c , and 46c for 30 minutes twice on days 6 and 10 , or three times on days 6 , 8 , and 10 ( figure 5(a ) ) . as shown in figures 5(b ) and 5(c ) , thermotherapy at 43c in group iv ( 43c , every other day for a total of three times on three days ) was the most effective for the growth inhibition of both the secondly transplanted , re - challenge melanoma and for improving the survival rates and duration of host mice . four of the nine mice in group iv ( 44.4% ) were protected completely against re - challenge with b16f1 melanoma on day 60 ( figure 5(c ) ) . our therapeutic conditions and their effects differ from those of magnetically mediated hyperthermia on the transplanted melanomas reported previously . mcl - mediated hyperthermia for b16 melanoma showed that hyperthermia at 46c once or twice led to regression of 40%90% of primary tumors and to 30%60% survival of mice , whereas hyperthermia at 43c failed to induce regression of the secondary tumors with 0% survival of mice . we then compared the effects of temperature and duration of nprcap / m - mediated hyperthermia at 43c for 15 minutes , 43c for 30 minutes , 46c for 15 minutes , and 46c for 30 minutes on the re - challenge with b16f1 melanoma ( figure 6(a ) ) . tumor volumes and survival rates and periods of treatment of mice clearly showed that hyperthermia at 43c elicited a more significant effect than that at 46c ( figures 6(b ) and 6(c ) ) . four of the eight mice ( 50% ) in group i ( 43c for 15 minutes ) and three of the seven mice ( 42.8% ) in group iii ( 43c for 30 minutes ) survived 60 days after a second transplantation of b16f1 ( figure 6(c ) ) , suggesting that nprcap / m with heat to the primary melanoma at 43c for 1530 minutes inhibits significantly the growth of distant metastatic melanoma , complete growth inhibition ( rejection ) of the second re - challenge melanoma being 42%50% . hyperthermia at 46c for 30 minutes strongly inhibited the growth of the b16f1 tumor but had little effect on the re - challenge tumor , whereas hyperthermia at 43c for 15 minutes hardly inhibited the growth of the primary tumor but strongly inhibited that of the second re - challenge tumor ( figure 6(d ) ) . these findings suggest that nprcap / m - mediated hyperthermia at 43c can be used most effectively to treat distant metastatic melanoma . heat shock protein forms a complex with intracellular peptides released from degrading tumor cells and presented by the mhc class i molecules of professional antigen - presenting cells . we analyzed hsp70 production in the primary tumor and cd4 and cd8 t cell infiltration into the re - challenge secondary tumor . figure 7(a ) shows the amounts of hsp70 in the tumors at 24 hours after the nprcap / m - mediated hyperthermia . among the six treatment groups , conditions of hyperthermia at 43c for 15 or 30 minutes and 46c for 15 minutes were equally effective for induction of hsp70 as those at 41c for 15 minutes or 30 minutes and at 46c for 30 minutes ( figure 7(a ) ) . we also investigated whether expression of hsp70 in the posttherapeutic tumors depended on the duration of amf exposure ( 15 minutes or 30 minutes ) , heating temperature ( 41c , 43c , or 46c ) , and time elapsed after exposure ( 24 hours , 48 hours , or 72 hours ) . figure 7(b ) shows that the amount of hsp70 in the treated b16f1 tumors was most abundant at 24 hours after hyperthermia at 43c , and over - expression of hsp70 was maintained at a significant level after 72 hours . although thermotherapy at 46c for 15 minutes could induce hsp70 as abundantly as that at 43c for 30 minutes ( figure 7(a ) ) , this condition failed to suppress the re - challenge melanoma transplant as efficiently as 43c thermotherapy ( figures 5(b ) and 5(c ) ) . this suggests that immunological factors other than hsps are at least in part responsible for growth inhibition and rejection of the re - challenge melanoma . hyperthermia at 43c for 1 hour mediated the expression of mhc class i molecules after 24 hours in association with enhanced expression of hsp70 . heat treatment of tumor cells permits enhanced cross - priming , possibly via up - regulation of both hsps and tumor antigen expression . by inducing hsp70 and possibly mhc class i we thus examined histochemically the immunological reaction against secondly transplanted , re - challenge b16f1 melanoma in hematoxylin and eosin ( he)- and cd4- and cd8-stained sections . in addition to neutrophilic leukocytes , macrophages , and plasma cells , cd4 and cd8 t cells were observed around and within the re - challenge tumors with necrotic lesions ( figures 8(a ) , 8(b ) , 8(c ) , and 8(d ) ) . these t cells were seen with a small number around the first transplant melanoma treated by nprcap / m with or without amf exposure but hardly observed around the naive b16f1 tumors in mice that were not treated by nprcap / m - mediated thermotherapy ( data not shown ) . this may indicate that melanoma - specific t cell immunity is likely involved in our nprcap / m therapy strategy . this study has provided the basis for developing a melanoma targeted chemo - immuno - thermotherapy ( cti ) strategy by conjugating melanogenesis substrate , nprcap with magnetite nanoparticles after exposure to alternating magnetic field . nprcap / m - mediated hyperthermia at a relatively low temperature ( 43c ) effectively inhibited the growth of second transplant , re - challenge melanoma . possibly , superficially bound nprcap possesses important roles in targeting nanoparticles to melanocytic cells and a chemotherapeutic effect on these cells . based upon the present animal therapeutic protocol , that is , three - every - other - day treatment at 43c , we have started preliminary clinical trials ( phase i / ii ) of nprcap / m cti therapy with a significant success to a limited number of advanced stages iii and iv melanoma patients . four patients entered in this trial after approval of the ethics committee of sapporo medical university and two of them showed pr and cr , still surviving and carrying out normal daily life for more than 24 months . lastly , it should be noted that melanin intermediates produce reactive oxygen species such as superoxide and h2o2 [ 4 , 32 , 33 ] . this unique biological property of melanin intermediates not only causes cell death but also may produce immunogenic properties . in fact , nprcap / m alone without heat was as effective as magnetite nanoparticles with amf exposure in inhibiting growth of re - challenge melanoma ( figures 3(b ) and 3(c ) ) . it would be interesting to know whether the growth of secondary re - challenge melanoma could be inhibited after treatment of nprcap alone onto the primary tumor . the molecular background of our nprcap / m cti therapy needs to be further studied .

melanogenesis substrate , n - propionyl - cysteaminylphenol ( nprcap ) , is selectively incorporated into melanoma cells and inhibits their growth by producing cytotoxic free radicals . magnetite nanoparticles also disintegrate cancer cells and generate heat shock protein ( hsp ) upon exposure to an alternating magnetic field ( amf ) . this study tested if a chemo - thermo - immunotherapy ( cti therapy ) strategy can be developed for better management of melanoma by conjugating nprcap on the surface of magnetite nanoparticles ( nprcap / m ) . we examined the feasibility of this approach in b16 mouse melanoma and evaluated the impact of exposure temperature , frequency , and interval on the inhibition of re - challenged melanoma growth . the therapeutic protocol against the primary transplanted tumor with or without amf exposure once a day every other day for a total of three treatments not only inhibited the growth of the primary transplant but also prevented the growth of the secondary , re - challenge transplant . the heat - generated therapeutic effect was more significant at a temperature of 43c than either 41c or 46c . nprcap / m with amf exposure , instead of control magnetite alone or without amf exposure , resulted in the most significant growth inhibition of the re - challenge tumor and increased the life span of the mice . hsp70 production was greatest at 43c compared to that with 41c or 46c . cd8+t cells were infiltrated at the site of the re - challenge melanoma transplant .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

the periodontium is a topographically complex organ consisting of epithelial tissue and soft and mineralized connective tissue . the structures comprising the periodontium include gingiva , cementum , bone and periodontal ligament . several diseases affect the composition and integrity of periodontal structures causing the destruction of the connective tissue matrix and cells , the loss of fibrous attachment and the resorption of alveolar bone following an intense inflammatory response to bacteria . such diseases are highly prevalent among different world populations , being an important impact factor in health oral programs . the main bacterial pathogens of periodontal diseases are gram - negative anaerobic species that express a number of potential virulence factors that stimulate an unbalanced production of several molecules and pro - inflammatory factors , being an important determinant in the disease outcome . innumerous studies have shown that , although largely nonspecific , basic treatment consisting of oral hygiene instruction and subgingival instrumentation is able to keep the microbial load at low levels and , if followed by daily self - performed oral hygiene and regular professional periodontal maintenance care , leads to periodontal health in most cases . however , due to its unique patient and site - specific characteristics , a small proportion of the population suffers from ' ' refractory '' types of periodontitis , showing inadequate resolution of inflammation and failure to return tissue to homeostasis . since knowledge of the pathways and processes underlying resolution of periodontal tissue inflammation have grown significantly , an increased interest in substances and/or drugs that may contribute to the restoration of tissue homeostasis has occurred as a more sophisticated biological treatment modality for recurrent types of periodontal disease . several biological systems are candidates to modulate the inflammatory process involved in periodontal disease . evidence from animal experiments and short - term clinical trials demonstrate that anti - inflammatory drugs are able to decrease the rate of periodontal bone destruction by local inhibition of pro - inflammatory molecules . in brazil , the leaves of species of mikania laevigata schultz bip . ex baker , popularly known as " guaco " , have been widely used as infusions or plasters , while the crude extract of this species is commonly commercialized as phytomedicine . guaco is also popular in brazil as an anti - inflammatory , antispasmodic and pain - reliever for rheumatism , arthritis , intestinal inflammation and ulcers . among the few pharmacological and phytochemical studies published , preparations obtained from aerial parts of mikania laevigata have been described as anti - ulcerogenic , antimicrobial , bronchodilatory and anti - inflammatory , possibly accounted for by the presence of several chemical constituents . our research group has recently assessed the pharmacological properties and the underlying molecular mechanisms of mikania laevigata , demonstrating that , in animals , its hydroalcoholic extract was able to reduce neutrophil migration and vascular permeability , and prevent the release of tnf- and il-1. since such findings could promote a protective effect in periodontal tissues against damage , the aim of this study was to evaluate the immunomodulatory action of systemic injection of a " guaco " extract in a model of experimental periodontitis in rats . the extract used in this study was previously obtained and tested as described elsewhere . briefly , the leaves of mikania laevigata were collected at the farm school of the university of uberaba ( tringulo mineiro , mg , brazil ) . a voucher specimen ( hufu 54.748 ) has been deposited at the herbarium of the federal university of uberlndia , brazil . , between days 15 and 17 of november and allowed to dry at 30c in an oven with air renovation for 15 days . the dry plant , triturated by knife mill , was extracted by maceration with 70% ethanol : water under continuous agitation ( shaker ) for three times during 7 days each totalizing 21 days [ the end ratio between plant and solvent was 1:8 ( w / v ) ] , obtaining a crude extract . the crude extract was dried and filtered using filter paper , concentrated in air - forced chamber at 30c until dry crude extract was obtained . forty male wistar rats ( 250 - 350 g ) were used in the study . the animals were kept in plastic cages with access to food and water ad libitum . prior to the surgical procedures , all animals were allowed to acclimatize to the laboratory environment for a period of 5 days . all experiments were conducted in accordance with national health guidelines for the welfare of experimental animals and were approved by the ethics committee of the university of uberaba ( protocol # 001/2008 ) . more specifically , under general anesthesia obtained by intramuscular administration of ketamine ( 1.0 ml / kg ) , ligature was placed and immobilized around both mandible first molars of each animal . the ligature was left in position for the whole experimental period so that inflammation could be constantly induced by the colonization of bacteria inside of it . one day following ligature placement , the animals were randomly assigned to one of the following groups ( n=10 ) : 1 ) animals without ligature placement receiving administration of empty vehicle ( control ) ; 2 ) animals without ligature placement receiving administration of mle ; 3 ) animals with ligature receiving administration of saline ; 4 ) animals with ligature receiving administration of subcutaneous mle ( 10 mg / kg / day ; 200 l . mle or vehicle was administered daily for 30 days . twenty - four hours after the last injection the current dose was chosen based on a previous study showing that it was effective to diminish the carrageenan - induced peritonitis . neutrophil infiltration to the gingival tissues of rats was evaluated by mpo kinetic - colorimetric assay as previously described . samples of gingival tissue were collected in 50 mm k2hpo4 buffer ( ph 6.0 ) containing 0.5% hexadecyl trimethylammonium bromide ( htab ) and kept at -80c until use . samples were homogenized using a polytron ( pt3100 ) and centrifuged at 13,000 g for 4 min . the resulting supernatant devoid of debris was subjected to mpo activity assay determined by spectrophotometer at 450 nm ( spectra max ; molecular devices inc . , briefly , 10 l of sample was mixed with 200 l of 50 mm phosphate buffer ph 6.0 , containing 0.167 mg / ml o - dianisidine dihydrochloride and 0.0005% hydrogen peroxide . the results were presented as mpo activity ( number of neutrophils x mg gingival tissue ) . the right and left jaws were dissected , fixed in 10% buffered neutral formalin for 48 h and decalcified in a decalcifying solution of ethylenediaminetetraacetic acid ( edta ) 10% for 3 months . after that , briefly washed in running tap water , dehydrated and embedded in paraffin wax . sections were mounted on glass slides and stained with hematoxylin and eosin ( he ) for the evaluation of bone resorption . using an image analysis system ( image j - national institute of health ) , the area of bone loss in the furcation region was histometrically determined as previously described . additional sections were mounted on glass slides pre - treated with 3-aminopropyltriethoxy - silane ( sigma chemical co. , st . sections were treated with 3% hydrogen peroxide ( h2o2 ) for 30 min to block endogenous peroxidase . to block the nonspecific binding of antibody , the sections were treated with phosphate buffered saline ( pbs)-1% bovine serum albumin ( vector laboratories , burlingame , ca , usa ) for 30 min at room temperature before incubation with the primary antibody ( polyclonal antibodies against rankl ( 1:50 ) ( santa cruz biotechnology , santa cruz , ca , usa ) for 24 h at 4c . biotinylated secondary antibody was used ( sigma;1:100 ) for 60 min at room temperature . the slides were treated with vectastain abc - ap kit ( diluted at 1:100 ; vector laboratories ) for 60 min at room temperature , and the specific reaction by each antibody was visualized using 3 , 3'diaminobenzidine ( dab ) . the slides were then counterstained with mayer 's hematoxylin , dehydrated through graded ethanol , cleared in xylene , and mounted in slides with the help of permount mounting media ( fisher scientific ) . at least 10 representative sections of each specimen were analyzed under a light microscope ( olympus bx50 , tokyo , japan ) . immunohistochemical analysis was performed individually by two previous calibrated examiners ( jcc and vjs - f ; kappa index=0.97 ) that were blind to the treatment conditions . the number of stained cells was counted and normalized in the area where bone resorption was taking place . statistical comparisons between groups were made using anova analysis of variance followed by bonferroni test . the extract used in this study was previously obtained and tested as described elsewhere . briefly , the leaves of mikania laevigata were collected at the farm school of the university of uberaba ( tringulo mineiro , mg , brazil ) . a voucher specimen ( hufu 54.748 ) has been deposited at the herbarium of the federal university of uberlndia , brazil . , between days 15 and 17 of november and allowed to dry at 30c in an oven with air renovation for 15 days . the dry plant , triturated by knife mill , was extracted by maceration with 70% ethanol : water under continuous agitation ( shaker ) for three times during 7 days each totalizing 21 days [ the end ratio between plant and solvent was 1:8 ( w / v ) ] , obtaining a crude extract . the crude extract was dried and filtered using filter paper , concentrated in air - forced chamber at 30c until dry crude extract was obtained . forty male wistar rats ( 250 - 350 g ) were used in the study . the animals were kept in plastic cages with access to food and water ad libitum . prior to the surgical procedures , all animals were allowed to acclimatize to the laboratory environment for a period of 5 days . all experiments were conducted in accordance with national health guidelines for the welfare of experimental animals and were approved by the ethics committee of the university of uberaba ( protocol # 001/2008 ) . more specifically , under general anesthesia obtained by intramuscular administration of ketamine ( 1.0 ml / kg ) , ligature was placed and immobilized around both mandible first molars of each animal . the ligature was left in position for the whole experimental period so that inflammation could be constantly induced by the colonization of bacteria inside of it . one day following ligature placement , the animals were randomly assigned to one of the following groups ( n=10 ) : 1 ) animals without ligature placement receiving administration of empty vehicle ( control ) ; 2 ) animals without ligature placement receiving administration of mle ; 3 ) animals with ligature receiving administration of saline ; 4 ) animals with ligature receiving administration of subcutaneous mle ( 10 mg / kg / day ; 200 l . mle or vehicle was administered daily for 30 days . twenty - four hours after the last injection , the current dose was chosen based on a previous study showing that it was effective to diminish the carrageenan - induced peritonitis . neutrophil infiltration to the gingival tissues of rats was evaluated by mpo kinetic - colorimetric assay as previously described . samples of gingival tissue were collected in 50 mm k2hpo4 buffer ( ph 6.0 ) containing 0.5% hexadecyl trimethylammonium bromide ( htab ) and kept at -80c until use . samples were homogenized using a polytron ( pt3100 ) and centrifuged at 13,000 g for 4 min . the resulting supernatant devoid of debris was subjected to mpo activity assay determined by spectrophotometer at 450 nm ( spectra max ; molecular devices inc . , sunnyvale , ca , usa ) with three readings within 1 min . briefly , 10 l of sample was mixed with 200 l of 50 mm phosphate buffer ph 6.0 , containing 0.167 mg / ml o - dianisidine dihydrochloride and 0.0005% hydrogen peroxide . the results were presented as mpo activity ( number of neutrophils x mg gingival tissue ) . the right and left jaws were dissected , fixed in 10% buffered neutral formalin for 48 h and decalcified in a decalcifying solution of ethylenediaminetetraacetic acid ( edta ) 10% for 3 months . after that , briefly washed in running tap water , dehydrated and embedded in paraffin wax . sections were mounted on glass slides and stained with hematoxylin and eosin ( he ) for the evaluation of bone resorption . using an image analysis system ( image j - national institute of health ) , the area of bone loss in the furcation region was histometrically determined as previously described . additional sections were mounted on glass slides pre - treated with 3-aminopropyltriethoxy - silane ( sigma chemical co. , st . sections were treated with 3% hydrogen peroxide ( h2o2 ) for 30 min to block endogenous peroxidase . to block the nonspecific binding of antibody , the sections were treated with phosphate buffered saline ( pbs)-1% bovine serum albumin ( vector laboratories , burlingame , ca , usa ) for 30 min at room temperature before incubation with the primary antibody ( polyclonal antibodies against rankl ( 1:50 ) ( santa cruz biotechnology , santa cruz , ca , usa ) for 24 h at 4c . biotinylated secondary antibody was used ( sigma;1:100 ) for 60 min at room temperature . the slides were treated with vectastain abc - ap kit ( diluted at 1:100 ; vector laboratories ) for 60 min at room temperature , and the specific reaction by each antibody was visualized using 3 , 3'diaminobenzidine ( dab ) . the slides were then counterstained with mayer 's hematoxylin , dehydrated through graded ethanol , cleared in xylene , and mounted in slides with the help of permount mounting media ( fisher scientific ) . at least 10 representative sections of each specimen were analyzed under a light microscope ( olympus bx50 , tokyo , japan ) . immunohistochemical analysis was performed individually by two previous calibrated examiners ( jcc and vjs - f ; kappa index=0.97 ) that were blind to the treatment conditions . the number of stained cells was counted and normalized in the area where bone resorption was taking place . statistical comparisons between groups were made using anova analysis of variance followed by bonferroni test . to assess whether mle would affect bone loss in the furcation region , he sections were histometrically analyzed . the resorption area measured after the experimental period demonstrated that ligature induced experimental periodontitis significantly increased bone loss ( p<0.05 ) ( figure 1c ) when compared with both vehicle ( 0.310.03 mm ) and mle ( 0.350.06 mm ) non - ligated groups , which were not different when compared with each other ( p>0.05 ) ( figures 1a and 1b ) . additionally , the results demonstrated that mle administration ( 10 mg / kg , daily ) was able to significantly inhibit ( 0.560.13 mm ; p<0.05 ) the volume of bone loss in ligated teeth ( figure 1d ) , however still higher when compared with the non - ligated teeth ( p<0.05 ) . the values of the resorption area of the three groups were represented in figure 1 . histology at the furcation of first molars sampled from the rats euthanized after 30 days of experiments is shown [ hematoxylin and eosin(he ) staining ] ; ( a ) non - ligated animals treated with vehicle , ( b ) non - ligated animals treated with ( mle ) ( 10 mg / kg / day for 30 consecutive days ) , ( c ) ligature - induced periodontitis treated with vehicle for 30 days , ( d ) ligature - induced periodontitis treated with subcutaneous mle ( 10 mg / kg/ day for 30 consecutive days ) . ( e ) results are expressed as mean area ( mm ) standard deviation of 10 animals in each group . different symbols indicate intergroup statistical significance ( anova followed by bonferroni s test ) . magnification at 40 objective ( scale bar=100 m ) next , the possible interference of mle on neutrophil migration was investigated . according to mpo assay , mle administration did not exert any significant effect on neutrophil accumulation on non - ligated teeth ( 55.7425.84 versus 53.1415.21 neutrophils x 10 ; p>0.05 ) by measuring gingival tissue mpo . on the other hand , the increased levels of neutrophil accumulation observed on ligated teeth ( 113.8238.96 neutrophils x 106 ; p<0.05 ) were significantly decreased by mle administration ( 61.2310.12 neutrophils x 10 ; p<0.05 ) ( figure 2 ) . thus , mle was able to reduce neutrophil accumulation and consequently inflammation in the gingival tissue . mikania laevigata extract ( mle ) decreases neutrophil migration to the gingival tissue . in order to estimate the relative numbers of infiltrating neutrophils in the gingival tissue , myeloperoxidase ( mpo ) activity present in the gingival tissue homogenates was measured . # p<0.05 compared with control animals ; * p<0.05 compared with ligature - induced periodontitis treated with vehicle ( anova followed by bonferroni 's test ) on the other hand , since a reduced bone resorption was observed in the mle - treated group , we tested the hypothesis that mle decreases the release of osteoclast regulatory factors . in this way , we used an immunohistochemistry assay to visualize the expression of rankl on periodontal tissues . as shown in figure 3 , animals with ligated - induced periodontitis treated with mle presented significantly less rankl - immunostained cells ( 76.2564.40 immunostained cells ; p<0.05 ) in the periodontal tissue when compared with ligated - animals treated with vehicle ( 242.0046.23 immunostained cells ) . non - ligated animals ( 49.6618.23 immunostained cells ) were not affected by mle administration ( 45.7522.42 immunostained cells ; p>0.05 ) . expression patterns of rankl in the periodontal tissue at the furcation of first molars sampled from the rats euthanized at 30 days are shown . low number of positive staining cells for rankl was observed in the furcation of non - ligated animals treated with vehicle ( a ) as well the non - ligated animals treated with mikania laevigata extract ( mle ) ( b ) . increased number of rankl positive cells were found in the furcation of the ligated - animals treated with vehicle ( c ) . lower number of rankl stained cells were found in the furcation of the ligated - animals treated with mle ( 10 mg / kg / day for 30 consecutive days ) . ( e ) data are presented as median standard deviation of the average rankl positive cells / per area of furcation . negative control staining was carried out by the incubation with secondary antibody alone that showed lack of immunostaining pattern in all experimental groups ( data not shown ) . # p<0.05 compared with control animals ; * p<0.05 compared with ligature - induced periodontitis treated with vehicle ( anova followed by bonferroni s test ) . the figure is representative of at least 4 different sections obtained from each group . scale bar at lower ( 40x ) magnification=100 m , and scale bar at higher ( 400x ) magnification=20 m to assess whether mle would affect bone loss in the furcation region , he sections were histometrically analyzed . the resorption area measured after the experimental period demonstrated that ligature induced experimental periodontitis significantly increased bone loss ( p<0.05 ) ( figure 1c ) when compared with both vehicle ( 0.310.03 mm ) and mle ( 0.350.06 mm ) non - ligated groups , which were not different when compared with each other ( p>0.05 ) ( figures 1a and 1b ) . additionally , the results demonstrated that mle administration ( 10 mg / kg , daily ) was able to significantly inhibit ( 0.560.13 mm ; p<0.05 ) the volume of bone loss in ligated teeth ( figure 1d ) , however still higher when compared with the non - ligated teeth ( p<0.05 ) . the values of the resorption area of the three groups were represented in figure 1 . histology at the furcation of first molars sampled from the rats euthanized after 30 days of experiments is shown [ hematoxylin and eosin(he ) staining ] ; ( a ) non - ligated animals treated with vehicle , ( b ) non - ligated animals treated with ( mle ) ( 10 mg / kg / day for 30 consecutive days ) , ( c ) ligature - induced periodontitis treated with vehicle for 30 days , ( d ) ligature - induced periodontitis treated with subcutaneous mle ( 10 mg / kg/ day for 30 consecutive days ) . ( e ) results are expressed as mean area ( mm ) standard deviation of 10 animals in each group . different symbols indicate intergroup statistical significance ( anova followed by bonferroni s test ) . next , the possible interference of mle on neutrophil migration was investigated . according to mpo assay , mle administration did not exert any significant effect on neutrophil accumulation on non - ligated teeth ( 55.7425.84 versus 53.1415.21 neutrophils x 10 ; p>0.05 ) by measuring gingival tissue mpo . on the other hand , the increased levels of neutrophil accumulation observed on ligated teeth ( 113.8238.96 neutrophils x 106 ; p<0.05 ) were significantly decreased by mle administration ( 61.2310.12 neutrophils x 10 ; p<0.05 ) ( figure 2 ) . thus , mle was able to reduce neutrophil accumulation and consequently inflammation in the gingival tissue . mikania laevigata extract ( mle ) decreases neutrophil migration to the gingival tissue . in order to estimate the relative numbers of infiltrating neutrophils in the gingival tissue , myeloperoxidase ( mpo ) # p<0.05 compared with control animals ; * p<0.05 compared with ligature - induced periodontitis treated with vehicle ( anova followed by bonferroni 's test ) on the other hand , since a reduced bone resorption was observed in the mle - treated group , we tested the hypothesis that mle decreases the release of osteoclast regulatory factors . in this way , we used an immunohistochemistry assay to visualize the expression of rankl on periodontal tissues . as shown in figure 3 , animals with ligated - induced periodontitis treated with mle presented significantly less rankl - immunostained cells ( 76.2564.40 immunostained cells ; p<0.05 ) in the periodontal tissue when compared with ligated - animals treated with vehicle ( 242.0046.23 immunostained cells ) . non - ligated animals ( 49.6618.23 immunostained cells ) were not affected by mle administration ( 45.7522.42 immunostained cells ; p>0.05 ) . expression patterns of rankl in the periodontal tissue at the furcation of first molars sampled from the rats euthanized at 30 days are shown . low number of positive staining cells for rankl was observed in the furcation of non - ligated animals treated with vehicle ( a ) as well the non - ligated animals treated with mikania laevigata extract ( mle ) ( b ) . increased number of rankl positive cells were found in the furcation of the ligated - animals treated with vehicle ( c ) . lower number of rankl stained cells were found in the furcation of the ligated - animals treated with mle ( 10 mg / kg / day for 30 consecutive days ) . ( e ) data are presented as median standard deviation of the average rankl positive cells / per area of furcation . negative control staining was carried out by the incubation with secondary antibody alone that showed lack of immunostaining pattern in all experimental groups ( data not shown ) . # p<0.05 compared with control animals ; * p<0.05 compared with ligature - induced periodontitis treated with vehicle ( anova followed by bonferroni s test ) . the figure is representative of at least 4 different sections obtained from each group . scale bar at lower ( 40x ) magnification=100 m , and scale bar at higher ( 400x ) magnification=20 m tooth loss , caused by poor periodontal health ( which affects up to 20% of the adult population worldwide ) can lead to significant morbidity and premature death . the economic impact of oral diseases is an important consideration with up to 10% of public health expenditure in developed countries related to curative dental care . in cases where traditional and basic treatments are not able to arrest periodontal disease progression , the adjunct use of alternative substances aimed at modulating the host immune response is desirable . however , despite several agents being commercially available , most of these chemicals may present undesirable side effects and limited scientific evidence so far . it is recognized that most of the new drugs discovered in the last few decades have originated from nature . chemical constituents obtained from medicinal plants and other natural products have been increasingly used to treat many inflammatory diseases . in the present study , it was demonstrated that an extract obtained from mikania laevigata , a popular medicinal plant in south american countries , was able to decrease alveolar bone loss , which may be explained , at least in part , by the lower expression of rankl and decreased neutrophil migration observed on mle - treated animals . a growing body of research suggests that chronic periodontal disease involves a failure of inflammation resolution pathways to restore tissue homeostasis . therefore , strategies to slow or arrest periodontal disease progression by the modulation of the host immune response have been investigated . among the commercially available substances earliest evaluated so far to modify resolution pathways , are the nonsteroidal anti - inflammatory drugs ( nsaids ) . these agents antagonize proinflammatory pathways and/or signaling , preventing the production arachidonic acid metabolites that are proinflammatory mediators implicated in a variety of bone resorptive and tissue degrading processes . although in vitro and experimental studies have shown promising results , periodontal clinical trials have not reached consistent clinical benefits when combined with conventional therapy besides having undesirable side effects when used in a long - term basis . hence , the search for alternative products continues and natural phytochemicals isolated from plants used in traditional medicine are considered as good alternatives to synthetic chemicals . more recently , the number of reports of the use of traditional plants and natural products for the treatment of periodontal disease has increased . in vitro studies evaluating the antimicrobial potential of substances in addition , in vivo studies in animals using compounds of natural plants such as baicalin and cannabinoids observed a protective role against periodontal tissue breakdown in ligature - induced periodontitis , which was partially explained by their inhibitory effects on prostaglandins ( cox-2 ) , interleukins ( il-1 and tnf- ) , nitric oxide and the bone related molecules rank and rankl . our results corroborate with such previous findings , with the administration of mle significantly decreasing alveolar bone resorption and promoting an inhibition of neutrophils influx and rankl production . in the progression of inflammatory diseases , the interactions of recruited neutrophils in the site of inflammation stimulate the local production of several inflammatory mediators that may further amplify the inflammatory response and injure the surrounding tissues . in fact , our group recently assessed the pharmacological properties and the underlying molecular mechanisms of the hydroalcoholic mle to confirm the popular wisdom as a putative anti - inflammatory drug . we could observe that the anti - inflammatory effect of mle was able to reduce neutrophil migration and vascular permeability in a peritonitis model . mle additionally prevented the release of both tnf- and il-1 and contributed to a reduction in leukocyte adhesion and transmigration across the endothelium as observed by intravital microscopy . taken together , the results suggest that use of the medicinal guaco extract may be able to suppress the development of inflammatory lesions , which are initiated by neutrophil recruitment including periodontal conditions . the discovery of the rank - rankl - osteoprotegerin ( opg ) system has brought rapid progress in the understanding of the regulatory mechanisms of osteoclast differentiation and activation exerted by the immune system . recent studies suggest the involvement of rankl and opg in the pathogenesis of periodontitis . not only osteoblasts but also other resident cells , including cells of the periodontal ligament , the findings of the present study , demonstrated that the administration of mle inhibited the expression of rankl . to the best of our knowledge , no direct effect of mle on the rank / rankl / opg system has been addressed in the literature . however , several investigations have addressed the close relation between the production of inflammatory mediators and the modulation of rankl / opg balance . during the progression of periodontal disease , high levels of pro - inflammatory cytokines have been positively related to rankl expression , suggesting that such inflammatory molecules play a major role in periodontal bone resorption . interestingly , a recent work demonstrated that lipopolysaccharide ( lps ) , a toll - like receptor 4 ligand , up - regulated the expression of membrane rankl in human blood neutrophils . besides , lps - activated human and murine neutrophils , cocultured with human monocyte - derived osteoclasts and raw 264.7 cells , respectively , stimulated bone resorption . therefore it is possible to suggest that the potent anti - inflammatory properties of mle may have caused the diminished production of rankl observed in mle - treated animals observed in the present study and indirectly participated in the decreased volume of bone loss during ligature - induced periodontal disease progression . there is considerable evidence that plant extracts have the potential to be developed into agents that can be used as preventative or treatment therapies for oral diseases . the results of the present investigation indicate that " guaco " extract may be useful to control bone resorption during progression of experimental periodontitis in rats in animal studies . while our results are encouraging , further controlled trial studies will be important to establish whether guaco is able to offer clinical therapeutic benefits .

objectivesthe mikania laevigata extract ( mle ) ( popularly known in brazil as " guaco " ) possesses anti - inflammatory properties . in the present study we tested the effects of mle in a periodontitis experimental model in rats . we also investigated possible mechanisms underlying such effects . material and methodsperiodontal disease was induced by a ligature placed around the mandibular first molars of each animal . male wistar rats were divided into 4 groups : non - ligated animals treated with vehicle ; non - ligated animals treated with mle ( 10 mg / kg , daily ) ; ligature - induced animals treated with vehicle and ligature - induced animals treated with mle ( 10 mg / kg , daily ) . thirty days after the induction of periodontal disease , the animals were euthanized and mandibles and gingival tissues removed for further analysis . resultsmorphometric analysis of alveolar bone loss demonstrated that mle - treated animals presented a decreased alveolar bone loss and a lower expression of the activator of nuclear factor-b ligand ( rankl ) measured by immunohistochemistry . moreover , gingival tissues from the mle - treated group showed decreased neutrophil migration myeloperoxidase ( mpo ) assay . conclusionsthese results indicate that mle may be useful to control bone resorption during progression of experimental periodontitis in rats .

INTRODUCTION MATERIAL AND METHODS Source of Mikania laevigata and preparation of the "guaco" extract Animals Experimental animal design MPO activity assay Histological and immunohistochemical analyses Statistical analysis RESULTS Effect of MLE on furcation bone loss Effect of MLE on neutrophil migration and RANKL expression DISCUSSION CONCLUSION ACKNOWLEDGEMENTS AND FUNDING

over the past year , 3 agents have been approved for the treatment of melanoma by the food and drug administration . these include pegylated interferon -2b for stage iii melanoma , vemurafenib for unresectable or metastatic melanoma with braf v600e mutation , and ipilimumab for unresectable or metastatic melanoma . we present here the case of a 65-year - old caucasian male diagnosed with advanced melanoma in april 2011 and treated with ipilimumab ( yervoy ) , a monoclonal antibody targeting cytotoxic t - lymphocyte - associated antigen 4 ( ctla-4 ) , as second - line treatment after progression with dacarbazine , for ( wild - type braf ) metastatic melanoma . the patient was started on first - line chemotherapy with dacarbazine 850 mg / m on day 1 , every 3 weeks . after 3 cycles , the patient showed disease progression with an increase in size of the skin metastasis . second - line treatment was started with ipilimumab 3 mg / kg on day 1 , every 3 weeks . at the end of the treatment , after 4 cycles , we documented a complete clinical response with total resolution of the skin metastasis . at the time of writing this paper , our patient had finished his treatment more than 9 months earlier and is still in complete remission . this is a paradigmatic case where , despite extensive metastatic disease , treatment with ipilimumab has confirmed its efficacy . it is still an open question why only a minority of patients have such a remarkable response , and further trials are warranted to address this important question . about 200,000 cases of melanoma are diagnosed and 48,000 melanoma - related deaths occur worldwide each year . among cancers in patients under 40 years of age , the incidence of melanoma is second only to that of breast cancer in women and leukemia in men . since 2010 , a real breakthrough has occurred in advanced melanoma therapy that had been anticipated for decades . molecular targeted therapy has been added to the treatment regimen of this disease . before 2010 , no systemic therapy had been shown to improve overall survival among patients with metastatic melanoma . ipilimumab , a monoclonal antibody targeting cytotoxic t - lymphocyte - associated antigen 4 ( ctla-4 ) , and vemurafenib , a selective braf inhibitor , have both been shown to improve survival among patients with metastatic melanoma in randomized trials [ 3 , 4 , 5 ] . here , we present the case of a 65-year - old caucasian male diagnosed with advanced melanoma in april 2011 and treated with ipilimumab , after dacarbazine failed , for ( wildtype braf ) metastatic disease . the patient was referred to us in april 2011 , presenting with several painful exophytic lesions on his right arm ( fig . 1 , fig . no evidence of any primary tumors was found despite confirmation of multiple lumps in his right arm . the patient was started on dacarbazine 850 mg / m on day 1 , every 3 weeks , but after 3 cycles he experienced disease progression with nearly all previous lesions increasing in size and a new one popping up . he was then started on ipilimumab 3 mg / kg and , after the whole treatment consisting of 4 cycles , which he tolerated very well , he achieved a complete clinical ( fig . the latest ct scan ( performed at the end of march 2013 ) did not show any metastatic disease . molecular targeted therapies ( e.g. braf inhibitors ) have reached high response rates but , unfortunately , rather short response duration ( progression - free survival : 6 months ) , while immunotherapy has shown slower but more durable results . reported that ipilimumab ( 3 mg / kg every 3 weeks or an initial dose of 3 mg / kg then 1 mg / kg every 3 weeks ) plus a peptide vaccine resulted in 2 complete responses and 5 partial responses among 56 patients with progressive stage iv melanoma . it is rare but not impossible to achieve a complete response , and some patients are currently in a state of complete response . to date , our patient has been free from cancer for 9 months . written informed consent was obtained from the patient for publication of this work and accompanying images . a copy of the written consent form is available for review from the editor - in - chief of this journal . this is a paradigmatic case where , despite extensive metastatic disease , the treatment with ipilimumab has confirmed its efficacy . it is still an open question why only a minority of patients have such a remarkable response , and further trials are warranted to address this important question . understanding the biology of melanoma is crucial for an accurate selection of patients who will be more likely to benefit from ipilimumab .

introductionover the past year , 3 agents have been approved for the treatment of melanoma by the food and drug administration . these include pegylated interferon -2b for stage iii melanoma , vemurafenib for unresectable or metastatic melanoma with braf v600e mutation , and ipilimumab for unresectable or metastatic melanoma.case presentationwe present here the case of a 65-year - old caucasian male diagnosed with advanced melanoma in april 2011 and treated with ipilimumab ( yervoy ) , a monoclonal antibody targeting cytotoxic t - lymphocyte - associated antigen 4 ( ctla-4 ) , as second - line treatment after progression with dacarbazine , for ( wild - type braf ) metastatic melanoma . the patient was referred to us for several painful lumps on his right arm . a biopsy of one of them revealed melanoma . ct and pet scans did not show any other lesions or a primary site . the patient was started on first - line chemotherapy with dacarbazine 850 mg / m2 on day 1 , every 3 weeks . after 3 cycles , the patient showed disease progression with an increase in size of the skin metastasis . second - line treatment was started with ipilimumab 3 mg / kg on day 1 , every 3 weeks . at the end of the treatment , after 4 cycles , we documented a complete clinical response with total resolution of the skin metastasis . at the time of writing this paper , our patient had finished his treatment more than 9 months earlier and is still in complete remission.conclusionthis is a paradigmatic case where , despite extensive metastatic disease , treatment with ipilimumab has confirmed its efficacy . it is still an open question why only a minority of patients have such a remarkable response , and further trials are warranted to address this important question .

Introduction Case Presentation Conclusion Introduction Case Report Conclusion Disclosure Statement

how did the idea of racial and ethnic difference in lung capacity become so widely accepted such that correction factors are actually programmed into the spirometer ? the notion that black and white lungs differ has a long history dating to the early years of the us slavery - based republic . in his influential notes on the state of virginia , former president and leading enlightenment intellectual thomas jefferson featured lung differences between slaves and white colonists . among the many physical distinctions that jefferson described to justify the condition of slaves in the republic , one was a difference of structure in the pulmonary apparatus ( 3 ) . jefferson s ideas about lungs would remain , however , in the realm of philosophical speculation without empirical foundation until the second half of the 19th century . interest in modern spirometers surged in europe in the 1840s after john hutchinson , a london - based physician , published several studies describing the technical features of the spirometer and its potential applications for monitoring the fitness of the police and armed forces , and life insurance applicants and for diagnosing tuberculosis , the great scourge of 19th - century industrializing nations . in a period of great enthusiasm for precision instrumentation and experimental interest in the functional features of the lungs , hutchinson avidly promoted his innovation , naming the spirometer , delineating vital capacity into discrete compartments , adapting the instrument to large - scale studies , and advocating for his technology to london s prestigious scientific societies ( 3 ) . hutchinson faced the same dilemma future researchers would encounter in ordering the wide variability in lung function . while he was most excited about his discovery that the relationship between height and lung capacity demonstrated what he considered to be a general law of nature , height did not completely account for the variability he observed . to capture more fully the potential he envisioned for his instrument , hutchinson further classified lung capacity measurements according to occupation ( 3 ) . however , occupational categories would remain an organizing principle for research on lung capacity measurements only into the early 20th century in britain ( 7 ) . knowledge of the spirometer spread quickly and hutchinson s innovations were adopted within a few years in germany and north america , where researchers worked to further refine its technical details and uses . perhaps the most significant experiments for the future of spirometry were those of plantation physician and slaveholder samuel cartwright in the us south . drawing explicitly on jefferson s interpretive framework , cartwright built his own spirometer to study difference in lung capacity in slaves and whites , and to quantify it precisely . according to cartwright , the deficiency in the negro was 20 per cent . defining difference as deficiency , cartwright established race as a key organizing principle of lung function measurements in the us ( 8) . jefferson s philosophical musings were to capture an even more solid empirical foundation in the 1860s when racial research examining lung capacity shifted to the northern us . in 1864 , the us sanitary commission asked benjamin apthorp gould to head a massive anthropometric survey of black and white soldiers at the end of the civil war . over several years , field workers collected detailed data regarding bodily characteristics of soldiers , which gould synthesized in his 1869 investigations in the military and anthropological statistics of american soldiers . for unclear reasons , he chose to devote an entire chapter to describing lung capacity measured using a spirometer according to race . without any adjustment for height or age , or attention to working and living conditions of newly emancipated slaves , gould reported that full blacks had lower lung capacity than whites . using ostensibly neutral language , he wrote ( 9 ) : the great difference of the mean volume found for the black race from that which seems to belong to the whites , can not fail to attract attention at the first glance . its bearings are perhaps better manifested by the more detailed tabulations which will follow.nearly 30 years later , frederick hoffman , chief statistician for prudential life insurance co. would turn to gould s data to make broad claims about the lack of fitness of african americans for freedom . according to hoffman , the smaller lung capacity of the colored race is in itself proof of an inferior physical organism ( 3 ) . the great difference of the mean volume found for the black race from that which seems to belong to the whites , can not fail to attract attention at the first glance . notably , leading african american intellectuals web dubois and kelly miller wrote trenchant critiques of hoffman s arguments over the inferiority of the negro . these critiques , however , failed to alter the narratives of difference embedded in lung capacity measurements , which would gain further scientific foundation in the 20th century ( 3 ) . beginning in the us in the 1920s , during a period when eugenic policies rooted in hereditarianism were popular , research documenting racial difference in lung function became an even broader global enterprise . in most studies , whites had higher lung capacity than blacks , chinese or indians ; explanations for findings centred on innate difference ( 10 ) . for example , wilson and edwards ( 11 ) published the first set of spirometry - based lung function standards according to race in 1922 , speculating that difference could be due to a possible racial factor . by 1925 , je myers published his reference handbook for clinicians , in which he reported differences among whites , blacks , chinese and filipinos as unquestioned fact ( 12 ) . thus , the idea of racial difference in lung capacity , first proposed by jefferson and further supported by cartwright and gould in the us , was firmly established by the early 20th century as an ostensible fact . future research would build on this framework . during the 1960s and continuing to the present interest in racial difference expanded to numerous populations across the world and researchers focused on developing standards for what they considered to be distinct populations . for historically specific reasons , the consequential technological innovation of a scaling factor for blacks in 1974 , however , was the result of the collaboration between charles rossiter of the pneumoconiosis unit in south wales and hans weill of tulane medical school in new orleans , louisiana ( 13 ) . a large proportion of the literature used an explanatory framework that emphasized innate or anthropometric difference . for the most part , researchers assumed racial identities to be straightforward ( 10 ) . in an article important to the history of spirometry , south african researchers questioned the interpretations of difference , arguing that previous research failed to account fully for social conditions ( 14,15 ) . north american and european researchers , however , failed to cite these articles and the idea that racial difference was innate remained firmly entrenched in the pulmonology literature . as demonstrated in a recent systematic review ( 16 ) , the exclusive racial framework continued into the 21st century . rather than a fluid , historically contingent system of classification , researchers treated race as a stable category , uncomplicated by social class , sex or geographical context . in fact , researchers only defined how they assigned individuals to racial categories in 17.3% of the articles ; 94% of the articles failed to include any measures of social class ( 10 ) . most recently , genomics studies have reinforced , rather than questioned , race - based models ( 17 ) . gould continues to be cited to the present day in prestigious us journals ( 18 . ) how can this brief history help us analyze the contemporary dilemmas in lung function research as it pertains to the use of race and ethnicity in pulmonary function tests ? at the very least , the idea that people labelled white naturally have higher lung capacity than other races throughout the world should be approached with some skepticism . the history of lung function suggests that we should be approaching spirometry differently . rather than using race in a routinized way that reflects assumptions of genetic difference research and clinical practice needs to devote more careful attention to the social nature of racial and ethnic categories and draw on more complex explanatory frameworks that incorporate disproportionate exposures to toxic environments , differential access to high - quality care and the daily insults of racism in every sphere of life that manifest biologically . across the globe , there is a continuum of human phenotypic and genetic variation that can not be apportioned into discrete categories . by featuring race with only marginal attention to the intersection of race and social class , we risk ignoring the complex and dynamic relationship of lung function and the environment . it is well - established that lower forced vital capacity is associated with social conditions , notably poverty ( 1,1922 ) . the specific details of how social class and race influence lung function physiologically , however , remains to be determined . it is time to rethink the problematic practice of race correction in light of this history .

spirometry is the most common lung function test and represents the cornerstone diagnostic and management tool for individuals with chronic respiratory diseases . variability and changing temporal trends in lung function measurements , however , have contributed to the problem of standardizing spirometry , especially with regard to race correction. this article examines the history of the practice , the dilemmas encountered by researchers and the implications of adhering to long - held beliefs without considering more complex explanatory frameworks .

RACIALIZING THE SPIROMETER CONCLUSION

according to ancient medical and veterinary literature , native information of herdsmen regarding the usage of medicinal plants in animal and human disorders is described as ethnoveterinary medicine ( evm ) . evm considers that traditional practices of veterinary medicine are legitimate and seeks to validate them . sources of medicinal plants and pharmaceuticals in many countries such as iran , china , ancient greece , india , and egypt have long been used in the treatment and diagnosis of different diseases ( ghasemi pirbalouti 2009 ; ghasemi pirbalouti et al . according to the world health organization ( who ) , plants are a supplier of medicines for human consumption ( ghasemi pirbalouti 2009 ; ghasemi pirbalouti et al . 2009 ; who 2001 ) . factors such as lack of access to the majority of chemical drugs and/or the cost of chemical drugs , as well as the adverse side effects of chemical drugs , lead to the advantages of using herbal drugs instead of chemical medicines ( ghasemi pirbalouti 2009 ; ghasemi pirbalouti et al . 2009 ) . herbal remedies used for hundreds of years by stock raisers could be put to commercial use , but scientists are demanding that traditional knowledge be validated , to verify the safety and efficacy of the treatments . this study was carried out to identify traditional plants used in the treatment of disorders based on iran s rich history in traditional and herbal medicine derived from the treatments of the famous iranian scientists such as avicenna , as well as the various customs of the different ethnic groups in iran . southern regions of ilam are part of a common market in pharmaceuticals and are involved in both the major cattle and sheep farmlands , and herd animals are often in transition from other regions . one of the main goals of this research was to investigate commonalities in ethnoveterinary medicine in these regions and to introduce new herbal drugs that can be appropriate in the treatment of disorders in human and domestic animals . ilam is located in the west of iran at latitude of 3338 north and longitude of 4626 east , in a cold , mountainous region 1,319 m above mean sea level . although this city is surrounded by mountains , its climate is also affected by deserts from the west and the south . heavy showers or heavy snow in the winter , and dusty , hot , dry weather in the summer are quite normal . abdanan is a county in ilam province , iran . at the time of the 2006 census , its population was 45,830 in 9,358 families . data collection and analysis of plants took place in the southern regions of ilam province from 2009 to 2010 . this paper describes a selection of the ethnoveterinary medicines that were used for herd dogs in these regions . herd dogs , used for protecting livestock , are quite important , particularly among traditional farmers . very little research has been conducted on ethnoveterinary medicine being used for dogs , and there are few comparative studies . in support of this purpose , the questionnaires for the five groups of diseases and syndromes such as gastrointestinal , cutaneous , parasitic , respiratory , and nervous disorders of sheepdog were designed . the questionnaires were distributed among 45 persons ( 7 men and 38 women ) of traditional farmers from 22 villages in the southern province of ilam with an age range of 5575 years . the questionnaire contents included information such as local name , plant parts used , traditional usage , and traditional therapeutic effects . in addition to achieving information regarding the plants being used for treatment and about the area , a sample of the herb that was used was collected . the plant - based remedies were evaluated for safety and efficacy with a nonexperimental method , prior to including them in the draft outline . published sources such as journal articles and books , and databases on pharmacology , ethnopharmacology , ethnobotany , and ethnomedicine available on the internet were searched to identify the plants chemical compounds and clinically tested physiological effects . the ethnoveterinary usages of locally available plants for herd dog in ilam province are summarized in table 1.table 1ethenoveterinary medicines used for dogs in southern regions of ilam provincescientific namefamilyplant part usednative namepersian nameuse ( ghasemi pirbalouti 2009)p . limonum fruitlimolimodiarrheaastakhys lavandili folia vahllabiataeaerial partkolparchaye kohidiarrheaceracus microcarpa ( c. a. mey . ) boiss.rosaseaeleafmalhomahlabantiseptic and disinfection of woundsc . colocynthis ( l. ) schardcucubitaceaefruit and flowergijalakhendavane abojahlantiseptic and wound healing ethenoveterinary medicines used for dogs in southern regions of ilam province ilam is located in the west of iran at latitude of 3338 north and longitude of 4626 east , in a cold , mountainous region 1,319 m above mean sea level . although this city is surrounded by mountains , its climate is also affected by deserts from the west and the south . heavy showers or heavy snow in the winter , and dusty , hot , dry weather in the summer are quite normal . abdanan is a county in ilam province , iran . at the time of the 2006 census , its population was 45,830 in 9,358 families . data collection and analysis of plants took place in the southern regions of ilam province from 2009 to 2010 . this paper describes a selection of the ethnoveterinary medicines that were used for herd dogs in these regions . herd dogs , used for protecting livestock , are quite important , particularly among traditional farmers . very little research has been conducted on ethnoveterinary medicine being used for dogs , and there are few comparative studies . in support of this purpose , the questionnaires for the five groups of diseases and syndromes such as gastrointestinal , cutaneous , parasitic , respiratory , and nervous disorders of sheepdog were designed . the questionnaires were distributed among 45 persons ( 7 men and 38 women ) of traditional farmers from 22 villages in the southern province of ilam with an age range of 5575 years . the questionnaire contents included information such as local name , plant parts used , traditional usage , and traditional therapeutic effects . in addition to achieving information regarding the plants being used for treatment and about the area , a sample of the herb that was used was collected . the plant - based remedies were evaluated for safety and efficacy with a nonexperimental method , prior to including them in the draft outline . published sources such as journal articles and books , and databases on pharmacology , ethnopharmacology , ethnobotany , and ethnomedicine available on the internet were searched to identify the plants chemical compounds and clinically tested physiological effects . the ethnoveterinary usages of locally available plants for herd dog in ilam province are summarized in table 1.table 1ethenoveterinary medicines used for dogs in southern regions of ilam provincescientific namefamilyplant part usednative namepersian nameuse ( ghasemi pirbalouti 2009)p . limonum fruitlimolimodiarrheaastakhys lavandili folia vahllabiataeaerial partkolparchaye kohidiarrheaceracus microcarpa ( c. a. mey . ) boiss.rosaseaeleafmalhomahlabantiseptic and disinfection of woundsc . colocynthis ( l. ) schardcucubitaceaefruit and flowergijalakhendavane abojahlantiseptic and wound healing ethenoveterinary medicines used for dogs in southern regions of ilam province one of the first studies to document gender was conducted by diana davis , who noted a difference in knowledge of evm of afghan pashtun nomads that paralleled the gender - based division in the society . davis found that women know more about healthcare for newborns and very sick animals that are taken care of near the home ( davis 2000 ) . since women prepare the carcass for consumption , they know twice as many types of internal parasites as men . women also help with dystocias and the manual removal of ectoparasites . in the present study , most of the information pertaining to the usages of herbal drugs was given by women , who are more knowledgeable about healthcare for sick animals that are taken care of near the home . in a research conducted in trinidad , it was noted that male farmers were using the reproductive knowledge of their female relatives to assist in the health care of their ruminants . female farmers were using the same plants for their animals that they used for themselves ( lans et al . it was found that in total , 22 plants were being used in the treatment of dog disorders ( table 1 ) . some of the plants in this study were poisonous . recognizing the components of each plant in all regions of a country can aid in our understanding of renewable natural resources and contribute effectively , in line with the characteristics and uses of medicinal plants and their uses ( ghasemi pirbalouti 2009 ; ghasemi pirbalouti et al . 2009 ) . in the present study , some plants which are used for dog disorders such as delphinium orientalis ( gay ) schrod , artemisia herba - alba , nicotina tabacum , echium strigosa labill , saturiya khuzistanica jamzed , aloe vera , scrophularia deserti del , and citrullus colocynthis ( l. ) schard have some new effects which have not previously been mentioned , but other plants which are used in the treatment of dog disorders such as a. vera and e. strigosa in new herbal medicine are mentioned ( amouzgar et al . 2008 ; jahanara and haerizadeh 2001 ; rojhan 2001 ; samsam shariat and moatar 1975 ; zargari 1994).table 2additional information about poisonous plantsscientific namefamilytoxic component ( ghasemi pirbalouti 2009)p . tabacumsolanaceaenicotine additional information about poisonous plants euphurbia graminifolius , salsola rigida , amygdalus arabica olivier , and a. herba - alba have been widely used for elimination of intestinal worms , and in most areas , these plants are used for the same purpose in other animals as well . a. vera , chenopodium album l. , d. orientalis ( gay ) schrod were used as a laxative in constipation , and can be lucrative for treatment of accumulation of rumen , reticulum , and omasum in ruminant rather than chemical drugs . the use of pistacia atlantica desf , pistacia khinjuk stocks , and quercus brantii lindl . persica ( jaub & spach ) zohary by farmers for the treatment and alleviation of coughing in dogs can be useful in the introduction of a new generation of herbal drugs for human and domestic animals . a. herba - alba is good fodder for grazing animals , mainly sheep and cattle . this species of sagebrush is widely used in folk and traditional medicine for its antiseptic and antispasmodic properties . a. herba - alba is reported as a traditional remedy of enteritis , and various intestinal disturbances . the essential oil from satureja khuzestanica jamzad ( skeo ) , an endemic plant from iran , was evaluated for its activity against inflammatory bowel disease ( ibd ) . skeo was examined on the experimental mouse model of ibd , which is acetic acid - induced colitis . peganum harmala is used as an analgesic and anti - inflammatory agent , as well as in the treatment of depression . it has antibacterial activity against drug - resistant bacteria and is used in the treatment of syphilis in india , fever in north africa , hysteria , neuralgia , parkinson s disease , prolapsed uterus , rheumatism , asthma , and eye irritation . p. harmala is an abortifacient and is effective against protozoa including malaria ( al - sharma et al . a. vera juice is used for consumption and relief of digestive issues such as heartburn and irritable bowel syndrome , although it bears significant potential to be toxic when taken orally . other uses for extracts of a. vera include the dilution of semen for the artificial fertilization of sheep , use as a fresh food preservative , and is used for water conservation in small farms . therapeutic effects of a. vera include treatment of arthritis , asthma , candidiasis , treatment of chronic fatigue , indigestion and intestinal disorders ( colitis and ulcerative colitis ) , skin disorders ( psoriasis , acne , burns , infections , foot fungus , skin damage caused by cold ) , sports injuries , and wounds . internal and external uses of a. vera have a long association with herbal medicine , although it is not known when its medical applications were first suspected . in addition to topical use in wound or burn healing , internal intake of a. vera has been linked in preliminary research with improved blood glucose levels in diabetics and with lower blood lipids in hyperlipidemic patients , as well as with acute hepatitis ( liver disease ) . in other diseases , preliminary studies have suggested oral a. vera gel may reduce symptoms and inflammation in patients with ulcerative colitis . in the present study , internal intake of a. vera in dogs compounds extracted from a. vera have been used as an immune stimulant that aids in fighting cancers in cats and dogs ; however , this treatment has not been scientifically tested in humans . and although anecdotally useful , it has not been proven to offer protection from sunburn . a. vera extracts might have antibacterial and antifungal activities , which could possibly help treat minor skin infections such as boils and benign skin cysts , and may inhibit growth of fungi causing tinea . inner leaf gel from a. vera was shown to inhibit the growth of streptococcus and shigella species in vitro ( chow et al . ( l. ) schard has been linked in preliminary research with improved blood glucose levels in diabetics and digestive disorders , and the laxative effects of this plant have been proven ( asfi 1994 ; tavakol afshari et al . 2008 ) , dizziness , and stomach inflammation , while almond was used to relieve acute urination and disorders of saliva . other usages of medical plants in ancient and traditional medicine included antimastitis effect of harmal , treatment of constipation , chronic wound healing , and treatment of infections of p. atlantica desf and p. khinjuk stocks . scrophularia scopolii was used for relief of pulmonary disorders , gangrenous wounds , and back pain , but in the present study , s. deserti del was used to disinfect wounds in skin and eyes ( ardakani yazdi 2006 ) . in a study by bahmani et al . ( 2011a , b ) , 35 plants were recognized for treatment of small ruminants disorders . in this study , the peanut plant was used for treatment of accumulation of rumen , q. brantii lindl . persica zohary for treatment of diarrhea , cough , and mouth ulcers ; d. orientalis ( gay ) schrod , coralinaceae , and a. vera as a laxative ( jeremy et al . 2005 ; ki et al.1999 ) ; p. atlantica desf for treatment of oestrus ovis larva ; and n. tabacum as an antileech drug . persica ( jaub & spach ) zohary was used for treatment of mastitis ( bahmani et al . ( 2011a , b ) and ghasemi pirbalouti ( 2009 ) demonstrated the anticandidiasis effect of s. deserti and scrophularia striata . in another study , ( 2010 , 2011a , b ) revealed the antileech effects of the tobacco plant . the present study confirmed the traditional medical effects of some plants , while revealing the unique medical effects of other plants .recognizing such plants and their effects can aid in the creation of new ideas for increasing knowledge in the modern pharmaceutical industry . since very few clinical trials have been conducted on plants that are native to ilam province , it is vital that more research be done to ensure the introduction of labeled and standardized products for human consumption . this ethnobotanical survey results revealed the wealth of indigenous knowledge and usage customs of traditional plants associated with the rural people of the southern regions of ilam . despite their use in traditional medicines , plant species renowned in the present fieldwork there was no written certificate of traditional healing knowledge , and the transfer of such knowledge to the future generation takes place only through oral communication . more detailed ethnopharmacological investigations need to be conducted in this area , particularly concerning conservation strategies and sustainable use of medicinal plants .

this paper describes a selection of the ethnoveterinary medicines used for herd dogs in the southern regions of ilam province , iran . traditional botanical medicine is the primary mode of healthcare for most of the rural population in ilam province . in this study , a questionnaire was distributed among 45 residential areas in 22 rural zones of the southern areas of ilam province . the objective of this study was the recognition of natural medicinal methods using medicinal plants , and the classification of ethnoveterinary applications and collection of domestic science . twenty - two medicinal plants from 16 families were identified . the main application of these plants was for the detection and treatment of digestive disorders using citrullus colocynthis , aristolochia clematis , scrophularia deserti , quercus brantii , ceracus microcarpa , echium strigosa , pistacia atlantica , and pistacia khinjuk which have been applied using euphurbia graminifolius , peganum harmala , salsola rigida , artemisia herba - alba , amygdalus arabica , jolbak of salt water , peganum harmala l. , and nicotina tabacum for external and internal parasite disorders . s. deserti for ophthalmic disorders , and p. atlantica , p. khinjuk , and q. brantii for respiratory disorders were applied . the present study confirmed the traditional medical effects of some plants and revealed the unique medical effects of other plants , which if recognized could be useful in the creation of new ideas and increasing knowledge for the modern pharmaceutical industry . since very few clinical trials have been conducted on plants native to ilam province , it is necessary that more research be conducted to ensure that labeled and standardized products are introduced for human consumption .

Introduction Material and methods Study area: south area of Ilam province Data collection Results and discussion

the discovery of intra - vascular foreign bodies is increasing and their associated danger is well recognized . endovascular retrieval of intravascular foreign bodies offers a high success rate with minimal morbidity and mortality . a variety of tools are available to the interventionist to aid in foreign body extraction ; however , a thorough knowledge of available devices and techniques is necessary to minimize the associated complications of endovascular retrieval . we describe the endovascular retrieval of an unusual intravascular foreign body , an irrigation cannula , from the thoracic aorta following cardiac surgery . an 81 year old male underwent coronary artery bypass grafting and aortic valve replacement . during the procedure , the ascending aorta had been irrigated several times with heparinized saline using a syringe capped with a stainless - steel olive tip irrigation cannula . when the syringe was handed back to the nursing staff , prior to closure of the sternum , the pericardium and the surgical field were searched again for the irrigating tip and thus the chest was closed . the missing instrument prompted a chest radiograph that demonstrated a metal irrigation cannula superimposed on the cardiac silhouette ( fig . right common femoral artery access was obtained and a thoracic aortogram was performed demonstrating the cannula to be lodged in the descending thoracic aorta ( fig . , we felt it was imperative to rule out an unidentified aortic abnormality at this location . an 8-french intravascular ultrasound ( ivus ) catheter ( volcano corporation , san diego , ca ) was then used to evaluate the descending thoracic aorta . no aortic abnormality was identified that may have prevented the caudal migration of the cannula . we surmised , then , that the cannula was lodged obliquely between the anterior and posterior aortic walls . a tri - lobed snare , en snare ( merit medical systems , south jordan , ut ) was used to grasp the cannula at its tip and withdraw it into the distal aorta . at the aortic bifurcation , order dislodge the cannula and allow retraction into the right iliac system , we advanced and rotated the snare . these maneuvers allowed further rotation of the cannula and successful withdrawal into the right iliac system . due to its large size , the irrigating cannula could not be retrieved further than the distal external iliac artery ( fig . 3 ) . the olive tip measures 45 mm in length and 5.5 mm in width . the cannula was successfully removed through a transverse arteriotomy in the distal right external iliac artery . he was discharged to a skilled nursing facility and had no adverse vascular complications noted on post - operative follow up . as the use of endovascular techniques and procedures have risen , so have the reports of intravascular foreign bodies . our report highlights an endovascular approach to retrieve an unusual aortic foreign body lost after open cardiac surgery . following our review of the literature , we believe this to be the only such case of an intra - aortic irrigation cannula successfully retrieved by an endovascular means . described the endovascular retrieval of a migrated patent foramen ovale occluder from the descending thoracic aorta . this case , while similar in some respect , differs from ours as the authors found it was necessary to use both a 5.5-french multipurpose biopsy forcep ( cordis corporation , a johnson & johnson company ; bridgewater , nj ) as well as an amplatz gooseneck snare ( ev3 peripheral vascular , part of covidien ; plymouth , minn ) to remove a large intra - aortic foreign body . we believe that our case , along with the report from basoor et al . , highlights that an endovascular snare is the preferred tool for intra - aortic foreign body extraction . this is also emphasized by the reports of others that have shown the snare to be useful in the retrieval of other , more common , foreign bodies such as fractured port catheters , introducer fragments , microcoils , dislocated stents , fragments of guidewires , pacemaker transducers , and vena cava filters , , , , . prior to these reports , the majority of the literature focused on the open surgical extraction of pedicle spinal fixation screws from the thoracic aorta . one unique case involved endovascular stent graft exclusion followed by extraction via a left anterolateral fourth intercostal space thoracotomy . while interesting as it allowed extraction without aortic cross clamping , this approach still subjected the patient to the morbidity associated with a thoracotomy . in our case , open extraction would have required a thoraco - abdominal incision to access the descending thoracic aorta and removal of the irrigation cannula . this would not only increase the potential morbidity of the case but also add substantial health care costs . lastly , we feel it important to mention that after the case , we discovered that a 22-french sheath would have been able to accommodate the irrigation cannula . in retrospect , a double - proglide ( abbott vascular , abbott park , illinois ) preclose technique followed by insertion of a long 22-french sheath insertion may have allowed successful extraction of the cannula percutaneously , thus avoiding a groin incision . endovascular retrieval of intravascular foreign bodies is minimally invasive , relatively simple , and carries minimal morbidity compared to conventional open surgical techniques . this unusual case of a thoracic aortic foreign body demonstrates the importance of a working knowledge of endovascular techniques and instruments necessary for safe and effective extraction of atypical intravascular foreign bodies . biju k. thomas : study concept , data collection , data interpretation , writing paper . james r. elmore : study concept , data collection , data interpretation , writing paper . robert p. garvin : study concept , data collection , data interpretation , writing paper . evan j. ryer : study concept , data collection , data interpretation , writing paper .

highlightsendovascular techniques to retrieve intra - vascular foreign bodies are a necessary component of the vascular surgeon s skill set.endovascular retrieval of intravascular foreign bodies is minimally invasive , relatively simple , and carries minimal morbidity compared to conventional open surgical techniques.it is important to have a working knowledge of techniques and instruments required for retrieval of intravascular foreign bodies .

Introduction Presentation of case Discussion Conclusion Conflict of interest Funding Ethical approval Author contribution Consent Guarantor

several methods of local anesthesia are routinely used in conventional extracapsular cataract extraction ( ecce ) or msics . of these , retrobulbar and peribulbar anesthesia are associated with globe perforation , retrobulbar hemorrhage , optic nerve injury , brain - stem anesthesia , and other serious complications.46 superior subconjunctival anesthesia produces adequate analgesia , but not adequate akinesia . its use has been reported in conventional extracapsular cataract surgery to avoid serious complications associated with other methods of anesthesia.7 we propose that msics can be performed under subconjunctival anesthesia . however , there are no reports of its use in msics , especially during residency training . we hypothesized that both subconjunctival anesthesia and retrobulbar block have similar levels of pain control in msics , and that subconjunctival anesthesia may have a lower pain score during injection than a retrobulbar block . to verify the effectiveness and safety of subconjunctival anesthesia in a residency training program , we conducted a clinical trial and compared pain control during msics with intraocular lens ( iol ) implantation achieved by superior subconjunctival anesthesia versus . retrobulbar anesthesia . a hospital - based , randomized , controlled clinical trial was conducted at prapokklao hospital , chanthaburi , thailand . this trial adhered to the tenets of the declaration of helsinki , and was approved by the prapokklao hospital ethics committee for human research . patients with cataracts who visited our center and planned to have cataract surgery performed by third - year residents between january 1 , 2011 , and december 31 , 2011 , were recruited . eligibility criteria included age > 40 years , and a visually significant cataract that was indicated for cataract surgery with iol implantation . patients with uveitis , glaucoma , or previous ocular trauma / surgery , or those who were unable to co - operate and communicate during surgery ( due to dementia , hearing impairment , etc ) were excluded . study participants were assigned randomly to receive superior subconjunctival anesthesia ( study group ) or retrobulbar anesthesia ( control group ) . stratified blocked randomization was used to reduce the variation in outcome due to chance disproportions in important baseline variables such as sex and second eye operation . the randomization code was allocated inside the operating room just before the surgery in a sealed envelope drawn by a nurse not involved in patient treatment . pain assessment was performed by a single skilled observer , blinded to the type of anesthesia . the primary outcome measure was pain scores during the operation , and during the injection . the secondary outcome measures were postoperative pain scores , operative time , anesthetic complications , and operative complications . pain visual analog scale ( vas ) scores ( 0 = no pain , and 100 = the worst imaginable pain ) were recorded by a well - trained research assistant after the patient was administered the anesthetic agent , and upon completion of the operation . was maintained throughout the study by ensuring that each surgeon carried out all the surgical procedures and anesthetic techniques accurately . all patients underwent msics using the modified blumenthal technique ( further described below).8 third - year residents from the university hospital in thailand performed the cataract surgeries under close supervision during the 2-week msics training course.9 each resident was required to have previous experience of having performed cataract surgery using ecce with iol implantation in at least 30 of the cases and of having performed cataract surgery using phacoemulisification in at least 10 cases , to ensure that they could perform capsulorhexis before they participated in this study . the 14 participating residents had performed retrobulbar anesthesia during their 2 years of residency training and had also received training in msics using the modified blumenthal technique for at least 1 week prior to operating on the patients enrolled in this study . patients in group one received superior subconjunctival anesthesia . within 5 minutes before surgery , a drop of 0.5% tetracaine hydrochloride was instilled into the lower conjunctival sac , followed by the administration of subconjunctival anesthesia consisting of 0.2 ml of xylocaine 2% with adrenalin 1:100,000 ( drocanil - a ; m and h manufacturing co , ltd , samutprakarn , thailand ) , injected under the superior bulbar conjunctiva . patients in group two received retrobulbar anesthesia . a drop of 0.5% tetracaine hydrochloride was instilled into the lower conjunctival sac , and a retrobulbar block with 2.0 ml of xylocaine 2% with adrenalin 1:100,000 ( drocanil - a ) was performed using the standard procedure . patients heart rate , respiratory rate , blood pressure , and oxygen saturation were monitored using a philips monitor ( mp40 model , philips healthcare , andover , ma ) . patients were advised to ask for supplemental anesthesia if they were unable to tolerate the procedure . they were also interviewed regarding the tolerance of the procedure 30 minutes after the surgery , by a trained interviewer blinded to the type of anesthesia received by the patients . patients who could not tolerate the procedure received an additional retrobulbar block or subconjunctival anesthesia as appropriate . successful anesthesia was defined as completion of the operation without requiring supplemental anesthesia . for the modified blumenthal technique,8 an anterior chamber maintainer was inserted through the 6 oclock side port . a 66.5 mm scleral tunnel incision was performed at the 12 oclock position and a one - side port was created at the 3 or 9 oclock position . a continuous circular capsulorhexis ( or capsulotomy ) was performed , followed by hydrodissection and nuclear dislocation into the anterior chamber . the nucleus was dislocated into the anterior chamber , and the lens glide was inserted below the nucleus . the cortex was removed , and then the iol was placed in the capsular bag . we used the data from a pilot study of 20 patients ( 10 patients in each group ) to determine the sample size . the intraoperative pain scores were 49 ( sd , 30 ) in the subconjunctival anesthesia group and 41 ( sd , 25 ) in the retrobulbar group . with a statistical power of 80% and the level of statistical significance set at p < 0.05 , we estimated that a minimum of 74 patients would be required in each group ( sample size calculator ; dss research , fort worth , tx ) . statistical analysis was performed using spss for windows ( version 11.5 ; ibm , armonk , ny ) . comparisons of operation time , pain scores , and requirement of supplemental anesthesia in the two groups were performed using the independent student s t - test , mann whitney u test , and fisher s exact test , respectively . patients with cataracts who visited our center and planned to have cataract surgery performed by third - year residents between january 1 , 2011 , and december 31 , 2011 , were recruited . eligibility criteria included age > 40 years , and a visually significant cataract that was indicated for cataract surgery with iol implantation . patients with uveitis , glaucoma , or previous ocular trauma / surgery , or those who were unable to co - operate and communicate during surgery ( due to dementia , hearing impairment , etc ) were excluded . study participants were assigned randomly to receive superior subconjunctival anesthesia ( study group ) or retrobulbar anesthesia ( control group ) . stratified blocked randomization was used to reduce the variation in outcome due to chance disproportions in important baseline variables such as sex and second eye operation . the randomization code was allocated inside the operating room just before the surgery in a sealed envelope drawn by a nurse not involved in patient treatment . pain assessment was performed by a single skilled observer , blinded to the type of anesthesia . the primary outcome measure was pain scores during the operation , and during the injection . the secondary outcome measures were postoperative pain scores , operative time , anesthetic complications , and operative complications . pain visual analog scale ( vas ) scores ( 0 = no pain , and 100 = the worst imaginable pain ) were recorded by a well - trained research assistant after the patient was administered the anesthetic agent , and upon completion of the operation . surgical standardization and consistency was maintained throughout the study by ensuring that each surgeon carried out all the surgical procedures and anesthetic techniques accurately . all patients underwent msics using the modified blumenthal technique ( further described below).8 third - year residents from the university hospital in thailand performed the cataract surgeries under close supervision during the 2-week msics training course.9 each resident was required to have previous experience of having performed cataract surgery using ecce with iol implantation in at least 30 of the cases and of having performed cataract surgery using phacoemulisification in at least 10 cases , to ensure that they could perform capsulorhexis before they participated in this study . the 14 participating residents had performed retrobulbar anesthesia during their 2 years of residency training and had also received training in msics using the modified blumenthal technique for at least 1 week prior to operating on the patients enrolled in this study . patients in group one received superior subconjunctival anesthesia . within 5 minutes before surgery , a drop of 0.5% tetracaine hydrochloride was instilled into the lower conjunctival sac , followed by the administration of subconjunctival anesthesia consisting of 0.2 ml of xylocaine 2% with adrenalin 1:100,000 ( drocanil - a ; m and h manufacturing co , ltd , samutprakarn , thailand ) , injected under the superior bulbar conjunctiva . patients in group two received retrobulbar anesthesia . a drop of 0.5% tetracaine hydrochloride was instilled into the lower conjunctival sac , and a retrobulbar block with 2.0 ml of xylocaine 2% with adrenalin 1:100,000 ( drocanil - a ) was performed using the standard procedure . patients heart rate , respiratory rate , blood pressure , and oxygen saturation were monitored using a philips monitor ( mp40 model , philips healthcare , andover , ma ) . patients were advised to ask for supplemental anesthesia if they were unable to tolerate the procedure . they were also interviewed regarding the tolerance of the procedure 30 minutes after the surgery , by a trained interviewer blinded to the type of anesthesia received by the patients . patients who could not tolerate the procedure received an additional retrobulbar block or subconjunctival anesthesia as appropriate . successful anesthesia was defined as completion of the operation without requiring supplemental anesthesia . for the modified blumenthal technique,8 an anterior chamber maintainer was inserted through the 6 oclock side port . a 66.5 mm scleral tunnel incision was performed at the 12 oclock position and a one - side port was created at the 3 or 9 oclock position . a continuous circular capsulorhexis ( or capsulotomy ) was performed , followed by hydrodissection and nuclear dislocation into the anterior chamber . the nucleus was dislocated into the anterior chamber , and the lens glide was inserted below the nucleus . the cortex was removed , and then the iol was placed in the capsular bag . we used the data from a pilot study of 20 patients ( 10 patients in each group ) to determine the sample size . the intraoperative pain scores were 49 ( sd , 30 ) in the subconjunctival anesthesia group and 41 ( sd , 25 ) in the retrobulbar group . with a statistical power of 80% and the level of statistical significance set at p < 0.05 , we estimated that a minimum of 74 patients would be required in each group ( sample size calculator ; dss research , fort worth , tx ) . statistical analysis was performed using spss for windows ( version 11.5 ; ibm , armonk , ny ) . comparisons of operation time , pain scores , and requirement of supplemental anesthesia in the two groups were performed using the independent student s t - test , mann whitney u test , and fisher s exact test , respectively . we recruited 150 patients with all types of cataracts for the trial ( figure 1 ) . seventy - five patients were allocated to the subconjunctival anesthesia group ( group 1 ) and 75 patients to the retrobulbar anesthesia group ( group 2 ) . their demographic data , including age and sex , as well as their associated conditions were similar at the time of recruitment ( table 1 ) . the mean operative time was 47.1 ( 9.9 ) min and 47.7 ( 10.9 ) min in groups 1 and 2 , respectively , which was not significantly different ( p = 0.74 ) . the vas - intraoperative pain ( vas - op ) , vas - postoperative pain ( vas - po ) , and vas - injection pain ( vas - in ) scores were rated on a 100-point vas . the median vas - in , vas - op , and vas - po scores are shown in table 2 . during the operation , a median ( interquartile range ) pain score of 40 ( range , 2070 ) vs 40 ( range , 2050 ) was seen in the subconjunctival group and the retrobulbar group , respectively . after the operation , a median ( interquartile range ) pain score of 20 ( range , 030 ) vs 10 ( range , 020 ) was observed in the subconjunctival group and the retrobulbar group , respectively . the pain score difference ( intraoperative and postoperative pain ) between the groups was not statistically significant ( figure 2 ) . the median injection pain score was significantly lower in group 1 as compared to group 2 ( p < 0.001 ) . supplemental anesthesia was required in nine of the 75 eyes ( 12% ) in the retrobulbar anesthesia group , but in only three of the 75 eyes ( 4% ) in the subconjunctival anesthesia group . the operation was successfully performed with supplemental anesthesia where required , without complications , in all patients . there was no difference in the operative complications , but the incidence of injection complications differed significantly ( table 2 ) . localized subconjunctival hemorrhage was observed in 19 of 75 eyes ( 25.3% ) in the subconjunctival group and in one of 75 eyes ( 1.3% ) in the retrobulbar group . iris prolapse tended to be more common in the retrobulbar group and was possibly related to the positive vitreous pressure , but the difference between the two groups was not statistically significant ( p = 0.209 ) . seventy - two of the 75 patients ( 96% ) in the subconjunctival group and 66 of the 75 patients ( 88% ) in the retrobulbar group tolerated the operation well . supplemental anesthesia was required in only three of the 75 patients ( 4% ) in the subconjunctival group and in nine of the 75 patients ( 12% ) in the retrobulbar group . however , there were only two levels of assessment ( yes / no ) . for more accuracy , the need for additional anesthesia should be evaluated in the questionnaire . the operative time was prolonged ( .47 minutes in both groups ) because it was performed by residents in training . the pain scores during the operation ( vas - op ) were not significantly different between the two groups , suggesting that subconjunctival anesthesia provides equal pain control as retrobulbar anesthesia during msics . the median injection pain score ( vas - in ) was significantly lower in group 1 than in group 2 , indicating that the administration of subconjunctival anesthesia causes less pain as compared to the retrobulbar block . unlike peribulbar or retrobulbar anesthesia , subconjunctival anesthesia eliminates the risk of globe perforation , retrobulbar hemorrhage , and optic - nerve trauma , and is associated with minimal discomfort.10 no serious intraoperative complications occurred in our study . posterior capsular tear with vitreous loss occurred in three of 75 patients ( 5.3% ) in the subconjunctival group , which is similar to the 5.01% rate reported previously.9 anesthetic complications such as localized subconjunctival hemorrhage are also more common when using this technique , which is in agreement with the results of tulvatana et al.7 the successful use of circumcorneal perilimbal anesthesia in extracapsular cataract surgery , and circumferential subconjunctival anesthesia and superior subconjunctival anesthesia with deep topical anesthesia has been reported.7,11,12 we used superior subconjunctival anesthesia , which permits the surgeon to perform procedures such as bridle suturing , subconjunctival peritomy , cautery , and wound construction . in addition , it allows the surgeon to manage a prolapsed iris or nucleus , enlarge the pupil , break the capsule , and perform a vitrectomy in the same manner as when injection anesthesia is used . residents in our 2-week training course of msics are wary about performing capsulorhexis , because , if the patients move their eyes during the capsulorhexis , while under subconjunctival anesthesia , an anterior capsular tear can occur . therefore , we suggest that the retrobulbar block is routinely utilized for anesthesia by residents in the first week of training , and the subconjunctival anesthesia later in the second week , by which time they have developed good surgical skills . patients with poor cooperation or communication during surgery ( because of hearing impairment , dementia , etc ) should not be selected for cataract surgery under subconjunctival anesthesia . however , this limitation could not be controlled because these residents from the university campus were on 2-week rotations for msics training . patient satisfaction as well as surgeon stress levels were not reported herein , and should be recorded in future studies .

purposeto evaluate the effectiveness of subconjunctival anesthesia as compared to retrobulbar anesthesia for pain control during manual small - incision cataract surgery ( msics ) performed by third - year residents.designa randomized , controlled trial.patients and methodsa total of 150 patients undergoing routine cataract surgery were randomly assigned to receive either subconjunctival anesthesia ( group 1 , n = 75 ) or retrobulbar anesthesia ( group 2 , n = 75 ) . third - year residents performed msics using the modified blumenthal technique . subconjunctival anesthesia was administered by injecting 2% xylocaine with adrenalin into the superior conjunctiva , and retrobulbar anesthesia by injecting 2 ml of 2% xylocaine with adrenalin into the retrobulbar space . we studied the following variables : intraoperative pain score rated on a 100-point visual analog scale ( vas ) , operative time , and injection and operative complications.resultsa mean age of 69 vs 70 years , an operative time of 47.1 ( sd , 9.9 ) min vs 47.7 ( 10.9 ) min , and a median ( interquartile range ) pain score of 40 ( range , 2070 ) vs 40 ( range , 2050 ) were observed in the subconjunctival and the retrobulbar groups , respectively . the injection complication of subconjunctival hemorrhage was significantly higher in the subconjunctival group ( 25.3% ) compared to the retrobulbar group ( 1.3% ) . the operative complication rate between groups was not different ( p > 0.05).conclusionboth , superior subconjunctival anesthesia and retrobulbar anesthesia were effective during msics when used in a residency training program .

Introduction Materials and methods Participants Study design Outcome measures Treatment procedure Sample size calculation Data analysis Results Discussion Conclusion

new , large genomic data sets are providing more in - depth insights into the diagnosis and treatment of disease . in the past decade , new and innovative methods have continued to add value to the underlying data and uncover the secrets of the genome . visual data inspection by experienced researchers is an important quality control element in the analytical process . unfortunately , this part of the process is tedious and time consuming , and the increasing volumes of high - throughput sequencing data of various types and platforms are proving to be a major analytical challenge . here , we report a visualization tool that allows researchers to explore their data at a very rapid speed and significantly reduce the burden of reviewing tens and hundreds of thousands of variant calls . areas with systematic read errors can be quickly identified , and inefficient attempts to verify results in noisy regions can be avoided . alview is a fast and portable visualization tool . the core code interfaces with heng li et al s samtools library1 for parsing bam files . the program is written in platform - independent c. peculiarities specific to an operating system are isolated with if defined ( ifdef ) directives ; so , for instance , when microsoft visual c provides alternate support for a portable operating system interface ( posix ) standard function , a handcrafted , native interface work around is supplied . for graphical user interface ( gui ) frameworks , alview uses win32 interface for windows , the gtk2 interface for linux , and bsd unix - based systems and cocoa for apple mac os x. samtools1 is written to posix standards , but different microsoft visual compilers provide various levels of support for these unix style standards . as a result , the source code for third - party libraries that were modified for windows is provided to facilitate compiling and linking alview on windows . the main code for alview , in the file alviewcore.cpp , is written to be portable between operating systems and emphasizes speed of execution . the code can be compiled as a stand - alone executable and must be linked with the zlib2 and samtools1 libraries . sequence reads are processed via custom samtools callback functions arranged in in - memory data structures and represented by an aesthetic , annotated image . the image is then output to the screen as a native graphics object or to the disk as a standard image format file . alview can also be compiled as a webserver daemon that uses the common gateway interface ( cgi)3 standard . the cgi version produces interactive html output and uses dynamic html54 features , including zoom in by selection via a jquery5 library . the cgi webserver alview version loads a list of permitted - to - access bam files from a user - maintained text file ; so custom lists of bam files of interest are easy to generate and use . the source code is free and open to modification so that users and local system operators can implement their own security . the alview cgi webserver version provides modifiable url access , so that , for instance , cells in a spreadsheet can link to viewable results for any sample or location . stand - alone alview accepts parameters that specify bam file name and genomic coordinates . invoking alview in a script can create a slideshow of interesting regions . for example , fields in a single nucleotide polymorphism ( snp ) detection output file can be used to specify a series of calls to alview to generate images for each purported polymorphism or mutation . the burden of reviewing ten and hundreds of thousands of mutation calls can therefore be significantly reduced . the source code is available at github.6 the readme file there points to links for selected executables and complete download packages that include the associated reference genome data . a live webserver version of alview for examining public human cancer short - read datasets is available at https://cgwb.nci.nih.gov/cgi-bin/alview . alview source code and executables for several operating systems are available at the national cancer institute ( nci)/national cancer informatics program s ( ncip s ) github site : https://github.com/ncip/alview . nci retains the copyrights to national cancer institute and associated images , which may not be used in forked projects . alview provides a solid substructure that allows for various types of access to short - read data across different operating systems . figure 1 . demonstrates the various navigation and information buttons available in the web version of alview and shows how selection via mouse provides zoom in capabilities . alview is a trim , fast , precise tool and complements existing programs such as the integrated genomics viewer ( igv),7 bamview,8 and gbrowse 2.0.9 the benefits of alview are extreme speed and a sharp focus on exploring short reads . different implementation philosophies can influence memory usage and performance but provide useful alternative paths to solving similar problems . igv provides much more functionality than alview by supporting many other input file types other than bam sequence read files . igv s java implementation provides write once , run anywhere portability via implementations of the java virtual machine . alview s implementation relies on low - level operating system and native gui toolkit api calls . igv requires registration for download for running off of disk , whereas alview does not . desktop igv may require internet for full , easy , simple operation , whereas alview does not require network connection ( though it may call user - invoked external webpages ) . 7 intel core i52400 cpu at 3.10 ghz and 8 gb ram , restarts of igv v2.3 took from 12 to 18 seconds . restarts of alview took a small fraction of one second . for a small view of a genomic region , the java platform se binary for igv took up 292 mb , while alview took up 11 mb .

the name alview is a contraction of the term alignment viewer . alview is a compiled to native architecture software tool for visualizing the alignment of sequencing data . inputs are files of short - read sequences aligned to a reference genome in the sam / bam format and files containing reference genome data . outputs are visualizations of these aligned short reads . alview is written in portable c with optional graphical user interface ( gui ) code written in c , c++ , and objective - c . the application can run in three different ways : as a web server , as a command line tool , or as a native , gui program . alview is compatible with microsoft windows , linux , and apple os x. it is available as a web demo at https://cgwb.nci.nih.gov/cgi-bin/alview . the source code and windows / mac / linux executables are available via https://github.com/ncip/alview .

Introduction Features and Methods Results

bentall and de bono introduced the initial standardized procedure for aortic root replacement ( arr ) in 1968 , and since then , technical improvements , surgical modifications , and development of new materials during the last 3 decades have made this procedure safer and more reproducible . in the current era , reported surgical outcomes following arr are very favorable in elective cases with hospital mortality rates of less than 5% [ 24 ] . with an increase in cases undergoing aortic root procedures , redo - arr is regarded as technically challenging , and related with high morbidity and mortality rates especially in emergency settings [ 57 ] . in the interest of reviewing outcomes of redo - arr in the current era , we sought to evaluate the early and late outcomes of redo - arr in our institution . from the institutional prospective cardiac surgical database , we identified 66 consecutive adult patients ( > 17 years old ) who underwent reoperative complete arr following surgery for the aortic valve ( av ) or an aortic root procedure between april 1995 and june 2015 . the arr was defined by complete replacement of the av and aortic sinus by valved conduits using mechanical protheses , bio - prostheses , or a homograft . all subject patients underwent coronary reattachment to a new aortic sinus using the coronary button technique . retrospective medical chart reviews were conducted to collect information on prior aortic root surgeries , baseline demographic and clinical profiles , details on redo - arr surgeries , and postoperative outcomes . major early adverse events included stroke , renal failure that required new dialysis , bleeding control surgery , pericardial effusion , surgical site wound problems , and permanent pacemaker insertion . primary outcomes of interest were death and valve - related complications that included hemorrhagic complications secondary to anticoagulation therapy , thromboembolic events , requirement for surgery in the operated aortic root , and infective endocarditis . emergency surgery was defined as an operation before the beginning of the next working day after the decision to operate based on the new euroscore ii guidelines . early death was defined as occurring within 30 days following surgery or during index hospitalization . categorical variables were expressed as percentages or frequencies , and continuous variables were expressed as meanstandard deviation or median with range . kaplan - meier curves were formulated to delineate the overall survival and event - free survival rates . differences in these rates between the groups were assessed using the log - rank test . to identify the predictors of primary adverse outcomes ( death and valve - related complications ) , cox - proportional hazard models were used . covariates listed in tables 1 , 2 , and 3 were evaluated in univariable cox models , and those with p - values of less than 0.1 in the univariable models were candidates for multivariable cox - hazard models . the multivariable analyses involve a stepwise backward elimination technique and variables with p - values of less than 0.1 were used in the final cox - hazard model . statistical analyses were performed using ibm spss statistics for windows ver . 21.0 software ( ibm co. , armonk , ny , usa ) . this study was approved by the asan medical center ethics committee / review board ( s2016 - 1344 - 0001 ) , who waived the need for patient consent . the mean age of subject patients in this study was 45.212.8 years ( range , 35.3 to 55 years ) and 29 cases ( 43.9% ) were emergency procedures . fifteen patients ( 22.7% ) presented with systemic vasculitis syndrome such as behcet disease ( n=9 ) and takayasu arteritis ( n=6 ) . among the patients , 53 patients ( 80.3% ) had undergone 1 previous cardiac operation , 11 ( 16.7% ) had undergone 2 , and 2 ( 3.0% ) had undergone 3 or more operations . the most common prior surgical procedure was an isolated aortic valve replacement ( avr ; n=28 , 42.4% ) , followed by a complete arr ( n=11 , 16.7% ) , and aortic root remodeling ( n=6 , 9.1% ) . indications for the redo - arr were aneurysm ( n=12 ) , pseudoaneurysm ( n=1 ) , or dissection ( n=6 ) of the residual native aortic sinus in 19 patients ( 28.8% ) , native av dysfunction in 8 patients ( 12.1% ) , structural dysfunction of an implanted bioprosthetic av in 19 patients ( 28.8% ) , and infection of a previously replaced av or previously placed proximal aortic grafts in 30 patients ( 45.5% ) ( table 3 ) . the mean interval between previous aortic root procedures and index redo - arr was 65.314.7 months . most patients received mechanical valved - conduits for redo - arr ( 74.2% ) while homograft and bioprosthetic valved conduits were used in 12.1% and 13.6% of patients , respectively . concomitant arch repair was undertaken in 14 patients ( 21.2% ) , while an additional 11 patients required circulatory arrest during redo - arr with a mean duration of 17.44.2 minutes . details of concomitant cardiac procedures , and durations of cardiopulmonary bypass ( cpb ) and cardiac ischemia during procedures are summarized in table 4 . there were 3 early deaths ( 4.5% , all of which occurred in emergency operations ) , in which the indications for redo - arr were residual aortic dissection in which the indications for redo - arr were as follows ; a dissection of residual native aorta in 1 patient and infective endocarditis of prosthetic av in 2 patients . no patients died following elective procedures . the former mortality case received redo - arr due to severe aortic regurgitation caused by residual aortic dissection in the native aortic root followed by supra - coronary aortic replacement in the setting of acute type a aortic dissection . the patient died of multi - organ failure despite redo - arr and postoperative extracorporeal membrane oxygenation support . the latter two mortality cases required emergency operations due to aggravation of infective endocarditis in prosthetic av . however , septic shock followed by multi - organ failure led to death in these two patients . overall , 18 patients ( 27.3% ) experienced major early complications , details of which are shown in table 5 . during follow - up ( median , 54.65 months ; quartile 13 , 17.93 to 95.71 months ) , there were 14 late deaths ( 21.2% ) , 9 valve - related complications including reoperation of the aortic root in 1 patient , infective endocarditis in 3 patients , and hemorrhagic events in 5 patients ( table 5 ) . there was 1 case of reoperation of the reoperated aortic root : trido bentall operation was conducted to treat recurred infective endocarditis . event - free survival rates at 5 and 10 years were 76.4%5.4% and 41.1%11.7% , respectively ( fig . five - year survival and event - free survival rates according to the baseline presentations were as follows : ( 1 ) emergency group ( n=29 ) ; , 74.9%8.2% , 68.2%9.8% , ( 2 ) vasculitis ( behcet , marfan , takayasu ) group ( n=23 ) ; 85.8%7.6% , 76.5%11.5% , and ( 3 ) av only avp or avr ( avp : aortic valve repair , avr : aortic valve replacement ) group ( n=31 ) ; 85.8% 9.4% , 84.6%10.0% , respectively ( fig . 2 ) . there was no statistical difference in overall survival according to the baseline settings of the patients ( fig . 2 ) . the emergency group had a lower event - free survival rate than that of non - emergency group ( p - value= 0.028 ) . among 66 patients , 17 had undergone previous arr either by valve - sparing or by valve - replacement , followed by redo - arr . there were no significant differences between those who had undergone arr and those who had undergone other procedures in overall survival ( p=0.160 ) and event - free survival ( p=0.310 ) ( fig . emergency surgery ( hazard ratio [ hr ] , 2.80 ; 95% confidence interval [ ci ] , 1.12 to 7.00 ; p=0.027 ) was the only significant and independent predictor of the major adverse outcomes ( death and valve - related complications ) while the baseline hemoglobin level was a marginally significant factor associated with the adverse outcomes ( hr , 0.80 ; 95% ci , 0.63 to 1.03 by 1 g / dl increment ; p=0.079 ) . the mean age of subject patients in this study was 45.212.8 years ( range , 35.3 to 55 years ) and 29 cases ( 43.9% ) were emergency procedures . fifteen patients ( 22.7% ) presented with systemic vasculitis syndrome such as behcet disease ( n=9 ) and takayasu arteritis ( n=6 ) . among the patients , 53 patients ( 80.3% ) had undergone 1 previous cardiac operation , 11 ( 16.7% ) had undergone 2 , and 2 ( 3.0% ) had undergone 3 or more operations . the most common prior surgical procedure was an isolated aortic valve replacement ( avr ; n=28 , 42.4% ) , followed by a complete arr ( n=11 , 16.7% ) , and aortic root remodeling ( n=6 , 9.1% ) . indications for the redo - arr were aneurysm ( n=12 ) , pseudoaneurysm ( n=1 ) , or dissection ( n=6 ) of the residual native aortic sinus in 19 patients ( 28.8% ) , native av dysfunction in 8 patients ( 12.1% ) , structural dysfunction of an implanted bioprosthetic av in 19 patients ( 28.8% ) , and infection of a previously replaced av or previously placed proximal aortic grafts in 30 patients ( 45.5% ) ( table 3 ) . the mean interval between previous aortic root procedures and index redo - arr was 65.314.7 months . most patients received mechanical valved - conduits for redo - arr ( 74.2% ) while homograft and bioprosthetic valved conduits were used in 12.1% and 13.6% of patients , respectively . concomitant arch repair was undertaken in 14 patients ( 21.2% ) , while an additional 11 patients required circulatory arrest during redo - arr with a mean duration of 17.44.2 minutes . details of concomitant cardiac procedures , and durations of cardiopulmonary bypass ( cpb ) and cardiac ischemia during procedures are summarized in table 4 . there were 3 early deaths ( 4.5% , all of which occurred in emergency operations ) , in which the indications for redo - arr were residual aortic dissection in which the indications for redo - arr were as follows ; a dissection of residual native aorta in 1 patient and infective endocarditis of prosthetic av in 2 patients . the former mortality case received redo - arr due to severe aortic regurgitation caused by residual aortic dissection in the native aortic root followed by supra - coronary aortic replacement in the setting of acute type a aortic dissection . the patient died of multi - organ failure despite redo - arr and postoperative extracorporeal membrane oxygenation support . the latter two mortality cases required emergency operations due to aggravation of infective endocarditis in prosthetic av . however , septic shock followed by multi - organ failure led to death in these two patients . overall , 18 patients ( 27.3% ) experienced major early complications , details of which are shown in table 5 . during follow - up ( median , 54.65 months ; quartile 13 , 17.93 to 95.71 months ) , there were 14 late deaths ( 21.2% ) , 9 valve - related complications including reoperation of the aortic root in 1 patient , infective endocarditis in 3 patients , and hemorrhagic events in 5 patients ( table 5 ) . there was 1 case of reoperation of the reoperated aortic root : trido bentall operation was conducted to treat recurred infective endocarditis . event - free survival rates at 5 and 10 years were 76.4%5.4% and 41.1%11.7% , respectively ( fig . five - year survival and event - free survival rates according to the baseline presentations were as follows : ( 1 ) emergency group ( n=29 ) ; , 74.9%8.2% , 68.2%9.8% , ( 2 ) vasculitis ( behcet , marfan , takayasu ) group ( n=23 ) ; 85.8%7.6% , 76.5%11.5% , and ( 3 ) av only avp or avr ( avp : aortic valve repair , avr : aortic valve replacement ) group ( n=31 ) ; 85.8% 9.4% , 84.6%10.0% , respectively ( fig . 2 ) . there was no statistical difference in overall survival according to the baseline settings of the patients ( fig . the emergency group had a lower event - free survival rate than that of non - emergency group ( p - value= 0.028 ) . among 66 patients , 17 had undergone previous arr either by valve - sparing or by valve - replacement , followed by redo - arr . there were no significant differences between those who had undergone arr and those who had undergone other procedures in overall survival ( p=0.160 ) and event - free survival ( p=0.310 ) ( fig . emergency surgery ( hazard ratio [ hr ] , 2.80 ; 95% confidence interval [ ci ] , 1.12 to 7.00 ; p=0.027 ) was the only significant and independent predictor of the major adverse outcomes ( death and valve - related complications ) while the baseline hemoglobin level was a marginally significant factor associated with the adverse outcomes ( hr , 0.80 ; 95% ci , 0.63 to 1.03 by 1 g / dl increment ; p=0.079 ) . due to the progress in surgical techniques , reoperation in cardiac surgery is increasingly performed . re - operative aortic root , ascending aorta , or both are challenging reoperations associated with high mortality rate [ 57 ] . due to great diversity among these operations , however , the reported mortality and morbidity have been highly variable among studies . in addition , there have been only a few studies that specifically evaluated clinical outcomes of redo - arr . the present study aimed to evaluate a homogeneous cohort who underwent redo - arr following av or aortic root procedures . although redo - arr had been reported to carry a high postoperative mortality in older studies , more recent series have demonstrated early mortality of 3% to 7% . our data showed similar outcomes with an in - hospital mortality rate of 4.5% ( n=3 ) . interestingly , these outcomes are very similar to those for an elective primary arr procedure , which varies from 4.5% to 7.5% . as the number of repeat - sternotomies increases , surgical risks of cardiac procedures are also reportedly increased with a strong positive association . in this study , the number of repeat sternotomies was not associated with any early or late adverse outcomes . of note , safe re - entry into the chest is thought to be the most important factor for favorable outcomes . advances in surgical strategies for re - exploration of the sternum promoted by the use of modern computed tomography imaging are believed to have contributed favorable results during re - entry into the chest . once high - risk patients have been identified with ct , utilization of cpb through other sites like the femoral or axillary vessels previous studies identified risk factors of in - hospital mortality , which were age of the patient ( 75 years ) , preoperative new york heart association functional class iv , emergency presentation , prior cardiac surgery , unplanned coronary artery bypass grafting , prolonged cpb time , and postoperative renal failure [ 57,9,10,1317 ] . in our study , emergency surgery was the only significant and independent predictor of the primary adverse outcomes ( hr , 2.80 ; 95% ci , 1.12 to 7.00 ; p=0.027 ) . among 66 patients , 29 patients had redo - arr in emergency presentation . in subgroup analysis , event - free survival rate was significantly lower ( log rank p - value=0.028 ) in the emergency group than in that of the non - emergency group . due to very limited numbers of early mortality cases ( n=3 out of 66 ) , as well as late adverse outcomes , studies on a larger cohort are needed to determine risk factors of redo - arr . in the present study 3 patients died in the early period , of which 2 patients died of acute prosthetic valve endocarditis ( pve ) . both patients had undergone a previous arr followed by the current redo - arr due to acute pve . a pve is known as a catastrophic complication , which carries operative mortality of 25% to 60% [ 18,19 ] . in this study , 13 patients presented with pve and 2 of them died , constituting 15.4% of the early mortality rate for pve , which seemed encouraging considering the challenging nature of such a condition . this study has several major limitations including the retrospective nature of the analyses and the small sample size . in addition , results are those of a high - volume tertiary academic center ; therefore , they may not be generalizable to other settings . despite technical challenges and a high rate of emergency conditions in patients requiring redo - arr , early and late outcomes following the procedure were acceptable in these patients . this study has several major limitations including the retrospective nature of the analyses and the small sample size . in addition , results are those of a high - volume tertiary academic center ; therefore , they may not be generalizable to other settings . despite technical challenges and a high rate of emergency conditions in patients requiring redo - arr , early and late outcomes following the procedure were acceptable in these patients .

backgroundgeneralization of standardized surgical techniques to treat aortic valve ( av ) and aortic root diseases has benefited large numbers of patients . as a consequence of the proliferation of patients receiving aortic root surgeries , surgeons are more frequently challenged by reoperative aortic root procedures . the aim of this study was to evaluate the outcomes of redo - aortic root replacement ( arr).methodswe retrospectively reviewed 66 patients ( 36 male ; mean age , 44.59.5 years ) who underwent redo - arr following av or aortic root procedures between april 1995 and june 2015.resultsemergency surgeries comprised 43.9% ( n=29 ) . indications for the redo - arr were aneurysm ( n=12 ) , pseudoaneurysm ( n=1 ) , or dissection ( n=6 ) of the residual native aortic sinus in 19 patients ( 28.8% ) , native av dysfunction in 8 patients ( 12.1% ) , structural dysfunction of an implanted bioprosthetic av in 19 patients ( 28.8% ) , and infection of previously replaced av or proximal aortic grafts in 30 patients ( 45.5% ) . there were 3 early deaths ( 4.5% ) . during follow - up ( median , 54.65 months ; quartile 13 , 17.93 to 95.71 months ) , there were 14 late deaths ( 21.2% ) , and 9 valve - related complications including reoperation of the aortic root in 1 patient , infective endocarditis in 3 patients , and hemorrhagic events in 5 patients . overall survival and event - free survival rates at 5 years were 81.5%5.1% and 76.4%5.4% , respectively.conclusiondespite technical challenges and a high rate of emergency conditions in patients requiring redo - arr , early and late outcomes were acceptable in these patients .

Introduction Methods Results 1) Baseline characteristics 2) Outcomes Discussion 1) Limitations 2) Conclusions

in the present issue of critical care , noveanu and colleagues evaluated brain natriuretic peptide ( bnp ) and n - terminal prohormone brain natriuretic peptide ( nt - probnp ) serial measurements as predictive of 30-day and 1-year mortality and readmission in patients admitted to the emergency department for acute decompensated heart failure ( adhf ) . the usefulness of measuring bnp and nt - probnp in the diagnosis and management of congestive heart failure is well known . both bnp and nt - probnp are useful for diagnosis in patients presenting with undifferentiated dyspnoea in the emergency department and have been shown to accurately reflect heart failure severity and prognosis [ 3 - 6 ] . moreover , in some studies , serial bnp evaluations have been demonstrated to be useful in clinical management . noveanu and colleagues showed during 1-year followup in a multivariate analysis that bnp at 24 hours ( mean 95% confidence interval ) ( 1.02 ( 1.01 to 1.04 ) , p = 0.003 ) , at 48 hours ( 1.04 ( 1.02 to 1.06 ) , p < 0.001 ) and at discharge ( 1.02 ( 1.01 to 1.03 ) , p < 0.001 ) independently predicted 1-year mortality , while only predischarge nt - probnp was predictive ( 1.07 ( 1.01 to 1.13 ) , p = 0.016 ) . comparable results could be obtained for the secondary endpoint of 30-day mortality but not for 1-year heart failure readmissions . these results from noveanu and colleagues ' paper are in accordance with data reported by our group . we demonstrated that a reduction of bnp > 46% at hospital discharge coupled with a bnp absolute value < 300 pg / ml resulted in a very powerful negative prognostic value for future cardiovascular outcomes in patients hospitalised with adhf . other studies demonstrated the usefulness of repeated measurements of natriuretic peptides during hospitalisation in predicting survival of adhf patients [ 8 - 11 ] . bnp variations during hospitalisation could give prognostic information , particularly at discharge , and could also suggest a qualitative variation of treatment ( intensification or decrement of drugs ) on the basis of natriuretic peptide levels . noveanu and colleagues have also demonstrated that the prognostic accuracy of bnp was comparable at 24 hours with 48 hours and with discharge . the authors suggested that bnp at 24 hours could be suitable to assess prognosis and to vary treatment in order to decrease mortality in patients with constant elevated levels of bnp . this suggestion is in accordance again with data from our laboratory , where we showed that a drop of bnp > 25% at 24 hours was a strong negative prognostic factor for future cardiovascular events , suggesting intensified treatment in patients who did not decrease their bnp > 25% at 24 hours . rapid change in bnp levels seems to reflect an adequate response to heart failure therapy , and could be considered very important for early risk stratification and therapy guidance . a lack of this response , assuming optimal medical treatment , implies a more complex and therapy - refractory disease , associated with an adverse long - term outcome . accordingly , if this change in bnp level does not occur , treatment intensification should be the consequence . in patients with a comparable decrease in bnp levels ( roughly 30% between admission and 24 hours ) , we would expect a favourable outcome ; however , future prospective studies need to evaluate a distinct cut - off point to allow more precise recommendations . moreover , from the data of noveanu and colleagues , bnp and nt - probnp seem to show a different response to treatment due to their different kinetics . this difference is probably due to the slower decrease of nt - probnp during treatment in adhf patients in comparison with bnp [ 9 - 12 ] . compared with nt - probnp , bnp could be more useful to determine initial clinical stabilisation of adhf patients , and to assess clinical improvement in hospitalisation as we also demonstrated . nt - probnp could be used to assess initial diagnosis but is of limited help for repeat measurements during hospitalisation because its variations are not as sensitive and rapid as those of bnp [ 9 - 12,14 ] . in conclusion , in patients admitted to the emergency department for adhf , serial measurements of bnp and nt - probnp are useful because they show a similar powerful predictive role for mortality in the short term and in the long term . interestingly , patients ' bnp and nt - probnp variations could help the physician to vary the therapeutic approach during the initial hours of hospitalisation in order to obtain favourable outcomes . nevertheless , when considering hospital readmissions after discharge it seems that the variation of the two bio - markers during hospitalisation at various time points is of no utility . logeart and colleagues showed that only predischarge bnp was a strong predictor of death , and also of readmissions for heart failure with a cut - off point of 350 ng / ml . previously published studies presuming this finding - including cheng and colleagues using bnp or bettencourt and colleagues using nt - probnp - used combined endpoints consisting of all - cause mortality and readmission for heart failure . although the results of noveanu and colleagues ' study are to be considered of importance for the role of natriuretic peptides in prognostic stratification for patients with adhf , multicentre studies on a larger number of patients should be carried out to better elucidate the real value of natriuretic peptides in avoiding readmission after hospital discharge in heart failure patients . adhf : acute decompensated heart failure ; bnp : brain natriuretic peptide ; nt - probnp : n - terminal prohormone brain natriuretic peptide .

acute decompensated heart failure is one of the most important causes of hospitalisation worldwide . natriuretic peptides have shown their usefulness in the diagnosis and management of heart failure . their variations during hospitalisation also appear useful to predict outcomes . in particular , data from the literature demonstrate that reduction from admission to discharge of brain natriuretic peptide and n - terminal prohormone brain natriuretic peptide in these patients is a predictor of future cardiovascular events .

None Abbreviations Competing interests

carcinoma of the thyroid gland is the most common endocrine malignancy , accounting in the usa for example for 95% of endocrine tumors . over the last 3 decades the incidence of well - differentiated thyroid cancer ( tc ) increased from 4.9 per 100 000 in 1975 to 14.3 per 100 000 in 2009 . in china , the standardized incidence of tc has increased from 1.6 per 100 000 in 1995 to 9.9 per 100 000 in 2010 . others are invasive early on , giving rise to clinical symptoms and metastasizing to lymph nodes . accurate and timely ultrasound ( us ) is the first - choice imaging modality for the diagnosis of tc . the diagnostic sensitivity , specificity , and accuracy rate are 92% , 72.9% , and 62~78% , respectively , for tc [ 57 ] . however , the sonograms of some lesions were overlapped between the benign and malignant as a result of the various ultrasonic appearances among different lesions . therefore , biopsy was relied on to get the final diagnosis , and is considered the diagnostic criterion standard [ 8 , 9 ] , but biopsy is invasive and has some risk for patients . previous studies showed that 4875% of patients who undergo biopsy are subsequently found to have benign lesions , the overall dissatisfaction rate for fna is between 1020% , and 2% of samples obtained by cnb are inadequate for histological diagnosis . how to reduce the number of unnecessary biopsies is an area of current research . contrast - enhanced ultrasound ( ceus ) scanning can detect differences in the distribution of blood and in the hemodynamics between tumors and surrounding tissues . it has found widespread application in many organs , such as the liver , gall bladder , and kidney . its use in assessing diseases of the thyroid gland has been the subject of a number of reports , although its precise role remains uncertain [ 1316 ] . ace / ame / eta ( american association of clinical endocrinologists / associazione medici endocrinology / european thyroid association ) guidelines in 2010 state that contrast agents provide only ancillary data for the diagnosis of malignant thyroid nodules and offer only a modest improvement over the information obtained with traditional color doppler or power doppler examinations , and that the use of us contrast agents should be restricted to defining the size of necrotic areas in lesions after us - guided ablation procedures . the 2013 nccn guidelines did not discuss the role of ceus scanning in diagnosing thyroid lesions . chinese tc diagnosis and therapy guidelines in 2012 state that the diagnostic value of ceus scanning should be explored in future studies . the enhancement patterns of thyroid nodules in previous studies were classified into low - enhancement , iso - enhancement , and high - enhancement , or homogeneous , heterogeneous , ring - enhancing , and no - enhancement , but no combination of them was reported in detail . in fact , variable thyroid nodules were observed during ceus scanning , and it may be that every each scan shows specific pathological changes . the peripheral ring of the lesion has been reported in previous papers , and ring enhancement was considered predictive of benign lesions . however , several kinds of peripheral rings were found in our clinical works , besides the common regular high - enhancement ring ( e.g. , irregular high - enhancement ring and no - enhancement ring ) . we decided to analyze the contrast enhancement patterns of the lesions and its peripheries on the basis of pervious research to assess the diagnostic value of ceus in thyroid nodules . the study was approved by the local ethics committee and those participating gave written informed consent . patients were eligible for inclusion if they were scheduled for thyroid biopsy as a consequence of having a palpable thyroid nodule or an abnormal ultrasound scan of the thyroid gland showing at least 1 of the following characteristics : hypoechogenicity , calcification , irregular or microlobulated margin , intranodular vascularity , and taller than wide . patients were excluded if they were known to be allergic to sulfur hexafluoride microbubbles ( sonovue ) or had a coagulation disorder . a total of 159 patients were identified , but 11 were excluded because no definite diagnosis was available . the mean sd age of the remaining 148 patients was 45.410.5 years ( range , 1671 years ) . between them a sequoia 512 ultrasound machine ( acuson , sequoia 512 encompass , siemens , usa ) and a 15l8w probe ( 814 mhz ) were used to image all patients . the contrast agent used was 59 mg dry powder sonovue ( bracco s.p.a inc . , this has a low mechanical index ( 0.200.23 ) and was administered intravenously at the elbow . the size , position , boundaries , internal structure , and blood supply of lesions were assessed . the ultrasound machine was then switched to ceus mode and the resulting images displayed on the monitor alongside the conventional gray - scale images . the real - time microbubble perfusion within lesions and surrounding tissues were observed for a minimum of 2 minutes and recorded on the ultrasound machine s internal hard drive . the recorded ceus images were reviewed and the degree of enhancement of the thyroid solid lesions was classified as : no - enhancement , low - enhancement , iso - enhancement , and high - enhancement . when enhancement was present , it was further assessed for the degree of homogeneity , producing the following lesion enhancement categories : ( 1 ) homogeneous low - enhancement , ( 2 ) heterogeneous low - enhancement , ( 3 ) homogeneous iso - enhancement , ( 4 ) heterogeneous iso - enhancement , ( 5 ) homogeneous high - enhancement , and ( 6 ) heterogeneous high - enhancement . the peripheral rings of nodules were divided into : ( 1 ) regular high - enhancement ring , ( 2 ) irregular high - enhancement ring , ( 3 ) regular no - enhancement ring , and ( 4 ) irregular no - enhancement ring . conversely , irregular rings were misshapen and their thickness was non - uniform . following ceus , a conventional biopsy of the thyroid lesion was performed on all patients , using an 18-gauge biopsy needle ( biopty ; bard , covington , ga , usa ) powered by an automatic biopsy device . the puncture position and direction were determined and monitored by ultrasound and non - enhancing areas on ceus were avoided . the biopsy specimens were examined by a single specialized thyroid pathologist with more than 15 years of experience . the ultrasonographer , the reviewer of the recorded ceus images , and the pathologist were all blinded to patient history and to each other s findings . all statistical analyses were performed using the statistical package for the social sciences ( spss ) software package , version 11.5 for windows ( spss inc . , the differences between lesion enhancement categories and the diagnostic value of conventional us and ceus were analyzed by pearson s chi - squared test ( ) . the relationship between the size of lesions and the lesion enhancement category was analyzed by f test . the study was approved by the local ethics committee and those participating gave written informed consent . patients were eligible for inclusion if they were scheduled for thyroid biopsy as a consequence of having a palpable thyroid nodule or an abnormal ultrasound scan of the thyroid gland showing at least 1 of the following characteristics : hypoechogenicity , calcification , irregular or microlobulated margin , intranodular vascularity , and taller than wide . patients were excluded if they were known to be allergic to sulfur hexafluoride microbubbles ( sonovue ) or had a coagulation disorder . a total of 159 patients were identified , but 11 were excluded because no definite diagnosis was available . the mean sd age of the remaining 148 patients was 45.410.5 years ( range , 1671 years ) . between them a sequoia 512 ultrasound machine ( acuson , sequoia 512 encompass , siemens , usa ) and a 15l8w probe ( 814 mhz ) were used to image all patients . the contrast agent used was 59 mg dry powder sonovue ( bracco s.p.a inc . , this has a low mechanical index ( 0.200.23 ) and was administered intravenously at the elbow . the size , position , boundaries , internal structure , and blood supply of lesions were assessed . the ultrasound machine was then switched to ceus mode and the resulting images displayed on the monitor alongside the conventional gray - scale images . the real - time microbubble perfusion within lesions and surrounding tissues were observed for a minimum of 2 minutes and recorded on the ultrasound machine s internal hard drive . the recorded ceus images were reviewed and the degree of enhancement of the thyroid solid lesions was classified as : no - enhancement , low - enhancement , iso - enhancement , and high - enhancement . when enhancement was present , it was further assessed for the degree of homogeneity , producing the following lesion enhancement categories : ( 1 ) homogeneous low - enhancement , ( 2 ) heterogeneous low - enhancement , ( 3 ) homogeneous iso - enhancement , ( 4 ) heterogeneous iso - enhancement , ( 5 ) homogeneous high - enhancement , and ( 6 ) heterogeneous high - enhancement . the peripheral rings of nodules were divided into : ( 1 ) regular high - enhancement ring , ( 2 ) irregular high - enhancement ring , ( 3 ) regular no - enhancement ring , and ( 4 ) irregular no - enhancement ring . following ceus , a conventional biopsy of the thyroid lesion was performed on all patients , using an 18-gauge biopsy needle ( biopty ; bard , covington , ga , usa ) powered by an automatic biopsy device . the puncture position and direction were determined and monitored by ultrasound and non - enhancing areas on ceus were avoided . the biopsy specimens were examined by a single specialized thyroid pathologist with more than 15 years of experience . the ultrasonographer , the reviewer of the recorded ceus images , and the pathologist were all blinded to patient history and to each other s findings . all statistical analyses were performed using the statistical package for the social sciences ( spss ) software package , version 11.5 for windows ( spss inc . , chicago , il , usa ) . the differences between lesion enhancement categories and the diagnostic value of conventional us and ceus were analyzed by pearson s chi - squared test ( ) . the relationship between the size of lesions and the lesion enhancement category was analyzed by f test . eighty - two lesions ( from 75 patients ) were malignant : papillary thyroid carcinoma ( 81 lesions ) , and medullary thyroid carcinoma ( 1 lesion ) . seventy - five lesions ( from 73 patients ) were benign : nodular goiter ( 43 lesions ) , follicular thyroid adenoma ( 15 lesions ) , hashimoto thyroiditis ( ht ) ( 10 lesions ) , chronic inflammation ( 3 lesions ) , subacute thyroiditis ( 2 lesions ) , oncocytic thyroid adenoma ( 1 lesion ) , and postoperative stitches ( 1 lesion ) . there were 137 solid lesions , and their contrast enhancement patterns are shown in table 1 . for each enhancement pattern , the difference between the benign and malignant lesions was significant ( =42.1334 ; p=0.000 ) . solid lesions that were malignant were found to have the following enhancement patterns on ceus scanning : 70.37% ( 57/81 ) low - enhancement ( including 75.44% heterogeneous patterns and 24.56% homogeneous patterns ) , 22.22% ( 18/81 ) iso - enhancement ( including 66.67% heterogeneous patterns and 33.33% homogeneous patterns ) , and 7.41% ( 6/81 ) heterogeneous high - enhancement . eighteen of 20 heterogeneous iso - enhancement lesions displayed iso - enhancement with focal low - enhancement regions ; the low - enhancement area comprised < 50% lesion , and 12 ( 66.67% , 12/18 ) lesions were malignant . twenty lesions were cystic - solid mixed ( figure 5 ) . among them , 15 lesions were showed iso - enhancement coexisting with no - enhancement ( 14 benign , 1 malignant ) , and 5 lesion was showed no - enhancement . in this group , all lesions with homogeneous high - enhancement and no - enhancement patterns were benign . with regards to lesions with low - enhancement appearance as malignant , the diagnostic sensitivity , specificity , and accuracy for tc were 84.15% , 65.33% , and 75.16% , respectively , for ceus scanning . no ring enhancement was seen in 117 lesions ( 74 malignant , 43 benign ) that had ill - defined margins . irregular ring high - enhancement was seen in 2 lesions ( figure 4c ) ; both were malignant . regular ring non - enhancement was seen in 6 lesions ( figure 3b ) ; 4 ( 67% ) were benign and the other 2 ( 33% ) were malignant . the differences of peripheral ring enhancement between malignant and benign lesions were significant ( =22.66 , p=0.000 ) . with regards to lesions with irregular rings as malignant , the diagnostic sensitivity , specificity , and accuracy for tc were 100% , 94.12% , and 95% , respectively , for ceus scanning . with regards to lesions with low - enhancement and/or a peripheral irregular ring as malignant criterion , the diagnostic sensitivity , specificity , and accuracy for tc were 88% , 65.33% , and 88.32% , respectively , for ceus scanning , whereas they were 98.88% , 42.67% and 71.97% , respectively , for tc by conventional us . the misdiagnosis rate was 57.33% for conventional ultrasound scanning and 34.67% for ceus scanning ( =7.76 , p=0.005 ) . there was a significant difference in the size of thyroid lesions between low - enhancement , iso - enhancement , high - enhancement , iso - enhancement with no - enhancement area , and no - enhancement ( f=11.44 , p=0.000 ) ( table 2 ) . no significant difference was found between lesions with high - enhancement and iso - enhancement with a non - enhancing area ( p>0.05 ) . eighty - two lesions ( from 75 patients ) were malignant : papillary thyroid carcinoma ( 81 lesions ) , and medullary thyroid carcinoma ( 1 lesion ) . seventy - five lesions ( from 73 patients ) were benign : nodular goiter ( 43 lesions ) , follicular thyroid adenoma ( 15 lesions ) , hashimoto thyroiditis ( ht ) ( 10 lesions ) , chronic inflammation ( 3 lesions ) , subacute thyroiditis ( 2 lesions ) , oncocytic thyroid adenoma ( 1 lesion ) , and postoperative stitches ( 1 lesion ) . there were 137 solid lesions , and their contrast enhancement patterns are shown in table 1 the difference between the benign and malignant lesions was significant ( =42.1334 ; p=0.000 ) . solid lesions that were malignant were found to have the following enhancement patterns on ceus scanning : 70.37% ( 57/81 ) low - enhancement ( including 75.44% heterogeneous patterns and 24.56% homogeneous patterns ) , 22.22% ( 18/81 ) iso - enhancement ( including 66.67% heterogeneous patterns and 33.33% homogeneous patterns ) , and 7.41% ( 6/81 ) heterogeneous high - enhancement . eighteen of 20 heterogeneous iso - enhancement lesions displayed iso - enhancement with focal low - enhancement regions ; the low - enhancement area comprised < 50% lesion , and 12 ( 66.67% , 12/18 ) lesions were malignant . twenty lesions were cystic - solid mixed ( figure 5 ) . among them , 15 lesions were showed iso - enhancement coexisting with no - enhancement ( 14 benign , 1 malignant ) , and 5 lesion was showed no - enhancement . in this group , all lesions with homogeneous high - enhancement and no - enhancement patterns were benign . with regards to lesions with low - enhancement appearance as malignant , the diagnostic sensitivity , specificity , and accuracy for tc were 84.15% , 65.33% , and 75.16% , respectively , for ceus scanning . no ring enhancement was seen in 117 lesions ( 74 malignant , 43 benign ) that had ill - defined margins . irregular ring high - enhancement was seen in 2 lesions ( figure 4c ) ; both were malignant . regular ring non - enhancement was seen in 6 lesions ( figure 3b ) ; 4 ( 67% ) were benign and the other 2 ( 33% ) were malignant . the differences of peripheral ring enhancement between malignant and benign lesions were significant ( =22.66 , p=0.000 ) . with regards to lesions with irregular rings as malignant , the diagnostic sensitivity , specificity , and accuracy for tc were 100% , 94.12% , and 95% , respectively , for ceus scanning . with regards to lesions with low - enhancement and/or a peripheral irregular ring as malignant criterion , the diagnostic sensitivity , specificity , and accuracy for tc were 88% , 65.33% , and 88.32% , respectively , for ceus scanning , whereas they were 98.88% , 42.67% and 71.97% , respectively , for tc by conventional us . the misdiagnosis rate was 57.33% for conventional ultrasound scanning and 34.67% for ceus scanning ( =7.76 , p=0.005 ) . there was a significant difference in the size of thyroid lesions between low - enhancement , iso - enhancement , high - enhancement , iso - enhancement with no - enhancement area , and no - enhancement ( f=11.44 , p=0.000 ) ( table 2 ) . no significant difference was found between lesions with high - enhancement and iso - enhancement with a non - enhancing area ( p>0.05 ) . ceus enhancement patterns were found to be different in benign and malignant thyroid lesions , but its contribution to the diagnosis of thyroid cancer is controversial . in our study , we found a variety of patterns of contrast enhancement in benign and malignant thyroid lesions . two of these patterns , iso - enhancement with a focal low - enhancement region within the lesion and a non - enhancing ring in the periphery of the lesion , have not been reported previously . we found the pattern of iso - enhancement with a focal low - enhancing area to be common with malignant lesions . on biopsy , both the focal low - enhancing area and the surrounding part of the lesion showing iso - enhancement contained malignant cells , and the focal low - enhancing area also showed evidence of interstitial fibrosis . hypo - vascularity is often observed in focal ht , which is associated with focal hypothyroidism with severe follicular degeneration . twenty lesions were benign , including 2 that were ht , 2 that were chronic inflammation , 1 case of subacute thyroiditis , and 1 postoperative suture . these lesions demonstrated heterogeneous low - enhancement within the lesion , without a peripheral ring . . the enhancement pattern in the periphery of lesions may provide useful diagnostic information , especially for those lesions with internal iso - enhancement appearances . regular high - enhancing rings were only found in benign lesions , especially in adenoma and nodular goiter . the vascularity of the surface capsule or the compressed tissue around lesion is the explanation for the high - enhancement ring at the periphery of the lesion . the ability of ceus to detect these features is useful in differentiating cancer from benign lesions . regular no - enhancement could be found both in benign and malignant nodules . in this group , 6 nodules had peripheral regular no - enhancement rings , in which 4 were benign ( 2 were adenoma ) and 2 malignant ( all were ptc ) . the presence of a non - enhancing ring in the periphery of lesions may reflect changes in tissues adjacent to the lesion , which might be a consequence of inflammatory exudate , interstitial edema , or mucoid degeneration because of strong pressure from the nodule . the pathological cause for an irregular non - enhancing peripheral ring is unclear , but it is possible that it reflects irregular invasion of cancerous tissue into adjacent tissues . peripheral irregular high - enhancement rings may result from interstitial angiogenesis hyperplasia caused by the cancerous cells invasion . generally , the visible range of lesions was larger on ceus than on gray - scale us in lesions with irregular high - enhancement ring . one of 15 cystic - solid mixed lesions in this group was ptc , and it showed iso - enhancement with non - enhancing areas . on conventional us , some tiny spots of blood signals were shown in the solid component of the lesion . with ceus scanning , the solid component showed heterogeneous iso - enhancement without a peripheral ring . the other 14 benign lesions showed homogeneous iso - enhancement in solid component with regular high - enhancing rings or non - enhancing rings . all lesions that failed to show any enhancement were benign . on conventional us scanning , they had a cystic - solid appearance . pathological results showed that many of these lesions were nodular goiters with areas of hemorrhage and the ceus appearances , suggesting that the apparently solid areas were not perfused by contrast agent . was related to the size of lesions , which is in agreement with a previous report . in our study , most malignant lesions showing low - enhancement were papillary thyroid microcarcinomas , perhaps due to the small or immature vascular network in microcarcinomas . the compression of blood vessels by cancer cells can increase resistance to blood flow and impair blood supply . conventional us detected tc with a high sensitivity , so the misdiagnosis rate is our concern . this study showed that ceus can clearly reduce the misdiagnosis rate compared with conventional us ( p<0.01 ) . our study shows that : ( 1 ) with ceus , the appearances of ht and other inflammatory disorders of the thyroid gland overlap with those of malignant thyroid disease . ( 2 ) ceus appearances of homogeneous hyper- , iso- , and no- enhancement within lesions and/or regular high - enhancement ring in the peripheries of lesions are suggestive of benign thyroid lesions . if there is doubt , conventional us scanning these appearances on ceus scanning may be helpful pre - operatively . ( 3 ) lesions showing iso - enhancement with a focal low - enhancement region should be considered malignant if inflammatory disease has been excluded . in patients with suspicious us characteristics , ceus had high specificity and contributed to establishing the diagnosis .

backgroundthe aim of this study was to investigate the accuracy of contrast - enhanced ultrasound ( ceus ) enhancement patterns in the assessment of thyroid nodules.material/methodsa total of 158 patients with suspected thyroid cancer underwent conventional ultrasound ( us ) and ceus examinations . the contrast enhancement patterns of the lesions , including the peripheries of the lesions , were assessed by ceus scans . the relationship between the size of the lesions and the degree of enhancement was also studied . us- and/or ceus - guided biopsy was used to obtain specimens for histopathological diagnosis.resultsthe final data included 148 patients with 157 lesions . seventy - five patients had 82 malignant lesions and 73 patients had 75 benign lesions . peripheral ring enhancement was seen in 40 lesions . the differences of enhancement patterns and peripheral rings between benign and malignant nodules were significant ( p=0.000 , 0.000 ) . the diagnostic sensitivity , specificity , and accuracy for malignant were 88% , 65.33% , and 88.32% , respectively , for ceus , whereas they were 98.33% , 42.67% , and 71.97% , respectively , for tc by conventional us . the misdiagnosis rate by conventional us was 57.33% and 34.67% by ceus ( p=0.005 ) . with regard to the size of lesions , a significant difference was found between low - enhancement , iso - enhancement , high - enhancement , iso - enhancement with no - enhancement area and no - enhancement ( p=0.000).conclusionsin patients with suspicious us characteristics , ceus had high specificity and contributed to establishing the diagnosis . therefore , ceus could avoid unnecessary biopsy .

Background Material and Methods Subjects Ultrasound imaging Image interpretation and analysis Thyroid biopsies Statistical analysis Results Pathological results Internal enhancement patterns of thyroid lesions Peripheral enhancement patterns of thyroid lesions Comparisons between conventional ultrasound scanning and CEUS scanning Enhancement patterns and size of thyroid lesions Discussion Conclusions

a 63-year - old woman with chronic atrial fibrillation treated with warfarin was admitted to emergency for coma and complete vertical gaze palsy . investigations : brain ct and mri , echo - colour doppler sonography of the supraaortic vessels , angio - ct of the intracranial vessels , eeg , transesophageal echocardiogram , biohumoral tests . brain ct and mri scans showed bilateral thalamic lesions with involvement of the right midbrain ; eeg showed a diffuse alpha rhythm prevalent on the posterior regions ; echo - colour doppler sonography of the supraaortic vessels showed marked reduction of blood flow in the right vertebral artery ; angio - ct scans showed occlusion of the right vertebral artery and a significant filling defect of the first part of the right posterior cerebral artery ( p1 ) from which the artery of percheron arises . a follow - up a 63-year - old , caucasian woman with chronic atrial fibrillation treated with warfarin was referred to our hospital for neurological evaluation on new year 's eve . the symptoms had begun while the patient had been sitting in front of the fireplace , with sudden onset of somnolence and inability to maintain an upright position . after a few minutes , the patient was helped into bed and presented sudden loss of consciousness . judging from a first neurological evaluation , she had a glasgow coma scale score of 7 . she moved the upper and lower limbs upon a painful stimulus without being able to localize it . the eyes were maintained closed and she appeared to be in a state of deep sleep , breathing heavily and snoring . the following laboratory tests were unremarkable : complete blood cell count , glycaemia , electrolytes , liver enzymes , creatinine , ammonia , arterial blood gas including carboxyhaemoglobin value , ethanol and benzodiazepine levels ; inr was 1.92 . brain ct scan performed on the day of admission was normal , in particular without early signs of ischemia . eeg performed on the following day showed a diffuse alpha rhythm prevalent on the posterior regions and a beta rhythm on the anterior regions with some occasionally interspersed theta waves . echo - colour doppler sonography of the supraaortic vessels showed orthodromic and asymmetric blood flow in the vertebral arteries with marked reduction in the right vertebral artery . a new brain ct scan performed 24 h later showed an ill - defined hypodensity in both thalami . a brain mri performed the same day demonstrated a large hyperintensity , on axial flair images , of the right mesencephalic peduncle and of the thalamic nuclei bilaterally ( fig . an angio - ct of the intracranial vessels , performed two days after the patient 's admission , confirmed occlusion of the right vertebral artery with a patent basilar artery but showed stenosis of the first part ( p1 ) of the right posterior cerebral artery ( fig . a second angio - ct done three weeks later confirmed occlusion of the right vertebral artery while the filling defect of the right posterior cerebral artery was no longer evident ( fig . transesophageal echocardiography was normal , showing no cardiac source of embolism and no patent foramen ovale . the patient presented an acute onset of coma and vertical gaze palsy with no other focal signs . blood examinations excluded a possible metabolic , toxic , infective , endocrine or iatrogenic aetiology of the comatose state . brain ct scan and mri showed bilateral thalamic lesions with involvement of the right midbrain that can be caused by several conditions : ischaemic stroke , deep cerebral vein thrombosis , thiamine deficiency , cerebral lupus , toxoplasmosis , cysticercosis , cerebral syphilitic gumma and tumours . presence of an alpha activity on the eeg can be seen in patients with vascular brainstem lesions , cerebral concussion , cerebral hypoxia and drug intoxication . it is known that alpha activity in comatose patients with drug intoxication is distributed predominantly over the frontal lobes . on the contrary , alpha activity in patients with brainstem lesions has similar features to the one of healthy normal subjects with a higher expression in the posterior regions . in our patient , alpha activity was predominant on the posterior regions supporting the hypothesis of a vascular brainstem lesion . angio - ct scans showed occlusion of the right vertebral artery and a filling defect of the p1 segment of the right posterior cerebral artery that disappeared after 25 days . on the basis of clinical , neuroimaging and neurovascular findings , the only possible diagnosis was an ischaemic stroke in the territory of the artery of percheron due to an embolic occlusion or hypoperfusion following a thrombosis of the right vertebral artery . the arterial supply to the thalamus is provided by perforating branches from the posterior cerebral artery and the posterior communicating artery . the p1 segment of the posterior cerebral artery , which connects the basilar artery with the posterior communicating artery , is anatomically a prolongation of the basilar artery . kaplan and ford named this arterial segment the mesencephalic artery ; percheron called it basilar communicating artery . the paramedian thalamic arteries arise from the basilar communicating artery and may share a common origin with the superior paramedian mesencephalic arteries that supply the medial areas of the upper brainstem . type i is a symmetrical arrangement : one paramedian thalamic artery arises on each side from the corresponding basilar communicating artery . type ii is asymmetrical : the right and left paramedian thalamic arteries arise from the same basilar communicating artery , separately or by a common trunk which then bifurcates . in this case , the two paramedian thalamic territories are supplied via only one basilar communicating artery . finally , in type iii , an arterial arcade connects the two basilar communicating arteries and gives rise to the paramedian arteries . in addition to the medial thalamic nuclei , these paramedian thalamic and mesencephalic arteries supply the interpeduncular nucleus , the decussation of the superior cerebellar peduncles , the medial part of the red nucleus , the third and fourth cranial nerve nuclei and the anterior portion of the periaqueductal grey matter . occlusion of type ii artery of percheron often results in bilateral infarctions in the middle aspects of thalami and brainstem . in fact , when paramedian thalamic infarcts are bilateral , there is either an unpaired thalamic perforator or the occlusion involves the bifurcation of the basilar artery . this condition is rare and none of the reports published through the years were able to demonstrate in vivo an occlusion of the artery of percheron , which could only be presumed in patients with bilateral thalamic lesions . were the only authors who demonstrated a patent unpaired thalamic perforator artery on superselective digital subtraction angiography in a patient with bilateral thalamic infarcts . patients with paramedian thalamic and midbrain infarctions have a specific clinical picture : loss of consciousness or somnolence at stroke onset , frequently followed by eye movement disturbances such as upward gaze palsy and/or skew deviation with pupillary abnormalities , but few motor or sensory deficits . long - term follow - up showed orientation and concentration deficits , memory dysfunction and behavioural disturbances . according to kumral et al . , the main cause of bilateral thalamic infarction is small artery disease followed by cardioembolism . aetiological diagnosis was done in our case only two days after the onset , as in most cases , because of complex and nonspecific clinical presentation ; nonetheless , the presence of residual stenosis of the basilar communicating artery followed by complete recanalization allowed us to diagnose an embolic occlusion . in our patient , the ischaemic lesions have been caused by a partial occlusion of the basilar communicating artery giving an occlusion or hypoperfusion of the artery of percheron . it is highly probable that the embolus departed during the thrombogenesis process of the ipsilateral vertebral artery . as in previous reports of paramedian thalamic and midbrain infarctions , symptom onset was characterized by somnolence , unsteadiness and subsequent coma with vertical gaze palsy . various names , such as top of the basilar syndrome and mesencephalothalamic syndrome , have been used to describe the clinical syndromes associated with paramedian thalamic and midbrain infarction . the clinical picture is variable and can present in various confusing ways , so careful attention to the differential diagnosis could be extremely important for the benefit of patients . thrombolysis was not done , even if intraarterial thrombolysis could have been considered . in any case , we suggest that an urgent vascular diagnosis , by means of urgent angio - ct , could have helped in the decision - making process . in this paper we present a case of bilateral paramedian thalamic and right mesencephalic infarction . when bilateral thalamic infarcts are found , occlusion of the artery of percheron should be considered as the main cause . in the majority of cases , the main clinical features of the occlusion of the artery of percheron are coma and vertical gaze palsy due to the involvement of the rostral midbrain tegmentum including the interstitial nucleus of cajal [ 10 , 11 ] . in our case , the occlusion of the artery of percheron was only indirectly diagnosed , since this artery is often below the resolution of the angio - ct . we think that performing an angio - ct could be a successful choice when a patient with unconsciousness and vertical gaze palsy is brought in for neurological evaluation . in these cases , the diagnosis is often made many hours or even days after the clinical onset , while the appropriate diagnosis should be done at the earliest possible stage , since the efficacy of thrombolysis decreases rapidly as time passes by . if the diagnosis is made in the shortest time possible , the thrombolytic therapy can still be done and the outcome can be fairly good . thus we strongly suggest to always consider angio - ct in the evaluation of emergency patients with sudden onset of coma and vertical gaze palsy .

backgrounda 63-year - old woman with chronic atrial fibrillation treated with warfarin was admitted to emergency for coma and complete vertical gaze palsy . investigations : brain ct and mri , echo - colour doppler sonography of the supraaortic vessels , angio - ct of the intracranial vessels , eeg , transesophageal echocardiogram , biohumoral tests . brain ct and mri scans showed bilateral thalamic lesions with involvement of the right midbrain ; eeg showed a diffuse alpha rhythm prevalent on the posterior regions ; echo - colour doppler sonography of the supraaortic vessels showed marked reduction of blood flow in the right vertebral artery ; angio - ct scans showed occlusion of the right vertebral artery and a significant filling defect of the first part of the right posterior cerebral artery ( p1 ) from which the artery of percheron arises . a follow - up angio - ct showed a complete recanalization of p1 . diagnosis : percheron artery syndrome . treatment and management : aspirin , neurorehabilitation .

Background The Case Discussion of Diagnosis Treatment and Management Conclusion

several epidemiological studies have confirmed the close relationship between diabetes and obesity ; more than 80% of patients with type 2 diabetes are estimated to be overweight or obese . as the prevalence of obesity increases , the risk of type 2 diabetes is likely to increase . between 1980 and 1990 , the majority of korean patients with diabetes were nonobese ; however , as the prevalence of obesity has increased in korea , the majority of patients with diabetes are now obese . according to the diabetes fact sheet in korea 2012 , the prevalence of type 2 diabetes was 10.1% in 2010 , and 75% of diabetic patients were overweight or obese ( i.e. , a mean body mass index [ bmi ] of 25.2 kg / m ) . type 2 diabetes and obesity have a complex relationship , and obesity is linked to insulin resistance , the precursor to type 2 diabetes . obese adults are at increased risk of several chronic diseases , including cardiovascular disease , stroke , cancer , and type 2 diabetes . furthermore , the risk of death is increased 20% to 40% in overweight adults and 2- to 3-fold in obese compared with normal weight adults . thus , treatment of obesity is crucial for the prevention of several health problems and for delayed onset of diabetes . moreover , weight reduction may affect the incidence of diabetes . according to the diabetes prevention program study , every kilogram of weight loss is correlated with a 16% reduction in the development of type 2 diabetes . moderate weight reduction in obese patients with type 2 diabetes is associated with a decrease in insulin resistance , improved glycemic parameters , and reductions in diabetic complications and several cardiovascular disease - related risk factors . the action for health diabetes ( look ahead ) study found that an 8.6% weight loss together with intensive lifestyle intervention was associated with significant improvements in cardiovascular risk factors in 5,145 overweight or obese participants with type 2 diabetes . it is difficult for patients with diabetes to reduce and maintain their weight with lifestyle changes alone . several oral hypoglycemic agents used to treat diabetes , such as sulfonylureas , glinides , and thiazolidinediones , and insulin are associated with significant weight gain that may impair metabolic conditions . thus , alternative antiobesity medications or bariatric surgery may be a necessary adjunct for obese patients with diabetes . several placebo - controlled studies have shown that various medications help promote weight loss in obese patients with type 2 diabetes . the focus of our review is antiobesity medications for the long - term treatment of obese patients with type 2 diabetes . the us food and drug administration ( fda ) approved the following drugs for use in adults with a bmi 30 ( obese ) or 27 ( overweight ) who had at least one weight - related condition such as type 2 diabetes , hypertension , or dyslipidemia . the recommended dose is 120 mg three times daily before meals . a lower dose ( 60 mg ) is available as an over - the - counter preparation in the united states . one- and 2-year studies evaluating orlistat treatment for obesity have shown significant improvements in glycemic control and in blood pressure and lipid profiles . in a large 4-year prospective study of 3,305 participants with bmi of 30 kg / m and normal or impaired glucose tolerance ( the xenical in the prevention of diabetes in obese subjects study ) , the orlistat - treated group exhibited significant mean weight loss ( 5.8 kg vs. 3.0 kg in the placebo group ; p<0.001 ) and reduced progression to type 2 diabetes ( risk reduction of 37.3% in the orlistat group ; p=0.0032 ) . a korean study in which patients were administered orlistat for 24 weeks reported a modest but significant mean weight loss of -2.73 kg ( mean weight change , -3.50%0.38% ) and significant improvements in lipid profiles , fasting insulin , waist circumference , blood pressure , fasting plasma glucose ( -10.414.62 mg / dl ) , and glycosylated hemoglobin ( hba1c , -0.87% ) . the most common adverse effects of orlistat include flatus with discharge and oily spotting or fecal urgency , and the drug interferes with the absorption of fat soluble vitamins , although not tremendously . lorcaserin is a selective serotonin 2c ( 5-ht2c ) receptor agonist that suppresses appetite via stimulation of melanocortin receptor 4 . the drug has low affinity for the 5-ht2b receptor , which is associated with the development of valvular heart disease and the cause of the withdrawal of agents such as dexfenfluramine . in 2012 , the fda approved lorcaserin 10 mg twice daily based on the findings of the pivotal randomized , placebo - controlled trials : behavioral modification and lorcaserin for overweight and obesity management ( bloom ) , behavioral modification and lorcaserin second study for obesity management ( blossom ) , and bloom - diabetes mellitus ( bloom - dm ) . the bloom and blossom trials investigated the effect of lorcaserin on overweight or obese nondiabetic patients and found a ~3% placebo - corrected weight change in the group treated with lorcaserin ( 20 mg / day ) ( bloom , -3.6% ; blossom , -2.9% ; both p<0.05 ) . moreover , a significantly higher proportion of patients in the lorcaserin group lost 5% of their baseline body weight compared with the placebo group ( approximately 47% vs. 20% , respectively in the bloom ; 47% vs. 25% , respectively in the blossom trials ) . furthermore , improvements in fasting glucose and triglyceride levels and blood pressure were observed in the lorcaserin groups . the bloom - dm study included 604 overweight and obese patients with type 2 diabetes who were treated with metformin and/or sulfonylurea and randomized to lorcaserin 10 or 20 mg / day or placebo groups for 52 weeks . consistent with the results of the bloom and blossom trials , the lorcaserin - treated groups achieved weight losses of -3.4% ( 10 mg ) and -3.1% ( 20 mg ) . a significantly higher proportion of patients in the lorcaserin groups lost 5% of their initial body weight ( 10 mg , 44.7% ; 20 mg , 37.5% ) compared with patients administered placebo ( 16.1% ) . the reduction in mean hba1c was significantly greater in the lorcaserin - treated groups than in the placebo group ( 10 mg , -1.0% change from baseline ; 20 mg , -0.9% change ; and placebo , -0.4% change ; p<0.001 ) . a significantly higher proportion of patients in the lorcaserin groups achieved hba1c 7% compared with patients treated with placebo ( 10 mg , 52.2% ; 20 mg , 50.4% ; and placebo , 26.3% ) . however , symptomatic hypoglycemia was more frequent in the lorcaserin groups ( 10 mg , 10.5% ; 20 mg , 7.4% ) than in the placebo group ( 6.3% ) . the adverse events associated with lorcaserin include headache , dizziness , fatigue , nausea , dry - mouth , and constipation . no statistically significant difference in the occurrence of fda - defined valvular disease was found between the lorcaserin and placebo groups . however , the postmarketing trials were required to evaluate the long - term cardiovascular effects of lorcaserin . phentermine / topiramate ( pt ) extended - release ( er ) is a combination of the fda - approved medications phentermine and topiramate in an er formulation . phentermine is indicated for short - term weight loss in overweight or obese adults who exercise and eat a reduced calorie diet . topiramate is indicated for the treatment of certain types of seizures in people who have epilepsy and to prevent migraine headaches . the exact mechanism underlying the weight loss action of topiramate is not known ; however , it may be associated with a combination of features such as an effect on sodium channels , enhancement of gaba - activated chloride channels , and inhibition of carbonic anhydrase isoenzymes . pt is available at a higher dose ( 15 mg phentermine/92 mg topiramate er ) for select patients . discontinuation or an increase in dosage is recommended for patients who have not lost 3% of their baseline weight . treatment should be discontinued if a weight loss > 5% of baseline is not achieved with 15/92 mg per day . fda approval of pt was based on three phase iii clinical trials performed to evaluate the efficacy of the drug ( equip , conquer , sequel ) . in the equip trial , 1,276 obese adults without diabetes were assigned randomly to a low- ( 3.75/23 mg / day ) or high- ( 15/92 mg / day ) dose pt or placebo group for 56 weeks . at the conclusion of the study , the patients in the low- and high - dose pt and placebo groups lost 5.1% , 10.9% , and 1.6% of their baseline body weight , respectively ( p<0.0001 ) . moreover , the results revealed improvements in waist circumference , triglyceride levels , and blood pressure . the conquer trial included 2,487 overweight and obese patients with two or more weight - related comorbidities such as hypertension , prediabetes , type 2 diabetes , dyslipidemia , and abdominal adiposity . the participants were assigned to pt ( 7.5/46 or 15/92 mg ) or placebo groups for 56 weeks . at the conclusion of the trial , the subjects in the 7.5/46 mg group achieved a -8.1 kg change in body weight ( mean change -7.8% ) , those in the 15/92 mg group a -10.2 kg change ( mean change -9.8% ) , and those in the placebo group a -1.4 kg change ( mean change -1.2% ) . a higher percentage of subjects in the pt groups achieved a 5% weight loss after 1 year compared with those receiving placebo ( 62% , 70% , and 21% in the 7.5/46 , 15/92 mg , and placebo groups , respectively ; p<0.001 for both groups vs. placebo ) . moreover , weight loss was maintained for a second year in the sequel trial , a 52-week extension of the conquer trial . the conquer trial revealed an overall significant improvement in hba1c ( % change from baseline : 0% , -0.1% , and 0.1% in the 7.5/46 , 15/92 mg , and placebo groups , respectively ; p<0.0001 for both groups vs. placebo ) and in fasting glucose levels ( % change from baseline : -0.01% , -0.07% , and 0.13% in the 7.5/46 , 15/92 mg , and placebo groups , respectively ; p=0.0047 for 7.5/46 mg vs. placebo and p<0.0001 for 15/92 mg vs. placebo ) at week 56 . the progression to type 2 diabetes among participants without diabetes was lower in the pt - treated patients ( 2.8% in the 7.5/46 mg and 1.7% in the 15/92 mg ) compared with those receiving the placebo ( 3.6% ) . the relative risk of type 2 diabetes versus placebo was 0.78 ( 95% confidence interval [ ci ] , 0.40 to 1.50 ) for the 7.5/46 mg and 0.47 ( 95% ci , 0.25 to 0.88 ) for the 15/92 mg doses of pt . an improvement in glycemic control was observed in the sequel trial . at the conclusion of the trial ( 108 weeks ) , fasting glucose levels were significantly reduced from baseline in the 15/92 mg dose group ( the least - squared change in fasting glucose was 0.1 in the 7.5/46 mg , -1.2 in the 15/92 mg , and 3.7 in the placebo groups ; p=0.0048 for 15/92 mg vs. placebo ) . in the participants without type 2 diabetes at baseline , the progression of type 2 diabetes decreased during the 1-year extension . the annualized incidence rates for progression to type 2 diabetes were 0.9% , 1.7% , and 3.7% in the 7.5/46 , 15/92 mg , and placebo groups , respectively , among the study participants without diabetes at baseline . these results revealed a 54% reduction in the progression to type 2 diabetes in the low- ( 7.5/46 mg ) and a 76% reduction in the high - dose ( 15/92 mg ) groups compared with the placebo group . among participants with type 2 diabetes at baseline , hba1c did not change markedly from baseline in the placebo group ( 0% ) , whereas treatment with 7.5/46 and 15/92 mg pt led to 0.4% and 0.2% reductions in hba1c , respectively . however , the results of the sequel trial should be interpreted with caution because , as an extension of the conquer trial , it may be subject to selection bias . common drug - related side effects include paresthesias , nausea , dizziness , constipation , and dry mouth . the reported neuropsychiatric adverse events include depression , anxiety , insomnia , and disturbances in attention . furthermore , pt may increase heart rate by two beats per minute ; however , the fda judged that the potential risk posed by an elevated heartbeat was outweighed by improvements in cardiovascular risk factors such as blood pressure . in september 2014 , the fda approved naltrexone / bupropion ( nb ) sustained - release ( sr ) tablets as a treatment option for chronic weight management . naltrexone was approved previously to treat alcohol and opioid dependence , and bupropion was approved for the treatment of depression or as an aid for smoking cessation . naltrexone is an opioid receptor antagonist , and bupropion acts on adrenergic and dopaminergic receptors in the hypothalamus . the combining of bupropion and naltrexone was based on the theory that naltrexone may block the compensatory mechanisms associated with bupropion that prevent sustained weight loss . the efficacy of nb was evaluated by the contrave obesity research ( cor ) program consisting of the cor - i , cor - ii , cor trial with intensive behavior modification ( cor - bmod ) , and cor - diabetes trials ( table 1 ) . the cor - i , cor - ii , and cor - bmod trials evaluated overweight / obese patients without diabetes . following treatment for 1 year , the group receiving naltrexone 32 mg / bupropion 360 mg lost 4 to 5 kg ( approximately -4% weight loss from baseline body weight ) more than the placebo group , and 48% to 66% of the patients in the nb group lost 5% of their baseline body weight . the cor - diabetes trial evaluated the safety and efficacy of nb in 505 overweight or obese patients with type 2 diabetes over a 56-week period . participants were assigned to the naltrexone 32 mg sr / bupropion 360 mg sr or placebo group . compared with the placebo group , the patients treated with nb lost significantly more weight ( -5.0% vs. -1.8% ; p<0.001 ) , and a higher proportion lost 5% of their body weight ( 44.5% vs. 18.9% ; p<0.001 ) . furthermore , baseline hba1c was significantly reduced in the patients receiving nb compared with the placebo group ( -0.6% vs. -0.1% , respectively ; p<0.001 ) , and 44.1% of patients receiving nb achieved < 7% hba1c compared with 26.3% in the placebo group ( p<0.001 ) . the cardiovascular safety of naltrexone 32 mg sr / bupropion 360 mg sr is currently under investigation ( clinical trial reg no . the most common adverse events include nausea , constipation or diarrhea , headache , vomiting , dizziness , insomnia , and dry mouth . the nb provides a boxed warning to alert users to the increased risk of suicidal thoughts and behaviors associated with bupropion . the nb has a dose - related risk of seizures and may elevate blood pressure and heart rate . liraglutide is a human glucagon - like peptide 1 analog that may be administered once a day because of its prolonged half - life of 13 hours . the efficacy of the drug was well established in the liraglutide effect and action in diabetes ( lead ) studies , a series of phase iii randomized , controlled trials . the approved doses for the treatment of diabetes are 1.2 and 1.8 mg in the united states and 0.6 and 0.9 mg in japan . the lead-3 trial comprised 746 patients with type 2 diabetes assigned to liraglutide ( 1.2 or 1.8 mg ) or glimepiride ( 8 mg ) treatment groups for 52 weeks . the hba1c reductions from baseline were -0.84% and -1.14% in the 1.2 and 1.8 mg groups , respectively , and body weight was reduced by -2.05 kg from baseline in the 1.2 mg group and -2.45 kg in the 1.8 mg group , whereas a significant weight gain ( 1.12 kg ) was observed in patients treated with glimepiride . the recommend dose is 3 mg in contrast to 1.2 or 1.8 mg for diabetes . the satiety and clinical adiposity - liraglutide evidence in nondiabetic and diabetic subjects ( scale ) trial consists of four clinical trials designed to demonstrate the safety and efficacy of liraglutide 3 mg for weight management . the scale obesity and prediabetes phase iiia trial involving 3,731 patients found that patients treated with liraglutide lost an average of 8% of their body weight in 56 weeks compared with 2.6% in the placebo group . moreover , significantly more patients in the liraglutide group lost 5% of their baseline body weight compared with those taking the placebo ( 63.5% vs. 26.6% , respectively ) , and 32.8% of the liraglutide group lost > 10% of their baseline body weight ( 10.1% in the placebo group ) . the scale - diabetes trial was a 56-week randomized , placebo - controlled trial designed to investigate the potential of liraglutide 3 mg in obese or overweight patients with type 2 diabetes . in this trial , 846 overweight or obese patients with diabetes were assigned randomly to liraglutide 3 mg or 1.8 mg or placebo groups . after 56 weeks , the 3 and 1.8 mg liraglutide groups achieved weight loses of 6% and 5% , respectively , compared with 2% in the placebo group . furthermore , 50% of the patients in the 3 mg group and 35% of those in the 1.8 mg group achieved 5% weight loss compared with 13% in the placebo group , and approximately 69% of the patients treated with liraglutide 3 mg achieved the hba1c target of < 7% ( clinical trial reg no . concerned by the lack of long - term data for liraglutide in the treatment of obesity and a variety of safety issues , such as gallbladder disease , pancreatitis , breast and thyroid cancers , and increased heart rate , the fda advisory panel recommended that these issues be addressed in postmarketing studies . however , the panel agreed that the ongoing cardiovascular outcomes trial of the liraglutide effect and action in diabetes : evaluation of cardiovascular outcome results study would be sufficient to characterize the cardiovascular risk of the liraglutide . the recommended dose is 120 mg three times daily before meals . a lower dose ( 60 mg ) is available as an over - the - counter preparation in the united states . one- and 2-year studies evaluating orlistat treatment for obesity have shown significant improvements in glycemic control and in blood pressure and lipid profiles . in a large 4-year prospective study of 3,305 participants with bmi of 30 kg / m and normal or impaired glucose tolerance ( the xenical in the prevention of diabetes in obese subjects study ) , the orlistat - treated group exhibited significant mean weight loss ( 5.8 kg vs. 3.0 kg in the placebo group ; p<0.001 ) and reduced progression to type 2 diabetes ( risk reduction of 37.3% in the orlistat group ; p=0.0032 ) . a korean study in which patients were administered orlistat for 24 weeks reported a modest but significant mean weight loss of -2.73 kg ( mean weight change , -3.50%0.38% ) and significant improvements in lipid profiles , fasting insulin , waist circumference , blood pressure , fasting plasma glucose ( -10.414.62 mg / dl ) , and glycosylated hemoglobin ( hba1c , -0.87% ) . the most common adverse effects of orlistat include flatus with discharge and oily spotting or fecal urgency , and the drug interferes with the absorption of fat soluble vitamins , although not tremendously . lorcaserin is a selective serotonin 2c ( 5-ht2c ) receptor agonist that suppresses appetite via stimulation of melanocortin receptor 4 . the drug has low affinity for the 5-ht2b receptor , which is associated with the development of valvular heart disease and the cause of the withdrawal of agents such as dexfenfluramine . in 2012 , the fda approved lorcaserin 10 mg twice daily based on the findings of the pivotal randomized , placebo - controlled trials : behavioral modification and lorcaserin for overweight and obesity management ( bloom ) , behavioral modification and lorcaserin second study for obesity management ( blossom ) , and bloom - diabetes mellitus ( bloom - dm ) . the bloom and blossom trials investigated the effect of lorcaserin on overweight or obese nondiabetic patients and found a ~3% placebo - corrected weight change in the group treated with lorcaserin ( 20 mg / day ) ( bloom , -3.6% ; blossom , -2.9% ; both p<0.05 ) . moreover , a significantly higher proportion of patients in the lorcaserin group lost 5% of their baseline body weight compared with the placebo group ( approximately 47% vs. 20% , respectively in the bloom ; 47% vs. 25% , respectively in the blossom trials ) . furthermore , improvements in fasting glucose and triglyceride levels and blood pressure were observed in the lorcaserin groups . the bloom - dm study included 604 overweight and obese patients with type 2 diabetes who were treated with metformin and/or sulfonylurea and randomized to lorcaserin 10 or 20 mg / day or placebo groups for 52 weeks . consistent with the results of the bloom and blossom trials , the lorcaserin - treated groups achieved weight losses of -3.4% ( 10 mg ) and -3.1% ( 20 mg ) . a significantly higher proportion of patients in the lorcaserin groups lost 5% of their initial body weight ( 10 mg , 44.7% ; 20 mg , 37.5% ) compared with patients administered placebo ( 16.1% ) . the reduction in mean hba1c was significantly greater in the lorcaserin - treated groups than in the placebo group ( 10 mg , -1.0% change from baseline ; 20 mg , -0.9% change ; and placebo , -0.4% change ; p<0.001 ) . a significantly higher proportion of patients in the lorcaserin groups achieved hba1c 7% compared with patients treated with placebo ( 10 mg , 52.2% ; 20 mg , 50.4% ; and placebo , 26.3% ) . however , symptomatic hypoglycemia was more frequent in the lorcaserin groups ( 10 mg , 10.5% ; 20 mg , 7.4% ) than in the placebo group ( 6.3% ) . the adverse events associated with lorcaserin include headache , dizziness , fatigue , nausea , dry - mouth , and constipation . no statistically significant difference in the occurrence of fda - defined valvular disease was found between the lorcaserin and placebo groups . however , the postmarketing trials were required to evaluate the long - term cardiovascular effects of lorcaserin . phentermine / topiramate ( pt ) extended - release ( er ) is a combination of the fda - approved medications phentermine and topiramate in an er formulation . phentermine is indicated for short - term weight loss in overweight or obese adults who exercise and eat a reduced calorie diet . topiramate is indicated for the treatment of certain types of seizures in people who have epilepsy and to prevent migraine headaches . the exact mechanism underlying the weight loss action of topiramate is not known ; however , it may be associated with a combination of features such as an effect on sodium channels , enhancement of gaba - activated chloride channels , and inhibition of carbonic anhydrase isoenzymes . pt is available at a higher dose ( 15 mg phentermine/92 mg topiramate er ) for select patients . discontinuation or an increase in dosage is recommended for patients who have not lost 3% of their baseline weight . treatment should be discontinued if a weight loss > 5% of baseline is not achieved with 15/92 mg per day . fda approval of pt was based on three phase iii clinical trials performed to evaluate the efficacy of the drug ( equip , conquer , sequel ) . in the equip trial , 1,276 obese adults without diabetes were assigned randomly to a low- ( 3.75/23 mg / day ) or high- ( 15/92 mg / day ) dose pt or placebo group for 56 weeks . at the conclusion of the study , the patients in the low- and high - dose pt and placebo groups lost 5.1% , 10.9% , and 1.6% of their baseline body weight , respectively ( p<0.0001 ) . moreover , the results revealed improvements in waist circumference , triglyceride levels , and blood pressure . the conquer trial included 2,487 overweight and obese patients with two or more weight - related comorbidities such as hypertension , prediabetes , type 2 diabetes , dyslipidemia , and abdominal adiposity . the participants were assigned to pt ( 7.5/46 or 15/92 mg ) or placebo groups for 56 weeks . at the conclusion of the trial , the subjects in the 7.5/46 mg group achieved a -8.1 kg change in body weight ( mean change -7.8% ) , those in the 15/92 mg group a -10.2 kg change ( mean change -9.8% ) , and those in the placebo group a -1.4 kg change ( mean change -1.2% ) . a higher percentage of subjects in the pt groups achieved a 5% weight loss after 1 year compared with those receiving placebo ( 62% , 70% , and 21% in the 7.5/46 , 15/92 mg , and placebo groups , respectively ; p<0.001 for both groups vs. placebo ) . moreover , weight loss was maintained for a second year in the sequel trial , a 52-week extension of the conquer trial . the conquer trial revealed an overall significant improvement in hba1c ( % change from baseline : 0% , -0.1% , and 0.1% in the 7.5/46 , 15/92 mg , and placebo groups , respectively ; p<0.0001 for both groups vs. placebo ) and in fasting glucose levels ( % change from baseline : -0.01% , -0.07% , and 0.13% in the 7.5/46 , 15/92 mg , and placebo groups , respectively ; p=0.0047 for 7.5/46 mg vs. placebo and p<0.0001 for 15/92 mg vs. placebo ) at week 56 . the progression to type 2 diabetes among participants without diabetes was lower in the pt - treated patients ( 2.8% in the 7.5/46 mg and 1.7% in the 15/92 mg ) compared with those receiving the placebo ( 3.6% ) . the relative risk of type 2 diabetes versus placebo was 0.78 ( 95% confidence interval [ ci ] , 0.40 to 1.50 ) for the 7.5/46 mg and 0.47 ( 95% ci , 0.25 to 0.88 ) for the 15/92 mg doses of pt . an improvement in glycemic control was observed in the sequel trial . at the conclusion of the trial ( 108 weeks ) , fasting glucose levels were significantly reduced from baseline in the 15/92 mg dose group ( the least - squared change in fasting glucose was 0.1 in the 7.5/46 mg , -1.2 in the 15/92 mg , and 3.7 in the placebo groups ; p=0.0048 for 15/92 mg vs. placebo ) . in the participants without type 2 diabetes at baseline , the progression of type 2 diabetes decreased during the 1-year extension . the annualized incidence rates for progression to type 2 diabetes were 0.9% , 1.7% , and 3.7% in the 7.5/46 , 15/92 mg , and placebo groups , respectively , among the study participants without diabetes at baseline . these results revealed a 54% reduction in the progression to type 2 diabetes in the low- ( 7.5/46 mg ) and a 76% reduction in the high - dose ( 15/92 mg ) groups compared with the placebo group . among participants with type 2 diabetes at baseline , hba1c did not change markedly from baseline in the placebo group ( 0% ) , whereas treatment with 7.5/46 and 15/92 mg pt led to 0.4% and 0.2% reductions in hba1c , respectively . however , the results of the sequel trial should be interpreted with caution because , as an extension of the conquer trial , it may be subject to selection bias . common drug - related side effects include paresthesias , nausea , dizziness , constipation , and dry mouth . the reported neuropsychiatric adverse events include depression , anxiety , insomnia , and disturbances in attention . , pt may increase heart rate by two beats per minute ; however , the fda judged that the potential risk posed by an elevated heartbeat was outweighed by improvements in cardiovascular risk factors such as blood pressure . in september 2014 , the fda approved naltrexone / bupropion ( nb ) sustained - release ( sr ) tablets as a treatment option for chronic weight management . naltrexone was approved previously to treat alcohol and opioid dependence , and bupropion was approved for the treatment of depression or as an aid for smoking cessation . naltrexone is an opioid receptor antagonist , and bupropion acts on adrenergic and dopaminergic receptors in the hypothalamus . the combining of bupropion and naltrexone was based on the theory that naltrexone may block the compensatory mechanisms associated with bupropion that prevent sustained weight loss . the efficacy of nb was evaluated by the contrave obesity research ( cor ) program consisting of the cor - i , cor - ii , cor trial with intensive behavior modification ( cor - bmod ) , and cor - diabetes trials ( table 1 ) . the cor - i , cor - ii , and cor - bmod trials evaluated overweight / obese patients without diabetes . following treatment for 1 year , the group receiving naltrexone 32 mg / bupropion 360 mg lost 4 to 5 kg ( approximately -4% weight loss from baseline body weight ) more than the placebo group , and 48% to 66% of the patients in the nb group lost 5% of their baseline body weight . the cor - diabetes trial evaluated the safety and efficacy of nb in 505 overweight or obese patients with type 2 diabetes over a 56-week period . participants were assigned to the naltrexone 32 mg sr / bupropion 360 mg sr or placebo group . compared with the placebo group , the patients treated with nb lost significantly more weight ( -5.0% vs. -1.8% ; p<0.001 ) , and a higher proportion lost 5% of their body weight ( 44.5% vs. 18.9% ; p<0.001 ) . furthermore , baseline hba1c was significantly reduced in the patients receiving nb compared with the placebo group ( -0.6% vs. -0.1% , respectively ; p<0.001 ) , and 44.1% of patients receiving nb achieved < 7% hba1c compared with 26.3% in the placebo group ( p<0.001 ) . the cardiovascular safety of naltrexone 32 mg sr / bupropion 360 mg sr is currently under investigation ( clinical trial reg no . the most common adverse events include nausea , constipation or diarrhea , headache , vomiting , dizziness , insomnia , and dry mouth . the nb provides a boxed warning to alert users to the increased risk of suicidal thoughts and behaviors associated with bupropion . the nb has a dose - related risk of seizures and may elevate blood pressure and heart rate . liraglutide is a human glucagon - like peptide 1 analog that may be administered once a day because of its prolonged half - life of 13 hours . the efficacy of the drug was well established in the liraglutide effect and action in diabetes ( lead ) studies , a series of phase iii randomized , controlled trials . the approved doses for the treatment of diabetes are 1.2 and 1.8 mg in the united states and 0.6 and 0.9 mg in japan . the lead-3 trial comprised 746 patients with type 2 diabetes assigned to liraglutide ( 1.2 or 1.8 mg ) or glimepiride ( 8 mg ) treatment groups for 52 weeks . the hba1c reductions from baseline were -0.84% and -1.14% in the 1.2 and 1.8 mg groups , respectively , and body weight was reduced by -2.05 kg from baseline in the 1.2 mg group and -2.45 kg in the 1.8 mg group , whereas a significant weight gain ( 1.12 kg ) was observed in patients treated with glimepiride . the recommend dose is 3 mg in contrast to 1.2 or 1.8 mg for diabetes . the satiety and clinical adiposity - liraglutide evidence in nondiabetic and diabetic subjects ( scale ) trial consists of four clinical trials designed to demonstrate the safety and efficacy of liraglutide 3 mg for weight management . the scale obesity and prediabetes phase iiia trial involving 3,731 patients found that patients treated with liraglutide lost an average of 8% of their body weight in 56 weeks compared with 2.6% in the placebo group . moreover , significantly more patients in the liraglutide group lost 5% of their baseline body weight compared with those taking the placebo ( 63.5% vs. 26.6% , respectively ) , and 32.8% of the liraglutide group lost > 10% of their baseline body weight ( 10.1% in the placebo group ) . the scale - diabetes trial was a 56-week randomized , placebo - controlled trial designed to investigate the potential of liraglutide 3 mg in obese or overweight patients with type 2 diabetes . in this trial , 846 overweight or obese patients with diabetes were assigned randomly to liraglutide 3 mg or 1.8 mg or placebo groups . after 56 weeks , the 3 and 1.8 mg liraglutide groups achieved weight loses of 6% and 5% , respectively , compared with 2% in the placebo group . furthermore , 50% of the patients in the 3 mg group and 35% of those in the 1.8 mg group achieved 5% weight loss compared with 13% in the placebo group , and approximately 69% of the patients treated with liraglutide 3 mg achieved the hba1c target of < 7% ( clinical trial reg no . concerned by the lack of long - term data for liraglutide in the treatment of obesity and a variety of safety issues , such as gallbladder disease , pancreatitis , breast and thyroid cancers , and increased heart rate , the fda advisory panel recommended that these issues be addressed in postmarketing studies . however , the panel agreed that the ongoing cardiovascular outcomes trial of the liraglutide effect and action in diabetes : evaluation of cardiovascular outcome results study would be sufficient to characterize the cardiovascular risk of the liraglutide . exercise and monitoring food intake are important components of a weight loss regime ; however , it is difficult for patients to reduce and maintain their weight by lifestyle changes alone ; thus , safe and effective long - term anti - obesity drugs may be a necessary adjunct to lifestyle modification . the five drugs currently approved or pending fda approval for long - term treatment of obesity are orlistat , lorcaserin , pt , nb , and liraglutide ( table 2 ) . these medications have been shown to reduce weight by 5% to 10% , improve glycemic profiles significantly , and reduce cardiovascular risk in patients with diabetes . however , possible adverse events should be monitored , and these medications must be considered as part of a comprehensive management regime for obese patients with diabetes . further study of neuropeptide - y , 3-adrenergic receptors , oxyntomodulin , and amylin analogues are warranted for the development of future antiobesity medications .

type 2 diabetes and obesity have a complex relationship ; obesity is linked to insulin resistance , the precursor to type 2 diabetes . the management of obesity is an important method to delay onset of diabetes and improve the glycemic durability of antidiabetic agents . however , insulin and some of the oral hypoglycemic agents used to treat diabetes cause significant weight gain , and it is difficult for patients with diabetes to reduce and maintain their weight by life - style changes alone . thus , antiobesity medications or bariatric surgery may be a necessary adjunct for certain obese patients with diabetes . in 2012 , the u.s . food and drug administration ( fda ) approved lorcaserin and phentermine / topiramate extended - release for the management of chronic weight , and approval for naltrexone / bupropion sustained - release as an adjunct to exercise and reduced caloric intake followed in 2014 . liraglutide is pending fda approval for antiobesity drug . here we review the efficacy of approved and new promising drugs for the management of obesity .

INTRODUCTION FDA-APPROVED MEDICATIONS FOR LONG-TERM TREATMENT OF OBESITY Orlistat Lorcaserin Phentermine/topiramate extended-release Naltrexone/bupropion sustained-release Liraglutide CONCLUSIONS

the first symptom of soft rot on apple fruit was a water - soaked appearance to the affected tissue . the diseased parts later disintegrated into a mushy mass of disorganized cells that sloughed off . white mycelia formed on infection sites of apples and gradually covered the fruit with tufted whisker - like gray sporangiophores and sporangia ( fig . sporangiospores , sporangia , and sporangiophores were observed under a light microscope ( table 1 ) . the fungal colonies that grew on potato dextrose agar were initially white and cottony , then became heavily speckled with sporangia , and finally became brownish - grey to blackish - grey and spread rapidly with stolons fired at various points to the substrate by rhizoids ( fig . the optimum temperature for mycelial growth was 30 , with good growth still apparent at 37. sporangiospores were unequal , numerous , irregular , sub - globose or oval , angular with striations , and 4~8 m ( fig . smooth - walled , simple or branched , non - septate , long , and arose from stolons opposite rhizoids usually in groups of 3~5 or more . sporangia were globose , white at first , and then turned black with many spores , mostly 40~200 m ( fig . 2c ) . columella were globose to sub - globose , pale brown , and mostly 85~110 m ( fig . twelve apple fruits were artificially inoculated with a representative fungus using the wound infection method . a conidial suspension ( 0.1 ml ; 3 10 conidia / ml ) of the causal fungus was placed on the surface of apple fruit . the inoculated fruit was kept in a moist chamber with 100% relative humidity at 30. after a 3 day incubation , the same fungal symptoms were reproduced : soft rot was observed on inoculated fruits that was identical to symptoms observed at the commercial markets ( fig . 1c and 1d ) . to confirm the identity of the causal fungus , the its rdna of the isolate was amplified and sequenced using its1 ( 5'-tccgtaggtgaacctgcgg-3 ' ) and its4 primers ( 5'-tcctccgcttattgatatgc-3 ' ) , as described by white et al . . a phylogenetic analysis was performed using mega4 with the neighbor - joining method and the tajima - nei distance model . previously published its sequences from r. oryzae strains were included for reference , and mucor miehei ( genbank accession no . the present isolate was placed within a clade comprising r. oryzae references isolates ( fig . soft rot of apple caused by r. stolonifer has been reported previously , but soft rot caused by r. oryzae has not been recorded in korea . the representative culture of the causal fungus was deposited in the korean agricultural culture collection ( kacc 45815 ) , national academy of agricultural science , suwon , korea . based on the mycological characters , molecular data , and pathogenicity testing of the host plant , the fungus was identified as rhizopus oryzae went & prisen geerligs .

soft rot in apple caused by rhizopus oryzae was found for the first time in korea . a detailed description of the specimen is given along with its internal transcribed spacer rdna sequence . the fungus was identified as rhizopus oryzae based on the mycological characteristics , molecular data , and pathogenicity testing .

Symptoms Mycological characteristics Pathogenicity testing Internal transcribed spacer (ITS) sequence analysis

hematohidrosis is known to be precipitated by stress , strain , or any sort of exertion , and it may occur in individuals with underlying bleeding disorders . it may occur in individuals suffering from extreme levels of stress . around the sweat glands , there are multiple blood vessels in a net - like form , which constrict under the pressure of great stress . then , as the anxiety passes , the blood vessels dilate to the point of rupture and goes into the sweat glands . as the sweat glands produce a lot of sweat , they push the blood to the surface , which comes out as droplets of blood mixed with sweat . we hereby report a case where bloody sweat discharged from the forehead , face , and body episodically in a healthy young girl who did not have any underlying disorders . a 12-year - old girl visited us with a history of bleeding from the intact skin over the forehead , scalp , cheek , nose , and trunk [ figures 1 and 2 ] for the last 2 years . the bleeding occurred in episodes , once or twice a day , sometimes more frequently , especially on waking up in the morning . no preceding history of stress or anxiety and no preceding episode of tingling sensation were found . each episode started with mild watery secretion over the forehead or other body parts , followed immediately with bright - red colored secretion . each episode lasted for about 10 - 15 min , and the patient remained perfectly alright during the post - episode period until the next episode . no history of ingestion of any anticoagulants , dyes , or other drugs was obtained from her . she did not have any history of major medical or surgical illness in the past . sweat admixed with blood from the right side of the face sweat admixed with blood from nose her general physical examination and systemic examinations did not reveal any abnormality . the skin over the forehead was normal . on gross examination , the secretion was bright - red in color , less viscous than blood , and it was not frank blood . on collection of the secretion and examination of its smeared preparation under a microscope , plenty of erythrocytes were observed ; the sample was benzidine test positive [ figure 3 ] and alkaptonuria negative . her routine hemogram , blood counts , platelet count , bleeding time ( 2 min ) , clotting time ( 3 min 30 sec ) , active partial thrombin time ( 25 sec ; normal range 24 - 32 sec ) , prothrombin time , liver function tests , and renal function tests were within normal limits . peripheral smear of the secretion showed multiple red blood cells ( rbcs ) along with numerous gram - positive cocci and bacilli [ figure 4 ] . benzidine test of the secretion , confirming presence of blood peripheral smear of the secretion showing rbcs and numerous cocci and bacilli psychiatric analysis revealed intelligent quotient ( i.q . ) between 60 and 70 and a loss of insight . the patient was given atropine transdermal patch over the involved sites and she noticed gradual improvement in both the severity and frequency of the episodes . we followed the patient for 2 months after stopping the atropine treatment . during this period hematohidrosis also known as hematidrosis , hemidrosis , and hematidrosis is a condition in which capillary blood vessels that feed the sweat glands rupture , causing them to exude blood ; it occurs under conditions of extreme physical or emotional stress . various causative factors have been suggested by holoubek , like component of systemic disease , vicarious menstruation , excessive exertion , psychogenic , psychogenic purpura , and unknown causes . acute fear and intense mental contemplation are the most frequent causes , as reported in six cases in a study . in our case , one hypothesis proposed for etiopathogenesis of hematohidrosis , as suggested by some authors , is that multiple blood vessels present in a net - like form around the sweat gland constrict under pressure of stress . as the anxiety passes out , the blood vessels dilate to the point of rupture . the blood goes into the sweat glands , which push it along with sweat to the surface , presenting as droplets of blood mixed with sweat . severe mental anxiety activates the sympathetic nervous system to invoke stress - fight reaction to such a degree as to cause hemorrhage of the vessels supplying the sweat glands into the ducts of the sweat glands . they concluded that pathological basis for hematohidrosis might be distinctive vasculitis , but direct immunofluorescence study did not reveal any abnormality in our case . biopsy during symptom free period did not reveal any blood - filled vascular spaces , intradermal bleeding or abnormality in hair follicle , sebaceous , or sweat glands . benzidine test is an important tool in diagnosis , where hemoglobin in blood reacts with hydrogen peroxide - liberating oxygen , which then reacts with organic reagent producing a green to blue colored compound . we believe that a further insight in the etiopathogenesis may help develop more comprehensive management protocol of this rare and unusual condition . 1 . in our case , no underlying precipitating factors such as stress , strain , or any bleeding disorder was identified .

hematohidrosis is a very rare condition in which an individual sweats blood . it may occur in an individual who is suffering from extreme levels of stress . various causative factors have been suggested like component of systemic disease , vicarious menstruation , excessive exertion , psychogenic , and unknown causes . fear and intense mental contemplation are the most frequent causes . it may also occur in bleeding disorders . we here report a case where bloody sweat was discharged from the forehead , face , and body episodically in a 12-year - old healthy girl with no bleeding disorder or any other underlying cause . all investigations done were within normal limits , except low intelligent quotient and loss of insight . the patient was given atropine sulphate transdermal patch with marked improvement in severity .

Introduction Case Report Discussion

our research is approved by the ethics committee of medical college hospitals , kolkata , west bengal , india . each of the authors for preparing this case report , dr . kallol dasbaksi ( corresponding author ) , dr . plaban mukherjee have contributed to the study concept or design , data collection , data analysis or interpretation , for writing the paper .

highlightsthis case presentation , emphasizes the typical manifestations of hepatic but associated asymptomatic pericardial hcs.hepatic hydatid cyst presenting as a painless abdominal lump preceded painful presentation for a few months.asymptomatic pericardial hydatid cyst could have presented with pericardial tamponade due to rupture , if ignored further.the origin of the pericardial hydatid cyst could have been due to trans diaphragmatic passage of scolices from the hepatic location , as the usual pulmonary involvement was absent .

Conflict of interest Funding Ethical approval Authors contribution Consent Guarantor Supplementary data

necrobiotic xanthogranuloma ( nxg ) was first described as a separate entity by kossard and winkelmann in 1980 . the disease is characterized by multiple , plaque- like , yellow - brown lesions , with a predilection for the periorbital area ( spectacle - like distribution ) . about 100 cases have been reported in world literature . to our knowledge , this is the first case of isolated cutaneous nxg being reported from india . a 65-year - old female presented with a 3-year history of gradually enlarging , periorbital , asymptomatic lesions . however , it recurred in a few months and was accompanied by new larger nodules . the patient 's medical history was not pertinent.on examination , there were multiple , yellowish brown , firm nodules distributed symmetrically in the periorbital area . the size of the nodules varied from 3 to 7 cm in their widest dimensions . the surface showed a few telangiectasias and mild atrophy [ figures 1 and 2 ] . yellow brown firm periorbital nodules superficial telangiectasias and mild atrophy with this history and clinical findings , a provisional diagnosis of a xanthomatous disorder was made . histopathological examination of a surgical wedge biopsy showed ill - defined large foci of dense granulomatous infiltrate of large histiocytes and histiocytic foamy cells with lymphocytes and few plasma cells . several histiocytic giant cells resembling touton giant cells were present [ figures 3 and 4 ] . however , typical findings of necrobiosis were not seen . dense granulomatous infiltrate of histiocytes laboratory investigations were normal for blood counts , electrolytes , glucose levels , lfts , except for s.triglycerides and s.cholesterol levels which were mildly elevated . immunohistochemistry was positive for cd 68 [ figure 5 ] and negative for cd 1a and s100 . thus , a final diagnosis of isolated cutaneous nxg without paraproteinemia or associated disorders was made . the patient was maintained on a regular follow - up of every 15 days for the first 2 months and then once a month for 9 months . nxg is a rare , progressive , histiocytic disease that features destructive cutaneous lesions , a close association with paraproteinemia , and multisystem extracutaneous manifestations . nxg is a disease of adults with an average age of onset in the sixth decade . nxg begins as papules and nodules that slowly enlarge into indurated plaques ranging from 0.5 to 25 cm . characteristic lesions have a red - orange or yellow color as well as telangiectasias , atrophy , scarsand ulcers . the most common site of involvement is the face , particularly the periorbital region ( 85% of cases ) ; the trunk and proximal extremities are other affected sites . ophthalmological complications affect approximately 50% of cases and include orbital masses , conjunctival involvement , keratitis , scleritis , ectropion , uveitis and blindness . nxg may involve other extracutaneous sites , including the heart , lungs , kidneys , liver , spleen , intestines , skeletal muscle and central nervous system . paraproteinemia is closely associated with nxg , with approximately 80% of patients demonstrating a monoclonal gammopathy of igg- on serum protein electrophoresis ; furthermore , 10% of these patients develop multiple myeloma . hepatosplenomegaly , lymphadenopathy , an increased erythrocyte sedimentation rate , leukopenia , hypocomplementemia , thrombocytopenia and cryoglobulinemia are other common findings . theories regarding pathogenesis include deposition of immunoglobulins and lipid complexes with a foreign body giant - cell reaction and monocyte activation with intracellular lipid accumulation . histopathological examination shows a palisading xanthogranuloma with areas of necrobiotic , degenerated collagen that infiltrates the mid - dermis with extension into the subcutaneous fat . multiple xanthomatized histiocytes , touton giant cells , other bizarre giant cells , cholesterol clefts and lymphoid follicles are often present within granulomas . improvement in skin lesions has been seen with low - dose chlorambucil , melphalan with or without prednisolone , methotrexate , localized radiotherapy , systemic glucocorticoids , interferon -2a , and plasmapheresis with hydroxychloroquine . topically nitrogen mustard , bncu , intralesional corticosteroids and intravenous immunoglobulin have also been tried . surgical excision has also been advocated . in our patient , we decided to surgically excise the lesions in view of absence of systemic involvement .

necrobiotic xanthogranuloma ( nxg ) is a very rare , progressive variant of non - langerhans cell histiocytosis . it is known to be associated with multisystem involvement and paraproteinemias . a 65-year - old female presented with chronic , slowly growing , asymptomatic periorbital nodules . the lesions had recurred after local excision elsewhere . no systemic involvement or paraproteinemias were detected . a provisional diagnosis of isolated cutaneous nxg was made which was confirmed by histopathology and immunohistochemistry staining . the lesions were surgically excised with excellent cosmetic and functional results . there was no recurrence over a period of 9 months . to our knowledge , this is the second case of nxg reported from india and the first without any systemic manifestations .

Introduction Case Report Discussion

the nasal cavity is susceptible to chemically induced injury as a result of exposure to inhaled irritants . some responses of the nasal mucosa to inhaled toxicants are species specific . these species - related differences in response may be due to variations in structural , physiologic , and biochemical factors , such as gross nasal cavity structure , distribution of luminal epithelial cell populations along the nasal airway , intranasal airflow patterns , nasal mucociliary apparatus , and nasal xenobiotic metabolism among animal species . this paper reviews the comparative anatomy and irritant - induced pathology of the nasal cavity in laboratory animals . the toxicologist , pathologist , and environmental risk assessor must have a good working knowledge of the similarities and differences in normal nasal structure and response to injury among species before they can select animal models for nasal toxicity studies , recognize toxicant - induced lesions in the nasal airway , and extrapolate experimental results to estimate the possible effects of an inhaled toxicant on the human nasal airway.imagesplate 1.plate 2 . aplate 2 . bplate 3 .

Images

the risk of rupture approximates 50% , most of which occur in the second trimester . was then performed with fetal intracardiac potassium chloride and intraplacental methotrexate . magnetic resonance imaging ( mri ) of the pelvis was obtained . while surgical resection of a rudimentary horn pregnancy is necessary , early diagnosis affords the opportunity to take steps that minimize surgical risks . mri assists surgical planning by demonstrating the form of attachment of the uterine horn to the unicornuate uterus . preoperative medical termination may decrease vascularity of the gestation , thereby decreasing operative blood loss . a unicornuate uterus results from unilateral failure of normal mllerian system development . in most instances , the abnormal mllerian duct has partially developed into a rudimentary uterine horn . in one series of 42 patients with unicornuate uterus , the anatomic variations of a rudimentary horn serve as the basis for classification of unicornuate uterus by the american fertility society : rudimentary horn with cavity , communicating with unicornuate uterus ( class iia ) , rudimentary horn with cavity , not communicating with unicornuate uterus ( class ii - b , the most common ) , rudimentary horn without cavity ( class ii - c ) , or unicornuate uterus without rudimentary horn ( class ii - d ) . pregnancy within a noncommunicating rudimentary uterine horn is a well - known complication , estimated to occur in 1 in 76,000 pregnancies . migration of ovum with sperm can also occur , as evidenced by the 8% prevalence of a corpus luteum on the side contralateral to the rudimentary horn containing the pregnancy . the prognosis for maternal and fetal outcome is poor , with a neonatal survival rate of 6% and uterine horn rupture risk of 50% . due to the high risk of this potentially catastrophic outcome , the classical teaching has been that all uterine horn pregnancies should be surgically removed immediately upon diagnosis . however , medical termination followed by delayed surgical resection is an option when diagnosis is obtained early and impending rupture seems unlikely , since most cases of rupture occur in the second trimester . we describe a case of noncommunicating rudimentary horn pregnancy that was medically terminated and subsequently resected along with the uterine horn and fallopian tube 6 weeks later . a 36-year - old female who had a known history of a left unicornuate uterus with a noncommunicating rudimentary horn presented to her obstetrician 8 weeks into her second pregnancy . her uterine anomaly was detected in her first pregnancy during a cesarean delivery at 34 weeks for failure to progress . however , an accurate description of the uterine anomaly was difficult given the gravid state . several months later , a hysterosalpingogram revealed a small uterine cavity deviating towards the left side , along with a patent left fallopian tube . an ultrasound at 8 weeks revealed a singleton pregnancy within the right uterine horn and a corpus luteum on the ipsilateral ovary ( figure 1 ) . the crown - rump length and quantitative b - hcg ( approximately 89,000 miu / ml ) were consistent with the gestational age , and cardiac activity was observed . the patient was referred to a perinatologist who confirmed the ectopic pregnancy diagnosis and counseled her about the risk of uterine horn rupture and poor prognosis for viability . the patient was also counseled that surgery would be necessary to remove the ectopic gestation and noncommunicating rudimentary horn . the perinatologist performed intracardiac injection of kcl along with intraplacental injection of methotrexate to prevent further gestational growth . the patient was seen weekly to follow b - hcg levels , which had decreased to 653 miu / ml after 6 weeks . four weeks after medical termination , we obtained an mri to further characterize the uterine anomaly and urinary system . the mri revealed a rudimentary horn attached to the unicornuate uterus by a small band of tissue , and no urinary system abnormalities were detected ( figures 2 and 3 ) . black arrow : right rudimentary horn containing pregnancy ; white arrow : left unicornuate uterus ; grey arrow : band of tissue connecting the rudimentary horn to the uterus . black arrow : right rudimentary horn containing pregnancy ; white arrow : left unicornuate uterus ; grey arrow : band of tissue connecting the rudimentary horn to the uterus . six weeks after medical termination , we performed laparoscopy with a 5-mm laparoscope in the umbilicus , a 10-mm trocar in the left lower quadrant , and a 5-mm trocar in the right lower quadrant . a 446-cm rudimentary horn was attached to the unicornuate uterus by a 15-mm diameter band of tissue ( figure 4 ) . we transected the right round ligament and opened the broad ligament by using a 5-mm ligasure ( valleylab , boulder , co ) . the anterior leaf of the broad ligament was dissected from the edge of the uterine horn , and a right salpingectomy was performed . the posterior leaf of the broad ligament was dissected off the edge of the uterine horn ( figure 5 ) , and the utero - ovarian ligament was transected . two pds endoloops ( ethicon endo - surgery , inc , cincinnati , oh ) were placed over the rudimentary horn and ligated around the fibrous band before it was transected with the ligasure . once the uterine horn and tube were free , the specimen was removed by using an endopouch ( ethicon endo - surgery , inc , cincinnati , oh ) . uterine horn ( black arrow ) is attached to the unicornuate uterus ( white arrow ) by a small band of tissue ( grey arrow ) . superficial lesions of endometriosis in the posterior culde - sac and on the left uterosacral ligament were fulgurated using bipolar cautery . estimated blood loss was less than 50ml and there were no complications . the patient was aware of the poor prognosis for carrying a pregnancy to term , yet she continued to express a strong desire for future childbearing . when ectopic pregnancy within a rudimentary uterine horn occurs , surgical resection is requisite due to the high risk of rupture . because most cases of uterine horn rupture occur in the second trimester , early diagnosis in the first trimester provides some time to take measures that can minimize surgical risks . in this case , medical termination was performed before surgery to decrease the vasculature to the ectopic gestation . for the nongravid patient , elective surgical removal of a rudimentary horn the patient was asymptomatic from the ectopic pregnancy and displayed no signs of impending rupture . termination with intracardiac kcl and intraplacental methotrexate was successful in reducing further development of the ectopic gestation , and the b - hcg levels decreased rapidly . single - dose intramuscular methotrexate has also been reported to terminate rudimentary horn pregnancy , which requires a larger dose of medication due to systemic adminstration . we delayed surgical resection until b - hcg levels declined significantly , hoping this would signify a decrease in blood flow to the rudimentary horn . the use of preoperative gnrh agonists to render the surgical field less vascular has been reported in cases without pregnancy . however , this was not used in our patient because we did not feel that a gnrh agonist would have a significant effect during pregnancy . characterizing the anatomic relation between the uterine horn and the unicornuate uterus with mri is helpful for planning the surgical approach . a rudimentary horn can be attached to the unicornuate uterus by either a band of tissue or a broad , firm attachment . a band of tissue is easily transected while a broad attachment requires much more dissection . separation is more difficult without a pedicle , and there is a higher risk of dissecting into the cavity of the uterus . an mri is also useful to determine whether any urinary abnormalities are present , given that approximately 38% of patients have coexisting renal abnormalities . unilateral renal agenesis is most commonly found , which is always ipsilateral with the rudimentary horn . mri is helpful for making this diagnosis and understanding the anatomy to plan the surgical approach for resection . when diagnosis is obtained early , medical termination before surgery can be used to potentially decrease blood flow to the gestation , facilitating surgical removal with limited blood loss .

background : pregnancy within a noncommunicating rudimentary horn is a known complication of unicornuate uterus . the risk of rupture approximates 50% , most of which occur in the second trimester.case:a rudimentary horn pregnancy was discovered at 8 weeks gestation . medical termination was then performed with fetal intracardiac potassium chloride and intraplacental methotrexate . magnetic resonance imaging ( mri ) of the pelvis was obtained . laparoscopic uterine horn resection 6 weeks after medical termination was performed.discussion:while surgical resection of a rudimentary horn pregnancy is necessary , early diagnosis affords the opportunity to take steps that minimize surgical risks . mri assists surgical planning by demonstrating the form of attachment of the uterine horn to the unicornuate uterus . preoperative medical termination may decrease vascularity of the gestation , thereby decreasing operative blood loss .

Background: Case: Discussion: INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

the louisiana state university health care services division ( lsuhcsd ) operates seven public hospitals and affiliated clinics in louisiana that provide quality medical care to the residents of louisiana regardless of their income or insurance coverage ( 1924 ) . overall , lsuhcsd facilities have served ~1.6 million patients ( 35% of the louisiana population ) since 1997 . administrative , anthropometric , laboratory , clinical diagnosis , and medication data collected at these facilities are available in electronic form for both inpatients and outpatients from 1997 . using these data , a cohort of patients with diabetes was established by using the icd-9 ( code 250 ) through the lsuhls database between 1 january 1999 and 31 december 2009 . both inpatients and outpatients internal diabetes disease - management guidelines call for physician confirmation of diabetes diagnoses by applying the american diabetes association criteria : a fasting plasma glucose level 126 mg / dl , 2-h glucose level 200 mg / dl after a 75-g 2-h oral glucose tolerance test , and one or more classic symptoms plus a random plasma glucose level 200 mg / dl ( 25 ) . the first record of diabetes diagnosis was used to establish the baseline for each patient in the present analyses owing to the design of the cohort study . before diagnosed with diabetes , the agreement of diabetes diagnosis was 97% : 20,919 subjects of a sample of 21,566 hospital patients with discharge diagnoses based on icd codes also had physician - confirmed diabetes by using the american diabetes association diabetes diagnosis criteria ( 25 ) . the current study included 35,368 patients with newly diagnosed diabetes ( 15,560 white and 19,808 african american ) who were 3094 years of age without a history of lea and with complete repeated data on all risk factor variables . in these patients with diabetes , ~77.3% qualify for free care ( by virtue of being low income and uninsured any individual or family unit whose income is 200% of federal poverty level ) , ~4.9% of patients are self - pay ( uninsured , but incomes not low enough to qualify for free care ) , ~5.2% of patients are covered by medicaid , ~10.4% of patients have medicare , and ~2.2% of patients are covered by commercial insurance . the study and analysis plan were approved by both the pennington biomedical research center and louisiana state university health sciences center institutional review boards . we did not obtain informed consent from participants involved in our study because we used anonymized data compiled from electronic medical records . the patient s characteristics , including age of diabetes diagnosis , sex , race / ethnicity , family income , smoking status , types of health insurance , body weight , height , bmi , blood pressure , total cholesterol , hdl cholesterol , ldl cholesterol , triglycerides , hba1c , estimated glomerular filtration rate ( egfr ) , history and incidence of peripheral arterial disease , ulcer , and foot deformity , and medication ( antihypertensive drugs , cholesterol - lowering drugs , and antidiabetes drugs ) within half a year after the diabetes diagnosis ( baseline ) and during follow - up after the diabetes diagnosis ( follow - up ) , were extracted from the computerized hospitalization records . foot risk factors were identified using icd-9 codes based on existing literature ( 26,27 ) , which included peripheral arterial disease ( icd-9 codes 443.81 , 440.2 , 440.20 , 440.21 , 440.22 , 440.23 , 440.24 , 440.29 , 440.8 , 440.9 , 442.2 , 442.3 , 443.0 , 443.1 , 443.81 , 443.89 , 443.9 , 444.22 , 444.81 , 2507.x , and 785.4 ) , ulcer ( icd-9 codes 707.1x and 707.9 ) , and foot deformity ( icd-9 codes 94.0 , 713.5 , 727.1 , 735.0 , 735.2 , and 735.4735.9 ) . the updated mean values of hba1c , ldl cholesterol , bmi , blood pressure , and egfr over time were measured first at baseline and second as an updated mean of annual measurement , calculated for each participant from baseline to each year of follow - up . for example , at 1 year the updated mean is the average of the baseline and 1-year values and at 3 years it is the average of baseline , 1-year , 2-year , and 3-year values . in case of an event during follow - up , the period for estimating updated mean value was from baseline to the year before this event occurred ( 10,28 ) . the average number of hba1c measurements during the follow - up period was 7.4 times . follow - up information was obtained from the lsuhls inpatient and outpatient database by using the unique number assigned to every patient who visits the lsuhcsd hospitals . the diagnosis of lea was the primary end point of interest of the study and was defined according to the icd-9 ( codes 84.1084.17 ) . since 1997 , diagnoses of lea were made by the treating physicians based on a clinical assessment and examinations as considered relevant by the clinician in charge of treatments . follow - up of each cohort member continued until the date of the diagnosis of lea , the date of the last visit if the subject stopped use of lsuhcsd hospitals , death , or 31 may 2012 . the association between hba1c and the risk of lea was analyzed by using cox proportional hazards models . hba1c was evaluated in the following two ways : 1 ) as six categories ( hba1c < 6.0% [ 42 mmol / mol ] [ reference group ] , 6.06.9% [ 4252 mmol / mol ] , 7.07.9% [ 5363 mmol / mol ] , 8.08.9% [ 6474 mmol / mol ] , 9.09.9% [ 7585 mmol / mol ] , and 10.0% [ 86 mmol / mol ] ) and different levels of hba1c were included in the models as dummy variables , and the significance of the trend over different categories of hba1c was tested in the same models by giving an ordinal numerical value for each dummy variable . all analyses were adjusted for age and sex and further for smoking , income , types of insurance , bmi , systolic blood pressure , ldl cholesterol , egfr , peripheral arterial disease , ulcer , and foot deformity at baseline and during follow - up , use of antihypertensive drugs , use of diabetes medications , and use of cholesterol - lowering agents . when we analyzed the association between updated mean of hba1c and lea risk , we adjusted for updated means of bmi , ldl cholesterol , systolic blood pressure , and egfr instead of baselines of these variables . for avoidance of the potential bias due to severe diseases at baseline , additional analyses were carried out excluding the subjects who were diagnosed with lea during the first 2 years of follow - up . statistical significance was considered to be p < 0.05 . all statistical analyses were performed with pasw for windows , version 20.0 ( ibm spss , chicago , il ) and sas for windows , version 9.3 ( sas institute , cary , nc ) . the patient s characteristics , including age of diabetes diagnosis , sex , race / ethnicity , family income , smoking status , types of health insurance , body weight , height , bmi , blood pressure , total cholesterol , hdl cholesterol , ldl cholesterol , triglycerides , hba1c , estimated glomerular filtration rate ( egfr ) , history and incidence of peripheral arterial disease , ulcer , and foot deformity , and medication ( antihypertensive drugs , cholesterol - lowering drugs , and antidiabetes drugs ) within half a year after the diabetes diagnosis ( baseline ) and during follow - up after the diabetes diagnosis ( follow - up ) , were extracted from the computerized hospitalization records . foot risk factors were identified using icd-9 codes based on existing literature ( 26,27 ) , which included peripheral arterial disease ( icd-9 codes 443.81 , 440.2 , 440.20 , 440.21 , 440.22 , 440.23 , 440.24 , 440.29 , 440.8 , 440.9 , 442.2 , 442.3 , 443.0 , 443.1 , 443.81 , 443.89 , 443.9 , 444.22 , 444.81 , 2507.x , and 785.4 ) , ulcer ( icd-9 codes 707.1x and 707.9 ) , and foot deformity ( icd-9 codes 94.0 , 713.5 , 727.1 , 735.0 , 735.2 , and 735.4735.9 ) . the updated mean values of hba1c , ldl cholesterol , bmi , blood pressure , and egfr over time were measured first at baseline and second as an updated mean of annual measurement , calculated for each participant from baseline to each year of follow - up . for example , at 1 year the updated mean is the average of the baseline and 1-year values and at 3 years it is the average of baseline , 1-year , 2-year , and 3-year values . in case of an event during follow - up , the period for estimating updated mean value was from baseline to the year before this event occurred ( 10,28 ) . the average number of hba1c measurements during the follow - up period was 7.4 times . follow - up information was obtained from the lsuhls inpatient and outpatient database by using the unique number assigned to every patient who visits the lsuhcsd hospitals . the diagnosis of lea was the primary end point of interest of the study and was defined according to the icd-9 ( codes 84.1084.17 ) . since 1997 , diagnoses of lea were made by the treating physicians based on a clinical assessment and examinations as considered relevant by the clinician in charge of treatments . follow - up of each cohort member continued until the date of the diagnosis of lea , the date of the last visit if the subject stopped use of lsuhcsd hospitals , death , or 31 may 2012 . the association between hba1c and the risk of lea was analyzed by using cox proportional hazards models . hba1c was evaluated in the following two ways : 1 ) as six categories ( hba1c < 6.0% [ 42 mmol / mol ] [ reference group ] , 6.06.9% [ 4252 mmol / mol ] , 7.07.9% [ 5363 mmol / mol ] , 8.08.9% [ 6474 mmol / mol ] , 9.09.9% [ 7585 mmol / mol ] , and 10.0% [ 86 mmol / mol ] ) and different levels of hba1c were included in the models as dummy variables , and the significance of the trend over different categories of hba1c was tested in the same models by giving an ordinal numerical value for each dummy variable . all analyses were adjusted for age and sex and further for smoking , income , types of insurance , bmi , systolic blood pressure , ldl cholesterol , egfr , peripheral arterial disease , ulcer , and foot deformity at baseline and during follow - up , use of antihypertensive drugs , use of diabetes medications , and use of cholesterol - lowering agents . when we analyzed the association between updated mean of hba1c and lea risk , we adjusted for updated means of bmi , ldl cholesterol , systolic blood pressure , and egfr instead of baselines of these variables . for avoidance of the potential bias due to severe diseases at baseline , additional analyses were carried out excluding the subjects who were diagnosed with lea during the first 2 years of follow - up . all statistical analyses were performed with pasw for windows , version 20.0 ( ibm spss , chicago , il ) and sas for windows , version 9.3 ( sas institute , cary , nc ) . general characteristics of the study population are presented by race in table 1 . during a mean follow - up period of 6.83 years a significantly increased risk of lea was observed among both african american and white patients with increasing baseline hba1c ( table 2 ) . after further adjustment for other confounding factors ( smoking , income , type of insurance , bmi , systolic blood pressure , ldl cholesterol , egfr , peripheral arterial disease , ulcer , and foot deformity at baseline and during follow - up and use of antihypertensive drugs , diabetes medications , and cholesterol - lowering agents ) , this graded association remained significant among white ( ptrend < 0.001 ) and african american ( ptrend < 0.001 ) patients with diabetes ( table 2 ) . each 1% increase in baseline hba1c was associated with a 13% ( 95% ci 1.081.17 ) increased risk of lea in african americans and a 15% ( 95% ci 1.091.21 ) increased risk of lea in whites . the risk of lea associated with hba1c was higher in white than african american patients with diabetes ( = 17.8 , df = 1 , p for interaction < 0.005 ) . baseline characteristics of african american and white patients with diabetes hazard ratio ( hr ) ( 95% ci ) of lea according to different levels of hba1c at baseline and during follow - up among african american and white patients with diabetes there was a significant interaction between sex and hba1c on lea risk ( table 3 ) . when stratified by sex , the graded association of hba1c at baseline with lea risk was present and more significant in female patients with diabetes than male ( p for interaction < 0.001 ) . when we stratified by age , smoking status , family income , blood pressure , ldl cholesterol , and bmi , the graded positive association of baseline hba1c with lea risk did not change ( table 3 ) . moreover , the graded positive association of hba1c with lea risk was also confirmed among patients with diabetes using glucose - lowering agents or not ( all p trend<0.01 ) ( table 3 ) . hazard ratio ( hr ) ( 95% ci ) of lea according to different levels of hba1c at baseline among various subpopulations when we did an additional analysis by using an updated mean of hba1c during follow - up , we found almost the same graded positive associations between baseline hba1c levels and updated mean levels of hba1c and lea risk among both african american and white patients with diabetes ( tables 2 and 4 ) . the mean of hba1c decreased from 8.0% ( 64 mmol / mol ) to 7.7% ( 61 mmol / mol ) for african americans and from 7.3% ( 56 mmol / mol ) to 7.2% ( 55 mmol / mol ) for whites , but the graded positive association between hba1c and lea did not change ( supplementary table 1 ) . hazard ratio ( hr ) ( 95% ci ) of lea according to different levels of hba1c during follow - up among various subpopulations after exclusion of the subjects who were diagnosed with lea during the first 2 years of follow - up ( n = 208 ) , the multivariable - adjusted hrs of lea associated with different levels of hba1c did not change ( data not shown ) . our study found a graded positive association between hba1c at baseline and during follow - up and the risk of lea among both african american and white patients with diabetes . this graded positive association was more significant in white than african american patients with diabetes . lea ranked first when participants rated their decrease in quality of life in the uk prospective diabetes study ( ukpds ) compared with other complications including blindness in one eye , stroke , heart failure , etc . health care providers encourage and strive to achieve good glycemic control for patients with diabetes ; however , there are limited data on the specific effect of glycemic control on lea risk among patients with diabetes . one meta - analysis of prospective studies demonstrated that there is a substantial increase in risk of lea associated with hyperglycemia in individuals with diabetes ( 30 ) ; however , small sample sizes ( 1,0443,642 participants ) , short follow - up ( 714 years ) , and few lea cases ( 44118 cases ) in each study limit the statistical power for subgroup analyses . there are limited clinical trial data on the specific effect of glycemic control on lea . in the prospective pioglitazone clinical trial in macrovascular events ( proactive ) study ( 18 ) , there were 28 amputations among 2,605 participants in the pioglitazone group and 26 of 2,633 in the placebo group , and the event rate was lower in ukpds than in proactive ( 15,17 ) . thus , it is quite difficult for a previous single prospective study or clinical trial to provide conclusive evidence about glucose lowering and lea risk ( 10,11,1518 ) . there is an urgent necessity to provide robust data to confirm the associations between glycemia control and lea risk in the population with diabetes . in the current study , during a mean follow - up of 6.8 years , 578 lea incident cases among 35,368 participants with diabetes were identified . we found a graded positive association by various hba1c intervals of clinical relevance or by using hba1c as a continuous variable at baseline and during follow - up with lea risk among both african american and white patients with diabetes . in addition , we found that this graded positive association was present in patients with diabetes with and without glucose - lowering agent treatment and in patients with diabetes with different age , sex , smoking status , family income , blood pressure levels , ldl cholesterol , and bmi groups . potential explanations for the increased risk of lea associated with hyperglycemia are likely to be mediated by number of mechanisms , which include but are not limited to peripheral sensory neuropathy , peripheral vascular disease , and soft - tissue sepsis . first , the importance of hyperglycemia in the development of peripheral neuropathy is well documented ( 31 ) . second , hyperglycemia contributes greatly to peripheral vessel disease in patients with diabetes ( 33 ) . finally , infection is present in the majority of foot ulcers , and hyperglycemia probably impairs host defense against infection . in the european study group on diabetes and the lower extremity ( eurodiale ) study ( 34 ) , peripheral arterial disease was diagnosed in 49% of the subjects and infection in 58% among patients with diabetic foot ulcers . patients with peripheral neuropathy often fail to notice minor trauma resulting in ulceration , infection , and nonhealing diabetic foot ulcers to the ultimate lea . improved glycemic control can potentially modify the risk of sensory neuropathy ( 35 ) and possibly the progression of peripheral arterial disease ( 36 ) . there are several strengths in our study , including the large sample size , high proportion of african americans , long follow - up time , and use of administrative databases to avoid differential recall bias . we have used both baseline hba1c levels and updated mean values of hba1c during follow - up in the analyses , which can avoid potential bias from a single baseline measurement . in addition , participants in this study used the same public health care system , which minimizes the influence from the accessibility of health care , particularly when comparing african americans and whites . one limitation of our study is that our analysis was not performed on a representative sample of the population , which limits the generalizability of the results ; however , lsuhcsd hospitals are public hospitals and cover > 1.6 million patients , most of whom are low - income persons in louisiana . the results of the current study will have wide applicability for the population with low income and without health insurance in the u.s . second , the validity of lea diagnoses in our study has not been confirmed by specialists . however , the method using hospital discharge registers to diagnose lea has been widely used in american and european cohort studies , such as the kaiser permanente medical care program ( 37 ) and the u.k . survey ( 38 ) . the validity of the diagnoses of lea by using hospital discharge registers in these cohort studies is available ( agreement 9799% ) ( 37,38 ) . third , even though our analyses adjusted for an extensive set of confounding factors , residual confounding due to the measurement error in the assessment of confounding factors and unmeasured factors such as physical activity , education , and dietary factors can not be excluded . in summary , our study demonstrates that there is a graded association between hba1c at baseline and during follow - up and the risk of lea among both african american and white patients with diabetes . in the absence of conclusive evidence from randomized intervention trials , our study provides further epidemiological support for glucose lowering as a strategy to reduce amputation in patients with diabetes .

objectivediabetes confers a very high risk of lower - extremity amputation ( lea ) ; however , few studies have assessed whether blood glucose control can reduce lea risk among patients with diabetes , especially in practice settings where low - income patients predominate.research design and methodswe performed a prospective cohort study ( 20002009 ) on patients with diabetes that included 19,808 african americans and 15,560 whites . the cohort was followed though 31 may 2012 . cox proportional hazards regression models were used to estimate the association of hba1c with lea risk.resultsduring a mean follow - up of 6.83 years , 578 lea incident cases were identified . the multivariable - adjusted hazard ratios of lea associated with different levels of hba1c at baseline ( < 6.0% [ reference group ] , 6.06.9 , 7.07.9 , 8.08.9 , 9.09.9 , and 10.0% ) were 1.00 , 1.73 ( 95% ci 1.072.80 ) , 1.65 ( 0.992.77 ) , 1.96 ( 1.143.36 ) , 3.02 ( 1.815.04 ) , and 3.30 ( 2.105.20 ) ( p trend < 0.001 ) for african american patients with diabetes and 1.00 , 1.16 ( 0.662.02 ) , 2.28 ( 1.353.85 ) , 2.38 ( 1.364.18 ) , 2.99 ( 1.715.22 ) , and 3.25 ( 1.985.33 ) ( p trend < 0.001 ) for white patients with diabetes , respectively . the graded positive association of hba1c during follow - up with lea risk was observed among both african american and white patients with diabetes ( all p trend < 0.001 ) . with stratification by sex , age , smoking status , blood pressure , ldl cholesterol , bmi , use of glucose - lowering agents , and income , this graded association of hba1c with lea was still present.conclusionsthe current study conducted in a low - income population suggests a graded association between hba1c and the risk of lea among both african american and white patients with type 2 diabetes .

RESEARCH DESIGN AND METHODS Baseline and follow-up measurements Prospective follow-up Statistical analyses RESULTS CONCLUSIONS Supplementary Material

magnesium ( mg ) is an important cofactor for the enzymes that are involved in carbohydrate metabolism : an important role of mg in insulin action has been reported [ 1 , 2 ] ; low serum and intracellular mg ( [ mg]i ) concentrations are associated with insulin resistance , impaired glucose tolerance , and decreased insulin secretion [ 3 , 4 ] . furthermore , lower dietary mg intake could cause insulin resistance both in children and adults [ 5 , 6 ] . based on these findings , we studied whether low [ mg]i in the fetus would be one of the critical abnormalities associated with small for gestational age ( sga ) . in addition , several studies have shown the association of size at birth or indices in poor fetal growth with later development of metabolic syndrome and insulin resistance . proposed that impaired glucose tolerance and type 2 diabetes might arise as a result of programming . programming is a term used to describe persistent changes in organ structure and function caused by exposure to adverse environmental influences during critical periods of development . hypothesis , which proposes that fetal adaptation to an adverse intrauterine environment affecting fetal growth may program life - long physiological changes . it has been proposed that various fetal growth patterns and intrauterine growth retardation ( iugr ) arise as a result of an early stimulus or insult . since both fetal weight and length gains are closely related , there is much overlap between sga and iugr . sga is defined as birth weight and/or length at least 2 standard deviations below the mean for gestational age . in this review , we discuss the potential contribution of aberrant mg regulation to sga and to the pathogenesis of metabolic syndrome later in life . it is reported that the amount of maternal mg intake is not only associated with pregnancy outcome but also with infant outcome . the mean daily intake of mg ( 284.3 mg / day ) during pregnancy was found to be lower than recommended . for example , mg deficiency is reported to occur in 44% of pregnant indian women . another study has demonstrated that mothers who drink water containing high amounts of mg have a reduced risk of having very low birth weight infants ( less than 1,500 g of birth weight ) . by the end of a normal pregnancy , the fetus is believed to have acquired approximately 28 g of calcium , 16 g of phosphorous , and 0.7 g of mg , mostly during the third trimester , also the time when 80% of fetal accretion of mg occurs . the fetal circulation contains higher levels of total calcium , ionized calcium , and mg compared to maternal blood . in the placental membrane , copper and selenium share the same transport pathway along a concentration gradient in the maternal - fetal direction , while mg and iron use predominantly an active transport pathway . in fact , evidence for an active transport mechanism for mg in the placenta has been demonstrated in cultured trophoblast cells in which low [ mg]i is maintained by the function of a na / mg exchanger . mg levels in umbilical cord blood are the lowest in preterm low birth weight babies , followed by term low birth weight babies and are the highest in term controls ( p < .05 ) . calcium and mg have an immediate effect on placental vascular flow . reduced placental vascular flow is at least , in part , responsible for placental insufficiency and fetal growth retardation ( fgr ) . a variety of hormones , cytokines , and growth factors produced by fetal membranes and the placenta can act locally on the myometrium . the ability of the uterine artery to dilate during pregnancy may be specifically related to the upregulation of multiple pathways for the production of nitric oxide . the activity of constitutive nitric oxide synthase is dependent on calcium and is inhibited by a reduction in the concentration of mg . blood mg levels were reportedly lower in women with severe pre - eclampsia than in healthy pregnant women ( 1.63 0.05 mg / dl versus 1.87 0.05 mg / dl ; p < .001 ) . therapeutic levels of mg have also been found to produce specific placental effects such as vasodilation . from an in vitro study of human umbilical artery resistance , mg sulfate was found to exert a relaxant effect on umbilical arterial tone attenuating the vasoconstrictor effect of angiotensin ii and endothelin-1 in the fetal - placental vasculature . mg sulfate used for the treatment of pre - eclampsia or hypertensive disease in pregnancy may have beneficial effects on the fetoplacental circulation . prenatal treatment with mg sulfate may influence calcium homeostasis and nonenzymatic antioxidant reserve in erythrocytes of preterm newborns . it is well known that plasma mg falls in pregnancy because of accumulation of ion in the placenta and fetus . mg is therefore widely given as a supplement during pregnancy , particularly in cases of preterm labor . there are several reports that oral mg supplementation in pregnancy is safe and that it has a positive effect on fetal morbidity . patients in preterm labor have significantly depressed serum mg levels , while in patients with pre - eclampsia , mg levels are not significantly different from that of the controls . mg supplementation was found to decrease the rate of fgr , premature rupture of membranes , and premature delivery in risk pregnancies treated with betamimetics . oral mg supplementation given before the 25th week of gestation is associated with a lower frequency of preterm births , a lower frequency of low birth weight , and fewer sga infants compared with the placebo . mg intake in 513 women who were near the end of their first trimester of pregnancy was determined from records of food consumption . mg intake correlated with weight , length , and head circumference at birth as well as length of gestation up to a threshold of around 3,200 g of birth weight . on the other hand , mg supplements ( 100 mg / day ) taken during the second and third trimesters had no effect on the outcome of the pregnancy . the effect of mg compared with placebo in a randomized double - blind controlled study of patients with pregnancy - induced hypertension was investigated . mg supplementation was found to be beneficial in the management of pregnancy - induced hypertension . at present , there is insufficient high - quality evidence to show that dietary mg supplementation during pregnancy is beneficial . it is important to determine the timing and dose of mg supplementation as both factors may alter the pregnancy outcome . we have previously reported that intracellular magnesium ( [ mg]i ) is lower in children with diabetes mellitus and in children who are obese . in the type 2 diabetes mellitus and obese groups , platelets responded well to insulin . under insulin - resistant states , [ mg]i decreases before the poor reactivity to insulin occurs in platelets . from these findings , we hypothesize that [ mg]i is decreased earlier than when poor reactivity to insulin develops in platelets exposed to an insulin resistant environment . this suggests that low [ mg]i may be an intrinsic abnormality in infants with low birth weight . to test this hypothesis , we studied the relationship of [ mg]i in cord blood platelets to birth weight . we found that mean basal [ mg]i , but not plasma magnesium , is lower in sga than in the appropriate for gestational age ( aga ) groups ( 323 162 mol / l versus 488 132 mol / l , p = .004 ) . mg]i significantly correlates with birth weight ( p < .0001 ) as well as cord plasma leptin ( p = .031 ) and igf-1 ( p < .001 ) . [ mg]i as well as leptin and igf-1 reflects the extent of fetal growth . also , [ mg]i significantly correlates with the quantitative insulin sensitivity check index ( quicki ) ) ( p < .001 ) . decreased [ mg]i in sga might underlie the initial pathophysiological events that lead to insulin resistance . for example , hyperglycemia may have an effect on mg transport and induce a decline of [ mg]i . reported that responses to hyperglycemia in vitro were blunted in adult hypertensive subjects and that these responses were closely linked to basal [ mg]i levels . in our study , we found no correlation between cord plasma glucose and basal [ mg]i levels . in addition , we did not find any significant difference in plasma glucose levels between the sga and aga groups . intrauterine glucose levels may have less of an effect on inducing sga or low [ mg]i . as [ mg]i plays a promotive role in fetal growth , low [ mg]i may partly be responsible for sga . as low [ mg]i is an intrinsic abnormality seen in infants with low birth weight , it is considered that fetal mg deficiency is an important determinant of insulin resistance in later life . in fact , our data was supported by a recent animal study demonstrating that maternal mg restriction irreversibly increases body fat and induces insulin resistance in pups by 6 months of age . the fetal origin hypothesis by barker et al . [ 810 ] states that fetal undernutrition in middle to late gestation leads to disproportionate fetal growth programs and later metabolic diseases . fetal programming is a phenomenon in which alterations in fetal growth and development in response to the prenatal environment have long - term or permanent effects . this theory was further established in the category of developmental origins of health and disease ( dohad ) . the mechanisms thought to be responsible for fetal programming include the direct effects on cell number , altered stem cell function , and the resetting of regulatory hormonal axes : hypothalamic - pituitary - adrenal axis [ 39 , 40 ] and growth hormone insulin - like growth factor axis . one of the underlying mechanisms for the postulated early - life programming is that of epigenetics . altered epigenetic regulation of genes in phenotype induction could possibly give rise to interventions that modify long - term disease risk associated with unbalanced nutrition in early life . reported that fasting insulin and homeostasis model assessment - insulin resistance ( homa - ir ) increased in offspring from mg - restricted dams at 6 months of age . maternal and postnatal mg status is important in the long - term programming of body adiposity and insulin secretion in rat offspring . although low birth weight and poor prenatal nutrition are strongly associated with metabolic syndrome in later life , postnatal catch - up growth was recently considered to also be a pivotal element associated with the development of various pathological conditions . the concept of a sensitive or crucial period that operates to cause long - term changes in development and adverse outcomes later in life is an intriguing one and should be the focus of more studies in the future . birth weight is only a crude index of early growth and indicates nothing about the success of a fetus in achieving its growth potential . [ mg]i may be a marker of early growth restriction , which may be of future diagnostic use as an early predictor of adult diseases . low [ mg]i , which may represent the prenatal programming of insulin resistance , has lifelong effects on metabolic regulation . a biological interpretation of the association between birth size and risk of insulin - resistant diseases should emphasize the possible underlying roles of [ mg]i .

magnesium deficiency in pregnancy frequently occurs because of inadequate or low intake of magnesium . magnesium deficiency during pregnancy can induce not only maternal and fetal nutritional problems , but also consequences that might last in offspring throughout life . many epidemiological studies have disclosed that small for gestational age ( sga ) is associated with an increased risk of insulin resistance in adult life . we reported that intracellular magnesium of cord blood platelets is lower in sga groups than that in appropriate for gestational age groups , suggesting that intrauterine magnesium deficiency may result in sga . taken together , intrauterine magnesium deficiency in the fetus may lead to or at least program insulin resistance after birth . in this review , we propose that intrauterine magnesium deficiency may induce metabolic syndrome in later life . we discuss the potential contribution of aberrant magnesium regulation to sga and to the pathogenesis of metabolic syndrome .

1. Introduction 2. Magnesium Deficiency During Pregnancy 3. Mg Supplements and Pregnancy Outcome 4. The Relation of SGA to Intracellular Magnesium in Cord Blood Platelets 5. Fetal Programming 6. Conclusion

the continuous monitoring and audit of clinical practice is an essential part of making improvements in medical science and enhancing patient care . this is true also in the management of neurosurgical cases , but no validated comparative tool was available until 2008 , when the physiological and operative severity score for the enumeration of mortality and morbidity ( possum ) and portsmouth - possum ( p - possum ) scoring systems were evaluated in indian patients undergoing elective craniotomy and concluded that the p - possum score was highly accurate in predicting overall mortality . initially designed the possum scoring system to evaluate morbidity and mortality in general surgical patients . it has also been modified as p - possum to improve the score 's accuracy . although the possum and p - possum scores are based on objective physiological and operative criteria , these vary across populations and healthcare systems and can not be immediately assumed to be valid across the countries . variations in mortality scores have been demonstrated before during such comparative studies in differing geographical regions . in fact , a fourfold difference in mortality in major surgical procedures was observed in uk and us patient population . we , therefore , evaluated the scores in patients undergoing elective and emergency craniotomies in a tertiary neurosurgical referral centre in the united kingdom to investigate how both systems performed in a different population and to explore the possibility of whether or not the p - possum and possum scoring system needed any further modification to allow its application in such patients . following approval from the local research ethical committee , data were collected from all neurosurgical patients undergoing craniotomy over a period of one year . the possum and p - possum scores are calculated using the following equations , which are a combination of weighted variables of physiological and operative data obtained for individual patients : predicted possum mortality ln [ r/(1r ) = 7.04 + 0 . 13 physiological score + 0.16 x operative score ; where r is the predicted mortality score . predicted p - possum mortality ln [ r/(1r ) ] = 9.37 + 0.19 physiological score + 0.15 x operative score ; we compared the observed in - hospital mortality with the predicted mortality obtained by a possum calculator . the online calculator records each of the 18 factors , which are weighted to a value of 1 , 2 , 4 or 8 depending on measured variables . preoperative physiological variables prior to induction of anaesthesia were collected from the clinical notes and investigations , and operative variables [ table 1 ] were collected after the surgery . the category both the surgery and anaesthesia were performed either by consultants or by specialist registrars under supervision of consultants . parameters used in possum scoring system we obtained in - hospital mortality data from hospital mortality records . patients were discharged from hospital at the discretion of the treating surgeon , as is usual practice in our institution . using the calculated possum and p - possum values , the observed to expected ratios were calculated where a value of 1 would represent the best prediction . . demographic values between survivors and non - survivors were compared by one - way anova for continuous variables and chi - square for categorical variables . microsoft excel worksheet statistics and spss 10.0 for windows were used for analysing the data . a total of 145 patients undergoing elective and emergency craniotomies had their clinical data analysed . descriptive characters of the patients the mean [ sd ] calculated physiologic score of the patients was 18.83 [ 5.07 ] and their mean [ sd ] operative score was 18.09 [ 3.75 ] . on comparing predicted and observed mortality with p - possum and possum , we noted that there was a statistically significant difference in the physiological score ( p = 0.005 ) , the operative score ( p = 0.005 ) in patients with observed in hospital mortality and survivors [ tables 3 and 4 ] . comparison of p - possum - predicted mortality with the observed mortality comparison of possum - predicted mortality with the observed mortality fifteen patients ( 10.3% ) died ; this was identical to the p - possum prediction . the difference between expected and observed frequencies over different predicted mortality range was not significant with p - possum score ( p = 0.122 ) . we noted that the prediction of mortality by p - possum was very accurate with an observed / predicted mortality ratio of 1.0 [ table 5 ] . comparison of p - possum data for urgency of surgery the prediction of mortality with possum score was poor in contrast to the favourable results achieved with p - possum . the expected mortality according to possum model was 28 patients ( 19.3% ) , which contrasts with the observed mortality of 15 patients . the goodness - of - fit chi - square test just showed statistically significant difference between expected and observed frequencies based on possum score ( p = 0.047 ) . we also compared the three categories of elective , emergency , immediate ( within 24 hours of stabilization ) surgeries using the p - possum scoring system [ table 5 ] . this suggests that p - possum was a better predictor for elective patients and for those undergoing immediate life - saving surgery although the difference was not significant [ p = 0.06 ] . auditing of practice and comparison of mortality data is essential to ensure that patients are well informed of risks and to improve quality of care in hospitals . several surgical outcome scores have been devised to help with this issue , such as the surgical apgar score , apache ii , but the possum [ physiological and operative severity score for the enumeration of mortality and morbidity ] and p - possum [ portsmouth - possum ] scores remain the most studied and most validated across specialities and patient populations . the possum and p - possum scores have been validated across many surgical procedures , but not until recently in craniotomies , which is an integral and important part of neurosurgical practice . the first validated and published tool was reported from an indian neurosurgical population in 2008 . when it was shown that that both the possum and the p - possum scores can be used to predict mortality accurately , with p - possum being the stronger score . this study set out to validate the scores in another patient population [ india ] to see whether the p - possum score could be used across different health care systems and patient populations , when compared to the uk . this would add weight to the tool and allow it to be used across geographical and healthcare boundaries . this is important as a general comment about predictive scores or models is one of universal applicability . however , that needed testing under clinical conditions , which is what this study has done . we , therefore , adhered to the method of the original study and collected all data just prior to surgery as that is more likely to affect outcomes than data collected at admission . we also selected in - hospital mortality as the end point as in the original study , as this reflects clinical practice individualised to the surgical and anaesthetics teams . overall , 15 patients ( 10.3% ) in the study died ; this was identical to the p - possum prediction of mortality . the difference between expected and observed frequencies over different predicted mortality range was not significant with p - possum score ( p = 0.122 ) . in common with work in other surgical specialities , p - possum was accurate at predicting overall risk rather than risk across the subgroups . the prediction of mortality with possum score was poor in contrast to the favourable results achieved with p - possum . the expected mortality according to possum model was 28 patients ( 19.3% ) , which contrasts with the observed mortality of 15 patients . we also compared the three categories of elective , immediate ( within 24 hours of stabilization ) or emergency surgery using the p - possum scoring system [ table 5 ] . this suggests that p - possum was a better predictor for elective patients and for those undergoing immediate life - saving surgery although the difference was not significant [ p = 0.06 . ] . we feel that this was due to the smaller number of patients in these categories and this deserves further work our patients were discharged home at the discretion of the surgical team , a process that lacks standardisation . we accepted that this was a more real reflection of surgical practice than to adopt other end points , such as 28-day mortality . additionally , a comparison of elective , stabilized and emergency patients did not reveal a significant difference , although this may be due to the small number of emergency procedures ( n = 14 ) in our centre over the year . the original work that validated p - possum for craniotomies study did not include emergency patients and as there was a small number only in our study population this time , it is not possible to draw any firm conclusions at this time . more importantly , it has not been validated outside of the australian population and system , whereas this study supports a familiar and a most popular and recognised system to demonstrate that it can be used across countries . therefore , it will be simpler to adopt the p - possum score into neurosurgical practice as it stands , rather than trying to improvise that and again looking at the validity of the new scoring system . we conclude that p - possum , but not possum , is a useful scoring system that can be used comparing mortality data for neurosurgical patients undergoing craniotomy in different populations and healthcare systems ( india and the uk ) . although p - possum seems to be a useful predictor in the emergency situation also , we would recommend further large scale work for its validation .

background and aims : continuous audit of clinical practice is an essential part of making improvements in medicine and enhancing patient care . validated tools are needed to gather evidence for comparisons . recently , physiological and operative severity score for the enumeration of mortality and morbidity ( possum ) and portsmouth - possum ( p - possum ) scores were evaluated in indian patients undergoing elective craniotomy and it was concluded that p - possum was highly accurate in predicting overall mortality . we wished to study whether this system could be used in a different country and health care system [ united kingdom , uk ] . we have evaluated these scores in patients undergoing elective and emergency craniotomies in a tertiary centre in the uk.methods:data was collected from all neurosurgical patients who underwent craniotomy overone year . preoperative variables were collected prior to induction of anaesthesia , and operative variables were also collected . chi - square test was used for expected and actual mortality differences . survivor and non - survivor demographics were compared by one - way anova for continuous and chi - square for categorical variables.results:one hundred and forty - five patients were studied . mean [ sd ] physiologic score of the patients was 18.83 [ 5.07 ] , and mean [ sd ] operative score was 18.09 [ 3.75 ] . p - possum was a better predictor for elective patients and for those undergoing immediate life - saving surgery.conclusion:this study confirms and validates the findings of previous work that p - possum is an accurate and reliable tool for estimating in - hospital mortality . it also confirms its usefulness in comparison of results across healthcare systems internationally . larger scale evaluations may be needed to examine its usefulness in emergency procedures .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

image - guided radiation therapy ( igrt ) during patient set - up prior to treatment delivery allows the patient to be positioned as closely as possible to the expected irradiation position . several recent igrt techniques include fan - beam ct [ 13 ] , ct - on - rails , cone - beam ct ( cbct ) [ 56 ] , electronic portal imaging device ( epid ) , ultrasound system and infrared marker . the first four of these techniques use x - rays in kilovoltage or megavoltage beam quality . depending on beam quality , however , these techniques may include features such as image contrast between bony structures and soft tissues , and the presence metallic artifacts . correct placement of the patient requires assurance of the performance of imaging devices in terms of both image quality and the lack of geometrical distortion related to the treatment room coordinates . mv - cbct uses the same x - ray source and gantry as those used for treatment . it also uses the same epid as for 2d imaging and thus eliminates the requirement for isocenter matching calibration . the linear accelerator ( linac ) vendor has proposed quality assurance ( qa ) instructions for mv - cbct that should be performed on a monthly basis ( referred to in siemens mvision physicist self - led training ) . qa frequency has also been proposed in the american association of physicists in medicine task group 142 report . this instruction includes a check of image quality and geometric distortion in the three dimensions of lateral , longitudinal and vertical . based on the vendor instructions , the measurement protocol is defined with 15 monitor units ( mu ) with low - energy photons for irradiation and the use of a filter named smoothing head and neck. a smoothing filter is applied to the reconstructed images to correct for the cupping effect due to the large amount of scatter inherent in the large field sizes in cone - beam geometry . pelvis or head and neck , which corrects the cupping effect based on the size of the respective anatomical site , and 3 ( head and neck region ) or 5 mu ( abdominal region ) are used for image guidance in clinical settings to minimize the absorbed dose the patient receives during visualization [ 12 , 13 ] . even when the results of monthly qa performed with 15 mu are within tolerance , occasional errors such as streak artifacts , tyre - track artifacts , image non - uniformity , and undesirable contrast resolution have been noted in clinical use . the high - quality protocol suggested by the vendor for mv - cbct at the linac for image quality assessment does not reflect the clinically recommended scan protocol , and subtle changes in imaging performance may occur within the 1-month testing period . in addition , if image quality does in fact decrease with time , a reduction in positioning or registration accuracy can be expected . here , to verify and track possible changes in qa results over periods of as long as one year , we performed the vendor - proposed qa on a weekly rather than monthly basis . an x - ray beam from an oncor impression plus dual photon energy linear accelerator ( 6 mv and 10 mv ; siemens medical systems , erlangen , germany ) was used . portal images were acquired with a siemens optivue 1000 epid ( siemens medical systems ) . the portal imager has matrices of 1024 1024 pixels with a physical size of 0.40 mm , giving an active area of 41 41 cm . a 3-mm copper plate overlays the sensitive layer of the epid to remove low - energy photons ; immediately beneath the copper plate is a scintillating layer of phosphor to transform incoming x - rays to visible photons , and then a pixel array implanted on the amorphous - si panel to capture visible photons and convert them to electric charges . the charge signals are then read out and digitized by a 16-bit analog - to - digital converter . source to image distance ( sid ) is changeable between 110 cm and 160 cm . qa measurements for mv - cbct were rotational irradiation , which started at a gantry angle of 270 to 110 at a fixed sid of 145 cm with a 27.4 cm 27.4 cm field size and low energy photons of 6 mv . sid was defined by the vendor . following insertion of the orthogonal tungsten wires ( which are named the xretic plate and are matched to the mechanical isocenter ) into the shadow tray , the siemens image quality phantom ( called the emma phantom , siemens medical systems ) was manually set to the isocenter using the wire shadow at gantry angles of 0 , 90 and 270 by matching the projection of the two orthogonal metal wires of the xretic plate with the reference lines of the phantom in the anterior and two lateral directions . after mv - cbct irradiation , the system automatically reconstructs the cbct image in a slice thickness of 1 mm and 256 256 matrices with filtered correction of cupping artifacts named smoothing head and neck , as described above . voxel size was 1.07 mm 1.07 mm 1.00 mm in the lateral , vertical and longitudinal directions , respectively . reconstructed image sets of a total of 274 slices were outputted and imported into our in - house software developed using codegear delphi 2007 . mv - cbct imaging by the phantom analysis described below was performed 100 times per week for 1 year . evaluation of image quality with regard to low - contrast resolution , high - contrast resolution , and contrast - to - noise ratio was done by displaying transverse slices using a 5-mm multiple plane reconstruction view on the in - house software to reduce noise . the emma phantom has three sets , each of four beads , that are used to check geometric distortion in three dimensions . the beads are distributed evenly around the circumference of the phantom with z coordinates of 100 mm , 0 mm and 100 mm for the superior , center and inferior slices , respectively . the four beads in each slice are located at the 3 , 6 , 9 and 12 o'clock positions , respectively . each of the four beads is separated from the center of the phantom by + 95.25 mm . four beads are evenly separated from the center of the phantom by 95.25 mm in transverse view . transverse planes are located at intervals of 100 mm along the z - axis . the emma phantom geometry and bead configuration in 3d and transverse views . four beads are evenly separated from the center of the phantom by 95.25 mm in transverse view . transverse planes are located at intervals of 100 mm along the z - axis . for the geometric distortion check , a reference point was manually placed at the center of each bead in the mv - cbct image of the phantom using the in - house software , and then the position was compared with that of the nominal position . minimum pixel resolution for analysis in the in - house software was 0.27 mm in the lateral and vertical axes , and 0.23 mm in the longitudinal axis . with regard to checking image quality , the emma phantom has a solid region that consists of four sections , namely : ( a ) a solid water section , ( b ) a low - contrast resolution section , ( c ) a spatial resolution section , and ( d ) a high - contrast resolution section . these sections in transverse view consist of ( a ) solid water section , ( b ) low - contrast resolution section , ( c ) spatial resolution section , and ( d ) high - contrast resolution section , respectively . these sections in transverse view consist of ( a ) solid water section , ( b ) low - contrast resolution section , ( c ) spatial resolution section , and ( d ) high - contrast resolution section , respectively . section ( a ) is a 40-mm uniform solid water cylinder that is used to check image noise and the uniformity of pixel values . on the central slice of this section , five circular regions of interest ( rois ) were automatically drawn on the image ; one in the center and four in the periphery at the 3 , 6 , 9 and 12 o'clock positions at an equidistance of 69.6 mm from the center of the phantom , as shown in fig . the distance of 69.6 mm was determined to be suitably close to the edge of the phantom and was used for all analyses . four peripheral rois are placed , evenly separated by 69.6 mm from the center of the phantom . four peripheral rois are placed , evenly separated by 69.6 mm from the center of the phantom . the mean pixel value and standard deviation for each roi were calculated , and the pixel value was then compared with the vendor specification . expected results for 6-mv acquisitions were as follows : the center roi , which is numbered 2 , should have ( i ) a standard deviation between + 26 and + 42 , and ( ii ) a mean value of pixels between 30 and + 42 . the difference between the mean pixel value of each peripheral roi and the mean pixel value of the central roi was calculated , and it was verified that the difference fell within the expected range of 80 to + 80 . image reconstruction artifacts due to dead pixels or wrong gantry rotation speed were also visually checked on each slice of this section . sections ( b ) and ( d ) , which contain inserts of different material rods with various diameters inside a solid water background , are shown in fig . 4.low-contrast resolution section ( b ) , and high - contrast section ( d ) of the emma phantom . each section has inserts of four different materials , namely ( b ) brain , liver , 1% sig , and 3% sig , and ( d ) air , cb2 - 50% , inner bone , and acrylic . the diameter of the five rods for each material is 2 , 1 , 0.7 , 0.5 and 0.3 cm , respectively . low - contrast resolution section ( b ) , and high - contrast section ( d ) of the emma phantom . each section has inserts of four different materials , namely ( b ) brain , liver , 1% sig , and 3% sig , and ( d ) air , cb2 - 50% , inner bone , and acrylic . the diameter of the five rods for each material is 2 , 1 , 0.7 , 0.5 and 0.3 cm , respectively . the physical density and relative electron density of each material with respect to the background are presented in table 1 . table 1.physical density and relative electron density of materials with respect to backgroundsectionmaterialphysical density ( g / cm)electron densityiibrain1.051.04iiliver1.091.06ii1% sig1.031.00ii3% sig1.051.02ivair0.000.00ivcb2 - 50%1.561.47ivinner bone1.141.08ivacrylic1.181.16sig = standard imaging grade of background material , cb2 - 50% , caco3 . physical density and relative electron density of materials with respect to background sig = standard imaging grade of background material , cb2 - 50% , caco3 . low - contrast resolution was qualitatively checked by adjusting the window level and window width to preset values , and by counting the number of inserts of each material that were visible on the image . after the set of roi included all inserts in each slice , the mean , maximum and minimum pixel values were calculated . the mean pixel value was used for the window level , and the difference between the minimum and maximum values was used for the window width in order to define the preset values for visual evaluation . table 2 lists the circles that should be visible in each of the eight groups in sections ( b ) and ( d ) under image acquisition at 6 mv . table 2.number of rods that should be visible in each of the eight groups in 6-mv image acquisitionsectionmaterialvisible rods countiibrain1iiliver2ii1% sig0ii3% sig0ivair5ivcb2 - 50%5ivinner bone4ivacrylic4sig = standard imaging grade of background material , cb2 - 50% , caco3 . number of rods that should be visible in each of the eight groups in 6-mv image acquisition sig = standard imaging grade of background material , cb2 - 50% , caco3 . different material rods in section ( d ) were also used to calculate the contrast - to - noise ratios ( cnr ) as presented by gayou et al . . the equation for cnr was : ( 1 ) where s and sbg are the mean pixel values in an insert and the background region surrounding the insert , respectively , and is the average standard deviation of the pixel value in the insert and the background . since this analysis was a quantitative evaluation and the position of each rod and of the background region was clearly defined , each roi of 2-cm diameter was automatically set to its position in the same manner as in pixel uniformity analysis , as shown in fig . after the image was rotated counterclockwise 20 , the roi at the same position was used for calculation . this section contained 11 bar groups , each group of which contained 5 bars , arranged so that each group had a different resolution , as shown in fig . 5 . fig . 5.spatial resolution section ( c ) of the emma phantom containing 11 bar groups with different numbers of line pairs per millimeter . h. spatial resolution section ( c ) of the emma phantom containing 11 bar groups with different numbers of line pairs per millimeter . this is a qualitative analysis based on the number of bars that are visible on the image in which we determine how many groups ( each with five line pairs ) are visible . the expected results for this test using 6-mv image acquisitions are that the largest to the sixth - largest line group ( corresponding to 0.30 lp / mm ) ; in other words , the nyquist frequency was calculated as 1 divided by twice the sampling frequency of 1.67 mm should be visible with all five dark lines distinctly visible , whereas lines 711 should not be resolvable , in accordance with the limitations of current imaging technology . table 3.specification of the chart for each bar group and nyquist frequency in line pairs per millimeter calculated by widthbar groupw ( mm)h ( mm)lp / mm17.512.00.06725.012.00.133.312.00.1542.512.00.252.012.00.2561.6712.00.371.2512.00.481.010.00.590.836.60.6100.625.00.8110.54.51.0w = width , h = height . specification of the chart for each bar group and nyquist frequency in line pairs per millimeter calculated by width w = width , h = height . an x - ray beam from an oncor impression plus dual photon energy linear accelerator ( 6 mv and 10 mv ; siemens medical systems , erlangen , germany ) was used . portal images were acquired with a siemens optivue 1000 epid ( siemens medical systems ) . the portal imager has matrices of 1024 1024 pixels with a physical size of 0.40 mm , giving an active area of 41 41 cm . a 3-mm copper plate overlays the sensitive layer of the epid to remove low - energy photons ; immediately beneath the copper plate is a scintillating layer of phosphor to transform incoming x - rays to visible photons , and then a pixel array implanted on the amorphous - si panel to capture visible photons and convert them to electric charges . the charge signals are then read out and digitized by a 16-bit analog - to - digital converter . source to image distance ( sid ) is changeable between 110 cm and 160 cm . qa measurements for mv - cbct were rotational irradiation , which started at a gantry angle of 270 to 110 at a fixed sid of 145 cm with a 27.4 cm 27.4 cm field size and low energy photons of 6 mv . sid was defined by the vendor . following insertion of the orthogonal tungsten wires ( which are named the xretic plate and are matched to the mechanical isocenter ) into the shadow tray , the siemens image quality phantom ( called the emma phantom , siemens medical systems ) was manually set to the isocenter using the wire shadow at gantry angles of 0 , 90 and 270 by matching the projection of the two orthogonal metal wires of the xretic plate with the reference lines of the phantom in the anterior and two lateral directions . after mv - cbct irradiation , the system automatically reconstructs the cbct image in a slice thickness of 1 mm and 256 256 matrices with filtered correction of cupping artifacts named smoothing head and neck , as described above . voxel size was 1.07 mm 1.07 mm 1.00 mm in the lateral , vertical and longitudinal directions , respectively . reconstructed image sets of a total of 274 slices were outputted and imported into our in - house software developed using codegear delphi 2007 . mv - cbct imaging by the phantom analysis described below was performed 100 times per week for 1 year . evaluation of image quality with regard to low - contrast resolution , high - contrast resolution , and contrast - to - noise ratio was done by displaying transverse slices using a 5-mm multiple plane reconstruction view on the in - house software to reduce noise . 1 . the emma phantom has three sets , each of four beads , that are used to check geometric distortion in three dimensions . the beads are distributed evenly around the circumference of the phantom with z coordinates of 100 mm , 0 mm and 100 mm for the superior , center and inferior slices , respectively . the four beads in each slice are located at the 3 , 6 , 9 and 12 o'clock positions , respectively . each of the four beads is separated from the center of the phantom by + 95.25 mm . four beads are evenly separated from the center of the phantom by 95.25 mm in transverse view . four beads are evenly separated from the center of the phantom by 95.25 mm in transverse view . transverse planes are located at intervals of 100 mm along the z - axis . for the geometric distortion check , a reference point was manually placed at the center of each bead in the mv - cbct image of the phantom using the in - house software , and then the position was compared with that of the nominal position . minimum pixel resolution for analysis in the in - house software was 0.27 mm in the lateral and vertical axes , and 0.23 mm in the longitudinal axis . with regard to checking image quality , the emma phantom has a solid region that consists of four sections , namely : ( a ) a solid water section , ( b ) a low - contrast resolution section , ( c ) a spatial resolution section , and ( d ) a high - contrast resolution section . these sections in transverse view consist of ( a ) solid water section , ( b ) low - contrast resolution section , ( c ) spatial resolution section , and ( d ) high - contrast resolution section , respectively . four solid sections of the emma phantom in sagittal and transverse view . these sections in transverse view consist of ( a ) solid water section , ( b ) low - contrast resolution section , ( c ) spatial resolution section , and ( d ) high - contrast resolution section , respectively . section ( a ) is a 40-mm uniform solid water cylinder that is used to check image noise and the uniformity of pixel values . on the central slice of this section , five circular regions of interest ( rois ) were automatically drawn on the image ; one in the center and four in the periphery at the 3 , 6 , 9 and 12 o'clock positions at an equidistance of 69.6 mm from the center of the phantom , as shown in fig . the distance of 69.6 mm was determined to be suitably close to the edge of the phantom and was used for all analyses . four peripheral rois are placed , evenly separated by 69.6 mm from the center of the phantom . four peripheral rois are placed , evenly separated by 69.6 mm from the center of the phantom . the mean pixel value and standard deviation for each roi were calculated , and the pixel value was then compared with the vendor specification . expected results for 6-mv acquisitions were as follows : the center roi , which is numbered 2 , should have ( i ) a standard deviation between + 26 and + 42 , and ( ii ) a mean value of pixels between 30 and + 42 . the difference between the mean pixel value of each peripheral roi and the mean pixel value of the central roi was calculated , and it was verified that the difference fell within the expected range of 80 to + 80 . image reconstruction artifacts due to dead pixels or wrong gantry rotation speed were also visually checked on each slice of this section . sections ( b ) and ( d ) , which contain inserts of different material rods with various diameters inside a solid water background , are shown in fig . 4.low-contrast resolution section ( b ) , and high - contrast section ( d ) of the emma phantom . each section has inserts of four different materials , namely ( b ) brain , liver , 1% sig , and 3% sig , and ( d ) air , cb2 - 50% , inner bone , and acrylic . the diameter of the five rods for each material is 2 , 1 , 0.7 , 0.5 and 0.3 cm , respectively . low - contrast resolution section ( b ) , and high - contrast section ( d ) of the emma phantom . each section has inserts of four different materials , namely ( b ) brain , liver , 1% sig , and 3% sig , and ( d ) air , cb2 - 50% , inner bone , and acrylic . the diameter of the five rods for each material is 2 , 1 , 0.7 , 0.5 and 0.3 cm , respectively . the physical density and relative electron density of each material with respect to the background are presented in table 1 . table 1.physical density and relative electron density of materials with respect to backgroundsectionmaterialphysical density ( g / cm)electron densityiibrain1.051.04iiliver1.091.06ii1% sig1.031.00ii3% sig1.051.02ivair0.000.00ivcb2 - 50%1.561.47ivinner bone1.141.08ivacrylic1.181.16sig = standard imaging grade of background material , cb2 - 50% , caco3 . physical density and relative electron density of materials with respect to background sig = standard imaging grade of background material , cb2 - 50% , caco3 . low - contrast resolution was qualitatively checked by adjusting the window level and window width to preset values , and by counting the number of inserts of each material that were visible on the image . after the set of roi included all inserts in each slice , the mean , maximum and minimum pixel values were calculated . the mean pixel value was used for the window level , and the difference between the minimum and maximum values was used for the window width in order to define the preset values for visual evaluation . table 2 lists the circles that should be visible in each of the eight groups in sections ( b ) and ( d ) under image acquisition at 6 mv . table 2.number of rods that should be visible in each of the eight groups in 6-mv image acquisitionsectionmaterialvisible rods countiibrain1iiliver2ii1% sig0ii3% sig0ivair5ivcb2 - 50%5ivinner bone4ivacrylic4sig = standard imaging grade of background material , cb2 - 50% , caco3 . number of rods that should be visible in each of the eight groups in 6-mv image acquisition sig = standard imaging grade of background material , cb2 - 50% , caco3 . different material rods in section ( d ) were also used to calculate the contrast - to - noise ratios ( cnr ) as presented by gayou et al . . the equation for cnr was : ( 1 ) where s and sbg are the mean pixel values in an insert and the background region surrounding the insert , respectively , and is the average standard deviation of the pixel value in the insert and the background . since this analysis was a quantitative evaluation and the position of each rod and of the background region was clearly defined , each roi of 2-cm diameter was automatically set to its position in the same manner as in pixel uniformity analysis , as shown in fig . after the image was rotated counterclockwise 20 , the roi at the same position was used for calculation . section ( c ) this section contained 11 bar groups , each group of which contained 5 bars , arranged so that each group had a different resolution , as shown in fig . 5 . fig . 5.spatial resolution section ( c ) of the emma phantom containing 11 bar groups with different numbers of line pairs per millimeter . h. spatial resolution section ( c ) of the emma phantom containing 11 bar groups with different numbers of line pairs per millimeter . h. table 3 shows the specification of the chart for each bar group . this is a qualitative analysis based on the number of bars that are visible on the image in which we determine how many groups ( each with five line pairs ) are visible . the expected results for this test using 6-mv image acquisitions are that the largest to the sixth - largest line group ( corresponding to 0.30 lp / mm ) ; in other words , the nyquist frequency was calculated as 1 divided by twice the sampling frequency of 1.67 mm should be visible with all five dark lines distinctly visible , whereas lines 711 should not be resolvable , in accordance with the limitations of current imaging technology . table 3.specification of the chart for each bar group and nyquist frequency in line pairs per millimeter calculated by widthbar groupw ( mm)h ( mm)lp / mm17.512.00.06725.012.00.133.312.00.1542.512.00.252.012.00.2561.6712.00.371.2512.00.481.010.00.590.836.60.6100.625.00.8110.54.51.0w = width , h = height . specification of the chart for each bar group and nyquist frequency in line pairs per millimeter calculated by width w = width , h = height . figure 6 shows positional errors of the measured positions of the 12 beads with respect to the expected positions from 100 measurements . for the x - axis , positional errors at 12 o'clock in all planes ( head 10 cm , center , and foot 10 cm ) were positive values , while those at 6 o'clock in all planes were negative . for the y - axis , positional errors at 3 o'clock in all planes were positive values , while those at 9 o'clock in all planes were negative . this pattern suggests that placement of the emma phantom might have been biased slightly toward the clockwise direction in each manual setup . for the z- axis , positional errors in the two center and head 10-cm planes were negative values , while those in the foot 10-cm plane were positive . however , uncertainties in the subjective , user - dependent placement of the reference point at the center of the bead might have contributed to these variations . 6.positional errors of measured positions of the 12 beads with respect to the expected positions from 100 measurements in the x , y and z axes . the vertical axes show the positional errors of relative lateral ( a : x - axis ) , vertical ( b : y - axis ) , and longitudinal ( c : z - axis ) measured positions of the 12 beads embedded in the emma phantom , with respect to their nominal values at their own bead positions . each bar presents the mean value from 100 measurements , and the error bar means one standard deviation . positional errors of measured positions of the 12 beads with respect to the expected positions from 100 measurements in the x , y and z axes . the vertical axes show the positional errors of relative lateral ( a : x - axis ) , vertical ( b : y - axis ) , and longitudinal ( c : z - axis ) measured positions of the 12 beads embedded in the emma phantom , with respect to their nominal values at their own bead positions . each bar presents the mean value from 100 measurements , and the error bar means one standard deviation . figure 7 shows the mean pixel value and one standard deviation for the roi at the center position , and the difference pixel value at each roi of 3 , 6 , 9 and 12 o'clock compared with the center position from 100 measurements . the measured values of two of the 100 measurements for mean pixel value were outside the range of 30 to + 42 ( shown in black arrows ) , at 40 and + 203 . all values were within tolerance after the adjustment in beam output for cone - beam ct acquisition . other values at the second black arrow for sd , 3 , 6 , 9 and 12 o'clock were also outside the tolerance range . after the adjustment of beam output for cone - beam ct acquisition , these values were within the tolerance range . apart from these two occurrences , other pixel values for the mean and sd at each roi position were within the tolerance range . 7.mean pixel value and 1 sd for the roi at the center position , and difference in pixel value at each roi of 3 , 6 , 9 and 12 o'clock compared with the center position from 100 measurements . the vertical axis shows the pixel value of the mean and 1 sd of the center roi and that of the roi at 3 , 6 , 9 and 12 o'clock from 100 measurements . the two black arrows indicate measurements in which the pixel values were out of tolerance . mean pixel value and 1 sd for the roi at the center position , and difference in pixel value at each roi of 3 , 6 , 9 and 12 o'clock compared with the center position from 100 measurements . the vertical axis shows the pixel value of the mean and 1 sd of the center roi and that of the roi at 3 , 6 , 9 and 12 o'clock from 100 measurements . the two black arrows indicate measurements in which the pixel values were out of tolerance . figure 8 shows the visible rod counts at the low- and high - contrast resolution sections for various materials from 100 measurements . for the low - contrast resolution section , all materials at up to the diameter specified by the vendor were visible . for the liver rod material , 95 of 100 measurements were more visible than expected , as were 83 of 100 measurements for the brain . for the high - contrast resolution section , all materials with up to the specified diameter by the vendor were also visible . fig . 8.visible rod counts at low- and high - contrast resolution sections for various materials from 100 measurements . the vertical axis shows the visible rod counts for each material at the ( a ) low - contrast resolution section , and ( b ) high - contrast resolution section from 100 measurements . for both contrast sections the visible rod counts were within the tolerance specified by the vendor . visible rod counts at low- and high - contrast resolution sections for various materials from 100 measurements . the vertical axis shows the visible rod counts for each material at the ( a ) low - contrast resolution section , and ( b ) high - contrast resolution section from 100 measurements . for both contrast sections the visible rod counts were within the tolerance specified by the vendor . figure 9 shows the cnr for the three different material rods of cb2 - 50% , inner bone , and acrylic from 100 measurements . the cnr value of cb2 - 50% was 15.9 0.8 ( mean sd ) , which was remarkably higher than those of inner bone ( 2.5 0.2 ) and acrylic ( 3.7 0.2 ) considering the electron density of each material . 9.contrast-to-noise ratio ( cnr ) for cb2 - 50% , inner bone and acrylic from 100 measurements . contrast - to - noise ratio ( cnr ) for cb2 - 50% , inner bone and acrylic from 100 measurements . for the evaluation of spatial resolution section by visual inspection , bars in group 6 , that is up to 0.3 lp / mm , were clearly visible for 100 measurements . imaging performance of mv - cbct was evaluated with regard to geometrical distortion and image quality for 1 year using 100 measurements . although the vendor has recommended that the qa used for this study should be performed every month , we changed this frequency to a weekly basis in order to identify subtle changes in characteristics , such as in geometrical distortion and imaging quality , and to evaluate whether the 1-month frequency was valid . in this regard , the aapm tg-147 report also states that the frequency of the test may be increased upon vendor recommendation . for geometrical distortion , relative position errors of all beads were within the 2-mm tolerance specified by the vendor . figure 10 shows a vector diagram of positional errors in the central plane in axial view . we performed the qa test 100 times over a period of 1 year , but the tendency identified in fig . although the error value was small , with a measurement uncertainty of 0.14 mm ( calculated from the minimum resolution of 0.27 mm divided by 2 ) , vector sums at the four positions of 3 , 6 , 9 and 12 o'clock might indicate a tendency to rotational error of 0.11 0.22 ( mean sd ) from the 100 measurements with respect to the z - axis in the clockwise direction . figure 11 shows axial and coronal views of a bucket filled with water taken by mv - cbct . we confirmed that the water plane was slightly tilted by 0.27 in the clockwise direction on the axial image , and that the image was asymmetric in the left - to - right direction in the coronal image . we interpret this strange phenomenon to mean that the geometry calibration for mv - cbct acquisition is not perfect . after the geometry phantom was carefully aligned around the machine isocenter , an image was acquired for each of the 200 gantry angles of the cone beam arc . the lookup tables , which are 4 4 matrices , consist of the rotation and translation for each projection image , and are automatically created to correct errors in the vertical and horizontal scale and skew of the detector that can be caused by detector sagging or mispositioning . when the geometry phantom is misaligned with a translational error of a few millimeters or a rotational error of a few degrees the lookup tables will not be updated because the offset values for the calibration are large and are regarded by the system as out of the tolerance range . the system does not allow users to determine the tolerance value for the geometric calibration , which means it is a black box. if the size of the error caused by misalignment is small , however , the calibration procedure is performed correctly . in the present study , we found that positional errors in geometrical distortion resulted in geometry calibration with slight misalignment errors , and that these would lead to residual error . 11.image of a water - filled bucket by mv - cbct acquisition in ( a ) axial and ( b ) coronal views . the coronal image was at the position of the water surface shown by the yellow line in the axial image . vector diagram of the positional errors in the central plane in axial view . dashed arrows show the mean translational errors in millimeters for 100 measurements . image of a water - filled bucket by mv - cbct acquisition in ( a ) axial and ( b ) coronal views . the coronal image was at the position of the water surface shown by the yellow line in the axial image . therefore , according to the results for 100 measurements , the new tolerance values in millimeters for bead positions were established in our department . table 4 summarized the new tolerance values at the four positions of 3 , 6 , 9 and 12 o'clock in each axis that were based on the 95% confidence interval ( i.e. mean 1.96 sd ) . those tolerance values were smaller than the vendor specification of 2 mm , and the maximum value for the results was 1.32 mm . although it was ideal that the tolerance values at any bead 's position in each axis were consistent , our tolerance values were considered to be valid , taking into consideration the residual error for the geometry calibration , the manual set - up error of the emma phantom , and the manual identification of the bead 's position . in terms of the frequency of qa for geometric distortion , the stable results for this study indicate that this can be reasonably performed on a monthly basis . table 4.the new tolerance values in each axis for bead position based on the results of 100 measurementsaxisbead position12 o'clock ( mm)3 o'clock ( mm)6 o'clock ( mm)9 o'clock ( mm)x0.390.830.451.131.321.120.890.58y0.830.400.831.310.480.940.900.63z0.810.060.980.000.93 0.020.870.06 the new tolerance values in each axis for bead position based on the results of 100 measurements image qualities for uniformity , low - contrast resolution , high - contrast resolution , spatial resolution and cnr were evaluated . for image uniformity , there were two incidents in 100 measurements in which pixel values of the roi were out of tolerance , both of which were solved by the adjustment of beam output for cone - beam ct acquisition . dose output calibration for cone - beam ct when used for image acquisition is performed in our department once a month , which is basically the same period as that used for calibration of the treatment beam for x - ray beams and electron beams . given the difficulty of predicting when such incidents might occur within the 1-month period between calibrations , it is important to check beam output for cone - beam ct as well as the treatment beam on a daily basis , or at least more frequently than on a monthly basis . in terms of the pixel uniformity at each roi position , the new tolerance values were also established from the results of 100 measurements . table 5 summarized the new tolerance values at each roi position of the center , 3 , 6 , 9 and 12 o'clock that were based on the 95% confidence interval ( i.e. mean 1.96 sd ) and the vendor specification for comparison . our tolerance values for the mean pixel value and the standard deviation at the center roi were slightly wider than those of the vendor specification . in contrast , our tolerance values at the four positions of 3 , 6 , 9 and 12 o'clock were narrower than those of the vendor specification . therefore , strictly based on the tolerance value , it was considered valid that the vendor specification would be applicable for the center roi , and that our tolerance values would be applicable for the other roi positions . table 5.the new tolerance values and the vendor specification for the pixel uniformity at each roi position based on the results of 100 measurementspixel value ( a.u.)roi positionevaluation itemour tolerance valuevendor specificationcentermean52403042centersd3142264212 o'clockmean difference from the center344880803 o'clockmean difference from the center444480806 o'clockmean difference from the center76480809 o'clockmean difference from the center52308080sd = standard deviation , a.u . the new tolerance values and the vendor specification for the pixel uniformity at each roi position based on the results of 100 measurements sd = standard deviation , a.u . , high - contrast , and spatial resolution , these are based on qualitative evaluation . all visual checks in this study were done by a single person , obviating the question of interobserver error . manipulation of window width and window level for cbct images invariably introduces a degree of subjectivity into visual inspection . for the purpose of quality control at least , qa should be performed using a consistent image acquisition protocol . however , the mu used for mv - cbct acquisition in clinical settings depends on treatment site , and efforts to reduce dosage mean that it will usually be < 15 mu . in general , however , the lower the mu , the worse the image quality , and an appropriate setting for image guidance should therefore be sought . with regard to cnr 9 , although two of all the mean pixel values were outside the tolerance , as shown in fig . 7 . even though the mean pixel values were outside the tolerance due to the out of calibrated beam output of mv - cbct , the difference between the mean value of the cb2 - 50% rod and the background region was close to that for the normal beam output of mv - cbct , and the standard deviation of the difference between the cb2 - 50% rod and the background region was also close to that for the normal beam output of mv - cbct . therefore the cnr value was not affected . gayou et al . presented cnr values of 8.8 , 3.0 and 4.2 for cb2 - 50% , inner bone and acrylic , respectively , using the same machine specifications as in our present study . these past results and our present results were similar for inner bone and acrylic , but our cnr findings for cb2 - 50% were higher as a result of the improvement of signal - to - noise ratio for the use of 5-mm multiple plane reconstruction . in the case of the use of a 1-mm reconstruction , our cnr results for cb2 - 50% were 9.7 0.8 ( mean sd ) , which are close to those of gayou et al . they reported that when field width in the longitudinal direction decreases from 27.4 to 5 cm , cnr increases by about 20% . given the balance between imaging dose and image quality , we consider that decreasing field size in the longitudinal direction is a convenient method for increasing cnr and reducing imaging dose . although there are no vendor specifications for the cnr , we have established the tolerance values of 14.417.5 , 2.12.8 and 3.34.2 for cb2 - 50% , inner bone and acrylic , respectively . these tolerance values were based on the 95% confidence interval ( i.e. mean 1.96 sd ) , using our results for the 100 measurements . here , we evaluated the long - term stability of an mv - cbct device during the evaluation period , and confirmed the suitability of the vendor 's qa process . based on our results , the new tolerance levels for bead position , pixel uniformity and the cnr were established . those tolerance levels will be useful data as the reference of periodical quality control in our department . our findings highlight the importance of recognizing the presence of a residual error in geometric distortion during geometric calibration of the mv - cbct . to ensure image quality , stability of the beam output

a linear accelerator vendor and the aapm tg-142 report propose that quality assurance testing for image - guided devices such megavoltage cone - beam ct ( mv - cbct ) be conducted on a monthly basis . in clinical settings , however , unpredictable errors such as image artifacts can occur even when quality assurance results performed at this frequency are within tolerance limits . here , we evaluated the imaging performance of mv - cbct on a weekly basis for 1 year using a siemens oncor machine with a 6-mv x - ray and an image - quality phantom . image acquisition was undertaken using 15 monitor units . geometric distortion was evaluated with beads evenly distributed in the phantom , and the results were compared with the expected position in three dimensions . image - quality characteristics of the system were measured and assessed qualitatively and quantitatively , including image noise and uniformity , low - contrast resolution , high - contrast resolution and spatial resolution . all evaluations were performed 100 times each . for geometric distortion , deviation between the measured and expected values was within the tolerance limit of 2 mm . however , a subtle systematic error was found which meant that the phantom was rotated slightly in a clockwise manner , possibly due to geometry calibration of the mv - cbct system . regarding image noise and uniformity , two incidents over tolerance occurred in 100 measurements . this phenomenon disappeared after dose calibration of beam output for mv - cbct . in contrast , all results for low - contrast resolution , high - contrast resolution and spatial resolution were within their respective tolerances .

INTRODUCTION MATERIALS AND METHODS Irradiation Phantom analysis RESULTS DISCUSSION CONCLUSION

the toxin a- and toxin b - specific recombination vectors pjir3051 and pjir3050 were constructed by cloning a 566 bp tcda ( accession m30307 ) pcr product ( nt 10711637 ) and the equivalent tcdb ( accession x53138 ) region , respectively , into the shuttle vector pjir1456 ( accession u90554 ) . these plasmids were transferred by conjugation to c. difficile strain jir8094 and the resultant chromosomal mutants analyzed by pcr , southern blotting and plasmid rescue13 . filtered supernatants of 2ty broth cultures ( 72 h ) were used for western blots , cytotoxicity and toxin neutralization assays . serial two - fold dilutions of these supernatants were used in vero and ht-29 cell cytotoxicity assays , with the end - point scored as the last dilution yielding complete cpe . toxins were neutralized with affinity purified polyclonal antibodies specific for toxin a and toxin b ; neutralization was carried out at the last dilution giving complete cpe . partially purified ammonium sulphate - precipitated toxin proteins were western blotted using toxin a - specific affinity purified polyclonal goat antibodies . clindamycin was administered orogastrically to render the hamsters susceptible to infection ( day zero ) . on day fecal pellets were collected daily for 12 days , then weekly until day 30 and inoculated onto selective tccfa plates to monitor colonization . fecal pellets were analyzed for the presence of toxin a using a toxin a - specific elisa and for the presence of toxin b using a human foreskin fibroblast cytotoxicity assay . full methods and any associated references are available in the online version of the paper at www.nature.com/nature .

clostridium difficile is the leading cause of infectious diarrhea in hospitals worldwide , because of its virulence , spore - forming ability and persistence1,2 . c. difficile - associated diseases ( cdad ) are induced by antibiotic treatment or disruption of the normal gastrointestinal flora3,4 . recently , morbidity and mortality resulting from cdad have increased significantly due to changes in the virulence of the causative strains and antibiotic usage patterns1,2,5,6 . since 2002 , epidemic toxinotype iii nap1/027 strains1,2 , which produce high levels of the major virulence factors , toxin a and toxin b , have emerged . these toxins have 63% amino acid sequence similarity7 and are members of the large clostridial glucosylating toxin family , which are monoglucosyltransferases that are proinflammatory , cytotoxic and enterotoxic in the human colon810 . inside host cells , both toxins catalyze the transfer of glucose onto the rho family of gtpases , leading to cell death8 , 11 . however , the role of these toxins in the context of a c. difficile infection is unknown . here we describe the construction of isogenic tcda and tcdb mutants of a virulent c. difficile strain and their use in the hamster disease model to show that toxin b is a key virulence determinant . previous studies showed that purified toxin a alone can induce most of the pathology observed following infection of hamsters with c. difficile8,9 , 12 and that toxin b is not toxic in animals unless it is co - administered with toxin a , suggesting that the toxins act synergistically12 . our work provides evidence that toxin b , not toxin a , is essential for virulence , which represents a major paradigm shift . furthermore , it is clear that the importance of these toxins in the context of infection can not be predicted exclusively from studies using purified toxins , reinforcing the importance of using the natural infection process to dissect the role of toxins in disease .

METHODS SUMMARY Supplementary Material

gastrointestinal stromal tumours ( gists ) are the most frequent mesenchymal tumours of the digestive tract , derived from the cajal interstitial cells . they are characterised by the presence of a gain - of - function mutation of the kit or pdgfra oncogenes . their incidence remains low , around 520 per million in different population - based studies , but their prevalence has been increasing during the last decade thanks to the therapeutic progress related to the use of tyrosine kinase inhibitors ( tkis ; imatinib , sunitinib , and regorafenib ) . gists can occur anywhere along the gastrointestinal tract , but the most frequent anatomical locations are the stomach ( 60% ) , jejunum , and ileum ( 30% ) , followed by the duodenum ( 5% ) , colorectum ( < 5% ) , and more rarely the oesophagus ( 12% ) and the omentum . about 40% of patients with gists will develop metastases , most commonly in the liver ( 65% ) or the peritoneum ( 50% ) , and less frequently in the lung . bone metastases of gists are a very rare event . in the small series in the literature , their proportion is low ( < 5% ) when compared with all secondary locations [ 5 , 6 , 7 , 8 ] . biologically , little is known about bone metastases of gists , as these metastatic sites are rarely biopsied . particularly , it is still unknown whether bone metastases keep the same kit / pdgfra mutations as the primary tumour or acquire new mutations . here , we present a case of gastric gist with synchronous liver and bone metastases that were fully documented by pathological and molecular analysis . a 66-year - old man , a retired oenologist with histories of asthma , non - insulin - dependent diabetes mellitus , and bilateral genu valgum consulted his general practitioner in november 2015 for asthenia lasting more than 6 months and left posterior intercostal pain . a thoraco - abdomino - pelvic ct scan ( fig . 1a ) revealed a 16-cm abdominal tumour lesion above the pancreas , associated with several suspect hepatic nodules , and multiple osteolytic lesions of the spine and pelvis . clinically , the patient 's weight was stable , and his who performance status was equal to 0 . the left posterior intercostal pain was imperfectly controlled by 3 g / day of paracetamol , and the patient had no digestive symptoms . physical examination found a painless voluminous tumour of the left hypochondrium ; neurological examination was normal . laboratory tests were normal , with the exception of a grade 1 increase in gt . gastric endoscopic ultrasound revealed a voluminous intra - abdominal 17-cm tumour , in contact with the stomach , mainly necrotic , as well as multiple suspect hepatic lesions , all enhanced after injection . pathological analysis of endoscopic ultrasound - guided fine - needle aspiration biopsies of the gastric tumour and 1 hepatic lesion revealed an epithelioid - cell gastric tumour and a spindle - cell liver tumour ( fig . , tumour cells were characterised by few mitoses ( < 5/50 high - power fields ) , no necrosis , and an immunohistochemistry ( ihc ) staining strongly positive for cd117 and dog1 , weakly positive for aml , and negative for desmin and ps100 . the gastric tumour was cd34-negative , whereas the hepatic lesion was cd34-positive ( table 1 ) . no kit or pdgfra mutation was found in the hepatic metastasis , whereas the gastric tumour harboured a kit exon 11 mutation ( c.1676_1714del , p.val559_ile571del ) , further confirming the diagnosis of gastric gist with hepatic metastasis . ct scan showed an increase in size of both the abdominal tumour ( 19 cm ) and the hepatic metastases as well as multiple osteolytic lesions involving notably the spine and the pelvis . 3 ) showed several bone uptakes located on the cervico - dorso - lumbar spine , sternum , scapula , pelvis ( sacrum and left sacroiliac joint ) , rib cage ( k5k7 ) , left femur , and right humerus . 1b , c ) confirmed the vertebral bone lesions , and notably identified a t1 and t9 epiduritis and a mild medullar compression in c7 , t1 , and t9 without intramedullary lesion . a ct scan - guided biopsy of a lesion of the right posterior iliac bone confirmed the diagnosis of bone metastasis by a spindle - cell gist showing the same morphological and ihc profile ( fig . 2c ) as the hepatic metastasis , and the same kit exon 11 mutation as the primary gastric tumour . the diagnosis of voluminous gastric gist with synchronous liver and bone metastases was retained . in february 2016 , no local treatment was initiated for the vertebral bone lesions because of the absence of any neurological symptoms , the expected efficacy of imatinib , and the control of dorsal pain by small doses of oxycodone . in may 2016 , after 3 months of treatment , ct scan showed a regression of both the primary tumour ( 14 cm ) and the hepatic and bone metastases . in august 2016 , after 6 months of treatment , ct scan showed further regression of the primary tumour ( 12 cm ) and the hepatic metastases ( recist 1.1 partial response with 33% decrease in target lesions when compared to baseline ) and stabilisation of bone metastases . in november 2016 , after 9 months of imatinib , ct scan confirmed the partial response of the primary and hepatic lesions , which had become more necrotic , and stabilisation of the bone lesions . currently , the patient continues imatinib , which is perfectly tolerated , without any digestive , bone , or neurological symptoms related to the tumour lesions . the classical metastatic locations of gists are the liver and peritoneum , whereas bone metastases are rare . we report here a case of gastric gist with synchronous hepatic and bone metastases at diagnosis , documented in all sites with respect to histology and molecular biology . in epidemiologic terms , the true incidence of bone metastases remains unknown , but was reported to be between 3.2 and 5.5% in 4 retrospective series of 53309 patients with metastatic gists [ 5 , 6 , 7 , 8 ] . to our knowledge , 37 cases , including the present case , have been reported in the english - language literature during the last decade , including 31 since 2011 . the incidence seems to be growing thanks to the longer survival of metastatic patients due to the increasing use of tkis , as observed in other cancers responding to medical therapies in their advanced stages . bone metastases mainly occurred in men ( n = 31/37 ) and patients older than 50 years ( n = 29/37 ) . like in our case , the location of the primary gist was mainly the stomach ( n = 12/36 informative cases ) , followed by the ileo - jejunum ( n = 10 ) , the rectum ( n = 8) , the duodenum ( n = 4 ) , the mesentery ( n = 1 ) , and the oesophagus ( n = 1 ) . of note , rectal gists were overrepresented in that population of bone metastases , with 8/36 patients ( 22% ) , whereas their frequency in overall gists is around 5% . a likely explanation might be the haematogenous spread via the non - portal vein drainage from the rectum . regarding the pathological characteristics , most of the primary tumours were spindle ( 11/20 informative cases ) or mixed ( 7/20 ) , and only 2 cases , including the present one , were purely epithelioid ( 2/20 ) . interestingly , contrary to our patient , most of the informative cases described a high number of mitoses of over 5/50 high - power fields ( n = 20/25 ) , suggestive of intermediate or high risk of relapse . the mutational status of the primary tumour was documented for only 5/37 cases and showed an exon 11 kit mutation , which is the most frequent mutation found in gists before any treatment . regarding the bone metastases , they occurred in 23/31 informative cases during the follow - up of treated primary gists , within a median delay of 48 months ( range , 4120 months ) after the diagnosis of the primary tumour . in 8 patients , including ours , the diagnoses of primary tumour and bone metastasis were synchronous , whereas 1 case corresponded to a prevalent bone metastasis . these rare observations suggest that bone metastasis can be the initial metastatic manifestation . like in our patient , the bone metastases mainly occurred concomitantly with or after other metastatic sites like the liver ( n = 27 ) , peritoneum ( n = 8) , lung ( n = 4 ) , or soft tissues ( n = 2 ) , likely through haematogenous spread via vein drainage from these metastatic sites . their topography was mainly the spine ( n = 17 ) , especially thoracic and lumbar , followed by the pelvis ( n = 11 ) , the ribs ( n = 11 ) , the femur ( n = 7 ) , and less frequently the shoulder ( n = 3 ) or the humerus ( n = 2 ) . interestingly , skull or face metastases were numerous ( n = 9 ) , including the temporal bone , clivus , or mandible , and led to complex diagnostic investigations and surgical procedures . the discovery of bone metastases was often based on clinical symptoms or was done incidentally during diagnostic or staging thoraco - abdomino - pelvic ct scan . the bone metastases in gist patients are classically lytic and well - defined lesions . with an expanded access to magnetic resonance imaging , it is likely that more bone metastases of gists will be diagnosed in the future . furthermore , bone metastases have an intense fluorodeoxyglucose uptake ; in case of response to tkis , the standardised uptake value decreases very early after the beginning of treatment , which makes pet scanning an interesting option for follow - up . the pathological type was spindle cells in all 10 informative cases , similar to our case ; all tested cases were kit - positive by ihc , and only 3 cases were sequenced , showing kit exon 11 mutation . comparison between the primary tumour and the bone metastasis was possible for some pairs ; the concordance was perfect for kit ihc ( 12/12 pairs ) and mutational analysis ( 2/2 pairs ) , suggesting clonal origin , whereas only 1 case ( ours ) showed discordant pathological type , with epithelioid type in the primary and spindle cells in the metastasis . the absence of mutation documented in the liver metastasis in our patient may reflect intratumour heterogeneity . because of the scarcity of data , it remains unclear whether a particular mutational status is associated with these unusual metastases . the systemic treatment was based on tkis , mainly imatinib ( n = 22 ) used in 1st line , sunitinib used in 2nd line ( n = 8) , and less frequently nilotinib ( n = 2 ) or sorafenib ( n = 1 ) , with several attitudes due to toxicity or long - term disease stabilisation , although not recommended today . regarding the local treatment of bone metastases , the literature review showed that surgery ( n = 8) was realised for oligometastatic patients for different skeletal localisations , or patients with functional impairment , sometimes associated with graft or prosthesis . some of these patients who underwent complex surgical bone resections with r0 margins benefitted from long - term disease - free survival [ 12 , 13 ] . even if gists , like soft tissue sarcomas , are often considered radio - resistant , external beam radiotherapy was associated with a significant improvement of bone pain in most of the cases . interestingly , 1 case report described the concomitant use of imatinib during radiotherapy , with good tolerance . bisphosphonates were used in 5 patients ( zoledronic acid ) , and 1 case of jaw osteonecrosis was described after imatinib and zoledronic combination . the clinical outcome after the diagnosis of bone metastasis was available in 26/37 patients : 9 patients died after a median time of 11 months ( range , 1103 ) , whereas 17 remained alive with a median follow - up of 19 months ( range , 2187 ) . thus , although bone metastases occurred preferentially in high - risk metastatic gists , their prognosis was not particularly dreadful . in conclusion , bone metastases of gist are rare but likely underdiagnosed , and their frequency is increasing because of the improvement of imaging techniques and of patients survival . physicians in charge of gist patients should be aware of this unusual metastatic site during the prolonged natural history of gists in the tki era . we recommend attention notably in case of gists > 5 cm , with a high mitotic index or with a rectal location . if bone metastasis remains a palliative disease , multiple therapeutics options ( like surgery , radiotherapy , tkis , and bisphosphonates ) are available to improve symptoms and prolong survival .

gastrointestinal stromal tumours ( gists ) are mesenchymal tumours of the digestive tract , derived from cajal interstitial cells . bone metastases are very rare , and there is no consensus regarding their treatment . here , we present the unusual case of a 66-year - old man with a gastric gist with synchronous bone and liver metastases , fully documented at the pathological and molecular levels with a kit exon 11 mutation . after 9 months of imatinib , the scanner showed a 33% partial response of target lesions . we also review the literature and describe the characteristics , treatment , and outcome of all cases previously reported .

Introduction Case Report Discussion Statement of Ethics Disclosure Statement

previous studies on the reproductive toxicity of boric acid have indicated that male rodents suffer testicular atrophy after treatment . there were , however , no studies of the potential effects on female fertility or on the neonate . in addition , no study described the development of the testicular lesion , thought to be related to the mechanism of toxicity . a reproductive assessment by continuous breeding ( racb ) study using mice exposed to boric acid at 1000 , 4500 , and 9000 ppm in the diet indicated that there are probably multiple sites of action , although male fertility appears very sensitive . possible effects on female fertility can not be separated from potential developmental toxicity and need additional investigation . decrements in sperm motility were observed at all exposure levels , and testicular atrophy was confirmed in high- and middle - dose - group males . this was investigated further by timed serial - sacrifice studies using 9000 ppm in the diet of rats , which found that the first lesion seen in the testis was an inhibition of spermiation ( release of mature spermatids ) . with continued dosing , this was followed by a disorganization of the normal ordered layering of the seminiferous epithelium , germ cell sloughing and death , and finally , atrophy . subsequent studies using additional doses ( 2000 , 3000 , 4500 , 6000 , and 9000 ppm ) found that it was possible to observe inhibited spermiation that did not progress to atrophy ( 4500 ppm and below ) within the 9-week exposure period.(abstract truncated at 250 words)imagesfigure 1 . afigure 1 . bfigure 1 . cfigure 1 . d

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alcohol consumption and abuse can have a variety of cutaneous manifestationsfamiliarization with the spectrum of cutaneous manifestations of alcohol abuse and alcoholic liver disease allows for early detection and treatment in an attempt to minimize the medical consequences . alcohol consumption and abuse can have a variety of cutaneous manifestations familiarization with the spectrum of cutaneous manifestations of alcohol abuse and alcoholic liver disease allows for early detection and treatment in an attempt to minimize the medical consequences . alcoholism is a multifactorial - genetically predisposed , environmentally and psychosocially influenced - chronic progressive , potentially fatal disease characterized by a tolerance of and physical dependency on ethanol drinking identifiable by impaired control over drinking , preoccupation with drinking alcohol despite continued adverse consequences , and distortions in thinking and/or diverse organ dysfunction . at - risk drinking the level that may over time lead to adverse health consequences is > 14 drinks / week or > 4 drinks per occasion in men , and > 7 drinks / week or > 3 drinks / occasion in women . intake of chronic high alcohol deserves greater attention as a public health problem than any other intoxicant as it affects society rather than just the individual . it may also modify the prevalence , presentation , severity , and responsiveness to therapy of some preexisting dermatoses such as psoriasis , rosacea , and seborrheic dermatitis alcohol - induced vasodilation and centrally altered vasomotor control of dermal vasculature produce the hallmark spider telangiectases . pinpoint telangiectases , ecchymoses , palmar erythema , corkscrew scleral vessels , caput medusae , etc . urticarial and anaphylactoid reactions , within minutes to hours of alcohol intake , and hyperpigmentation have also been associated . alcohol - induced malnutrition may be primary , due to displacement of essential nutrients from the diet , or secondary , to malabsorption and hepatic cellular injury and may manifest as protein / calorie deficiency syndromes of kwashiorkor / marasmus and deficiencies of specific nutrients and/or trace elements , namely , phrynoderma , xerosis , angular stomatitis , cheilosis , glossitis , seborrhea - like dermatitis , pellagra , and scurvy . malnutrition also contributes to increased rate of infection in alcoholics as do increased propensity to trauma and suppressed immunity . as regards endocrine changes in male alcoholics , hypogonadism manifests by the loss of libido , impotence , testicular atrophy , reduced fertility , reduced facial hair growth , etc . , and hyperestrogenism , by gynecomastia , vascular spiders , changes in fat distribution , loss of body hair , and female distribution of pubic hair . the patients of alcoholism , in common with those of any other substance abuse , even when asked , seldom acquiesce and the treating dermatologists , often unaware , are usually reluctant to ask . the resultant delay or absence of consultation with a specialist leads to nonaddressal of alcohol abuse and perpetuation of its related morbidity . while countless surveys regarding harmful systemic effects of chronic alcoholism exist in the literature , the dermatological spectrum thereof is seldom documented . we enrolled 196 males ( > 18 years ) attending the de - addiction center ( of our charitable tertiary care hospital run by a trust ) fulfilling the criteria of royal college of psychiatry for chronic drinking , i.e. , > 20 units ( 1 unit = 10 ml of pure alcohol ) of alcohol / week and referred for dermatological consult ; lack of alcoholism in our semi - urban study population from the state of maharashtra ( india ) precluded enrollment of female cases . after clearance by the institutional ethical committee , written informed consent of the patients was obtained , and they were subjected to detailed history taking , clinical examination , liver function testing , and blood sugar profile . complete hemogram , renal function tests , anti - human immunodeficiency virus ( hiv)/hepatitis b surface antigen antibodies , and histopathological examination were carried out where required . data were tabulated , classified , and compared with a similar number of age-/sex - matched teetotalers attending dermatology outpatient department . chi - square , independent t and spearman 's rank correlation tests were performed using the statistical package for the social sciences version 22 ( spss inc . , chicago , il , usa ) as appropriate . a post hoc power analysis with the program g*power the age of our study patients ranged from 24 to 65 ( mean : 43.30 8.926 ) years . their alcohol intake / week was 2277 ( mean : 39.51 7.7.69 ) units spanning 340 ( mean : 20.01 9.322 ) years . dermatologist consultation was sought approximately 2 months after onset by a majority ( 128 ; 59.5% ) of cases . smoking was practiced by 106 ( 54.1% ) patients and 94 ( 47.9% ) controls ; 65 ( 33.2% ) cases and 59 ( 30.1% ) controls chewed tobacco [ table 1 ] . involvement of skin , skin and mucosa , and ( only ) mucosa was present in 182 ( 92.9% ) , 34 ( 17.3% ) , and 14 ( 7.1% ) patients , respectively . generalized pruritus was significantly more in cases ( 135 ; 68.7% ) than in controls ( 13 ; 6.63% , odds ratio [ or ] : 31.15 , p < 0.001 ) , a post hoc power analysis revealed the two - tailed power to be > 99% . infections were the presenting complaint in 73 ( 37.2% ) patients and comprised bacterial ( 28 ; 38.4% ) , fungal ( 27 ; 36.9% ) , and viral ( 18 ; 24.7% ) [ table 2 ] . bacterial infections were more significant in cases than controls ( 28 vs. 10 , or : 3.1 , p = 0.002 ) as a group , rather than as specific entities ; this too was associated with a favorable post hoc power of 92.6% . the increased prevalence of superficial fungal infections in controls ( 41 ; 48.8% ) than in cases ( 27 ; 36.9% ) was not statistically significant . of the 24 ( 12.2% ) cases with infestations , 23 ( 95.8% ) had scabies ; remaining one , pediculosis . comparison of infections in cases and controls forty - five ( 22.9% ) patients suffered from eczema [ table 3 ] ; the most common , seborrheic , seen in 22 ( 48.9% ) was , surprisingly , related inversely to duration of alcohol intake ( 12 6.094 vs. 21.02 9.179 years , p < 0.001 ) , i.e. , more in those with lesser years of intake and of lesser age ( 35.68 5.093 vs. 44.26 8.852 years ; p it was also more significant in cases ( 22 ) than controls ( 2 , or : 12.26 , p < 0.001 ) ( post hoc power > 99% ) . comparison of dermatoses other than infections in cases and controls dermatoses associated with duration ( years ) and quantum ( units / week ) of alcohol intake generalized hyperpigmentation was the most common ( 28 ; 14.2% ) of the pigmentary disorders and was significantly correlated with the patients ' age ( 47.25 8.686 vs. 42.64 8.819 years , p = 0.011 ) , units / week ( 43.95 4.377 vs. 38.77 7.97 , p < 0.001 ) , and the duration ( 24.0 7.736 vs. 19.35 9.417 years , p = 0.007 ) of alcohol intake . xerosis was significantly more common in patients than in controls ( 17 vs. 5 , or : 3.62 , p = 0.008 ) ( post hoc power : 84.7% ) and was significantly associated with the duration ( 25.65 6.819 vs. 19.47 9.365 years , p = 0.002 ) as well as the weekly alcohol intake ( 44.23 4.026 vs. 39.06 7.895 units , p < 0.001 ) . four ( 2% ) patients presented with erythroderma : two , secondary to psoriasis ; remaining two , idiopathic . psoriasis , also seen in another 21 ( 10.7% ) cases , was significantly associated with the duration ( 26.81 8.542 vs. 19.19 9.097 years , p < 0.001 ) and inversely with alcohol intake per week : those drinking less ( 32.08 6.249 vs. 40.40 7.463 units , p < 0.001 ) being more susceptible . abnormalities directly due to alcohol abuse seen were spider nevi , in 34 ( 17.3% ) ; gynecomastia , in 19 ( 9.7% ) ; and pellagroid dermatitis , in 8 ( 4.1% ) [ table 3 ] ; none of these occurred in controls . a statistically significant association emerged between spider nevi and the mean duration ( 24.15 8.486 vs. 19.14 9.28 years , p = 0.004 ) and quantum ( 44.85 7.802 vs. 38.39 7.303 units / week , p < 0.001 ) of alcohol intake as well as raised transaminases ( 26 ; 76.5% , p < 0.001 ) . gynecomastia , too , was significantly associated with the quantum of alcohol ( 43.20 4.318 vs. 39.27 7.889 units / week , p = 0.011 ) and increased liver transaminases ( 11 vs. 1 , p < 0.001 ) but not with the duration of alcohol consumption . the presence of pellagroid dermatitis was significantly associated only with the quantum of alcohol ( 45.74 4.504 vs. 39.25 7.775 units / week , p = 0.02 ) . oral changes were observed in 14 ( 7.1% ) patients ; glossitis ( 7 ; 50% ) , being the most frequent . the most common of the nail changes was nail plate discoloration ( 64 ; 32.7% ) . of the 76 ( 38.8% ) cases of alopecia , 62 ( 81.6% ) had diffuse and 14 ( 18.4% ) , patchy . systemic abnormalities commonly seen were an alcoholic liver disease ( 45 ; 22.9% ) , diabetes mellitus ( 23 ; 11.7% ) , and peripheral neuropathy ( 19 ; 9.7% ) . serum bilirubin , aspartate transaminase , and alkaline phosphatase were raised in 58 ( 29.6% ) , 51 ( 26.0% ) , and 47 ( 24.0% ) cases , respectively . our study population , like of other studies , comprised mostly of young to middle - aged adults , however , unlike the previous study by rao , majority ( 176 ; 89.8% ) had been imbibing alcohol longer ( > 10 years ) and four times . more than half ( 106 ; 54.1% ) smoked cigarettes ( 106 ; 54.1% ) and a third ( 65 ; 33.2% ) , chewed tobacco , habits reported almost in a similar proportion by rao . the delay in reporting ( > 2 months ) for consultation with a dermatologist by the majority of our cases could indicate their disregard for self . duration and quantum of alcohol consumption had no relationship with each other , those drinking over a longer duration , not necessarily imbibing a significantly greater quantum . spider nevi seen in 34 ( 17.3% ) were significantly associated with duration and quantum of alcohol intake as well as increased transaminases . their emergence as a reliable clinical marker of alcohol consumption as well as of liver damage was corroborative of their similar eminence in previous studies . the rarity of palmar erythema ( 2 ; 1% ) and flushing ( 1 ; 0.5% ) in our study could probably be due to the dark brown skin of our cases . pruritus detected in 135 ( 68.7% ) of our cases solely by the presence of excoriations was higher than the estimated occurrence in 40% of patients with liver disease . pruritus , known to precede the development of cirrhosis by several years , did not correlate significantly with either the amount or the duration of alcohol intake in our study . out of the multiple nail changes , mostly nonspecific , the well - known changes of clubbing and terry 's nails are reported in 15% and 80% of cirrhotics in the literature . nail bed discoloration , pitting , and longitudinal ridging seen in 64 ( 32.7% ) , 28 ( 14.5% ) , and 7 ( 3.6% ) patients respectively in our study were banal . terry 's nails occurred in 7 ( 3.6% ) of our cases but were seen neither in the study by rao nor in the brazilian one . clubbing of fingernails , seen in 10 ( 5.1% ) of our cases , was reported in 16% of his patients by rao . our study reaffirms the significance of generalized hyperpigmentation as a marker for the duration as well as the quantity of alcohol intake . however , periorbital ( 3 ; 1.5% ) and palmar crease pigmentation ( 1 ; 0.5% ) were uncommon as was the case in an earlier indian study . there was no association between the presence of squamous cell carcinoma ( 4 ; 2% ) and tobacco or alcohol intake , possibly due to the small size of our study . the solitary case of basal cell carcinoma in our study population , too , may be corroborative of its lack of association with chronic alcoholism . glossitis , which possibly occurs due to vitamin b deficiency , was the most common ( 7 ; 3.6% ) oral change observed ; its incidence in the previous indian study was 7% . the typical lingual syndrome consisting of a thickened tongue , atrophy of the lingual mucosa and lacquer edges was not seen in our study . generalized xerosis was the most common of the probable alcohol - induced deficiency disorders in our study as well as that of parish and fine ; its significant association with both the duration and amount of alcohol intake should raise an alert for other signs of liver damage . the significant association of pellagra with the amount of alcohol intake is probably consequent to primary malnutrition . the prevalence of gynecomastia seen in 19 ( 9.7% ) in our study was detected to be 7% in his study by rao . the significant association of gynecomastia with the quantum , rather than the duration , of alcohol in our study , could probably be related to the direct inhibitory effect of ethanol on the testicular germinal epithelium as well on the increased metabolic clearance rate of testosterone . the significant association of gynecomastia with increased liver enzymes could be due to increased peripheral conversion of androstenedione to estrone and of testosterone to estradiol and may be related to changes in microsomal enzymes due to liver damage . almost half of our cases had either an infection or an infestation ; scabies , the most common , followed by hiv and pityriasis versicolor . the prevalence of bacterial ( 28 ; 14.3% ) and dermatophytic ( 12 ; 6% ) infections was slightly higher than that observed by rao ( 9% and 4.5% ) while the prevalence of pityriasis versicolor was lower ( 10 ; 5.1% vs. 28 ; 14% ) . in the brazilian study , only 2.8% of patients were found to have scabies , much less than our study ( 23 ; 11.7% ) , but the prevalence of tinea pedis ( 26.32% ) was much more ( 3 ; 1.5% ) . the lesser incidence of scabies could probably be due to the better socioeconomic standard of the brazilians , while higher incidence of tinea pedis , probably due to their increased use of closed footwear . our study had more psoriatics ( 21 ; 10.7% ) than that in the brazilian study ( 6% ) . however , the similar prevalence of psoriasis in cases and controls ( or : 1.56 , p = 0.21 ) could be due to a lot of referrals to our tertiary center . the significant association of psoriasis with the duration of alcohol intake and inversely with the amount of alcohol consumption in our study is surprising and probably indicates that chronicity , rather than the quantum , of alcohol intake predisposes , through unknown mechanisms , to the development of psoriasis thereby directly contradicting the findings of the study by chaput et al . in whose study , 5.3% of cirrhotics drinking > 50 g alcohol / day had psoriasis compared to only 0.7% cirrhotics drinking < 50 g. the rarity of rosacea ( 3 ; 1.5% ) in ours as well as in some earlier studies could be because of the tendency of rosacea patients to avoid alcohol entirely because of the uncomfortable flushing . the prevalence ( 22 ; 11.2% ) of seborrheic dermatitis in our study and an earlier indian study ( 11.5% ) was less than in the brazilian ( 15.8% ) study . the significantly higher risk of seborrheic dermatitis ( or : 12.26 ) in alcoholics , as compared to controls , could be due to vitamin b deficiency presenting as seborrhoea - like dermatitis ; its significant inverse association with the duration of alcohol intake and patients ' age could be explained by its occurrence in the typical distribution in the younger age group . spider nevi , generalized hyperpigmentation , and generalized xerosis emerged to be associated with the duration as well as the quantum of alcohol intake in this study . in contrast , pruritus a very common marker that occurs before onset of liver damage was associated neither with the duration nor the quantum of alcohol intake . the association of gynecomastia with raised liver enzymes and amount , rather than the duration , of alcohol intake in our study , probably indicates that binge drinking , rather than chronic intake , may lead to liver damage sufficient enough to cause gynecomastia . in contrast , the presence of psoriasis was associated with a longer duration as well as a lesser quantum of alcohol intake . however , small sample size , nonreliable history of alcohol intake and the inability to completely eliminate the confounding bias due to factors such as smoking , tobacco , and hiv infection , are the limitations of this study . familiarization with the broad spectrum of these dermatoses would : first , allow early detection , second , facilitate proactive inquiry by the dermatologist regarding alcohol abuse from the patient , and finally , probable timely mitigation , if not elimination , of this substance abuse hopefully before irreversible liver damage and adverse consequences - medical / accident - related . generalized pruritus , xerosis , and seborrheic dermatitis were significantly more common in cases than controlsgeneralized hyperpigmentation and xerosis correlated significantly with the duration as well as the quantum of alcohol intakespider nevi , gynecomastia , and pellagroid dermatitis were seen exclusively in alcoholics . generalized pruritus , xerosis , and seborrheic dermatitis were significantly more common in cases than controls generalized hyperpigmentation and xerosis correlated significantly with the duration as well as the quantum of alcohol intake spider nevi , gynecomastia , and pellagroid dermatitis were seen exclusively in alcoholics . generalized pruritus , xerosis , and seborrheic dermatitis were significantly more common in cases than controlsgeneralized hyperpigmentation and xerosis correlated significantly with the duration as well as the quantum of alcohol intakespider nevi , gynecomastia , and pellagroid dermatitis were seen exclusively in alcoholics . generalized pruritus , xerosis , and seborrheic dermatitis were significantly more common in cases than controls generalized hyperpigmentation and xerosis correlated significantly with the duration as well as the quantum of alcohol intake spider nevi , gynecomastia , and pellagroid dermatitis were seen exclusively in alcoholics . generalized pruritus , xerosis , and seborrheic dermatitis were significantly more common in cases than controlsgeneralized hyperpigmentation and xerosis correlated significantly with the duration as well as the quantum of alcohol intakespider nevi , gynecomastia , and pellagroid dermatitis were seen exclusively in alcoholics . generalized pruritus , xerosis , and seborrheic dermatitis were significantly more common in cases than controls generalized hyperpigmentation and xerosis correlated significantly with the duration as well as the quantum of alcohol intake spider nevi , gynecomastia , and pellagroid dermatitis were seen exclusively in alcoholics .

background : chronic alcohol intake impacts skin directly , through organ dysfunction or by modifying preexisting dermatoses . however , dermatoses afflicting chronic alcoholics figure in a few studies only.aim:this study aims to correlate the spectrum of dermatoses in chronic alcoholics with the quantum / duration of alcohol intake and raised liver transaminases.materials and methods : adult males , totaling 196 , ascertained to fulfill the royal college of psychiatry criteria for chronic alcoholism by the de - addiction center and referred for dermatological consult were enrolled as cases , and similar number of age-/sex - matched teetotallers , as controls . data emanating from detailed history , clinical examination , and routine liver functions tests were summarized and subsequently analyzed , including statistically using the chi - square , independent t and spearman 's rank correlation tests , and compared with data from previous studies.results:majority ( 104 ) drank 4150 units of alcohol / week since 340 ( mean : 20.01 9.322 ) years . generalized pruritus ( odds ratio [ or ] : 31.15 , p < 0.001 ) , xerosis ( or : 3.62 , p = 0.008 ) , and seborrheic dermatitis ( or : 12.26 , p < 0.001 ) were significantly more common in cases than controls . infections ( 73 ; 37.2% ) , eczemas ( 45 ; 22.9% ) , and generalized hyperpigmentation ( 28 ; 14.2% ) were the major presenting complaints . spider nevi , gynecomastia , and pellagroid dermatitis were present in 34 ( 17.3% ) , 19 ( 9.7% ) , and 8 ( 4.1% ) respectively exclusively in cases only . commonly seen systemic abnormalities were an alcoholic liver disease ( 45 ; 22.9% ) , diabetes mellitus ( 23 ; 11.7% ) , and peripheral neuropathy ( 19 ; 9.7%).conclusion : knowledge of cutaneous manifestations of chronic alcoholism could prompt in - depth history taking of alcohol intake , lead to specialist referral and thereby enable timely de - addiction , hopefully before serious adversities in the chronic alcoholics .

Introduction Materials and Methods Results Discussion Conclusion None Financial support and sponsorship Conflicts of interest

candida albicans is the most common fungal pathogen causing invasive and bloodstream infections in immunocompromised patients , although in recent years , several non - albicans species and other yeasts have also emerged as major opportunistic pathogens ( 1,2 ) . studies in the us identify candida glabrata as the second most common candida species involved in invasive fungal infections . moreover , antifungal drug resistance , especially to azoles , is common among c. glabrata clinical strains isolated from patients with prior azole treatment ( 1 ) . the availability of genome sequences for these pathogenic fungi has made it possible to study genes that play a role in pathogenesis and drug resistance in candida species , thereby increasing our understanding of the mechanisms of virulence in fungal pathogens . the candida genome database ( cgd , http://www.candidagenome.org/ ) is an online resource for the scientific research community studying fungal molecular biology and pathogenesis . the primary mission of cgd is to facilitate and accelerate candida research by providing both an extensively curated compendium of candida gene , protein and sequence information , and easy - to - use web - based tools for accessing , analyzing and exploring these data . when the cgd project began in 2004 , our initial efforts focused on curation of c. albicans , because it is the best - characterized species of the group and has the largest corpus of gene - specific scientific literature . we have now expanded the scope of the project to include other candida species , and provide an extensive suite of tools and resources that have been redesigned to facilitate the analysis of multiple species concurrently . the cgd locus summary page ( lsp ) has been updated with information about the identity of orthologous genes in c. glabrata , and with orthology - based functional predictions and gene descriptions . we currently display both manual and computational gene , protein and sequence information about c. albicans and the recently added species , c. glabrata . we also provide genomic and protein sequence downloads and blast ( 3 ) resources for multiple candida species and strains , including c. albicans strains sc5314 ( 4 ) and wo-1 ( 5 ) , c. dubliniensis ( 6 ) , c. guilliermondii ( 5 ) , c. lusitaniae ( 5 ) , c. parapsilosis ( 5 ) , c. tropicalis ( 5 ) , debaryomyces hansenii ( 7 ) and lodderomyces elongisporus ( 5 ) . we will be adding curated information for all these other candida species in the future . we also have an extensive suite of online user documentation , and provide advice and user support by e - mail at [email protected] . at cgd , phd level curators perform ongoing manual curation of the scientific literature to collect , organize , summarize and present a comprehensive picture of each characterized gene . manual curation includes the recording of gene names , addition and updates to our summary gene descriptions , capture of mutant phenotype data and the assignment of relevant go annotations with evidence and citations . the manual curation of the previously published literature pertaining to genes of c. albicans and c. glabrata is now complete ( table 1 ) . we have combed the scientific literature for gene - specific information and gene bibliographies ; gene ontology ( go ) annotations describing the function , role and localization of gene products ; and mutant phenotypes . these are now reported in cgd for all of the genes for which this information is available . at this time , there are 6203 predicted c. albicans protein - encoding genes localized to chromosomes in the current ( assembly 21 ) reference gene set , 22% with manually annotated gene and protein information . for c. glabrata , the reference annotation set contains 5212 predicted genes , each of which has a lsp ( figure 1 ) , and 3% of which have manually curated annotations . cgd now includes a detailed genome snapshot for c. glabrata in addition to c. albicans , which provides a graphical and tabular summary of information about the total number of chromosomal features and feature types , changes to the reference sequence and a distribution of gene products by functional categories and cellular localization ( figure 2 ) . lsps for both c. albicans and c. glabrata now feature new expanded orthology information sections , orthology - based description lines for uncharacterized genes , orthology - based go term predictions and protein domain - based go term predictions . pie chart from the cgd genome snapshots , comparing the current extent of the characterization of the predicted protein - coding genes in the c. albicans and c. glabrata genomes . dubious have no experimental characterization and appear to be indistinguishable from random non - coding sequences ( 5 ) . table 1.cgd curation statisticscandida albicanscandida glabratanumber of orfs61085212number of trnas156230verified orfs1403178uncharacterized orfs47055034dubious orfs152n / amanual go annotations46974689features with manual go annotations13 7072622orthology - based go annotations13 24619 655features with orthology - based go annotations30994157protein - domain ( interpro)-based go annotations60485087features with protein - domain ( interpro)-based go annotations29632583features with orthology - based description lines13523982 updates to the cgd locus summary page ( lsp ) . lsps for both c. albicans and c. glabrata now feature new expanded orthology information sections , orthology - based description lines for uncharacterized genes , orthology - based go term predictions and protein domain - based go term predictions . pie chart from the cgd genome snapshots , comparing the current extent of the characterization of the predicted protein - coding genes in the c. albicans and c. glabrata genomes . dubious have no experimental characterization and appear to be indistinguishable from random non - coding sequences ( 5 ) . cgd curation statistics in addition , cgd curators have composed in - depth descriptive locus summaries for 272 selected c. albicans genes , which , in contrast to the very concise locus descriptions , are more detailed enumerations of the characteristics of each gene , presented in a bullet - point format on the cgd lsps . they provide additional experimental details and gene regulatory information that can not be accommodated within the space limits of the locus description line . these lists are displayed in the locus summary section located near the bottom of the page and are fully searchable through the cgd text search tool . the curation of the entire body of scientific literature for these organisms is a large and ongoing endeavor as new papers are published , and we welcome suggestions from users as to papers that should be prioritized or other data that should be included . we greatly appreciate the beneficial interactions with members of the candida research community who have already volunteered to review specific lsps and provide feedback on the curation content for specific genes . the comments we have received have resulted in refinement of description lines , improvements to phenotype and go annotations , and addition of new references that we had not encountered in our literature searches improvements that benefit the entire community of cgd users . cgd was originally modeled after the saccharomyces genome database ( sgd ) ( 8) , a database that provides the saccharomyces cerevisiae reference sequence with literature curation , and gene , protein and sequence analysis tools for the s. cerevisiae research community . sgd , and initially cgd , were designed to store and display data for only a single species at a time . to accommodate the incorporation of additional species in the database , user interface and analysis tools , significant design modifications to the software and the cgd search tools , such as quick search , text search , gene / sequence resources , ortholog search and pattern match have been redesigned to search multiple species . in order to accommodate search results for multiple species , the new results page for the cgd quick search and text search tools now displays three sections . search results that apply to all species ( e.g. go terms , authors and reference information , colleagues ) are displayed at the top , with sections for species - specific search results displayed below . all of the tools that perform species- or sequence - specific searches ( e.g. gene / sequence resources , pattern match , advanced search , batch download , restriction mapper , go term finder , go slim mapper ) have been updated , and they now prompt users to select the species of interest . the ortholog search now retrieves ortholog and best - hit matches among all of the species in cgd and sgd ( currently c. albicans , c. glabrata and s. cerevisiae ) . blast searches at cgd have also been redesigned to allow queries against any combination of the several candida species for which we have complete sequence sets ( c. albicans , c. glabrata , c. dubliniensis , c. guilliermondii , c. lusitaniae , c. parapsilosis , c. tropicalis , debaryomyces hansenii and lodderomyces elongisporus ) . in addition , the curation tools have been extensively modified to facilitate the curation of multiple species . each gene in cgd is represented on a lsp , which is the central organizing unit of the cgd web site . the lsp contains the basic information that describes the gene and provides access to tools for retrieval , analysis and visualization of gene data . lsps for each c. albicans and c. glabrata gene now feature an expanded orthology section , by which the lsps of each c. albicans gene are hyperlinked to the lsps of their c. glabrata orthologs , and vice versa . the lsp for c. glabrata genes also provide external links to gene pages available at ge nolevures ( http://www.genolevures.org/cagl.html # ) this section also serves as a gateway to information about the orthologs in saccharomyces cerevisiae , providing hyperlinks to the lsp of each ortholog in the sgd . including s. cerevisiae ortholog information is especially useful for the c. glabrata lsps : the evolutionary divergence between c. glabrata and s. cerevisiae is considerably more recent ( 100 - 300 million years ago ) ( 7,9 ) than the divergence between these two species and c. albicans ( 700 - 800 million years ago ) ( 10 ) , and thus c. glabrata shares a larger number of orthologs with s. cerevisiae than with c. albicans , 4372 and 3201 , respectively ( as predicted by inparanoid ) . to define orthology relationships , we use the inparanoid algorithm , which identifies reciprocal best blast hits between species ( 11 ) . these mappings and links are updated quarterly in order to reflect changes in gene models and annotations at cgd and sgd . in addition to the new orthology relationships displayed in cgd , another level of similarity - based information is provided via the new protein tab on the lsp of each protein - coding gene ( figure 3 ) . this tab opens the protein information page that provides descriptions and a graphical display of conserved protein domains and motifs identified using interproscan software ( 12,13 ) . the protein information pages also display the structure of the most similar protein in the protein data bank ( 14 ) , and contain information about the predicted protein length , molecular weight , sequence and a link to a table of calculated physicochemical properties . the protein information page provides data including structural information inferred from homologs in pdb ( rcsb protein data bank ) , an interactive domains / motifs browser , protein sequence and physicochemical property details , blastp search against other cgd sequences and links to external protein resources such as uniprot . the protein information page provides data including structural information inferred from homologs in pdb ( rcsb protein data bank ) , an interactive domains / motifs browser , protein sequence and physicochemical property details , blastp search against other cgd sequences and links to external protein resources such as uniprot . the go is a structured vocabulary that is used to describe three aspects of gene products : their molecular function or activity , the broader biological process in which they participate , and the cellular location in which they reside ( 15 ) . a gene product can be annotated with any number of terms about any of the three aspects , depending on the available data . each go term assignment is associated with an evidence code that describes the type of data the assignment is based on , and with a reference to its source . the go is in wide use in genomic research and because it is rigorously structured , it ensures consistency in capturing of functional information about genes from different organisms and thus enables reliable analysis of biological significance of genomic data ( 1521 ) . for the fully curated species , c. albicans and c. glabrata , all of the available gene - related literature pertaining to these two species has been read and all possible go assignments from these papers have been made . to augment the manual curation , we have leveraged the orthology relationships to infer go annotations for genes having an experimentally characterized ortholog in sgd or cgd . predictions for c. albicans are made based on s. cerevisiae and c. glabrata orthologs , whereas predictions for c. glabrata are based on orthologs from s. cerevisiae and c. albicans . despite the evolutionary distances between c. albicans , c. glabrata and s. cerevisiae , the use of orthology relationships to infer go annotations between c. albicans and c. glabrata allow the transfer of a significant number of important pathogenesis - related terms to be transferred between these two fungal pathogens . candidate go annotations to be used as the basis for these inferences are limited to those with experimental evidence , i.e. associated with evidence codes of inferred from direct assay ( ida) , inferred from physical interaction ( ipi) , inferred from genetic interaction ( igi) , or inferred from mutant phenotype ( imp). any annotations that are themselves predicted in s. cerevisiae or in candida , either based on sequence similarity or by some other methods , are excluded from this group to avoid transitive propagation of predictions . also excluded from the predicted annotation set are annotations that are redundant with existing , manually curated annotations , or those that assign a related but less specific go term other than candidate annotations . these orthology - based go assignments are associated with evidence code inferred from electronic annotation ( iea) and displayed with the source species and gene name they are derived from along with a hyperlink to the appropriate lsp at cgd or sgd . cgd has also taken advantage of protein domain and motif homology to assign go annotations for c. albicans and c. glabrata genes . we systematically predict conserved domains in cgd protein sequences using interproscan ( 12 ) , and then use the interpro - to - go mappings ( 12,13 ) provided by the go consortium to provide molecular function annotations for those proteins . these annotations are assigned the evidence code iea and are displayed with the interpro identifier of the protein that serves as the basis for the annotation . the identifier is linked to the embl - ebi database to provide access to more extensive information about each structural domain . we have also used the trnascan - se software to predict trna genes , and have inferred predicted go annotations for these trnas ( 22 ) . the new annotations that have been transferred from s. cerevisiae to c. albicans and c. glabrata , and between c. albicans and c. glabrata , are summarized in table 1 . in addition to having the evidence code iea , all these orthology - based annotations are identified as being derived computationally , rather than manually extracted from the scientific literature . predictions are updated several times a year to make sure they remain current with annotation updates and new curation in cgd , sgd and in the protein domain datasets . now that all literature - based go assignments for c. albicans and c. glabrata , and all orthology - based and protein domain - based predictions have been made , we consider curation of both species to be go - complete. for the remaining uncharacterized genes , we have explicitly assigned unknown annotations to indicate that to the best of our knowledge no data are available . we have also used the multispecies information to create informative descriptions for those candida genes that lack any experimental characterization , and which therefore have no literature - based description on the lsp , incorporating orthology relationships and orthology - based functional predictions into the gene description in cases where there would otherwise be no information available . additional cgd resources for the candida research community include a new collection of bibliographies on topics relevant to candida biology , which is accessible under community resources from the navigation sidebar on the cgd home page . these highlights in candida biology contain lists of important references , including many key reviews , and are designed to provide an overview of selected subject areas in c. albicans and c. glabrata biology . as new species are curated at cgd , highlights in candida biology will expand to include bibliographies on these species as well . we have also curated a directory of strains , which provides descriptions and references for commonly used candida laboratory strains , along with a lineage diagram that graphically depicts the relationship among these strains . this resource is especially important for researchers because differences in strain background are known to have a significant impact on observed mutant phenotypes . in some cases , genes have been found to be lethal in one genetic background while successful gene disruption is possible in another . an example of this is the c. albicans ume6 gene , for which homozygous mutants are viable in the sn152 genetic background ( 23 ) yet inviable in the bwp17 strain background ( 24 ) . because of its importance , we also provide all available strain background information along with all of the curated phenotypes for each gene . additional cgd resources for the candida research community include a new collection of bibliographies on topics relevant to candida biology , which is accessible under community resources from the navigation sidebar on the cgd home page . these highlights in candida biology contain lists of important references , including many key reviews , and are designed to provide an overview of selected subject areas in c. albicans and c. glabrata biology . as new species are curated at cgd , highlights in candida biology will expand to include bibliographies on these species as well . we have also curated a directory of strains , which provides descriptions and references for commonly used candida laboratory strains , along with a lineage diagram that graphically depicts the relationship among these strains . this resource is especially important for researchers because differences in strain background are known to have a significant impact on observed mutant phenotypes . in some cases , genes have been found to be lethal in one genetic background while successful gene disruption is possible in another . an example of this is the c. albicans ume6 gene , for which homozygous mutants are viable in the sn152 genetic background ( 23 ) yet inviable in the bwp17 strain background ( 24 ) . because of its importance , we also provide all available strain background information along with all of the curated phenotypes for each gene . now that the underlying database has been re - tooled to accommodate the curation of multiple species , we will add curated information for other candida - related species including c. dubliniensis , c. guilliermondii , c. lusitaniae , c. parapsilosis , c. tropicalis , debaryomyces hansenii and lodderomyces elongisporus . in order to facilitate navigation across multiple genomes , we will provide links to an interactive comparative visualization tool , which will allow users to explore ortholog clusters in their genomic context . recent advances in genomics technologies have created a deluge of information that poses a significant challenge of making all these data organized and readily available to researchers . we have adapted our genome browser , gbrowse , to enable users to visualize unannotated transcripts in c. albicans that have been identified by rnaseq ( 2527 ) . these transcripts are aligned to the reference genome and displayed alongside the existing set of features in the reference annotation . we will further develop and/or integrate existing software to incorporate and visualize more types of data and more data sets from high - throughput studies . funding for open access charge : national institute of dental and craniofacial research at the us national institutes of health ( grant no .

the candida genome database ( cgd , http://www.candidagenome.org/ ) is an internet - based resource that provides centralized access to genomic sequence data and manually curated functional information about genes and proteins of the fungal pathogen candida albicans and other candida species . as the scope of candida research , and the number of sequenced strains and related species , has grown in recent years , the need for expanded genomic resources has also grown . to answer this need , cgd has expanded beyond storing data solely for c. albicans , now integrating data from multiple species . herein we describe the incorporation of this multispecies information , which includes curated gene information and the reference sequence for c. glabrata , as well as orthology relationships that interconnect locus summary pages , allowing easy navigation between genes of c. albicans and c. glabrata . these orthology relationships are also used to predict go annotations of their products . we have also added protein information pages that display domains , structural information and physicochemical properties ; bibliographic pages highlighting important topic areas in candida biology ; and a laboratory strain lineage page that describes the lineage of commonly used laboratory strains . all of these data are freely available at http://www.candidagenome.org/. we welcome feedback from the research community at [email protected] .

INTRODUCTION LITERATURE CURATION FOR MULTIPLE TOOLS FOR SEARCH AND DISPLAY OF MULTISPECIES INFORMATION IN CGD LEVERAGING MULTISPECIES INFORMATION IN CGD: HOMOLOGY-BASED FUNCTIONAL PREDICTIONS CURATED INFORMATIONAL PAGES AT CGD FUTURE DIRECTIONS FUNDING

urinary incontinence affects up to 50% of women , resulting in a significant medical , social , and economic burden . the annual direct costs of incontinence in the united states were estimated to be more than $ 16 billion . among women with incontinence , 50% to 80% are identified as having stress urinary incontinence ( sui ) , or involuntary leakage of urine resulting from physical exertion or sneezing and coughing . . similar to its frequency in western countries , about 37.8% of korean adult women suffer from sui . although the initial treatment of stress incontinence is often nonsurgical ( behavioral therapy , pelvic - floor exercises , or incontinence devices ) , surgical treatment is considered for patients who are bothered by persistent symptoms . an estimated 4% to 10% of women in the united states undergo surgery intended to restore continence , and this rate has increased steadily during the past 20 years . at present , one of the most successful treatments for sui is provided by surgery . burch corposuspension was once the standard technique , but tension - free vaginal tape ( tvt ) is now the gold standard . tvt is a minimally invasive procedure with a cure rate comparable to burch corposuspension in an appropriate randomized control study . however , tvt has the risk of serious complications associated with the ' blind ' course of the needle through the retropubic space . therefore , bladder perforation as well as vascular and bowel injuries are occasionally reported [ 10 - 12 ] . for obviation of needle passage through the retropubic space , the transobturator sling procedure demonstrated a comparable cure rate but fewer complications associated with needle passage through the retropubic space . however , the trans - obturator sling can induce entrapment of the obturator nerve , and groin and upper thigh pain occasionally occur . therefore , further improvement of the surgical methods for the treatment of sui is needed . the tvt - secur system was developed to reduce the invasiveness of the surgical procedures and to avoid passing the needle through the retropubic or obturator regions . the advantages of this technique are related to the short course of the needles , which minimizes the risk of vascular , nerve , or visceral injury . the prosthetic implant is placed under the midurethra and can be fixed in the hammock ( h ) position into the obturator internus muscle or in the u - shaped position into the connective tissue of the urogenital diaphragm behind the pubic bone . some concerns may arise regarding the strength of the fixation point and the tensioning maneuver . herein , we conducted a prospective , randomized , comparative study to compare effectiveness and safety between the h and u approaches of the tvt - secur procedure for the treatment of female sui . this was a 12-month , randomized trial involving one experienced surgeon at a single medical center . participants received detailed information about the study procedure and provided written consent before study entry . consenting patients were randomly allocated to either the u - type or the h - type method immediately . women who were 18 years old or older and presented with urodynamic sui for at least 3 months were selected for this study . patients with a history of prior anti - incontinence surgery or stage 2 pelvic organ prolapse were excluded . baseline measures included patient demographics , detailed medical and surgical history , and obstetric and gynecological history . all women underwent a physical examination for urethral hypermobility , as measured by the q - tip test , and pelvic - organ prolapse . preoperatively , we assayed women by a urodynamic study , sandvik questionnaire , incontinence quality of life questionnaire ( i - qol ) , bristol female lower urinary tract symptoms - scored form ( bfluts - sf ) questionnaire , incontinence visual analogue scale ( i - vas ) , 3-day voiding diary , and uroflowmetry . just after the decision of surgery , changes in the i - qol questionnaire , bfluts - sf questionnaire , i - vas , 3-day voiding diary , and uroflowmetry were assessed ; complications were also assessed . patients ' perception of treatment benefit , satisfaction , and willingness to undergo retreatment or recommend treatment were also evaluated . cure was defined as no leakage during stress after surgery at the 12-month postoperative visit . the primary endpoint was the difference in the cure rate of sui between the h- and u - type approaches . the secondary endpoints included differences in 1 ) postoperative changes in i - qol , bfluts - sf , i - vas , uroflowmetry , and voiding diary ; 2 ) patients ' perception of treatment benefit , satisfaction , and willingness to undergo retreatment or recommend treatment ; 3 ) perioperative parameters ( operative time , bleeding amount , and immediate postoperative pain vas ) ; and 4 ) return to normal activities . overall cure rate and complications were also evaluated . our institutional review board approved this study , and written informed consent was obtained from all participants before enrollment . random allocation to either the u or the h approach was performed after obtaining informed consent . all operations were conducted at a day care surgery center by the same experienced surgeon . under a combination of local anesthesia and light sedation , patients were positioned in the dorsal lithotomy position . after infiltrating mixed normal saline with lidocaine and 1:200,000 epinephrine into the anterior vaginal wall , an approximately 1.5 cm midline vertical vaginal incision was made from 1 cm below the external urethral meatus , and paraurethral dissections were processed bilaterally . for the u - type procedure , the inserter was grasped without the protective cover and using the needle holder , and the inserter was introduced into the previously dissected paraurethral incision orienting the inserter to 45 from the sagittal midline and toward the ipsilateral shoulder . the inserter was advanced upward until the back edge of the pubic bone was reached , keeping the inserter tip against the backside of the pubic bone . the needle driver was disconnected from the first inserter and was connected to the second inserter after removing the protective cover . the second inserter and device were placed in the back side of the pubic bone in the same manner . after disconnecting the needle holder from the second inserter after the final adjustment , the release wire was pulled and the inserters were removed . for the h approach , dissection was made laterally , towards the ischiopubic ramus and parallel to the floor on both sides , and the inserter and device were positioned firmly into the obturator internus muscle . urethral catheters were not used , and patients were discharged after several instances of self - voiding . random allocation to either the u or the h approach was performed after obtaining informed consent . all operations were conducted at a day care surgery center by the same experienced surgeon . under a combination of local anesthesia and light sedation , patients were positioned in the dorsal lithotomy position . after infiltrating mixed normal saline with lidocaine and 1:200,000 epinephrine into the anterior vaginal wall , an approximately 1.5 cm midline vertical vaginal incision was made from 1 cm below the external urethral meatus , and paraurethral dissections were processed bilaterally . for the u - type procedure , the inserter was grasped without the protective cover and using the needle holder , and the inserter was introduced into the previously dissected paraurethral incision orienting the inserter to 45 from the sagittal midline and toward the ipsilateral shoulder . the inserter was advanced upward until the back edge of the pubic bone was reached , keeping the inserter tip against the backside of the pubic bone . the needle driver was disconnected from the first inserter and was connected to the second inserter after removing the protective cover . the second inserter and device were placed in the back side of the pubic bone in the same manner . after disconnecting the needle holder from the second inserter after the final adjustment , the release wire was pulled and the inserters were removed . for the h approach , dissection was made laterally , towards the ischiopubic ramus and parallel to the floor on both sides , and the inserter and device were positioned firmly into the obturator internus muscle . urethral catheters were not used , and patients were discharged after several instances of self - voiding . from march 2007 to july 2008 , 115 women with sui underwent the tvt - secur procedure . of those , 53 underwent the u - type surgery , and 62 underwent the h - type surgery . initially , 133 women were enrolled and randomly allocated to either the u - type ( n=66 ) or the h - type ( n=67 ) procedure . of those , 13 women in the u - type group and 5 women in the h - type group withdrew informed consent and did the mean age was 55.219.34 years in those receiving the u - type surgery and 56.68.68 years in those undergoing the h - type approach ( p= 0.410 ) . there were no significant differences in preoperative patient characteristics or perioperative parameters between the u - type and h - type approaches ( table 1 , 2 ) . cure rates were 88.7% ( 47/53 ) for the u - type approach and 87.1% ( 54/62 ) for the h - type approach ( p=0.796 ) , with an overall cure rate of 87.8% after 12 months postoperatively . the i - qol ( total and all subscale scores ) , filling , incontinence , sexual function , and qol sum ( bfluts - sf ) , and incontinence vas were significantly improved with both approaches , but the degree of improvement did not differ significantly between the two approaches ( table 3 ) . regarding the voiding diary , micturition episodes were reduced with both approaches , but urgency episodes decreased significantly with only the h - type approach ( table 3 ) . the maximal flow rate did not change with either approach , but post - void residuals increased in women treated with the h method ( table 3 ) . approximately 82.9% of women treated with the u - type approach and 84.3% of those treated with the h - type method reported that they were satisfied with the surgical outcome ( p=0.858 ) . a total of 80.5% of those treated with the u - type method and 86.3% of those treated with the h - type approach reported a benefit from the surgery ( p=0.455 ) . about 87.8% and 88.0% treated with the u - type and the h - type approaches , respectively , reported that they would recommend the surgery to others who had sui ( p=0.442 ) . also , 87.8% and 86.3% treated with the u - type and with the h - type methods reported that they would undergo the same surgery if they had been in same condition ( p=0.800 ) . regarding intra - operative parameters , the mean duration of the operation was 14.86.3 min for the u - type approach and 13.45.1 min for the h - type approach ( p=0.114 ) . the mean amount of blood loss was 56.445.3 ml for the u - type approach and 66.471.6 ml for the h - type approach ( p=0.645 ) . immediate postoperative pain vas was 2.22.0 for the u - type approach and 2.11.8 for the h - type approach ( p=0.926 ) . the time required for patients to return to normal activities was not significantly different between the approaches ( table 4 ) . there were no patient complaints about dyspareunia or loss of libido after either type of procedure . there were 3 cases of intra - operative vaginal wall perforations during the h - type approach . all 3 cases were immediately repaired , and the wound healed without infection or erosion . immediate postoperative retention was observed in 2 women treated with the u - type approach and in 1 woman treated with the h - type method . one woman had tape release and additional cutting procedures for persistent and large amounts of residual urine . overall , 4 women underwent additional midurethral sling procedures for persistent urine leakage . in detail , transobturator tape ( tot ) was applied for 3 women as an additional sling procedure and tvt was applied for 1 woman . our study demonstrated that there was no significant difference in the cure rate between the u - type and the h - type methods of tvt - secur , with an approximately 88% overall cure rate after 1 year of follow - up , compared with previous studies reporting 1-year cure rates of 91% for tvt , 92% for tot , and 86% for tvt - obturator ( tvt - o ) procedures . to our knowledge , no comparative studies between tvt - secur and other midurethral sling procedures such as tvt and tot have been performed . also , although there are some reports on tvt - secur , no prospective comparative study of the u and h methods has been carried out . in our study , both types of surgery demonstrated comparable cure rates and patient - reported outcomes at the 1-year follow - up . our study demonstrated a comparable cure rate with the other sling methods , including tvt , the gold standard treatment for female sui . a few studies have reported unfavorable outcomes for tvt - secur [ 19 - 21 ] . recently , however , a prospective study reported a 1-year cure rate as high as 93.5% . in addition , miniarc , other types of midurethral slings with single - incision systems , have resulted in up to 91% 1-year cure rates . an important point for obtaining continence is the disconnection of the inserter from the mesh . this entails a gentle twist of the handle while gently pushing the device into the patient 's body . this maintains the ideal tension of the mesh , which should be left abutting the urethra , forcing the periurethral tissues to protrude slightly through the mesh pores . otherwise , the mesh will not be firmly attached to the connective tissue and will fail to provide adequate tension to the urethra . tvt - secur is an 8 cm long polypropylene mesh with the absorbable end able to be fixed to the obturator internus internal fascia that can be inserted through a single 1.5 cm vaginal incision . the extension of the dissection required to create a passage for the sling is very limited . because neither sutures nor inserting needles are needed , the risk of internal injury inherent to the blind passage of inserting needles is hypothetically minimized . because mortalities have been reported after midurethral slings associated with perineal fasciitis as well as vascular and bowl injury [ 10 - 12 ] , safety must be considered when choosing the type of midurethral sling . in our study , 6 cases of complication were reported . there were no cases of bladder perforation , which occurs in up to 11% of tvt procedures . nor were there any cases of vessel injury , which usually results from the blind course of the needles and occurs in up to 4.0% of those treated with tvt . furthermore , no patient suffered from postoperative persistent groin or thigh pain due to obturator nerve entrapment by the mesh , which occurs in up to 14.3% of those treated with the transobsturator sling series . the technique for placing the tvt - secur can be rapidly learned by the average surgeon qualified to treat sui . however , caution is required when placing the tvt - secur extremities in the fibrous attachment of the internal obturator muscle , because this might not be quickly achieved by inexperienced surgeons . the tvt - secur needle dimension requires a wider tunnel , and surgeons probably need some experience with the procedures to find it . the operator must be careful to avoid vaginal perforation . in this study , there were 3 cases of intra - operative vaginal wall perforation with the h - type approach . tvt - secur , especially the h - type , requires a wider tunnel to prevent dragging of the vaginal submucosal connective tissue when placing the end of the device into the fibrous tissue of the internal obturator muscle . neuman also reported a 10% incidence of vaginal tape protrusion , a 5% incidence of tape removal , a 2% incidence of bladder outlet obstruction , and a 1% incidence of paravesical hematoma . changes in post - void residuals were significantly larger with the h - type approach than with the u - type approach . tvt induces urethral obstruction more frequently than do trans - obturator procedures , as evidenced by ultrasonographic and urodynamic findings . currently , it is unclear why the h - type approach resulted in significantly larger post - void residuals in our study than the u - type approach ; clinically , however , the increase is thought to be insignificant . furthermore , of the 3 patients with immediate postoperative urinary retention , none needed additional treatment for persistent large post - void residuals or voiding symptoms after removal of the temporary urethral catheter . generally , studies comparing tvt and tot reported no difference in de novo frequency and urgency interestingly , midurethral sling procedures are effective in improving preexisting overactive bladder symptoms in half of the patients with mixed incontinence . contact with urine in the proximal urethra can induce a reflex contraction of the detrusor muscle . thus , eliminating urinary leakage may decrease this reflex after anti - incontinence surgery . at present , improvement in overactive bladder symptoms , including urgency and de novo urgency , after tvt - secur need further study . statistically considered sample size calculation for randomized controlled trials was not applied for the design of this study . for the calculation of an optimistic sample size , the investigator must have some information on both the variability of response to therapy and the assumed degree of effectiveness of therapy . but , because only a few reports provided information about the variability and effectiveness of each subtype of tvt - secur before our study , statistically considered sample size calculation was not applicable for this study . at baseline , the abdominal leak point pressure ( alpp ) of the h - type approach group was significantly higher than that of the u - type group . but , because the absolute difference in mean value was just 7.46 cmh2o and the proportion of intrinsic sphincter deficiency ( isd , alpp<60 cmh2o ) between the u - type ( 0.37% ) and h - type ( 0.48% ) groups was not significantly different ( p=0.87 ) , the difference in alpp may be clinically insignificant . however , this unbalanced baseline characteristic could be a prognostic factor having a negative effect on the u group . if random allocation had been more balanced and clinically significant effect - size and power had been calculated , the surgical outcome of the u - type approach could have been superior to that of the h - type . at this time , we can not explain the exact reasons for persistent urine leakage after tvt - secur . all 4 women who underwent additional midurethral sling because of persistent sui after tvt - secur were cured . therefore , one possible explanation for failure is suboptimal urethral tension of tvt - secur . the goal of a minimally invasive surgical procedure should be to provide an acceptable cure rate that is comparable to that of standard methods as well as a relatively low incidence of complications . in the context of the acceptable cure rate and low rate of complications compared with tvt and tot , our study demonstrates that with relatively short - term follow - up , both the u - type and the h - type approaches of the tvt - secur procedure are equally effective and safe for the treatment of female sui . both the u - type and the h - type approaches of the tvt - secur system are effective procedures in terms of cure rate and patient - reported outcomes for the treatment of female sui with 1 year of follow - up . there were no severe complications related to either approach .

purposewe compared outcomes of the u- and h - type approaches of the tension - free vaginal tape ( tvt)-secur procedure for the treatment of female stress urinary incontinence ( sui).materials and methodsfrom march 2007 to july 2008 , 115 women with sui underwent tvt - secur by a single surgeon . patients were randomly assigned to either the u- or the h - type approach . after 12 months , postoperative changes in the sandvik questionnaire , incontinence quality of life questionnaire ( i - qol ) , bristol female lower urinary tract symptoms - scored form ( bfluts - sf ) , and postoperative patient satisfaction were evaluated . cure was regarded as no leakage on the sandvik questionnaire . complications were also evaluated.resultsof 115 women , 53 were treated with the u approach , and 62 women were treated with the h approach . at 12 months , 88.7% of those treated with the u approach and 87.1% of those treated with the h approach were cured ( p=0.796 ) . the i - qol and filling , incontinence , sexual function , and qol sum ( bfluts - sf ) scores were improved with both approaches , and there were no significant differences in the degree of improvement between approaches . approximately 83.7% and 82.9% of the women treated with the u and h approaches , respectively , were satisfied with the outcome ( p=0.858 ) . there were 3 cases of intra - operative vaginal wall perforation in the h - type group . immediate postoperative retention was observed in 2 women in the u - type group and 1 woman in the h - type group . one woman in the u - type group underwent tape releasing and cutting procedures for persistent large post - void residuals.conclusionsthe u- and the h - type approaches of the tvt - secur procedure provided comparable effectiveness for the treatment of female sui .

INTRODUCTION MATERIALS AND METHODS 1. Surgical procedure RESULTS DISCUSSION CONCLUSIONS

this study was approved by the ethics committee of the armed forces medical command , which waived the requirement of informed patients consent for this retrospective study . from july 18 to july 30 , 2009 , 18 out of 57 patients who presented with acute febrile respiratory illness ( fever of 38 or higher , and/or a cough or shortness of breath ) ( 8) , were confirmed with the pandemic h1n1 2009 influenza virus infection in a tertiary military hospital in south korea . we retrospectively reviewed the medical charts , as well as the laboratory and radiologic findings of these patients . all patients were young male soldiers with a mean age of 20 years ( age range : 19 to 22 years ) . fifteen of the 18 patients confirmed with the pandemic h1n1 2009 influenza virus infection were from the same barrack , and they visited the emergency department due to acute febrile respiratory illness from july 18 to july 20 , 2009 . the other three patients were from different barracks and were admitted with suspected cases of rhabdomyolysis after a long distance march ( n = 1 ) , acute gastroenterocolitis ( n = 1 ) , and acute febrile respiratory illness with leukocytopenia and thrombocytopenia ( n = 1 ) . all three patients came into contact with one or more of the previously - mentioned patients lying in nearby beds in our hospital . among the 15 patients who presented to the emergency department with acute febrile respiratory illness , two patients had returned from a brief visit to their hometowns one week prior to the outbreak of acute febrile respiratory illness and were suspected as the source of the illness . specimens from a nasopharyngeal swab ( n = 17 ) , and tracheal aspirate ( n = 1 ) were collected at the emergency department or wards when the 15 patients of the same barrack visited the emergency department , or when symptoms developed in the remaining three patients who came into contact with them . reverse - transcriptase - polymerase - chain - reaction ( rt - pcr ) tests and additional viral cultures were performed in accordance with published guidelines from the who in all patients ( 8) . a malaria smear and triple antibody tests of hantann , leptospirosis , and tsutsugamushi virus were also performed to rule out other potential causes of fever , and revealed no remarkable abnormalities . for the review of the clinical features , initial symptoms such as fever , cough , sputum , rhinorrhea , sore throat , myalgia , headache , dizziness , vomiting , and diarrhea were recorded . in turn , patient symptom progression while under treatment was also reviewed . for review of the laboratory data , the hematological analysis of the white blood cell ( wbc ) count , red blood cell ( rbc ) count , hemoglobin level , hematocrit level , platelet count , neutrophil count and the biochemical analysis of alkaline phosphatase ( alp ) , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , creatine kinase ( ck ) , erythrocyte sedimentation rate ( esr ) , and c - reactive protein ( crp ) were recorded . chest radiographs and thin - section ct examinations were obtained from 18 and 12 patients , respectively , prior to the administration of antiviral agents . two out of the 12 patients undergoing thin - section cts received follow - up thin - section ct examinations a week after starting antiviral therapy . all radiographic examinations were performed using digital radiographic equipment ( tdr4600-f80 ; gold mountain medical system , seoul , korea ) and a standardized technique ( 110 kv , 2 mas , and a 180-cm film - focus distance for the posteroanterior views ) . thin - section cts of the thorax were performed using a 16 channel ct scanner ( brightspeed ; ge medical systems , wi ) , with the following parameters : 1.25 mm slice thickness with a 2.5 mm gap , supine position , scanning during inspiration , 6 seconds scan time , 120kv , auto ma . all chest radiographs and thin - section ct images were reviewed by three experienced chest radiologists ( 8 , 10 , and 16 years of experience in chest radiology , respectively ) using a picture archiving and communication systems ( pacs ) viewer . the presence of nodular opacity , consolidation ( focal , multifocal , or bilateral ) or interstitial patterns , distribution of findings including central ( inner two - thirds of lung ) or peripheral ( outer one - third of lung ) and upper , middle or lower lung zones , atelectasis , mediastinal abnormalities , and the presence or absence of a pleural effusion were analyzed ( 15 ) . thin - section ct findings were interpreted using the descriptors proposed by the fleischer society nomenclature committee ( 16 ) . ground - glass opacity ( ggo ) was defined as an increase in the lung parenchymal attenuation that did not obscure the underlying vascular architecture . a nodule was defined as round opacity , at least moderately well - defined , and no more than 3 cm in diameter . consolidation was defined as increased lung attenuation that obscured the underlying vasculature ( 16 ) . the lesion size was described as small ( diameter , less than 1 cm ) , medium ( diameter , 1 to 3 cm ) , large ( diameter , 3 cm to 50% of the segment ) , or segmental ( 50% to 100% of the segment ) . the sites were described by the name and number of involved segments of the lung . location of the lesion was defined as peripheral if it was in the outer one - third of the lung ; otherwise , it was defined as central ( 17 ) . attention was also paid to the presence of other abnormalities such as pleural effusion , lymphadenopathy , cavitation , calcification , or septal thickening . from july 18 to july 30 , 2009 , 18 out of 57 patients who presented with acute febrile respiratory illness ( fever of 38 or higher , and/or a cough or shortness of breath ) ( 8) , were confirmed with the pandemic h1n1 2009 influenza virus infection in a tertiary military hospital in south korea . we retrospectively reviewed the medical charts , as well as the laboratory and radiologic findings of these patients . all patients were young male soldiers with a mean age of 20 years ( age range : 19 to 22 years ) . fifteen of the 18 patients confirmed with the pandemic h1n1 2009 influenza virus infection were from the same barrack , and they visited the emergency department due to acute febrile respiratory illness from july 18 to july 20 , 2009 . the other three patients were from different barracks and were admitted with suspected cases of rhabdomyolysis after a long distance march ( n = 1 ) , acute gastroenterocolitis ( n = 1 ) , and acute febrile respiratory illness with leukocytopenia and thrombocytopenia ( n = 1 ) . all three patients came into contact with one or more of the previously - mentioned patients lying in nearby beds in our hospital . among the 15 patients who presented to the emergency department with acute febrile respiratory illness , two patients had returned from a brief visit to their hometowns one week prior to the outbreak of acute febrile respiratory illness and were suspected as the source of the illness . specimens from a nasopharyngeal swab ( n = 17 ) , and tracheal aspirate ( n = 1 ) were collected at the emergency department or wards when the 15 patients of the same barrack visited the emergency department , or when symptoms developed in the remaining three patients who came into contact with them . reverse - transcriptase - polymerase - chain - reaction ( rt - pcr ) tests and additional viral cultures were performed in accordance with published guidelines from the who in all patients ( 8) . a malaria smear and triple antibody tests of hantann , leptospirosis , and tsutsugamushi virus were also performed to rule out other potential causes of fever , and revealed no remarkable abnormalities . two authors retrospectively reviewed the clinical and laboratory findings . for the review of the clinical features , initial symptoms such as fever , cough , sputum , rhinorrhea , sore throat , myalgia , headache , dizziness , vomiting , and diarrhea were recorded . in turn , patient symptom progression while under treatment was also reviewed . for review of the laboratory data , the hematological analysis of the white blood cell ( wbc ) count , red blood cell ( rbc ) count , hemoglobin level , hematocrit level , platelet count , neutrophil count and the biochemical analysis of alkaline phosphatase ( alp ) , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , creatine kinase ( ck ) , erythrocyte sedimentation rate ( esr ) , and c - reactive protein ( crp ) were recorded . chest radiographs and thin - section ct examinations were obtained from 18 and 12 patients , respectively , prior to the administration of antiviral agents . two out of the 12 patients undergoing thin - section cts received follow - up thin - section ct examinations a week after starting antiviral therapy . all radiographic examinations were performed using digital radiographic equipment ( tdr4600-f80 ; gold mountain medical system , seoul , korea ) and a standardized technique ( 110 kv , 2 mas , and a 180-cm film - focus distance for the posteroanterior views ) . thin - section cts of the thorax were performed using a 16 channel ct scanner ( brightspeed ; ge medical systems , wi ) , with the following parameters : 1.25 mm slice thickness with a 2.5 mm gap , supine position , scanning during inspiration , 6 seconds scan time , 120kv , auto ma . all chest radiographs and thin - section ct images were reviewed by three experienced chest radiologists ( 8 , 10 , and 16 years of experience in chest radiology , respectively ) using a picture archiving and communication systems ( pacs ) viewer . decisions were reached by consensus . to review chest radiographs , the presence of nodular opacity , consolidation ( focal , multifocal , or bilateral ) or interstitial patterns , distribution of findings including central ( inner two - thirds of lung ) or peripheral ( outer one - third of lung ) and upper , middle or lower lung zones , atelectasis , mediastinal abnormalities , and the presence or absence of a pleural effusion were analyzed ( 15 ) . thin - section ct findings were interpreted using the descriptors proposed by the fleischer society nomenclature committee ( 16 ) . ground - glass opacity ( ggo ) was defined as an increase in the lung parenchymal attenuation that did not obscure the underlying vascular architecture . a nodule was defined as round opacity , at least moderately well - defined , and no more than 3 cm in diameter . consolidation was defined as increased lung attenuation that obscured the underlying vasculature ( 16 ) . the lesion size was described as small ( diameter , less than 1 cm ) , medium ( diameter , 1 to 3 cm ) , large ( diameter , 3 cm to 50% of the segment ) , or segmental ( 50% to 100% of the segment ) . the sites were described by the name and number of involved segments of the lung . location of the lesion was defined as peripheral if it was in the outer one - third of the lung ; otherwise , it was defined as central ( 17 ) . attention was also paid to the presence of other abnormalities such as pleural effusion , lymphadenopathy , cavitation , calcification , or septal thickening . the clinical course and outcome of all patients are summarized in figure 1 . in the analysis of 15 patients who visited the emergency department , the mean interval between symptom onset and emergency room visit was 1.7 days 1.5 days ( range , 0 to 5 days ) . in the analysis of all 18 patients , the mean interval between symptom onset and the start of oseltamivir phosphate ( tamiflu , roche , basel , switzerland ) administration was 3.9 1.6 days ( range , 1 to 7 days ) . in addition , the mean interval between symptom onset and symptom improvement was 4.7 1.7 days ( range , 2 to 8 days ) . the clinical characteristics of the 18 patients in our study are summarized in tables 1 and 2 . all patients presented with high fever ( > 38.0 , median value and range ; 38.5 and 38.2 - 39.5 ) , with common accompanying symptoms including coughing ( 78% ) , rhinorrhea ( 89% ) , sore throat ( 78% ) , myalgia ( 78% ) , vomiting ( 50% ) or diarrhea ( 56% ) . seventeen out of the 18 patients showed wbc counts within normal limits , whereas one patient showed leukocytopenia . in addition , thrombocytopenia and lymphocytopenia were present in three of 18 ( 17% ) and 12 of 18 patients ( 67% ) , respectively . no abnormality was noted for the rbc count , hemoglobin level , hematocrit level , platelet count , and neutrophil count . however , all patients showed elevated crp values . after the diagnosis of the pandemic h1n1 2009 influenza virus infection through rt - pcr tests and additional viral cultures , all patients were treated with oseltamivir phosphate . of the 18 patients , 16 rapidly recovered after the administration of the antiviral agent . in the two remaining patients , the symptoms started to improve prior to oseltamivir phosphate treatment . among our study population , one patient presented with high fever , severe nocturnal cough , and blood tinged sputum , and the respiratory symptoms eventually progressed into acute respiratory distress syndrome ( ards ) , which further led him to receive mechanical ventilation ( case no . a retrospective medical chart review revealed that this patient was a solider serving in a different unit from the majority of the other patients and had been in contact with a patient with pandemic h1n1 2009 influenza virus infection lying in a nearby bed in our hospital prior to symptom aggravation . the laboratory data of this patient showed leukocytopenia , thrombocytopenia , and an increased ck level . he was initially treated with antibiotics such as macrolide and a 3rd generation cephalosporin under suspicion of bacterial pneumonia , which was never confirmed . five days after contact with a patient with pandemic h1n1 2009 influenza viral infection , severe respiratory distress presented and a laboratory analysis from the tracheal aspirate performed during endotracheal intubation confirmed the pandemic h1n1 2009 influenza virus infection . following the administration of oseltamivir phosphate and antibiotics , the patient 's symptoms improved and was taken off mechanical ventilation five days after antiviral agent administration . sixteen out of 18 patients in our study population showed no abnormalities on chest radiographs . moreover , only two patients in our study showed abnormal findings of a small central nodular opacity in the right upper lung zone in one ( fig . 2a ) , and consolidations in the left middle lung and both lower lungs in the other ( fig . of these six patients , five ( 83% ) had small ggos ; four patients had them in the upper lobe of the lung and one had a small ggo discovered in the lower lobe of the lung . also , in four out of the five patients with small ggos ( 80% ) , the lesions were localized in the periphery of the lung , and two of the five patients also showed bilateral involvement . for segmental involvement in terms of lesion size , three patients showed one segment , one patient showed two segments , one patient showed three segments , and one patient showed eight segments . nodules were observed in all patients ( 100% ) , which were small in size with showing poorly defined margins ( fig . in addition , the nodule was a solitary finding in one patient ( 17% ) , which was located in the peripheral area of the right upper lobe . the patient who progressed into ards had bilateral lobar consolidations combined with ggos , nodules , and bilateral pleural effusion ( fig . one other patient showed right paratracheal lymphadenopathy . in our study , there was no evidence of calcification , mass , cavitation , or septal thickenings in any of the patients . the thin - section ct findings of the 12 patients are summarized in table 3 . in two patients , follow - up thin section ct images acquired one week after the start of antiviral therapy showed complete resolution or a remarkable decrease in size and opacity of the previously observed ggos and nodules ( fig . the clinical course and outcome of all patients are summarized in figure 1 . in the analysis of 15 patients who visited the emergency department , the mean interval between symptom onset and emergency room visit was 1.7 days 1.5 days ( range , 0 to 5 days ) . in the analysis of all 18 patients , the mean interval between symptom onset and the start of oseltamivir phosphate ( tamiflu , roche , basel , switzerland ) administration was 3.9 1.6 days ( range , 1 to 7 days ) . in addition , the mean interval between symptom onset and symptom improvement was 4.7 1.7 days ( range , 2 to 8 days ) . the clinical characteristics of the 18 patients in our study are summarized in tables 1 and 2 . all patients presented with high fever ( > 38.0 , median value and range ; 38.5 and 38.2 - 39.5 ) , with common accompanying symptoms including coughing ( 78% ) , rhinorrhea ( 89% ) , sore throat ( 78% ) , myalgia ( 78% ) , vomiting ( 50% ) or diarrhea ( 56% ) . seventeen out of the 18 patients showed wbc counts within normal limits , whereas one patient showed leukocytopenia . in addition , thrombocytopenia and lymphocytopenia were present in three of 18 ( 17% ) and 12 of 18 patients ( 67% ) , respectively . no abnormality was noted for the rbc count , hemoglobin level , hematocrit level , platelet count , and neutrophil count . however , all patients showed elevated crp values . after the diagnosis of the pandemic h1n1 2009 influenza virus infection through rt - pcr tests and additional viral cultures , all patients were treated with oseltamivir phosphate . of the 18 patients , 16 rapidly recovered after the administration of the antiviral agent . in the two remaining patients , the symptoms started to improve prior to oseltamivir phosphate treatment . among our study population , one patient presented with high fever , severe nocturnal cough , and blood tinged sputum , and the respiratory symptoms eventually progressed into acute respiratory distress syndrome ( ards ) , which further led him to receive mechanical ventilation ( case no . a retrospective medical chart review revealed that this patient was a solider serving in a different unit from the majority of the other patients and had been in contact with a patient with pandemic h1n1 2009 influenza virus infection lying in a nearby bed in our hospital prior to symptom aggravation . the laboratory data of this patient showed leukocytopenia , thrombocytopenia , and an increased ck level . he was initially treated with antibiotics such as macrolide and a 3rd generation cephalosporin under suspicion of bacterial pneumonia , which was never confirmed . five days after contact with a patient with pandemic h1n1 2009 influenza viral infection , severe respiratory distress presented and a laboratory analysis from the tracheal aspirate performed during endotracheal intubation confirmed the pandemic h1n1 2009 influenza virus infection . following the administration of oseltamivir phosphate and antibiotics , the patient 's symptoms improved and was taken off mechanical ventilation five days after antiviral agent administration . sixteen out of 18 patients in our study population showed no abnormalities on chest radiographs . moreover , only two patients in our study showed abnormal findings of a small central nodular opacity in the right upper lung zone in one ( fig . 2a ) , and consolidations in the left middle lung and both lower lungs in the other ( fig . of these six patients , five ( 83% ) had small ggos ; four patients had them in the upper lobe of the lung and one had a small ggo discovered in the lower lobe of the lung . also , in four out of the five patients with small ggos ( 80% ) , the lesions were localized in the periphery of the lung , and two of the five patients also showed bilateral involvement . for segmental involvement in terms of lesion size , three patients showed one segment , one patient showed two segments , one patient showed three segments , and one patient showed eight segments . nodules were observed in all patients ( 100% ) , which were small in size with showing poorly defined margins ( fig . in addition , the nodule was a solitary finding in one patient ( 17% ) , which was located in the peripheral area of the right upper lobe . the patient who progressed into ards had bilateral lobar consolidations combined with ggos , nodules , and bilateral pleural effusion ( fig . one other patient showed right paratracheal lymphadenopathy . in our study , there was no evidence of calcification , mass , cavitation , or septal thickenings in any of the patients . the thin - section ct findings of the 12 patients are summarized in table 3 . in two patients , follow - up thin section ct images acquired one week after the start of antiviral therapy showed complete resolution or a remarkable decrease in size and opacity of the previously observed ggos and nodules ( fig . with increasing concern over the spread of the pandemic h1n1 2009 influenza virus infection worldwide , detailed knowledge of both its clinical and radiologic features can assist the clinical practice and decision - making of physicians facing this emerging pandemic infectious disease . in addition , preparation for this pandemic infectious disease among institutionalized populations in semi - closed settings such as schools , dormitories , military barracks , or prisons should be made since this infection can spread very rapidly and may quickly become unmanageable without prompt and proper management in the early stages of disease spread . in this study , we attempt to describe one incidence of a military outbreak of the pandemic h1n1 2009 influenza virus with a focus on the clinical and radiological features of these patients , as well as the outcome of treatment . we found common clinical features for all patients with the pandemic h1n1 2009 influenza virus infection in the present study , which included a high fever with accompanying symptoms of cough , rhinorrhea , sore throat , myalgia , or gastrointestinal symptoms such as vomiting or diarrhea . these findings are similar to the symptoms of other influenzas ( 8) and those found in previous studies that dealt with unspecified populations ( 3 , 5 , 9 - 13 ) . generally , laboratory tests , with the exception of serology and culture examinations , are not useful for the specific diagnosis of influenza . leukocyte counts are variable according to stage ; frequently low during the early stages of illness , and later become normal or slightly elevated ( 15 ) . in the present series , there were no patients with elevated wbc counts and 67% of patients showed lymphocytopenia . these findings are consistent with those found in previous studies that dealt with unspecified populations ( 3 , 5 ) . one additional diagnostic laboratory feature we were able to find was an elevated crp level while both the esr and wbc counts remained within a normal range in all patients in our study with pandemic h1n1 2009 influenza virus infection . we believe that elevated crp levels , combined with a lack of leukocytosis or elevated esr , may be characteristic of patients with the pandemic h1n1 2009 influenza virus infection , although these laboratory tests might generally not be able to render a specific diagnosis of this influenza viral infection ( 14 ) . further detailed comparative studies with a larger number of patients would be required to fully explore this issue . most patients in our study did not show a severe hospital course , although one patient 's symptoms aggravated into ards and needed mechanical ventilation before laboratory tests revealed that he had the pandemic h1n1 2009 influenza virus infection . we considered the possibility that co - infection of the pandemic h1n1 2009 influenza virus with other unknown respiratory pathogens under the leukocytopenia status could have attributed to this symptom aggravation ( 9 , 10 ) , particularly as he was a soldier serving in a different unit from the majority of other patients . in addition , the symptom aggravation had occurred after he had been in contact with a patient infected with the pandemic h1n1 2009 influenza virus and there were no pandemic h1n1 2009 influenza virus cases in the unit the patient had been enrolled in . however , since his respiratory symptoms and high fever did not respond to other previous antibiotic treatment , there is still the possibility that the disease course in this case could have been solely due to a severe manifestation of the pandemic h1n1 2009 influenza virus infection . in the present study , chest radiographs were abnormal in two of 18 patients with h1n1 , which was less frequent than that of a previous report , which showed a 42% rate of chest radiograph abnormality ( 12 ) . this suggests that negative findings on chest radiographs can not rule out the possibility of the pandemic h1n1 2009 influenza virus infection , particularly in its early stage or for an uncomplicated influenza infection ( 14 ) . thin - section ct examination results revealed that abnormal findings were found in six out of 12 patients ( 50% ) . the most common ct abnormalities included small ggos in five out of six patients and nodules in all six patients . however , in the previous studies ( 12 , 13 ) , multifocal consolidations ( two of seven patients and 50% ) and ggos ( two of seven patients and 25% ) were the most common ct findings of the pandemic h1n1 2009 influenza virus infection . the differences between the present study and previous studies could be attributed to the fact that radiological examinations in the present study were performed at the initial presentation , and probably the early stages of the disease ; whereas in previous studies ( 12 , 13 ) , radiological examinations were performed at various times after symptom onset . in our study , there was one patient with bilateral lobar consolidations , which progressed into ards . other investigators ( 3 ) also reported similar radiographic features in patients who progressed into ards . although extensive consolidations in the lung might be severe manifestations of primary viral pneumonia from the pandemic h1n1 2009 influenza virus infection , there still remains the possibility of combined pneumonia caused by other pathogens or pulmonary manifestations of ards ( 14 ) . the clinical courses and outcomes of patients in our study with the pandemic h1n1 2009 influenza virus infection were much better than those of previously reported cases ( 3 , 4 ) . furthermore , according to military rule , any problems concerning health must be notified to the medical department immediately , and thus , our patients may have been detected at an earlier stage of the disease course than would be found for the general population . in comparison with a previous report ( 3 ) that showed a high mortality rate in patients with the pandemic h1n1 2009 influenza virus infection , the interval between symptom onset and the emergency department visit ( mean 1.7 days in 15 among 18 patients ) and symptom onset and the start of antiviral agent therapy ( mean 3.9 days ) were shorter . therefore , we conclude that an early diagnosis and early antiviral management can improve the clinical course and outcome of the pandemic h1n1 2009 influenza virus infection . however , to understand the detailed relationship between symptom onset to admission or antiviral management and clinical courses or outcomes , further study , including a comparative analysis with a large case series , is warranted . in our series , most patients ( 15 out of 18 ) were from the same barrack , a semi - closed setting ; whereas , the three remaining patients from different barracks were thought to be infected from these 15 patients . clinical and laboratory results from this homogeneous study population can be helpful in understanding the clinical characteristics of the pandemic h1n1 2009 influenza virus infection in semi - closed settings and in institutionalized populations composed of a young healthy population , such as in schools , dormitories , prisons , or military units . furthermore , this report demonstrated that a prompt diagnosis and treatment resulted in excellent outcomes and could provide as an example for the detection and management strategy for cluster outbreaks of this virus . however , there may be limitations in applying these results to the larger general population . in conclusion , in a population of healthy young adults , elevated crp with normal esr and wbc levels combined with ggos and nodules on thin - section ct scans may indicate early signs of infection by the pandemic h1n1 2009 influenza virus .

objectiveto describe detailed clinical and radiological features of the pandemic h1n1 2009 influenza viral infection among healthy young males in a semi - closed institutionalized setting.materials and methodsa total of 18 patients confirmed with the pandemic h1n1 2009 influenza virus infection from july 18 to july 30 , 2009 were enrolled in this study . each patient underwent an evaluation to determine detailed clinical and radiological features.resultsall patients presented with high fever ( > 38.0 ) , with accompanying symptoms of cough , rhinorrhea , sore throat , myalgia and diarrhea , and increased c - reactive protein ( crp ) values with no leukocytosis nor elevated erythrocyte sedimentation rate ( esr ) . all patients , including one patient who progressed into acute respiratory distress syndrome , were treated with oseltamivir phosphate and quickly recovered from their symptoms . chest radiographs showed abnormalities of small nodules and lobar consolidation in only two out of 18 patients . however , six of 12 patients who underwent thin - section ct examinations showed abnormal findings for small ground - glass opacities ( ggos ) in addition to poorly - defined nodules with upper lobe predominance.conclusionin a population of healthy young adults , elevated crp with normal esr and white blood cell levels combined with ggos and nodules on thin - section ct scans may indicate early signs of infection by the pandemic h1n1 2009 influenza virus .

MATERIALS AND METHODS Patients Laboratory Confirmation Review of Clinical and Laboratory Characteristics Radiologic Examinations Review of Chest Radiographs and Thin-Section CT Images RESULTS Clinical and Laboratory Characteristics of Patients Chest Radiograph and Thin-Section CT Findings DISCUSSION

briefly , we injected 12 ng of morpholino into 4-cell stage x. laevis embryos to knock down rfx2 expression ; the morpholino sequence has been previously reported . we then prepared 100 animal caps ( ectodermal explants of stage 10 x. laevis embryos , dissected with forceps ) , both for control samples and rfx2 morphants , and cultured them until stage 20 . the stage of animal caps was estimated by comparison against embryos from the same clutch . total rna was collected using the trizol method , and then processed using a non - strand - specific illumina rna - seq library preparation kit with poly - a enrichment ( truseq v2 ) . we sequenced these libraries in a 2 50 bp paired - end configuration using an illumina hiseq 2000 . the x. laevis genome project was ongoing when we collected these data , so for this study we used a draft genome sequence ( jgi version 6.0 genome scaffolds ; available at ftp://ftp.xenbase.org/pub/genomics/jgi/xenla6.0/ ) and annotation ( oktoberfest version of putative transcripts , mainly derived from rna - seq de novo assembly and then confirmed against jgi version 6.0 genome scaffolds ; see http://www.marcottelab.org/index.php/xenla_oktoberfest for more details ) . all scaffolds and transcripts are available at xenbase ( ftp://xenbaseturbofrog.org/sequence_information/uta/ ) and our supplementary website ( http://www.marcottelab.org/index.php/chungkwon2013_rfx2 ) . because it is easier for gene - level expression analysis , we conducted rna - seq mapping against putative transcripts rather than the whole genome . using bowtie1 ( version 0.12.7 ) , we mapped our rna - seq reads to the oktoberfest models ( which contain 25,537 putative transcripts for each gene ) using the longest transcript model for each locus . then we used edger to identify differentially expressed genes , focusing on genes with greater than 2-fold difference and a false discovery rate less than 0.05 . one of the challenges in x. laevis rna - seq analysis is the presence of homoeologs , i.e. duplicated genes that arise as a result of allotetraploidy . using an allowance of 2 mismatches within a 50-bp read ( the -v 2 option in bowtie1 ) we used two datasets for this test : ( 1 ) 827 gene pairs previously identified by a variety of labs and curated at xenbase using an -a/-b gene name suffix , and ( 2 ) 2218 assembled est pairs identified as involved in a trio relationship with xenopus tropicalis . as shown in fig . 1 , 6875% of reads we were particularly interested in the differential expression between wild - type embryos and rfx2 morphants . thus , in order to maximize the expression signals in our analysis , we allowed for all possible hits in mapping with the -a option ( i.e. interchangeably mapped reads would be counted twice ) , and then conducted differential expression analysis . ultimately we found no major differences in differential analysis between these approaches ( data not shown ) . out of 24,089 x. laevis transcripts detected in our rna - seq experiments , 3209 transcripts were down - regulated in the rfx2 knockdown condition , and 1523 transcripts were up - regulated . to perform functional network analysis using humannet , we converted these gene lists to human orthologs ( based on ensembl version 69 ) . note that initial orthology assignments are already captured by the x. laevis oktoberfest transcript gene names , because as part of the transcript set construction , all x. laevis protein sequence candidates were compared to the reference proteome of five different species ( human , mouse , zebrafish , chicken , and x. tropicalis ) for the purpose of assigning gene names consistent with the human orthologs . for homoeologs , if only one of two duplicated genes was determined to be significantly differentially expressed but not the other , we still assigned the corresponding human gene as being differentially expressed . after converting all x. laevis genes into human orthologs , we identified 2750 human candidate genes transcriptionally regulated by rfx2 . the detailed description of our chip - seq sample preparation has been previously reported . briefly , we injected mrna encoding gfp - tagged rfx2 into 4-cell stage x. laevis embryos and then pulled down the tagged protein with -gfp antibody ( ab290 ) from 600 whole embryos ( stage 20 ) . before immunoprecipitation , we cross - linked rfx2-genomic dna complexes with 1% formaldehyde and fragmented them with a branson 450 sonifier ( expected fragment size was from 200 to 500 bp ) . as a control , we injected gfp messenger rnas alone and conducted the same immunoprecipitation procedure . dna fragments were extracted with phenol chloroform and purified with a qiaquick pcr purification kit ( qiagen ) . sequencing libraries were prepared with a standard illumina genomic library construction kit ( truseq ) and sequenced with an illumina hiseq 2000 in 1 50 bp configuration . briefly , we injected 12 ng of morpholino into 4-cell stage x. laevis embryos to knock down rfx2 expression ; the morpholino sequence has been previously reported . we then prepared 100 animal caps ( ectodermal explants of stage 10 x. laevis embryos , dissected with forceps ) , both for control samples and rfx2 morphants , and cultured them until stage 20 . the stage of animal caps was estimated by comparison against embryos from the same clutch . total rna was collected using the trizol method , and then processed using a non - strand - specific illumina rna - seq library preparation kit with poly - a enrichment ( truseq v2 ) . we sequenced these libraries in a 2 50 bp paired - end configuration using an illumina hiseq 2000 . the x. laevis genome project was ongoing when we collected these data , so for this study we used a draft genome sequence ( jgi version 6.0 genome scaffolds ; available at ftp://ftp.xenbase.org/pub/genomics/jgi/xenla6.0/ ) and annotation ( oktoberfest version of putative transcripts , mainly derived from rna - seq de novo assembly and then confirmed against jgi version 6.0 genome scaffolds ; see http://www.marcottelab.org/index.php/xenla_oktoberfest for more details ) . all scaffolds and transcripts are available at xenbase ( ftp://xenbaseturbofrog.org/sequence_information/uta/ ) and our supplementary website ( http://www.marcottelab.org/index.php/chungkwon2013_rfx2 ) . because it is easier for gene - level expression analysis , we conducted rna - seq mapping against putative transcripts rather than the whole genome . using bowtie1 ( version 0.12.7 ) , we mapped our rna - seq reads to the oktoberfest models ( which contain 25,537 putative transcripts for each gene ) using the longest transcript model for each locus . then we used edger to identify differentially expressed genes , focusing on genes with greater than 2-fold difference and a false discovery rate less than 0.05 . one of the challenges in x. laevis rna - seq analysis is the presence of homoeologs , i.e. duplicated genes that arise as a result of allotetraploidy . using an allowance of 2 mismatches within a 50-bp read ( the -v 2 option in bowtie1 ) , we evaluated how many reads were mapped interchangeably between homoeologs . we used two datasets for this test : ( 1 ) 827 gene pairs previously identified by a variety of labs and curated at xenbase using an -a/-b gene name suffix , and ( 2 ) 2218 assembled est pairs identified as involved in a trio relationship with xenopus tropicalis . as shown in fig . 1 , 6875% of reads we were particularly interested in the differential expression between wild - type embryos and rfx2 morphants . thus , in order to maximize the expression signals in our analysis , we allowed for all possible hits in mapping with the -a option ( i.e. interchangeably mapped reads would be counted twice ) , and then conducted differential expression analysis . ultimately we found no major differences in differential analysis between these approaches ( data not shown ) . out of 24,089 x. laevis transcripts detected in our rna - seq experiments , 3209 transcripts were down - regulated in the rfx2 knockdown condition , and 1523 transcripts were up - regulated . to perform functional network analysis using humannet , we converted these gene lists to human orthologs ( based on ensembl version 69 ) . note that initial orthology assignments are already captured by the x. laevis oktoberfest transcript gene names , because as part of the transcript set construction , all x. laevis protein sequence candidates were compared to the reference proteome of five different species ( human , mouse , zebrafish , chicken , and x. tropicalis ) for the purpose of assigning gene names consistent with the human orthologs . for homoeologs , if only one of two duplicated genes was determined to be significantly differentially expressed but not the other , we still assigned the corresponding human gene as being differentially expressed . after converting all x. laevis genes into human orthologs , we identified 2750 human candidate genes transcriptionally regulated by rfx2 . briefly , we injected mrna encoding gfp - tagged rfx2 into 4-cell stage x. laevis embryos and then pulled down the tagged protein with -gfp antibody ( ab290 ) from 600 whole embryos ( stage 20 ) . before immunoprecipitation , we cross - linked rfx2-genomic dna complexes with 1% formaldehyde and fragmented them with a branson 450 sonifier ( expected fragment size was from 200 to 500 bp ) . as a control , we injected gfp messenger rnas alone and conducted the same immunoprecipitation procedure . dna fragments were extracted with phenol chloroform and purified with a qiaquick pcr purification kit ( qiagen ) . sequencing libraries were prepared with a standard illumina genomic library construction kit ( truseq ) and sequenced with an illumina hiseq 2000 in 1 50 bp configuration . similar to the rna - seq data analysis , we conducted chip - seq analysis to discriminate between homoeolog genes . we applied a more stringent criteria for chip - seq read mapping , requiring uniquely mapped reads to the genome scaffold ( jgi version 6.0 ) and a maximum of 2 mismatches within the seed sequence ( i.e. the -m 1 -n 2 options in bowtie1 ) . for peak calling , we used macs ( version 1.4.2 ) with default options . we initially determined significant rfx2-bound peaks by using a false discovery rate ( fdr ) cutoff ( < 0.05 ) reported by macs . however , as shown in fig . 2 , only a few peaks demonstrated an fdr above 0.05 if the fold enrichment of the peak was greater than 20 , so we included these peaks as well in our further analysis . for each peak , we assigned the closest protein - coding gene as its target gene , so long as it was within 10 kb . most of these peaks were located less than 1000 bp from the transcript start site of their assigned gene , suggesting that , if anything , our criteria for associating chip - seq peaks to target genes were over - generous . out of 29,448 peaks identified in total , 6646 peaks were selected for further study that exhibited either an fdr < 5% or a fold - enrichment > 20 , and 5024 of those peaks were assigned to their neighboring genes . as with our rna - seq data analysis , we converted the 5024 x. laevis target gene ids to human genes , collapsing duplicated genes into a single human ortholog based on their names . this analysis resulted in a final set of 911 putative directly bound rfx2 target genes that also showed significantly differential gene expression after rfx2 knockdown . a list of all 911 genes is available in supplemental file 1 in our previous report .

recently , using the frog xenopus laevis as a model system , we showed that the transcription factor rfx2 coordinates many genes involved in ciliogenesis and cell movement in multiciliated cells ( chung et al . , 2014 ) . to our knowledge , it was the first paper to utilize the genomic resources , including genome sequences and interim gene annotations , from the ongoing x. laevis genome project . for researchers who are interested in the application of genomics and systems biology approaches in xenopus studies , here we provide additional details about our dataset ( ncbi geo accession number gse50593 ) and describe how we analyzed rna - seq and chip - seq data to identify direct targets of rfx2 .

Direct link to deposited data Experimental design, materials and methods RNA-seq experiments RNA-seq analysis ChIP-seq experiments ChIP-seq analysis

atrial fibrillation ( af ) with progressive atrial dilation combined with inadequate anticoagulation exhibits a high risk of thromboembolic events . we present the unusual case of a ball thrombus originating from the left atrium ( la ) with remaining continuity to the left atrial appendage ( laa ) causing syncope and cerebral ischemia . an 81-year - old female presenting with syncope and aphasia was admitted to the emergency department of our hospital . the patient was diagnosed with permanent af four years prior to presentation and was managed with rate - control therapy . however , for unknown reasons , there was no anticoagulation at the time of presentation . so far , except for af and arterial hypertension ( treated by angiotensin receptor blockers and diuretics ) , the patient was free from other independent risk factors for la thrombus formation ( no previous cardiac or embolic history ) . the head computed tomography scan of the patient , who was normotensive at this time , revealed acute focal ischemia at the left temporal lobe . transthoracic echocardiography showed the presence of a 4 3.5 cm measuring , free - floating , nonhomogeneous mass in the la . there was a rate - dependent dislocation of this mass to the mitral valve orifice ( fig . this caused intermittent obstruction of the left ventricular outflow tract , and was associated with presyncopal symptoms . in addition , echocardiography demonstrated severely dilated atria , presumed to be due to long - standing af . the lv showed concentric hypertrophy with a preserved ejection fraction . after excluding significant coronary stenosis by coronary angiography , the primary operative finding was a spherical purple gelatinous mass measuring 4.5 4 cm , partially free floating in the la , but attached to the laa by a thin bridge ( fig . excision of the mass including the shaft was performed , followed by suture ligation of the laa . histopathological examination confirmed a layered , partly necrotic , mostly organized , parietal thrombus ( not displayed ) . surgical inspection confirmed the echocardiographic findings of moderate mitral regurgitation , so additional valve repair was not indicated . moreover , a maze procedure was considered , but because of the patient s critical status and cerebral ischemia , it was deferred . at the 6-month follow - up evaluation , there were no signs of relevant postoperative complications or neurologic postischemic sequelae . the patient was anticoagulated during this period , and no new thrombus formation was identified on repeat echocardiographic examination ( fig . in this report , we present the case of a patient with syncope and acute cerebral ischemia due to embolization from an unusual la ball thrombus with preserved attachment to the laa not visible on transthoracic echocardiography . thrombus formation and embolization are the most threatening complications of an inadequate anticoagulation in the presence of af . as in this case , the presence of a mass in the la often presents a diagnostic challenge for cardiologists . echocardiography can help distinguish between solid benign or malignant mass , and organized or disorganized thrombus formation . shape and echogenicity are important characteristics that help to distinguish among these potential etiologies of atrial mass . to date , there have been few reports of the presence of an la ball thrombus without mitral valve disease.13 most frequently , the presence of a ball thrombus suggests mass formation by continuous increase in size independently from the la wall . therapeutic decision - making and optimal treatment strategy selection , ie , thrombolysis versus surgical intervention , require prompt evaluation of clinical parameters and evaluation of the thrombus morphofunctional characteristics . several authors have reported recurrent ischemic episodes after thrombolysis in the presence of la thrombus.4,5 when a free - floating thrombus is identified in the la , emergent cardiac surgery for embolectomy is often required because of the high risk for sudden circulatory collapse or systemic embolization.3,6 as in the case we present here , accurate trans thoracic echocardiographic evaluation of the mass provides critical information required for initiating timely therapeutic procedures.7 continuous anticoagulation treatment for patients with af remains extremely important to prevent embolic complications.8 selection of the optimal treatment strategy for atypical la thrombus is a challenging clinical scenario . unfortunately , no guidelines exist to direct the selection of the most appropriate evaluation and treatment for these cases . the use of transthoracic echocardiography in emergency situations can be a cost - effective and time - saving tool impacting immediate therapeutic decision - making .

few cases of a left atrial thrombus without mitral valve disease have been reported . we present an unusual case in which a patient presented to the emergency department with syncope and acute cerebral ischemia caused by a ball thrombus originating from the left atrium ( la ) . an emergency bedside echocardiogram showed the la ball thrombus intermittently obstructing the mitral orifice and , at times , compromising the left ventricular outflow tract . this thrombus was determined to be the source of cerebral embolization resulting in acute ischemia . surgical excision of the mass was performed . at operation , the thrombus was found to be tethered to the left atrial appendage . this tethering was not apparent on the echocardiographic images , where the thrombus appeared to be free floating . this case demonstrates the utility of transthoracic echocardiography in establishing the etiology of emergent conditions seemingly unrelated to acute cardiac disease , in this situation a neurologic presentation with syncope and cerebral ischemia .

Introduction Case Report Discussion Conclusions

gynecologic malignancies , including cervical and vaginal cancers , often require a multidisciplinary approach that combines radiation , chemotherapy and/or surgery . imaging modalities that can accurately assess the extent of the disease and the effect of treatments have become an integral component in evaluating the success of therapy . radiation therapy is an essential component in the management of unresectable locally advanced disease . more specifically , external beam radiation therapy ( ebrt ) and brachytherapy ( bt ) are the two radiation modalities that are used sequentially to reduce the risk of local recurrence and improve survival . at diagnosis , positron emission tomography / computed tomography ( pet / ct ) with the tracer 2-[fluorine-18]fluoro-2-deoxy - d - glucose ( fdg ) however , studies have indicated that , when fdg - pet is used immediately after ebrt to assess treatment response , it may be difficult to differentiate between residual or recurrent active tumor and inflammation in the area treated with radiation using this tracer ( choi et al . , 2014 ) . therefore , fdg - pet is not a reliable assessment tool to monitor residual tumor and to direct bt doses immediately after ebrt . another available pet tracer used for tumor assessment , f-18-fluorothymidine ( flt ) , has the potential to improve detection of metabolically active tumors ( wang et al . , 2015 , hoshikawa et al . , 2015 ) . this relatively new radiotracer has attracted attention for evaluation of post - radiation response in , head / neck , esophageal , breast , lung , and rectal cancers though its role in the evaluation of gynecologic cancers after ebrt is unknown ( yue et al . , 2010 , , 2012 , wieder et al . , 2007 , kahraman et al . , 2011 , menda et al . , 2009 ) . the fact that only proliferating cells ( i.e. malignant tissues ) enable thymidine incorporation in dna synthesis makes flt imaging a particularly attractive tool when differentiating between residual / recurrent malignancy and inflammation ( rasey et al . , 2002 , the goals of this study were to compare metabolic parameter standard uptake values ( suvs ) of flt with fdg pet in patients with gynecologic cancer at diagnosis and to determine if flt suvs are affected by inflammation immediately following ebrt . between august 2012 and april 2014 , six patients with newly diagnosed gynecologic cancers treated with concurrent chemoradiotherapy ( chemort ) were enrolled on a prospective clinical trial for image - guided brachytherapy . all tumors were staged according to the international federation of gynecology and obstetrics ( figo ) 2009 staging system ( pecorelli , 2009).the eligibility requirements included : histopathologically confirmed primary lesions ( five were cervical cancers and one was vaginal cancer ) ; mri and/or pet - ct scan within 4 months before registration ; age 18 ; ecog performance status 2 ; and no uncontrolled illness that would limit compliance with study requirements . the study was approved by the institutional review board , and all participants signed an informed consent form . the bwh cyclotron and radiopharmacy produced flt in a nucleophilic synthesis box using in - process controls such as high - purity solvents to synthesize the radiopharmaceutical and automated software that controls the temperature and timing parameters and records the information from the production run . an integrity test was performed on the sterilizing filter in addition to a sterility test on the final drug product ; the process achieved a radiochemical purity of more than 95% . for both fdg and flt imaging , pet / ct was performed after at least 4 h of fasting and 60 10 min after the intravenous administration of 370480 megabecquerels of fdg or 296370 megabecquerels of flt . pet imaging was performed in 3-dimensional mode on a discovery st pet / ct scanner ( general electric , milwaukee , wi ) from the skull base through the mid thighs . non - contrast helical ct imaging was performed over the same range without breath - hold for attenuation correction of pet images and anatomic correlation . the review of fdg- and flt - pet / ct images and measurements of the tumor suvmax were performed on hermes workstation ( hermes medical solutions ab , stockholm , sweden ) . the wilcoxon rank sum test was used to compare the suvs of flt and fdg at diagnosis . between august 2012 and april 2014 , six patients with newly diagnosed gynecologic cancers treated with concurrent chemoradiotherapy ( chemort ) were enrolled on a prospective clinical trial for image - guided brachytherapy . all tumors were staged according to the international federation of gynecology and obstetrics ( figo ) 2009 staging system ( pecorelli , 2009).the eligibility requirements included : histopathologically confirmed primary lesions ( five were cervical cancers and one was vaginal cancer ) ; mri and/or pet - ct scan within 4 months before registration ; age 18 ; ecog performance status 2 ; and no uncontrolled illness that would limit compliance with study requirements . the study was approved by the institutional review board , and all participants signed an informed consent form . the bwh cyclotron and radiopharmacy produced flt in a nucleophilic synthesis box using in - process controls such as high - purity solvents to synthesize the radiopharmaceutical and automated software that controls the temperature and timing parameters and records the information from the production run . an integrity test was performed on the sterilizing filter in addition to a sterility test on the final drug product ; the process achieved a radiochemical purity of more than 95% . for both fdg and flt imaging , pet / ct was performed after at least 4 h of fasting and 60 10 min after the intravenous administration of 370480 megabecquerels of fdg or 296370 megabecquerels of flt . pet imaging was performed in 3-dimensional mode on a discovery st pet / ct scanner ( general electric , milwaukee , wi ) from the skull base through the mid thighs . non - contrast helical ct imaging was performed over the same range without breath - hold for attenuation correction of pet images and anatomic correlation . the review of fdg- and flt - pet / ct images and measurements of the tumor suvmax were performed on hermes workstation ( hermes medical solutions ab , stockholm , sweden ) . the wilcoxon rank sum test was used to compare the suvs of flt and fdg at diagnosis . the median age at diagnosis for the six patients was 61 years ( range , 3372 ) . the five cervical cancers were staged as ib2 ( n = 1 , 20% ) , iib ( n = 1 , 20% ) , iiib ( n = 1 , 20% ) and iva ( n = 2 , 40% ) and the one vaginal cancer was stage iiib ( n = 1 ) . the most common histology was squamous cell carcinoma ( n = 3 , 50% ) followed by adenocarcinoma ( n = 2 , 33% ) and clear cell adenosquamous carcinoma ( n = 1 , 17% ) . median follow - up time was 24 months ( range , 630 months ) . all six patients received concurrent weekly chemotherapy ( cisplatin for 5 and carboplatin for 1 ) with radiation therapy ( table 1 ) . all received a total radiation dose of 45 gy in 25 fractions of 1.8 gy to the pelvis and para - aortic nodes . patient a received an additional 6 gy boost to the right inguinal nodes , and patient b received an additional 10 gy boost to the right iliac nodes . all patients underwent flt - pet and fdg - pet scanning at diagnosis ; these scans were performed 13 weeks before chemort . all patients also had post - chemort flt - pet / ct scans before receiving brachytherapy , except patient c due to scanner malfunction ( table 2 ) . all patients had post - chemort fdg - pet , but are not reported due to diffuse uptake . at last follow - up , five patients were alive and one had died with the most recent mri exam showing significant tumor shrinkage 3-months prior to death but the cause was undeterminable . two patients with primary cervical cancer were known to have had recurrence : 1 in the vaginal cuff ( patient c ) and 1 with metastatic disease to the lung ( patient d ) . at diagnosis , fdg uptake was visually more vivid in the primary tumor in all patients than flt uptake ( fig . the median suvmax at diagnosis was 6.9 ( range , 3.914.2 ) for flt - pet and 11.6 ( range , 5.923.2 ) for fdg - pet . suvs were 1.53.5 times higher for fdg than for flt in 4 patients ; the other two patients only had minor differences between the suvs of the two tracers . the difference between the medians was not statistically significant ( p = 0.15 by wilcoxon rank sum test ) , which may be due to the small sample size . overall , flt accumulation in primary showed significant decrease after chemort both visually and quantitatively . in 4 of the 5 patients who underwent flt pet after chemort , suvmax of flt declined moderately to markedly . these declines measured 54% ( patient a , from suvmax of 6.9 to 3.2 ) , 70% ( patient d , from 3.9 to 1.2 ) , 81% ( patient b , from 8.7 to 1.7 ) , and 85% ( patient e , from 6.0 to 0.9 ) ( fig . the remaining patient f was assessed to have complete resolution of all tumors on both ct and flt pet , and therefore suvmax was not measured . the median age at diagnosis for the six patients was 61 years ( range , 3372 ) . the five cervical cancers were staged as ib2 ( n = 1 , 20% ) , iib ( n = 1 , 20% ) , iiib ( n = 1 , 20% ) and iva ( n = 2 , 40% ) and the one vaginal cancer was stage iiib ( n = 1 ) . the most common histology was squamous cell carcinoma ( n = 3 , 50% ) followed by adenocarcinoma ( n = 2 , 33% ) and clear cell adenosquamous carcinoma ( n = 1 , 17% ) . median follow - up time was 24 months ( range , 630 months ) . all six patients received concurrent weekly chemotherapy ( cisplatin for 5 and carboplatin for 1 ) with radiation therapy ( table 1 ) . all received a total radiation dose of 45 gy in 25 fractions of 1.8 gy to the pelvis and para - aortic nodes . patient a received an additional 6 gy boost to the right inguinal nodes , and patient b received an additional 10 gy boost to the right iliac nodes . all patients underwent flt - pet and fdg - pet scanning at diagnosis ; these scans were performed 13 weeks before chemort . all patients also had post - chemort flt - pet / ct scans before receiving brachytherapy , except patient c due to scanner malfunction ( table 2 ) . all patients had post - chemort fdg - pet , but are not reported due to diffuse uptake . at last follow - up , five patients were alive and one had died with the most recent mri exam showing significant tumor shrinkage 3-months prior to death but the cause was undeterminable . two patients with primary cervical cancer were known to have had recurrence : 1 in the vaginal cuff ( patient c ) and 1 with metastatic disease to the lung ( patient d ) . at diagnosis , fdg uptake was visually more vivid in the primary tumor in all patients than flt uptake ( fig . the median suvmax at diagnosis was 6.9 ( range , 3.914.2 ) for flt - pet and 11.6 ( range , 5.923.2 ) for fdg - pet . suvs were 1.53.5 times higher for fdg than for flt in 4 patients ; the other two patients only had minor differences between the suvs of the two tracers . the difference between the medians was not statistically significant ( p = 0.15 by wilcoxon rank sum test ) , which may be due to the small sample size . overall , flt accumulation in primary showed significant decrease after chemort both visually and quantitatively . in 4 of the 5 patients who underwent flt pet after chemort , suvmax of flt declined moderately to markedly . these declines measured 54% ( patient a , from suvmax of 6.9 to 3.2 ) , 70% ( patient d , from 3.9 to 1.2 ) , 81% ( patient b , from 8.7 to 1.7 ) , and 85% ( patient e , from 6.0 to 0.9 ) ( fig . the remaining patient f was assessed to have complete resolution of all tumors on both ct and flt pet , and therefore suvmax was not measured . in this small pilot evaluation of the role of flt - pet in imaging gynecologic tumors , we found that flt uptake in gynecologic cancers before treatment was slightly lower than that of fdg but high enough to be detected and assessed visually and quantitatively . furthermore , there was a considerable decrease in flt uptake after chemort in all patients who had post - chemort scans , suggesting no significant influence of radiation - induced inflammation on flt uptake . greatly increased cell proliferation is a characteristic of many malignant lesions and this cancer - specific process has been exploited in tracer imaging . retention of flt , an analog of thymidine , has been shown to correlate with thymidine uptake and thymidine kinase-1 ( tk1 ) activity in cancer cells ( rasey et al . , 2002 , toyohara et al . , 2002 ) . the fact that only proliferating cells enable thymidine incorporation in dna synthesis ( as opposed to glucose utilization , which is increased in both malignant tissue and inflammatory tissue ) makes flt imaging a particularly attractive tool when differentiating between residual / recurrent malignancy and inflammation . flt has previously been shown to be not as significantly affected by post - radiation inflammation and a more specific tracer than fdg when assessing response to radiation therapy , whereas fdg uptake is higher than flt uptake at diagnosis in most tumors ( chen et al . , for example , in laryngeal cancers , fdg uptake was higher than flt uptake at diagnosis both visually and quantitatively ( been et al . , 2009 ) . on the other hand , kishino et al . found on their mid - treatment scans performed after 40 gy of radiation therapy that abnormal flt uptake disappeared in 63% of head - and - neck squamous cell carcinoma lesions whereas fdg uptake disappeared in only 16% of the lesions ; specificity was 72% for flt compared to 19% for fdg . the specificity of flt - pet ( 80% ) performed after completion of chemoradiation was also higher than that of fdg - pet ( 48% ) . however , the sensitivities of mid - treatment and post - rt flt - pet scans ( 87.5% and 89% , respectively ) were both lower than those of the equivalent fdg - pet scans ( 100% for both ) ( kishino et al . , 2012 ) . van waarde et al . found similar results in an animal study ; using an acute - inflammation model in rats , they demonstrated that flt is a more cancer - specific pet tracer than fdg , showing no uptake in inflammatory tissues ( van waarde et al . , 2004 ) . however , the usefulness of flt imaging in gynecologic cancers has not been evaluated to date in the setting of post - radiation inflammation . our results show similar trends when compared to those in other malignancies reported in the literature , such as those for laryngeal and head - and - neck cancer cited above ( been et al . , 2009 , kishino et al . , 2012 , van waarde et al . , 2004 ) . although there was a visually significant difference between flt and fdg uptake by tumors at diagnosis in 4 of 6 patients , the overall difference in suv did not reach statistical significance , likely due to the small sample size . also , although our results indicate the feasibility of flt and its potential usefulness in assessing chemort response in gynecologic cancer , exact sensitivity and specificity of post - rt flt - pet / ct will need to be evaluated in future studies . furthermore , larger samples are needed to confirm the validity of our findings and to determine the diagnostic accuracy of flt in this patient population , with the goal of developing quantitative imaging tools that will provide feasible method for triaging treatment and a reliable measurement of the changes after radiation therapy . overall , flt uptake in gynecologic cancers was lower than that of fdg at initial diagnosis but was high enough to be detected visually and to be quantifiable in all patients . flt may be a useful tool when assessing the effects of chemort on gynecologic cancers and for optimizing post - chemort brachytherapy planning .

objectivethe role of f-18-fluorothymidine ( flt ) pet - ct imaging in the evaluation of gynecologic cancers has not been established . we sought to evaluate ( flt ) pet - ct imaging in gynecologic cancers by comparing standard uptake values ( suvs ) of flt with f-18-fluorodeoxyglucose ( fdg ) pet in the primary tumor at diagnosis , and assess flt uptake immediately following concurrent chemoradiotherapy ( chemort).methodsin this pilot study , patients treated for cervical ( 5 ) or vaginal ( 1 ) cancer underwent flt - pet and fdg - pet scanning at diagnosis ( flt1 and fdg1 ) . five patients ( 4 cervical and 1 vaginal ) also underwent flt - pet within 13 weeks after chemort before brachytherapy ( flt2 ) . wilcoxon rank - sum test was used to compare the flt1 and fdg1 parameters.resultsmedian age at diagnosis was 61-years ( range , 3372 ) . cervical cancers were staged as ib2 ( n = 1 , 20% ) , iib ( n = 1 , 20% ) , iiib ( n = 1 , 20% ) and iva ( n = 2 , 40% ) and the single vaginal cancer was staged iiib . the most common histology was squamous cell carcinoma ( n = 3 , 50% ) followed by adenocarcinoma ( n = 2 , 33% ) and clear - cell adenosquamous carcinoma ( n = 1 , 17% ) . median tumor suvmax at diagnosis was 7.8 on flt1-pet ( 3.914.2 ) versus 11.6 ( 5.923.2 ) on fdg1-pet ( p = 0.15 ) . tumor suvmax of flt declined 54%100% after chemort.conclusionthe tumor suv of flt at diagnosis was lower than that of fdg - pet . flt uptake was markedly decreased after chemort . results indicate that there may not be a significant effect of inflammation on flt uptake in gynecologic cancers . flt may be a useful tool when assessing the effects of chemort on gynecologic malignancies and planning for postchemort brachytherapy treatments .

Introduction Material and methods Patients FLT synthesis PET/CT acquisition Statistical analysis Results Patient and tumor characteristics Comparison of FLT and FDG uptake at diagnosis Changes in FLT uptake after chemoRT Discussion Conclusion Disclosure

recent years have seen growing numbers of cervical disc replacements and fusions due to improved surgical techniques.1,2,3 diagnostics and differential diagnostics have been highly standardized;4 however , recurrent cervical symptoms frequently occur.5 possible vertebrogenic causes lie within the treated or adjacent segments ( table 1).6 . primarily , local diagnostics of these causes are required ( table 2 ) ; yet , no algorithm for the diagnostic assessment of recurrent cervical symptoms has been established to date.7 this bears the risk that non - vertebrogenic causes of cervical symptoms are diagnosed at a late stage . a 53-year - old nonsmoking man presented to our outpatient clinic with a new episode of cervicobrachialgia after a 2-year symptom - free interval . two years ago , because of bilateral foraminal stenosis at c5/6 and c6/7 with increasing cervicobrachialgia , decompression and fusion at the severest level c5/6 ( pina titan cage 8 mm 14 16 mm ) and disc replacement at c6/7 ( prodisc - c xl 8 mm ) had been performed due to different degenerative changes . in the preoperative routine check - up routine cervical x - ray had shown a stable fusion and a beginning kyphosis of the disc prosthesis ( fig . two years after surgery the patient reported recurrent cervicobrachialgia ; however , cervical magnetic resonance imaging ( mri ) including stir sequences showed no pathological findings except for incipient foraminal stenosis at levels c3/4 and c4/5 due to uncovertebral arthrosis ( fig . therefore , no further imaging or invasive investigations such as computed tomography ( ct)-myelography were indicated . ( a ) postoperative cervical x - ray , lateral view . ( b ) postoperative cervical x - ray , anteroposterior view . a 2-year postoperative cervical mri , t2-weighted sagittal view . during the next 6 months his symptoms got worse with increasing numbness and weakness of the right arm . repeated cervical mri revealed bone metastases of the fourth and fifth vertebrae with intraspinal tumor growth ( fig . 3 ) . thoracic x - ray and ct demonstrated an extensive small cell bronchial carcinoma ( figs . 4,5 ) . beginning paraparesis required immediate treatment comprising debulking , radiotherapy as well as etoposide and cisplatin chemotherapy . subsequent to deterioration of his general state of health , the patient died 2 weeks after the cancer diagnosis of cardiopulmonary decompensation . ( b ) a 2.5-year postoperative cervical mri , t1-weighted axial view . a 2.5-year postoperative thoracic x - ray , anteroposterior view . a 2.5-year postoperative thoracic ct , axial view . good - to - excellent clinical results with a low complication rate are reported for ventral cervical fusion and disc replacement.8,9,10 this applies likewise to disc replacement in combination with fusion.11,12 yet , postoperative cervicobrachialgia is not uncommon.5,13,14 especially after a symptom - free interval of 2 years recurrent cervical symptoms are often believed to result from vertebrogenic causes . metal artifacts complicate the evaluation of mri and ct scans.15,16,17 the absence of neurological deficits demands a nonsurgical treatment leading to a successful outcome in most patients.6 however , recurrent cervicobrachialgia without pathological cervical imaging requires interdisciplinary diagnostics . to our knowledge , there is only one report of a similar case in the literature . pillai et al 18 presented the case of a patient developing metastases at the site of the arthroplasty 1 year after fusion at c3/4 to caution spine surgeons about the possibility that metastases might occur at the site of a cervical fusion and that a metastatic tumor should be accounted for in the differential diagnosis of recurrent cervicobrachialgia . basic diagnostic tests , such as thoracic x - ray , erythrocyte sedimentation rate , c - reactive protein , and electrocardiogram may assist to exclude severe nonvertebrogenic pathologies . this case illustrates the need for interdisciplinary diagnostics in recurrent cervicobrachialgia without pathological cervical imaging . there are three important learning points here : not everything that looks like a horse is a zebra . however , just because disc degeneration is so very common should not mean that we operate on all patients with cervical imaging abnormalities . the principle of evaluating a patient clinically and then connecting clinical findings with insights gained by imaging remains the time - honored principle of spinal management for a good reason . for patients with neck / arm symptoms a differential diagnosis of other pathoentities , such as primary shoulder problems , compressive neuropathies , brachial plexus lesions , radiculitis or parsonage - turner syndrome and in rare cases malignancy ( pancoast tumor ! ) or infection are important considerations to keep in mind . the use of electrodiagnostic tests , such as electromyography , is frequently shunned in this era of painless advanced imaging but is essential to differentiate a radiculopathy from a brachial neuritis and a compressive neuropathy . symptom recurrence after spine surgery : melloh and barz correctly point out that symptom recurrence after neural decompression surgery is not uncommon and is multifactorial . a structured approach toward symptom recurrence is indeed helpful . the ebsj editors recommend the methodical five - step approach suggested by the late henry bohlman : was the index surgery done for the right reason in the right patient at the right time ? was the surgery done well ? has everything healed well ? has something new occurred , such as recurrent stenosis , breakdown above or below , infection , neoplasia , neurodegenerative disorder , metabolic disease , and so on ? the commentary by john g devine might help settle some of the rampant misperceptions , especially regarding use of mri following arthroplasty and invite a more systematic approach to be formulated . department of orthopaedic surgery eisenhower army medical center melloh and barz describe an interesting case of recurrent cervicobrachialgia after a 2-year symptom - free period . the patient had undergone a two - level anterior cervical discectomy with a fusion at the c5/6 level and an arthroplasty at the c6/7 level . the initial work - up included cervical x - rays and magnetic resonance imaging ( mri ) including stir sequences that revealed no obvious pathological findings . as his symptoms progressed over the next 6 months , a repeated cervical mri was obtained revealing metastatic lesions of the fourth and fifth cervical vertebrae with extension of the tumor into the canal . eventual diagnosis was made after thoracic computed tomography ( ct ) revealed the origin of the metastasis to be small cell bronchial carcinoma . the authors point out that there is only one other reported case in the literature.1 however , this is not an uncommon clinical scenario . in addition to a medical history and physical examination , first - line imaging should include x - ray evaluation and mri . titanium devices allow for satisfactory imaging of the adjacent and index levels , but non - titanium devices ( cobalt - chrome - molybdenum alloys ) create significant image distortion , preventing accurate imaging at the index and adjacent levels.2 more recently , it has been demonstrated that magnet strength affects the artifact from cobalt - chrome alloys . using a lower strength magnet , such as the 0.2 tesla magnet found in many of the open scanners , the reduction in artifact allows for adjacent segment imaging without a significant reduction in quality.3 however , the index levels are still significantly distorted . in cases when symptoms persist without an obvious explanation using mri - myelography followed by ct - myelogram is the imaging modality of choice . the image distortion can be minimized , while allowing better visualization of the vertebral morphology , and earlier detection of trabecular destruction in the case of metastasis . additionally , the presence of neural compression can be appreciated centrally and in the proximal nerve roots at every level , including the index level after arthroplasty . last , other imaging modalities can be used to detect the presence of occult metastasis in the setting of disc arthroplasty when the index of suspicion is high on the differential , or if there is already a diagnosis of malignancy and staging is required . technetium-99 m bone scan is useful to identify areas of amplified metabolic activity and has a high sensitivity for detecting lesions . positron emission tomography uses fluorodeoxyglucose to define sites of increased metabolic activity and is more sensitive and specific in detecting bone metastasis . as melloh and barz make clear , in cases when no obvious pathology is identified in the initial mri in patients presenting with recurrent symptoms after disc arthroplasty , interdisciplinary diagnostics may be required . in addition to basic diagnostic tests , such as erythrocyte sedimentation rate and c - reactive protein , x - rays and electrocardiogram , i would also recommend ct - myelogram in the setting of a preexisting disc arthroplasty .

recurrent cervical symptoms frequently occur after cervical disc replacement and fusion . to date , no algorithm for the diagnostic assessment of these symptoms has been established . we present a case report and review of the literature to illustrate the need for interdisciplinary diagnostics in recurrent cervicobrachialgia without pathological cervical imaging . the hospital chart , medical history , physical examination , and imaging of a single patient were reviewed . a 53-year - old man with preexisting cervical disc replacement and fusion presented with a new episode of cervicobrachialgia after a 2-year symptom - free interval . cervical magnetic resonance imaging ( mri ) showed no pathological findings . six months later the patient reported increasing symptoms including numbness and weakness of the right arm . repeated cervical mri and thoracic computed tomography revealed cervical metastases with intraspinal tumor growth and an underlying extensive small cell bronchial carcinoma . in recurrent cervicobrachialgia , without pathological cervical imaging , interdisciplinary diagnostics are needed . basic diagnostic tests may assist to exclude severe non - vertebrogenic pathologies .

Introduction Report of a Case Discussion Conclusion Editorial Perspective Commentary

stroke is the second highest cause of death in the world and the main cause of disability in people in productive ages . in indonesia , the average age of stroke patients is 58.8 years old and stroke is the leading cause of high mortality rate in people above 50 years old . statistics of heart disease and stroke2013 update reveals that number of stroke patients aged between 2045 years has increased significantly in recent years . according to world health organization , stroke is rapidly developing clinical signs of focal ( or global ) disturbance of cerebral function . the symptoms last in 24 hours or longer and may lead to death , with no apparent cause other than of vascular origin . more than 87% of stroke cases are caused by ischemia due to thrombosis or cardio embolisms . ischemic stroke is atherosclerosis - associated complication [ 35 ] which is progressive in process . it requires 3 to 4 decades or more since the period of endothelial dysfunction and carotid intimamedia thickness ( cimt ) to develop until clinical manifestations appear . a study on american children aged 1014 years old who died from motorcycle accidents showed that 50% of them have early atherosclerosis evidence . similarly , bogalusa heart study revealed that 50% of its 204 subjects aged 215 years old had fatty streak in their coronary arteries . thus , detecting atherosclerotic vascular changes in childhood helps identifying high - risk groups which is very useful for the early stroke management . cimt is a surrogate marker that reveals atherosclerosis in general and constitutes a predictor of blood vessels condition in the future . the increase of cimt has been proven to be related with the raise of ischemia stroke risk [ 1113 ] . in indonesia , cimt measurement has not yet become a routine screening procedure for atherosclerosis prevention . to date , there is no normal value data of cimt for population in indonesia . , this sign becomes very useful to be intermediate phenotype in genetic studies [ 14 , 15 ] . epidemiological data show that the increase of cimt is influenced by genetic factors , with the prediction that 30% to 40% is heritable . in 2006 , genetique de l'infarctus cerebral [ genic ] study conducted a research on genetic factors related with cimt of cerebral infraction patients . the study examined polymorphism association with vascular pathologies which incorporate inflammation , hypertension , coagulation , and lipid metabolism . the findings revealed that there was a significant relationship between cimt with monocyte chemoattractant protein-1 [ mcp-1 ] and osteopontin ( opn ) polymorphisms . mcp-1 is synthesized by various cells associated with atherosclerosis , including endothelial cells , muscle cells , fibroblasts , and macrophages . in early stages of atherogenesis , mcp-1 is primary chemokine which recruits monocytes into arterial subendothelium . mcp-1 protein and rna are highly evidenced in atherosclerotic vessels , but not in normal vessels . the mcp-1 polymorphism in the forms of snp-957 , snp-2518 , and snp-2578 causes earlier occurrence of cimt faster progression [ 12 , 1619 ] in chinese , japanese , french , and slovakian people . however , the mechanism of earlier and faster cimt development could not be identified yet . some researchers suggest that ethnic and geographic factors give different effects on genetic polymorphisms taking in mcp-1 polymorphism . thus far , there has never been a study on mcp-1 polymorphism and its contribution toward cimt conducted for indonesian population . it functions in atherosclerosis , in cell - mediated immunity , and in macrophage recruitment and activation . in early stages of atherosclerosis , opn attracts inflammatory cells , promotes the releases of proteinolytic enzymes , and stimulates smooth muscle cell proliferation . the opn promoter polymorphism is studied in several areas , such as in snp-443 and snp-66 [ 12 , 21 ] . this research attempts at explaining the effect of mcp-1 and opn promoter polymorphism on cimt changes in javanese indonesian children . subjects were 54 javanese indonesian children who were classified into two groups : case and controlled . the case group consisted of 27 children aged 1021 years whose parents had ischemic stroke historical background . the controlled group consisted of 27 javanese children aged 1021 years whose parents had no ischemic stroke history . all participants were required to complete a standardized questionnaire designed to obtain information about family history . plasma concentrations of total cholesterol , high - density lipoprotein [ hdl ] , low - density lipoprotein [ ldl ] cholesterol concentrations , triglycerides , and fasting blood glucose were measured in accordance with standardized protocols in the physiology laboratory of medical faculty , university of brawijaya malang . the research had been approved to be conducted by ethical committee , medical faculty , university of brawijaya , malang , indonesia . to measure cimt , all subjects went through carotid ultrasound process in the radiology department of saiful anwar general hospital in malang , indonesia . high resolution carotid ultrasound measurements were performed using logiq s6 ultrasound ( ge healthcare ) , with 10 mhz linier transducer . the measurement was done by scanning the far wall of common carotid arteries in 1.0 cm distal . the crest at the origin of the bifurcation was used as an anatomical mark to identify segment to be visualized . in each examination , the sonographer used three different scanning angles , namely , transversal , anterolateral , and posterolateral angles , to record the greatest cimt ( figure 1 ) [ 2224 ] . genomic dna was isolated from 1 ml blood using extraction kit ( wizard genomic dna purification kit / promega ) . mcp and opn promoters were amplified by using specific primer mcp forward 5-ccgagatgttcccagcacag-3 and mcp reverse : 5-ctgctttgcttgtgcctctt-3 . amplified dna was sequenced by employing automated sequencer ( macrogen , seoul , korea ) . data characteristics , laboratory test results and ultrasound results were tested with data normality test ( kolmogorov - smirnov ) and data homogeneity test ( levence statistics ) prior to comparison test by using independent t - test . the calculation of mutation influence on carotid artery toward cimt - increasing risks was done using the odd ratio [ or ] in a 2 2 table . each sample received the same treatment in which right and left common carotid arteries were checked , and cimt and diameter of the carotid artery were measured and calculated . result of correlation analysis on the average cimt with clinical characteristic variables is presented as in table 2 . result of correlation test carried on using pearson product moment test showed that there was no correlation between average cimt with age , height , weight , body mass index , fasting glucose , total cholesterol , hdl , and ldl , as well as triglycerides . mutations took place in base number-2138 in which adenine base had substituted to thymine base . the mutations occurred in two controlled samples ( 14.3% ) and one case sample ( 5% ) ( figure 2 ) . further mutations took place in base number-2464 in which guanine base mutated into adenine base . the calculation of mutation influence on cimt - increasing risks measured through odd ratio [ or ] using a 2 2 table appears as in table 3 . based on previous research [ 11 , 24 , 26 ] , 0.5 mm was set as the normal value of cimt for odd ratio analysis . this means that children with mutation have 1.471 times higher tendency of having cimt thickening than children without mutation . all samples were analyzed for identifying t-66 g opn polymorphism promoter , but there was no mutation found in all samples . from the research , two types of point mutation in mcp-1 gene promoters were found , that is , a-2138 t and g-2464a . either base-2138 or base-2464 was located in promoter site . the promoter determines the efficiency of rna polymerase binding and , thus , it also determines the transcription efficiency . the transcription factor is attached to the binding site in the promoter region and stimulates rna polymerase to bind with the promoter site . in japan , mcp-1a-2138 t polymorphism has been reported to be identified in myocardial infarction patients above 65 years old . it was found out that the mcp-1 a-2138 t polymorphism was significantly associated with mcp-1 serum level and incidence of myocardial infarction . in this research , mcp-1a-2138 t polymorphism was found in children from parents with ischemic stroke history and children whose parents do not have ischemic stroke history as well . the polymorphism has never been reported to be found in people with no ischemic stroke history previously . children with a-2138 t polymorphism , either from the case or the controlled group , had a thicker cimt than the normal value . the two children with a-2138 t polymorphisms had a family history of myocardial infarction disease with body mass index over 25 . ( 2006 ) informed that in japan a-2138 t polymorphism was found in patients with body mass index of more than 25 kg / m with myocardial infarction . , the polymorphism was found in the case sample of a 19-year - old girl who inherited it from the mother suffering from ischemic stroke at the age of 32 . her grandfather also suffered from ischemic stroke with hypertension , while her grandmother suffered from hypertension . her total cholesterol was 208 mg / dl and ldl cholesterol was 136 mg / dl , which indicated dyslipidemia . her average cimt was 0.60 mm , or thicker than normal average cimt [ 11 , 26 ] . results of this research reveal that children with polymorphism had thicker cimt than children without mutation . risk measured with odd ratio shows that children with mcp-1 polymorphism promoter had 1.471 times possibility to have thicker cimt than children without polymorphism . these results confirm the importance of genetic examination , especially the mcp-1 polymorphism promoter analysis in children with atherosclerosis risk factors . result of this interaction is mcp-1/ccr2 messenger system increasing activity which leads to the local recruitment of monocytes at the site of injury in the arterial wall . this , in turn , will lead to increasing atherosclerosis incidence [ 29 , 30 ] . the relation of mcp-1 promoter polymorphism with increasing cimt has been reported in several studies , in which the mcp-1 g-927c and a-2578 g polymorphisms are significantly associated with the increasing cimt in patients with ischemic stroke . the mcp-1 g-362c polymorphism in black population is associated with the increasing cimt and atherosclerosis risk . the mcp-1 g-928c polymorphism is associated with the increased cimt and the atherosclerosis risk [ 29 , 30 ] . in contrast to some other studies conducted on atherosclerosis - related conditions , this research did not result in any finding on mcp-1 a-2518 g polymorphism [ 16 , 31 ] . the difference among the above genetic polymorphism findings is likely to be due to the ethnic and geographical differences . high mcp-1 transcription rate contributes to the severity of stroke [ 32 , 33 ] . the mcp-1 transcription rate is influenced by several risk factors such as hypertension , hypercholesterolemia , smoking habit , and diabetes . in summary , two mcp-1 promoter polymorphisms were found in the research , namely , a-2138 t and g-2464a . children with polymorphism demonstrate increasing cimt and have 1.471 times higher possibility to have thicker cimt . cimt of javanese indonesian children from parents with ischemic stroke is thicker than cimt of children from healthy parents .

carotid intima media thickness ( cimt ) is clearly associated with atherosclerosis . studies in ischemic stroke patients reveal that there is a significant association between cimt with monocyte chemoattractant protein-1 ( mcp-1 ) and osteopontin ( opn ) promoter polymorphism . this research aims to explain the effect of mcp-1 and opn promoter polymorphism toward cimt changes identified in javanese indonesian children . subjects were 54 children : 27 were from parents with ischemic stroke ( cases ) , and 27 were from healthy parents ( controlled ) . the cimt was examined by utilizing high resolution b - mode ultrasound . physical examination and genotyping analysis of mcp-1 promoter were conducted by employing pcr method . research results indicate that two polymorphisms were obtained , that is , a-2138 t and g-2464a , respectively . a-2138 t polymorphism was found in 5% of case children and in 14.3% of controlled children . g-2464a polymorphism was found in 5% of case children . cimt of case children was significantly different from that of controlled children ( 0.61 0.012 mm versus , 0.52 0.015 mm , p = 0.021 ) . subjects with mcp-1 promoter polymorphism have 1.471 times higher tendency to have thicker cimt than subjects with no polymorphism in mcp1 promoter . opn promoter t-66 g was also studied but it did not indicate occurrence of polymorphism in samples .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion

, early evidence characterizing spontaneous physical activity in children has shown that children take frequent ( 83 to 89 bouts per hour ) , short - duration ( mean duration of 20 to 21 s ) bouts of movement of variable but mostly low intensity , interspersed by comparatively long intervals of nonactive time [ 2 , 3 ] . this pattern of movement was termed the tempo of physical activity , that is , the frequency with which an activity event occurs and the intervals between these . it has been suggested that these pattern characteristics may be physiologically more important than composite measures of physical activity . for instance , dynamic heart rate recovery times were noted to be of similar length to the nonactive intervals between bouts of movement . more recent evidence has shown that bouts of movement were shorter and less intense in overweight compared to lean boys . evidence from spontaneous walking in adults has also shown that the duration of each walking bout resulted in obese adults walking about 2 hrs per day less than the lean , equivalent to approximately 3.5 fewer miles walked per day . in the same study , participants were overfed for a period of 8 weeks resulting in weight gain and a decrease in the distance walked per day in the obese , suggestive of a mechanistic link between obesity , weight control , and spontaneous physical activity . whereas various social , psychological , and environmental factors have been shown to explain interindividual differences in childhood physical activity levels , intra - individual variation in free - living movement enables scrutiny of the extent to available data suggests that total physical activity shows low intra - individual variation , with values between 20 and 25% in both children and adults [ 9 , 10 ] . similarly , a lack of variation in daily physical activity in response to changes in the physical environment in children has been used to support the argument that there is a physical activity phenotype . what is not known is the degree to which the tempo of spontaneous physical activity varies within a child and how the tempo of physical activity differs with weight status . the primary objective of this study , therefore , was to provide information on the pattern characteristics of physical activity in lean and obese girls and boys during the school day and over the weekend and the intra - individual variation in these . specifically , we hypothesized that ( i ) lean children would have more frequent and intense movement bouts and shorter intervals between these in comparison to the obese , independent of sex and location ( school versus home ) ; ( ii ) intra - individual variability in the pattern characteristics of physical activity would be similar in the lean and obese , independent of sex and location . to achieve this , we utilized cluster recognition algorithms to characterize the tempo of physical activity from second - by - second triaxial accelerometer output . the algorithms are based on the work of veldhuis and johnson , which were originally used to characterize the pulsatile patterns of hormone release but have since been used to identify activity pattern characteristics . chinese children aged 7 to 9 years were recruited from three government primary schools in hong kong . from a potential of 600 7- to 9-year olds , 180 volunteered to participate . height and weight were assessed in school in all 180 pupils , and those above the 90th percentile for age- and sex - specific bmi cutoffs , as well as a group of age- and sex - matched lean children , were invited to join the study . eighty - four children ( 42 lean and 42 obese , 50% male ) agreed to have physical activity assessed for three weeks . the experimental protocols were approved by the institutional review board for human ethics and written parental consent was obtained for all children . following recruitment , the children met with research staff in the early morning at school prior to the start of lessons . baseline anthropometric measurements were taken on the first day , and the children were instructed as to the correct care and usage of the accelerometer . all children were asked to wear the accelerometer for three weeks with 9 of the possible 15 weekdays and 3 of the possible 6 weekend days randomly chosen for each child and used for the analyses . body mass was determined barefoot and in light clothing with an accuracy of 0.1 kg using digital scales ( tanita tbf-401 , japan ) . height was measured with an accuracy of 0.1 cm on a fixed stadiometer ( invicta 2007246 , leicester , uk ) . waist circumference was measured at the narrowest point between the bottom of the ribcage and the top of the iliac crest and taken at the end of expiration . hip circumference was measured at the level of the greater trochanters , where the buttocks protruded most . each circumference was taken twice and recorded to the nearest 0.1 cm . a triaxial accelerometer ( rt3 , stayhealthy inc . this small , light piezoelectric device is designed to detect accelerations in three axes and combine these to provide a triaxial vector magnitude count . the rt3 has been shown to be a valid and reliable device for assessing physical activity in caucasian and chinese children [ 1517 ] . intermonitor variability , however , does exist , and in an attempt to counter this we ensured that each child used the same device throughout the measurement period ( changed only if the device was not working satisfactorily ) . additionally , before commencement and at the finish of each measurement day , every rt3 device was checked to determine the deviation in activity counts for each axis . devices were passed if all axes were kept within 5 counts , which ensured minimal intermonitor variability . the study began with 40 rt3 devices , and four failed the intermonitor variability check during the course of the study , leaving 36 devices by the end of the study . after the intermonitor variability check , devices were initialized according to manufacturer specifications and set in the vector magnitude , 1-s epoch mode . the rt3 was placed in a close - fitting bag , attached to a belt , and fastened firmly on the right hip , immediately superior to the iliac crest on the midaxilla line . the children were asked to wear the device at school from 7.30 am until they returned home , which was usually 4.30 pm . on the weekend , they were asked to wear the device from the moment they got out of bed until when they went to bed in the evening . a daily monitoring period of 9 hours was delineated by the memory capacity of the accelerometer . to try to control wear time , we considered periods of consecutive zeros exceeding 60 minutes as nonwear time . four children recorded a day with more than 60 minutes of consecutive zeros , and when asked about these periods they all reported taking the monitor off . this method involves using a series of algorithms to search for significant increases and decreases within the data series . these peaks and troughs identification and classification is based upon the threshold values which we have previously published , where nonactive behavior is defined as < 7.0 rt3vmag s ; low - intensity activity is defined as 7.0 to < 31 rt3vmag s , moderate intensity as 31 to < 68.5 rt3vmag s , and vigorous intensity activity is defined as 68.5 rt3vmag s. a bout was , therefore , defined as a continuous segment whose element value was above h , the preset nonactive threshold 7.0 rt3vmag s. the duration of each bout was denoted as td , and the duration of the nonactive interval between two adjacent bouts was denoted as ti ; the maximum element value or peak amplitude of an activity bout was denoted as vp . the variables computed by the program include the total number of bouts per day , mean duration of daily bouts in seconds ( s ) , mean duration of the rest intervals between bouts in seconds ( s ) , and the mean peak amplitude of daily bouts ( rt3vmag s ) . we also report the total minutes spent sedentary , in low , moderate , and vigorous activity for lean and obese . baseline anthropometric data were compared between the lean and obese using one - way analyses of variance . factorial analyses of variance with repeated measures were used to determine the main effects of weight status ( lean and obese ) , sex and location ( school versus home ) on activity pattern characteristics , and the variance in these activity pattern characteristics ( expressed as the coefficient of variation ) . eighty - four children ( 42 lean and 42 obese ) completed the anthropometric assessment . there was no significant difference in age , although the obese were taller and heavier and had a greater waist and hip circumference compared to the lean . physical activity pattern characteristics derived from the bout identification algorithms are presented in table 2 . all 84 children completed the school - day monitoring , whilst 40 lean and 39 obese children completed the weekend monitoring . factorial anovas revealed significant main effects for location ( school versus home ) for the number of activity bouts ( f(1,75 ) = 79.58 ; p < .001 , 0.515 ) , the mean length of activity bouts ( f(1,75 ) = 9.93 ; p < .01 , 0.117 ) , and the rest interval between bouts ( f(1,75 ) = 21.76 ; p < .001 , 0.225 ) . followup analyses indicated that the number and length of activity bouts were greater at school than at home , whilst the rest intervals were longer at home compared to school ( see table 2 ) . a significant main effect for weight status was apparent for the number of activity bouts per day ( f(1,75 ) = 38.48 ; p < .001 , 0.275 ) , with the lean engaging in more bouts of activity during school and at home in comparison to the obese ( p < .05 ) . a significant main effect for weight status was also apparent for the duration of rest intervals between bouts ( f(1,75 ) = 11.74 ; p < .01 , 0.135 ) , with the obese taking longer rest intervals than the lean both at school and at home ( p < .05 ) . there were no significant location by sex interactions for any variable , or weight status by sex , but there was a significant location by weight status interaction for the duration of the rest intervals between bouts ( f(1,75 ) = 8.12 ; p < .05 , 0.098 ) . followup analyses showed that between - bout intervals were significantly longer in the obese and lean at home compared to school , but the difference between home and school was much more pronounced in the obese ( see table 2 ; p < .05 ) . the mean minutes per day spent sedentary or in low , moderate , or vigorous physical activity are provided in table 3 . there was no main effects for weight status , sex , or location for the total minutes spent sedentary , engaged in low , moderate , or vigorous activity ( p > .05 ) . the intra - individual variation in day - to - day activity pattern characteristics are shown in table 4 . the factorial anova revealed significant main effects for location for the variance in the number of activity bouts per day ( f(1.71 ) = 55.9 ; p < .001 , 0.441 ) , the duration of bouts ( f(1.71 ) = 25.3 ; p < .001 , 0.263 ) , interval between bouts of activity ( f(1.71 ) = 82.9 ; p < .001 , 0.539 ) , and bout intensity ( f(1.71 ) = 13.5 ; p < .001 , 0.159 ) . followup analyses showed that variance increased at home compared to the school regardless of sex ( p < .05 ) . the only interaction was for bout peak intensity , where a significant location by sex interaction was present ( f(1,71 ) = 7.70 ; p < .01 , 0.098 ) . followup analyses showed that intra - individual variance in the peak intensity of activity bouts was constant in boys whether at school or at home ( p > .05 ) . in girls , however , variance in the peak intensity of activity bouts increased significantly at home in comparison to school ( p < .05 ) . a significant main effect for sex in intra - individual variance existed for the mean duration of an activity bout ( f(1,71 ) = 3.97 ; p < .05 , 0.053 ) , where variance was greater in the girls ( p < .05 ) . similarly , significant main effect for sex in intra - individual variance in the peak intensity of an activity bout was apparent ( f(1,71 ) = 14.22 ; p < .001 , 0.167 ) , with variance greater in the girls when at home ( p < .05 ) . significant main effects for weight status were apparent in the intra - individual variance in the number of bouts per day ( f(1.71 ) = 5.00 ; p < .05 , 0.066 ) and the rest interval between these activity bouts ( f(1,71 ) = 6.1 ; p < .05 , 0.079 ) . in both cases , variance was higher in the obese than the lean ( p < .05 ) . free - living movement in children has been shown to comprise combinations of movements of varying intensities that together form bouts of activity . these bouts have previously been found to be transient and largely composed of low - intensity walking . the activity pattern characteristics that we report for the school day are remarkably similar to the previous observation studies [ 2 , 3 ] in terms of the number of bouts per hour , the duration of the rest interval between bouts , and the intensity of bouts . the average physical activity bout length in our study of between 10 and 17 s corresponds well with bailey et al . 's finding that 95% of children 's physical activity bouts last less than 15 s . previous work using heart rate monitoring has already confirmed that children rarely sustain 10- or 20-minute bouts of moderate - intensity activity [ 1921 ] . the data from our study provide further evidence that children 's free - living movement patterns are comprised of many , low - intensity , very short - duration bouts . in contrast to the work of stone et al . , we found that the length and intensity of activity bouts were similar in the lean and obese children , and these showed low intra - individual variation , regardless of location . we found that the intra - individual variance in activity pattern characteristics during school days were generally modest , varying from 9 to 15% in the lean and 8 and 26% in the obese . these are not dissimilar from previous findings of intra - individual variability in composite markers of physical activity , which are reported to be between 20 and 25% in children . less frequent movement bouts , coupled with longer rest periods , distinguished the obese from the lean . it is noteworthy that the length of time spent resting between bouts of movement shows the greatest intra - individual variation . at the weekend , the frequency of activity bouts declined in both the lean and obese , girls and boys alike . similarly , the rest interval between movement bouts increased over the weekend in both the lean and obese , but this increase was substantially greater in the obese . this is the first study , to our knowledge , that has reported the variance in the duration of sedentary intervals between bouts of activity in lean and obese children , and our findings suggest that the amount of time spent resting between movement bouts appears to be a key characteristic differentiating lean from obese children . obese adults have been found to sit for about 2 hours more than the lean , and our findings suggest that a similar , albeit less pronounced , difference may exist in lean and obese children . the cross - sectional design of the present study limits the ability to fully understand the relationship between rest intervals and weight status in the young , and experimental or longitudinal work is recommended . the likelihood of increasing aspects of physical activity that are uncommon in daily movement patterns , such as longer duration bouts of more intense physical activity , is probably poor . similarly , aspects of physical activity that vary little within an individual are less likely to respond to intervention . attention may be better focused upon decreasing the periods of rest between bouts of movement and increasing the number of short - duration bouts of movement per day . the primary limitation of this study is the relatively small sample which may increase the chance of type ii errors . the data are also restricted to 79-year - old hong kong chinese children , and this may limit the generalisability of the findings . it is notable that our primary findings are similar to those of bailey et al . ; however , direct comparisons of our findings to other data sets are challenging because data collection approaches are not uniform . the advent of more sophisticated and affordable monitoring tools , such as triaxial accelerometers with high sampling rates and large memory capacity , will certainly improve this situation and should encourage greater harmonization of accelerometry methodology . we choose not to assess the physical activity patterns at home during the weekday because we felt hong kong children were less likely to be active at this time given the homework that is completed during the week ; however , these data may provide valuable information about the interplay between movement bouts and inactivity during periods of likely high sedentary behavior and should be considered in future studies . to conclude , this study has shown that , similar to previous findings , chinese children engage in many predominantly low - intensity bouts of short - duration activity . small lifestyle changes are quite likely to have considerable impact on activity pattern characteristics and identifying the biological and environmental ( both physical and sociocultural ) antecedents of this pattern of movement is an important next step . the obese child engages in fewer activity bouts and takes longer rest intervals between bouts , both during the school day and at home . as the prevalence of obesity continues to rise , efforts should also be directed toward understanding the consequences of extended rest periods between bouts of movement and fewer movement bouts a day , as well as finding ways to reverse these .

physical activity and sedentary behavior are central components of lifetime weight control ; however , our understanding of dimensions of these behaviors in childhood is limited . this study investigated free - living activity pattern characteristics and the individual variability of these characteristics in 84 lean and obese chinese children ( 79 y ) during the school day and over the weekend . activity pattern characteristics were established from triaxial accelerometry ( stayhealthy rt3 ) . results indicated that children 's free - living activity is characterized by many short - duration , low - intensity bouts of movement . obese children take longer rest intervals between bouts and engage in fewer activity bouts both at school and at home . intraindividual variability in activity patterns was low during school days but high for the rest intervals between bouts and number of activity bouts per day at the weekend . finding ways to reduce the rest time between bouts of movement and increase the number of movement bouts a child experiences each day is an important next step .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

multidisciplinary therapeutic techniques are employed in the rehabilitation of patients with advanced disease of the stomatognathic and craniofacial structures [ 1 , 2 ] . although surgical intervention can halt the disease process , prosthodontic rehabilitation is often needed to restore mastication , speech , and esthetics . ultimately , the goals are to optimize function and cosmetic results to minimize morbidity and allow reestablishment of self - esteem [ 35 ] . current materials proposed for external maxillofacial applications experience some serious problems , particularly low tear strength [ 6 , 7 ] . thus , it is necessary to have a material with satisfactory tear strength , tensile properties , and appropriate hardness . the ideal material should be similar to the missing facial tissue to optimally match a patient 's articulate features of mastication , speech resonance , and facial gesture . consequently , there is a need for improved materials with superior physical and mechanical properties that are comparable to those of human tissues and skin [ 8 , 9 ] . although numerous advances in maxillofacial prosthetic materials have been made in the past several years , the need for improvement continues . research is ongoing to develop new polymeric materials with superior mechanical properties , such as high tear strength and low hardness . a great deal of research has been devoted to developing a new class of polymeric materials by incorporating nanofillers into the organic polymer matrix , creating a nanocomposite that combines at the same time the strength of the filler and flexibility of the organic matrix . silicone elastomer has achieved a wide clinical acceptance , due to its many advantageous properties that consecrate it as the most appropriate material for facial prostheses such as biocompatibility , low chemical reactivity , ease of manipulation , and optical transparency . furthermore , it can be pigmented to simulate skin tone ; therefore , it enhances the aesthetic outcome of the prosthetic device [ 11 , 12 ] . . the required physical and mechanical characteristics of the fabricated silicone elastomer depend on the type and the concentration of the filler used , which has to be tailored to meet the requirements of strong yet elastic material with mechanical properties that meet the clinical requirements [ 1315 ] . silicon dioxide nanoparticles ( sio2 ) have increasingly been exploited for numerous biomedical and biotechnological applications . sio2 nanoparticles are characterized by their small size , large interface area , active function , and strong interfacial interaction with the organic polymer . therefore , they can improve the physical , mechanical , and optical properties of the organic polymer and provide resistance to environmental stress - caused cracking and aging . it is imperative to maintain the nanofillers content at a proper level because of their higher surface energy and chemical reactivity ; otherwise , the nanoparticles may agglomerate . when the silicone elastomer is under external forces , the agglomerated particles act as stress concentrating centers in the silicone elastomer matrix , thereby decreasing the mechanical strength of the silicone elastomer . hence , it becomes crucial to incorporate well - dispersed nanofillers into the polymer to obtain beneficial mechanical and physical properties [ 21 , 22 ] . many efforts have been devoted to prevent the agglomeration and aggregation of nanoparticles , such as applying silane coupling agent between nanosilica particles and the polymer [ 21 , 23 ] . coating the inorganic filler with a silane coupling agent can link the inorganic filler and the organic matrix chemically . . these silanol groups may chemically react with dimethyldichlorosilane to render the silica hydrophobic ( figure 1 ) . taking this as departure point , the aim of this study was to evaluate clinically the important mechanical properties of a silicone elastomer that is used for extraoral maxillofacial prosthesis after adding different concentrations of surface treated sio2 nanoparticles in order to help in the design of an improved maxillofacial prosthetic material with optimum mechanical properties . 147 specimens were prepared in strict compliance with the manufacturer 's instructions and divided into seven groups , each of 21 specimens , such that one group ( gi ) was prepared without adding sio2 nanoparticles to the silicone elastomer ( a-2186 , platinum catalyzed , vinyl terminated rtv silicone , obtained from factor ii inc . , lakeside , az , usa ) . this will act as a control group , and six experimental groups were prepared by combining the silicone elastomer with various weight percentage amounts of the sio2 nanoparticles ( hydrophobic sio2 coated with silane coupling agent ; average particle size : 15 nm and specific surface area : 650 m / g , obtained from mknano , mississauga , canada ) , such as ( gii ) 0.5% , ( giii ) 1.0% , ( giv ) 1.5% , ( gv ) 2.0% , ( gvi ) 2.5% , and ( gvii ) 3.0% by weight as suggested by previous relevant study . to prepare specimens for the experimental groups , sio2 nanopowder was weighed by using the analytical balance ( citizen , cx 301 , usa ) then added to the base of the preweighed silicone elastomer gradually . the modified base was then mixed for 20 minutes using the mechanical mixer ( eurostar , power control - visc , ika - werke , germany ) at mixing speed of 150 rpm . then , the silicone cross - linking agent was added according to the manufacturer 's recommended ratio of 10 : 1 by weight ( base : cross - linking agent ) and mixed with the modified base . the mixtures were placed in the vacuum oven ( barnstead lab - line , 3618 - 6ce , usa ) for 40 minutes at pressure ( 948.1 mbar ) , as recommended by the manufacturer to get rid of the incorporated air bubbles . the vacuum was applied to a container four times the volume of the material to prevent overflow of the bubbles . the mixture was allowed to reach its maximum capacity and fall to the bottom of the container . eventually , the mixture was loaded into machined split copper molds lined with petroleum jelly , in specific dimensions required by each standardized test method . the material was allowed to polymerize at room temperature ( 23 1c ) for 24 hours , after which the molds were carefully separated , specimens removed , and the flash trimmed away with a sharp scalpel . the control group was prepared in the same way as described before for the experimental groups except for adding the nanopowder . all specimens were evaluated for tensile strength , percentage elongation , and tear strength using the universal testing machine ( instron 3382 , usa ) . shore a hardness was measured by the shore type a digital durometer ( std 226 , satra , uk ) . all tests were performed at room temperature ( 23 1c ) and relative humidity ( 50% 5% ) . the selection criteria of specimens for testing were absence of tears at borders , absence of air bubbles , and absence of surface irregularities . the thickness and width of each specimen were measured at three different locations using a vernier caliper with digital readout ( absolute digimatic caliper , mitutoyo , usa ) and the average value was entered as input data which was used in calculating the specimen cross - sectional area via the computer software ( bluehill 2 ) . the specimen was placed under tension in the grips of the universal testing machine and carefully adjusted symmetrically to distribute the tension equally over the cross - section . the lower member of the universal testing machine remained fixed , while the upper member moved at a constant rate of 500 mm / min cross - head speed . the maximum amount of force immediately prior to breaking ( n ) and elongation measurements were recorded electronically using the computer software and the resulting stress - strain curves were constructed . tensile strength ( mpa ) was calculated using the following equation : ( 1)ts = fa , where ts is the tensile strength ( mpa ) ; f is the force magnitude prior to breaking ( n ) ; a is the cross - sectional area of unstrained specimen ( mm ) . the original length was measured before testing using the digital caliper by placing benchmarks on the dumbbell - shaped specimen 25 mm apart , equidistant from the center and perpendicular to its long axis . the additional distance between the benchmarks upon sample failure , was recorded by the computer software . the percentage elongation was calculated from the equation : ( 2)e=100lblolo , where : e is the percentage elongation ; lb is the length at specimen break ; lo is the original length . the thickness of the specimen was measured in three different sites across the width of the specimen near its center , and the average value was recorded and entered as input data which was used in the calculations . the specimen was then placed in the grips of the universal testing machine and stretched at constant cross - head speed of 500 mm / min , until the specimen was ruptured ; the force required to break the specimen ( n ) was recorded by the computer software . from these measurements , tear strength ( n / mm ) was calculated using the following equation : ( 3)t = fd , where t is the tear strength ( n / mm ) ; f is the maximum force ( n ) ; d is the thickness of the specimen ( mm ) . each specimen was of at least 6 mm thickness and its lateral dimensions were 12 mm from any edge . the digital durometer was placed in a vertical position and the presser foot was applied parallel to the surface of the specimens as rapidly as possible without shock . three sites were measured per each specimen , and the mean value was recorded as the hardness of each specimen . scanning electron microscopic ( sem ) examination was performed using analytical scanning electron microscope ( jsm 636ola ; joel , tokyo , japan ) to monitor the dispersion of sio2 nanoparticles within the silicone elastomer matrix . thin cross - sections were cut from torn tensile strength specimens and mounted rigidly on specimen holders . since the silicone elastomer is nonconductive , accordingly the specimens were coated with an ultrathin coating of gold , by sputter coating using the ion sputtering device ( jfc-1100e ; jeol , tokyo , japan ) . the gold coating is important to prevent charge build up on the specimen , but , at the same time , the thickness of the gold layer should be small enough to prevent masking of the surface layer and impairment of resolution . specimens were observed at 10,000 magnification and at an accelerating voltage of 30 kv . data from quantitative studies of the experimental groups were collected and compared to the control group using one - way analysis of variance ( anova ) with concentration as main variable for tensile strength , percentage elongation , tear strength , and shore a hardness . when significant differences were observed , the scheffe test was used as post hoc test to identify differences among the groups at a significance level of = 0.05 for all tests . all statistical tests were performed using a statistical software spss ( statistical package for social sciences , version 20.0 , spss inc . , chicago , il , usa ) . the mean values of tensile strength in ( mpa ) of all the studied groups containing an increasing concentration of sio2 nanofiller are shown graphically in ( figure 2(a ) ) . there was a significant increase in the tensile strength ( p < 0.001 ) in the formulations prepared at 3% sio2 nanofiller concentration ( 3.62 0.69 mpa ) when compared with that of the control group ( 2.78 0.36 mpa ) . there was no significant difference ( p > 0.05 ) in the tensile strength of formulations prepared at 0% and 0.5% concentration . the mean values of percentage elongation of all the studied groups containing an increasing concentration of sio2 nanofiller are shown graphically in ( figure 2(b ) ) . the greatest value was in the 1.5% formulation ( 754.8 4.06 ) , and there was a small but significant decrease in the percentage elongation as the concentration of sio2 nanofiller increased from 2 to 3% . the mean values of tear strength in ( n / mm ) of all the studied groups containing an increasing concentration of sio2 nanofiller are shown graphically in ( figure 2(c ) ) . the tear strength of the formulations prepared at 3% sio2 nanofiller concentration ( 45.90 1.94 n / mm ) was significantly ( p < 0.001 ) greater than that of the control group 0% ( 19.32 1.90 n / mm ) . the mean values of shore a hardness of all the studied groups containing an increasing concentration of sio2 nanofiller are shown graphically in ( figure 2(d ) ) . there was no significant difference ( p > 0.05 ) in the hardness of formulations prepared at 0% and 0.5% . there was also no significant difference when the sio2 nanofiller increased from 0.5 to 1.5% , and there was no statistically significant difference between the 2.5% and the 3% sio2 nanofiller concentration . there was a small but significant increase in the hardness as the concentration of the sio2 nanofiller was increased from 0% ( 28.09 0.32 ) to 3% ( 29.97 0.38 ) . sem images demonstrate the homogenous dispersion of the spherical and whitish sio2 nanoparticles within the silicone elastomer specimens as shown in ( figure 3 ) . sem examination indicated that all the nano - sio2 concentrations were distributed uniformly throughout the silicone specimens . the aim of this study was to develop an improved maxillofacial prosthetic material with optimum mechanical properties . the main focus was to enhance the tensile strength , tear strength , and the percentage elongation . to accomplish this , formulations were developed by incorporating different concentrations of surface treated sio2 nanofiller , followed by evaluation of the mechanical properties , in view of the fact that testing of the mechanical properties is an important step towards the modification of the current material or acceptance of a new material . the addition of silica filler is a vital factor in the physical and mechanical properties of silicone elastomers , because the unfilled cross - linked polydimethylsiloxane ( pdms ) has very low mechanical properties , since a very high cross - link density produces an inelastic brittle material [ 11 , 27 ] . that made using silica fillers essential for enhancing the mechanical properties . the addition of surface treated silica fillers can augment the tensile strength of the cross - linked polymer by up to 40 times . the reason behind the increased strength is the strong physical and chemical bonds between the vulcanized polymer and the silica filler . thus , polymer / filler interactions are maximized [ 26 , 28 , 29 ] . the hydrophobic surface treatment of the filler is also essential to prevent water absorption into the cured pdms elastomer , since finished facial prostheses is subjected to sebum , sebaceous , and perspirations from the underlying living human skin which may lead to deterioration of the prosthesis . surface treated silica fillers are also better at dispersion into the silicone elastomer and have a reduced base viscosity compared to nonsurface treated silica fillers . under deformation , these surface treated fillers help to increase the strength of the elastomer by allowing the polymer chains to uncoil and slide past neighboring filler particles increasing the crystallization between neighboring pdms chains . as depicted in the statistical analysis , results of this study revealed significant improvement in the tensile strength and tear strength with the use of 3% concentration of surface treated sio2 nanofiller , given that the values of the experimental group ( gvii ) were found to be significantly higher than those of the control group ( gi ) . the percentage elongation was found to increase with increasing the nanofiller concentration till reaching its maximum value at 1.5% ( giv ) then started to decrease with increasing the concentration of the nanofiller but still the 3% has a higher value of elongation than the control group . this should not pose any serious problem , because clinically the value of the elongation obtained is accepted as satisfactory for the use of maxillofacial prosthesis . the improvement in the mechanical properties may be attributed to the enhanced polymer adsorption by the nanosilica 's large surface area . moreover , the higher surface energy and chemical reactivity of the nanoparticles allowed them to interact with the silicone elastomer matrix and form a 3-dimensional network by chemical bonding in the presence of the surface treatment [ 31 , 32 ] . by this means , silicone elastomer with high tear strength , tensile strength , and elongation percentage is produced . a high percentage elongation and high tear strength produce the most desirable combination [ 27 , 33 ] . the surface treatment furthermore improved the incorporation and dispersion of the nano - sio2 in the silicone elastomer matrix ; this was supported by the sem images which revealed uniform dispersion of the nano - sio2 in specimens of all the six concentrations tested . results revealed as well a small but significant increase in the hardness as the concentration of the nanofiller increased from 0.5 to 3% but still within the clinically acceptable range ( 2535 shore a ) . as described in the literature [ 34 , 35 ] , higher filler loading may result in further increase in the hardness . the study performed by han et al . recommended the incorporation of nanooxides of ti , zn , or ce ( nonsurface treated ) at concentrations of 2 to 2.5% by weight into a-2186 silicone elastomer ; these concentrations improved the mechanical properties . their results indicated that when the concentration was increased to 3% , the tear strength , tensile strength , and elongation decreased . contrasting with this , the results of the present study indicated that with the use of 3% surface treated nano - sio2 , there was more improvement in the tear strength , tensile strength , and elongation . sem performed by han et al . revealed that , at a concentration of 2% , particles of all the three nanooxides were well distributed in the silicone elastomer matrix . however , when the concentration was increased to 3% , all the three nanooxides had agglomerated which resulted in a decrease in the mechanical properties of silicone elastomer . on the other hand , results from the present study demonstrated that , with the use of 3% surface treated nano - sio2 , sem showed no particles agglomeration and thus improvement in the mechanical properties ; this may be attributed to the surface treatment of the nanoparticles . according to the results of the present study , the surface treated nano - sio2 filler evenly dispersed within the silicone matrix and consequently improved the mechanical properties of the silicone elastomer . therefore , it is possible to affirm that , apart from the increase in filler / polymer interaction , the surface treatment could improve the dispersion of nanosilica within the silicone matrix by reduction in silica agglomeration , which was supported by the sem images . it is proposed that they interact with each other by van der waals force and hydrogen bonds . the addition of the surface treated sio2 nanofiller in 3% concentration did not affect the viscosity of silicone base . a further increase in filler concentration may lead to filler overloading , producing the highest base viscosity . in other words , the increase in loading of silica might result in a difficulty in the mixing process . the addition of that nanofiller did not also influence the translucency of silicone elastomer considerably , contrasting with the fact that the addition of the same concentration ( 3% ) of nano - tio2 turned the specimen white . accordingly , the present study recommends the use of 3% surface treated nano - sio2 , since the concentration is appropriate for reinforcing the mechanical properties of maxillofacial silicone elastomer ( a-2186 ) without much affecting the hardness , the translucency , or the viscosity . future work should be planned to investigate the effect of surface treated nano - sio2 on the ability to provide skin - colored prostheses and its color stability . the effect of artificial accelerated weathering and the influence of chemical disinfection on the mechanical and physical properties of silicone elastomer modified with surface treated nano - sio2 require evaluation as well . under the conditions of this study and with the specific materials used , the following conclusions can be derived.the incorporation of surface treated sio2 nanoparticles for the reinforcement of maxillofacial silicone elastomer a-2186 provided it with more favorable mechanical properties , especially in terms of tear strength.surface treatment of the sio2 nanoparticles improved its distribution within the silicone matrix and prevented its agglomeration . the incorporation of surface treated sio2 nanoparticles for the reinforcement of maxillofacial silicone elastomer a-2186 provided it with more favorable mechanical properties , especially in terms of tear strength . surface treatment of the sio2 nanoparticles improved its distribution within the silicone matrix and prevented its agglomeration .

current materials used for maxillofacial prostheses are far from ideal and there is a need for novel improved materials which mimic as close as possible the natural behavior of facial soft tissues . this study aimed to evaluate the effect of adding different concentrations of surface treated silicon dioxide nanoparticles ( sio2 ) on clinically important mechanical properties of a maxillofacial silicone elastomer . 147 specimens of the silicone elastomer were prepared and divided into seven groups ( n = 21 ) . one control group was prepared without nanoparticles and six study groups with different concentrations of nanoparticles , from 0.5% to 3% by weight . specimens were tested for tear strength ( astm d624 ) , tensile strength ( astm d412 ) , percent elongation , and shore a hardness . sem was used to assess the dispersion of nano - sio2 within the elastomer matrix . data were analyzed by one - way anova and scheffe test ( = 0.05 ) . results revealed significant improvement in all mechanical properties tested , as the concentration of the nanoparticles increased . this was supported by the results of the sem . hence , it can be concluded that the incorporation of surface treated sio2 nanoparticles at concentration of 3% enhanced the overall mechanical properties of a-2186 silicone elastomer .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

using a scanning tunneling and atomic force microscope combined with in - vacuum atomic hydrogen cleaning we demonstrate stable scanning tunneling spectroscopy ( sts ) with nanoscale resolution on electrically active nanowire devices in the common lateral configuration . we use this method to map out the surface density of states on both the gasb and inas segments of gasb inas esaki diodes as well as the transition region between the two segments . generally the surface shows small bandgaps centered around the fermi level , which is attributed to a thin multielement surface layer , except in the diode transition region where we observe a sudden broadening of the bandgap . by applying a bias to the nanowire we find that the sts spectra shift according to the local nanoscale potential drop inside the wire . importantly , this shows that we have a nanoscale probe with which we can infer both surface electronic structure and the local potential inside the nanowire and we can connect this information directly to the performance of the imaged device .

Supplementary Material

novel biological agents , including anti - epidermal growth factor receptor ( egfr ) , anti - vegf targeting therapies , and small - molecule multikinase inhibitors , have recently changed the standard of care of metastatic colorectal cancer ( mcrc ) patients . while the use of positive predictive biomarkers for biological therapy would be desirable in order to identify specific subsets of responsive patients , to date the only validated negative predictive biomarker for mcrc is the mutational status of all members of the ras gene family , which identifies mcrc patients not eligible to monoclonal antibody ( moab ) anti - egfr therapies . emphasizing the limitations of negative predictive biomarkers , unfortunately only a subgroup of wt ras mcrc patients respond to anti - egfr drugs , being the molecular mechanism / s underlying resistance to anti - egfr treatment not fully understood . activating mutations in other members of the ras - braf - mek and pi3k - akt pathways , both acting downstream of the egfr signaling cascade , are being investigated as further potential predictive biomarkers . apparently , no specific target treatment seems to be available for wt ras and anti - egfr resistant mcrc patients . indeed , the inhibition of the braf oncoprotein by the small - molecule drug vemurafenib , which is highly effective in melanoma , showed a very limited response in the mcrc setting . coherently , only a prognostic significance has been attributed to braf mutations in crc , so far . interestingly however , preclinical studies have indicated that egfr reactivation contributes to insensitivity of braf - mutant crc to vemurafenib . thus , the association of braf and egfr inhibitors might effectively target braf mutant colon cancers . we report here the first case of a patient with braf - mutant , amplified egfr ( double positive ) and kras / pik3ca wt , not - amplified her2 ( triple negative ) mcrc whose disease had progressed on standard lines of treatment , but successfully responded to a new combination therapy consisting of vemurafenib ( zelboraf ) and panitumumab ( vectibix ) . a 55-y - old man was admitted to our oncology department in july 2007 for a poorly - differentiated adenocarcinoma of the transverse colon . preoperative carcinoembryonic antigen ( cea ) and ca19.9 serum levels were 1.2 ng / ml and 63 u / ml , respectively . the patient was staged as iiib and adjuvant standard treatment with folfox4 ( 6 mo ) was performed . eleven months later , the patient developed peritoneal carcinomatosis and was treated with folfiri - bevacizumab ( 9 cycles ) , discontinued for pulmonary embolism , followed by cytoreductive surgery plus hyperthermic intraperitoneal chemotherapy . after a 12 mo disease - free interval , an increment of ca19.9 and a ct scan revealed a peritoneal progression . at this time the patient was characterized for wild - type kras mutational status and high egfr expression by immunohistochemistry and underwent several lines of treatment , such as irinotecan cetuximab , a second peritoneal cytoreductive surgery , capecitabine every disease progression was exclusively peritoneal and marked by a significant increase in ca19.9 and cea . an additional line of treatment with regorafenib demonstrated a good control of the disease for 9 mo in an expanded access program . subsequently , the patient showed a significant rise in serum markers ( ca19.9 and cea ) and a multivisceral disease progression ( peritoneum , liver , and lung ) accompanied by important clinical troubles including diffuse abdominal pain , weight loss , and episodes of sub - ileus . in order to find additional treatment opportunities dictated by tumor biology , the molecular profile of the tumor was evaluated on a liver metastasis biopsy performed at the time of the latest progression and on previously collected tumor material ( primary lesion and peritoneal implants ) . all samples concordantly revealed the following status : non - amplified her2 , kras wt , pik3ca wt , amplified egfr , and mutant braf . the lack of other established therapeutic alternatives together with the above mentioned molecular characterization and preclinical data , met the ethical constraints of a tailored therapy , and prompted us to propose a vemurafenib - panitumumab combined treatment . the protocol was approved by the institutional review board ( policlinico umberto i , sapienza university of rome ) on june 2013 , and the patient provided written informed consent . patient restaging before vemurafenib panitumumab treatment showed hepatic , pulmonary and peritoneal disease measurable at spiral ct scan ( fig . cea and ca19.9 serum levels were 558 ng / ml and 4800 u / ml , respectively ( fig . 2 ) . q - pcr analysis of plasma dna indicated the presence of circulating tumor dna ( ctdna ) via the identification of the braf mutation ( fig . 3 ) . ct scans of the patient before and after panitumumab - vemurafenib treatment for metastatic crc . tumor masses ( arrow ) can be seen in the liver of the patient before initiation of panitumumab - vemurafenib treatment ( a ) . the masses ( arrow ) became hypodense , homogenous and significantly reduced in size on ct obtained 3 and 6 mo after treatment ( b and c ) , indicating good response to combination treatment . ( a ) electropherogram showing the heterozygous braf mutation in dna isolated from patient 's crc tissue . ( b ) allele - specific q - pcr detection of the braf mutation in plasma free dna reveals the presence of circulating tumor dna before treatment ( t0 ) but not 12 wk after treatment initiation ( t1 ) . data are reported as averages of the threshold cycles ( ct ) obtained in two different q - pcr for the braf amplicon and the reference gene amplicon . the patient was treated with panitumumab 6 mg / kg iv every 14 d and vemurafenib 960 mg orally twice daily . soon after 4 wk , a significant clinical benefit with complete regression of the clinical symptoms occurred . twelve weeks later , the programmed disease restaging with ct scan showed a strong reduction of all metastatic lesions ( pr according to recist1.1 criteria ) ( fig . consistent with the ct scan , tumor markers also showed a significant decrease : cea , 51 ng / ml , ca19.9 , 221 u / ml ( fig . 2 ) . , the patient is still asymptomatic 28 wk after starting the treatment when the ct scan showed a further reduction of all metastatic lesions ( pr according to recist1.1 criteria ) ( fig . tumor markers are stably low ( fig . 2 ) and circulating braf dna absent . treatment regimen is being well tolerated and the patient only presents minor skin - related toxicity ( maximum grade 2 ) . in particular no cutaneous toxicity ( such as squamous cell carcinomas and keratoacanthomas ) typically associated with vemurafenib are observed . our work represents the first clinical evidence that the combination of an inhibitor of braf ( vemurafenib ) and an egfr antagonist ( panitumumab ) is well tolerated and results in a highly effective control of the disease , in a mcrc patient extensively pretreated with several standard and off - label lines of treatment . braf belongs to a gene family also including craf ( or raf1 ) and araf , encoding serine threonine kinases that heterodimerize each other and constitute , together with downstream mek and erk proteins , a powerful mitogen - activated protein kinase ( mapk ) pathway . raf kinases are activated by several receptor tyrosine kinases ( rtks ) such as egfr . notably , while mutations of araf and craf are rare in human cancer , mutated braf frequently occurs in papillary thyroid carcinoma , melanoma , hairy cell leukemia , hepatocellular carcinoma , and 11% of crc . to this regard , braf is an ideal druggable target in braf mutated cancers ( i.e. , crc ) since it is active as a monomer in an upstream egfr / ras - independent way , thus providing an explanation to the observed resistance to inhibitors of egfr / ras pathway ( i.e. , anti - egfr antibodies ) ( fig . 4 , left panel ) . the resistance to egfr - targeted therapies eventually developing along progression of lung , colorectal , pancreatic , or head and neck cancers , is challenging because of the various mechanisms underlying anti - egfr insensitivity ( summarized in fig . 4 , right panel ) . understanding these various resistance pathways would provide an opportunity to develop new inhibitors to prevent or overcome therapeutic resistance . in this context , it can be predicted that braf - containing crcs would be inhibitable by braf antagonists ( fig . 4 , right panel ) . likewise a short - term sensitivity to anti - braf monotherapy has been reported in melanoma . model of braf - dependent cell growth and mechanisms of resistance to braf inhibition and restoring of drug sensitivity through combination therapies . braf mutation confers constitutive pathway activation ( red arrows ) independent of upstream rtks ( egfr , igf-1r)/ras signaling ( left panel ) , providing an explanation to the observed insensitivity to anti - egfr antibody treatment ( panitumumab , cetuximab ) in braf mutant crc . such a process is blunted by braf ( braf ) inhibitors ( blue symbols and drugs ) ( right panel ) . additional mechanisms of egfr primary or secondary resistance described in lung , head - neck cancer and crc ( i.e. , egfr mutation ; oncogenic shift or activation of a bypass pathway such as kras , braf , pik3ca , secondary egfr mutation or a parallel / alternative pathway [ pten , igf1 ] is also indicated ) . right panel also illustrates how bypass and resistance to braf - inhibitors may occur through the rescue of the braf - mediated attenuation of erk negative feedback induced by braf inhibitors and subsequent reactivation of ligand - dependent signaling from egfr or igf-1r or via wild type ras / raf or pik3ca / akt or pik3ca or egfr or mek1 or further braf gain - of - function mutations ( pik3ca or asterisks ) in several tumor types . drugs under development in preclinical models or clinical trials to overcome braf inhibitor resistance are labeled in blue . several mechanisms have been reported to underlie the resistance to braf inhibitors , as summarized in figure 4 . while inhibitors of mutant braf monomers suppress the erk signaling , they relieve erk - dependent negative feedback and reactivate ligand - dependent egfr / ras signaling upon wild - type raf dimers in melanoma cells . likewise , recent preclinical evidence of the feedback reactivation of egfr in crc by vemurafenib - mediated blockade of braf has been reported ( fig . in turn , this triggers sustained ras- and craf - mediated mapk signaling and cell proliferation . blocking egfr activity with a monoclonal antibody to egfr has been shown to restore the sensitivity to vemurafenib in preclinical models , providing a strong rationale for the combination of vemurafenib and anti - egfr antibodies in braf - mutated crc . resistance to braf inhibitors has also been reported through further activation of ras / mapk pathway ( e.g. , mutations of nras or mek1 , loss of nf1 , alternatively spliced variants of braf , amplification of craf or braf ) as well as activation of additional parallel signaling pathways such as the igf1r / pi3k / akt ( e.g. , resulting from pten loss ) or pdgfr pathways ( fig . 4 ) . although this drug resistance mechanism and its overcoming by combined treatments is potentially operating in several other tumors harboring braf mutations , no clinical efficacy has been reported either . in addition to the clinical efficacy of the combined therapy with vemurafenib and panitumumab described here , al - marrawi et al . recently reported that treatment of a single patient with a combination of sorafenib and cetuximab led to a mixed radiographic response with some areas showing dramatic improvement and other areas showing stable disease over a 7-mo period . these findings are consistent with the modest disease control we also observed in our case treated with panitunumab and sorafenib . however , sorafenib , a multi - kinase inhibitor with limited activity on braf , can not be considered an optimal drug for the specific purpose . indeed , weak inhibition of braf by sorafenib would transactivate raf dimers maintaining a still consistent downstream mek activity . our case report raises several considerations that need to be exploited in subsequent studies performed on large series . the first one is related to the observation of braf mutation together with egfr amplification , suggesting that vemurafenib might be an effective drug in braf mutant mcrc patients if used in association with egfr inhibitors this would blunt escaping signaling pathways ( i.e. , pi3k - akt ) downstream of the amplified egfr responsible for the maintenance of the disease progression , suggesting that the dramatic response observed in our case might have been further influenced by egfr amplification . thus , a careful selection of the patients eligible to this treatment might be performed on the basis of two distinct positive predictive markers . however , vemurafenib toxicity spectrum , ( e.g. , squamous cell carcinomas / keratoacanthomas , maculopapular rashes , hyperkeratosis ) is significantly distinct from that of panitumumab ( acneiform rash , paronychia , xerosis ) . in the case described , no cutaneous toxic effects typically associated with vemurafenib were observed . in particular , squamoproliferative lesions appearing at early times ( median time 8 wk ) during vemurafenib treatment therefore the vemurafenib - panitumumab combination appears to limit the typical toxicity of single agents . besides overcoming braf inhibitor resistance through co - targeting activated parallel or upstream rtk mitogenic or survival pathways ( i.e. , egfr , met , igf1r ) , additional therapeutic strategies include the combined use of mek or erk or akt inhibitors as well as the intermittent administration of braf inhibitors described in preclinical models . however , to date no clinical evidence substantiates this hypothesis . here we report the first clinical evidence that the combination of an anti - egfr ( panitumumab ) and an inhibitor of braf ( vemurafenib ) is well tolerated and results in a highly effective disease control in an extensively pretreated mcrc patient . the possibility to select patients eligible to this therapeutic approach on the basis of two distinct positive predictive markers ( egfr gene amplification / overexpression and mutant braf ) and the apparent reciprocal limitation of the side effects of the two drugs are interesting hypotheses to be tested in prospective studies . furthermore , unraveling the various mechanisms of resistance to braf inhibition is expected to pinpoint novel combination therapies . to this regard , recent advances in crc patients classification based on gene expression profiles may help identifying new strategies for personalized treatment .

as the knowledge on cancer genetic alterations progresses , it fosters the need for more personalized therapeutic intervention in modern cancer management . recently , mutations in kras , braf , and pik3ca genes have emerged as important mechanisms of resistance to egfr - targeted therapy in metastatic colorectal cancer ( mcrc ) . here we report the first case of a mcrc patient whose disease had progressed on standard lines of treatment and for which we devised a personalized therapeutic approach consisting of vemurafenib ( zelboraftm ) and panitumumab ( vectibixtm ) , based on the following molecular profile : brafv600e - mutant , amplified egfr ( double positive ) and wt kras , wt pik3ca , not - amplified her2 ( triple negative ) . this new combination therapy was well tolerated and resulted in a strong control of the disease . in particular , the vemurafenib - panitumumab combination appears to limit the typical toxicity of single agents , since no cutaneous toxic effects typically associated with vemurafenib were observed . here we report the first clinical evidence that the combination of an anti - egfr ( panitumumab ) and an inhibitor of brafv600e ( vemurafenib ) is well tolerated and results in a strong disease control in an extensively pretreated mcrc patient .

Introduction Case Report Discussion Conclusions and Future Directions

primary hyperparathyroidism ( php ) is an endocrine system disease , mostly seen in older population . the cause of php is solitary parathyroid adenoma ( pa ) in 80 - 85% of the cases . number of imaging techniques have been used for preoperative localization of parathyroid adenoma including technetium-99 m labelled methoxyisobutyl isonitrile ( tc-99m - mibi ) , ultrasound ( us ) , magnetic resonance imaging ( mri ) and computed tomography ( ct ) . although some authors use us alone as the primary modality to diagnose parathyroid adenoma and hyperplasia , it is dependent on the physician 's experience and has a suboptimal detection rate in multinodular goiter , also in the mediastinum , tracheoesophageal groove and retroesophageal regions . tc-99m - mibi is a widely used and accurate scintigraphy technique for the preoperative localization of pa in php . single - photon emission computed tomography ( spect ) imaging provides information about localizing parathyroid lesions , differentiating thyroid from parathyroid lesions , detecting and localizing ectopic parathyroid tissues . the aim of this study was to compare the value of tc-99 m pertechnetate , dual - phase mibi and mibi - spect images in the determination and localization of parathyroid lesions and was to compare both planar and spect images with lesion based . ninety - tree patients who referred to parathyroid scintigraphy with the preliminary diagnosis of hyperparathyroidism were included in this study . after positive parathyroid lesion imaging and clinical findings , 38 of these patients were operated . the mean pth level was 258 161.5 pg / ml , calcium level was 11.26 0.85 mg / dl and phosphor level was 2.56 0.71 meq / l . for all patients , two patients had history of surgery due to parathyroid adenoma and imaging was performed for recurrent hyperparathyroidism . single head gamma camera was used for planar imaging and dual head gamma camera ( mediso nucline spirit , hungary ) was used for spect imaging . twenty minutes after the intravenous injection of 185 mbq tc-99 m pertechnetate , planar imaging and directly after , spect imaging of the neck with dual head gamma camera was performed for all patients . then 20 min after the injection of 740 - 925 mbq tc-99 m mibi , 10 minutes of thyroid static images and anterior mediastinum were performed . after planar imaging , mibi spect images were performed with the same parameters as early and late imaging . tc-99 m pertechnetate and mibi , early and late mibi spect images of all patients were interpreted by two independent physicians who were blinded to clinical and other data . the relatively increased activity in mibi images but not be seen in pertechnetate images were considered as pathological parathyroid tissue . tc-99 m pertechnetate planar and spect images were used to determine the boundaries of thyroid gland and discriminate the parathyroid gland from thyroid tissue . for quantitative assessment , region of interest ( roi ) was drawn on each tc-99 m mibi early and late spect images according to color scale where the highest level of activity and on the background in every patient . the average number of the count taken from late spect images were divided to the average number of the count from early spect images . single head gamma camera was used for planar imaging and dual head gamma camera ( mediso nucline spirit , hungary ) was used for spect imaging . twenty minutes after the intravenous injection of 185 mbq tc-99 m pertechnetate , planar imaging and directly after , spect imaging of the neck with dual head gamma camera was performed for all patients . then 20 min after the injection of 740 - 925 mbq tc-99 m mibi , 10 minutes of thyroid static images and anterior mediastinum were performed . after planar imaging , mibi spect images were performed with the same parameters as early and late imaging . tc-99 m pertechnetate and mibi , early and late mibi spect images of all patients were interpreted by two independent physicians who were blinded to clinical and other data . the relatively increased activity in mibi images but not be seen in pertechnetate images were considered as pathological parathyroid tissue . tc-99 m pertechnetate planar and spect images were used to determine the boundaries of thyroid gland and discriminate the parathyroid gland from thyroid tissue . for quantitative assessment , region of interest ( roi ) was drawn on each tc-99 m mibi early and late spect images according to color scale where the highest level of activity and on the background in every patient . the average number of the count taken from late spect images were divided to the average number of the count from early spect images . out of 38 patients , 30 of them were diagnosed to have pa , 2 patients had ph on planar images , whereas 6 patients were evaluated as normal with planar images figure 1 . according to tc-99 m mibi spect images , 34 of 38 patients were diagnosed to have pa . according to both spect and planar images , 4 of these 38 patients histopathologic evaluation of the lesions which were surgically removed from the same areas confirmed diagnosis of ph . characteristics and results of 38 patients tc-99 m po4 , tc-99 m mibi early and late planar images of 38-year - old female patient six patients were diagnosed as normal via tc-99 m mibi and pertechnetate planar images and four of these patients reported to have positive status for parathyroid adenoma in early mibi spect . thyroid usg revealed mng in four of these six patient and the other two patients were diagnosed to have solitary ng . out of these thirty - eight patients , 16 were diagnosed with mng and 12 of them with solitary ng . according to planar mibi images , out of these 38 patients , 30 had positive in terms of pa , 2 had ph , whereas 6 had negative . early mibi spect imaging findings matched with pathology results in 34 patients as pa . in one patient , even though scintigraphy showed to have pa at lower right lobe , pa was found to be on the middle right lobe in the surgical operation . according to pathology results , 34 of the 38 patients were diagnosed to have pa and 4 patients were diagnosed to have ph . for the other pa cases , tc-99 m pertechnetate scintigraphy showed two thyroid nodules , one of them was large and hyperactive whereas the other one was small and normoactive . thyroidectomy was applied to the patient and the pathology result showed multicentric thyroid papillary carcinoma . according to these results , the sensitivity of tc-99 m mibi planar and pinhole imaging is 88.2% , tc-99 m mibi spect sensitivity is 100% in detecting parathyroid adenoma figure 2 . tc-99 m mibi spect images of another patient showed increased uptake in the right inferior adjacent area of the thyroid gland dual - phase tc-99 m mibi parathyroid scintigraphy is a valuable method based on the differential washout rate between the thyroid tissue and pa . pas have very high metabolic rate despite of their small size and they show intense tc-99 m mibi uptake . oxyphilic cells which are enriched by mitochondria and the increased vascularity are the main causes of this uptake . bilateral exploration of the parathyroid glands with excision of the abnormally enlarged glands and biopsy of at least one normal size gland has been the traditional standard approach for the surgical management of php . experienced endocrine surgeons successfully cure the php by bilateral neck dissection in 95% of patients without needing any preoperative imaging . benard f et al . hypothesized that mitochondria - rich oxyphil cells constitute a logical target for the prolonged retention of tc - mibi usually observed in abnormal parathyroids because tissues with a low mitochondrial content would not be expected to significantly accumulate the tracer . in such cases , rapid washout of tc-99 m mibi from abnormal tissue with low number of mitochondria pathologic parathyroid tissue activity can be observed at early mibi images probably because of increased flow or cellularity in the tumor . spect images are useful for localization of preoperative parathyroid adenomas , which have negative planar imaging and especially for the cases localized in mediastinum and may increase the sensitivity . as already stated in our study , early spect have extra contribution to the patients that have been diagnosed with parathyroid adenoma via planar imaging . to improve the lesion detectability , some studies using a pinhole collimator for imaging reported better localization of adenomas than images acquired by a parallel - hole collimator or delayed spect . it is also reported that a combination of early and delayed pinhole imaging and spect maximize parathyroid lesion detection . pinhole spect was shown to be a complementary to planar scintigraphy , especially in secondary hyperparathyroidism . using us combination with scintigraphy is common method for lesion localization and to exclude thyroid nodules in the thyroid glands . using a hybrid spect / ct scan can further enhance localization by providing better resolution of surrounding structures and this system allows for the combined anatomic information from ct and the physiologic three - dimensional information from spect . however spect / ct is high cost modality and many departments can - not afford this system . the sensitivity of tc-99 m mibi for localization of preoperative parathyroid adenoma was found different in various studies . found that the sensitivity of tc-99 m mibi late spect imaging in abnormal parathyroid tissue detection in the patients who had not underwent surgery as 87% , in the patients who had been operated is 92% ; and in patients with hyperplasic glands is 45% . perez - monte et al . compared the early and late tc-99 m mibi spect images of 37 patients who had parathyroid adenoma and had been operated . pathologic reports confirmed parathyroid adenoma in 34 patients ( 92% ) and pa in 3 patients ( 8% ) . 74% of parathyroid adenoma were detected by late spect images and 32% of adenoma were correctly localized in this study . early tc-99 m mibi spect imaging was found better than late tc-99 m mibi imaging for localization of parathyroid adenomas according to these researchers . the reason of this rapid washout rate of mibi from adenomas in late images increases the false negativity . another study comparing the early and late spect images of 52 patients found that 41 parathyroid adenoma ( 79% ) were detected by planar imaging and 50 were ( 96% ) by early spect imaging . this study showed that spect imaging is superior than planar imaging in pa localization especially in patients with ectopic adenomas and multinodular goiter disease . a study with 82 php patients , published by pinhas et al . reported that the sensitivity of planar imaging for parathyroid adenoma was 78% and early spect was 96% for parathyroid adenoma localization detection . in our study , early mibi spect correctly found pa localization in 34 out of 38 patients , mibi planar images found pa localization in 30 out of 38 patients . also the washout rate of the mibi was calculated to be 0.686 in quantitative evaluation which had been performed with early and late images of pa adenomas . on the other hand , there are some authors reporting that spect does not bring additional information and does not improve the detection rate for adenomas . chen et al . found that the sensitivity of tc-99 mibi imaging for pa detection was 95% , the sensitivity of late planar imaging is 98% and the late spect imaging of tc-99 mibi was 98% in 55 hyperparathyroid patients . they conclude that the latter images subtraction images and spect images does not necessarily bring much information other than early images with tc-99 m pertechnetate . performed planar images with tc-99m - mibi , spect , us , and subtraction scintigraphy with tl-201 and tc99m - pertechnetate on patients with thyroid disease ( 50% of the cases ) and previous cervical surgery ( 21% of the cases ) and compared the images with the histopathology of the surgical specimen . they found that spect provide additional information about adenoma localization in 39% of patients with thyroid disease or previous cervical surgery . however in recent years , spect / ct technology gives more information about this kind of mng patients . in many centers such as our department , us is being used for excluding thyroid nodules . the reason for that is that localization of parathyroid adenomas are more commonly behind thyroid gland . in mng patients , it is hard to differentiate thyroid nodules from parathyroid nodules if thyroid nodule is mibi avid . even though oblique imaging is advised when parathyroid adenomas and thyroid nodules are crossed with each other , it is not useful for parathyroid adenomas . we found that early spect is superior to the planar imaging in mng patients in concordance with the other studies . it is also suggested that in persistent hyperparathyroidism , the accuracy of tc-99 m mibi spect for identification of residual hyperactive glands is considerably lower before reoperation than before initial surgery . in addition , hipervitaminosis d are more likely to cause false positive results on a sestamibi scan . this study showed that early mibi spect imaging is superior than both planar tc-99 m pertechnetate and planar mibi scintigraphy in pa localization , especially in mng patients . since this protocol takes long time , early mibi spect should be performed only if the mibi planar imaging is negative or suspicious . our study demonstrated that quantitative statistics of early and late spect imaging is necessary . as a result , our present study suggest an important additive contribution of the spect study not only in localization of the parathyroid adenoma but also in the differentiation between a suspect thyroid nodule and a parathyroid gland localization when spect / ct is not achieved or us is not performed .

background : the purpose of this study was to evaluate the value of tc-99 m pertechnetate planar , dual - phase mibi and mibi - spect images in the determination and localization of parathyroid lesions.materials and methods : in this study , 38 patients who underwent operation for primary hyperparathyroidism were included . tc-99 m pertechnetate planar - pinhole imaging of the neck and then mibi planar and spect images in supine position was performed . late spect images were acquired 120 minutes after the injection . early and late mibi images were quantitatively evaluated.results:of the 38 patients , 30 of them had adenoma , 2 patients had hyperplasia and 6 of them were normal on planar images . thirty - four of 38 patients were positive on spect images . spect images of the patients with positive results were matched with pathology results.conclusion:as a result , tc-99 m pertechnetate planar - pinhole , tc-99 m mibi planar and spect images are useful for localization of parathyroid lesions especially in multinodular thyroid gland . however , us or ct images are necessary for more accurate localization and tc-99 m pertechnetate images are useful for interpreting and comparing with the early and late mibi images .

I M Imaging Image interpretation Quantitative assessment R D

the database from 2 randomized controlled phase iii trials comparing vancomycin with fidaxomicin for the treatment of cdi was used to assess the predictive value of fever , leukocyte count , and serum creatinine level [ 9 , 10 ] . patients were recruited in the united states , canada , and europe ( clinicaltrials.gov registry number nct00314951 , april 2006july 2008 , united states and canada ; and clinicaltrials.gov registry number nct00468728 , april 2007november 2009 , united states , belgium , canada , france , germany , italy , spain , sweden , and united kingdom ) . patients with cdi , defined on the basis of diarrhea ( > 3 unformed bowel movements [ ubms ] per day ) and a positive stool toxin test for c. difficile , were randomly assigned to receive 125 mg of vancomycin 4 times daily or 200 mg of fidaxomicin twice daily for 10 days . the numbers and times of ubms were recorded during treatment and for 2 days after an end - of - therapy visit . for patients with rectal collection devices , volume was converted to number of ubms by dividing the volume by 60 ml and rounding up to the nearest whole number . at the end - of - therapy visit , clinical failure was defined as the persistence of diarrhea , the need for additional therapy for cdi , or both on the basis of the opinion of the investigator . recurrence of cdi ( determined by use of the same criteria as for enrollment [ ie , > 3 ubms per 24 hours and a positive stool toxin test result ] ) was assessed during the mean follow - up duration ( sd ) of 28 2 days after completion of therapy . at enrollment , temperature , leukocyte count , and serum creatinine level were collected . to assess whether the timing of laboratory measurements could influence their prognostic value , we used the database of a prospective cohort study performed at leeds teaching hospital in 2007 . in this database , 104 consecutive adult inpatients with cdi ( defined on the basis of the presence of unformed stool and a positive c. difficile toxin test result ) were included . on days 3 to + 3 relative to day 0 ( the day the diarrheal sample was collected ) , data from a minimum of 2 leukocyte counts and creatinine levels on different days were required for patients to be included in the analyses . in both analyses , we defined fever as a core body temperature > 38.5c and leukocytosis as a leukocyte count > 15 10 leukocytes / l . because the pre - cdi serum creatinine level was not known for each patient , we substituted the 50% creatinine level increase with a fixed value of the creatinine level > 133 mol / l ( > 1.5 mg / dl ) . the intention - to - treat population that received at least 1 dose of study medication was used for the analysis . distributions of the continuous variables of temperature , leukocyte count , and creatinine level were compared for patients with and patients without clinical treatment failure and recurrence . risk ratios ( rrs ) and 95% confidence intervals ( cis ) were calculated for the associations of fever , leukocytosis , and renal failure with the outcome parameters . kaplan - meier survival curves were constructed to investigate the association of fever , leukocytosis , and renal failure with the time to resolution of diarrhea ( expressed in hours from the first dose of fidaxomicin or vancomycin ) . the log - rank test was used to test the difference between the survival curves . receiver operating characteristic curves were constructed to assess the validity of the cutoffs used to define categorical variables . variability of leukocyte counts and serum creatinine levels were compared within patients and expressed in absolute differences . all analyses were carried out in spss for windows software , version 17.0 ( spss , chicago , il ) . the database from 2 randomized controlled phase iii trials comparing vancomycin with fidaxomicin for the treatment of cdi was used to assess the predictive value of fever , leukocyte count , and serum creatinine level [ 9 , 10 ] . patients were recruited in the united states , canada , and europe ( clinicaltrials.gov registry number nct00314951 , april 2006july 2008 , united states and canada ; and clinicaltrials.gov registry number nct00468728 , april 2007november 2009 , united states , belgium , canada , france , germany , italy , spain , sweden , and united kingdom ) . patients with cdi , defined on the basis of diarrhea ( > 3 unformed bowel movements [ ubms ] per day ) and a positive stool toxin test for c. difficile , were randomly assigned to receive 125 mg of vancomycin 4 times daily or 200 mg of fidaxomicin twice daily for 10 days . the numbers and times of ubms were recorded during treatment and for 2 days after an end - of - therapy visit . for patients with rectal collection devices , volume was converted to number of ubms by dividing the volume by 60 ml and rounding up to the nearest whole number . at the end - of - therapy visit , clinical failure was defined as the persistence of diarrhea , the need for additional therapy for cdi , or both on the basis of the opinion of the investigator . recurrence of cdi ( determined by use of the same criteria as for enrollment [ ie , > 3 ubms per 24 hours and a positive stool toxin test result ] ) was assessed during the mean follow - up duration ( sd ) of 28 2 days after completion of therapy . at enrollment , temperature , leukocyte count , and serum creatinine level were collected . to assess whether the timing of laboratory measurements could influence their prognostic value , we used the database of a prospective cohort study performed at leeds teaching hospital in 2007 . in this database , 104 consecutive adult inpatients with cdi ( defined on the basis of the presence of unformed stool and a positive c. difficile toxin test result ) were included . on days 3 to + 3 relative to day 0 ( the day the diarrheal sample was collected ) , data from a minimum of 2 leukocyte counts and creatinine levels on different days were required for patients to be included in the analyses . in both analyses , we defined fever as a core body temperature > 38.5c and leukocytosis as a leukocyte count > 15 10 leukocytes / l . because the pre - cdi serum creatinine level was not known for each patient , we substituted the 50% creatinine level increase with a fixed value of the creatinine level > 133 mol / l ( > 1.5 mg / dl ) . the intention - to - treat population that received at least 1 dose of study medication was used for the analysis . distributions of the continuous variables of temperature , leukocyte count , and creatinine level were compared for patients with and patients without clinical treatment failure and recurrence . risk ratios ( rrs ) and 95% confidence intervals ( cis ) were calculated for the associations of fever , leukocytosis , and renal failure with the outcome parameters . kaplan - meier survival curves were constructed to investigate the association of fever , leukocytosis , and renal failure with the time to resolution of diarrhea ( expressed in hours from the first dose of fidaxomicin or vancomycin ) . the log - rank test was used to test the difference between the survival curves . receiver operating characteristic curves were constructed to assess the validity of the cutoffs used to define categorical variables . variability of leukocyte counts and serum creatinine levels were compared within patients and expressed in absolute differences . all analyses were carried out in spss for windows software , version 17.0 ( spss , chicago , il ) . patients treated with vancomycin ( n = 566 ) or fidaxomicin ( n = 539 ) had similar median values for temperature , leukocyte count , and serum creatinine level and were evenly distributed across the groups with respect to dichotomized continuous variables ( data not shown ) . fever was rare ; only 1.2% of patients ( 13 of 1102 ) had a temperature > 38.5c . overall , 143 patients ( 13% ) experienced clinical treatment failure at the end of treatment . of the 962 patients who were cured after treatment , 194 ( 20% ) experienced recurrence the median leukocyte count and creatinine level were significantly higher in patients with clinical treatment failure ; temperature distributions in patients with and those without treatment failure were almost identical . in addition , dichotomous categories of fever , leukocytosis , and renal failure all showed significant correlation with treatment failure ( table 1 ) . the median creatinine level was significantly higher in patients with recurrence , and this parameter was the only significant predictor of recurrence ( table 2 ) . different cutoffs for the continuous variables of temperature , leukocyte count , and creatinine level , assessed by receiver operating characteristics , did not lead to higher relative risks and therefore better performance in the prediction of clinical treatment failure or recurrent cdi . table 1.determinants of clinical treatment failure among patients with clostridium difficile infectionvariablemedian valueiqrpcontinuous , outcometemperature ( c ) failure36.836.437.2.180 cure36.736.437.1leukocyte count ( 10 leukocytes / l ) failure10.56.817.4.002 cure8.96.512.1creatinine level ( mol / l ) failure80 62150.005 cure71 6297categorical , cutofffailurerr95% cifever , temperature > 38.5c4/132.451.075.61 38.5c137/1089leukocytosis , leukocyte level > 15 10 leukocytes / l38/1532.291.633.21 15 10 leukocytes / l90/829renal failure , creatinine level 133 mol / l41/1602.521.823.50 < 133 mol / l91/896abbreviations : ci , confidence interval ; iqr , interquartile range ; rr , risk ratio . creatinine conversion : 1 mol / l is equal to 0.0113 mg / dl . therefore , 133 mol / l is equal to 1.50 mg / dl . table 2.determinants of clostridium difficile infection recurrencevariablemedian valueiqrpcontinuous , outcometemperature ( c ) no recurrence36.736.437.1.827 recurrence36.736.437.0leukocyte count ( 10 leukocytes / l ) no recurrence8.86.512.1.276 recurrence9.16.612.8creatinine level ( mol / l ) no recurrence716297.008 recurrence8062115categorical , cutoffrecurrencerr95% cifever , temperature > 38.5c1/90.55.093.51 38.5c192/952leukocytosis , leukocyte level > 15 10 leukocytes / l22/1151.00.671.50 15 10 leukocytes / l141/739renal failure , creatinine level 133 mol / l32/1191.451.052.02 < 133 mol / l149/805abbreviations : ci , confidence interval ; iqr , interquartile range ; rr , risk ratio . creatinine conversion : 1 mol / l is equal to 0.0113 mg / dl . therefore , 133 mol / l is equal to 1.50 mg / dl . determinants of clinical treatment failure among patients with clostridium difficile infection abbreviations : ci , confidence interval ; iqr , interquartile range ; rr , risk ratio . creatinine conversion : 1 mol / l is equal to 0.0113 mg / dl . therefore , 133 mol / l is equal to 1.50 mg / dl . . determinants of clostridium difficile infection recurrence abbreviations : ci , confidence interval ; iqr , interquartile range ; rr , risk ratio . creatinine conversion : 1 mol / l is equal to 0.0113 mg / dl . therefore , 133 mol / l is equal to 1.50 mg / dl . the probability of resolution of diarrhea within 10 days of treatment was slightly lower in patients with renal failure , compared with patients without renal failure ( hr , 0.83 ; 95% ci , .681.02 ; figure 1 ) . neither fever ( hr , 1.08 ; 95% ci , .611.91 ) nor leukocytosis ( hr , 1.02 ; 95% ci , .841.24 ) was associated with a lower probability of resolution of diarrhea . although creatinine level distributions were similar between patients treated with fidaxomicin and those treated with vancomycin , we repeated the analysis of renal failure as a predictor of resolution of diarrhea stratified according to treatment group and found similar results ( vancomycin : hr , 0.80 [ 95% ci , .611.05 ] ; fidaxomicin : hr , 0.88 [ 95% ci , .661.19 ] ) . because recurrences occurred less often in patients treated with fidaxomicin figure 1.kaplan-meier analysis of time to resolution of diarrhea for patients with and without renal failure . kaplan - meier analysis of time to resolution of diarrhea for patients with and without renal failure . clinical treatment failure rates were similar in the fidaxomicin and vancomycin treatment groups regardless of clinical status , using the 3 severity factors . recurrence was significantly more frequent following vancomycin treatment , compared with fidaxomicin treatment . in patients without renal failure , 93 of 402 patients ( 23.1% ) cured by vancomycin therapy had a recurrence , whereas only 56 of 403 ( 13.9% ) experienced a recurrence after successful fidaxomicin treatment ( p < .001 ) . in patients with renal failure at baseline , fidaxomicin therapy was associated with a 60% reduction in the frequency of recurrences ( 8 of 54 [ 14.8% ] ) relative to vancomycin ( 24 of 65 [ 36.9% ] ; p = .007 ) . likewise , in patients categorized as having leukocytosis or severe cdi , the incidence of recurrence was more than double for patients cured with vancomycin , compared with those treated successfully with fidaxomicin ( p < .01 for each comparison ) . because leukocytosis and renal failure at the time of diagnosis were shown to be the strongest predictors , we investigated the stability of these parameters during a 6-day interval around diagnosis . in the population from the database of leeds teaching hospital , the highest mean leukocyte count ( 13.4 10 leukocytes / l ) was found on the day of cdi diagnosis . within the interval from 3 days before to 3 days after the diagnosis of cdi , the mean difference between the highest and lowest leukocyte counts was 6.4 10 > 10 10 leukocytes / l , and 33 ( 38.4% ) patients had a minimum to maximum leukocyte count range that included the cutoff of 15 10 leukocytes / l ; therefore , a difference in timing of a single blood sample around diagnosis could have led to a different severity classification . the mean serum creatinine concentration was 147 mol / l on the day of diagnosis . nineteen of 93 patients ( 20.4% ) had a minimum to maximum range in creatinine levels that included the cutoff of 133 mol / l , which could have led to a different classification in the case of different timing . only renal failure showed a trend toward longer duration of diarrhea during treatment and was correlated significantly with recurrence after successful treatment . fever was found to be too infrequent in our study to be a useful predictor , but its associated relative risk was significant . in previous studies , leukocytosis and renal failure were also associated with complications and recurrence of cdi [ 3 , 1113 ] . however , because of the variable nature of these values around the time of cdi diagnosis , a strict definition is needed before incorporating these parameters in a prediction rule . both fever and leukocytosis are thought to reflect more severe inflammation of the bowel wall . however , fever was too rare in our patient population to be of use as a predictor . renal failure may reflect loss of effective circulating volume due either to dehydration because of diarrhea or to shock in the context of a systemic inflammatory response . unfortunately , the predictive value of these parameters may decrease because of underlying illnesses and comorbid conditions . renal failure was present in 14% of clinical patients and was the only significant predictor of recurrence , and it was the only parameter associated , albeit nonsignificantly , with a longer time to resolution of diarrhea . thus , creatinine level may be a good predictor , also because of its relatively greater stability around the time of cdi diagnosis in comparison to leukocytosis . strengths of this study are the large number of patients with cdi in the database with a well - described definition of diarrhea and a consistent measure of ubms . one limitation is that other potential predictors of severe cdi , such as age , serum albumin level , or use of concomitant antibiotics , were not included in this analysis . therefore , we were not able to develop a complete risk score . another limitation is the absence of a baseline creatinine level for each patient , precluding us from distinguishing between chronic and acute renal failure . the results of our study suggest that both leukocytosis and renal failure predict clinical treatment failure , whereas only renal failure is a predictor of recurrence after therapy . however , these predictors are highly dependent on the timing of their determination , hampering their use in clinical practice . we need better and more closely defined predictors to construct a reliable prediction score for complicated and recurrent cdi that is applicable in clinical practice .

nonsevere clostridium difficile infection ( cdi ) and severe cdi , which carries a higher risk than nonsevere cdi for treatment failure and cdi recurrence , are difficult to distinguish at the time of diagnosis . to investigate the prognostic value of 3 markers of severe cdi suggested by recent guidelines ( fever , leukocytosis , and renal failure ) , we used the database of 2 randomized controlled trials , which contained information for 1105 patients with cdi . leukocytosis ( risk ratio [ rr ] , 2.29 ; 95% confidence interval [ ci ] , 1.633.21 ) and renal failure ( rr , 2.52 ; 95% ci , 1.823.50 ) were associated with treatment failure . fever , although associated with treatment failure ( rr , 2.45 ; 95% ci , 1.075.61 ) , was rare . renal failure was the only significant predictor of recurrence ( rr , 1.45 ; 95% ci , 1.052.02 ) . different timing of measurements of leukocyte count and serum creatinine level around the cdi diagnosis led to a different severity classification in many cases . in conclusion , both leukocytosis and renal failure are useful predictors , although timing of measurement is important .

METHODS Databases Analyses RESULTS DISCUSSION

supramolecular self - assembly represents a key technology for the spontaneous construction of nanoarchitectures and for the fabrication of materials with enhanced physical and chemical properties [ 14 ] . in addition , a significant asset of supramolecular self - assemblies rests on their reversible formation , thanks to the kinetic lability of their noncovalent interactions . this dynamic nature can be exploited for the development of self - healing and smart materials towards the tuning of their functional properties upon various external factors [ 58 ] . one particular intriguing objective in the field is to reach a high level of control over the shape and size of the supramolecular architectures , in order to produce well - defined functional nanostructures by rational design . in this direction , many investigations have been pursued toward the construction of self - assembled objects from numerous low - molecular weight scaffolds [ 1018 ] , for instance , by exploiting multiple directional hydrogen - bonding interactions . in particular , nucleobases have been used as supramolecular synthons as a result of their efficiency to code for noncovalent interaction motifs . among nucleobases , guanine represents the most versatile one , because of its different h - bond donor ( nh and nh ) and acceptor sites ( o , n , and n ) which display self - complementary patterns of interactions . interestingly , and depending on the environmental conditions , guanosine derivatives can form various types of structures . most of the supramolecular architectures reported so far from guanosine derivatives require the presence of a cation ( alkali metal , earth - alkali , or lanthanide ions ) [ 2031 ] which stabilizes , via dipole - ion interactions , the macrocyclic g - quartet that can , in turn , stack in columnar g - quadruplex arrangements ( figure 1(a ) ) . in addition , guanosine can polymerize via hydrogen bonding to give a variety of supramolecular networks including linear ribbons such as a- and b - types ( figures 1(b ) and 1(c ) ) . this complex supramolecular behaviour confers to the guanine - guanine interactions their upper interest among all the homonucleobases studied [ 20 , 32 ] . they have been subjected to intense investigations in various areas ranging from structural biology and medicinal chemistry guanine - rich sequences are abundant in telomeric ends of chromosomes and promoter regions of dna and are capable of forming g - quartet based structures [ 3335]to material science and nanotechnology [ 36 , 37 ] . in 1998 , it was reported that , even in the absence of metal cations , lipophilic guanosine derivative 1 ( figure 2 ) is able to self - assemble in ribbon - like structures leading to the formation of a lyotropic liquid crystalline phase . in the following years , the structures of the ribbons obtained from different guanosine derivatives have been characterized in solution ( by nmr and esi - ms ) , in the solid state ( by x - ray diffraction and nmr ) , and at graphite - solution interface ( by stm ) [ 4046 ] . two different types of ribbons , with different patterns of hydrogen bonds , have been resolved in solution . the first species ( ribbon - a , figure 1(b ) ) is characterised by nho and nhn hydrogen bonds , and the second one ( ribbon - b , figure 1(c ) ) by nho and nhn hydrogen bonds . in the case of 1 , ribbon - a is detected in anhydrous chloroform solutions ( c > 10 m ) soon after dissolving the polycrystalline powder but subsequently undergoes a structural transition towards a thermodynamically more stable ribbon - b . interestingly , in the case of the closely related chemical structure of 2 , only ribbon - b has been detected in chloroform solution ( even soon after dissolution ) . for derivative 1 , the critical concentration for the formation of the gel - like phase was measured 0.596 m in chloroform , and for derivative 2 0.125 m in the same solvent . in both ribbon - like polymers a and b however , the two ribbons possess a different symmetry and , as a consequence , while ribbon - a does present a permanent dipole moment , it is not the case for ribbon - b . this feature has been exploited in organic electronic prototype devices [ 4749 ] , and more generally , linear ribbons from guanosine derivatives are considered as potential scaffolds for functional materials [ 5054 ] . although the formations of the linear ribbons and their h - bonding patterns have been demonstrated in solution [ 40 , 41 ] ( as well as in the crystal state and on graphite surface ) , only very few investigations have been performed so far toward the determination of the length of these supramolecular polymers , as well as on their possible hierarchical self - assemblies in higher scale structures . nmr spectroscopic indications of the supramolecular polymerisation only come from the shifting and broadening of the resonance signals upon concentration of the solution . in addition , the observation of negative enhancements ( or positive cross - peaks ) in noe ( or noesy ) experiments indicates that guanosine derivatives behave as large molecules with mw > 1000 ( c > 1 ) [ 3840 ] . in this paper we report on the investigation of the supramolecular polymerisation of 1 and 2 in chloroform by using light scattering technique as well as transmission electronic microscopy ( tem ) . this line of studies reveals the formation of supramolecular polymers with high molecular weights that produce hierarchical structuring of very soft self - assemblies displaying either fibrillar ( 1 ) or lamellar ( 2 ) organizations . the multiangle laser light scattering is the common technique [ 55 , 56 ] for determining the shape of the polymers through the mean square radius of gyration rgz and their structure through the particle scattering factor pz(q ) , the molecular weight mw , and the second virial coefficient a2 . theory took great interests long time ago giving statistical sense to these parameters [ 5561 ] . when a2 is near zero and solutions considered as athermal according to general polymer theories one may deal with the apparent molecular weight mw , app and use the so - called statistical dimensions determined at discrete concentrations [ 59 , 60 ] instead of the one extrapolated at c 0 . looking at the variation of mw , app versus concentration or temperature allows equilibrium constant k0 to be evaluated which open fields to dynamics of the scaffolds and to their assembly ability . the form factor pz(q ) may be also useful to determine structural changes of the objects during the diffusion process of elementary unimers [ 59 , 60 ] . the form factor may derive from calculations especially when the structure of the coil is well known . theoretical aspects are shortly reviewed at the end of the paper ( section 4 ) . there is shown typical processes for data calculations leading to zimm plot which yields to the molecular dimensions and solution thermodynamics [ 5558 ] . all the data obtained for guanosines 1 and 2 are summarized in table 1 . the zimm plot obtained from guanosine 1 at 25c in chcl3 for different concentrations , the apparent molecular weight was determined to be relatively stable below the gelation threshold . the linear regression reveals a modest molecular weight of 3620 ( corresponding to an aggregation number of 6 to 7 units ) but with a radius of gyration of 57 nm which indicates the presence of large objects . the discrepancy between these two parameters is explained by the high polydispersity of the supramolecular polymer with an average of small ribbons together with a smaller population of large objects . indeed , the gyration radius being determined from mw , the importance of the large structures becomes predominant . the molecular weight of the polymers depends largely on the concentration range and a study between 2.88 10 and 2.88 10 gml reveals a power law of 0.75 ( see figure 4 ) which is higher than the value ( 0.6 ) for regular swollen coils and which is the signature for rigid objects of about 200 nm in length [ 57 , 62 ] . finally , the association constant k0 between two monomers in the supramolecular b - type ribbon ( see figure 1(c ) ) was determined by plotting the apparent molecular weight as a function of the concentration ( figure 5 ) and shows a relatively small value k0 = 400 l mol [ 59 , 60 ] . when the polymer is homogeneous , flory krigbaum derived the more realistic model for a2 introducing the enthalpy and entropy parameter , respectively . by comparison with the flory - huggins theory these parameters are seen to be related by = ( 1/2 ) where is the flory huggins interaction parameters . for ideal behaviour = and a2 = 0 so the theta point is reached , with theta temperature being given by = t(/ ) where t is the absolute temperature . the theory leads to some basics which are the equivalent size of the solvent and monomers , respectively , and a number of association for statistical calculations . these statistical basics hold for guanosine 2 but comments on guanosine 1 may lie in the expression of idealized lattice model for second virial coefficient . it is obvious that one of the optimal conditions for association is an athermal solution which is what we have found for guanosine 2 ( table 1 ) . in the case of guanosine 2 , the light scattering reveals a quite different behavior of the self - assembly process . the zimm plot in figure 6 shows a smaller polidispersity than what is measured for guanosine 1 together with higher molecular weights ( mw = 5.93 10 , and the degree of polymerisation dp = 1250 ) , but with a close radius of gyration ( 43 nm ) which might correlate with the formation of more compact objects this expectation is confirmed by plotting the mw in the range of concentration 3.4 10 and 6.82 10 g ml . the experimentally determined power law of 0.3 ( see figure 7 ) is the signature for very compact objects such as spheres [ 57 , 62 ] . finally , the determination of the equilibrium constant [ 59 , 60 ] for guanosine 2 shows a strong association process ( k0 = 6.75 10 l mol ) and a complex behaviour of a sigmoid type ( figure 8) . this nonlinear effect indicates a cooperative process and the hierarchical association between the linear supramolecular ribbons with their organization in higher - ordered self - assembled structures that in turn stabilize the primary association . structural observation of self - assembled system can easily be achieved by tem approaches , as widely used and described in the literature [ 65 , 66 ] . guanosine derivative 1 in bromoform ( 1.44 10 g ml ) forms small short fibers ( figure 9(a ) ) . bromoform was used for technical reasons regarding the evaporation rate at room temperature which is too high for chloroform thus changing the concentration of the sample during the preparation step . the dielectric constants are very close to each other for chloroform and bromoform ( 4,8 and 4,4 at 20c , resp . ) , and the gelation concentration was observed to be similar in both cases . these fibers present a diameter of 6 nm and a length of approximatively 200 nm . indeed , the precise length can not be determined as the fibers present a high entanglement ratio . this length corresponds to the distance between the nodes of the networks and was also observed from time to time on individual filaments ( see figure 9(b ) ) . the small individual fibers self - assemble in larger ones to form bundles of 30 nm . these latter can be formed during the preparation step as the diameter of the bundles varies from one experiment to another . the distribution of the fibers within a clear network illustrates the organogelator properties of guanosine 1 . guanosine derivative 2 in bromoform ( 4 10 g ml ) forms small aggregates and small lamellar structures . on figure 10 , large aggregates consisting of the superposition of small ( 100 nm ) lamellar structures are observed ( see arrows ) . these structures show no define contours and are the results of the aggregations of the smaller domains . no internal features inside the small domains are visible at the resolution of the technique . the aggregation within highly compact lamellar structures is also in good agreement with the light scattering experiments that indicate mainly the presence of pseudospherical objects with a strong cooperative effect occurring between the ribbons and their higher - scale self - assemblies . we have determined the hierarchical polymeric natures of the self - assemblies obtained from the ribbon forming [ 40 , 41 ] guanosine derivatives 1 and 2 . the light scattering measurements appeared as an appropriate technique for the molecular weight determination versus the structure of these objects that are both concentration sensitive . lipophilic guanosine 1 forms very soft fibrillar objects of 6 nm of diameter and 200 nm in length and that in turn produces bundles of networked fibers with 30 nm of diameter . despite its closely related structure , guanosine 2 forms much longer ribbons with molecular weights up to 6 10 that in turn fold in very compact aggregates with lamellar structures . this process has been shown to be highly cooperative by the determination of the molecular weight as a function of the concentration . interestingly , while both derivatives show the same motif of h - bonding in the formation of the first level of supramolecular assembly ( the b - type ribbon ) , their hierarchical organization is quite different . this finding points out the role of other structural aspects in addition to the h - bond recognition pattern ( e.g. , the number and length of tails ) in determining the shape and dimension of the object obtained at the nanoscale level . these investigations furnish an explanation to the gelation properties of these two derivatives and can be used to rationalize the synthesis of functional guanosine - based soft materials . 3,5-o - didecanoyl-2-deoxyguanosine 1compound 1 was prepared in 94% yield starting from 2-deoxyguanosine ( fluka ) ( 1 mmol ) and decanoic anhydride ( 2.2 mmol ) according to the literature procedure for 3,5-o - dipropanyl-2-deoxyguanosine . h nmr ( 300 mhz , [ d6]dmso ) : = 0.84 0.86 ( tt , 6h ; 2 ch3 ) , 1.18 1.40 ( m , 24h ; 12 ch2 ) , 1.40 1.60 ( m , 4h ; 2 ch2ch2co ) , 2.35 - 2.36 ( tt , 4h ; 2 ch2co ) , 2.42 and 2.93 ( mm , 2h , h-2/h-2 ) , 4.19 4.38 ( m , 3h ; h-4/h-5/h-5 ) , 5.35 ( m , 1h ; h-3 ) , 6.17 ( m , 1h ; h-1 ) , 6.45 ( bs , 2h ; nh2 ) , 7.9 ( s , 1h ; h-8 ) , 10.65 ( s , 1h ; nh ) ; es - ms : m / z ( % ) : 576.8 ( 100 ) [ 1+h ] . compound 1 was prepared in 94% yield starting from 2-deoxyguanosine ( fluka ) ( 1 mmol ) and decanoic anhydride ( 2.2 mmol ) according to the literature procedure for 3,5-o - dipropanyl-2-deoxyguanosine . h nmr ( 300 mhz , [ d6]dmso ) : = 0.84 0.86 ( tt , 6h ; 2 ch3 ) , 1.18 1.40 ( m , 24h ; 12 ch2 ) , 1.40 1.60 ( m , 4h ; 2 ch2ch2co ) , 2.35 - 2.36 ( tt , 4h ; 2 ch2co ) , 2.42 and 2.93 ( mm , 2h , h-2/h-2 ) , 4.19 4.38 ( m , 3h ; h-4/h-5/h-5 ) , 5.35 ( m , 1h ; h-3 ) , 6.17 ( m , 1h ; h-1 ) , 6.45 ( bs , 2h ; nh2 ) , 7.9 ( s , 1h ; h-8 ) , 10.65 ( s , 1h ; nh ) ; es - ms : m / z ( % ) : 576.8 ( 100 ) [ 1+h ] . 2,3-o - isopropylidene-5-decanoylguanosine 2compound 2 was prepared in 86% yield starting from 2 , 3-o - isopropylideneguanosine ( sigma ) ( 0.77 mmol ) and decanoic anhydride ( 1.2 equiv ) according to the literature procedure . compound 2 was prepared in 86% yield starting from 2 , 3-o - isopropylideneguanosine ( sigma ) ( 0.77 mmol ) and decanoic anhydride ( 1.2 equiv ) according to the literature procedure . the light scattering experiments were carried out with an in - house apparatus equipped with ( i ) a red he - ne laser of wavelength 0 = 632.8 nm in vacuum , ( ii ) a discrete - angle goniometer acting within the range from 20 to 155 , ( iii ) a hamamatsu type photomultiplier as detector , ( iv ) a photocounting device , and ( v ) a toluene matching bath . the vertical polarization of the incident beam with respect to the scattering plane has been used and is written v. the analyzer , arranged between the measuring cell and the photomultiplier , is vertically oriented and is written vv . this optical setup allows measurement of the isotropic vv scattering intensity . the excess of light scattering intensity vv(q ) = vvsolution vvsolvent was measured as a function of scattering vector q = ( 4n/0)sin ( /2 ) with an accuracy of 1% ( the scattering angle , n the solvent refractive index ) . the values of rayleigh excess scattering intensity r(q ) were obtained through the calibration of vv(q ) with a benzene standard . the intensities vv(q ) , after normalization of the raw data , can be written rv for the vertically polarized scattering light through an analyzer . use the following formula for stray light rv : ( 1)rv = kvcmwp(q)s(q ) , where mw is the molecular weight , c is the concentration of the polymer , p(q ) is the form factor , and s(q ) the long range interference from distant scatterers , where for dilute solution s(q ) ~ 1 and kv is the optical factor for the system including the refractive index increment of the polymer as follows , where kv = kvbenzene(dn / dc ) is the optical contrast calibrated with benzene standard ( the suffix gives the polarization of incident beam and stray light ) . the calibration of the spectrometer was made by evaluating the optical constant kv or kv , v as follows : ( 2)kv=42nref2rv , refna04.na is avogadro number , 0 is the wavelength in vacuum , nref is the benzene refractive index , and rv , ref is the benzene rayleigh ratio for vertically polarised incident light . the chloroform refractive index and the average refractive index increment of the assembly for 1 to chcl3 are equal to n = 1.4459 and dn / dc = 0.0769 ml / g , respectively . the chloroform refractive index and the average refractive index increment of the assembly for 2 to chcl3 are equal to n = 1.4459 and dn / dc = 0.0830 ml / g respectively . according to yamakawa the ratio kvc / rv may be read versus q as ( 3)kvcrv=1mwp(q)(1+a2mwc)2 , where rv is the normalized scattering intensity from vertically incident beam analyzed vertically , and a2 is the second virial coefficient . using the formalism of debye and zimm , ( 2 ) gives the following : ( 4)kvcrv = mw1p1(q)+2a2c . the inverse of the form factor p(q ) leads after a maclaurin transform at low q vector to ( 5 ) . here we get rgz the second moment of the mass distribution which is the statistic z average mean radius of gyration [ 57 , 58 ] as follows : ( 5)kvcrv = mw1(1+q2r2z3+)+2a2c . for extrapolation at q 0 and c 0 ( 4 ) leads to the determination of the molecular mass and the second virial coefficient a2 . when the thermodynamic forces applied on the coil equilibrate , we can write a2 = 0 . this athermal condition for binary mixture allows the determination of the apparent molecular mass mw , app for each concentration [ 59 , 60 ] : ( 6)kvcrv = mw , app1(1+q2r2z3). equation ( 6 ) contains all information on the shape and the conformation of the isolated polymer in solution . the determination of the radius of gyration is only valid in the guinier range qr < 1 where the architectures of the polymers are barely distinguishable . the plot of ( 5 ) versus kq + kc , called zimm - plot , allows a simultaneous extrapolation to q = 0 and c = 0 , which yields mw(g mol ) as the ordinate intercept and rgz as the initial slope of ( 6 ) and the second viral coefficient a2 from ( 5 ) . the value of the second viral coefficient a2 is in our case of an athermal binary mixture and thus neglected : ( 7)lim(q0 ) kcrv = mw , app1 . so we get a good evaluation of the variation for mw , app versus the concentration c. following the calculation of huglin and elias we obtain ( 8)mw , app2mw , app , i2=4000(k0mw , app , i)c . here mw , app , i is the molecular weight of the initial monomer and mw , app is the weight average molecular weight determined through ( 7 ) . measurements of each sample have been carried out after various dilutions with precise volume of clarified solvent obtained by filtration through millipore filters size 0.45 m . all the samples are directly processed in the measurements vial to avoid dust pollution .

here we investigate the supramolecular polymerizations of two lipophilic guanosine derivatives in chloroform by light scattering technique and tem experiments . the obtained data reveal the presence of several levels of organization due to the hierarchical self - assembly of the guanosine units in ribbons that in turn aggregate in fibrillar or lamellar soft structures . the elucidation of these structures furnishes an explanation to the physical behaviour of guanosine units which display organogelator properties .

1. Introduction 2. Results and Discussion 3. Conclusions 4. Materials and Methods

for more than two decades , the functional role of ca - activated cl channels ( caccs ) in vascular smooth muscle cells ( vsmcs ) has been actively investigated . this research focus was initiated by the first demonstration in 1987 of a ca - activated cl current ( icl(ca ) ) in smooth muscle cells , which naturally led to the suggestion that caccs are also present in vsmcs . although icl(ca ) has subsequently been well - characterized in patch clamp studies of isolated vsmcs these studies have not linked the icl(ca ) to its function in the vascular wall . since cl is actively accumulated in vsmcs , opening of caccs at resting membrane potentials will lead to cl efflux and membrane depolarization . these cacc properties integrate intracellular calcium ( [ ca]i ) increases with membrane potential changes and suggest an amplifying link for agonist - induced contraction as well as several other important properties of the vascular wall , e.g. , vascular rhythmicity and myogenic tone . uncertainties in the mechanistic explanation of this amplifying link are , at least in part , due to the lack of specific pharmacological tools and uncertainty about the molecular structure of caccs . significant progress in this field has occurred over the last years as putative cacc proteins have been identified . four members of the bestrophin family can produce icl(ca ) , although their direct association with the endogenous icl(ca ) has been questioned . tmem16a and some other members of the tmem16 protein family are also shown to be essential for icl(ca ) . there is no universal profile for the biophysical and pharmacological properties of icl(ca ) reported in vsmcs . the caccs responsible for these currents are suggested to vary in their ca - sensitivity and the mechanism of ca activation , voltage- and time - dependency , halide permeability and sensitivities to blockers . these variable characteristics could partially be attributable to differences in the methods of registration , protocols for current activation , solutions and origins of vsmcs . it is , however , most probable that the caccs represent a heterologous group of channel proteins unified by their anion sensitivity and ca - dependence . several subgroups of caccs have been suggested based upon their activation mechanism , e.g. , the caccs activated directly by [ ca]i and those that need additional activators such as camkii or cgmp - dependent protein kinase ( pkg ) . the current , general classification of icl(ca ) is based on biophysical and pharmacological characteristics obtained under variable experimental conditions . there is a substantial need to improve this classification based on an understanding of the molecular structures that are associated with the specific characteristics . evidence for the association between icl(ca ) with different properties and different putative cacc proteins is needed . such associations are beginning to appear , e.g. , in the vasculature , where the classical icl(ca ) co - exists with another icl(ca ) , which has an absolute requirement for cyclic gmp for its activation and several other biophysical and pharmacological characteristics distinct from the classical icl(ca ) . the cgmp - dependent icl(ca ) depends upon the expression of bestrophins while the classical icl(ca ) does not . it is therefore of interest to know whether tmem16a is important for the classical icl(ca ) and what relation this protein has to the cgmp - dependent icl(ca ) in vsmcs . interestingly , tmem16a is essential for both the classical icl(ca ) and the cgmp - dependent icl(ca ) . this complicates the description of icl(ca ) with a certain characteristic to a specific protein or protein family . although the association of bestrophins and tmem16a with caccs has been originally suggested in heterologous expression experiments , detailed molecular - based studies of the functional importance of caccs in the vascular wall in vitro and in vivo have been made possible by novel approaches to studying vascular tissue ; sirna - induced downregulation of the protein of interest , tissue specific knockdown in mice , molecular cloning and mutagenesis and drug - screening strategies . an sirna approach suggests that the bestrophin - associated cgmp - dependent icl(ca ) has no significance for tonic contractile response in small arteries . these findings are further supported by a comprehensive study suggesting minor or little importance of the cl conductance for arterial contraction in rat mesenteric small arteries . however , suppressing tmem16a expression reduces arterial contraction and lowers arterial blood pressure . a detailed analysis of the functional consequences of tmem16a expression changes suggests that tmem16a is more than a channel - forming protein and likely has several other modulatory and expression - related cellular functions . in spite of significant progress in our understanding the role cacc has in the vasculature , several significant incongruities between various reports as well as between working hypotheses and experimental results remain to be resolved . 1 ) the role of cl conductances for arterial contraction ; 2 ) whether the two caccs are represented by a single channel - forming protein family ; 3 ) how tmem16a and bestrophins interact with each other to produce different ca - activated cl conductances ; 4 ) other putative functions of cacc - associated proteins in the vascular wall . the existence of a depolarizing conductance that can be activated by cgmp and [ ca]i was previously hypothesized based on the model for generation of vasomotion . a depolarizing current dependent on or activated by cgmp however , the cgmp - dependent icl(ca ) in vsmcs in rat mesenteric small arteries has been reported by two independent laboratories . the functional characterization of this conductance clearly indicates that it is a unique current in comparison with the well - described classical icl(ca ) . in addition to an absolute cgmp - dependence of only one of these two icl(ca ) , these conductances differ in their ca- and voltage - dependence as well as in their pharmacological profile . the inhibitors which have been shown to be effective , at least in patch clamp experiments , for the classical icl(ca ) , e.g. , niflumic acid , 5-isothiocyanato-2-[2-(4-isothiocyanato-2-sulfophenyl)ethenyl]benzene-1-sulfonic acid ( dids ) and indanyloxyacetic acid 94 ( iaa94 ) , affected the cgmp - dependent icl(ca ) only at high concentrations . in contrast , the cgmp - dependent icl(ca ) was shown to be sensitive to extracellular zn in concentrations that are without effect on the classical icl(ca ) . differences in blocker sensitivity and distinction in cgmp - activity make it possible to differentiate these two icl(ca ) in vsmcs during voltage - clamp experiments . until recently this was the approach to isolate and study the different icl(ca ) in vsmcs and pharmacological differentiation was limited in situ due to off - target effects of the blockers . when inhibitors are tested in patch clamp experiments , the experimental conditions are created to emphasize the current of interest , e.g. , the icl(ca ) . in contrast , in situ experiments do not often provide such possibility and several conductances can therefore be affected by the same blocker . thus , a commonly used cacc inhibitor , niflumic acid , is also known to activate the big conductance ca - activated k channels ( bkca ) and to inhibit l - type ca channel ( ltcc ) . these effects would have the same consequences for vsmc membrane potential and vascular tone as inhibition of the icl(ca ) . this limited specificity of the conventional cacc inhibitors limits their use for studies of the importance of caccs for regulation of vascular tone . the pharmacological overlap between icl(ca ) and cation currents has led to the suggestion of molecular interaction between different groups of channels and even that some molecules may be shared in the molecular complexes important for these currents . the overlapping pharmacological profile does not in itself prove protein - protein interaction nor molecular sharing . although the observation that cacc blockers can modulate bkca and ltcc , and that the modulation of caccs by bkca and ltcc can also occur , together with localization of these channels in the same membrane microdomains , does suggest a close physical interaction . greenwood and co - authors have addressed this question suggesting the potential co - localization of bkca and caccs in cholesterol - rich lipid rafts in vsmc membrane . while bkca are shown to localize in cholesterol - rich lipid rafts and their properties are modulated by interaction with caveolin and cholesterol depletion , the pharmacological overlap between caccs and bkca can not be explained by a simple co - localization of these channels . the putative cacc tmem16a is shown to be distributed more evenly in the vsmc plasma membrane having only slight overlap with bkca localization . this is consistent with the finding that cholesterol depletion omits the effects of bkca blockers on icl(ca ) but has no major consequences for the effects of niflumic acid . this suggests that the interaction between bkca and caccs is more subtle or complex than simple consolidation in a microdomain . in contrast , a close proximity of caccs to voltage - gated ca channels ( vgccs ) in the membrane of hippocampal neurons was previously shown . this co - localization is , however , tissue specific as caccs were shown to be functionally and structurally separate from vgccs in other types of neurons : caccs were instead shown to interact specifically with ryanodine receptors in avian sensory neurons and with ins(1,4,5)p3 receptors in dorsal root ganglia neurons . this variation in the interacting partner for caccs could be the reason for variability of side - effects for different pharmacological agents in different types of cells . an interesting approach to differentiate icl(ca ) in vsmcs was recently used where inhibitory antibodies against two putative cacc proteins tmem16a and bestrophin importantly , antibodies raised against bestrophin or tmem16a inhibited the cgmp - dependent icl(ca ) and the classical icl(ca ) , respectively . while the authors did not test whether the anti - tmem16a antibody affects the cgmp - dependent icl(ca ) it is tempting to suggest that these two ca - activated cl conductances have different molecular identities . knowledge of molecular candidates significantly improves the efficiency for drug - screening that has led to the generation of novel , small molecule inhibitors . the recently developed inhibitor t16ainh - a01 effectively inhibits the tmem16a - associated icl(ca ) without effect on the bestrophin - associated cgmp - dependent icl(ca ) in vsmcs . this inhibitor is now widely used for pharmacological identification of tmem16a - associated icl(ca ) and its functions , including vsmc contraction . drug - screening also generated another inhibitor , caccinh - a01 , which has been shown to inhibit tmem16a - associated icl(ca ) . this inhibitor has , however , some peculiar properties , since it was shown to affect tmem16a in cancer cells in two distinct ways : in addition to its inhibitory action on icl(ca ) , caccinh - a01 reduces protein level in the membrane by facilitating reticulum - associated , proteasomal turn - over of tmem16a . the latter action of caccinh - a01 is complex because after prolonged exposure of a cell culture to caccinh - a01 a pool of caccinh - a01 resistant cells developed , where the inhibitor could still block the icl(ca ) but tmem16a remained in the membrane . importantly , the initial caccinh - a01-induced retrieval of tmem16a from the membrane is associated with inhibition of cell proliferation and the authors provided evidence that the anti - proliferative effect could be ascribed to the disappearance of tmem16a . this interpretation was consistent with observation that t16ainh - a01 , which only blocks the icl(ca ) , has no effect on proliferation . these findings strongly suggest a role for tmem16a in proliferation , which is only partially dependent upon its channel function . this conclusion is further complicated , however , by the lack of direct evidence for binding of t16ainh - a01 and caccinh - a01 to tmem16a and by the lack of knowledge about the mechanism of their inhibitory action . a structurally different inhibitor , monna , was recently shown to have high potency and selectivity for the tmem16a - associated icl(ca ) , while it was ineffective toward inhibition of bestrophin-1 associated icl(ca ) . the fact that these novel small molecular inhibitors can inhibit the tmem16a - associated icl(ca ) without an effect on the bestrophin - associated icl(ca ) is supportive evidence for different proteins forming these two conductances . unfortunately , a specific inhibitor for the bestrophin - associated icl(ca ) is still lacking . importantly , although the effects of these inhibitors on cation membrane conductances have not been analyzed in detail , t16ainh - a01 has been shown to relax different types of vasculature in accordance with the hypothesized role of caccs in agonist - induced arterial contraction . an in - depth analysis of the effects of t16ainh - a01 on voltage - gated ca currents and k currents in vsmc membrane is , however , necessary to substantiate this conclusion . the other small molecular inhibitors , caccinh - a01 and monna , have not been evaluated on vascular tissue . the cgmp - dependent icl(ca ) in rat vsmcs is absent after knockdown of both tmem16a and bestrophin-3 proteins . however , expression of bestrophin-3 is required only for the cgmp - dependent icl(ca ) . is it possible that bestrophin is a channel subunit that confers the cgmp - dependence ? in fact , a cgmp - dependence has previously been shown for human bestrophin-1 but the mechanism behind it has not been analyzed . the data from rat vsmcs are inconsistent with the results from a heterologously expressed mouse heart variant of bestrophin-3 , which can produce the icl(ca ) of significant amplitude without any presence of cgmp under whole - cell configuration . importantly , bestrophins are known to be expressed in various splice variants , sometimes present in the same cell type , and this could explain the inconsistent experimental findings . this possibility is supported by a study showing that expression of the full - length clone of mouse bestrophin-3 does not produce any significant cl current due to autoinhibition by the c - terminus . this autoinhibitory cytoplasmic c - region has been predicted to contain sites for pkg - dependent phosphorylation as well as for other protein - protein interactions . moreover , mouse bestrophin-3 with the c - terminus deleted loses its autoinhibitory capacity and produces a large cl current when expressed heterologously . these studies indirectly suggest that the cgmp - dependency of bestrophin-3-associated icl(ca ) could be a consequence of the splice variant expressed . although the presence of the autoinhibitory c - terminal domain has been suggested for all members of the bestrophin family , the exact autoinhibitory phosphorylation site remains to be determined . there is no evidence for direct pkg - phosphorylation of the bestrophin protein : this effect might be indirect and involve several additional phosphorylation and dephosphorylation steps . thus , the c - terminal autoinhibitory domain of mouse bestrophin-3 has been suggested to interact directly with negatively - charged membrane phospholipids and this interaction dynamically regulates the bestrophin-3-associated cacc activation via the phosphatidyl inositol 3-kinase . however , this modulation is not essential for channel activation and other regulatory sites have been suggested . indeed , it has been previously shown that [ ca]i might activate human bestrophin-3 without going through a freely diffusible messenger or through protein phosphorylation . this conclusion was , however , reached based upon very slow activation and deactivation kinetics of the cacc and can not exclude the involvement of membrane - associated messenger(s ) acting on the autoinhibitory c - terminal . accordingly , bestrophin-1 activation was shown to be dependent on a camkii - dependent phosphorylation . this phosphorylation is further regulated by a modulatory action of protein phosphatase 2a , which has been found physically - associated with bestrophin . finally , the regulation of bestrophin - associated caccs could involve the phosphorylation of an accessory subunit , which is as yet unidentified . very recently , however , novel evidence has been presented to suggest that bestrophins can also form homotetrameric channels and perform highly - regulated cacc activity independently of other proteins . a recent study of sirna - induced downregulation of tmem16a demonstrated that both the bestrophin - associated cgmp - dependent icl(ca ) and the classical icl(ca ) disappeared . based on these results it is tempting to suggest that tmem16a might be the channel - forming protein and association with bestrophin generates the cgmp - dependent icl(ca ) ( fig . 1 ) . this suggestion is , however , complicated by the demonstrated ability of tmem16a to modulate the expression of other membrane proteins including bestrophins and l - type ca channels . it is therefore not clear how the expression of bestrophins is essential for the cgmp - dependent icl(ca ) and whether tmem16a downregulation affects this current indirectly via reduction in bestrophin expression . other experimental models , e.g. , tmem16a knockout mice , will be helpful in addressing this question . agonist - induced ca - release activates the icl(ca ) which in term depolarises vsmcs leading to influx of ca through voltage - gated l - type ca channels ( ltcc ) and consequent force development . it has been suggested that tmem16a is the pore - forming subunit responsible for icl(ca ) . bestrophin has been suggested to act as sarcoplasmic reticulum ( sr)-located channel that serves as a countercurrent for ca movement ( a ) . other data suggest that bestrophin may be either a subunit of a tmem16a - formed channel , which modifies the biophysical and pharmacological characteristics of the tmem16a - associated caccs or forms the caccs with characteristics distinct from the tmem16a - associated caccs ( b ) . the mechanism by which tmem16a affects expression of other genes and proteins is unknown but might involve protein kinase - dependent signaling pathways . in fact , bestrophin-3 expression in renal epithelial cells has been shown to be modulated by extracellular - signal - regulated kinases ( erk1/2 ) . this erk1/2 signaling is in turn known as the mechanism involved in tmem16a - induced cancer cell proliferation . interestingly , the small - molecular inhibitors ( t16ainh - a01 and caccinh - a01 ) have been shown to abrogate the tumorigenic action of tmem16a , but their mechanism of action is not fully understood , as discussed above . while the effect of caccinh - a01is independent from cacc activity , the pore - forming region is necessary for successful inhibition of erk1/2 signaling by t16ainh - a01 . the involvement of erk1/2 signaling for the control of bestrophin expression by tmem16a remains to be shown ; nevertheless , erk1/2 signaling is known to be active in vsmcs . it is tempting to consider that similar mechanism(s ) have a place in regulation of the function and growth of the vascular wall . kb ( nf - kb ) , which mediates the expression of numerous genes . whether bestrophins are also modulated by nf - kb is unknown . moreover , whether nf - kb signaling in vsmcs is under control of tmem16a is also unknown . there is no molecular evidence to date that tmem16a can work directly as a transcription regulator but it is possible that proteolytically processed part(s ) of tmem16a may act as gene transcription factor . a similar function has previously been suggested for a c - terminal segment of voltage - gated l - type ca channels in neurons and vsmcs . recent evidence from sirna - mediated downregulation of tmem16a suggests that tmem16a can modulate the expression of l - type ca channels . this is inconsistent with a recently reported tamoxifen - induced vsmc - specific tmem16a knockout where voltage - dependent ca influx was unaffected . however , in this knockout study the icl(ca ) was abolished suggesting a defective cacc function but a truncated tmem16a protein was still present in vsmcs and this could potentially be sufficient for gene transcription regulation . finally , it is possible that the expression - related effect of tmem16a is indirectly mediated via changes in intracellular spatio - temporal ca signaling , affecting the ca - dependent transcription factors in vsmcs . although it remains to be found which gene(s ) are modulated by tmem16a and the mechanism behind this modulation , lessons from cancer cell proliferation studies and the recent demonstration of tmem16a regulation of gene expression in vsmcs clearly indicate that tmem16a serves as a modulator for gene transcription . although the role of tmem16a in modulation of gene expression is completely unexplored , the importance of this mechanism can be highlighted by the involvement of tmem16a expression in several physiological and pathophysiological changes in the vascular wall . thus , it has been shown that hypoxia - induced pulmonary artery hypertrophy and experimental pulmonary hypertension are associated with an increase in tmem16a - associated icl(ca ) and tmem16a protein expression . whether these changes are primary or secondary to the remodeling - associated changes in gene expression is not known , but tmem16a has been suggested to be a negative regulator of vsmc proliferation in hypertension - induced cerebrovascular remodeling . this importance of tmem16a for arterial structure can vary between different vascular beds in addition to being a stimulus - specific response . interestingly , no structural changes in peripheral arteries have been reported for the recently published tmem16a knockout mice . there are several potential modes of interaction between tmem16a and bestrophins ( fig . 1 ) . one possibility is that bestrophin are intracellular channels in the sarcoplasmic reticulum membrane providing countercurrent for ca release , which in turn stimulates tmem16a - formed caccs . however , voltage - clamp studies where bestrophin - associated icl(ca ) was measured do not provide experimental support for this model . indeed , the bestrophin - associated cgmp - dependent icl(ca ) was elicited independently from the sarcoplasmic reticulum by elevating [ ca]i via patch pipette solution and via membrane permeabilization for ca . although it can not entirely be ruled out that the high cytosolic ca leads to increased release of ca from the sarcoplasmic reticulum these experiments together with single channel recordings , suggest that bestrophin is directly associated with a plasma membrane conductance . there are two possibilities for bestrophin functions in the cell membrane ( fig . 1 ) . bestrophins could be responsible for atypical icl(ca ) currents , i.e. , the cgmp - dependent icl(ca ) , while tmem16a proteins could be responsible for the classical icl(ca ) . this possibility can not be tested easily as the expression of tmem16a modulates bestrophin expression , as discussed above . moreover , the bestrophin expression profile has not yet been compared between wild - type and tmem16a knockout mice . another possibility is that bestrophin forms a regulatory subunit modifying the biophysical and pharmacological properties of a tmem16a - formed channel ( fig . 1 ) : similar modulation has been shown for the inward - rectifying k channels , which change their electrophysiology and pharmacology substantially after binding to the sulfonylurea receptor subunit to form atp - dependent k channels . testing of this possibility for interaction between tmem16a and bestrophin is difficult due to technical difficulties in independently modulating the expression of tmem16a and bestrophin proteins . the majority of cells used for heterologous expression studies possess an endogenous cacc and express either bestrophins or tmem16a or both . a cacc - free expressional system , such as axolotl ( ambystoma mexicanum ) oocytes , would be suitable for addressing this question . importantly , recent single - molecule subunit analysis suggested that bestrophins are preferentially homotetrameric channels which have little or no interaction with each other and tmem16a . since cl is actively pumped into vsmcs and opening of cl channels consequently produces membrane depolarization , the caccs have been suggested to participate in the excitation - contraction coupling where they link [ ca]i and membrane potential changes . an agonist - induced ca increase leads to cacc - dependent membrane depolarization , opening of voltage - gated ca channels and further increases in [ ca]i . this amplifying mechanism has been suggested to be important for agonist - induced contraction of vsmcs . if the cl conductance is important for contraction , the contraction should depend on the cl gradient across the plasma membrane . several studies have shown that replacement of extracellular cl with impermeable anions acutely amplifies agonist - induced depolarization and contraction but this is not always the case . substitution of extracellular cl was shown to have only mild effect on both resting membrane potential and membrane potential in noradrenaline - stimulated rat mesenteric small arteries and no significant effect on contraction . nevertheless , a variable significance of cl for vsmc contraction depending on the type of vasculature and stimulation can be suggested . in fact , this is consistent with recent results from tmem16a knockout mice showing a lack of effect of knockout in mesenteric arteries , while in other blood vessels , e.g. , aorta and carotid artery , the contractile response was significantly diminished by tmem16a knockout . the lack of the effect of downregulation of bestrophin - associated cgmp - dependent icl(ca ) on force development supported the suggestion that cl conductance has little role for agonist - induced contraction of rat mesenteric small arteries . however , when the tmem16a - associated classical icl(ca ) and cgmp - dependent icl(ca ) were downregulated , the arteries depolarized significantly less to noradrenaline , and the [ ca]i increase and contraction were reduced as was the response to vasopressin in these arteries . if this effect was due to lower cacc - dependent depolarization , it should be rescued by depolarizing the arterial wall but this was not the case : kcl - induced depolarization and contraction were significantly reduced in the tmem16a - downregulated arteries . furthermore , in the same experimental series , when the membrane potential was similar , [ ca]i was lower in the arteries where tmem16a was downregulated . taken together these functional results suggest that tmem16a may modify voltage - dependent ca influx independently of the membrane potential . importantly , these results do not exclude caccs from having a role in vsmc depolarization but rather suggest that this role is not exclusive , at least in rat mesenteric arteries . in contrast , the agonist - induced contractions of other arteries have been shown to be strongly dependent on cacc depolarizations with no indication of the involvement of voltage - gated ca channels . thus , in functional studies on mouse thoracic aorta and mesenteric artery as well as human abdominal visceral adipose artery a novel blocker of tmem16a - associated caccs , e.g. , t16ainh - a01 , relaxed preconstricted arteries in vitro . t16ainh - a01 was without significant effect on arterial contraction induced by 60 mm kcl . this could be due to a chronic effect of sirna - induced downregulation that reduces voltage - gated ca channels vs. an acute pharmacological effect of the blocker . however , sirna - induced downregulation of tmem16a in rat cerebral artery organ culture does not support this suggestion . rat cerebral arteries downregulated for tmem16a with sirna demonstrate a reduction in pressure - induced depolarization and myogenic vasoconstriction , but the contraction to 60 mm kcl depolarization was unaffected . however , the authors suggested that ca influx via mechanosensitive nonselective cation channels stimulates tmem16a - associated channels to induce myogenic vasoconstriction and demonstrated no importance of voltage - dependent ca - influx or ca - release for activation of tmem16a - associated channels . the reason for this inconsistency is unclear and could reflect different roles of cacc in different vascular beds . in fact , it has recently been shown that mouse mesenteric small arteries have a very weak expression of tmem16a and an icl(ca ) of low amplitude . consistently , an inducible vsmc - specific knockout of tmem16a in mice was without any effect on mesenteric small artery contractility . in contrast , larger blood vessels , e.g. , aorta and carotid artery from tmem16a knockout mice , had reduced contractility to angiotensin ii and u46619 ( thromboxane analog ) compared with their wild type counterparts . these larger arteries from wild type mice have a large tmem16a - associated icl(ca ) . it is of interest to determine whether the responses to adrenergic agonists are also reduced in the larger vessels . knockout of tmem16a had no effect on the contraction to 60 mm kcl suggesting that the expression of voltage - gated ca channels was unaffected in this model . however , as mentioned above , although the icl(ca ) was gone a significant part of the truncated tmem16a protein was still present in the vsmcs which could be relevant for function . the current evidence indicates that tmem16a is essential for generation of icl(ca ) suggesting that this protein is a channel - forming subunit of caccs . it remains to be determined whether tmem16a is associated with different forms of caccs and whether other icl(ca)-associated proteins , e.g. , bestrophins , serve as regulatory proteins that modify the channel s properties . this challenge is hampered by the surprising finding that tmem16a expression is important for the expression of other genes , such as bestrophins , which are associated with some forms of the icl(ca ) , and voltage - gated ca channels . these non - contractile functions of tmem16a protein in vsmcs further complicate the analysis of cacc function in the vascular wall . it seems that the expression and the function ( either membrane depolarization or modulation of voltage - gated ca channels ) of tmem16a vary in different vascular beds and thus may greatly influence experimental results . there is however a general consensus that the tmem16a - associated icl(ca ) is an important component of vsmc function and is important for maintenance of total peripheral resistance and , thus , blood pressure .

the presence of ca2 + -activated cl currents ( icl(ca ) ) in vascular smooth muscle cells ( vsmcs ) is well established . icl(ca ) are supposedly important for arterial contraction by linking changes in [ ca2+]i and membrane depolarization . bestrophins and some members of the tmem16 protein family were recently associated with icl(ca ) . two distinct icl(ca ) are characterized in vsmcs ; the cgmp - dependent icl(ca ) dependent upon bestrophin expression and the classical ca2 + -activated cl current , which is bestrophin - independent . interestingly , tmem16a is essential for both the cgmp - dependent and the classical icl(ca ) . furthermore , tmem16a has a role in arterial contraction while bestrophins do not . tmem16a s role in the contractile response can not be explained however only by a simple suppression of the depolarization by cl channels . it is suggested that tmem16a expression modulates voltage - gated ca2 + influx in a voltage - independent manner and recent studies also demonstrate a complex role of tmem16a in modulating other membrane proteins .

Introduction Two Functionally Distinct Is Bestrophin still a Putative CaCC Protein? The Expression of Bestrophins in Vascular Wall is Modulated by TMEM16A: A Challenge for Structure-Function Analyses TMEM16A and Bestrophins can Interact in the Vascular Wall What is the Role of the Ca The Functional Significance of TMEM16A in the Vascular Wall Concluding Remarks

a 35-year - old man presented with a 7.0 cm anterior mediastinal mass on a chest radiograph during a routine medical evaluation at an outside hospital . the patient was transferred to the department of thoracic and cardiovascular surgery for further evaluation and treatment . the patient did not present with any specific medical problems or symptoms and had no family history of cancer or prior surgeries except for an operation due to a pneumothorax that was performed 12 years prior to presentation . fluorodeoxyglucose ( fdg)-positron emission tomography / computed tomography ( ct ) revealed two heterogenic hypermetabolic foci in the anterior mediastinum , with standard uptake values of 3.3 and 3.4 . no significant fdg uptake was observed in the lymph nodes or anywhere else in the body to suggest distant metastasis , except for physiological uptake in both testes ( fig . contrast - enhanced chest ct images showed multiple tumors in the anterior mediastinum . specifically , a 5.0 cm mass compressing the superior vena cava , a 1.5 cm nodule , and a 1.0 cm nodule were observed ( fig . ultrasonography of the testes revealed a normal shape and echogenicity without focal mass or infiltrative lesions . the serum levels of human chorionic gonadotropin ( 0.1 iu / l ) and -fetoprotein ( 12.8 ng / ml ) were in the normal range . a biopsy was performed in the operating room , and the diagnosis of thymoma was determined based on frozen tissue sections . the largest mass ( 5.05.0 cm in size ) showed a lobulated , homogeneous , firm , cut surface with a partial hemorrhage . in the normal thymic tissue surrounding the largest tumor , two smaller masses ( 1.51.2 cm and 1.00.5 cm in size ) were seen , which had a well - defined , round , homogeneous , soft appearance . the medium - sized mass was located 1.5 cm from the largest tumor , and the smallest mass was 0.5 cm away ( fig . , low - power magnification of the largest mass showed a well - circumscribed , thick - walled capsule with a partially incomplete area , lobular architecture , and perivascular spaces containing proteinaceous fluid and lymphocytes . the tumor was composed of a dual population of lymphocytes and epithelial cells in equal proportions . under high - power magnification , the epithelial cells had a bland nuclear appearance with small inconspicuous nucleoli . the lymphocytes that were intermixed with the epithelial cells were small and mature - appearing . the two small masses without a capsule revealed clusters or islands of large epithelioid cells with prominent nucleoli and pale cytoplasm that were intermingled with small , infiltrating lymphocytic cells . interestingly , these two small tumors revealed a prominent lymphoid follicular hyperplasia with the formation of germinal centers , some microscopic cysts , and hassall s corpuscles in the surrounding area of the seminomatous component ( fig . , the epithelial cells of the thymoma showed diffuse positive reactions for cytokeratin 19 and p63 and negative reactions for placental - like alkaline phosphatase , cd5 , cd117 , and d2 - 40 ( fig . in contrast , the epithelial cells of the seminoma - like tumors showed diffuse positive reactions for cd117 , partially strong positive reactions for placental alkaline phosphatase ( plap ) and d2 - 40 , and negative reactions for -fetoprotein , cytokeratin 19 , and p63 ( fig . epithelial membrane antigen staining was observed in hassall s epithelial cells of the thymoma and in microcystic epithelial cells of the seminomas . in summary , histological and immunohistochemical analyses of the anterior mediastinal masses revealed a type b2 invasive thymoma and two seminomas . the simultaneous occurrence of a thymoma and seminoma in the thymus has not yet been reported except for two cases described by weissferdt et al . in 2014 . however , they reported a thymoma with a component of a thymic seminoma combined within a single tumor . the seminomatous component showed seminoma - like epithelioid cells with distinct hyalinization adjacent to the area of the conventional thymoma . our case included one large thymoma and two small seminomas in the anterior mediastinum , with distances of 1.5 cm and 0.5 cm between the thymoma and the seminomas . because multiple thymic masses containing a thymoma and small seminomas are very rare , the possibility of metastasis from another organ or a morphological transformation of the thymic epithelial tumor cells into thymic carcinoma should be considered . the two small masses showed negative reaction for cd5 and pan - cytokeratin and positive reaction for plap and d2 - 40 by immunohistochemical staining . although cd5 loss in thymic carcinoma is known , the other immunohistochemical findings favor seminoma rather than thymic carcinoma . we could not find a case of a thymic carcinoma with a positive reaction for plap or d2 - 40 in the literature . our case showed site - associated features such as the presence of multiple cysts , reactive remnant thymic epithelium , epithelioid granulomatous reaction , fibrosis , and follicular lymphoid hyperplasia . in the seminomatous tumor fortunately , site - associated morphologic features of mediastinal masses are rare in gonadal tumors . although a biopsy of the testes was not performed , metastatic seminoma in the mediastinum from an intratubular germ cell neoplasia is unusual in the absence of retroperitoneal lymph node metastasis . these clinical and histological findings support the conclusion that these seminomas are of mediastinal origin rather than due to metastasis . primary mediastinal seminomas were first described in 1955 by woolner et al . and have been well recognized for over 50 years . however , it is believed that mediastinal seminomas originate from extragonadal germ cells in the thymic gland due to chromosomal abnormalities . chromosome 12p abnormalities , which are specific genetic alterations found in testicular seminomas , are also present in mediastinal seminomas . specifically , przygodzki et al . reported a unique pattern of a kit exon 17 mutations in mediastinal seminomas , which are rare in testicular seminomas . this may imply that the mediastinal seminoma develops through different pathways than that of its gonadal counterpart . regardless of the tumor location , seminomas show kit protein expression , which is also seen in thymic carcinomas but not in thymomas . histopathological analyses show that thymomas have heterogeneous morphological features such as spindle cells , lymphocytic infiltrate , and rhabdomyomatous components . additionally , cytokeratin expression in seminoma tumor cells , which reflects the differential potential or the morphological transformation of the seminoma into another nonseminomatous gcts , has been reported . moreover , a thymic carcinoma or seminoma may occur in combination with a thymoma within a single tumor . therefore , we cautiously hypothesize that thymoma , thymic carcinoma , and seminoma occur from cells of the same origin , such as embryonic stem cells or primordial germ cells , which have the ability to differentiate into a diverse population of cells . they obtain chromosomal and molecular abnormalities such as chromosome 12p abnormalities and kit mutations and transform from thymic epithelial tumors into gcts ( seminoma ) or from seminomas into thymic epithelial tumors . the etiology and pathogenesis of thymomas and seminomas in the thymus are unknown , and further research is required in this area . follicular lymphoid hyperplasia of the thymus can be seen in reactive , autoimmune diseases like myasthenia gravis , follicular lymphoid hyperplasia may be associated with specific antigens produced by the microenvironment of the seminoma . weissferdt and moran postulated that the specific distribution of antigen - presenting dendritic cells in mediastinal seminomas is related to the pathogenesis of follicular lymphoid hyperplasia . in short , we report a case of one thymoma and two mediastinal seminomas that developed simultaneously in the thymus of a young adult . the seminomas revealed florid follicular lymphoid hyperplasia and microcystic changes that do not appear in testicular seminomas . in addition , no clinical or radiological evidence of gcts was found elsewhere . therefore , even though multiple masses were present , we considered these tumors to be synchronous primary thymic tumors rather than metastases from an occult tumor of the testis . to our knowledge , this is the first description of a synchronous occurrence of a separate thymoma and mediastinal seminomas within the thymus .

thymoma is the most common neoplasm of the anterior mediastinum and has malignant potential . germ cell tumors ( gcts ) found in the anterior mediastinum are usually benign , and malignant gcts , such as seminomas , are rare . histologically , mediastinal seminoma is indistinguishable from testicular seminoma except for site - associated morphological features such as lymphoid follicular hyperplasia . therefore , excluding metastasis is very important . recently , we treated a young adult patient with multiple thymic masses that occurred simultaneously . the patient underwent a thymectomy for the removal of the mediastinal masses , one of which was diagnosed as type b2 invasive thymoma , and two of which were diagnosed as primary mediastinal seminomas with massive follicular hyperplasia . the patient received adjuvant chemotherapy after surgical resection . to our knowledge , this is the first description of a thymoma and a mediastinal seminoma occurring simultaneously in the thymus . we present this case along with a literature review .

CASE REPORT DISCUSSION

the causes of posterior shoulder dislocations are usually a strong traumatic event or a sudden violent internal rotatory muscles contraction such as what may happen during a convulsive crisis or electrocution [ 13 ] . in most of the cases , the dislocation is subacromial and posterosuperior ; the subspinatus and subglenoidea forms are very uncommon . anteromedial humeral head impaction fractures ( mclaughlin lesion or reverse hill sacs ) always complete this scenario . although posterior capsular or labral laceration seems to be constant , posterior tears of the rotatory cuff are not so much described in the literature [ 46 ] . most of the cases can be misunderstood because of being without a correct x - ray projection or the not - always clear presentation of the clinical signs , which essentially consist of pain and unability to do complete elevation and external rotation . anyway a posterior dislocation must be suspected when there is a lesser tuberosity fracture or a fixed internal rotation of the arm . the treatment depends on the delay of the diagnosis , on the associated bony and cartilaginous lesions , on the age and functional request of the patient , and finally on the surgeon 's experience . most of the authors agree that a dislocation can be considered inveterate if it is discovered 6 weeks after the trauma . it is very important to establish if it is inveterate or not because trying to reduce an inveterate luxation can lead to an epiphyseal fracture [ 610 ] . the goal of the treatment of the acute posterior dislocation of the shoulder must be to restore the patient 's ability to do his previous activities and to prevent posterior instability . we present the case of a 57-year - old man with right hemiparesis and epilepsy secondary to a stroke . the patient presented pain and impossibility at the elevation and extrarotation of his left shoulder soon after a convulsive crisis during his first hospitalization in the department of neurology of our hospital . it shows humeral head fractures with some very little fragments and internal rotation of the humerus . in order to confirm the posterior dislocation and to discover the associated fractures and the dimension of the reverse hill - sachs lesion it shows posterior dislocation and a damage of the cartilaginous surface of humeral head with loss of the bone that amounts between 20 and 30% of the articular surface . in order to prevent instability and to obtain an anatomical restoration that allows early mobilization and complete range of movement , we reduced the dislocation through a deltoid - pectoral incision , we stabilized the lesser tuberosity with a cancellous bone screw , and then we filled the reverse hill sachs with crioconserved femoral head contoured in the right shape to reconstruct the patient 's humeral head , maintaining his original bending radius . the allograft was fixed by using 2 herbert screws ( figure 3 ) . finally , we reduced and stabilized the posterior glenoid rim fracture with a herbert screw , through a posterior miniapproach . this treatment allowed the patient an early mobilization , and so he obtained a range of motion very similar to his previous condition . at 8-month followup , there are no signs of avascular necrosis ( figures 4 and 5 ) and the patient is satisfied with our operation . we did not perform the constant score because of the right hemiparesis . however , the patient is able to keep his left arm above his head and to rotate internally till sacral bone ( figures 6 and 7 ) . although the surgical techniques used for chronic posterior instability are quite the same for acute traumatic dislocations , it is important to make the right diagnosis soon after the trauma to avoid avascular necrosis [ 710 ] . sometimes it is quite difficult to obtain correct x - ray projection because of the pain that limits the patient 's mobility . a posterior dislocation must be suspected when there is a fracture of the anteromedial humeral head and when the arm is fixed in internal rotation . ct scan is always necessary to study the osteocartilaginous surface damage [ 5 , 6 ] . mclaughlin first had explained the relevance of the percentage of the articular humeral head cartilage loss to decide the treatment [ 7 , 8 ] . for less than 25% , hawkins reported his outcomes of 7 cases with no treatment . as shown by loebenberg , the decision depends on the age and on the functional request of the patient . for a cartilaginous loss that is between 25 and 40% , there is no universal agreement with reduction and osteosynthesis or prosthesis , while total or hemiarthroplasty is considered necessary for damage superior to 40% [ 4 , 7 , 8 ] . reconstruction of the shape of the humeral head by elevation of depressed cartilage and subchondral buttressing with cancellous bone graft has been advocated as a joint - preserving alternative [ 4 , 5 ] . dubouesset proposed the use of crioconserved allograft to restore the anatomy and to preserve the bone stock , preparing , that way , the patient for a probable future joint replacement . they had excellent results in 3 cases and 1 avascular necrosis at 1-year followup . beside the case discussed here , we treated this way other 2 chronic posterior shoulder dislocations , and in both of them we achieved satisfying results , so it is our opinion that using crioconserved bone to reconstruct the right shape of humeral head is a very valid method , and it is good in prevision of a future prosthesis . when crioconserved bone is not available , it is our opinion that using autologous transplantation from iliac crest is a good solution . maybe no delay in diagnosis can help to avoid avascular necrosis , but we believe that further studies are necessary .

posterior dislocation of the shoulder is an unfrequent event that often occurs as a consequence of a direct trauma or epileptic crisis . frequently the posterior dislocations are misunderstood , so they become chronic lesions . we reported a case of an acute posterior left shoulder dislocation with lesser tuberosity fracture and reverse hill - sachs lesions which involved more than 25% of the articular surface of the humeral head , in a 57-old - year man with right hemiparesis . we performed a synthesis of the lesser tuberosity with a screw , and we restored the shape of the humeral head with allograft . we achieved a good result that allows the patient to be able to do his previous activities of daily living .

1. Introduction 2. Case Report 3. Discussion

the data presented here were obtained from an ongoing prospective multicentre clinical study to search for biomarkers of periodontal disease progression . a consecutive sample of 329 participants was recruited between january 2012 and april 2015 at four centres in the united states : the forsyth institute ( cambridge , ma ) , new york university college of dentistry ( new york , ny ) , southern illinois university school of dental medicine ( alton , il ) , and the university at buffalo , state university of new york ( amherst , ny ) . participants were examined clinically by calibrated examiners every 2 months for 12 months to monitor for periodontal disease progression based on cal measurements . the study was approved by the institutional review board at each centre prior to initiation . the inclusion and exclusion criteria can be found in the online supporting information , further details can be obtained at clinicaltrials.gov ( https://clinicaltrials.gov/ct2/home ) under the identifier nct01489839 . from 29,189 replicate sitespecific cal measurements from the 329 participants baseline data , we computed the average standard deviation ( sd ) of cal within each subject and across subjects . data obtained from 16 clinical examiners based on 318,237 replicate measures across all visits were used to calculate intraexaminer agreement . periodontal assessments performed on each subject included up to 168 sites per subject ( 6 sites per tooth mesiobuccal , buccal , distobuccal , mesiolingual , lingual and distolingual for up to 28 teeth excluding third molars ) and included : presence or absence of gingival redness and plaque ; probing depth ( pd ) ; measurement of distance from the cementoenamel junction to the free gingival margin ( b measure ) ( in case of recession , a negative value was assigned ) ; cal ( calculated by subtracting the b measure from the pd ) ; presence or absence of plaque , gingival redness , bop and suppuration . pd and the b measure were measured using a north carolina manual periodontal probe ( pcpunc 15 hufriedy co , chicago , il ) , rounding down to the nearest millimetre and at premolars and the first and second molars these variables were measured twice . cal was calculated for each pass by the electronic data capturing ( edc ) system . if the difference between the 2 measurements was 2 mm , the examiner was prompted by the edc to obtain pd and the b measure a third time . subjects with 6 sites with cumulative loss of attachment 2 mm from baseline during monitoring phase had their monitoring interrupted , and proceeded to treatment . participants displaying 4 mm of cal increase at a given site received periodontal rescue therapy at such sites and continued with monitoring . after the monitoring phase , periodontally healthy subjects received professional dental prophylaxis and exited the study , whereas participants with periodontal disease received nonsurgical mechanical periodontal therapy . the dataset used in this report consisted of participants who had enrolled in the study up until april 12 , 2015 . if a subject received rescue therapy in some but not all sites , data for such sites were removed from the analysis and the subject was otherwise retained in the analysis for any remaining sites . in addition , sites with extreme variations in cal ( i.e. a difference between the minimum cal and maximum cal > 5 mm ) were also excluded . the analyses proceeded in three stages : we ( i ) calculated the observed proportion of sites with progression and regression according to different thresholds ; ( ii ) performed an alternating logistic regressions ( alr ) implementation of the generalized estimating equations ( gee ) procedure to assess whether the proportion of progressing sites changes over time and ( iii ) applied lmms to predict subjectspecific trends in cal for each site and from which classifications of progression and regression were made . stage 1 the observed proportion of sites with changes in cal from the baseline values greater or equal to 1 , 2 and 3 mm , respectively , were summarized for every visit in each clinical group separately . in addition , we also summarized the proportion of progressed sites with reversals at the subsequent visit ( i.e. being progressed a visit j , and then notprogressed at visit j + 1 ) . for these calculations , missing data points in the observed cal measurements were imputed by locf ( lastobservationcarriedforward ) from the previous visit . in contrast , all lmms and the gee analysis described below were conducted without imputation of missing data points . stage 2 the alr ( carey et al . 1993 ) implementation of gee for populationaveraged modelling was performed using all the sites simultaneously to assess whether the populationaveraged proportions of observed cal increases 1 mm from baseline to 2 , 4 , 6 , 8 , 10 and 12 months changed over time , while adjusting for disease cohort ( healthy , mild , severe ) . for additional details on this model stage 3 for each of the 168 tooth sites , a separate linear mixed effects model ( laird & ware 1982 , holditchdavis et al . 1998 ) with a cubic polynomial for time ( months ) was fitted to quantify the course of progression within individuals . specifically , the model for calit , the value of attachment loss from the i subject at time = t ( for t = 0 , 2 , 4 , 6 , 8 , 10 or 12 months ) iscalit=0+b0i+1agei+2femalei+3bcali+(4+b1i)time+(5+b2i)time2+6time3+7bcali2+8ageitime+9femaleitime+10bcalitime+it the model includes fixed effects for age , gender ( with males as the reference group ) , time , timesquared and timecubed , the twoway interactions age by time and gender by time , mean baseline cal for the subject ( bcali ) and its square , as well as the interaction of bcali and ( linear ) time . this is a fairly rich model for fixed effects with respect to inclusion of polynomial and interaction terms . the rationale was to account for betweensubject variability and thereby reduce error in the prediction of subjectspecific trends , which in turn should improve accuracy of classification of withinsubject change . to this end , the model additionally includes random effects b0i , b1i and b2i for subjects , time and timesquared respectively ; these and the random error term ij , are independent and normally distributed with unknown variances . the random effects component for each subject is the difference between the subject 's regression and the populationaveraged regression ( the latter determined from fixed effects ) . this random effects component is a measure of how progression of periodontal disease at the site for each subject systematically differs from the typical course of progression in the whole population after accounting for age , gender and baseline cal . furthermore , each tooth site of each subject has his or her own regression curve for cal given by the sum of the fixed effects and random effects components . from the subjectspecific curves generated from the regression models ( one model per site ) , predicted values of cal were computed at baseline and at 2 , 4 , 6 , 8 , 10 and 12 months for each site and person . model assessment was based on the predicted residual error sum of squares ( press ) residual e(i)t , which is the difference between observed and predicted cal from the ith subject at the tth visit based on the fit of the model that leaves out that subject . the prediction accuracy of the lmm for each site was calculated using two summaries of press residuals , the press statistic ( liu et al . 1999 ) and the sum of absolute predicted residual errors , each divided by the total number of observations across all subjects and visits for the site . these statistics enable identification of sites where prediction of cal is best and where it is poorest ( for additional details , see online supplementary material ) . we developed a threshold for progression empirically based on the prediction standard errors from a second series of lmms ( again , one per site ) fitted to calit , which is the change in cal value from baseline to time = t ( for t = 2 , 4 , 6 , 8 , 10 or 12 months ) for subject i. these models are identical to the models described above , except that the outcome is calij . the threshold for change was based on the 75 percentile of the distribution of the standard errors for subjectspecific predicted calij . sites were then classified as progressing based on the predictions from the first series of lmms using the threshold established from the second series . in particular , considering that the halfwidth of a 95% prediction interval for predicted cal is 1.96q75 ( mm ) , we grouped sites based on changes in pcal ( pcal ) into : ( i ) regressing sites ( pcal < 2q75 ) ; ( ii ) stable sites ( 2q75 pcal 2q75 mm ) ; ( iii ) intermediate sites ( 2q75 < pcal < 4q75 mm ) and ( iv ) progressing sites ( pcal 4q75 ) ( for additional details on this model , see online supplementary material ) . the high threshold of 4q75 for progression was motivated by the desire for high specificity in the classification of progression of periodontitis potentially at the expense of lower sensitivity . to show the effect of classification of sites based upon lmm prediction , the mean observed and predicted cal values were calculated at each visit across all sites and subjects within each category of disease progression . in addition , the observed proportion of sites with changes in observed cal from the baseline values greater or equal to 1 , 2 and 3 mm , respectively , were summarized for every visit in each site category of progression . we also summarized the proportion of progressed sites with reversals at the subsequent visit . to illustrate further the fluctuation of cal over time for individual sites and the smoothing effect of the lmm on the profile of longitudinal changes in cal measurements , we selected a participant ( subject x ) with a large number of sites ( n = 30 ) with increase in cal 2 mm from baseline to 12 months . the observed and estimated values for cal over time and the data presented here were obtained from an ongoing prospective multicentre clinical study to search for biomarkers of periodontal disease progression . a consecutive sample of 329 participants was recruited between january 2012 and april 2015 at four centres in the united states : the forsyth institute ( cambridge , ma ) , new york university college of dentistry ( new york , ny ) , southern illinois university school of dental medicine ( alton , il ) , and the university at buffalo , state university of new york ( amherst , ny ) . participants were examined clinically by calibrated examiners every 2 months for 12 months to monitor for periodontal disease progression based on cal measurements . the study was approved by the institutional review board at each centre prior to initiation . the inclusion and exclusion criteria can be found in the online supporting information , further details can be obtained at clinicaltrials.gov ( https://clinicaltrials.gov/ct2/home ) under the identifier nct01489839 . from 29,189 replicate sitespecific cal measurements from the 329 participants baseline data , we computed the average standard deviation ( sd ) of cal within each subject and across subjects . data obtained from 16 clinical examiners based on 318,237 replicate measures across all visits were used to calculate intraexaminer agreement . periodontal assessments performed on each subject included up to 168 sites per subject ( 6 sites per tooth mesiobuccal , buccal , distobuccal , mesiolingual , lingual and distolingual for up to 28 teeth excluding third molars ) and included : presence or absence of gingival redness and plaque ; probing depth ( pd ) ; measurement of distance from the cementoenamel junction to the free gingival margin ( b measure ) ( in case of recession , a negative value was assigned ) ; cal ( calculated by subtracting the b measure from the pd ) ; presence or absence of plaque , gingival redness , bop and suppuration . pd and the b measure were measured using a north carolina manual periodontal probe ( pcpunc 15 hufriedy co , chicago , il ) , rounding down to the nearest millimetre and at premolars and the first and second molars these variables were measured twice . cal was calculated for each pass by the electronic data capturing ( edc ) system . if the difference between the 2 measurements was 2 mm , the examiner was prompted by the edc to obtain pd and the b measure a third time . subjects with 6 sites with cumulative loss of attachment 2 mm from baseline during monitoring phase had their monitoring interrupted , and proceeded to treatment . participants displaying 4 mm of cal increase at a given site received periodontal rescue therapy at such sites and continued with monitoring . after the monitoring phase , periodontally healthy subjects received professional dental prophylaxis and exited the study , whereas participants with periodontal disease received nonsurgical mechanical periodontal therapy . the dataset used in this report consisted of participants who had enrolled in the study up until april 12 , 2015 . if a subject received rescue therapy in some but not all sites , data for such sites were removed from the analysis and the subject was otherwise retained in the analysis for any remaining sites . in addition , sites with extreme variations in cal ( i.e. a difference between the minimum cal and maximum cal > 5 mm ) were also excluded . the analyses proceeded in three stages : we ( i ) calculated the observed proportion of sites with progression and regression according to different thresholds ; ( ii ) performed an alternating logistic regressions ( alr ) implementation of the generalized estimating equations ( gee ) procedure to assess whether the proportion of progressing sites changes over time and ( iii ) applied lmms to predict subjectspecific trends in cal for each site and from which classifications of progression and regression were made . statistical significance was defined as p < 0.05 throughout . all statistical analyses were conducted using sas software . stage 1 the observed proportion of sites with changes in cal from the baseline values greater or equal to 1 , 2 and 3 mm , respectively , were summarized for every visit in each clinical group separately . in addition , we also summarized the proportion of progressed sites with reversals at the subsequent visit ( i.e. being progressed a visit j , and then notprogressed at visit j + 1 ) . for these calculations , missing data points in the observed cal measurements were imputed by locf ( lastobservationcarriedforward ) from the previous visit . in contrast , all lmms and the gee analysis described below were conducted without imputation of missing data points . stage 2 the alr ( carey et al . 1993 ) implementation of gee for populationaveraged modelling was performed using all the sites simultaneously to assess whether the populationaveraged proportions of observed cal increases 1 mm from baseline to 2 , 4 , 6 , 8 , 10 and 12 months changed over time , while adjusting for disease cohort ( healthy , mild , severe ) . for additional details on this model stage 3 for each of the 168 tooth sites , a separate linear mixed effects model ( laird & ware 1982 , holditchdavis et al . 1998 ) with a cubic polynomial for time ( months ) was fitted to quantify the course of progression within individuals . specifically , the model for calit , the value of attachment loss from the i subject at time = t ( for t = 0 , 2 , 4 , 6 , 8 , 10 or 12 months ) iscalit=0+b0i+1agei+2femalei+3bcali+(4+b1i)time+(5+b2i)time2+6time3+7bcali2+8ageitime+9femaleitime+10bcalitime+it the model includes fixed effects for age , gender ( with males as the reference group ) , time , timesquared and timecubed , the twoway interactions age by time and gender by time , mean baseline cal for the subject ( bcali ) and its square , as well as the interaction of bcali and ( linear ) time . this is a fairly rich model for fixed effects with respect to inclusion of polynomial and interaction terms . the rationale was to account for betweensubject variability and thereby reduce error in the prediction of subjectspecific trends , which in turn should improve accuracy of classification of withinsubject change . to this end , the model additionally includes random effects b0i , b1i and b2i for subjects , time and timesquared respectively ; these and the random error term ij , are independent and normally distributed with unknown variances . the random effects component for each subject is the difference between the subject 's regression and the populationaveraged regression ( the latter determined from fixed effects ) . this random effects component is a measure of how progression of periodontal disease at the site for each subject systematically differs from the typical course of progression in the whole population after accounting for age , gender and baseline cal . furthermore , each tooth site of each subject has his or her own regression curve for cal given by the sum of the fixed effects and random effects components . from the subjectspecific curves generated from the regression models ( one model per site ) , predicted values of cal were computed at baseline and at 2 , 4 , 6 , 8 , 10 and 12 months for each site and person . model assessment was based on the predicted residual error sum of squares ( press ) residual e(i)t , which is the difference between observed and predicted cal from the ith subject at the tth visit based on the fit of the model that leaves out that subject . the prediction accuracy of the lmm for each site was calculated using two summaries of press residuals , the press statistic ( liu et al . 1999 ) and the sum of absolute predicted residual errors , each divided by the total number of observations across all subjects and visits for the site . these statistics enable identification of sites where prediction of cal is best and where it is poorest ( for additional details , see online supplementary material ) . we developed a threshold for progression empirically based on the prediction standard errors from a second series of lmms ( again , one per site ) fitted to calit , which is the change in cal value from baseline to time = t ( for t = 2 , 4 , 6 , 8 , 10 or 12 months ) for subject i. these models are identical to the models described above , except that the outcome is calij . the threshold for change was based on the 75 percentile of the distribution of the standard errors for subjectspecific predicted calij . sites were then classified as progressing based on the predictions from the first series of lmms using the threshold established from the second series . in particular , considering that the halfwidth of a 95% prediction interval for predicted cal is 1.96q75 ( mm ) , we grouped sites based on changes in pcal ( pcal ) into : ( i ) regressing sites ( pcal < 2q75 ) ; ( ii ) stable sites ( 2q75 pcal 2q75 mm ) ; ( iii ) intermediate sites ( 2q75 < pcal < 4q75 mm ) and ( iv ) progressing sites ( pcal 4q75 ) ( for additional details on this model , see online supplementary material ) . the high threshold of 4q75 for progression was motivated by the desire for high specificity in the classification of progression of periodontitis potentially at the expense of lower sensitivity . to show the effect of classification of sites based upon lmm prediction , the mean observed and predicted cal values were calculated at each visit across all sites and subjects within each category of disease progression . in addition , the observed proportion of sites with changes in observed cal from the baseline values greater or equal to 1 , 2 and 3 mm , respectively , were summarized for every visit in each site category of progression . we also summarized the proportion of progressed sites with reversals at the subsequent visit . to illustrate further the fluctuation of cal over time for individual sites and the smoothing effect of the lmm on the profile of longitudinal changes in cal measurements , we selected a participant ( subject x ) with a large number of sites ( n = 30 ) with increase in cal 2 mm from baseline to 12 months . the observed and estimated values for cal over time and the longitudinal changes in cal were then plotted for every progressing site . out of the 533 participants who attended a baseline visit , 51 subjects had their monitoring interrupted due to rescue therapy , whereas 350 had already completed the 12month monitoring ( fig 21 were excluded because of a change in examiner , resulting in 329 participants in the final analysis . periodontally healthy subjects tended to be younger , more likely to be female , and to have fewer missing teeth than subjects with mild or severe periodontal loss ( table 1 ) . one can also observe that subjects in the healthy category presented less plaque , gingival redness , bop and suppuration than the periodontitis groups . periodontally healthy were not necessarily periodontally intact and had an average cal of 1.2 mm . this was the result of our inclusion criteria , which allowed for the presence of recessions , with the intent of allowing for the recruitment of older subjects in this category . among these participants , 48 sites were excluded due to a fluctuation in cal greater than 5 mm and 107 sites were excluded due to rescue therapy for a final number of 52,441 sites included in analyses . flow chart of subject recruitment for the study : 2,533 subjects were telephone screened for this study ; 1,072 subjects were enrolled ( consented ) in the study ; 549 enrolled subjects were deemed eligible for the study after clinical screening ; and 533 subjects attended a baseline visit . of those , 51 subjects were moved to the treatment phase due to rescue therapy and 350 subjects completed their 12month visit by april 12 , 2015 . twentyone of these individuals were excluded due to change in the examiner during the monitoring phase , resulting in 329 subjects ( 93 periodontally healthy ; 113 with mild periodontal loss and 123 with severe periodontal loss ) . demographic and clinical parameters of study subjects in the three clinical categories : periodontally healthy subjects , subjects with mild periodontal loss and subjects with severe periodontal loss aa , african american ; c , caucasian ; nd , not disclosed . the subjectlevel mean sd of duplicate measures was 0.26 mm ( range 0 to 0.79 mm ) . the intraexaminer agreement for the 16 clinical examiners was : exact agreement ( sd ) a total of 2,192 sites ( 4.2% ) that had missing cal data points had data carried forward from the previous visit in the first ( descriptive ) stage of the analysis . within 12 months , overall , 21.2 , 2.8 and 0.3% of sites progressed according to the thresholds of 1 , 2 and 3 mm respectively . the proportion of sites that progressed according to simple thresholds increased over time , with a high proportion of reversals , irrespective of the diagnostic threshold and the clinical group ( table 2 ) . the data indicate a higher proportion of progressing sites in the periodontal disease groups compared to healthy subjects for all three thresholds . the proportion of sites reversing was relatively independent of the duration of followup , though the proportion of sites classified as reversing within a given time period increased greatly as the progression threshold increased . observed percentage of sites with progression and percentage of progressed sites with reversals at the subsequent visit ( i.e. no longer being in the state of progression ) based on changes in cal from baseline greater than or equal to 1 , 2 and 3 mm at each visit ; data for all subjects and stratified for the three clinical groups all three groups healthy , mild and severe periodontal disease experienced a statistically significant increase in progression of periodontitis over time ( fig . 2 ) . plots of probabilities that clinical attachment loss ( cal ) 1 mm against time for the three disease categories : periodontally healthy , mild periodontal loss and severe periodontal loss , calculated using alternating logistic regression . the distribution of mean values ( i.e. minimum , 25 quartile , median , 75 quartile and maximum ) for the press statistics and absolute value of press residuals for each of the 168 sites measured were : 0.51 ; 0.79 ; 0.95 ; 1.22 ; 2.12 and 0.53 ; 0.64 ; 0.71 ; 0.79 ; 1.06 respectively . the latter set of results indicate that for the site with the best prediction , site 445 , the average absolute press residual was 0.53 mm . for the site with the worst prediction , site 273 , the mean lack of fit was 1.06 mm ( see online supplementary material for additional details ) . from the lmm for changes in cal , the 75 percentile for the standard errors of prediction was 0.238 and the width of the 95% prediction interval for predicted cal 0.47 mm ( see online supplementary material ) . using this threshold to classify sites according to their progressing state , progression occurred in 0.2% , 0.9% and 0.7% of the sites for healthy , mild and severe subjects ( table 3 ) . line plots of mean observed and predicted cal ( sd ) for sites in the four categories of progression illustrate how the classification scheme resulted in distinguishable patterns of changes in cal for both observed and predicted values of cal ( fig . the proportion of progressing and reversing sites based on observed cal measurements , stratified for the 4 categories of progression , revealed that progressing sites had the highest proportions of sites crossing the thresholds for cal change and the lowest percentages of reversal from the progressing state ( table 4 ) . one can also observe that for the 1 and 2 mm thresholds , the rates of reversals decreased over time in progressing sites . number of subjects and number and percentage of sites ( in parenthesis ) for each of the four categories of progression from baseline to month 12 with classifications based on linear mixed model predictions of cal for subjects in the three distinct clinical groups thresholds for categories of progression : ( i ) regressing sites ( pcal < 0.47 mm ) ; ( ii ) stable sites ( 0.47 mm pcal 0.47 mm ) ; ( iii ) intermediate sites ( 0.47 mm < pcal < 0.94 mm ) ; and ( iv ) progressing sites ( pcal 0.94 mm ) . line plots of mean changes in clinical attachment loss ( cal ) for observed and predicted values over time for sites grouped in the four categories of progression based in the linear mixed models : regressing 1,918 sites from 238 subjects ; stable 46,661 sites from 329 subjects ; intermediate observed percentage of sites with progression and percentage of progressed sites with reversals at the subsequent visit ( i.e. no longer being in the state of progression ) based on changes in cal from baseline greater than or equal to 1 , 2 and 3 mm at each visit and stratified for the four site categories of progression there were no progressing sites at 10 months and , therefore , no subsequent reversal . using all sites from a single participant ( subject x ) with changes 2 mm at 12 months to illustrate , plotting the observed cal measurements or their changes from baseline does not distinguish profiles for the different sites ( figs s1a and b respectively ) . in contrast , the lmm smoothed the longitudinal profiles of cal measurements , helping to differentiate profiles from different sites ( fig . for instance , the three curves that had the greatest change in pcal , including the one highlighted in red , were the only three classified as being progressed based on the lmm approach ( fig . the subjectlevel mean sd of duplicate measures was 0.26 mm ( range 0 to 0.79 mm ) . the intraexaminer agreement for the 16 clinical examiners was : exact agreement ( sd ) a total of 2,192 sites ( 4.2% ) that had missing cal data points had data carried forward from the previous visit in the first ( descriptive ) stage of the analysis . within 12 months , overall , 21.2 , 2.8 and 0.3% of sites progressed according to the thresholds of 1 , 2 and 3 mm respectively . the proportion of sites that progressed according to simple thresholds increased over time , with a high proportion of reversals , irrespective of the diagnostic threshold and the clinical group ( table 2 ) . the data indicate a higher proportion of progressing sites in the periodontal disease groups compared to healthy subjects for all three thresholds . the proportion of sites reversing was relatively independent of the duration of followup , though the proportion of sites classified as reversing within a given time period increased greatly as the progression threshold increased . observed percentage of sites with progression and percentage of progressed sites with reversals at the subsequent visit ( i.e. no longer being in the state of progression ) based on changes in cal from baseline greater than or equal to 1 , 2 and 3 mm at each visit ; data for all subjects and stratified for the three clinical groups all three groups healthy , mild and severe periodontal disease experienced a statistically significant increase in progression of periodontitis over time ( fig . 2 ) . plots of probabilities that clinical attachment loss ( cal ) 1 mm against time for the three disease categories : periodontally healthy , mild periodontal loss and severe periodontal loss , calculated using alternating logistic regression . the distribution of mean values ( i.e. minimum , 25 quartile , median , 75 quartile and maximum ) for the press statistics and absolute value of press residuals for each of the 168 sites measured were : 0.51 ; 0.79 ; 0.95 ; 1.22 ; 2.12 and 0.53 ; 0.64 ; 0.71 ; 0.79 ; 1.06 respectively . the latter set of results indicate that for the site with the best prediction , site 445 , the average absolute press residual was 0.53 mm . for the site with the worst prediction , site 273 , the mean lack of fit was 1.06 mm ( see online supplementary material for additional details ) . from the lmm for changes in cal , the 75 percentile for the standard errors of prediction was 0.238 and the width of the 95% prediction interval for predicted cal 0.47 mm ( see online supplementary material ) . using this threshold to classify sites according to their progressing state , progression occurred in 0.2% , 0.9% and 0.7% of the sites for healthy , mild and severe subjects ( table 3 ) . line plots of mean observed and predicted cal ( sd ) for sites in the four categories of progression illustrate how the classification scheme resulted in distinguishable patterns of changes in cal for both observed and predicted values of cal ( fig . 3 ) . the proportion of progressing and reversing sites based on observed cal measurements , stratified for the 4 categories of progression , revealed that progressing sites had the highest proportions of sites crossing the thresholds for cal change and the lowest percentages of reversal from the progressing state ( table 4 ) . one can also observe that for the 1 and 2 mm thresholds , the rates of reversals decreased over time in progressing sites . number of subjects and number and percentage of sites ( in parenthesis ) for each of the four categories of progression from baseline to month 12 with classifications based on linear mixed model predictions of cal for subjects in the three distinct clinical groups thresholds for categories of progression : ( i ) regressing sites ( pcal < 0.47 mm ) ; ( ii ) stable sites ( 0.47 mm pcal 0.47 mm ) ; ( iii ) intermediate sites ( 0.47 mm < pcal < 0.94 mm ) ; and ( iv ) progressing sites ( pcal 0.94 mm ) . line plots of mean changes in clinical attachment loss ( cal ) for observed and predicted values over time for sites grouped in the four categories of progression based in the linear mixed models : regressing 1,918 sites from 238 subjects ; stable 46,661 sites from 329 subjects ; intermediate 3,535 sites from 319 subjects ; and progressing 327 sites from 147 subjects . observed percentage of sites with progression and percentage of progressed sites with reversals at the subsequent visit ( i.e. no longer being in the state of progression ) based on changes in cal from baseline greater than or equal to 1 , 2 and 3 mm at each visit and stratified for the four site categories of progression there were no progressing sites at 10 months and , therefore , no subsequent reversal . using all sites from a single participant ( subject x ) with changes 2 mm at 12 months to illustrate , plotting the observed cal measurements or their changes from baseline does not distinguish profiles for the different sites ( figs s1a and b respectively ) . in contrast , the lmm smoothed the longitudinal profiles of cal measurements , helping to differentiate profiles from different sites ( fig . for instance , the three curves that had the greatest change in pcal , including the one highlighted in red , were the only three classified as being progressed based on the lmm approach ( fig . the use of lmms to predict periodontal disease progression showed several advantages over traditional methods described in the literature . first , the lmms could be applied to all visits from all subjects simultaneously while accounting for variation in cal measurements within subjects over time and between subjects . specifically , by introducing subjectspecific intercepts , slopes and quadratic trends ( i.e. random effects ) , the models accounted for the random fluctuations in cal measurements over time , while providing smoothed predicted profiles of sitespecific change in predicted cal for each site . the predictions were additionally based on fixed effects , which are similar to the more familiar coefficients from standard linear regression models . in particular , the model adjusted for potential influences on cal changes from age , gender and baseline mean cal ( subject level ) differences . using a cutoff based on the standard errors of predicted change in cal , we classified a small percentage of sites as progressing . although we can not verify the biological or clinical accuracy of this classification , this proportion agrees with literature suggesting that periodontal disease progression affects a very small percentage of examined sites ( lindhe et al . 1983 , 1989 , 2003 ) . also in accord with the literature , we classified as progressing a higher percentage of sites in subjects with versus without periodontitis ( le et al . different approaches in the literature have been used to overcome the influence of error in measurement of cal in classifying periodontal disease progression ( haffajee et al . most strategies focus on identifying thresholds of change in cal that would compensate for variance in cal assessments , as determined by pairs of crosssectional measurements . however , given the high level of errors in cal measurements in periodontitis subjects , detection of changes in cal are subjected to high rates of falsepositives ( corraini et al . another weakness of this approach is its lack of accounting for longitudinal sources of variation in cal measurements ( espeland et al . an additional challenge is that apparent longitudinal fluctuation in cal that is partly due to measurement error may be indistinguishable from actual reversal in cal . as illustrated by the high proportion of sites demonstrating reversal after crossing the threshold of 3 mm of cal increase in our dataset , raising the threshold for progression does not avoid issues associated with reversal of changes in cal and in fact was associated with a high overall proportion ( 7080% ) classified as reversing within any time interval . indeed , one of the first questions raised after we detected relatively high rates of reversal was if disease progression had occurred at all in this population . to globally address this issue we tested if the proportion of sites crossing certain thresholds of increases in cal would accumulate over time the use of alr provided strong evidence that the percentage of sites with increases in cal accumulated over time in all clinical groups . that the apparent high rate of reversal did not nullify increases in cal noteworthy , when the proportions of sites crossing the preset thresholds of cal increase and the rates of reversal were computed for sites stratified in the four categories of progression , progressing sites had the highest proportions of sites crossing the three thresholds of increases in cal and the lowest rates of reversal . these results indicate that our classification of progression was able to identify sites with a high likelihood of having undergone irreversible progression . because the diagnosis of periodontal disease progression relies on changes in cal , which are known to have many sources of error ( corraini et al . 2013 ) , we made every effort to minimize the error in the cal measurements . values for the average sd of duplicate sitespecific cal measurements reported in the literature have varied from 0.63 mm ( zappa et al . our results compared favourably with these figures with an overall mean sd of replicate cal measurements of only 0.28 mm . this could be partly explained by the inclusion of periodontally healthy subjects in this study population ( most studies examined only subjects with periodontitis ) and a lower level of disease in the subjects with periodontitis . some investigators have reported intraexaminer cal or relative attachment level agreement 1.0 mm for replicate measurements of cal varying from 93% ( baelum et al . perhaps the main limitation of the analysis was that it was impossible to determine the diagnostic reliability of the proposed method , as the true diagnosis of disease progression remains unknown . in fact , because we can not separate biologically or clinically meaningful fluctuation from measurement error , it is possible that we smoothed relevant changes in cal along with noise . this problem is not unique to the proposed approach , and we anticipate that as the field accrues more wellcollected longitudinal data , we can begin to develop gold standard measures of progression . further , computational difficulties associated with model complexity , specifically with respect to the number of variance components requiring estimation , prohibited the simultaneous fitting of all sites . nonetheless , the lmm applied to individual sites leveraged repeated measurements data from all participants and should better account for errors in cal measurements than previously proposed approaches . in summary , the lmm accounted for several sources of error in longitudinal cal measurement with the goal of enabling a more accurate identification of progressing sites . the results corroborate previous investigations suggesting that the diagnosis of disease progression based on a pair of visits is prone to a high rate of false positives . figure s1 . ( a ) line plot of clinical attachment loss ( cal ) measurements for 30 sites with changes 2 mm in observed cal measurements from baseline to 12 months from a single participant ( subject x ) . ( c ) predicted cal values from the linear mixed models for the same 30 sites over time . the blue profile highlights an example of a site classified as stable , whereas the red profile illustrates a progressing site based in the lmms . log odds ratio estimates ( standard errors ) from the alr analysis with dichotomous outcome cal 1 mm versus cal < 1 mm with change relative to baseline cal . sites with the highest and the lowest mean absolute press residuals . table s4 .

abstractaimthe goal of this study was to identify progressing periodontal sites by applying linear mixed models ( lmm ) to longitudinal measurements of clinical attachment loss ( cal).methodsninetythree periodontally healthy and 236 periodontitis subjects had their cal measured bimonthly for 12 months . the proportions of sites demonstrating increases in cal from baseline above specified thresholds were calculated for each visit . the proportions of sites reversing from the progressing state were also computed . lmm were fitted for each tooth site and the predicted cal levels used to categorize sites regarding progression or regression . the threshold for progression was established based on the modelestimated error in predictions.resultsover 12 months , 21.2% , 2.8% and 0.3% of sites progressed , according to thresholds of 1 , 2 and 3 mm of cal increase . however , on average , 42.0% , 64.4% and 77.7% of progressing sites for the different thresholds reversed in subsequent visits . conversely , 97.1% , 76.9% and 23.1% of sites classified as progressing using lmm had observed cal increases above 1 , 2 and 3 mm after 12 months , whereas mean rates of reversal were 10.6% , 30.2% and 53.0% respectively.conclusion lmm accounted for several sources of error in longitudinal cal measurement , providing an improved method for classifying progressing sites .

Material and Methods Study design Study population Standard deviations of duplicate measures of CAL and intraexaminer reproducibility Clinical examination Rescue therapy Subjects and sites included in analyses Data analyses Results Standard deviations of duplicate measures of CAL and intraexaminer reproducibility Proportion of sites with progression and reversals Alternating logistic regressions Linear mixed models for CAL measurements Discussion Supporting information

gardner 's syndrome ( gs ) is an autosomal dominant disorder localized to a small region on the long arm of chromosome 5 ( 5q21 - 22).1,2,3 menzel first described adenomatosis of the colon in 1721 , and in 1863 , cripps discovered the heredity of colon polyposis and termed it familial adenomatosis.4 devic and bussy in 1912 described a triad of intestinal polyps , soft tissue tumors , and multiple osteomas of the skull.5 in 1943 fitzgerald described a case of intraosseous osteomas , abdominal desmoids , and multiple colon polyposis and defined the oral and facial aspects of the disease.6,7 in the early 1950s , the syndrome was defined by gardner . in 1962 , he also discovered the dental abnormalities and skeletal alterations of these patients.8 oral and facial anomalies are commonly observed in patients affected with this disease complex , and intestinal polyps predominantly cause malignancy . dentists should be aware that oral and facial anomalies may play an important role in the early diagnosis of gs . this article presents a case of gs and briefly reviews the oral and maxillofacial considerations of this disease . a 21-year - old patient was referred to the department of oral surgery at istanbul university , istanbul , turkey , for the management of craniofacial manifestations of gs . the patient came to our attention due to a primarily esthetic complaint about the right angle of the mandible . an initial clinical examination showed a nodular formation that was palpable along the mesial portion of the right mandibular angle ( figure 1 ) . a panoramic radiograph and an anteroposterior radiography showed the presence of multiple round radiopaque lesions in both the maxilla and mandible , multiple impacted teeth ( upper right first , second premolar , upper right third molar , upper left canine , upper left first and second premolar , upper third molar , lower left canine , first and second premolar , and lower right canine , first and second premolar ) , and multiple odontomas , each measuring approximately 0.5 to 2 cm in diameter ( figure 2 ) . on palpation , these lesions were hard , well limited , and non - adherent to the skin . no deviation of the mandible was observed , and no pain on mouth opening was evident . dental volumetric tomographic ( dvt ) images showed multiple osteomas of the buccal cortex of the right mandibular angle and left mandibular angle ( figure 3,4 ) . additionally , in the coronal and sagittal sections of the mandible condyle , a huge osteoma that limited mouth opening was diagnosed ( figure 5 ) . cephalometrically , he showed a slightly retrusive maxilla with an anb angle of 1and a relatively normal mandibula in anterioposterior direction . his mandibular plane angle ( s - n / go - me : 25 ) and articular angle ( s - ar - go : 133 ) were reduced ( table 1 ) . according to the steiner 's s line his lips were in normal position ( figure 6 ) . histopathologic examination revealed that the specimens displayed a normal - appearing dense compact lamellar bone with minimal marrow spaces and rare irregular haversian canals that did not show osteoclasts or osteoblasts ( h&e x100)(figure 7 ) . due to the dental anomalies , the osteomas in the mandible and the familial history of the patient , a 21-year - old patient was referred to the department of oral surgery at istanbul university , istanbul , turkey , for the management of craniofacial manifestations of gs . the patient came to our attention due to a primarily esthetic complaint about the right angle of the mandible . an initial clinical examination showed a nodular formation that was palpable along the mesial portion of the right mandibular angle ( figure 1 ) . a panoramic radiograph and an anteroposterior radiography showed the presence of multiple round radiopaque lesions in both the maxilla and mandible , multiple impacted teeth ( upper right first , second premolar , upper right third molar , upper left canine , upper left first and second premolar , upper third molar , lower left canine , first and second premolar , and lower right canine , first and second premolar ) , and multiple odontomas , each measuring approximately 0.5 to 2 cm in diameter ( figure 2 ) . on palpation , these lesions were hard , well limited , and non - adherent to the skin . no deviation of the mandible was observed , and no pain on mouth opening was evident . dental volumetric tomographic ( dvt ) images showed multiple osteomas of the buccal cortex of the right mandibular angle and left mandibular angle ( figure 3,4 ) . additionally , in the coronal and sagittal sections of the mandible condyle , a huge osteoma that limited mouth opening was diagnosed ( figure 5 ) . cephalometrically , he showed a slightly retrusive maxilla with an anb angle of 1and a relatively normal mandibula in anterioposterior direction . his mandibular plane angle ( s - n / go - me : 25 ) and articular angle ( s - ar - go : 133 ) were reduced ( table 1 ) . his profile was straight due to the slightly retruded maxilla position . according to the steiner 's s line histopathologic examination revealed that the specimens displayed a normal - appearing dense compact lamellar bone with minimal marrow spaces and rare irregular haversian canals that did not show osteoclasts or osteoblasts ( h&e x100)(figure 7 ) . due to the dental anomalies , the osteomas in the mandible and the familial history of the patient , the patient was diagnosed as gs . following resection of the osteomas that caused discomfort , gardner 's syndrome is inherited as an autosomal dominant disorder with an incidence ranging between 1 in 4,000 and 1 in 12,000 , depending on the region.5,9 gs is known to be caused by a gene mutation located at chromosome 5.10 the patient in this study displayed a familial history of the disease ; the mother and sister had both died due to gs . the intestinal polyps of the disease typically occur prior to puberty and become generalized in the 20 - 40-year age group . the polyps display the potential for malignant change.3,6 these multiple polyps can be observed at any location in the gastrointestinal tract , particularly in the distal colon , and must be completely removed due to the high rate of transformation to adenocarsinoma.6 gastric fundic polyps occur in 90% of affected individuals , but they are not malign lesions . they are premalignant lesions for periampullary carcinoma.2,6 an increased number of peptic ulcers have also been observed.8 soft - tissue lesions such as fibromas , neurofibromas , keloids , sebaceous cysts , leiomyomas , and lipomas are also observed.8 desmoid tumors are considered locally invasive , non - malignant , and non - encapsulated and may occur in the skin of the anterior abdominal wall or intra - abdominally . they display no malignant potential and have a slow growth pattern.2,3,6 these lesions occur in approximately 10% of patients and are three times more common in women.11 the majority of cutaneous lesions observed in gs present as multiple epidermoid cysts on the face , scalp , and extremities . they are benign lesions that occur at an early age , around puberty.3,6 other neoplasias such as papillary carcinoma , adrenal adenoma , adenocarcinoma , hepatocellular carcinoma , osteosarcoma , chondrosarcoma , osteochondroma , tiroid tumors , liver tumors , and less commonly , hypertrophy of the pigment layer of the retina have also been reported.12 dental abnormalities are present in 30 - 75% and osteomas in 68 - 82% of gs patients . the osteotomas are generally located in the paranasal sinuses and mandible , display slow growth , and vary from a slight thickening to a large mass.13 osteomas predominantly affect the mandible and maxilla5,12 but can additionally affect the skull and long bones . calvarium osteomas , maxillary osteomas , and hypercementosis also occur.14 osteomas are an essential component of gs , forming a slight thickening to a large mass that may affect all parts of the skeleton . the frontal bones are the most frequent osteoma site.6 exosteoses osteomas , also called peripheral osteomas , and enosteoses osteomas can be palpable , and enostoses may be detected by routine radiography.3,6 in the mandible , two types of osteomas occur : central or lobulated . centrally located osteomas are characteristically near the roots of the teeth , and lobulated types arise from the cortex and most commonly observed at the mandibular angle.6 we saw a huge lobulated osteoma at the right condyle and many central osteomas throught both maxilla and mandible . seventy percent of gs patients display dental anomalies such as impacted or unerupted teeth , congenitally missing teeth , supernumerary teeth , hypercementosis , dentigerous cysts , fused molar roots , long and tapered molar roots , hypodontia , compound odontomes , and multiple caries.3,5 difficulties in extraction due to ankylosis have also been reported.6 our patient presented several dental abnormalities including multiple impacted teeth in the mandible and maxilla and osteomas throughout the mandible , maxilla , and both condyle and coronoid processes . osteomas are predominantly detected by routine panoramic radiography.15 pain is rarely observed , and the disease is predominantly asymptomatic , but the lesions can cause facial asymmetry as a result of expansion.13 in addition to clinical palpation , dental panoramic radiography is an effective means of detecting multiple osteomas of the jaws that are characteristic of gs . radiologically , the lesions are radio - opaque . in our patient , radio - opacities on the angle of the mandible were observed , and several impacted teeth in all segments of the jaws were evident . increased difficulty of tooth extraction was present due to the high density of the alveolar bone , the limited mouth opening , and the loss of the periodontal space.our diagnosis was performed by ct radiographs to detect osteomas and dental anomalies . in conclusion this case demonstrated the characteristic oral and maxillofacial features of gs , but the patient displayed no signs of intestinal polyps or cutaneous lesions . we report this case due to the absence of intestinal polyps , a rare occurrence in gs patients . according to lew in contrast , the oral and maxillofacial manifestations of this syndrome appear many years prior to the intestinal lesions , meaning our patient may present intestinal polyps in the future . osteomas that limit mandibular movement or cause esthetic problems were removed , but the possibility of recurrence exists . in such situations , the maxillofacial surgeon must perform an early gs diagnosis . the patients should always be in contact with a gastroenterologist , an oncologist , and an oral surgeon

gardner 's syndrome is a variant of familial adenomatosis polyposis ( fap ) with a triad consisting of polyps of the colon , multiple osteomas and surface tumors of soft and hard tissue . the intestinal polyps have a % 100 risk of undergoing malignant transformation , therefore early identification of this disease is very important . there are several symptoms of gardner 's syndrome in the oral and maxillofacial surgery , which can be discovered during routine dental examination . we report a case of a 25-year old male patient with gardner 's syndrome who has not any intestinal polyps but osteomas in the mandible and jaw deformalities .

Introduction Case Discussion

on december 8 , 2003 , as president george w. bush prepared to sign the mma into law , he took a moment to look back over the work that went into the legislation . with the medicare act of 2003 , our government is finally bringing prescription drug coverage to the seniors of america , he said . the challenges facing seniors on medicare [ to be addressed by the act ] were apparent for many years . and those years passed with much debate and a lot of politics , and little reform to show for it . ( milbank , 2003 . ) indeed , the addition of a prescription drug benefit to medicare has been a topic of considerable debate since before the program 's creation . that such coverage was not included until almost four decades after medicare was inaugurated was the result of a number of factors which speak to the difficulties in policymaking for the nation 's most important health care program . medicare 's passage under president lyndon johnson was the consummation of years of debate over a national health insurance policy dating back to the truman administration . like all legislation , it was the product of compromise , and proponents of the broadest possible program realized it did not meet all the requirements they had pressed for . ironically , the two major counterproposals to medicare during the policy debates of 1965 both included prescription drug coverage while medicare did not . the most important alternative proposal offered in 1965 was eldercare , introduced by curtis ( r - mo ) and herlong ( d - fl ) and supported by the american medical association ( ama ) , which objected to medicare as government interference with the traditional doctor - patient relationship . eldercare was a proposed extension of the already existing kerr - mills program , a federal - state initiative enacted in 1960 to support the medically indigent elderly , which included drug coverage . eldercare 's proponents in congress and the ama proposed that matching federal grants would help the states pay for the program . most observers believe that the ama supported eldercare mostly as a mechanism to defeat the passage of medicare , particularly by making clear to congress and the public how expensive expanded medical benefits under federal health insurance would be . in addition to medicare and eldercare , another proposal in the 1965 debates was offered by house ways and means committee member byrnes ( r - wi ) . byrnes offered a voluntary plan which would have created an insurance program with costs shared equally by the government and the insured . under his plan , 80 percent of the cost of prescription drugs would have been covered . byrnes ' bill , which drew on the existing plan for active and retired federal employees in its enumeration of benefits , helped frame what became the part b portion of medicare in the final bill , but the prescription drug language did not survive the drafting process in the house . thus , medicare 's proponents on capitol hill and in the administration were well aware of the possibility of including prescription drugs in the benefits of the program . such a benefit had not been a feature of previous medicare proposals , dating back to the kennedy administration , but could easily have been included as part of the legislative process in 1965 . when the senate took up the debate on the administration 's medicare bill that year , javits ( r - ny ) proposed that a drug benefit be included . his proposed amendment was supplanted in the senate 's version of the bill by language calling for a study of drug coverage . even this watered - down commitment to only study the matter was dropped in the house - senate conference that led to the final wording of the bill that president johnson signed into law on july 30 , 1965 . although there has been speculation that javits ' amendment to include prescription drugs under medicare was an effort , like the eldercare proposal by the ama , to drive up costs and thus make the entire program politically untenable , this seems unlikely given javits ' cosponsorship of similar legislation even after the program was passed . the reasons why medicare 's proponents in congress and the administration did not press to include an outpatient prescription drug benefit for the elderly ( except those hospitalized or in nursing homes under the provisions of part a ) are not fully clear . there was little debate on this particular facet of medicare ; instead , it was subsumed under the larger national debate over whether or not the federal government should be getting into the health insurance business at all . a number of possible reasons why prescription drug coverage was not included have been raised by those who have studied the matter ; most likely , the confluence of these circumstances was the determining factor at the time . costs were the primary concern : the fear was that drug costs would be a particularly unpredictable element in years to come , and there was no way to gauge how beneficiaries ' usage of prescription drugs under the program in , presumably , very large numbers , would drive these costs in the future . these and other concerns conspired to keep a prescription drug benefit out of the original medicare program . most likely , medicare 's proponents might have fought harder for its inclusion , but the sense in congress and the administration was that medicare needed to be passed , with whatever limitations necessary to the political exigencies of the moment . once the program was created , incremental adjustments over time could fix whatever problems existed or add whatever was missing . in this as in other areas , president johnson sensed that he had a rare , but probably brief , mandate to expand the federal government 's role in ways that would surpass even the programs of franklin roosevelt . his main concern was to set the agenda for what would follow : get the programs created , with whatever flaws were included in the legislative process . after medicare was enacted without a prescription drug benefit , its supporters and critics in congress and the public promptly set about to fill in the gap . as early as 1966 , douglas ( d - il ) and others introduced an amendment to a social security bill to add a prescription drug benefit to medicare . although the senate passed douglas ' amendment , the language was deleted in the house - senate conference , much as had been the case a year earlier with javits ' language calling for a study of prescription drug coverage under medicare . however , by early 1967 , president johnson requested hew secretary john gardner to convene the task force on prescription drugs , chaired by assistant secretary for health and scientific affairs philip lee and including nine other members , the fda commissioner , the surgeon general , and the social security commissioner . while gardner emphasized that the task force has no prior commitment to recommend for or against the inclusion of outpatient drugs in the medicare program , few observers doubted that the johnson administration expected the task force 's final report to make a case strongly for inclusion ( u.s . department of health , education , and welfare , 1969 ) . the complexity of the job before the task force , however , led their studies into a number of valuable , but time - consuming , areas . while gardner had asked for recommendations within 6 months , the task force instead offered five interim reports , beginning in march 1968 , before a final report was issued in february 1969 . this report argued in order to improve the access of the elderly to high quality health care , and to protect them where possible against high drug expenses which they may be unable to meet , there is a need for an out - of - hospital drug insurance program under medicare . department of health , education , and welfare , 1969 ) . in creating such a program , the task force recommended limiting the number of drugs to be covered at first , due to costs and the complexity of administration , to only those this in effect meant limiting coverage to chronic disease treatments and leaving acute cases outside of coverage . it also suggested limiting the drug benefit to beneficiaries age 70 or over and advocated cost - sharing provisions ( including coinsurance , copayments , and a deductible ) . the task force strongly came down on the side of generic drugs , to be encouraged wherever this is consistent with high - quality health care . it spoke strongly in favor of the formulary approach , calling it not a mark of second - class medicine but rather an approach associated with the provision of the highest quality of medicine in the outstanding hospitals in the nation . a successful medicare prescription drug program , in the task force 's judgment , would be difficult if not impossible to achieve without a formulary ( u.s . department of health , education , welfare , 1969 ) . moreover , while noting that the idea deserved further study , the task force argued against the direct purchase of drugs by the federal government for medicare beneficiaries . while some task force recommendations led to specific medicare enhancements , on the fundamental issue of adding a prescription drug benefit to medicare , the task force did not help bring about any such program , or even specific legislative initiatives to accomplish that goal . turning a 6-month mandate into a 20-month process certainly meant the task force issued its final report in a distinctly changed political environment . president johnson had called for gardner to undertake the study in early 1967 . instead , the task force 's interim report , issued in march 1968 , was forwarded to a chief executive who could count on little support in congress due to the crisis in vietnam , and who announced at the end of that month he would not stand for re - election . by the time the final report was issued , in february 1969 , president nixon had been inaugurated and most observers assumed this holdover from his predecessor would be swept away with the change in administration . rather than ignore the task force recommendations , nixon 's new hew secretary , robert finch , named a committee to study its recommendations and report to him . this review committee , chaired by john t. dunlop included 16 non - governmental members and representatives of the health care and pharmacy industries , reported its findings in july 1969 . dunlop 's committee supported the task force recommendation to have an outpatient prescription drug program under medicare . on more specific recommendations , however , dunlop 's group disagreed with the task force . the review committee considered the task force 's recommendation of excluding acute care drug coverage ( focusing instead on chronic cases only ) both inadvisable and difficult to administer . the committee strongly declared that [ a]n age limitation other than over 65 is undesirable . on the matter of beneficiary payment , it described coinsurance provisions as less desirable than copayment features . also , if a deductible were imposed , the committee noted the undesirability of requiring beneficiaries to keep records on their payment thereof , rather than government administrators . the committee also took issue with the task force 's proposals for a formulary approach . trade association representatives of pharmacists , retail druggists , and pharmaceutical manufacturers offered specific reservations about the effects of the reimbursement mechanism or the formulary approach on their members , but in general the review committee was surprisingly positive in its response to the overall framework the task force had proposed ( coster , 1990 ) . further impetus for a prescription drug benefit under medicare came later in nixon 's first term from a 1971 report by a commission studying social security chaired by arthur flemming . a program with a flat cost - sharing payment of $ 2 per new prescription and $ 1 for refills , with financing to be on an equal tripartite basis from workers , employers , and general revenues . ( coster , 1990 . ) the white house conference on aging that same year also called for including prescription drugs in medicare , in this case proposing a program to be entirely financed by payroll taxes and subsidies from general revenues ( myers , 1972 ) . the nixon administration thus had many reasons to urge the coverage of prescription drugs initiated by lyndon johnson in 1967 . however , the issue was largely thrust aside by a number of other health initiatives that the nixon administration increasingly focused on . cost - containment efforts for medicare were a particular concern as health care costs began their spiral , ahead even of the high overall inflation rate of the decade , making support for a potentially costly prescription drug initiative less likely . in 1972 , robert myers , chief actuary of the social security administration , announced that the administration had moved away from a broad - based prescription drug program under medicare ( myers , 1972 ) . with the white house backing away from a comprehensive prescription drug initiative under medicare , the best chance for such an effort had been lost for some time . for the rest of the 1970s , members of congress , especially in the senate , took the lead , pressing the case in proposed legislation . the two most consistent in the late 1960s and early 1970s were long ( d - la ) , and montoya ( d - nm ) . the 1972 long - montoya amendment turned out to be the closest proponents of a prescription drug benefit came to enactment for the rest of the decade . a number of factors played a role , including the distractions of the watergate scandal , the increasingly precarious state of the u.s . so , although approximately 50 different pieces of legislation supporting prescription drug benefits under medicare were proposed in the early post - enactment years , proponents had nothing to show for their efforts . later proposals , including those sponsored by church ( d - id ) , thurmond ( r - sc ) and kennedy ( d - ma ) , and pepper ( d - fl ) and obey ( d - wi ) , met similar fates later in the 1970s . instead of medicare reform , the national health care discussion centered for most of the 1970s on the prospects for national health insurance ( nhi ) , an idea seriously discussed at least since the truman administration , but now strongly revived . calls for prescription drug coverage under medicare were largely put aside as redundant ; under most nhi proposals the entire population would receive prescription drug coverage , either for all drugs or for those drugs most needed for the sort of chronic care required by the elderly . in hindsight , the failure of the nixon , ford , or carter administrations to pass any form of nhi legislation further hindered the cause of a more specific prescription drug element within medicare . with the election of ronald reagan in 1980 , the chances for either sweeping nhi legislation or a separate medicare prescription drug benefit seemed strikingly diminished . however , supporters of health care reform adjusted their sights and focused on smaller , more incremental changes in medicare . in particular , the cause of a prescription drug benefit was pressed by some members of congress and interest groups such as aarp . numerous states had also implemented some form of prescription drug assistance programs by the mid-1980s , offering a range of models to draw on in the development of a national program , appropriate to the more state - oriented focus of reagan administration policies . one result of the smaller scale focus of reformers was the addition of coverage of immunosuppressive drugs under medicare in 1987 . its discussions then laid the groundwork for the immunosuppressive drug therapy act , cosponsored by kennedy ( d - ma ) and hatch ( r - ut ) . authorized $ 15 million in block grants to the states for a period of 3 years [ for fiscal years 1987 - 1989 ] for the purchase of immunosuppressive drugs . the legislation fit an incremental model of medicare expansion and was partially the result of new attention to organ transplantation in the early years of aids . reagan administration officials , led by hcfa administrator william roper , argued that there was little evidence to support the contention that the cost of immunosuppressive therapy was an impediment to patients obtaining necessary transplantation procedures . despite cost concerns , the measure passed , eventually leading medicare to cover the costs of immunosuppressive drugs within the first year of a medicare - approved organ transplantation . during reagan 's second term , supporters of a prescription drug benefit under medicare took advantage of the unique opportunity presented by the president 's call for catastrophic coverage under medicare to append their program to the administration 's . the result was the shortlived medicare catastrophic coverage act ( mcca ) , passed in 1988 and repealed the following year . president reagan made the issue of catastrophic coverage one of his top health care priorities in his 1986 state of the union address , calling on hhs secretary dr . recommendations on how the private sector and government can work together to address the problems of affordable insurance for those whose life savings would otherwise be threatened when catastrophic illness strikes . ( u.s . government printing office , 1986 . ) advocates of medicare expansion saw the catastrophic legislation as an opportunity to open the program into other areas , including prescription drugs . house speaker jim wright met with democratic leaders of the key committees in may to encourage the addition of a prescription drug benefit to the legislation . wright 's goal was to put a democratic stamp on the legislation and ensure that the president would only get catastrophic coverage if he were willing to swallow the drug benefit as well . later that month , henry waxman , chairman of the house energy and commerce subcommittee on health introduced a catastrophic coverage bill with a prescription drug benefit attached and held hearings on the drug benefit . waxman 's bill was approved by the subcommittee and later the full energy and commerce committee in june . in the senate , bentsen ( d - tx ) , who chaired the finance committee , introduced a version of the catastrophic legislation in may 1987 , which did not include the drug benefit . by september of that year , however , bentsen agreed to support a prescription drug amendment , but only if it was phased in , ( himelfarb , 1995 . ) president reagan made the bill the subject of a weekly radio address on july 25 , 1987 , charging that bowen 's original proposal had been converted into a massive program that will impose a new tax on the elderly and soon threaten to bankrupt the medicare trust fund . costs were a major concern for critics of the addition of the drug benefit , although estimates varied wildly . hhs estimated the 1989 cost of the house ways and means plan at $ 6.4 billion , while the u.s . congressional budget office estimated the same plan 's cost as only $ 750 million . ) attempts by fiscal conservatives to eliminate the prescription drug benefit were blunted , however , by the threat of an even more expensive alternative embodied in legislation proposed by pepper ( d - fl ) in 1987 and 1988 . the lobbying efforts of aarp was also crucial in the transformation of reagan and bowen 's original proposal . the influential organization had objected to the financing mechanism of bowen 's original plan ( which would have been met by the elderly population alone , rather than being spread throughout the general population ) and had vigorously called for the inclusion of a prescription drug benefit . senator bentsen 's willingness , during negotiations with the white house , to place cost containment measures in the final version of the bill that mostly affected the actual catastrophic coverage portions of the program , helped administration officials come to terms with it financially . aarp and other advocates of the elderly accepted these changes as a fair price to pay for the prescription drug benefit under medicare . the prescription drug benefit created in the mcca of 1988 was designed to be phased - in over a number of years . the broadest prescription drug benefit , covering all drugs , was set to begin in 1991 . patients in 1991 would meet a deductible of $ 600 after which medicare would pay 60 percent of the costs of prescription drugs . the annual deductible was set to rise to $ 652 in 1992 , but medicare would also cover more of the after - deductible costs ( 80 percent ) , and in the years following medicare would cover 80 percent of patient drug costs after patients met the catastrophic deductible . this deductible was set to be indexed each year , with the intent that 16.8 percent of beneficiaries would qualify for the prescription drug benefit . overall , the mcca ( including its numerous provisions outside the scope of the prescription drug benefit ) was a major expansion of the medicare system . a large and well - coordinated public outcry against the legislation , particularly its funding mechanisms , led to a series of confrontations . fears about its potential costs had led lawmakers to develop new funding mechanisms for the overall program while phasing - in many of its benefits , especially the drug program , over time . thus , many elderly americans foresaw a scenario where they would be forced to pay up - front for benefits they were skeptical they would really receive . in addition , many already received drug benefits from their former employers . the house of representatives bowed to constituent pressure and repealed the entire mcca in october 1989 , and senate supporters could not keep the program alive . both houses of congress , supporters of the drug benefit tried to offer counterproposals that would have accepted repeal of the overall catastrophic program , but kept some or all of the prescription drug coverage , but their efforts were unavailing . the 1989 repeal of the catastrophic coverage program , before its prescription drug benefit had even begun its phase - in , began a frustrating period for supporters of a drug benefit under medicare . at no time during the remainder of the george h.w . the election of bill clinton , in part due to his focus on health care issues , seemed to bode well , but debate over a drug benefit under medicare was largely subsumed within the larger debate over the failed clinton health plan in 1993 and 1994 . although shorter in duration , the situation was similar to that of the debates over national health insurance in the 1970s , where a much broader set of proposals meant there was less attention for the specifics of prescription drug coverage under medicare . the architects of the clinton health plan did include a prescription drug plan similar to the one under mcca in their proposals , but it failed when the larger effort failed . in clinton 's second term , however , public attention to the idea of a prescription drug benefit gained force . the president made regular calls for a drug benefit under medicare a part of his health care stump speech after it was clear that the wholesale reform he had proposed was not going to occur . we must find a way through medicare , he told audiences , to provide a prescription drug benefit . clinton proposed the following in 1999 : medicare will pay for half of all the prescription drug costs , over the next few years , up to a ceiling of $ 5,000 . the clinton plan would have included a co - pay and monthly premiums ( except for people at or just above the poverty rate ) with no deductible . although clinton 's plan made no headway in congress , his emphasis of the issue both reflected and fed rising public support for the addition of a prescription drug plan under medicare . as a result , the topic of drug coverage under medicare became one of most important health care issues in the 2000 presidential election , with both candidates pledging to act on the matter if elected . the concurrent resolution on the budget in 2000 set aside $ 40 billion over the next 5 years to address medicare prescription drugs . house republicans offered the medicare drug 2000 act , designed to provide a voluntary prescription drug program which was to be administered by a new agency within hhs , the medicare benefits administration ( which would also , under the bill , administer the medicare+choice [ m+c ] program ) . this bill was favorably reported by the ways and means committee to the house of representatives , although it did not pass the senate . while passage of a prescription drug plan under medicare was hardly insured the long and tortuous history of the idea since before medicare 's passage in 1965 is testament to that fact president george w. bush made medicare reform , including the addition of a prescription drug benefit , one of his centerpiece domestic issues , and his administration entered into negotiations with the crucial congressional committees and interest groups such as aarp . the final result of these lengthy negotiations , the 2003 mma , was signed into law by president bush on december 8 , 2003 . the act reformed medicare in a number of significant ways , although most of the public discussion of the legislation centered on the prescription drug elements under title i of the act . the legislation first provided for a transitional measure , medicare - endorsed drug discount cards provided by the private sector , available to enrollees in either parts a or b of medicare after june 1 , 2004 . lower - income seniors ( not already receiving drug benefits from the federal government or medicaid ) became eligible for up to $ 600 for prescription drugs during this phase of the plan . the full prescription drug benefit is legislated to begin on january 1 , 2006 . under the full plan , eligible seniors have the option of a part d medicare , administered by private companies with cms oversight . seniors have the choice of at least one prescription drug plan and one integrated plan in each region . beneficiaries are responsible for both a premium ( about $ 35 per month in the program 's first year ) and a deductible ( $ 250 in 2006 ) , with medicare covering three - quarters of drug costs above the deductible , up to a ceiling of $ 2,250 . beneficiaries have to pay the full amount of drug costs above that ceiling , but a catastrophic drug coverage provision is triggered once total out - of - pocket costs reached $ 3,600 , above which medicare covers 95 percent of all drug costs . as in the transitional drug discount card phase of the plan , other titles of the act transmuted m+c into a new medicare advantage program , provided improvements in rural health care , and amended existing provisions under parts a and b of medicare . in the 2 years since the passage of the mma full implementation of the prescription drug aspect of the law will not be completed for some time , and the final assessment of its impact on the medicare population and on the nation awaits a history yet to be written . certainly , the prescription drug benefit signed into law in 2003 differed markedly from many earlier iterations of a drug benefit under medicare . nevertheless , supporters of prescription drug coverage were entitled to look back with some satisfaction that they had finally managed to make happen what had been stymied so many times , so many ways , in the preceding four decades .

this article describes the lengthy background and debate leading up to the passage of the medicare prescription drug , improvement and modernization act of 2003 ( mma ) . full implementation of the prescription drug aspect of the law will not be completed for some time , and final assessment of its impact awaits a history yet to be written . instead , this article summarizes the efforts of supporters until they finally managed to succeed after being stymied so many times in the preceding four decades .

Introduction

it is a symptom that expresses a disorder in the transport of food and endogenous secretions ( saliva ) through the upper digestive tract . oropharyngeal dysphagia ( od ) is a more anatomically restricted term referred to alterations in the transfer of the bolus from the mouth to the esophagus ( that means , in bolus propelling from the mouth to the pharynx , in the pharyngeal reconfiguration during the swallow , or in the opening of the upper esophageal sphincter ) . od is an inescapable concern in the management of patients with laryngeal and hypopharyngeal cancer . being as a symptom at presentation , as an adverse effect during whatever the treatment , or as sequelae compromising the quality of life of the patients , swallowing disorders have to be adequately anticipated and dealt with . swallowing is one of the vital functions that the larynx is involved in . for an outcome to be considered functional , the patient has to be able to swallow in an effective and safe manner . actually , preserving a functional deglutition is usually the most important goal of the different function - preserving surgical techniques on the larynx and the hypopharynx , as a larynx that does not prevent aspiration even though od has been specifically classified in the latest versions of the international classification of diseases , it has not yet been given the attention it deserves . od is clearly underdiagnosed , and consequently under - treated , in spite of the high rate of complications it entails . although the highest prevalence corresponds to the elderly and patients with neurological disorders , head and neck cancer patients are a population where the disease directly affects the anatomy and function of the structures involved in deglutition . the functional outcome of the patients with laryngeal and hypopharyngeal cancer will ultimately depend upon an accurate diagnosis , treatment , and rehabilitation . to accomplish breathing without a tracheostomy , oral feeding and close to normal phonation may become a difficult challenge . in many cases a function - preserving surgery will not be such without adequate rehabilitation . moreover , chances of function - preserving surgery might be underestimated if a comprehensive swallowing rehabilitation protocol is not available . normal swallowing is a complex series of neuromuscular events which has both reflexive and voluntary controls ( the later making rehabilitation possible ) . although swallowing is a smooth and continuous process , it is conventionally divided into three phases in which timing is controlled by different central pattern generators of the brainstem . the food enters the oral cavity and is mixed with saliva and masticated to form a cohesive bolus . lips , tongue , teeth , mandible , and palate are involved in the preparation and propulsion of the bolus . the pharyngeal phase is initiated when the tongue pushes the bolus towards the posterior pharyngeal wall , eliciting a series of programmed responses : the soft palate elevates to prevent nasal reflux , pharyngeal constrictor muscles contract to push bolus through the pharynx , laryngeal sphincter mechanisms close to prevent aspiration ( epiglottis inverts and true and false vocal folds adduct ) , the larynx is pulled in an anterior and superior direction , and so cricopharyngeal muscle ( upper esophageal sphincter ) is opened . the recent research in the physiology of swallowing has shown that this response can be modulated . the esophageal phase is completely involuntary : peristaltic waves propel the bolus to the stomach . anatomic structures involved in deglutition have a complex motor and sensory innervation by the cranial nerves ( cn ) . the trigeminal nerve ( cn v ) is responsible for the general sensation of the face and for the motor supply to the main muscles involved in mastication . facial nerve ( cn vii ) gives motor function to the lips and taste to the anterior two thirds of the tongue . the glossopharyngeal nerve ( cn ix ) gives general sensation to the posterior third of the tongue and motor supply to the pharyngeal constrictor muscles . the vagus nerve ( cn x ) gives motor function to the soft palate , pharynx , larynx and esophagus , and general sensation to the larynx . this includes the lingual side of the epiglottis , and important sensory site that always triggers deglutition ( a basic mechanism to protect the airway ) . finally , the hypoglossal nerve ( cn xii ) controls most of the muscles involved in tongue motility . od may be caused by anomalies involving the oral cavity , pharynx , and larynx that can be either structural or functional . the site of the primary tumor will determinate symptoms due to alterations of different phases of deglutition , sometimes similar to those that will have to be managed after the treatment . although we focus our interest on the larynx that has certainly a central critical role in deglutition , an isolated discussion on it would be absurd , as patient situation is usually more complex : there are different factors conditioning od , locally advanced tumors will extent to neighboring sites , the neck will have to be treated in many instances , and treatment schemes are usually mixed . in general , lesions in the oral cavity will impair bolus preparation , containment , and posterior movement of the bolus to the pharynx . pharyngeal and laryngeal lesions may show variably altered swallow responses that may condition laryngeal penetration or even tracheal aspiration . the three modalities of treatment used for head and neck malignancies , radiation therapy , chemotherapy , and surgery will have different effects on swallowing that can be additive . external - beam irradiation has early effects due to mucositis that may cause superficial ulceration and pain . fibrosis due to radiation therapy may be expressed as strictures , sometimes requiring dilation or even surgery , muscle changes , and different mechanical changes that will affect deglutition : fixation of the hyolaryngeal complex , reduced tongue motion , and insufficient glottis closure . there are some methods to prevent or minimize radiation sequelae , like shielding or modified radiation protocols ( like intensity modulation ) . in general , irradiated patients will show reduced oral and pharyngeal functions , with longer transit times , more pharyngeal residue , and reduced cricopharyngeal opening time . actually , postoperative radiation therapy seems to be the main factor influencing worse functional results after partial surgery of the larynx . virtually all of the patients on chemotherapy schemes for head and neck cancer will show some degree of mucositis . it will be clinically significant in up to 40% of the patients and in 100% of those under chemoradiation . on the other hand , patients may have nausea and vomiting along with extreme weakness that might impair swallowing . effects on swallowing caused by surgical treatment are of particular interest because it is precisely the severity of those that will allow the surgery to be considered a function - preserving one . effective and safe swallowing is a requirement for oral alimentation , and so it is one of the main objectives pursued by the different surgical procedures . in general , surgical treatment will alter the structure and function of different anatomical sites in the oral cavity , pharynx , and larynx . in many cases structure can be restored in a close - to - normal anatomic way by different reconstructive procedures . treatment of tumors of the oral cavity will cause a range of predictable problems depending on its location , size , and type of reconstruction performed . the function of the oral ( labial ) sphincter may be affected by local resection or by lesions of the marginal branch of the facial nerve . resections of the floor of the mouth may lead to the loss of the glossoalveolar sulcus or fixation of the tongue . this may cause a variety of problems : they may obstruct bolus passage if they are too bulky , they have no motor function and so there might be a loss of propulsive force , and as they are usually nonsensate flaps , they will lack the normal sensation required to guide the bolus to the oropharynx . tumors affecting the tongue base are probably the ones in this region most prone to cause dysphagia , as tongue base is critical in initiating swallow , propelling the bolus through the pharynx , and obtaining an efficient pharyngeal peristalsis . resections of up to one third of the tongue are well tolerated ; also preserving some neural control ( and thus some movement , at least in one side ) is critical . if the tongue gets fixed to the floor of the mouth or the hypoglossal nerve is damaged , dysphagia will worsen , as it will be difficult to control and direct the bolus inside the mouth , to chew , and to propel the bolus posteriorly . the primary and evolutionarily original function of the larynx is related to the fact that there is an aerodigestive confluence and thus a need for a swallowing act that guides the food in the right direction . the larynx is elevated and moved anteriorly while its sphincters close , thus preventing food to enter the airway . supraglottic laryngectomy will alter some of the sphincter mechanisms of the larynx , and possibly laryngeal excursion during swallowing . in the classic open surgery this late problem ( and partially the former one ) is minimized by laryngeal suspension . transoral laser surgery causes a much smaller damage to the extrinsic mechanism involved in laryngeal movement , and even though the technique does not in any way suspend in extended procedures involving portions of the tongue base , hyoid bone , and others , swallowing prognosis might be worse . supracricoid laryngectomies , a more aggressive type of partial horizontal laryngectomy , pose problems on the airway ( and thus on the possibility of decannulation ) , on phonation and on swallowing , that may vary depending on every particular case ( figure 1 ) . vertical hemilaryngectomy requires and increased effort for laryngeal adduction and frequently facilitating maneuvers , as one side of the larynx in loss and reconstructed with more or less static structures . patients undergoing total laryngectomy usually do not have significant swallowing problems after surgery ( although they usually do have at diagnosis , so frequently from the patient 's view in this regard function is improved ) . occasionally they may have problems with bolus propelling or strictures in the pharyngeal suture , as well as alterations in the cricopharyngeal sphincter and esophageal motility . it is associated with a rate of morbimortality and impairment in the quality of life . it has a maximum incidence in the elderly , patients with neurological disorders and patients with head and neck cancer . patients in these and other clinical situations may be candidates to be evaluated for swallowing disorders . there is growing evidence that screening for dysphagia is advisable in different groups of patients . a high percentage of patients with head and neck cancer , and virtually all of those with advanced stage tumors , will suffer dysphagia before , during , or after treatment . the assessment of od should include a detailed history focusing on the medical status of the patient , and a comprehensive clinical examination . from the clinical point of view , although the efficiency of oral nutrition might be impaired ( which would lead to malnutrition and dehydration if no alternative route is used ) , it is the possibility of aspiration what focuses the most pressing interest . usually patients with impaired safety of the swallow show cough ( and ultimately aspiration and pneumonia ) , but silent aspiration is not unusual . swallowing trials may be performed on a bedside fashion . the accuracy of the volume - viscosity swallow test for clinical screening of od and aspiration has been demonstrated . it assesses a series of items on the effectiveness and safety of the swallowing in a systematic fashion with different volumes ( 5 , 10 , and 20 ml ) and viscosities ( liquid , nectar , and pudding ) . the main goals of the clinical assessment are screening for the presence of dysphagia , and if so , determining the risk of aspiration and the feasibility of oral nutrition , therefore avoiding the most usual complications , namely , malnutrition and aspiration pneumonia . modifications of the diet , facilitating maneuvers , and other therapeutic measures might be recommended , but if efficiency and safety are not warranted , nonoral nutrition should be advised . finally , dysphagia - related quality of life of the patients can also be assessed by specific questionnaires . virtually every patient with head and neck cancer will show some degree of at least temporary dysphagia that will impact his quality of life . also some conditions associated with the swallowing disorder , like requiring a nasogastric feeding tube , are felt as particularly troublesome for the patients . instrumental assessment of swallowing provides useful information on both the structure and function of the swallowing mechanism , also when anatomy has been changed by the surgical procedure . there are two main diagnostic procedures to be used in the assessment of od . a high - resolution video is used to record a movie that can be later measured and timed on slow motion or static pictures ( figure 2 ) . the modified barium swallow is the technique of choice for the diagnosis of oropharyngeal dysphagia when there is an attributable cause ( as it does happen in head and neck cancer patients ) . the test allows the examiner to observe the interactions between the different swallowing phases and assesses the whole dynamics of the process ( figure 3 ) . a high - resolution video is used to record a movie that can be later measured and timed on slow motion or static pictures ( figure 2 ) . the modified barium swallow is the technique of choice for the diagnosis of oropharyngeal dysphagia when there is an attributable cause ( as it does happen in head and neck cancer patients ) . the test allows the examiner to observe the interactions between the different swallowing phases and assesses the whole dynamics of the process ( figure 3 ) . fiberoptic endoscopic evaluation of swallowingit is a way of directly observing the act of swallowing by means of a flexible endoscope passed through the nose and situated at the nasopharynx facing down towards the hypopharynx . different consistencies and volumes of a colored substance are used for the test . in the swallow test both oral and pharyngeal phases the examination provides particularly complete information on the structure and function of the pharyngeal phase . it can also assess palatal function and the normal movement of the larynx in respiration and phonation . dribble will indicate lip incompetency , and dripping to the hypopharynx will show incompetency of the palatoglossus closure , posing a risk of predeglutition aspiration . when asking the patient to swallow , tongue base movement should be observed to assess propulsion . there might remain residues in the mouth or exist nasopharyngeal reflux if there is nasopharyngeal incompetency . the sequence and synchrony of the movements of the pharyngeal phase should be observed , as well as eventual penetration ( food enters the laryngeal vestibule but remains over the glottic plane ) or aspiration ( there is food in the airway under the vocal folds ) ( figure 4 ) . a sensitivity test can also be performed either with the tip of the endoscope or , ideally , with air pulses . it also gives valuable information on the management of secretions , and it can also be used as a feedback in retraining therapy . it is a way of directly observing the act of swallowing by means of a flexible endoscope passed through the nose and situated at the nasopharynx facing down towards the hypopharynx . different consistencies and volumes of a colored substance are used for the test . in the swallow test both oral and pharyngeal phases the examination provides particularly complete information on the structure and function of the pharyngeal phase . it can also assess palatal function and the normal movement of the larynx in respiration and phonation . dribble will indicate lip incompetency , and dripping to the hypopharynx will show incompetency of the palatoglossus closure , posing a risk of predeglutition aspiration . when asking the patient to swallow , tongue base movement should be observed to assess propulsion . there might remain residues in the mouth or exist nasopharyngeal reflux if there is nasopharyngeal incompetency . the sequence and synchrony of the movements of the pharyngeal phase should be observed , as well as eventual penetration ( food enters the laryngeal vestibule but remains over the glottic plane ) or aspiration ( there is food in the airway under the vocal folds ) ( figure 4 ) . a sensitivity test can also be performed either with the tip of the endoscope or , ideally , with air pulses . it also gives valuable information on the management of secretions , and it can also be used as a feedback in retraining therapy . a number of other procedures can provide additional information on selected cases : other endoscopic procedures like conventional upper digestive tract endoscopy or transnasal esophagoscopy , esophagic manometry , 24-hour ph - metry . historically , the systematic therapeutic approach to patients with od was first attempted by speech pathologist , as they realized that they could not treat their patients with cerebral palsy if patients were not able to adequately manage oral secretions . the particular organization and composition of the therapeutic team may differ in every institution although a multidisciplinary team will be required , ideally in specific units devoted to the treatment of dysphagia . most of the swallowing disorders can be improved or solved with an adequate personalized training depending on the patient 's condition . this means that the patient can rely on oral diet and will not have aspiration . patients should be informed before the oncologic treatment on the possibility of dysphagia and on the eventual need for rehabilitation . although dysphagia is a usual symptom at diagnosis ( and depending on the tumor it may improve with the treatment ) , pretreatment counseling on the expected swallowing difficulties will help prepare the patient for rehabilitation . some prophylactic measures may be undertaken . when radiation therapy is to be given , it is useful counseling on oral hygiene , avoidance of alcohol and tobacco , hydration , and artificial saliva when required . chemotherapy - related mucositis would be given a symptomatic treatment , usually in the form or mixtures for mouthwashes . severe mucositis can require hospitalization and intravenous treatment or even modifications of the scheme of treatment . when planning a function - preserving surgery , the surgeon has to make certain that , with the preserved or reconstructed structures , deglutition ( without aspiration ) will be possible with adequate rehabilitation . otherwise , alternative options should be considered . a detailed description of treatment modalities for swallowing impairment is beyond the scope of this paper . there is a wide array of therapeutic medical procedures that will fall in some of the following categories . modifications of the environmentthe patient will need a quiet environment and enough time to eat . different specific instruments to introduce the food in the mouth may be preferable in different situations , including specifically designed ones like the glossectomy spoon . although supervision is adequate , the patient should be encouraged to self - feeding ( when possible ) . different specific instruments to introduce the food in the mouth may be preferable in different situations , including specifically designed ones like the glossectomy spoon . although supervision is adequate , the patient should be encouraged to self - feeding ( when possible ) . diet modificationsvolume and consistency of the bolus should be modified according to the findings in the clinical tests . sour bolus has been found to significantly shorten pharyngeal transit time in patients with head and neck cancer . volume and consistency of the bolus should be modified according to the findings in the clinical tests . sour bolus has been found to significantly shorten pharyngeal transit time in patients with head and neck cancer . muscular rehabilitationdifferent physiologic exercises may be advisable , like motion exercises or resistance exercises for the jaw , lips , oral tongue , tongue base , laryngeal elevation , laryngeal closure . different physiologic exercises may be advisable , like motion exercises or resistance exercises for the jaw , lips , oral tongue , tongue base , laryngeal elevation , laryngeal closure . a variety of choices are available : thermal stimulation by altering food temperature , tactile stimulation by applying pressure to the tongue , sensory stimuli ( anterior facial arch ) to elicit the oropharyngeal phase , introducing mastication ( when possible) . a variety of choices are available : thermal stimulation by altering food temperature , tactile stimulation by applying pressure to the tongue , sensory stimuli ( anterior facial arch ) to elicit the oropharyngeal phase , introducing mastication ( when possible) . they also allow the patient to voluntarily modify the dimensions and relationships of the different anatomic structures . this may be used alone or in combination.chin-to-chest maneuver : holding the chin down against the chest facilitates the contact of the tongue base with the posterior pharyngeal wall . it is advisable whenever there is a delay in the swallowing reflex.head extension : helps nasopharyngeal closure and facilitates oral and pharyngeal transit when there is a deficit in the lip or nasopharyngeal closure , or impaired lingual propulsion . adequately preserved laryngeal closure and elevation are imperative to prevent aspiration during the maneuver.head rotation : to one side helps the bolus pass down through the opposite pyriform sinus and closes a damaged pharynx or a paralyzed larynx.head tilt : makes gravity help the bolus down through the healthy side.lying supine or lateral : minimizes the effect of gravity in the bolus when there is poor voluntary control of the mouth to pharynx passage . they also allow the patient to voluntarily modify the dimensions and relationships of the different anatomic structures . this may be used alone or in combination.chin-to-chest maneuver : holding the chin down against the chest facilitates the contact of the tongue base with the posterior pharyngeal wall . it is advisable whenever there is a delay in the swallowing reflex.head extension : helps nasopharyngeal closure and facilitates oral and pharyngeal transit when there is a deficit in the lip or nasopharyngeal closure , or impaired lingual propulsion . adequately preserved laryngeal closure and elevation are imperative to prevent aspiration during the maneuver.head rotation : to one side helps the bolus pass down through the opposite pyriform sinus and closes a damaged pharynx or a paralyzed larynx.head tilt : makes gravity help the bolus down through the healthy side.lying supine or lateral : minimizes the effect of gravity in the bolus when there is poor voluntary control of the mouth to pharynx passage . chin - to - chest maneuver : holding the chin down against the chest facilitates the contact of the tongue base with the posterior pharyngeal wall . head extension : helps nasopharyngeal closure and facilitates oral and pharyngeal transit when there is a deficit in the lip or nasopharyngeal closure , or impaired lingual propulsion . adequately preserved laryngeal closure and elevation are imperative to prevent aspiration during the maneuver . head rotation : to one side helps the bolus pass down through the opposite pyriform sinus and closes a damaged pharynx or a paralyzed larynx . lying supine or lateral : minimizes the effect of gravity in the bolus when there is poor voluntary control of the mouth to pharynx passage . specific swallowing maneuversswallowing maneuvers are designed to alter the physiology of the swallow.supraglottic swallow : closes the vocal folds before and during the swallow to prevent aspiration . this is obtained by a voluntary apnea before the swallow . a voluntary after - swallowing it is useful when there is weakness in the tongue base or an altered pharyngeal peristalsis . it can be assisted by applying the hand on the patient 's forehead and instructing him to press while swallowing.super-supraglottic swallow : is an it is used when laryngeal closure is deficient.mendelsohn maneuver : enhances anterior - superior displacement of the larynx to facilitate cricopharyngeal opening . it is performed by manual displacement and holding of the larynx , and it is indicated when the normal physiologic excursion is impaired or when deglutition is uncoordinated . it improves the transit of the bolus and reduces residues.masako maneuver : ( tongue holding maneuver by biting it ) facilitates the movement of the tongue base and its contact with the posterior pharyngeal wall.repeated swallow : dry swallow reduces residues . swallowing maneuvers are designed to alter the physiology of the swallow.supraglottic swallow : closes the vocal folds before and during the swallow to prevent aspiration . this is obtained by a voluntary apnea before the swallow . a voluntary after - swallowing it is useful when there is weakness in the tongue base or an altered pharyngeal peristalsis . it can be assisted by applying the hand on the patient 's forehead and instructing him to press while swallowing.super-supraglottic swallow : is an it is used when laryngeal closure is deficient.mendelsohn maneuver : enhances anterior - superior displacement of the larynx to facilitate cricopharyngeal opening . it is performed by manual displacement and holding of the larynx , and it is indicated when the normal physiologic excursion is impaired or when deglutition is uncoordinated . it improves the transit of the bolus and reduces residues.masako maneuver : ( tongue holding maneuver by biting it ) facilitates the movement of the tongue base and its contact with the posterior pharyngeal wall.repeated swallow : dry swallow reduces residues . supraglottic swallow : closes the vocal folds before and during the swallow to prevent aspiration . this is obtained by a voluntary apnea before the swallow . a voluntary after - swallowing it is useful when there is weakness in the tongue base or an altered pharyngeal peristalsis . it can be assisted by applying the hand on the patient 's forehead and instructing him to press while swallowing . mendelsohn maneuver : enhances anterior - superior displacement of the larynx to facilitate cricopharyngeal opening . it is performed by manual displacement and holding of the larynx , and it is indicated when the normal physiologic excursion is impaired or when deglutition is uncoordinated . masako maneuver : ( tongue holding maneuver by biting it ) facilitates the movement of the tongue base and its contact with the posterior pharyngeal wall . repeated swallow : dry swallow reduces residues . depending on the surgical procedure , and on the swallowing alterations observed in the clinical evaluation , patients will require a personalized therapeutic program that will include a number of the abovementioned resources ( figures 5 and 6 ) . the first one is the importance of a meticulous care in the technique of the function - preserving surgery , with particular attention in the surgical steps specifically directed to improve or preserve swallowing . there are a number of defined entities with specific surgical treatment like procedures for vocal cord medialization . there are also nutritional concerns in the treatment of patients with head and neck cancer , not only before treatment , but also afterwards . patients with mucositis , xerostomia , dysgeusia , odynophagia , or those on liquid diet might be unable ( or unwilling ) to meet their nutritional requirements . nutritional support will improve functional outcomes and the patient 's sense of wellbeing . finally , if oral nutrition is not possible , an alternative method of enteral nutrition should be offered . when a long - term need is expected , gastrostomy should be the option taken . in this situation the airway needs to be protected ; this may be achieved by a tracheostomy with a cuffed cannula ( although deglutition will be further impaired with this measure ) or by means of laryngeal exclusion ( usually by laryngectomy ) , which would of course destroy the expectations of a functional treatment , but would perhaps save the life of the patient . oropharyngeal dysphagia is a critical concern in any function - preserving surgical procedure in patients with laryngeal and hypopharyngeal cancer . this may be done either by clinical or instrumental methods , depending on every particular situation . swallowing rehabilitation is imperative in most aggressive procedures , to the extent that the functional outcome may rely on it .

on considering a function - preserving treatment for laryngeal and hypopharyngeal cancer , swallowing is a capital issue . for most of the patients , achieving an effective and safe deglutition will mark the difference between a functional and a dysfunctional outcome . we present an overview of the management of dysphagia in head and neck cancer patients . a brief review on the normal physiology of swallowing is mandatory to analyze next the impact of head and neck cancer and its treatment on the anatomic and functional foundations of deglutition . the approach proposed underlines two leading principles : a transversal one , that is , the multidisciplinary approach , as clinical aspects to be managed in the oncologic patient with oropharyngeal dysphagia are diverse , and a longitudinal one ; that is , the concern for preserving a functional swallow permeates the whole process of the diagnosis and treatment , with interventions required at multiple levels . we further discuss the clinical reports of two patients who underwent a supracricoid laryngectomy , a function - preserving surgical technique that particularly disturbs the laryngeal mechanics , and in which swallowing rehabilitation dramatically conditions the functional results .

1. Introduction 2. Physiology of Deglutition 3. Diagnosis of Dysphagia 4. Treatment 5. Conclusion

diabetic nephropathy ( dn ) is one of the most devastating microvascular complications of diabetes , which remains the most common cause for end stage renal disease ( esrd ) . the prevalence of diabetes and the patients suffering from diabetic microvascular complications is increasing worldwide . nearly one - third of patients with diabetes develop nephropathy , and early diagnosis is critical in preventing long - term kidney loss . however , the mechanisms that cause dn have not been completely clarified , and the treatment options are limited . hyperglycemia plays a pivotal role in activating various inflammatory pathways in the development and progression of dn . it induces the fibrotic factor transforming growth factor- ( tgf- ) and fibronectin ( fn ) , the renin - angiotensin - aldosterone system ( raas ) , and advanced glycation end products both directly and via gene transcription , which leads to thickening of the glomerular and tubular basement membranes , progressive accumulation of extracellular matrix ( ecm ) proteins , interstitial fibrosis , and glomerulosclerosis [ 36 ] . fn is one of the main ingredients of ecm and an important symbol of cell injury . clinical trials have reported that strict glycemic control reduces the progression of diabetic complications over time . diabetic complications , including chronic kidney disorders such as dn , have been shown to persist and progress even after glucose control has been achieved , possibly as a result of prior episodes of hyperglycemia that are considered the epigenetic metabolic memory . preliminary work using endothelial cells has shown that transient episodes of hyperglycemia can induce changes in gene expression that are dependent on the modification of histone tails ( i.e. , methylation ) . the persistence of such modifications has not been fully explained . additional studies regarding the epigenetic mechanisms involved are necessary to provide valuable new insights into the pathology of dn . posttranslational modifications of the aminoterminal tails of nucleosomal histones , including acetylation , methylation , ubiquitination , phosphorylation , and sumoylation , play key roles in modulating the chromatin structure and gene transcription that have been implicated in regulating the metabolism of diabetic complications . the modification of histones by ubiquitination is a prominent epigenetic mark that may influence changes in gene expression and involves a variety of chromatin - based events , such as gene silencing and repair of dna damage . the majority of histone ubiquitination occurs on chromatin by the addition of a single ubiquitin molecule via isopeptide linkage to a specific lysine residue on the c - terminal tail of histones h2a and h2b . to a lesser extent , histones h1 , h3 , and h4 can be ubiquitylated in vivo , and ubiquitination of different histones has distinct functions . however , the effects of histone ubiquitination on dn are unclear . recent research has indicated that histone modification is directly or indirectly related to diabetic attacks [ 1215 ] . histone acetylation can activate the tgf- signaling pathway , which plays an important role in dn renal fibrosis . similarly , dn is associated with increased renal h3k9 and h3k23 acetylation , h3k4 dimethylation , and h3 phosphorylation at serine 10 , which enhances chromatin unfolding and gene expression [ 16 , 17 ] . to date , it is unknown whether histone ubiquitination is involved in interstitial fibrosis and glomerulosclerosis in dn or whether the effects of hyperglycemia on such epigenetic events can be mediated through tgf- signaling pathways . mg132 , a proteasome inhibitor , is suggested to attenuate hypertension - induced cardiac remodeling and dysfunction by downregulating the levels of tgf-1 . whether ubiquitin proteasome inhibitors can inhibit renal fibrosis which was followed by activation of the tgf- signaling pathway in diabetic nephropathy remain unclear . so , additional research to develop new treatments for dn is necessary . in this study , we evaluated the influence of high glucose on the induction of histone h2a ubiquitination , reduced histone h2b ubiquitination in gcms , and changes in the expression of tgf- followed by abnormal histone ubiquitination . mg132 , which acts as a ubiquitin proteasome inhibitor , may prevent the alterations in h2a and h2b ubiquitination induced by high glucose . rat glomerular mesangial cells ( hbzy-1 ) were purchased from the preservation center at wuhan university and maintained in low glucose dmem with 10% fetal bovine serum at 37c and 5% co2 . gmcs were used between the 2nd and 5th passages for all experiments and randomly divided into the following six groups : the normal control group ( nc group ) , with medium containing 5.6 mmol / l glucose , the 10 mmol / l glucose group ( hg1 group ) , with medium containing 10 mmol / l glucose , the 20 mmol / l glucose group ( hg2 group ) , with medium containing 20 mmol / l glucose , the 30 mmol / l glucose group ( hg3 group ) , with medium containing 30 mmol / l glucose , the osmotic pressure group as a control ( op group ) , with medium containing 5.6 mmol / l glucose + 22.4 mmol / l mannitol , the mg132 intervention group ( mi group ) , with medium containing 30 mmol / l glucose + 1 mol / l mg132 . the normal control group ( nc group ) , with medium containing 5.6 mmol / l glucose , the 10 mmol / l glucose group ( hg1 group ) , with medium containing 10 mmol / l glucose , the 20 mmol / l glucose group ( hg2 group ) , with medium containing 20 mmol / l glucose , the 30 mmol / l glucose group ( hg3 group ) , with medium containing 30 mmol / l glucose , the osmotic pressure group as a control ( op group ) , with medium containing 5.6 mmol / l glucose + 22.4 mmol / l mannitol , the mg132 intervention group ( mi group ) , with medium containing 30 mmol / l glucose + 1 mol / l mg132 . each group had been cultured for 12 hr , 24 hr , and 48 hr . total proteins were isolated from gmcs using a total protein extraction kit ( kaiji , shanghai , china ) . the protein concentration was determined using bca analysis ( beyotime , shanghai , china ) . immunoblot analysis was performed using anti - ubiquityl - histone h2a ( mouse , 1 : 1000 ; millipore , usa ) , anti - ubiquityl - histone h2b ( rabbit , 1 : 1000 ; cst , usa ) , and anti - gapdh and actin ( rabbit , 1 : 1000 ; beyotime , shanghai , china ) . horseradish peroxidase - conjugated secondary antibodies ( anti - rabbit and anti - mouse ) were obtained from the beyotime institute of biotechnology , shanghai , china . proteins were detected using the enhanced chemiluminescence system and ecl hyperfilm ( millipore , usa ) . total rna was extracted from gmcs using an rna extraction kit ( tiangen biotech , beijing , china ) . a total of 500 ng rna was reverse transcribed at a final volume of 20 l using a takara rna pcr kit ( baoshengwu , dalian , china ) . four microliters of cdna were amplified in a gradient thermal cycler ( eppendorf , germany ) using the pcr master mix ( baoshengwu , dalian , china ) . the results were determined using a uv transilluminator and normalized to glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) gene expression . the primer sequences were as follows : histone h2a ubiquitination ( uh2a ) , forward , 5-gca ccc tga cct tgc cta t -3 , reverse , 5-cct tcc cag act cca cca t-3 , histone h2b ubiquitination ( uh2b ) , forward , 5-cgc ctg gct cat tac aac-3 , reverse , 5-ctt ggt ttc cga ca-3 , transforming growth factor- ( tgf- ) , forward , 5-atg gtg gac cgc aac aac-3 , reverse , 5-gag cac tga agc gaa agc-3 , fn , forward , 5- tgc cga atg tag atg agga -3 , reverse , 5-aaa tga cca ctg cca aagc -3 , and gapdh , forward , 5-cct caa gat tgt cag caa t -3 , reverse , 5-cca tcc aca gtc ttc tga gt-3. cells were grown on coverslips in 6-well plates . after overnight adherence , the cells were treated with media containing high glucose , mannitol , and the mi media for 24 the cells were fixed in 4% paraformaldehyde ( pierce ) and permeabilized in 0.2% tween 20 ( sigma ) for 10 min after being washed briefly with pbs . the cells were blocked with 5% goat serum for 1 hr at room temperature and incubated overnight with primary antibodies followed by washes with pbs . the cells were incubated for 40 min with the appropriate secondary antibody conjugated to the fitc fluorescent dye . the coverslips were washed and mounted onto slides using fluorescent mounting medium ( beyotime , shanghai , china ) . images were taken with a dmire2 laser scanning confocal microscope ( leica , germany ) . data were expressed as the mean sd ( x s ) , which were analyzed using spss 11.5 statistical software . statistical differences were calculated using one - way analysis of variance ( one - way anova ) to compare more than two groups , followed by the lsd test for multiple comparisons . the cellular morphology in different glucose culture medium is not significantly changing ( figure 1 ) , but the expression of fn increased in the high glucose group over time , especially in hg3 group for 48 hr ( figure 2 ) . after 48 hr in culture , western blot analysis showed low h2a ubiquitin expression in the nc group . expression was higher in the high glucose group compared to the nc group ( p < 0.01 ) in a concentration dependent manner . the strongest expression was in the 30 mmol / l high glucose group ( p < 0.01 ) . there were no significant differences in h2b ubiquitination expression in the 10 mmol / l high glucose group compared to the nc group ( p = 0.327 ) . expression was lower in the 20 and 30 mmol / l high glucose groups compared to the nc group ( p < 0.01 ) and was the weakest in the 30 mmol / l high glucose group . there were no differences between the op and nc groups regarding the ubiquitination of h2a and h2b ( p > 0.05 ) ( figure 3 ) . the expression of the uh2a protein increased at various time intervals in the hg3 group , particularly after 24 hr in culture . the increased expression of uh2a at various time points was statistically significant ( p < 0.01 ) . expression of the uh2b protein was significantly reduced in a time dependent manner ( p < 0.01 ) ( figure 4 ) . after mg132 intervention , the expression of uh2a was significantly decreased compared to expression in the 30 mmol / l glucose group ( p < 0.05 ) . in contrast , the expression of uh2b was recovered in the mi group compared to the hg3 group ( p < 0.05 ) ( figure 5 ) . the same results were detected using cell immunofluorescent staining and laser scanning confocal microscopy applications ( figure 6 ) . the expression of tgf- mrna increased in the high glucose group compared to the nc group ( p < 0.01 ) in a concentration and time dependent manner ( figure 7 ) . and it decreased along with the normalization of uh2a and h2b protein expression ( p < 0.05 ) , although the mrna levels of uh2a and h2b were not statistically different in each group ( figure 8) . hyperglycemia plays an important role in the development and progression of dn , which can induce the expression of fn and cause cell injury , but the dna sequence changes can not solely explain the heritable patterns of gene expression . however , hyperglycemic memory may explain why intensive glucose control has failed to improve cardiovascular outcomes in patients with diabetes , although the molecular mechanisms of this phenomenon remain to be elucidated . the current study found histone modifications can change the state of evacuation and aggregation in chromatin by affecting compatibility between the histones and double - stranded dna and influencing the affinity of transcription factors for structural gene promoters , which can regulate the expression of genes . recent studies have shown that diabetes - induced epigenetic changes can affect gene expression in vascular endothelial cells and vascular smooth muscle cells and long - lasting changes in epigenetic modifications at key inflammatory gene promoters following exposure to diabetic conditions [ 7 , 21 ] . histone acetylation attenuates epidermal growth factor signaling , which has a key role in the development of dn , and genome - wide studies have shown cell - type specific changes in histone methylation patterns under conditions of dn [ 22 , 23 ] . and in diabetic retinopathy , histone acetylation was significantly increased in retinas from diabetic rats and contributed to the hyperglycemia - induced upregulation of proinflammatory proteins and thereby to the development of diabetic retinopathy . in dn , histone acetylation , specific histone acetyl transferases , and histone deacetylases significantly enhanced tgf-1-induced gene expression in rat mesangial cells and in glomeruli from diabetic mice and augmented glomerular dysfunction linked to diabetic nephropathy . histone methylation has also gained much attention as potential molecular mechanisms underlying metabolic memory and dn . the specific set7 methyltransferase is the best characterised lysine enzyme , which showed high expression in dn . furthermore , the contribution of set7 to the aetiology of diabetic complications may extend to other transcriptional events through methylation of nonhistone substrates [ 26 , 27 ] . however , little is known about histone ubiquitination in diabetic nephropathy . in this study , we found high glucose may cause cell damage , induce the ubiquitination of histone h2a , and reduce the ubiquitination of histone h2b in gmcs . the results indicate that histone h2a and h2b ubiquitination may be involved in the development and progression of dn as an epigenetic mechanism . although the mechanisms of action vary for different histones , ubiquitination of histone h2a k119 may induce dn and ubiquitination of histone h2b k120 has been shown to delay the onset of dn . tgf- has been implicated in various human disorders , including vascular and renal diseases , and is a primary fibrotic factor . diabetic nephropathy ( dn ) is a chronic renal complication characterized by thickening of the glomerular and tubular basement membranes and progressive accumulation of extracellular matrix ( ecm ) proteins , such as type i and type iv collagens , fibronectin , and laminin in the tubular interstitium and mesangium . tgf- increases ecm accumulation and plays a major role in the development of chronic renal diseases through the induction of a downstream effector , which is a connective tissue growth factor , and by decreasing matrix degradation through the inhibition of proteases or activation of protease inhibitors . the tgf- signaling pathway is controlled by many factors , including histone modification and epigenetic chromatin marks , such as histone h3 lysine methylation in tgf-1-induced gene expression in rat mesangial cells under normal and high glucose conditions . tgf-1 has been shown to increase the expression of ecm - associated genes , the connective tissue growth factor collagen-1 , and plasminogen activator inhibitor-1 . increased levels of histone h3 k4 methylation associated with active genes and decreased levels of histone h3 k9 methylation at these gene promoters accompany changes in expression . tgf-1 also increased the expression of h3 k4 methyltransferase set7/9 and recruitment to these promoters . set7/9 gene silencing with sirnas significantly attenuated tgf-1-induced ecm gene expression . in this study , we did not examine changes in the mrna levels of uh2a and uh2b as a consequence of uh2a and uh2b proteins stimulation by high glucose . this implies that there is no difference in the gene order of histones h2a and h2b , except for posttranslational modifications , including histone ubiquitination . we observed that the mrna level of tgf- dramatically increased followed by changes in uh2a and uh2b proteins . in summary , changes in uh2a and uh2b protein expression induced by high glucose in gmcs may enhance the activation of tgf- and influence the pathogenesis of dn . mg132 exerts an antifibrotic effect by simultaneously downregulating type i collagen and a tissue inhibitor of metalloproteinase-1 and upregulating metalloproteinase-1 production in human dermal fibroblasts . tubular injury in a rat model of type 2 diabetes was shown to be prevented by mg132 by reducing renal tubule interstitial fibrosis . several studies have shown that mg132 has an effect on mitigating renal fibrosis by inhibiting the expression of kidney fibronectin mrna in rats with early diabetic nephropathy and could improve proteinuria and other symptoms . research on histone ubiquitination is scarce , and inhibitors that can effectively and specifically block the ubiquitination of histones have not been described . mg132 is a specific ubiquitin proteasome inhibitor that can inhibit activation of the tgf- signaling pathway , which is important in the development of fibrosis in dn . however , there is not any evidence in the literature about whether mg132 can inhibit histone ubiquitination disorders or eliminate epigenetic metabolic memory to treat dn . our experiments show that disorders involving histone h2a and h2b ubiquitination can exhibit an apparent reversal trend based on treating rat glomerular mesangial cells with mg132 and 30 mmol / l high glucose . after eliminating the dysfunction of histone ubiquitination this suggests that ubiquitin proteasome inhibitors may have a positive function in the treatment of diabetic nephropathy by inhibiting the disorders involving histone h2a and h2b ubiquitination that affects gene expression of tgf-. the proteasome inhibitor mg132 induces apoptosis . for example , mg132 inhibited the pi3k / akt and nfb pathways , promoted mitochondrial depolarization , and decreased the concentration of mitochondrial antiapoptotic protein . mg132 also mediated activation of p38-jnk1/2 signaling and enhanced selective apoptosis in glioblastoma cells [ 34 , 35 ] . mg132 is promising for cancer treatment because it markedly inhibited the growth of malignant tumor cells and arrested cells in the g2/m phase of the cell cycle , and the cells become apoptotic . strom and panlsen reported that mg132 inhibited the ubiquitin proteasome , which degrades the apoptosis inducer ngfi - b and is a nuclear receptor . thakur suggested that mg132 inhibits histone deacetylation by increasing the degradation of histone deacetylase induced by green tea polyphenols in a prostate cancer cell line , followed by cell arrest and apoptosis . possible answers regarding the causes of histone ubiquitination were identified in this study , but the detailed mechanisms involved will require additional research . determining the specific inhibitors of histone ubiquitination and discovering the role of histone ubiquitination in diabetic nephropathy renal fibrosis remain a challenge . in conclusion , we demonstrated that the high glucose may induce the ubiquitination of histone h2a and reduce the ubiquitination of histone h2b in gmcs . the changes of histone ubiquitination in gmcs could activate tgf- signaling pathway involved in the pathogenesis of diabetic nephropathy .

background . hyperglycemia plays a pivotal role in the development of diabetic nephropathy ( dn ) and may be related to epigenetic metabolic memory . one of the most crucial epigenetic mechanisms is histone modification , which is associated with the expression of a fibrosis factor in vascular injury . aim .in this study , we investigated the ubiquitination of histones h2a and h2b to explore the epigenetic mechanisms of dn . materials and methods . the gmcs were cultured as follows : normal group , high glucose group , mannitol group , and intervention group . after 12 hr , 24 hr , and 48 hr , histones ubiquitination , transforming growth factor- ( tgf- ) , and fibronectin ( fn ) were measured using wb , rt - pcr , and if . result . high glucose can induce the upregulation of fn . h2a ubiquitination in gmcs increased in high glucose group ( p < 0.01 ) , whereas it decreased significantly in intervention group ( p < 0.05 ) . in contrast , h2b ubiquitination decreased with an increasing concentration of glucose , but it was recovered in the intervention group ( p < 0.05 ) . expression of tgf- changed in response to abnormal histone ubiquitination . conclusions . the high glucose may induce h2a ubiquitination and reduce h2b ubiquitination in gmcs . the changes of histone ubiquitination may be due in part to dn by activating tgf- signaling pathway .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

obesity , influenced by environmental and genetic factors , is one of the important factors in the etiology of metabolic syndrome , cardiovascular disease ( cvd ) , and cancer [ 13 ] . obesity is related to increased level of inflammatory markers such as crp ( c - reactive protein ) that are associated with metabolic syndrome . according to evidence of 2005 , obesity is significantly correlated with an increase in dietary energy density and also it can elevate the risk of cvd and metabolic syndrome [ 6 , 7 ] . adherence to the healthy dietary pattern and high intake of fruits and vegetables are inversely related to metabolic syndrome . moreover , the consumption of plant proteins , such as beans , drives a significant reduction in the inflammatory markers such as crp , while meat consumption can increase inflammatory markers in bloodstream [ 10 , 11 ] . a study showed that the risk of cancer in obese women is 50% more than women with normal weight . according to published studies , obesity , inflammation , insulin resistance , metabolic syndrome , cardiovascular disease , and cancer plasma adiponectin levels in normal healthy individuals are 10 g / ml where it accounts 0.01% of all plasma proteins . this hormone presents in plasma as two important epimers : low molecular weight ( lmw ) or trimeric complex and high molecular weight ( hmw ) or oligomeric complex [ 15 , 18 ] . it also has an impact on pathogenesis of diabetes through the regulation of glucose and free fatty acid metabolisms and insulin sensitivity in the epithelial cells [ 19 , 20 ] . adipo r1 receptors are predominantly expressed in muscle and adipo r2 receptors are mainly expressed in the liver . although the hormone is secreted from adipose tissue , as the weight increases the plasma level of adiponectin decreases . higher adiponectin concentrations in women are more likely due to estrogen or androgen in their bloodstream and are independent of fat mass . while testosterone in men causes a reduction in concentrations of serum adiponectin and prevents production of hmw in adipose tissue . several studies suggest that individuals with higher levels of adiponectin show generally higher concentrations of high density lipoprotein ( hdl ) and lower levels of low density lipoprotein ( ldl ) , triglycerides ( tg ) , and total cholesterol . this hormone also plays an important role in the secretion of estrogen and insulin - like growth factor ( igf ) , which are important cancer risk factors . studies indicate that cancer cells express adiponectin receptors thus attaching adiponectin to its receptors may limit the proliferation of cancer cells [ 18 , 26 ] . since obesity and insulin resistance are risk factors of cancer , adiponectin may act as an anticancer agent specially in breast cancer due to its significant effect on obesity and insulin resistance . the relationship between adiponectin concentrations and prevention of cancer cells proliferation and its anticancer role [ 1 , 27 ] , and also the concentration of this hormone in patients with different types of cancers such as endometrial , breast , prostate , and colorectal cancer , has been investigated in several recent studies [ 2629 ] which indicates a close relationship between adiponectin and types of cancers . most studies [ 1 , 27 ] but not all of them suggest that the relationship between adiponectin and cancer is correlated with hormones , including estrogen , igf1 , obesity , and insulin resistance . this present study aims to review available evidence related to the serum adiponectin and breast , prostate , endometrial , and gastrointestinal tract cancers . in order to examine the relationship between serum adiponectin levels and breast cancer , endometrial cancer , prostate cancer , and colorectal cancer , 153 articles between 2002 and october 2011 were accessed by using pubmed search engine and using keywords such as : cancer , malignancy , cell proliferation , and adiponectin . 45 articles with case control , cross - sectional , and prospective cohort design were reviewed . other articles were excluded owing to lack of direct relation with the issue , duplication , and lack of access to full text articles . studies that investigated among association between levels of adiponectin and cancer are shown in table 1 . findings from several studies indicate an inverse relationship between serum adiponectin levels and breast cancer [ 18 , 19 , 26 ] . in a case control study on comprising 102 women with primary breast cancer patient and 100 healthy women , in the age of 5155 years , those in the lowest tertile of serum adiponectin levels ( 9.6 g / ml ) had a significantly higher risk of having breast cancer compared with women in the highest tertile ( 10.6 g / ml ) . this relationship was observed in women both in pre- and postmenopause ( p < 0.005 ) . another study showed , that risk of breast cancer in cases who were in the highest quartile of serum adiponectin levels were 77% less than those who were in the lowest quartile of hormone ( p = 0.02 ) . patients with cancer had 6.21% and 3.12% lower levels of adiponectin and hmw , respectively . there was no significant relationship between hormone levels and risk of breast cancer in pre- and postmenopausal women ( ptrend = 0.09 and ptrend = 0.08 , resp . ) . in another case - control study on women with cancer and healthy women , aged 2965 years , adiponectin levels in the cases were significantly ( p < 0.005 ) lower than the controls . also , obesity and physical inactivity can increase risk of breast cancer by 3 and 25 times , respectively . low levels of adiponectin , obesity , and physical inactivity were reported as important risk factors for breast cancer in malaysian women . study on 41 women with cancer and 45 control women suggested that serum adiponectin levels in the cases were nonsignificantly lower than control group ( p = 0.37 ) . 15.6% reduction in proliferation of mcf-7 breast cancer cells was reported by 10 g / ml adiponectin levels , during the 96 hours , in comparison with controls . to conclude , the findings of different studies suggest that risk of breast cancer in low adiponectin concentrations increases independent of bmi , leptin , igf1 , and menopausal status [ 19 , 20 ] . according to a survey in 2004 , higher bmi and lower serum adiponectin levels increased risk of endometrial cancer , 6.5 times , more than women with normal bmi and high adiponectin concentrations . in this study , adiponectin was considered as a mediator to explain the relationship between insulin resistance , obesity , and endometrial cancer . in a prospective nested case - control study which was conducted on over 284 women with endometrial cancer and 548 healthy women , those who had the highest adiponectin levels were significantly at lower risk of endometrial cancer compared with the women with the lowest levels of hormone ( ptrend = 0.006 ) . also women with the lowest concentrations of adiponectin were at risk of endometrial cancer 11 fold more than women who have the highest level of the hormone . the results of most epidemiological studies suggest an inverse relationship between hormone levels and risk of prostate cancer [ 28 , 42 ] . in a prospective study on 654 men with prostate cancer and 644 healthy men , the cases in the highest quintile of serum adiponectin had lower risk of fatal prostate cancer , compared with the lowest quintile of adiponectin concentration . this relationship was not substantial after adjusting the effect of bmi and c - peptide . based on this study , adiponectin is considered as a possible mediator between obesity and prostate cancer . in another study on 25 patients with benign prostatic hyperplasia and 43 prostate cancer patients , also in a study , serum adiponectin was called as a potential antiprostate cancer . in another case - control study , different types of adiponectin gene in african american men with prostate cancer , who participated in a community - based case - control study , did not show significant association with prostate cancer . it seems that there is an inverse relationship between serum adiponectin concentrations , colon , and colorectal cancer [ 30 , 47 , 48 ] . in a cohort study , low levels of adiponectin were considered as a more important risk factor than bmi and tg in early stage of colorectal cancer . the risk of colorectal cancer in men who had high plasma adiponectin concentrations was 60% lower than those with lower levels of adiponectin after adjusting for some potential confounding ( bmi , waist circumference , and physical activity ) . in a nested prospective case - control study on 381 men with colorectal cancer and 381 healthy men , no significant association between hormone levels and colorectal cancer was found . on the other hand , in another study on men and women aged 4880 with colon cancer , with and without cachexia , a higher level of adiponectin was observed in those with 5% or more bmi reduction in 6 months . in this study results from several case - control colorectal cancer studies represent important role of a variety of adiponectin gene ( adipoq ) at increasing the risk of colorectal cancer or reduce its risk . results of a meta - analysis did not show any relationship between adipoq change and colorectal cancer . in another case - control study on 420 cancer patients with colorectal cancer and 555 control subjects , matched by age and sex factors , substantial association was found between adipo r1 receptor and risk of colorectal cancer . in a study on 62 cancer patients and 30 healthy individuals , in the age of 2083 years , adiponectin levels were significantly lower ( p < 0.001 ) in patients with oesophageal cancer . this inverse relationship was significant in those blood samples that are collected over 5 years ( ptrend = 0.031 ) . while the results of two small case - control studies on pancreatic cancer , a conflicting correlation show between adiponectin levels and pancreatic cancer both in men and women [ 52 , 53 ] . the evidence from studies suggests a significant inverse relationship between serum adiponectin levels and risk of prostate , breast , endometrial , and colorectal cancer . hormone levels are usually reduced in cancer patients [ 1 , 30 , 33 , 43 ] . adiponectin is inversely associated with obesity and insulin resistance and also it stimulates insulin sensitivity of peripheral tissues . on the other hand , adiponectin reduction is associated with insulin resistance which leads to an increase in the levels and activity of igf1 . high concentrations of circulating igf1 can increase the risk of breast cancer [ 2 , 27 ] . inverse relationship between adiponectin and breast cancer in post - menopausal women confirms the main effect of adiponectin in the pathogenesis of insulin resistance - related cancers such as breast cancer caused by obesity [ 27 , 54 ] . igf1 and insulin are effective in carcinogenesis through binding to their receptors which leads to an increase in cell proliferation and inhibit apoptosis in tissues which regulate the secretion of vascular endothelial growth factor ( vefg ) . this factor is restrained by increasing the level of adiponectin through stimulating the ligand of peroxisome proliferator activated receptor ( ppar ) . thus low levels of adiponectin leads to reduction in ppar activity and thus increase the risk of cancer . according to a study , the relationship between adiponectin and breast cancer risk is independent of the possible effects of igf major system components , leptin , bmi , and menopausal status [ 18 , 27 ] . adiponectin plays an effective role in glucose metabolism , so that augmentation of glucose level lead to reduction of adiponectin . importance of adiponectin on glucose and lipid metabolism is through the adiponectin receptors ( r1 and r2 ) . relationship between obesity and breast cancer is mainly due to the estrogen receptors [ 2 , 59 ] ) . . low level of adiponectin in obese patients and high level of estrogen increase the risk of breast cancer . while in another study the inverse relationship between hormone levels and breast cancer risk has been considered independent of estrogen levels during menopause . in a case - control study on 70 women with cancer , low levels of adiponectin associated with obesity and lack of physical activity throughout life was considered as important risk factors for breast cancer as physical activity can decrease the cancer risk by 25 times . physical activity can delay the first menstrual cycle by affecting a secretory production of ovarian hormones that reduce the risk of breast cancer . overweight and reduction of adiponectin concentrations are associated with endometrial cancer substantially by increasing estrogen and insulin resistance . ampk and ppar pathway activation leads to increasing insulin sensitivity and free fatty acid oxidation . on the other hand , ampk has direct effects on insulin resistance , cell proliferation , and apoptosis and it can interfere in tumor growth . a research found that hmw has an inhibitory activity on prostate cancer cell growth and proliferation . furthermore , another study indicates a reduction of expression of adipo r1 and r2 receptors in prostate cancer tissues compared to benign prostatic hyperplasia ( bph ) and healthy tissue . natural concentrations of adiponectin inhibit cancer cell growth , metastasis , and cancer cell lines and also inhibit dehydrotestosterone that stimulates cell proliferation [ 42 , 62 ] . adiponectin reduces the growth of androgen of prostate cancer cells through activation of ampk up to 90% . the hormone inhibits inflammation by inhibiting the activity of mature phagocytic macrophages which are associated with prostate cancer . in addition , it can reduce adiponectin levels and ampk activity which increase the risk of prostate cancer [ 28 , 43 ] . adiponectin has an inhibitory effect on transcription factor which is involved in regulation of vegf . the reduction of the igf binding protein ( igf1 bp ) and increasing igf1 in low levels of adiponectin concentrations can increase cell proliferation and inhibits apoptosis in colorectal cancer cells . r1 and r2 adiponectin receptor expression in pancreatic -cells expresses the relationship of adiponectin with the endocrine activity of pancreas . on the other hand , positive correlation between adiponectin concentrations and risk of pancreatic cancer in two case - control studies with small sample volumes was found [ 52 , 53 ] . furthermore , the elevation of hormone secretion in cancer cells may be due to reducing hormone receptors . however , in a study on 81 patients with pancreatic cancer and 81 healthy men and women r1 and r2 receptors on cancer cells were significantly expressed . adiponectin receptor agonist that is called 355adp was identified by isolating 149166 peptide of adiponectin sequence . 355adp adiponectin with similar behaviour to adiponectin was discussed as a recommended way to treat obesity , insulin resistance , and cancer . adherence to mediterranean dietary pattern and healthy dietary pattern can increase circulating adiponectin levels [ 6769 ] . this association is shown not only in cohort and cross - sectional studies [ 6870 ] , but also in clinical trials . consumption of diet rich in fiber , low fat dairy products , whole grain , reducing energy intake from saturated fatty acids and refined grain , and increased consumption of fish and fish products and vegetable proteins may be effective to improve adiponectin levels [ 67 , 68 ] . low calorie diet with 10% weight reduction , in overweight and obese individuals , can influence on improvement of hormone levels [ 21 , 72 , 73 ] . as the importance of dietary pattern and dietary components on circulating adiponectin level , more investigations this study aimed to review the results of studies on the relationship between serum adiponectin levels and risk of cancer . findings from different studies represent an inverse relationship between hormone levels and the risk of breast cancer [ 1 , 27 , 31 ] , independent of bmi and menopausal status . this association was observed in prospective and case - control studies [ 27 , 31 ] , both in pre- and post - menopausal women . the risk of endometrial cancer in women who had high circulating adiponectin levels was less than those with lower adiponectin levels . also , it seems that the risk of prostate cancer and benign prostatic hyperplasia in men with low adiponectin level is higher than others [ 44 , 46 ] . several cohort and case - control studies suggest an inverse relationship between serum levels of adiponectin , genomic changes , and colorectal cancer risk [ 36 , 48 ] . result of a study on esophageal cancer indicates a low level of hormones in the patients . it seems that there is an inverse relationship between plasma adiponectin and pancreatic cancer in men . however , the results are conflicting in the men and women [ 52 , 53 ] .

background . adiponectin , an adipokine secreted from adipose tissue , has antiobesity , anti - insulin resistance , and anticancer roles . the present study aimed to review the epidemiologic evidence about the association between adiponectin and cancers . method . we searched in pubmed from 2002 to october 2011 by using the following key words : cancer , malignancy , cell proliferation , and adiponectin . finally , 45 articles were recruited to review in the present paper . findings . several findings suggested inverse association between concentration of hormone and breast cancer risk . low levels of adiponectin increase the risk of endometrial cancer in women . adiponectin levels were significantly associated with prostate cancer in men . it seems that there is an inverse relationship between levels of adiponectin or its gene and colorectal cancer . significant association between hormone and pancreatic cancer was found . conclusion . several findings suggested the negative correlation between adiponectin and risk of cancers . this relationship was more elucidated by the correlation between the hormone with obesity and insulin resistance . suppression of growth and proliferation of cancer cells by adiponectin were explained via several mechanisms .

1. Introduction 2. Methods 3. Discussion 4. Conclusion

this study was approved by institutional review boards ( irb ) of the national centre of excellence in molecular biology and the combined neuroscience ( cns ) irb at the national institutes of health . ophthalmological examinations were performed at the layton rehmatullah benevolent trust hospital in lahore , pakistan . presence and types of cataract in both affected and unaffected individuals of the families were confirmed by slit lamp biomicroscopy . a cohort of 143 cc consanguineous families was collected over a period of 10 years . genome - wide linkage analyses using 384 highly polymorphic microsatellite markers and sanger sequencing had identified a molecular diagnosis in 3 unpublished and 30 previously reported families . from the remaining 110 unlinked families , 83 were selected for homozygosity screening analysis based on the availability of dna samples and an ar inheritance pattern that in addition included consanguineous matings . in addition , control dna samples were available from 96 unrelated , ethnically matched pakistani individuals . blood samples were obtained from study participants , and dna was extracted using standard methods , as previously described . thirty - three candidate genes and loci involved in cc and related disorders or based on expression and function were selected for screening ( table 1 ) . each was screened for homozygosity by genotyping 1 or 2 microsatellite markers ( total of 51 markers ; table 1 ) . the screening algorithm and a summary of the results the microsatellite markers were selected based on reported high heterozygosity ( 0.75 or more ) and were located within 1 to 2 megabases ( mb ) of the candidate gene . if a single marker with 75% or greater heterozygosity was not available , two markers were genotyped . information on the pcr primer reaction conditions , heterozygosity , and location was obtained from the unists human genome database and national center for biotechnology information ( ncbi ) mapview databases . the detection of homozygosity at a given locus in an affected family member was followed by genotyping a second affected family individual at the locus . a variant of the multiplexing short tandem repeat with tailed primers approach described by oetting et al . using fluorescently labeled tagged primers homologous to extensions on initial primers in a two - pcr approach was used to genotype these microsatellite markers . the pcr products were multiplex electrophoresed on an abi 3130 genetic analyzer ( applied biosystems , foster city , ca , usa ) , and fragment sizes were determined by genemapper version 4.0 ( applied biosystems ) . if the second individual also was homozygous , linkage was carried out with all available family members to confirm cosegregation of markers with disease . polymerase chain reaction products were separated on an abi 3130 dna analyzer ( applied biosystems ) , and alleles were assigned with genemapper software version 4.0 ( applied biosystems ) . haplotype comparisons used the cyrillic 2.1 program ( cyrillic software , wallingford , oxfordshire , uk ) for inspection to identify homozygous regions common to affected individuals in each family . two - point linkage analyses were performed with the fastlink version of mlink from the linkage program package . maximum logarithm of the odds ( lod ) scores were calculated with ilink from the linkage program package . autosomal recessive cataracts were analyzed as a fully penetrant trait with a disease allele frequency of 0.0001 , and mutation frequency of 0 . the marker order and physical distances between the markers were obtained from the marshfield database and the ncbi chromosome sequence maps . mutation screening of candidate gene coding regions used pcr amplification of exons and adjacent intronic regions . primer pairs for individual exons in the critical interval were designed online with the primer3 program ( http://primer3.sourceforge.net/ , in the public domain ) . polymerase chain reaction primers for each exon were used for bidirectional sequencing with big dye terminator ready reaction mix per instructions of the manufacturer ( applied biosystems ) . sequencing was performed using abi prism 3130 automated sequencers ( applied biosystems ) and analyzed using mutation surveyor ( soft genetics , inc . , state college , pa , usa ) and the seqman program of dnastar software ( dnastar , inc . , madison , wi , usa ) . sequence changes observed were checked for cosegregation in the family and for presence or absence in at least 96 healthy control individuals as well as the 1000 genomes ( http://www.internationalgenome.org/home , in the public domain ) and exac ( http://exac.broadinstitute.org/ , in the public domain ) databases , although low frequencies of heterozygous changes were not considered to exclude pathogenicity . a mutation was considered novel if it was not present in the human mutation database ( http://www.hgmd.cf.ac.uk/ac , in the public domain ) or the ncbi dbsnp database ( http://www.ncbi.nlm.nih.gov/projects/snp/index.html , in the public domain ) and not in cat - map ( http://cat-map.wustl.edu , in the public domain ) . a sequence variation was considered pathogenic when it cosegregated with the disease in the family ; was not present in 96 randomly selected controls from the pakistani population ; altered a well - conserved amino acid , preferably in a conserved region ( http://www.ebi.ac.uk/tools/clustalw2/index.html , in the public domain ) ; and it was judged significant in computational tests for pathogenicity . missense variants were assessed for possible causality with the online programs sorting intolerant from tolerant ( sift ; http://sift.jcvi.org/www/sift_enst_submit.html , in the public domain ) and polymorphism phenotyping ( polyphen-2 ; http://genetics.bwh.harvard.edu/pph2/ , in the public domain ) , as well as condel ( http://bg.upf.edu/fannsdb/ , in the public domain ) , which uses input from multiple programs in its assessment . this study was approved by institutional review boards ( irb ) of the national centre of excellence in molecular biology and the combined neuroscience ( cns ) irb at the national institutes of health . ophthalmological examinations were performed at the layton rehmatullah benevolent trust hospital in lahore , pakistan . presence and types of cataract in both affected and unaffected individuals of the families were confirmed by slit lamp biomicroscopy . a cohort of 143 cc consanguineous families was collected over a period of 10 years . genome - wide linkage analyses using 384 highly polymorphic microsatellite markers and sanger sequencing had identified a molecular diagnosis in 3 unpublished and 30 previously reported families . from the remaining 110 unlinked families , 83 were selected for homozygosity screening analysis based on the availability of dna samples and an ar inheritance pattern that in addition included consanguineous matings . in addition , control dna samples were available from 96 unrelated , ethnically matched pakistani individuals . blood samples were obtained from study participants , and dna was extracted using standard methods , as previously described . thirty - three candidate genes and loci involved in cc and related disorders or based on expression and function were selected for screening ( table 1 ) . each was screened for homozygosity by genotyping 1 or 2 microsatellite markers ( total of 51 markers ; table 1 ) . the screening algorithm and a summary of the results the microsatellite markers were selected based on reported high heterozygosity ( 0.75 or more ) and were located within 1 to 2 megabases ( mb ) of the candidate gene . if a single marker with 75% or greater heterozygosity was not available , two markers were genotyped . information on the pcr primer reaction conditions , heterozygosity , and location was obtained from the unists human genome database and national center for biotechnology information ( ncbi ) mapview databases . the detection of homozygosity at a given locus in an affected family member was followed by genotyping a second affected family individual at the locus . a variant of the multiplexing short tandem repeat with tailed primers approach described by oetting et al . using fluorescently labeled tagged primers homologous to extensions on initial primers in a two - pcr approach was used to genotype these microsatellite markers . the pcr products were multiplex electrophoresed on an abi 3130 genetic analyzer ( applied biosystems , foster city , ca , usa ) , and fragment sizes were determined by genemapper version 4.0 ( applied biosystems ) . if the second individual also was homozygous , linkage was carried out with all available family members to confirm cosegregation of markers with disease . polymerase chain reaction products were separated on an abi 3130 dna analyzer ( applied biosystems ) , and alleles were assigned with genemapper software version 4.0 ( applied biosystems ) . haplotype comparisons used the cyrillic 2.1 program ( cyrillic software , wallingford , oxfordshire , uk ) for inspection to identify homozygous regions common to affected individuals in each family . two - point linkage analyses were performed with the fastlink version of mlink from the linkage program package . maximum logarithm of the odds ( lod ) scores were calculated with ilink from the linkage program package . autosomal recessive cataracts were analyzed as a fully penetrant trait with a disease allele frequency of 0.0001 , and mutation frequency of 0 . the marker order and physical distances between the markers were obtained from the marshfield database and the ncbi chromosome sequence maps . mutation screening of candidate gene coding regions used pcr amplification of exons and adjacent intronic regions . primer pairs for individual exons in the critical interval were designed online with the primer3 program ( http://primer3.sourceforge.net/ , in the public domain ) . polymerase chain reaction primers for each exon were used for bidirectional sequencing with big dye terminator ready reaction mix per instructions of the manufacturer ( applied biosystems ) . sequencing was performed using abi prism 3130 automated sequencers ( applied biosystems ) and analyzed using mutation surveyor ( soft genetics , inc . , state college , pa , usa ) and the seqman program of dnastar software ( dnastar , inc . , madison , wi , usa ) . sequence changes observed were checked for cosegregation in the family and for presence or absence in at least 96 healthy control individuals as well as the 1000 genomes ( http://www.internationalgenome.org/home , in the public domain ) and exac ( http://exac.broadinstitute.org/ , in the public domain ) databases , although low frequencies of heterozygous changes were not considered to exclude pathogenicity . a mutation was considered novel if it was not present in the human mutation database ( http://www.hgmd.cf.ac.uk/ac , in the public domain ) or the ncbi dbsnp database ( http://www.ncbi.nlm.nih.gov/projects/snp/index.html , in the public domain ) and not in cat - map ( http://cat-map.wustl.edu , in the public domain ) . a sequence variation was considered pathogenic when it cosegregated with the disease in the family ; was not present in 96 randomly selected controls from the pakistani population ; altered a well - conserved amino acid , preferably in a conserved region ( http://www.ebi.ac.uk/tools/clustalw2/index.html , in the public domain ) ; and it was judged significant in computational tests for pathogenicity . missense variants were assessed for possible causality with the online programs sorting intolerant from tolerant ( sift ; http://sift.jcvi.org/www/sift_enst_submit.html , in the public domain ) and polymorphism phenotyping ( polyphen-2 ; http://genetics.bwh.harvard.edu/pph2/ , in the public domain ) , as well as condel ( http://bg.upf.edu/fannsdb/ , in the public domain ) , which uses input from multiple programs in its assessment . in the 83 unlinked arcc families , fluorescently labeled microsatellite markers flanking each of the 33 genes or loci were genotyped to test for homozygosity . in the first stage , a single affected individual from each family was screened , and all markers tested for the 33 loci were heterozygous in two unlinked arcc families . in the second stage , a second affected individual in each of the 81 remaining families was screened in regions that were homozygous in the first affected individual , and all markers were heterozygous in 49 families . in the remaining 32 families , two - point linkage analyses and haplotype analyses were performed with closely spaced microsatellite markers in regions that were homozygous in both the affected individuals tested in stages 1 and 2 . an lod less than 2 was obtained in 11 of the families , leaving 21 families requiring sequence analysis of candidate genes , of which mutations were identified in 10 . the work flow is summarized in figure 1 and the results of the linkage analysis are shown in figure 2 and table 2 . the 13 arcc pedigrees collected from pakistan including 10 families that were mapped through homozygosity mapping and 3 families that were mapped by genome - wide linkage analysis ( denoted by asterisks ) . filled symbols denote affected individuals . the blackened bars correspond to affected haplotypes with alleles that cosegregate with the disease and that are homozygous in affected individuals . two - point lod scores of known cataract gene markers in the 13 arcc families twenty - one families had homozygous regions containing 1 or more of the 33 known genes with lod scores higher than 2 at = 0 ( table 2 ) . sequence analysis of these genes in the corresponding families revealed nine mutations ( eight of which were novel ) cosegregating with and likely to be causative for cc in 10 of the respective families ( supplementary fig . , mutations were identified in three families that had undergone unpublished genome - wide linkage analysis , including previously described mutations in fyco1 and galk1 and a novel mutation in hsf4 , marked as u in table 3 . pathogenicity of mutations was evaluated using a detailed in silico analysis ( table 3 ) . domain structures of the encoded genes and the location of the mutations in them are shown in figure 3 , as are the sequence conservation in nine species ranging from humans to zebrafish for missense mutations . known cataract gene mutations in pakistani arcc families domain structure and evolutionary conservation of proteins with missense mutations . graphic overview of the proteins encoded by genes in which mutations were identified in epha2 , foxe3 , fyco1 , tdrd7 , aqp0 , hsf4 , galk1 , and cryba4 . structural or functional domains are depicted , as well as the position of the mutation . amino acid sequence conservation around residues affected by missense mutations are shown for the five known cataract genes identified in this study . the sequences of proteins or predicted translation products from nine species from humans to zebrafish have been compared and aligned . mutations identified included a novel homozygous substitution in epha2 exon 10 ( c.1814c > t , p.[thr605ile ] ) in families 60061 and 60157 ; a novel homozygous substitution in foxe3 ( c.307g > a , p.[glu103lys ] ) in family 60039 ; a known homozygous substitution in fyco1 exon 8 ( c.2206c > t ; p.[gln736 * ] ) , in families 60218 and 60228 ; a c.1129delg frameshift mutation predicted to result in premature termination , p.(ala377profs*2 ) , in family 60152 ; novel homozygous substitution in exon 1 ( c.67t > a ; p.[tyr23asn ] ) in family 60090 ; a c.1067t > c p.(leu356pro ) missense mutation in family 60133 ; and a novel homozygous c.440g > t ( p.[gly147val ] ) substitution in cryba4 exon 5 in family 60038 . this variant ( rs753345828 ) has a reported minor allele frequency of 0.00005 in dbsnp ( https://www.ncbi.nlm.nih.gov/projects/snp/ ) and was not seen in 192 ethnically matched control chromosomes ( 96 individuals ) . families 60061 and 60157 share a common haplotype of 11 consecutive snp markers across epha2 , suggesting that they derive the mutant allele from a common ancestor ( supplementary table s2 ) . the thr605 residue is conserved among species from humans to chickens , but not in the zebrafish ( fig . the sift score for this change was 0 , predicting that it is deleterious to the protein . however , the polyphen-2 program predicts this mutation to be damaging using the humdiv dataset but benign using the humvar dataset for comparisons . thus , although likely to be pathogenic , the significance of this sequence change is currently uncertain . twenty - one families had homozygous regions containing 1 or more of the 33 known genes with lod scores higher than 2 at = 0 ( table 2 ) . sequence analysis of these genes in the corresponding families revealed nine mutations ( eight of which were novel ) cosegregating with and likely to be causative for cc in 10 of the respective families ( supplementary fig . , mutations were identified in three families that had undergone unpublished genome - wide linkage analysis , including previously described mutations in fyco1 and galk1 and a novel mutation in hsf4 , marked as u in table 3 . pathogenicity of mutations was evaluated using a detailed in silico analysis ( table 3 ) . domain structures of the encoded genes and the location of the mutations in them are shown in figure 3 , as are the sequence conservation in nine species ranging from humans to zebrafish for missense mutations . known cataract gene mutations in pakistani arcc families domain structure and evolutionary conservation of proteins with missense mutations . graphic overview of the proteins encoded by genes in which mutations were identified in epha2 , foxe3 , fyco1 , tdrd7 , aqp0 , hsf4 , galk1 , and cryba4 . structural or functional domains are depicted , as well as the position of the mutation . amino acid sequence conservation around residues affected by missense mutations are shown for the five known cataract genes identified in this study . the sequences of proteins or predicted translation products from nine species from humans to zebrafish have been compared and aligned . mutations identified included a novel homozygous substitution in epha2 exon 10 ( c.1814c > t , p.[thr605ile ] ) in families 60061 and 60157 ; a novel homozygous substitution in foxe3 ( c.307g > a , p.[glu103lys ] ) in family 60039 ; a known homozygous substitution in fyco1 exon 8 ( c.2206c > t ; p.[gln736 * ] ) , in families 60218 and 60228 ; a c.1129delg frameshift mutation predicted to result in premature termination , p.(ala377profs*2 ) , in family 60152 ; novel homozygous substitution in exon 1 ( c.67t > a ; p.[tyr23asn ] ) in family 60090 ; a c.1067t > c p.(leu356pro ) missense mutation in family 60133 ; and a novel homozygous c.440g > t ( p.[gly147val ] ) substitution in cryba4 exon 5 in family 60038 . this variant ( rs753345828 ) has a reported minor allele frequency of 0.00005 in dbsnp ( https://www.ncbi.nlm.nih.gov/projects/snp/ ) and was not seen in 192 ethnically matched control chromosomes ( 96 individuals ) . families 60061 and 60157 share a common haplotype of 11 consecutive snp markers across epha2 , suggesting that they derive the mutant allele from a common ancestor ( supplementary table s2 ) . the thr605 residue is conserved among species from humans to chickens , but not in the zebrafish ( fig . 3 ) , suggesting that it is essential for protein function . the sift score for this change was 0 , predicting that it is deleterious to the protein . however , the polyphen-2 program predicts this mutation to be damaging using the humdiv dataset but benign using the humvar dataset for comparisons . thus , although likely to be pathogenic , the significance of this sequence change is currently uncertain . here , we describe the results of screening 83 unlinked arcc families for homozygosity at 33 genes or loci known to be involved in arcc . nine disease - causing mutations were identified in 10 families , and in 11 families no mutations were identified in the linked gene ( supplementary table s3 ) . we also describe the results of genome - wide linkage analysis in three families for which the mutation had been identified by using a standard linkage approach , but for which the results had not yet been published . overall , including previously and newly identified mutations , causative genes or loci were identified in 37.1% of the entire set of families studied as part of this project . the high degree of genetic heterogeneity in arcc makes genetic screening and gene identification expensive and time - consuming . although this can be approached efficiently by using high - throughput sequencing , this approach is generally more expensive than homozygosity mapping and requires full knowledge of the causative genes and their structure . in contrast to cc in european populations studied , 87% of the families in this project , collected in an unbiased fashion , had arcc , whereas only 13% had adcc or an ambiguous pedigree ( fig . because of these considerations and the high levels of consanguinity in our families , we chose to use homozygosity testing disease gene loci for arcc . this enables relatively rapid and economical screening of many loci and is particularly useful in analysis of consanguineous families in which regions of several centimorgans adjacent to the disease gene are expected to be identical by descent . screening of 33 genes or loci in the present study identified putative pathogenic alterations in seven different genes in 10 ( 12% ) of 83 families . five missense , one nonsense , two frame shift , and one splice site mutations were detected , of which eight were novel . it is unclear why 11 of the 21 families remaining after linkage analysis did not show a mutation in the included candidate gene . the most likely explanation is that these families were too small to yield a statistically significant lod score ( table 2 ) , so that the homozygosity is fortuitous and the true locus has yet to be mapped . another possibility is that these families harbor mutations that might be missed by sanger sequencing , either in introns or currently unidentified exons or control regions . also , while studying offspring of consanguineous matings should decrease compound heterozygosity , it is possible that this is responsible for the dropout of some families during homozygosity mapping . epha2 ( omim 176946 ) belongs to the a - subclass of receptor tyrosine kinase and interacts with its cognate membrane - anchored ligands to activate cell bidirectional signaling pathway . first described as a cause of ad cataracts in a caucasian family , homozygous recessive mutations were subsequently implicated in arcc in a pakistani family . to date , nine different mutations in epha2 have been reported ( see cat - map ) in 15 families , and epha2 has also been implicated in age - related cataract . here , we report a novel homozygous epha2 missense mutation in two consanguineous pakistani families . dna sequencing revealed the transition c.1814c > t , p.(thr605ile ) in exon 10 located near the protein tyrosine kinase domain of the protein ( fig . 3 ) , suggesting it might alter the tyrosine kinase activity of the epha2 protein . the gene foxe3 ( omim 601094 ) , on chromosome 1p33 , is a member of the forkhead box gene family , consisting of a single exon encoding a 319amino acid dna - binding transcription factor , consistent with a role in the development of the lens placode . the c.307g > a , p.(glu103lys ) , mutation reported in this study is a novel homozygous missense mutation associated with posterior subcapsular cataract . this mutation occurs in a highly conserved amino acid located in the fork head domain ( fig . fyco1 contains 18 exons and encodes for a coiled coil protein comprising 1478 amino acids ( 167 kda ) . expressed widely including the eye ( unigene , https://www.ncbi.nlm.nih.gov/unigene , in the public domain ) , it comprises an -helical run domain following by a long coiled - coil region , an fyve zinc - finger domain , an lc3-interacting region , and a golgi dynamics domain . fyco1 interacts directly with lc3 , affecting the maturation of p40phox phagosomes , to participate in autophagosomal trafficking . the mutation seen in family 60173 , c.2206c > t , p.(gln736 * ) , table 3 , was identified in three previously published families ( 60003 , 60012 , and 60069 ) and one unpublished family ( 60237 ) . p.(ala1051aspfs*27 ) mutation identified in family 60228 results in the inactivation of a splice acceptor site . all are predicted to cause nonsense - medicated decay of the fyco1 mrna and a loss of fyco1 function in the face of the requirement for turning over large amounts of protein and organelles as part of fiber cell differentiation . tdrd7 ( omim 611258 ) belongs to a large family of tudor domain containing proteins , and as an rna granule component interacts with methylated arginine residues and rna to control the levels of mrnas posttranscriptionally . tdrd7 is highly expressed in differentiating fiber cells of the lens . to date , only two mutations in tdrd7 have been reported : a balanced chromosomal rearrangement disrupting the tdrd7 gene causing juvenile cataracts in an isolated patient , and as a cause of arccs in a family . , we report a novel homozygous c.1129delg , p.(ala377profs*2 ) tdrd7 frameshift mutation cosegregating with cataracts in a consanguineous pakistani family ( 60152 ; table 3 ) , possibly resulting in nonsense - medicated decay of the tdrd7 mrna . the aqp0 ( major intrinsic protein [ mip ] , omim 154050 ) gene on chromosome 12q13 is a member of the aquaporin family , a ubiquitous family of membrane water transport proteins that confers rapid movements of water across cell membranes . this 263amino acid intrinsic membrane protein is expressed only in terminally differentiated fiber cells , constituting more than 50% of the total membrane protein in the lens . members of the aquaporin family are predicted to share a unique structure with six transmembrane bilayer - spanning domains ( tm1tm6 , fig . fourteen different mutations in mip have been identified in 15 families with different types of ad cataract , as listed in cat - map . however , this novel homozygous missense mutation is the first to be associated with ar cataracts . the c.67t > a , p.(tyr23asn ) mutation occurs in a highly conserved amino acid located within the first transmembrane region of the protein ( fig . 3 ) , suggesting that it might alter the water pore channel function , possibly through affecting water - permeability properties or trafficking . consistent with the ar cataracts resulting from a loss of function , a knockout mouse model also shows bilateral cataracts . hsf4 ( omim 116800 ) mutations were originally identified in ad cataract , and later in ar cataract families . a novel missense mutation , c.433g > c , p.(ala145pro ) , in the sixth exon of hsf4 was found to cosegregate with the disease phenotype in this ar congenital nuclear cataract family ( fig . galk1 ( galactokinase , omim 604313 ) contains eight exons and is located on chromosome 17q25.1 . mutations in galk1 cause recessive cataracts , and two mutations , c.410delg , p.(gly137valfs*27 ) , and c.416t > c , p.(leu139pro ) , were reported in two pakistani families . family 60248 in this study showed ac.766c > t , p.(arg256trp ) , mutation previously reported by asada et al . , and family 61133 showed a novel c.1067t > c , p.(leu356pro ) , mutation at the junction of the second atp binding site , which might be important for atp binding . the -crystallin gene family includes three basic ( crybb ) and four acidic ( cryba ) crystallin proteins , believed to derive from a common -crystallin ancestor . the c.440g > t , p.(gly147val ) mutation ( table 3 ) is the first cryba4 mutation to be associated with ar cataracts , suggesting a lack of function in cryba4 causes the cataracts , and further that cryba4 might have a functional role in the lens beyond that of a structural crystallin . 3 ) , changes a highly conserved amino acid , and thus probably damages the protein structure . this glycine residue is a critical part of a tryptophan corner motif , occurring at the junction of a greek key and the following -strand of the barrel , and thought to aid in folding of the greek key . the glycine residue is two amino acids to the n - terminal of a tryptophan residue ( w-2 ) , which forms a hydrogen bond with the hydrophilic w-3 residue ( supplementary fig . s2 ) . in total , members in 21 of the 83 families had at least one homozygous region harboring a mutation in a known cc gene . in 10 probands , the causative mutation was identified in the included genes . in the remaining 11 arcc families , sequencing of the known genes in the mapped loci did not reveal a mutation that cosegregated with the disease phenotype ( supplementary table s3 ) . clarifying the origin of cataracts in these families next - generation sequencing ( ngs ) has already proven valuable in identifying novel disease genes , both through whole exome or whole genome sequencing and targeted sequencing of linkage intervals or specific genomic regions . however , because of the expense of ngs , homozygosity mapping remains effective in terms of cost and time for localizing mutations in patients with arcc in populations with a high frequency of consanguineous matings , such as the pakistani population . taken together with our previous work , mutations and loci were identified in 43 of 116 pakistani arcc families . fyco1 was implicated most commonly , with causative mutations identified in 13.8% ( 16/116 ) of arcc families , whereas crybb3 accounted for 5.2% ( 6/116 ) of arcc in the families studied . in addition , the percentage of arcc cases that can be attributed to the other genes in our study cohort is approximately 3.4% for galk1 ( 4/116 ) , approximately 2.6% for epha2 ( 3/116 ) , approximately 1.7% each for cryab ( 2/116 ) and sil1 ( 2/116 ) , and approximately 0.9% each for foxe3 ( 1/116 ) , tdrd7 ( 1/116 ) , mip ( 1/115 ) , hsf4 ( 1/116 ) , and cryba4 ( 1/116 ) . figure 4 summarizes the genetic causes of arcc in the pakistani families studied . in all , the 11 genes are responsible for approximately 32.8% of inherited cataracts in these 116 pakistani families , with cataracts in the remaining families excluded from these loci . pie chart showing the frequencies of cataract gene mutations in the pakistani population as seen in this study and our previous studies . overall , this work demonstrates that homozygosity mapping is an efficacious and economical initial step in localizing genetic defects of consanguineous arcc families , allowing insight into the genetic architecture of arcc in the pakistani population . in addition , these results lay the groundwork for screening larger groups of arcc families by using a similar approach followed by ngs to identify the causative genes in all families . the current advances in conventional and genetic therapies mean that knowledge of the genetic causes of disease in these patients is becoming increasingly valuable for their medical treatment . genbank : epha2 mrna , nm_004431.3 ; epha2 protein , np_004422.2 . foxe3 mrna , nm_012186.2 , protein , np_036318.1

purposeto identify the genetic origins of autosomal recessive congenital cataracts ( arcc ) in the pakistani population.methodsbased on the hypothesis that most arcc patients in consanguineous families in the punjab areas of pakistan should be homozygous for causative mutations , affected individuals were screened for homozygosity of nearby highly informative microsatellite markers and then screened for pathogenic mutations by dna sequencing . a total of 83 unmapped consanguineous families were screened for mutations in 33 known candidate genes.resultspatients in 32 arcc families were homozygous for markers near at least 1 of the 33 known cc genes . sequencing the included genes revealed homozygous cosegregating sequence changes in 10 families , 2 of which had the same variation . these included five missense , one nonsense , two frame shift , and one splice site mutations , eight of which were novel , in epha2 , foxe3 , fyco1 , tdrd7 , mip , galk1 , and cryba4.conclusionsthe above results confirm the usefulness of homozygosity mapping for identifying genetic defects underlying autosomal recessive disorders in consanguineous families . in our ongoing study of arcc in pakistan , including 83 arcc families that underwent homozygosity mapping , 3 mapped using genome - wide linkage analysis in unpublished data , and 30 previously reported families , mutations were detected in approximately 37.1% ( 43/116 ) of all families studied , suggesting that additional genes might be responsible in the remaining families . the most commonly mutated gene was fyco1 ( 14% ) , followed by crybb3 ( 5.2% ) , galk1 ( 3.5% ) , and epha2 ( 2.6% ) . this provides the first comprehensive description of the genetic architecture of arcc in the pakistani population .

Subjects and Methods Ascertainment of Families and Clinical Analysis Homozygosity Mapping Linkage Analysis DNA Sequencing Pathogenicity Assessment of Identified Variants Results Mutation Analysis of Known Cataract Genes in the Homozygous Regions None Discussion Supplementary Material

clinical data including age , sex , presenting symptoms and clinical course , duration of hospitalization , medication , and neurologic status at discharge were collected in all patients . routine csf analyses included leukocyte count , csf protein concentration , qalb , csf / serum igg ratio ( qigg ) , csf culture , csf serology ( herpes simplex virus [ hsv ] igg / igm , varicella - zoster virus [ vzv ] igg / igm , measles igg / igm , mumps igg / igm , borrelia burgdorferi igg / igm , syphilis , hiv ) , and pcr analysis for neurotropic viruses ( hsv 1/2 , vzv , cmv , epstein - barr virus , enterovirus , human herpesvirus 6 ) . brain barrier dysfunction and intrathecal synthesis of igg were determined based on the method of reiber . the resulting data are presented in reibergrams ( figure 1 ) , discriminating the qigg from the igg / serum albumin ratio with hyperbolic reference range . the upper limit of the reference range for an intact brain barrier function is age - dependent and was calculated for each patient according to the formula qalb < ( 4 + age/15 ) 10 . brain barrier dysfunction was classified as mild ( qalb < 10 10 ) , moderate ( qalb 1020 10 ) , or severe ( qalb > 20 10 ) . increasing albumin quotients reflect increasing blood - brain barrier dysfunction ( x - axis ) . the cutoff value for brain barrier dysfunction depends on the patient 's age and is therefore not marked . on the y - axis , the blue line separates blood - borne igg ( below the line ) and intrathecal igg synthesis ( above the line ) . in our patient cohort , the csf / serum albumin ratio ( qalb ) was significantly higher in patients with leptomeningeal enhancement on postcontrast fluid - attenuated inversion recovery ( flair ) imaging ( red ) than in those without leptomeningeal enhancement ( green ) . clinical data and csf analyses of the included patients mri studies were performed on a 3 t mri system ( magnetom skyra ; siemens healthcare , erlangen , germany ) using a standardized protocol including t2-weighted flair echo planar diffusion - weighted imaging including apparent diffusion coefficient calculations and subsequent contrast - enhanced t1-weighted and flair sequences . the mri data were analyzed with a structured reporting scheme independently by 2 reviewers unaware of clinical detail and experienced with diagnostic and research studies using postcontrast flair mri . statistical analysis was performed using spss version 22.0.0.0 ( ibm , armonk , ny ) . group comparisons between patients with and without leptomeningeal postcontrast flair hyperintensity regarding the severity of brain barrier dysfunction and csf protein concentration , qalb , and qigg were calculated by mann - whitney u test and 2-tailed student t test , as appropriate . a p value < 0.05 was considered to be statistically significant . this study was approved by the local institutional review board ( medizinische ethikkommission ii der medizinischen fakultt mannheim , university of heidelberg ) and performed in accordance with the ethical standards of the 1964 declaration of helsinki and its later amendments . patient consent was not required by our institutional review board due to the retrospective nature of the study and the lack of patient interaction . this study was approved by the local institutional review board ( medizinische ethikkommission ii der medizinischen fakultt mannheim , university of heidelberg ) and performed in accordance with the ethical standards of the 1964 declaration of helsinki and its later amendments . patient consent was not required by our institutional review board due to the retrospective nature of the study and the lack of patient interaction . during the analyzed period , 15 patients with a diagnosis of aseptic meningitis were examined with a standardized 3 t mri protocol including postcontrast flair sequences and were potentially eligible for the study . one patient had to be excluded due to incomplete csf workup ( qalb missing ) . fourteen patients ( 8 men , age range 1858 years , all immunocompetent ) were included in our study . all patients presented with moderate to severe new - onset headache ; 8/14 had fever at presentation . in the clinical course , 2 patients developed cranial nerve palsy ( abducens nerve in 1 , facial and vestibulocochlear nerve in 1 ) . on the initial mri , no parenchymal signal alterations were detected in precontrast sequences including native flair imaging in any of the patients . on postcontrast flair images , 7/14 patients showed leptomeningeal and/or sulcal contrast enhancement . in 4/7 , a distinct leptomeningeal enhancement was present along each cerebral lobe of both hemispheres and the cerebellum ; in 3/7 , contrast enhancement was more subtle and confined to the supratentorial leptomeninges . an example of ubiquitous contrast enhancement of the meninges in a patient with meningitis caused by vzv is given in figure 2 . on contrast - enhanced t1-weighted images , no parenchymal or meningeal signal abnormalities were noted . mri of a 54-year - old patient with viral meningitis caused by varicella - zoster virus . ( a ) unenhanced precontrast fluid - attenuated inversion recovery ( flair ) images . on postcontrast flair images ( b ) , sulcal contrast enhancement and a fine hyperintense lining can be seen , probably involving the leptomeninges in the absence of obvious white or gray matter lesions on both flair and postcontrast t1-weighted mri ( c ) . csf analyses were performed a mean of 2.6 days ( 2.1 days , range 08 days ) prior to mri and showed lymphocytic pleocytosis ( mean 206 cells/l , range 21491 ) indicating aseptic meningitis in all patients . there was no evidence of noninfectious aseptic meningitis ( postvaccinal , drugs , systemic inflammatory disease , neoplastic disorder ) in any of the patients . of these , the 3 patients with leptomeningeal enhancement were infected by herpesviridae ( vzv , hsv2 , cmv ) , whereas the 4 patients without leptomeningeal enhancement were positive for enterovirus rna . in relation to qigg , qalb accounted for a dysfunction of the brain barriers that was classified as severe in 3/13 , moderate in 5/13 , and mild in 5/13 . patients with leptomeningeal enhancement on postcontrast flair imaging showed significantly higher values of brain barrier dysfunction ( p = 0.001 ) . leptomeningeal enhancement on postcontrast flair imaging was present in all but 1 patient with moderate or severe brain barrier dysfunction , whereas none of the patients with mild or no disturbance of the brain barriers showed contrast enhancement of the leptomeninges . the presence of postcontrast leptomeningeal enhancement was associated with higher qalb ( p = 0.003 ) and qigg ( p = 0.002 ) as well as a higher csf protein concentration ( p = 0.016 ) . leukocyte count did not differ between patients with or without postcontrast leptomeningeal enhancement ( p = 0.449 ) . clinical outcome was favorable , with resolution of symptoms within 2 weeks in all but 1 patient . this patient showed marked leptomeningeal enhancement and moderate brain barrier disturbance on csf analysis and had several complications , including sixth cranial nerve palsy , undulating leukocyte count > 100/l over 4 weeks , and increase of intracranial pressure requiring continuous lumbar csf drainage for 7 days . in patients without leptomeningeal contrast enhancement , symptoms regressed more quickly than in those showing postcontrast flair signal enhancement . as a result , the average length of hospital stay was longer in patients with leptomeningeal contrast enhancement ( mean 19 days vs 7 days in those without enhancing leptomeninges ) . postcontrast flair mri sequences have been shown to be valuable in the detection and evaluation of different leptomeningeal diseases , including subarachnoid hemorrhage , meningeosis , and infectious meningitis . the nulling of the csf signal due to the inversion time facilitates an optimized delineation of hyperintense pathology adjacent to the csf in the sulcal and leptomeningeal space while the t1 shortening in flair sequences is responsible for the highly sensitive delineation of pathologic contrast enhancement following application of gadolinium . in a large cohort of infants with bacterial meningitis , contrast enhancement of the leptomeninges was the most common pathologic finding on mri . from a pathophysiologic point of view , leakage from damaged pial vessels leading to focal extravasation of contrast agent into the csf adjacent to the brain is the likely cause for leptomeningeal enhancement on flair images . contrast enhancement of the leptomeninges on postcontrast flair sequences may thus be a surrogate for dysfunction of the bcsfb and , more indirectly , a marker of bbb dysfunction . in contrast to patients with ischemic stroke or cerebral amyloid angiopathy , in whom localized brain barrier breakdown can be observed , in patients with meningitis a more generalized pattern of contrast enhancement was seen , involving both hemispheres and also the infratentorial space in some patients . we substantiate this hypothesis by correlating the csf findings with the presence of leptomeningeal enhancement in patients with aseptic meningitis . contrast enhancement of the meninges occurred only in patients with moderate to severe dysfunction of the brain barriers and not in those with mild or no brain barrier disturbance . moreover , we demonstrate a positive correlation between the qalb as a quantitative marker for brain barrier dysfunction and the occurrence of leptomeningeal enhancement . partly in line with these findings , an association of log10-transformed csf protein and a leptomeningeal enhancement score has been reported in patients with infectious meningitis . however , log10-transformed csf protein is only an approximate value for brain barrier disturbance , as it is not corrected for serum protein content . the positive correlation may thus be primarily based on the inclusion of patients with bacterial meningitis and marked csf protein elevation . severe damage to the brain barriers , particularly as seen in bacterial meningitis , may predispose to complications and poor prognosis . accordingly , a positive correlation between the extent of leptomeningeal enhancement on postcontrast flair images and the occurrence of complications has been demonstrated in a study of 43 patients with meningitis of various etiologies . however , complications affected only 3 patients with bacterial meningitis and 1 patient with fungal meningitis , while the clinical course of patients with viral meningitis was benign overall . our study focused on patients with aseptic meningitis , characterized by mostly mild to moderate brain barrier dysfunction . of interest , although only 1 patient in our cohort had a complicated clinical course , the presence of leptomeningeal enhancement still accounted for a delayed resolution of clinical symptoms and a longer hospital stay . on the other hand , a mild increase in brain barrier permeability in patients with viral meningitis may be a precondition for lymphocyte infiltration and subsequent effective viral clearance . furthermore , the extent of brain barrier dysfunction may vary depending on the specific viral agent . viral infections are characterized by individual patterns of chemokine secretion that promote vascular permeability . in our cohort , 3 patients with leptomeningeal enhancement were infected by herpesviridae ( vzv , hsv2 , cmv ) , coated double - stranded dna viruses , whereas 4 patients without leptomeningeal enhancement were positive for enteroviruses , uncoated double - stranded rna viruses . although data on hsv2 are lacking , hsv1 has been shown to affect the bbb mainly via increased mmp2 and mmp9 activity , leading to collagen type iv degradation . instead , in vitro studies have shown that infection of choroid plexus cells with echovirus 30 , a member of the enterovirus family , leads to enhanced secretion of cxcl1 - 3 , chemoattractants for t lymphocytes . this is motivation to study the potential association between viral agents and the occurrence of leptomeningeal enhancement in future cohorts with more patients . because contrast - enhanced mri was performed after csf analysis , it is theoretically possible that leptomeningeal enhancement was caused or enhanced by the lumbar puncture . however , meningeal enhancement seems to occur only in the case of post lumbar puncture headache or intracranial hypotension , which did not occur in any of our patients . in patients with aseptic meningitis , leptomeningeal enhancement on postcontrast flair images is closely related to the extent of leakage of blood - borne proteins into the csf . both leptomeningeal enhancement and qalb angelika alonso : conceptualization of the study , data acquisition , analysis and interpretation of the data , drafting and revising the manuscript . philipp eisele : data acquisition , analysis and interpretation of the data , revising the manuscript . anne d. ebert : analysis and interpretation of the data , revising the manuscript , statistical analysis . martin griebe : acquisition , analysis , and interpretation of the data , revising the manuscript . kristina szabo : acquisition , analysis , and interpretation of the data , revising the manuscript . achim gass : conceptualization of the study , data acquisition , analysis and interpretation of the data , drafting and revising the manuscript . b. engelhardt is on the scientific advisory board for swiss multiple sclerosis society , arsep , cluster of excellence for systems neurology synergy , and zentrum fr molekulare neurobiologie ; is on the editorial board for european journal of immunology , fluids and barriers of the cns , and acta neuropathologica ; and received research support from swiss national science foundation , swiss multiple sclerosis society , arsep , bangerther rhyner foundation , and schweizerische herzstiftung . k. szabo is on the editorial board for cerebrovascular diseases and case reports in neurology and received research support from german research foundation . hennerici is an editor for cerebrovascular diseases and interventional neurology , is a consulting editor for international journal of stroke , receives publishing royalties from thieme publishers , and received research support from tiaregistry . gass received travel funding and/or speaker honoraria from bayer schering , biogen idec , merck serono , novartis , and teva neurosciences and is on the editorial board for cerebrovascular diseases and journal of neuroimaging .

objective : to investigate the blood - csf barrier ( bcsfb ) dysfunction in aseptic meningitis.methods:in our case series of 14 patients with acute aseptic meningitis , we compared mri characteristics with csf findings.results:contrast enhancement in the sulcal space in a leptomeningeal pattern was visualized in 7 patients with bcsfb dysfunction categorized as moderate to severe as evidenced by the csf / serum albumin ratio ( qalb ) but was not present in those with mild or no barrier disturbance ( p = 0.001 ) . the qalb as a marker for the leakiness of the bcsfb and , more indirectly , of the blood - brain barrier ( bbb ) was positively correlated with the incidence of leptomeningeal contrast enhancement seen on postcontrast fluid - attenuated inversion recovery ( flair ) mri ( p = 0.003 ) . patients with a more pronounced brain barrier dysfunction recovered more slowly and stayed longer in the hospital.conclusions:the severity of meningeal bbb disturbance can be estimated on postcontrast flair mri , which may be of diagnostic value in patients with aseptic meningitis .

METHODS Standard protocol approvals, registrations, and patient consents. RESULTS DISCUSSION AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

neuromyelitis optica ( nmo ) is an inflammatory disorder restricted to the spinal cord and optic nerves . contrary to multiple sclerosis ( ms ) , relapses of nmo are often strikingly severe and most nmo patients present stepwise neurological impairments . nmo treatments are aimed to prevent the relapses with the administration of various promising immunosuppressive drugs . since the largely used steroid treatment usually fails to control severe attacks , specific add - on treatments have to be considered in order to limit the stepwise increase of residual impairment . given that a strong humoral response characterizes nmo physiology , one might assume plasma exchange ( plex ) to be particularly well adapted in severe nmo relapses . we here propose to outline the rationale of the plex treatment based on physiological grounds and summarize the relevant data of plex studies in the setting of nmo spectrum disorder , assessing the results obtained in each type of attacks . finally we will try to build up an original concept linking the inner dynamic of the lesion , the timing of plex , onset , and the expected clinical results . a vasculocentric pattern of activated complement and immunoglobulin of igg and igm types is observed that mirrors the normal expression of aqp4 . aqp4 expression is definitely reduced in normal appearing white matter and lost throughout the lesions . these modifications are the hallmark of nmo and could occur alone or associated with a wide range of lesions from mild demyelination to large necrosis . this pattern of lesion was classified in the pattern ii of the lassmann classification of the inflammatory lesions [ 1 , 2 ] . contrary to ms , t cells are rare in nmo lesions and probably have no major effect on the formation of the lesions . however , t cells are probably involved upstream in physiopathological cascade in the earlier phases of the disease where a complex interplay leads to antigen sensitization and possibly in the initial opening of the blood - brain barrier ( bbb ) . moreover , the pattern associating vasculocentric deposition of ig and complement , cells ( eosinophils / lymphocytes ) infiltration and aqp4 loss is sometimes fully dissociated from demyelination . this antibody is detected with tissue - based immunofluorescence assays with a sensitivity and specificity for clinically defined nmo of more than , respectively , 60% and 90% . clinically diagnosed nmo patients share clinical common and evolutional characteristics regardless of their nmo - igg status . beyond the surrogate marker value of nmo - igg , this marker is now used as a major diagnostic criterion and delineates the nmo spectrum disorders that gather in a same entity both typical nmo and unusual or truncated clinical forms . aqp4 is a transmembrane protein expressed in the apical domain of the membrane feet expansions of the astrocytes close to the surrounded blood vessels . this protein is critically involved in the homeostasis of the water in the brain and interfaces with blood vessels , especially in the clearance of free water . loss of perivascular aqp4 in the basal state results in cellular swelling , ostensibly due to a failure to eliminate water generated from cellular metabolism . thus in nmo , since the interaction of nmo - igg and aqp4 leads to a functional knockout phenotype of aqp4 , edema develops as a result of functional impairment of aqp4 although bbb is expected to be still intact , which may explain the paradoxical lack of gadolinium enhancement in most nmo lesions . apart from water homeostasis , the removal of aqp4 from astrocytes membrane is associated with an impaired homeostasis of glutamate via the loss of function of eaat2 , a major glutamate transporter associated with aqp4 in a macromolecular complex . the disruption of glutamate homeostasis initiates an excitotoxic mechanism damaging oligodendrocytes and ultimately leading to demyelination . virtually all the cns astrocytes express aqp4 , however , some regions are enriched in aqp4 . those regions are the spinal cord gray matter , the posterior optic nerve , the floor of the fourth ventricle and the circumventricular organs especially the area postrema , explaining the restriction of the sites of lesion characterizing nmo . interestingly circumventricular organs are also the only sites of the cns expressing fenestrated capillaries favoring local passive diffusion of circulating antibodies nmo - igg detection is a strong predictor of recurrence after an initial spinal or optic attack [ 1315 ] . in few patients , nmo - igg was high during flares and became negative during the stabilized disease following treatment , and , in contrary , an initially seronegative patient became positive during a further attack [ 16 , 17 ] . that is to say that nmo - igg negative sera are not always nmo - igg negative patients on long term . in the seminal work of takahashi et al . , nmo - igg levels were positively correlated with both clinical severity ( i.e. , blindness ) and radiological severity . moreover , a strong positive correlation was obtained between the nmo - igg titres at the nadir of exacerbations and the spinal cord lesion length on mri . in contrast , low nmo - igg titres were observed during remission induced by immunosuppressive maintenance therapy . in vitro , the binding of nmo - igg to the extracellular domain of aqp4 reversibly downregulates its plasma expression . in the presence of active complement , nmo serum igm is not aqp4-specific and abundant igm deposits in the nmo lesions may have passively diffused after the bbb disruption by the seminal focal complement activation initiated by nmo - igg . in an animal model of eae with passive transfer of nmo - igg , the transfer exacerbated eae signs and the typical pathological characteristics were reproduced in treated rats [ 20 , 21 ] . direct injection of nmo - igg in mice brains could reproduce the pathology , but only when complement is coinjected . the nmo - igg ability to lesion aqp4-transfected cells in the presence of complement the percentage of cells lesioned by complement was strongly higher in presence of sera from patients with severe attacks , although lesion induced by sera from patients with mild attacks did not differ from negative controls or ms patients . thus , the severity of the disease may be partly determined by intrinsic nmo - igg characteristics to activate the complement . as we already described , nmo lesions are associated with a strong igg , igm and complement deposition , typical of the pattern ii in the lassmann classification . the nmo - igg is involved in a complement - dependant toxicity against the astrocytes . all of these components igg , igm , and complement are targeted by plasma exchanges . by means of 5 exchanges , all the exchanged molecules will drop to less than 20% of their initial level [ 24 , 25 ] . by this way , antibodies and complement , which are the core of the pattern ii lesions , are excluded from the circulating pool and can not migrate anymore to the lesions . although plex has long been used in various demyelinating disorders , there is some clue that the pattern is a key determinant of plex efficiency . in a retrospective study , keegan et al . reported that all the patients suffering from demyelinating disorders and improved by plex had a biopsy proven pattern ii lesion . however all these patients were ms without nmo - igg and none were nmo . all the aforementioned findings stress that circulating nmo - igg and complements are the two main actors of the nmo pathogeny and why clearing them from blood with plex should be appropriate for special benefits . plex or plasmapheresis is the filtration of the plasma , which is removed , replaced by artificial plasma and reinfused to the patient the plasma exchange . basically , the goal of the filtration step is to remove a given volume of patients plasma and to return an artificial plasma substitute in its place . two high flow rate accesses are mandatory : an input line from patient to device ( artery ) and a return line from device to patient ( vein ) . in continuous filtration , two needles are placed in both arms or groins in order to drawn out the blood of the body through an extracorporeal line connected to one needle , then blood is processed and reinfused continuously through the other needle . in case of discontinuous filtration , the separation and remixing are done in small batches through a single venous access in the groin where in and out cycles may alternate . anticoagulation ( citrate or heparin ) is added to the blood preplasma filter to prevent from clotting . the removed blood is processed ( apheresis procedure ) in a cell separator that continuously separates plasma from cellular components ( consisting of red and white blood cells and platelets ) either by a centrifugation ring with permanent in and out flow , or by filtration through a porous membrane . small molecules like cytokines as well as large molecules , such as albumin and immunoglobulin , are easily extruded from the blood compartment with a reported sieving coefficient > 0.95 at a blood flow rate of 100 ml / min . the cleared cellular components are then combined with the replacement fluid ( donor plasma or artificial albumin mixed with a saline solution ) and returned to the patient through the needle in the other arm . a plex session is usually performed in 2 to 6 hours , depending on patient 's height , weight , viscosity of the blood , and technical parameters . all the targeted components are distributed in the interstitium ( extra - vascular compartment ) by variable part . the relation curve of the achieved concentration of a plasma component ( c ) after a unique exchange of a given plasma volume is exponential inverse , following a single kinetics . the larger volume of plasma exchanged during each session clears a larger amount targeted circulating component . an exchange of one body plasma volume leads to the immediate clearance of 50% of the circulating component . a 1.3 body mass volume exchange that removes about 72% of the concentration is generally agreed . beyond , the volume to process increases massively for too little gain . however , according to the distribution of c in the interstitium , the achievement of the clearance of c will necessitate the use of multiple plex sessions separated by the time necessary for the equilibration of c concentration between interstitium and vascular spaces . the number and frequency of sessions should be evaluated according to the biological characteristics of the components to remove ( synthesis level , vascular distribution , diffusion ability ) . the durability of the immunomodulatory effect after plex is difficult to assess and will depend on the turnover rate of the targeted humoral components . concomitant intensive immunosuppressive therapy ( i.e. , steroids , mitoxantrone , mycophenolate mofetil , and rituximab ) will be required to sustain the obtained depletive effect . plex are contraindicated in case of ongoing infectious disease , precarious hemodynamics , and active hemorrhage ( heparin ) . immediate side effects are related to the extracorporeal line : hemodynamic instability , vasovagal syndrome , numbness or tingling , venous puncture hazards with excessive local bleeding , septicaemia , or allergy . since blood coagulation factors are all depleted by plex , hemostasis is affected in variable ways : first , a hypocoagulation state is immediately achieved by the global depletion of all the coagulation factors for half a day ; at day 2 , short - life procoagulant factors are regained but antithrombin - iii synthesis is delayed leading to a hypercoagulable state until day 3 . persistently low fibrinogen levels have been described with the concomitant use of high dosage steroid infusion . in summary , methylprednisolone pharmacokinetics is characterized by a very short half - life ( about 2 hours ) and although steroid binding to plasma protein is about 75% , the volume of distribution is very large ( about 1.5 l / kg ) . these factors are the key of the negligible plex effect upon steroids biodisponibility [ 30 , 31 ] . when used the same day as plex procedure , steroids were infused at the end of each plex session . various regimens of high doses of intravenous methylprednisolone are used in first line of treatment ranging from 3 g infused in 3 days , to 10 g in 5 to 10 days , depending on authors . there is no evidence in favor of one regimen or another and efficacy assessment has never been addressed . moreover , even if steroids reduce the inflammatory cellular response by triggering apoptosis of lymphocytes , they are clearly not sufficient because poor outcomes are still a common issue even when steroid treatment is given immediately after onset . we wish to develop here the evidence for the effectiveness of plex that we have been largely using as an add - on therapy for more than 10 years . plex proved to be efficient in central demyelinating diseases in a randomized sham - controlled study [ 32 , 33 ] . reviewed the clinical data from 59 patients who received plex for inflammatory demyelinating diseases , including 10 nmo and 6 acute transverse myelitis ( atm ) cases . a moderate or marked improvement was obtained in half of nmo and atm patient groups . the late final outcome at one year was more or less obtained during the first month after treatment in both groups , without regard to success or failure of treatment [ 26 , 34 ] . a small number of case reports and few small studies were reported with variable issues . judging improvement is even more complex due to the subjective classification of improvement in mild / moderate / marked instead of a quantified clinical exam [ 26 , 3436 ] . moreover the natural history of single spinal relapse in nmo has never been addressed , so any improvement bias after plex is inappreciable in the absence of a control group . finally , most authors used plex as a rescue treatment given late after the onset . for example plex was delayed from onset by a mean of 33 30 days in brunot et al . and a median of 30 days ( 6 to 90 days ) in llufriu et al . . although a synergistic effect of steroids and plex was long expected due to their complementary action , none of these studies compared conventional steroid treatment alone with add - on plex - treated attacks . we previously refined these results in a study of outcome after severe spinal attacks associated with nmo spectrum disorders . we included 96 spinal attacks from 43 patients , divided in two groups : ( 1 ) a steroid - only group designed from historical patients treated with steroids alone ; ( 2 ) an active group treated with both plex and steroids . plex decision was raised at the same time and started as soon as possible during the two days later . as a major difference with other groups , plex was never initiated as a delayed rescue treatment after a standard steroid treatment failure . since plex therapy is mainly expected to minimize residual impairment , we used the edss ( calculated as the difference between residual and basal edss ) as the main outcome . if we except 5 plex delayed due to difficult medevac reasons , plex was initiated by a mean of 5.4 3.1 days after attack onset with a median of 4 sessions . there was no significant difference between the plex - treated and steroid - only groups for basal and acute edss ( 3.9 2.9 versus 4.2 2.9 , and 7.9 1.0 versus 8.0 1.4 ; p = ns ) , however , residual edss ( 5.1 2.4 versus 6.8 1.9 , p < 0.01 ) and mean edss ( 1.2 1.6 versus 2.6 2.4 , p < 0.01 ) were significantly lower in the plex - treated group than in the steroid - only group . basal edss dramatically influenced therapy outcome ( table 1 ) . during the first attack , although acute edss were similar in both groups ( 7.6 1.2 versus 7.1 1.5 , p = ns ) , edss and residual edss were dramatically reduced in the plex - treated group ( 2.1 1.9 versus 5.9 2.0 , p < 0.01 ) given that acute edss was similar in this sub - group . in the two other sub - groups of basal impairment ( edss 1.0 to 5.5 and edss 6.0 ) , residual edss and edss tended to be lower in plex - treated attacks but no statistical signification could be obtained due to the small size of these groups . the classical lazarus effect , defined as a very short - term dramatic improvement , was rather unusual in our group but our study was not designed to analyse short - term improvement . the patients who experienced this effect have all received a very early treatment ( less than 2 days ) . however , in magaa et al . 's paper , patients who exhibited functional improvement did so within a median of 4 days ( third plex ) , although a minority ( 6% ) exhibited a delayed response ( more than 2 months ) . minor side effects occurred in 24% of plex - treated attacks and resulted in plex interruption once ( 84-year - old patient with pulmonary embolism ) . in summary , plex - treated patients achieved a significantly better outcome after a spinal attack , especially if plex was given during the first attack . the exact effect of plex in previously impaired patients should be validated in a larger multicentric cohort . as plex proved to be a promising treatment in spinal attacks , it would now be unethical to design a study with a sham - treated control group . predictors of good outcome were studied in a large group of plex including 26 nmo patients . surprisingly a short plex delay was associated with a good outcome in a first study but had no effect in a second study , although one should remind that median plex delay was long ( 23 days ) in this later compared to our group . the same plex response rate was obtained irrespective of nmo - igg serostatus in our cohort and in the mayo clinic cohort . as a practical consequence , faced with a patient suffering from a severe relapse , the knowledge of nmo - igg status should not be required to start plex as soon as possible , since plex was found efficient in nmo - igg negative patients . we previously showed that an immediate unilateral blindness occurred in a third of patients after the first optic neuritis ( on ) , and generally two attacks are sufficient to cause a definitive loss of vision . few plex were undertaken after on and a quick dramatic recovery is usual as we also observed [ 41 , and unpublished results ] . depending authors , plex were used immediately or as a delayed add - on therapy [ 4245 ] . after pooling severe ( acute visual acuity < 1/10 ) on patients ( either nmo or in the nmo spectrum ) from available studies [ 41 , 42 , 45 , 46 ] with ours ( unpublished results ) , data were gathered for 39 eyes . plex were given in a median of 19 days in patients who recovered a visual acuity more than 1/10 ( considered here as a treatment success ) but 41 days in treatment failure . a clear effect of plex delay was observed since success rate was 8/8 ( 100% ) during the first 11 days , then 4/7 ( 57% ) from days 12 to 22 , and 7/13 ( 53% ) from days 23 to 73 . moreover , even when patients recovered , averaged residual va tended to be lower in delayed plex patients . interestingly , the spontaneous recovery ( > 1/10 ) after severe on treated by steroids alone was about 40% in our cohort ( from ) , which is very close to the recovery obtained in the two last groups of late plex . in conclusion , strong clues support that plex change the outcome of severe on only when they are given early , however broader studies using carefully chosen patients are still lacking to confirm this hypothesis . apart from opticospinal attacks , severe brain attacks are described in nmo , especially involving hypothalamus and medulla oblongata . those lesions are usually severe and associated with blindness , central endocrine disorders , or quadriplegia with respiratory failure . nonspecific telencephalic lesions are common but are mostly asymptomatic . however , symptomatic lesions involving supratentorial white matter are exceptional and extensive . even if a favourable outcome after plex has been reported in a few severe cases [ 48 , 49 ] , comparative data are still lacking . posterior reversible encephalopathy syndrome ( pres ) is an encephalopathy with consciousness and visual disturbances with rapidly reversible changes on mri consistent with vasogenic edema . it seems to occur more often than coincidental in nmo patients : 5 out of 70 consecutive nmo - igg patients evaluated at mayo clinic ; 2 out of 5 in hadassah medical school , israel . authors proposed that the autoimmune - mediated disruption of the aqp4 water channel function may predisposes to pres at comparable levels of acute illness . plex was involved as a trigger in one case with a good final outcome . in few cases , plex were implemented as curative treatment with an overall good outcome [ 50 , 51 ] . besides knowing plex are effective and safe , the central question remains : is plex necessary as soon as and as often as possible ? prospective , randomised , multicentre clinical trials would be required to definitively answer the question . for most authors , to date plex are considered as an add - on rescue treatment after steroid failure . the european recommendation from efns is to start with an early steroid course no matter the severity . early escalation with plex is only recommended after a failure of a second course of steroids , that is to say that plex initiation may be postponed for more than a week . as we demonstrated before , plex efficiency depends on the timing of initiation , ranging from immediate dramatic improvement ( the lazarus effect ) to no effect according to whether they are given early or very late . we propose to regress to the dynamic of the inflammatory nmo lesions to explain why plex efficiency is strongly dependant of the timing of their onset . as we described above , lesion is the consequence of a cascade of reversible events , susceptible to an external action . one could abruptly divide this cascade into two main points : ( 1 ) firstly , the binding of nmo - igg to aqp4 triggers the functional impairment of astrocytes , quickly completed by a complement - mediated cell destruction ; ( 2 ) the dysfunction of eaat2 transporters , as a bystander effect , impairs the clearance of free glutamate which progressively accumulates and initiates an excito - toxic mechanism upon oligodendrocytes , ultimately leading to oligodendrocytes apoptosis and demyelination [ 11 , 53 ] . this excito - toxicity is reversed in vitro by adding a competitive antagonist of the nmda receptor . the time sequence of these events was studied in lesions induced by direct mouse brain injection with nmo - igg and complement . loss of aqp4 and gfap , and myelin breakdown were evident 7 h following the injection . finally , one could suppose that a very early intervention targeting astrocytes dysfunction may prevent the progression to the bystander effect . the influence of treatment delay upon outcome has been addressed in a single study of first on receiving steroid treatments . the outcomes were both visual acuity and the width of the retinal fibers layer evaluated with optic tomography ( oct ) . patients were divided into two groups : one group with a good visual outcome , including a high residual visual acuity and high rfl ; a second group with a poor visual outcome in terms of low acuity and low rfl . very interestingly , the two groups were similar in all the parameters except one : patients with a good outcome received steroids with a lower mean delay after on onset , by a mean of 1.8 1.1 days compared to 7.8 3.8 days in patients with a poor outcome . this study is the first proof that a delayed infusion of steroids is associated with a poorer outcome . a similar effect of treatment delay , treated with plex , early initiation of treatment was one out of the predictors of good outcome . in a larger study encompassing attacks from various demyelinating disorders , success rates were stratified by delay : improvement occurred in 83% when given before day 15 , but fell to 43 after 2 months . moreover the dramatically very short - term improvement , called the lazarus effect , is sometimes observed after severe attacks receiving a very early treatment with plex and steroids . however , this earliness responsibility on the lazarus effect remains elusive since no study is available on this rather unusual effect . in the light of the available data , we postulate a link between the staging of nmo lesion and the plex effect upon clinical and radiological outcome ( figure 1 ) . stage 1 ( first hours ) : acute attack provokes for hours an astrocyte dysfunction ( by nmo - igg binding on aqp4 leading to internalization ) mainly expressed by an edema . this purely edematous lesion could be immediately reversible by the clearance of nmo - igg preventing the loss of astrocytes and the excitotoxic cascade . stage 2 ( days ) : the underexpression of eaat2 induces an excitotoxic effect of glutamate on oligodendrocytes leading progressively to demyelination and axonal loss . astrocytes loss initiates a self - sustained excitotoxic process henceforth independent from nmo - igg persistence . even if the extraction of nmo - igg and complement by plex ends the astrocytes aggression . a variable amount of them has been already lost and excitotoxic effects upon oligodendrocytes are evident . stage 3 ( weeks ) : atrocytes , oligodendrocytes , and axonal loss are prominent , engulfed in large areas of necrosis . we propose to reconsider plex as a major part of the treatment of severe nmo attacks and suggest that plex could be given systematically in severe relapses of nmo , extended transverse myelitis or bilateral severe on resistant to steroids . since nmo - igg positivity is both predictive of attacks and severity , achieving a low concentration of plasmatic antibodies remains a goal to achieve . besides immunosuppressive drugs , weekly plex have been used to achieve a sustained depletion of nmo - igg and complement . miyamoto and kusunoki proposed to use plex as preventive treatment as an add - on therapy after immunosuppressive drugs failure . the natural history of nmo leads all the patients to a deep impairment in a stepwise fashion without progressive phase . in our study , 5 years after the onset , 70% of patients suffered from a unilateral loss of vision and almost half of them from a bilateral loss of vision . after 8 years , half of the patients had suffered from a severe myelitis and had become chairbound . the mortality rate was very high before immunosuppressive drugs but dramatically dropped since they are largely used . the exact role of recurring plex along the remaining attacks to tune the outcome to a low impairment has not yet been addressed but remains most probable considering this striking epidemiological change of mortality in french west indies . immunosuppressive drugs are necessary to prevent further relapses but no recommendation is yet available concerning therapeutic escalation . since the lesion severity mostly depends on the initial and definitive depth of the loss of aqp4 and astrocytes , future treatments strategies may be directed upon aqp4 preservation . small molecules or monoclonal antibodies could be used to prevent nmo - igg binding to aqp4 and to block the physiopathological cascade upstream . another strategy may deplete pathogenic antibodies by apheresis using dedicated immunoadsorption systems as previously described in myasthenia gravis and in various extra neurological disorders . however , if the value of this technique is less clear in disorders like ms [ 60 , 61 ] , where pathology is broader than a specific antibody , it could be especially suitable to nmo since anti - aqp4 seems to be the pathological core . the strength of these techniques is the specificity of the antigenic absorption with no regard to the class or subclass of antibodies and no fluid replacement . this alternative therapy may be considered in plex - resistant relapses and should be better studied in severe relapses as an alternative of steroids and plex . a complementary approach may target the complement system with newly developed anticomplement recombinant antibodies at various levels , with preliminary promising results . plex , in synergy with steroids , could be a major treatment of relapses , aimed at preventing cumulative disability . these preliminary results suggest that plex may modify the short prognostic of nmo relapses , as long as given early . those models should be able to confirm that early therapeutic intervention directed to halt the ongoing lesions should be even more dramatic in an early narrow therapeutic window . the next steps should be to concentrate upon large multicentric therapeutic trials in order to validate the therapeutic procedure and to determine the time opportunity window . however , we are aware that good trials against placebo could now be difficult to accept since this is an extremely devastating disease .

background . neuromyelitis optica ( nmo ) attacks are poorly controlled by steroids and evolve in stepwise neurological impairments . assuming the strong humoral response underlying nmo attacks , plasma exchange ( plex ) is an appropriate technique in severe nmo attacks . objective . presenting an up - to - date review of the literature of plex in nmo . methods . we summarize the rationale of plex in relation with the physiology of nmo , the main technical aspects , and the available studies . results . plex in severe attacks from myelitis or optic neuritis are associated with a better outcome , depending on plex delay ( time is cord and eyes ) . nmo - igg status has no influence . finally , we build up an original concept linking the inner dynamic of the lesion , the timing of plex onset and the expected clinical results . conclusion . plex is a safe and efficient add - on therapy in nmo , in synergy with steroids . large therapeutic trials are required to definitely assess the procedure and define the time opportunity window .

1. Introduction 2. Physiopathology of NMO 3. Plasma Exchange Procedure 4. PLEX in Severe Attacks 5. Timing of PLEX: Evolving to a Key Concept 6. PLEX as Preventive Treatment 7. Preventive Treatments and Future Avenues 8. Conclusion

more than a decade ago , the unusual catalytic activity of the dna repair enzyme alkb was demonstrated by sedgwick , falnes , and co - workers , whereby oxygen is added to foreign , unnatural carbon atoms attached to nitrogen bases , leading to the liberation of an aldehyde and restoration of the canonical purine or pyrimidine base structures within dna . initially , the results pointed to a major role for alkb in protecting cells against low molecular weight alkylating agents that react with nitrogen atoms on the watson crick base - paring side of purine and pyrimidine dna bases forming covalent adducts , such as the positively charged 1-methyladenine ( m1a ) , 3-methylcytosine ( m3c ) , and 3-ethylcytosine ( e3c ) lesions ( figure 1a ) . the methylated nucleobases 1-methylguanine ( m1 g ) , 3-methylthymine ( m3 t ) , and n - methyladenine ( m6a ) , uncharged at physiological ph , were also discovered to be substrates of alkb . more complex dna adducts formed either by oxidative stress - derived lipid byproducts or exogenous vinyl chloride exposure , such as 1,n - ethenoadenine ( ea ) and 3,n - ethenocytosine ( ec ) , as well as the 1,n - ethanoadenine ( ea ) adduct formed by the anticancer agent bcnu ( bis - chloroethylnitrosourea ) , are also repaired by alkb both in vitro and in vivo ( figure 1a ) . although we explored the possibility of alkb acting upon certain alkyl - adducts of oxygen ( o - methylguanine ( omg ) ) and carbon ( 5-methylcytosine ( m5c ) ) , no such evidence was found . several crystal structures of alkb and homologues bound to alkyl substrates have provided detailed information on their substrate recognition and repair specificity . the substrate scope and properties of alkb chemical structures and abbreviations of dna lesions investigated as possible repair substrates by the alkb protein using esi - tof mass spectrometry . ( a ) lesions in this panel are known substrates for alkb ; ( b ) lesions in this panel were tested as potential substrates for alkb in this study . many scenarios of alkylation damage have been explored in order to find possible substrates for alkb with respect to base , position of alkylation , and type of alkyl group damage ( figure 1a ) . however , there are still two positions on the watson crick base - paring interface of the four bases that , if alkylated , have not yet been investigated as possible substrates for alkb , namely , the n position of guanine and the n atom of cytosine . to provide a complete picture of the substrate range of alkb , we examined the possibility that this enzyme can act upon the following series of n - alkylguanine adducts : n - methylguanine ( m2 g ) , n - ethylguanine ( e2 g ) , n - furan-2-yl - methylguanine ( ff ) , n - tetrahydrofuran-2-yl - methylguanine ( hf ) , and n - methylcytosine ( m4c ) ( figure 1b ) . the spectrum of lesions chosen for this analysis was motivated by biological relevance gleaned from several studies . the m2 g lesion is formed by a reaction of the n - amino group of guanine with formaldehyde , a human and rodent carcinogen . the e2 g lesion is formed by a similar reaction of deoxyguanosine with acetaldehyde , a chemical that occurs both endogenously from the metabolic oxidation of ethanol and from lipid peroxidation products , as well as from environmental sources such as tobacco smoke , vehicle exhaust , and food flavorings . importantly , acetaldehyde has been shown to be carcinogenic in rodents and can elicit a variety of dna lesions in human cells . the ff lesion is a structural model of the principal dna adduct formed by nitrofurazone ( nfz ) , a topical antibacterial agent used for treating burns and skin grafts in patients and animals . postreplicative modification of cytosine by dna methylases produces m4c , which is an epigenetic marker for bacteria . in this study , we used high resolution electrospray ionization time - of - flight ( esi - tof ) mass spectrometry to analyze the reaction products of alkb and the five alkyl adducts mentioned above ( figure 1b ) . we demonstrate that these lesions are substrates for alkb , leading to the conclusion that all simple n - alkyl lesions , such as methyl adducts , at the watson crick base - pairing interface on the four bases can be dealkylated by alkb in single stranded dna . sixteen - mer oligonucleotides of the sequence 5-gaagacctxggcgtcc-3 , where x is the lesion of interest ( m2 g , e2 g , ff , hf , and m4c ) , were synthesized and purified as described . the calculated monoisotopic molecular weight ( mw ) for each oligonucleotide is shown in table 1 . to create double - stranded substrates , complementary 17-mer oligonucleotides of the sequence 5-tggacgccyaggtcttc-3 these were one base longer to avoid overlap with the ms signal from 16-mer lesion - containing oligonucleotides . y denotes the base placed opposite the lesion x ( c for n - alkylg lesions and g for m4c ) . dna concentration was measured by uv absorbance using a calculated extinction coefficient ( ) at 260 nm . the extinction coefficient used for an alkylated base was that of its unmodified counterpart , due to the negligible difference between the values in the context of 16-mer dna . all assays were performed with alkbn11 protein , a truncated version of alkb in which the first 11 residues were deleted . alkbn11 protein was purified as described and shown previously to have activity similar to that of the wild - type protein . reactions were performed at 37 c in 45 mm hepes ( ph 8.0 ) , 0.9 mm -ketoglutarate , 67 m fe(nh4)2(so4)2 , and 1.8 mm ascorbate , followed by dry ice storage until esi - tof ms analysis . a typical reaction was performed with all the necessary cofactors , 5 m lesion - containing dna , and either 2.5 m alkb protein or protein purification buffer in a 10-l volume for 1 h. where indicated , complementary dna ( 6 m ) was added and annealed at 65 c for 5 min to form a dsdna substrate for the alkb reaction . oligonucleotide analyses were performed on an agilent 6510 esi - tof mass spectrometer ( palo alto , ca ) . nitrogen gas was set with drying at 10 l / min , with the heated capillary at 325 c and the nebulizer set at 15 psig . liquid chromatographic separations were performed using a zorbax sb - aq column ( 2.1 150 mm ; 3.5 m ; agilent technologies , palo alto , ca ) with a flow rate of 0.2 ml / min . solvent a was 10 mm ammonium acetate in water ( ph 7 ) ; solvent b was 100% acetonitrile . a linear gradient was carried out in the following steps : 98 to 70% a over 30 min , 70 to 98% a over 5 min , and 98 to 98% a over 10 min . lc - ms / ms analyses were conducted on an agilent 6510 qtof system equipped with an esi source . operating parameters were as follows : gas temperature , 350 c ; esi capillary voltage , 4000 v ; nebulizer pressure , 50 psi ; drying gas flow , 10 l / min ; fragmentation energy , 20 v. the theoretical m / z values , as illustrated in figure s29 ( supporting information ) , were calculated using mongo oligo mass calculator , v2.06 ( http://library.med.utah.edu/masspec/mongo.htm ) . the ability of alkb to modify the five alkylated bases was tested in vitro using a previously described experimental procedure . briefly , n - dg and n - dc adducts were incorporated into 16-mer oligonucleotides in a site - specific manner . the modified oligonucleotides were then incubated with purified alkb for 1 h at 37 c , and the reaction products and intermediates ( scheme 1 ) were analyzed by high resolution esi - tof mass spectrometry . for each lesion , experiments were conducted both in the presence and absence of alkb with all necessary cofactors . additionally , the repair experiments were also carried out in the presence of a 17-mer complementary strand ; the ng lesions were placed opposite a cytosine , and the m4c lesion was placed opposite a guanine . the 16-mer oligonucleotides demonstrated a good signal in the 4 charge envelope of the mass spectra ( figure 2 ) . to illustrate the method of analysis , the molecular weight ( mw ) of the 16-mer carrying an m2 g lesion is calculated as 4918.87 da ( table 1 ) ; its monoisotopic peak ( all c , n , o , etc . ) in the 4 charge state has a calculated m / z of 1228.71 . experimentally , we observed a peak at an m / z of 1228.62 ( figure 2a and figure s1 ( supporting information ) ) . esi - tof mass spectra from incubations of n - alkyl dna adducts with the alkb protein . data represent the intensity vs mass / charge ( m / z ) values for the 4 charge envelopes , with the monoisotopic ( all c , n , o , etc . ) peak labeled above each envelope . calculated and observed monoisotopic molecular weights of oligonucleotides and intermediates present in the alkb reactions ( a ) m2 g ; ( b ) m2 g + alkb ; ( c ) e2 g ; ( d ) e2 g + alkb ; ( e ) ff ; ( f ) ff + alkb ; ( g ) hf ; ( h ) hf + alkb ; ( i ) m4c ; and ( j ) m4c + alkb . using the method described above , we found that in 1 h , 38% of m2 g is converted to guanine in the presence of alkb ( figure 2b , 4 charge m / z 1225.11 ) , leaving 62% of the m2 g unchanged ( m / z 1228.62 ) ( figure 2b ) . similarly , alkb converted 64% of the e2 g base to undamaged guanine ( m / z 1225.24 , figure 2d ) in 1 h. the oligonucleotide containing ff has an m / z of 1245.25 in the absence of alkb ( figure 2e and table 1 ) . incubation with alkb resulted in the majority of the starting material being converted into two new species ( figure 2f ) , with masses being consistent with undamaged guanine at m / z 1225.24 and ff-2h at 1244.74 ( scheme 1c ) . the isotopic distribution pattern observed for the assigned ff-2h structure ( figure s9 , supporting information ) was similar to the ff starting material ( figure s7 , supporting information ) , providing confidence that little to no ff starting material was buried underneath the ff-2h signal . a possible mechanism for ff transformation is that alkb first oxidizes the furfuryl methylene group to generate an ho - ff intermediate ( scheme 1c ) , mirroring the oxidative hydroxylation mechanism alkb uses on other dna alkyl adducts , such as the formation of ho - m6a from m6a . the ho - ff intermediate can then either directly decompose to undamaged guanine or lose water to form the imine ff-2h . because ff-2h is a schiff s base , it is susceptible to be hydrolyzed to the undamaged guanine and furfurylaldehyde . in the experiment with hf , an m / z of 1246.26 was observed for the starting material in the absence of alkb ( figure 2 g ) . addition of alkb resulted in four peak envelopes in the mass spectrum ( figure 2h ) . unreacted starting material ( 35% remaining after 60 min incubation ) was clearly identified at an m / z of 1246.26 , while unmodified guanine ( 8% ) was present at 1225.24 ( figure s11 , supporting information ) . the peak envelope at 1250.26 ( 56% ) matched the calculated mw of the ho - hf ( 1250.22 , scheme 1d and figure s13 , supporting information ) . for the ho - hf intermediate , the reaction could proceed either to form a dehydration product hf-2h or to decompose to guanine ( scheme 1d ) . the fourth peak , albeit a small one at 1% , corresponded to the schiff s base type intermediate hf-2h at m / z of 1245.75 ( figure 2h and figure s12 , supporting information ) , which can be hydrolyzed to the undamaged guanine and unconjugated thf - aldehyde ( scheme 1d ) . for the incubation of m4c without alkb , an m / z of 1218.70 was observed for the 16-mer oligonucleotide containing m4c ( figure 2i ) , which corresponds well with the calculated m / z 1218.71 ( table 1 ) . there was another small peak envelope at m / z of 1215.45 ( figure s15 , supporting information ) in the spectrum , which corresponds to the 16-mer containing a uracil base at the lesion site ( calculated m / z 1215.45 , table 1 ) . the uracil base in the oligonucleotide could be generated from the deamination reaction of m4c . in the presence of alkb , about 10% of m4c is converted to cytosine after 1 h of incubation ( figure 2j and figure s16 , supporting information , 4 charge m / z 1215.20 ) , with approximately 90% of m4c remaining unchanged ( figure 2j ) . the mw of the oligonucleotide containing cytosine ( 4864.85 ) is 1 da less than that of the oligonucleotide containing uracil ( 4865.83 ) . the mw difference between the two oligonucleotides at 4 charge state corresponds to an m / z difference of 0.25 unit , which is clearly demonstrated by the observed m / z of 1215.45 for uracil and 1215.20 for cytosine . these observations strongly support the conclusion that alkb indeed repairs the m4c adduct to unmodified cytosine . to confirm the identity of the oligonucleotides containing lesions , repair intermediates , and products , ms / ms analysis was performed on each of the 5 alkb reactions . in each case , both the unreacted oligonucleotide and the product oligonucleotide were chosen for collision - induced - dissociation ( cid ) . the results , presented in detail in the supporting information , confirmed the identity of the corresponding oligonucleotide species ( figures s18s29 and tables s2s12 , supporting information ) . the results of the repair reactions of the five lesions carried out in the presence of the complementary strand are summarized in table s1 ( supporting information ) . a control experiment to test repair efficiency in both ssdna and dsdna was setup using the known alkb substrate m1a , which was 20% converted to a in 1 h in both contexts . unlike the reactions in ssdna , the n - aklyl - g and m4c lesions in dsdna were left essentially untouched by alkb . this study completes our analysis of the sites on dna that alkb can access in order to repair single stranded dna . the data , taken together , show that the enzyme repairs alkylation damage at all possible nitrogens involved in the normal base - pairing of guanine , cytosine , adenine , and thymine . here , we show that alkb repairs the simple alkyl lesions m2 g , e2 g , and m4c , as well as the bulky lesions ff and hf when present in ssdna , but not in dsdna . the modified oligonucleotides were incubated with or without purified alkb enzyme in vitro , with or without the complementary strand , and the reaction products were analyzed by esi - tof mass spectrometry and ms / ms . these assay conditions were not developed to provide kinetic constants for the alkb reactions . rather , they show qualitatively whether or not a given lesion has the chemical properties needed to be a substrate . if the lesion is chemically competent for repair , it is introduced into cells in subsequent studies to determine if alkb acts upon the lesion in vivo . the finding that alkb can act on the m4c dna adduct suggests an additional biological function for alkb , besides its normal role as a dna repair enzyme . the m4c adduct , which is similar to m6a , is normally not considered a deleterious lesion . the results presented above suggest that alkb might not only act as a repair enzyme to defend against dna alkyl damage but also play a role in controlling gene expression and/or dna replication and segregation . the present study also highlights the versatility of alkb in dealing with different sizes of alkyl adducts at the n position of guanine . the enzyme can modify not only the small alkyl lesions m2 g or e2 g but also the bulky ff and hf lesions . interestingly , the ff and hf lesions , which are structurally similar to each other , displayed very different amounts of alkb - induced repair intermediates ( figure 2 ) . conversion of ff to undamaged guanine was also much greater than the conversion for hf . reasons for these observations may be that the ff lesion possesses an aromatic furan ring , which may aid in oxidation of the aliphatic carbon to form ho - ff ( analogous to the oxidation of a chemically reactive benzylic position ) , as well as dehydration of ho - ff to ff-2h . also , the furfurylaldehyde released enjoys conjugation between the aldehyde carbonyl group and the aromatic furan ring , which is lacking for the ring - saturated thf - aldehyde released from the abundant ho - hf intermediate . interestingly , a major intermediate , ff-2h ( scheme 1c ) , consistent with being a schiff s base dehydration product , was much more prevalent for the ff alkb incubation than was hf-2h for the analogous hf - derived incubation . meanwhile , the amount of ho - ff was very small compared to the amount of ho - hf . one possible explanation is that the dehydration of ho - ff is driven by the formation of the intermediate ff-2h ( scheme 1c ) , a very stable extensively conjugated system throughout the entire guanine and furan rings . however , hf-2h ( scheme 1d ) lacks such conjugation leading to the accumulation of larger amounts of ho - hf ( figure 2h ) . alkb is known to repair lesions , such as m1a and m3c , in both ssdna and dsdna , with ssdna being the preferred substrate . crick base - pairing interactions , thus facilitating their recognition by alkb and its homologues in both ssdna and dsdna . interestingly , while all the lesions tested in this work were repaired by alkb in ssdna , they were not good substrates for repair by alkb when presented in dsdna ( table s1 , supporting information ) . one possible explanation for these observations relies on the fact that the n - alkyl - dg and m4c lesions display alkyl - groups on the exocyclic amino groups of dg or dc ; these alkyl groups can rotate in the minor or major groove to allow for unimpeded watson several crystal structures of alkb with different dna alkyl lesions in ssdna and dsdna have been published . we modeled the ng lesions based on the structures of alkb with m1a and ea and modeled the m4c lesion based on the m3c structure . among the different conformers generated from the free - rotation of the exocyclic amino groups in of ng and m4c lesions , we chose the conformers that directed the n - attached lesion carbon closest to the fe(ii ) center . the analyses showed that the distance between this carbon and the fe(ii ) center was about 5 , which was similar to the distance between the n - attached lesion carbon and fe(ii ) center in the crystal structures of oligonucleotides containing m1a , m1 g , ea , and m3c with alkb . on the basis of the structural analyses , the five exocyclic lesions tested in this work should be able to adopt conformations that place the alkyl groups in the vicinity of the catalytic fe(ii ) center of alkb , allowing the enzyme to oxidize and dealkylate the modified bases . while alkb may appear to be a panacea for wiping the nucleobase n - alkylation damage slate clean at all watson crick base - pairing positions , the relative extents of alkylation removal will depend not only on the position of the alkylated nitrogen atom for each base but also on other factors , which include the alkb binding specificity for a given lesion , the relative conformation of the alkyl group in the active site , and the rate of decomposition of the oxidized intermediates to products . further kinetic studies are needed to establish the rank order of alkylation repair by alkb and its homologues for the diverse constellation of their alkyl lesion substrates .

the alkb enzyme is an fe(ii)- and -ketoglutarate - dependent dioxygenase that repairs dna alkyl lesions by a direct reversal of damage mechanism as part of the adaptive response in e. coli . the reported substrate scope of alkb includes simple dna alkyl adducts , such as 1-methyladenine , 3-methylcytosine , 3-ethylcytosine , 1-methylguanine , 3-methylthymine , and n6-methyladenine , as well as more complex dna adducts , such as 1,n6-ethenoadenine , 3,n4-ethenocytosine , and 1,n6-ethanoadenine . previous studies have revealed , in a piecemeal way , that alkb has an impressive repertoire of substrates . the present study makes two additions to this list , showing that alkyl adducts on the n2 position of guanine and n4 position of cytosine are also substrates for alkb . using high resolution esi - tof mass spectrometry , we show that alkb has the biochemical capability to repair in vitron2-methylguanine , n2-ethylguanine , n2-furan-2-yl - methylguanine , n2-tetrahydrofuran-2-yl - methylguanine , and n4-methylcytosine in ssdna but not in dsdna . when viewed together with previous work , the experimental data herein demonstrate that alkb is able to repair all simple n - alkyl adducts occurring at the watson crick base pairing interface of the four dna bases , confirming alkb as a versatile gatekeeper of genomic integrity under alkylation stress .

Introduction Experimental Procedures Results Discussion

historically , the sweetness of urine and other body fluids in diabetic patients suggested that glucose had an important role in the physiopathology of this common disease . thus , glucose metabolism and the insulin - glucose axis were the leading fields for scientific investigations . to emphasize this concept , not only hyperglycaemia is crucial for the diagnosis of diabetes and the development of clinical complications , but also increasing evidence demonstrated the involvement of insulin in the physiopathology of this disease . in fact , diabetes is a metabolic disease characterized by hyperglycaemia resulting from either defects in insulin secretion or insulin properties , or both . in the present review , we focus on defects of insulin properties , with particular regard to insulin resistance , which can be defined as a state of reduced responsiveness to normal circulating levels of insulin . this condition is a feature of various disorders , such as type 2 diabetes , which may range from predominantly insulin resistance with relative insulin deficiency to a predominatly secretory defect of insulin . insulin resistance is also implicated in impaired glucose tolerance ( igt ) or impaired fasting glucose ( ifg ) , both considered as prediabetes by the expert committee on the diagnosis and classification of diabetes mellitus [ 24 ] , as well as in obesity , hypertension , dyslipidaemia ( all disorders clustering in the so - called metabolic syndrome ) , other endocrinopathies , but also in different diseases , such as cancer , infections or rheumatic , and autoimmune diseases [ 711 ] . therefore , given the association between insulin resistance and different diseases , no epidemiological data are available , specifically focused on insulin resistance syndrome prevalence or incidence . furthermore , only recently ( in 1997 ) the who have accepted obesity as an epidemic public health burden in adults , without evaluating a well - defined method to monitoring the problem in children [ 1215 ] . finally , still few clinical studies on asian and african cohorts focused on insulin resistance and related diseases have been published [ 1619 ] . for all these reasons , to better define insulin resistance epidemiology syndrome requires more investigations insulin resistance was initially recognized as an allergy to insulin , with the production of antibodies anti - insulin [ 20 , 21 ] . further investigations showed that metabolism of both nonesterified fatty acids ( nefa ) and free fatty acids ( ffas ) was a crucial step in the development of insulin resistance [ 22 , 23 ] . on the basis of these new evidences , shafrir and raz suggested in 2003 that diabetes should be now called lipidus instead of mellitus . given the importance of physiological effects of insulin during atherogenesis [ 2527 ] , there is a need to better clarify the complexity of mechanisms underlying insulin resistance . insulin is an anabolic essential hormone for the maintenance of glucose omeostasis , tissue growth , and development . it is well known that insulin is secreted by the pancreatic cells mainly in response to increased blood levels of glucose and aminoacids after the meals ( extrinsic rhythm ) . in addition , the concentration of insulin in the blood displays regular variations independently from the food intake . in fact , two rhythms with periods of 510 minutes and 60120 minutes have been documented ( intrinsic rhythm ) [ 3133 ] . the extrinsic rhythm was found altered in a lot of diseases including gestational diabetes , maturity onset diabetes of the young ( mody ) 1 [ 35 , 36 ] , mody 3 , and chagas disease . furthermore , an altered plasma insulin secretory response has been also observed as an effect of aging processes . on the contrary , the intrinsic rhythm has been found altered in various diseases , such as type 2 diabetes ( i.e. , mody 2 as well as maternally inherited diabetes and deafness ( midd ) ) [ 40 , 41 ] , obesity , and hypertension . several genetic and molecular studies have been performed to investigate the causes of the dysregulated plasma insulin pattern . although genetic mutations account for a minor role in the large part of insulin resistance , an alteration of insulin signal transduction , which may be due to genetic mutations , could contribute to the impairment of insulin secretory profile and insulin resistance . thus , mutations of glucokinase phosphorylated glucose , hepatic nuclear factor-4 alpha , hepatic nuclear factor-1 alpha , mitochondrial trnaleu(uur ) , and also insulin receptor genes have been found [ 35 , 37 , 4446 ] . for instance , a mutation in the insulin receptor gene of the pancreatic cells has been correlated with a defective insulin - mediated intracellular signal transduction . on the other hand , obesity and increased ffa levels mediate insulin resistance by inducing a decreased irs-1-associated phosphatidylinositol 3-kinase ( pi3-k ) activity . in line with these findings , it has been shown that insulin resistance was reversed when obese persons lose weight . however , this weight loss did not restore normal insulin pulsatiliy in type 2 diabetes patients . these data suggest that the defective insulin - mediated intracellular signal transduction is not the only cause responsible for insulin resistance , and that the molecular mechanisms of insulin resistance are not completely understood . we will discuss in the following the potential mechanisms contributing to insulin resistance which are currently under investigation . it is a member of the receptor tyrosine kinase family , composed of two -subunits and two -subunits linked together by disulphide bonds . two isoforms of insulin receptors are known , exhibiting different affinity for insulin and distribution within tissues . although it is tempting to suggest that differences in binding activity could contribute to insulin resistance , experimental evidence for the involvement of receptor isoforms or receptor hybrids remains controversial . apart from the insulin binding step to its receptor , the receptor intracellular tyrosine kinase domains ( capable of intrinsic kinase activity ) have been investigated in view of their possible implication in insulin resistance . a variety of scaffolding proteins , including insulin receptor substrate ( irs ) proteins , casitas b lineage lymphoma ( cbl ) , or cbl associated protein ( cap ) , bind to intracellular receptor sites and become phosphorylated [ 5153 ] . irs-1 and -2 are considered the most important proteins in regulation of glucose metabolism . as shown in knockout mouse models , irs-1 or irs-2 inactivation causes insulin resistance [ 55 , 56 ] . in addition , in vitro experiments showed an increased serine phosphorylation of irs by tumor necrosis factor - alpha ( tnf- ) or ffa stimulation , thereby causing impaired insulin signal transduction [ 54 , 57 ] . finally , a prolonged exposure to insulin ( a typical condition in hyperinsulinemic patients ) may result in a degradation of irs protein . all together , these data support irs-1 and irs-2 as crucial players in the development of insulin resistance . furthermore , numerous studies aimed to identify downstream elements of irs proteins in the insulin - mediated signal transduction pathway . three different isoforms of this kinase have been identified : ia , pi3-k / akt , capable of generating phosphatidylinositol 4,5-bisphosphate ( pip2 ) and phosphatidylinositol 3,4,5-trisphosphate ( pip3 ) ; ib , g protein regulated kinase ; ii , incapable of generating pip2 and pip3 . as shown in figure 1(a ) , activated pi3-k is responsible for the beginning of a complex phosphorylation cascade , involving the phospholipids pip2 , pip3 , the phosphoinositide - dependent kinase 1 ( pdk1 ) , the protein kinase b ( pkb , also called akt ) , as well as the protein kinase c ( pkc ) . akt mediates the effects of insulin on glucose transport ( glut ) , glycogen synthesis , protein synthesis , lipogenesis , and suppression of hepatic gluconeogenesis . once activated , akt detaches from the plasma membrane and translocates into the nucleus through a still unknown mechanism , or activates different substrates , such as glycogen synthase kinase-3 ( gsk-3 ) and transcription factors of the foxo - family . all these proteins and phospholipids are likely to be implicated in insulin resistance . for instance , a reduced pi3-k activity has been reported in skeletal muscle and adipocytes of patients with insulin resistance [ 6264 ] . in addition , akt activation has been found reduced in several diseases associated with insulin resistance [ 65 , 66 ] . however , akt involvement in insulin resistance is controversial , since other studies did not show any alteration of akt activation in insulin resistance associated syndromes [ 67 , 68 ] . on the other hand , insulin - induced pkc activation has been found altered in type 2 diabetic or obese patients . therefore , although other studies are needed , all these observations suggest that a reduction of insulin - mediated intracellular signaling is crucial for the establishment of insulin resistance . another possible mechanism leading to insulin resistance might be an upregulation of protein - tyrosine phosphatases ( ptpases ) , capable of functioning as negative regulators of the insulin - triggered pathway . among various proteins , ptp 1b has been shown as a key regulator of insulin signaling [ 7173 ] . other phosphatases , such as ectonucleotide pyrophosphatase phosphodiesterase 1 ( enpp1 , also known as pc-1 ) , sh-2-containing inositol 5-phosphatase 2 ( ship2 ) , and phosphatase and tensin homolog deleted on chromosome 10 ( pten ) have been shown to interfere with insulin sensitivity [ 7476 ] . although further investigations are warranted to very verify these hypotheses , these proteins may represent future potential targets for the treatment of insulin resistance . glucose uptake into muscle and fat tissue depends on glucose transporter 4 ( glut4 ) expression on the cell membrane . insulin reduces glycaemia , mainly by inducing the secretion of this molecule by muscle and fat cells . however , glut4 polymorphisms or mutations inactivating glut4 gene were not associated with insulin resistance . in addition , glut4 concentrations in skeletal muscle of insulin resistant patients were not reduced . this suggests that alterations in glut4 expression are not a primary cause for the development of insulin resistance . in this context in fact , glut4 upregulation represents the final event of insulin signaling cascade [ 8082 ] . among various kinases ( figure 1(a ) ) , akt has been shown to play an essential role . recently , in vitro and in vivo studies suggested that pkb alterations or disruption are responsible for the reduction of insulin - induced glucose uptake . consistent with these data , recent studies in humans detected a missense mutation in the kinase domain of pkb ( akt 2 ) associated with severe insulin resistance . these data suggest that insulin signaling also plays crucial role in the regulation glucose homeostasis . recent evidence suggests that inflammation might be crucial for the development of insulin resistance . proinflammatory cytokines and acute - phase reactants are positively correlated with insulin resistance in metabolic syndrome patients . among these soluble mediators , interleukin ( il)-1 and il-1 receptor antagonist ( ra ) have been implicated in the development of insulin resistance in humans [ 88 , 89 ] and in rodents [ 90 , 91 ] . il-1ra is anaturally occurring cytokine and a member of the il-1 family whose only function is to prevent a biologic response to il-1 . in humans , the blockade of il-1 with il-1 ra improves glycaemia and beta - cell secretory function and reduces markers of systemic inflammation . accordingly , il-1 has been shown to induce insulin resistance mainly by inhibiting insulin - mediated signaling [ 94 , 95 ] . thus , il-1 has to be considered as an important factor involved in insulin resistance . tnf- and il-6 are also of particular interest , because of their increased expression in adipose tissue and their capacity to induce insulin resistance . further evidence for the link between tnf- and insulin resistance was provided by a study using blocking anti - tnf- antibodies in obese rodents or tnf- knockout obese mice . in these animals , a reduced insulin resistance was obtained by the suppression of tnf-. the possible molecular mechanism of tnf--induced insulin resistance may involve irs-1 . surprisingly , the infusion of anti - tnf- antibody in humans did not affect insulin resistance . further investigations are needed to better understand these opposite results obtained in human and mice . on the other hand , the role of il-6 in insulin resistance il-6 interferes with the metabolism of both adipose and skeletal muscle tissues [ 99 , 100 ] , but has also a positive effect on skeletal muscle cell insulin sensitivity . in addition , il-15 has been shown to play a possible role in myocyte - adipocyte crosstalk , but only few studies are published at present to better clarify its role in insulin resistance . moreover , it is now established that hormones from adipose tissue hormones contribute to insulin resistance . for instance , leptin has been shown to reverse insulin resistance in mice with congenital lipodystrophy . administration of leptin to patients with lipodystrophy can increase the body fat content and reverse insulin resistance . resistin , a new adipocyte hormone , may be another important link between increased fat mass and insulin resistance . resistin decreases insulin - dependent glucose transport in vitro and increases fasting blood glucose concentrations and hepatic glucose production in vivo [ 106109 ] . very recently , adiponectin has been showed as an anti - inflammatory and immunomodulatory molecule [ 110 , 111 ] . in humans , mice deficient in adiponectin are insulin resistant and the administration of adiponectine to obese and insulin resistant mice has been shown to improve insulin sensitivity [ 113115 ] . in addition , inflammatory mediators such as the proinflammatory chemokine monocyte chemotactic protein-1 ( mcp-1 ) are believed to play a role in the pathogenesis of insulin resistance . recent in vitro evidence suggests that mcp-1 induces insulin resistance in both adipocytes and skeletal muscle cells . finally , retinol - binding protein ( rbp)-4 and tissue inhibitors of metalloproteinases ( timp)-1 were recently described to contribute to insulin resistance in vivo , but the underlying mechanism remains unclear [ 117119 ] . in conclusion , at the present state of knowledge , insulin resistance has to be defined as a complex syndrome involving not only glucose and lipids , but also several proinflammatory molecules . lipid abnormalities , such as increased circulating free fatty acids , are frequently associated with insulin resistance . the excessive fat intake causes an increased influx of triglycerides into the blood and an excess of plasma levels of ffas , which induce insulin resistance , with consequent hyperglycaemia . the increased levels of glucose stimulate pancreatic cells to secrete more and more insulin , generating hyperinsulinemia , which further triggers the elevation of triglycerides and closes the vicious circle . when insulin secretion is not sufficient and elevated glucose levels prevail , diabetes becomes overt . defective insulin secretion is a result of chronic exposure to elevated levels of fatty acids , which inhibits insulin gene expression by functioning as true toxic agents for pancreatic cells . lipotoxicity depends on the interference with insulin - mediated intracellular signaling in various cell types ( figure 1 ) . in particular , ffas have been shown to activate pkc ( figure 1(a ) ) , which not only interferes with insulin signaling ( by inducing insulin resistance ) , but also is implicated in promoting proatherogenic mechanisms , such as endothelial dysfunction , growth , migration , and apoptosis of vascular smooth muscle cells , induction of adhesion molecules and oxidized low - density lipoprotein uptake of oxidized low - density lipoprotein by monocyte - derived macrophages . furthermore , it was recently shown that ffas induce insulin resistance in muscle through the activation of inhibitor b kinase ( ikk ) and c - jun n - terminal kinase ( jnk ) ( figure 1(a ) ) . therefore , ffas induce insulin resistance in hepatocytes and skeletal muscle cells through the activation of different kinases ( figures 1(a ) and 1(b ) ) . both ffas from plasma and those released from stored triglycerides ffas are also involved in modulating insulin production by pancreatic -cells and cytokine secretion by hepatocytes , adipocyte , muscle cells , and inflammatory cells ( figure 1(c ) ) . the development of atherosclerotic plaques is dependent on the interaction of multistep biochemical processes that lead to the plaque formation , maturation , and complication . plaque instability , rupture , and thrombosis are crucial events in the acute artery occlusion , which causes dramatic ischemic consequences in the heart , brain , and also peripheral tissues . insulin resistance is considered to be a pivotal event in the increased risk of plaque instability through different pathways [ 127 , 128 ] . high concentrations of insulin directly increase proinflammatory activities of leukocytes , which are involved in atherosclerotic plaque instability . in particular , insulin directly increases neutrophil and monocyte in vitro migration in response to chemokines secreted in atherosclerotic plaques [ 129 , 130 ] . furthermore , insulin induces in vivo production of matrix metalloproteinase-9 ( mmp-9 ) , which is responsible for plaque instability and rupture [ 132134 ] . the pharmacologic or behavioral treatments to reduce insulin resistance have been shown to inhibit mmp-9 secretion [ 126 , 135 , 136 ] . on the other hand , insulin could also induce a serious atherothrombotic state , by increasing platelet resistance to antiaggregating agents and production of procoagulatory factors , such as plasminogen activator inhibitor-1 ( pai-1 ) , factor vii , factor xii , fibrinogen , and tissue plasminogen activator . these evidences strongly support an emerging role of insulin in plaque instability and rupture . further investigations are needed to better clarify the specific roles and the interactions of insulin and lipids on inflammatory cells . in the last years , the standard definition of insulin resistance has been shifted from a traditional glucocentric to a new lipocentric view . several soluble mediators are involved in the development of insulin resistance , through generating insulin signaling dysfunction . among these , ffas have to be considered as proatherosclerotic agents , capable of interfering with insulin signaling and provoking insulin resistance . new and selective therapies contrasting ffa effects may be promising targets for the treatment of insulin resistance . a possible promising strategy capable of reducing the consequences of excessive lipolysis and reorient ffa flux toward adipose tissue might be represented by peroxisome proliferator - activated receptor ( ppar)- and - agonists . ppar- agonists have been recently shown to regulate trygliceride lipase in adipocytes in vitro and in vivo . furthermore , ppar- has been shown as crucial in the control of differentiation of human monocytes in m2 macrophages , the subset of macrophages resident in atherosclerotic plaques with anti - inflammatory activity . in vivo studies have also demonstrated that ppar- agonists treatment in patients with type 2 diabetes mellitus is associated with a reduction in plasma nefa levels [ 142145 ] . however , two recent important clinical studies have shown an increase of acute cardiovascular outcomes induced by treatment with thiazolidinediones ( ppar- agonists ) [ 146 , 147 ] . on the other hand , although ppar- has been shown to have vascular and metabolic beneficial effects , the activity of ppar- agonists on lipid metabolism is still controversial [ 148 , 149 ] . therefore , further trials are needed to recommend the use of these pharmacological agents for reducing lipid - mediated insulin resistance .

the dysregulation of the insulin - glucose axis represents the crucial event in insulin resistance syndrome . insulin resistance increases atherogenesis and atherosclerotic plaque instability by inducing proinflammatory activities on vascular and immune cells . this condition characterizes several diseases , such as type 2 diabetes , impaired glucose tolerance ( igt ) , impaired fasting glucose ( ifg ) , obesity , hypertension , dyslipidemia , and other endocrinopathies , but also cancer . recent studies suggest that the pathophysiology of insulin resistance is closely related to interferences with insulin - mediated intracellular signaling on skeletal muscle cells , hepatocytes , and adipocytes . strong evidence supports the role of free fatty acids ( ffas ) in promoting insulin resistance . the ffa - induced activation of protein kinase c ( pkc ) delta , inhibitor kappab kinase ( ikk ) , or c - jun n - terminal kinase ( jnk ) modulates insulin - triggered intracellular pathway ( classically known as pi3-k - dependent ) . therefore , reduction of ffa levels represents a selective target for modulating insulin resistance .

1. EPIDEMIOLOGY OF INSULIN RESISTANCE 2. MECHANISMS OF INSULIN RESISTANCE 3. ROLE OF INSULIN RESISTANCE IN ATHEROSCLEROTIC PLAQUE INSTABILITY 4. CONCLUSION REMARKS

zinc is an essential trace element for living organisms , because it is required for the catalytic activity of numerous metalloenzymes ( 1 ) and can also serve as a key structural component of a large number of zinc - dependent proteins 2 . , 3 .. zinc homeostasis in the cells , therefore , is achieved through the coordinate regulation of zinc influx , efflux , and distribution to intracellular organelles ( 4 ) . zinc transporters have essential functions in such processes and a number of zinc transporters have been identified in many organisms 4 . , 5 . , 6 . , 7 .. zinc transporters are largely classified into two metal transporter families , the zip ( zrt / irt - like protein ) and cdf ( cation diffusion facilitator ) families 4 . , 5 . , 7 .. in bacteria , the abc transporters and p - type atpases have been shown to function as zinc transporters ( 8) , but neither of them plays a physiological role in zinc transport in eukaryotes ( 5 ) . the zip and cdf families are also assigned as solute carrier 39 ( slc39a ) and slc30a families 9 . both seem to have a very ancient origin because they are identified in diverse organisms from archeae and eubacteria to eukaryotes ( 5 ) . zip family transporters function in zinc influx into the cytosol , while cdf family transporters mobilize zinc in the opposite direction . all members of both families are thought to transport zinc across the biological membranes , but certain proteins are speculated to transport other metals such as iron , nickel , and manganese as a major substrate . in fact , zip and cdf transporters have been shown to transport these metals as physiologically important substrates in certain plants 11 . manganese not zinc is described as a more selective substrate of zip8/bigm103 in the competitive assay of cadmium uptake ( 14 ) . recently , interesting functions of zip and cdf transporters have been found in various organisms . a comprehensive deliberation on these functions together with integrative comparison of the sequence similarity within each zip and cdf transporter family would provide a clue to speculate functions of the uncharacterized zip and cdf proteins and to elicit further functions of the characterized ones . here we review the physiological and cellular functions of zip and cdf transporters with emphasis on these matters . to date , fourteen zip proteins have been molecularly characterized or identified in human and mouse 4 . , 9 .. the zip family is divided into subfamilies i , ii , liv1/lzt , and gufa , based on their degrees of sequence conservation 5 . the liv1/lzt subfamily is characterized by having a metalloprotease motif ( hexphexgd ) around the membrane - spanning domain v. although the initial histidine in the hexphexgd motif is thought to be requisite for the zinc transport activity of liv1/lzt transporters , zip8/bigm103 and zip14 lacking it have zinc transport activity 19 . , 22 .. zip proteins are predicted to have eight membrane - spanning domains with a membrane topology in which the n- and c - terminal ends are located outside the plasma membrane , and have a cytoplasmic his - rich loop between membrane - spanning domains iii and iv , which is thought to function as a zinc - binding site . however , zip11 , zip12 , and zip13 lack the his - rich loop . table 1 shows the zip proteins found in the genome sequences of human , mouse , chicken , zebrafish , fruit fly , nematode , and yeast according to the similarity to the human zip proteins . as shown in table 1 , most liv1/lzt proteins in the indicated organisms are arranged to each human liv1/lzt member except in yeast , where the liv1/lzt protein is only found as the homologous protein to zip7/ke4 . in liv1/lzt subfamily , zip12 and zip4 , zip8/bigm103 and zip14 , zip10 and zip6/liv1 , or zip13 and zip7/ke4 are similar ( figure 1 ) , but not completely homologous . for example , zip12 lacks the his - rich loop between membrane - spanning domains iii and iv while zip4 has ; zip13 lacks histidine residues in n - terminal portion and between membrane - spanning domains ii and iii , or iii and iv , but zip7/ke4 has many histidine residues in these portions . zip8/bigm103 and zip14 , or zip10 and zip6/liv1 have a high identity ( 48% or 38% identity , respectively ) and are homologous in the length of amino acid sequence , the property of the his - rich loop , and the distribution of histidine residues in their sequences 19 . , 23 .. the expression of only one or the other of zip8/bigm103 and zip14 in nematode , or zip10 and zip6/liv1 in fruit fly and nematode , may be sufficient for the biological function , judging from the genome sequences ( table 1 ) . compared with liv1/lzt subfamily , zipii subfamily has similar amino acid length , topology , and subcellular localization ( at the plasma membrane ) 4 . , 5 . , 24 .. interestingly , the numbers of homologous proteins of zip1 , zip2 , or zip3 found are : one in chicken and zebrafish , two in yeast , and six in nematode ( table 1 ) , which may be linked to the fact that nematode has extra cdf proteins ( see table 2 and below ) . all eukaryotes have proteins with similarity to zip9 of zipi subfamily or zip11 of gufa subfamily , which suggests that they may have retained important functions during evolution . zebrafish zip6/liv1 is essential for epithelial - mesenchymal transition ( emt ) , which is one of the central events of embryonic development , organ and tissue remodeling , and cancer metastasis , by regulating the nuclear localization of the zinc - finger transcription factor snail , a master regulator of emt , because it represses the transcription of e - cadherin ( 25 ) . the expression of zebrafish zip6/liv1 is dependent on stat3 , which is required for the cell migration , and this characteristic is conserved in human and mouse zip6/liv1 ( 25 ) . as zip6/liv1 was identified as an estrogen - regulated gene in breast cancer cells ( 26 ) and was shown to be significantly associated with the spread of breast cancer to the lymph nodes ( 27 ) , the presented function of zip6/liv1 is very interesting in that it may be a novel therapeutic target for improving tumor therapy ( 28 ) . foi , a homologous protein of zip10 in fruit fly , can act as a zinc transporter ( 29 ) , and is required for both germ cell ensheathment and gonad morphogenesis in order to control germ cell migration without affecting gonad cell identity ( 30 ) . it controls the level of e - cadherin in the gonad that is essential for the cell - cell adhesion ( 31 ) . foi was reported as the closely related protein to zip6/liv1 ( 30 ) , but its sequence is the most homologous to zip10 ( table 1 ) . zip10 and zip6/liv1 are homologous in amino acid sequence ( 38% identity ) and the property of many histidine residues in the his - rich loop or n - terminal portion , therefore they are likely to have very similar functions . another liv1/lzt protein , catsup , a fruit fly zip7/ke4 orthologue , down - regulates tyrosine hydroxylase activity ( 32 ) . interestingly , iar1 , an arabidopsis zip7/ke4 homologue , is supposed to regulate auxin conjugate hydroxylase activity by exporting inhibitory metal ( zinc ) out of the secretory pathway ( 33 ) . actually , zip7/ke4 is localized to the endoplasmic reticulum ( er ) and the golgi apparatus 34 . , 35 . and transport zinc out of the golgi apparatus ( 35 ) . since the expression of mouse zip7/ke4 cdna complements the defects of iar1 mutant ( 33 ) , zip7/ke4 and all of its orthologues may export zinc out of the secretory compartments to fine - tune the activity of zinc - requiring enzymes and other metal - requiring enzymes like hydroxylases . in yeast , a high - affinity zinc uptake transporter , zrt1 , is rapidly endocytosed from plasma membrane through a ubiquitin - mediated mechanism and degraded in vacuoles in response to high levels of extracellular zinc 36 . , 37 .. this type of posttranslational distribution operates in mammalian zip proteins ; not only in the zrt1 homologous proteins zip1 and zip3 38 . , 39 . , 41 .. these characteristics of zip transporters indicate that the traffic of zip proteins in response to extracellular zinc would be essential for physiological and cellular zinc homeostasis . to date , fourteen zip proteins have been molecularly characterized or identified in human and mouse 4 . , 9 .. the zip family is divided into subfamilies i , ii , liv1/lzt , and gufa , based on their degrees of sequence conservation 5 . the liv1/lzt subfamily is characterized by having a metalloprotease motif ( hexphexgd ) around the membrane - spanning domain v. although the initial histidine in the hexphexgd motif is thought to be requisite for the zinc transport activity of liv1/lzt transporters , zip8/bigm103 and zip14 lacking it have zinc transport activity 19 . , 22 .. zip proteins are predicted to have eight membrane - spanning domains with a membrane topology in which the n- and c - terminal ends are located outside the plasma membrane , and have a cytoplasmic his - rich loop between membrane - spanning domains iii and iv , which is thought to function as a zinc - binding site . however , zip11 , zip12 , and zip13 lack the his - rich loop . table 1 shows the zip proteins found in the genome sequences of human , mouse , chicken , zebrafish , fruit fly , nematode , and yeast according to the similarity to the human zip proteins . as shown in table 1 , most liv1/lzt proteins in the indicated organisms are arranged to each human liv1/lzt member except in yeast , where the liv1/lzt protein is only found as the homologous protein to zip7/ke4 . in liv1/lzt subfamily , zip12 and zip4 , zip8/bigm103 and zip14 , zip10 and zip6/liv1 , or zip13 and zip7/ke4 are similar ( figure 1 ) , but not completely homologous . for example , zip12 lacks the his - rich loop between membrane - spanning domains iii and iv while zip4 has ; zip13 lacks histidine residues in n - terminal portion and between membrane - spanning domains ii and iii , or iii and iv , but zip7/ke4 has many histidine residues in these portions . zip8/bigm103 and zip14 , or zip10 and zip6/liv1 have a high identity ( 48% or 38% identity , respectively ) and are homologous in the length of amino acid sequence , the property of the his - rich loop , and the distribution of histidine residues in their sequences 19 . , 23 .. the expression of only one or the other of zip8/bigm103 and zip14 in nematode , or zip10 and zip6/liv1 in fruit fly and nematode , may be sufficient for the biological function , judging from the genome sequences ( table 1 ) . compared with liv1/lzt subfamily , zipii subfamily has similar amino acid length , topology , and subcellular localization ( at the plasma membrane ) 4 . , 5 . , 24 .. interestingly , the numbers of homologous proteins of zip1 , zip2 , or zip3 found are : one in chicken and zebrafish , two in yeast , and six in nematode ( table 1 ) , which may be linked to the fact that nematode has extra cdf proteins ( see table 2 and below ) . all eukaryotes have proteins with similarity to zip9 of zipi subfamily or zip11 of gufa subfamily , which suggests that they may have retained important functions during evolution . zebrafish zip6/liv1 is essential for epithelial - mesenchymal transition ( emt ) , which is one of the central events of embryonic development , organ and tissue remodeling , and cancer metastasis , by regulating the nuclear localization of the zinc - finger transcription factor snail , a master regulator of emt , because it represses the transcription of e - cadherin ( 25 ) . the expression of zebrafish zip6/liv1 is dependent on stat3 , which is required for the cell migration , and this characteristic is conserved in human and mouse zip6/liv1 ( 25 ) . as zip6/liv1 was identified as an estrogen - regulated gene in breast cancer cells ( 26 ) and was shown to be significantly associated with the spread of breast cancer to the lymph nodes ( 27 ) , the presented function of zip6/liv1 is very interesting in that it may be a novel therapeutic target for improving tumor therapy ( 28 ) . foi , a homologous protein of zip10 in fruit fly , can act as a zinc transporter ( 29 ) , and is required for both germ cell ensheathment and gonad morphogenesis in order to control germ cell migration without affecting gonad cell identity ( 30 ) . it controls the level of e - cadherin in the gonad that is essential for the cell - cell adhesion ( 31 ) . foi was reported as the closely related protein to zip6/liv1 ( 30 ) , but its sequence is the most homologous to zip10 ( table 1 ) . zip10 and zip6/liv1 are homologous in amino acid sequence ( 38% identity ) and the property of many histidine residues in the his - rich loop or n - terminal portion , therefore they are likely to have very similar functions . either of them may function as a backup system if expressed simultaneously . another liv1/lzt protein , catsup , a fruit fly zip7/ke4 orthologue , down - regulates tyrosine hydroxylase activity ( 32 ) . interestingly , iar1 , an arabidopsis zip7/ke4 homologue , is supposed to regulate auxin conjugate hydroxylase activity by exporting inhibitory metal ( zinc ) out of the secretory pathway ( 33 ) . actually , zip7/ke4 is localized to the endoplasmic reticulum ( er ) and the golgi apparatus 34 . , 35 . and transport zinc out of the golgi apparatus ( 35 ) . since the expression of mouse zip7/ke4 cdna complements the defects of iar1 mutant ( 33 ) , zip7/ke4 and all of its orthologues may export zinc out of the secretory compartments to fine - tune the activity of zinc - requiring enzymes and other metal - requiring enzymes like hydroxylases . in yeast , a high - affinity zinc uptake transporter , zrt1 , is rapidly endocytosed from plasma membrane through a ubiquitin - mediated mechanism and degraded in vacuoles in response to high levels of extracellular zinc 36 . , 37 .. this type of posttranslational distribution operates in mammalian zip proteins ; not only in the zrt1 homologous proteins zip1 and zip3 38 . , 39 . , 41 .. these characteristics of zip transporters indicate that the traffic of zip proteins in response to extracellular zinc would be essential for physiological and cellular zinc homeostasis . to date , ten cdf proteins designated as znt ( zn transporter ) proteins of human or murine origin have been molecularly characterized or identified 7 . , 10 . , 42 .. cdf transporters are divided into three subgroups , cdf subfamilies i , ii , and iii , based on their sequence similarities ( 5 ) . most eukaryotic members are assigned to subfamilies ii and iii ( 5 ) but znt9/huel and its homologous proteins are classified into cdf subfamily i ( figure 1 ) , which contains mostly prokaryotic members from both eubacterial and archeael sources ( 5 ) . there are sequence similarities among znt2 , znt3 , znt4 , and znt8 , between znt1 and znt10 , and between znt5 and znt7 ( figure 1 ) , which suggests that these closely related proteins have similar functions in the cells . cdf transporters have the same predicted membrane topology of six membrane - spanning domains with both n- and c - terminal ends thought to reside intracellularly and a cytoplasmic his - rich loop between membrane - spanning domains iv and v , although znt5 and its homologous proteins have a long n - terminal portion with extra membrane - spanning domains ( 43 ) . in znt6 and its orthologues , the his - rich loop is not rich in histidine residues but retains serine residues instead ( 44 ) . in znt10 and its orthologues , the loop lacks histidine residues but bears a long loop rich in serine and basic amino acid residues ( 42 ) . the his - rich loop is thought to function as a metal - binding site and is shown to have essential functions ( 45 ) . znt9/huel has a cation efflux domain ( pfam01545 ) ; therefore , it has been assigned to the cdf family 7 . , 10 .. however , znt9/huel and its homologous proteins have significant homology to the dna - binding domain and the nuclear receptor interaction motif ( 46 ) . furthermore , znt9/huel is predominantly localized to the cytoplasm and translocates to the nucleus in a cell cycle - dependent manner ( 46 ) . the cdf proteins found in the human , mouse , chicken , zebrafish , fruit fly , nematode , and yeast genome sequences are arranged in table 2 according to the similarity to human znt proteins as in table 1 . compared with zip proteins , cdf proteins in the indicated organisms are arranged to each human znt member except for znt3 . znt3 is specifically expressed in the brain , which suggests that znt3 has important neural functions in mammals ( 47 ) . since the zinc transported by znt3 into the synaptic vesicles is implicated in -amyloid plaque formation ( 48 ) , the expression level of znt3 may be an important factor in the incidence of alzheimer s disease . the sequences of znt5 and znt6 are found simultaneously in all organisms except for fruit fly ( table 2 ) , which is consistent with their characteristic to form hetero - oligomeric complexes ( 49 ) . in fruit fly , the znt7 homologous protein is found ( table 2 ) . as znt5 and znt7 have similar functions in the secretory pathway ( see below and ref . 45 ) , the expression of either znt5 ( with znt6 ) or znt7 would be sufficient in fruit fly , nematode , and yeast . mft1 and mft2 , which were identified as mitochondrial iron transporters in yeast ( 50 ) , and sur7 , which is the nematode cdf protein involved in ras signaling ( 51 ) , are not classified into human members because of low homology and different subcellular localization and functions ( table 2 ) . nematode has five more cdf proteins that fail to show similarity to human members ( table 2 ) . further investigation is needed to identify their functions and the relationship between these unclassified cdf proteins and mammalian members . like the zip proteins , interesting cdf functions have been found in the model organisms . in yeast , msc2 and zrg17 form hetero - oligomeric complexes and have essential functions to maintain homeostasis in the er by transporting zinc into the er ( 52 ) . they are counterpart proteins of znt5 and znt6 , although msc2 is homologous to znt5 only in the c - terminal portion including six membrane - spanning domains 43 . , 53 . and zrg17 is the distant homologue of znt6 ( 52 ) . the hetero - oligomeric formation of znt5 and znt6 has been evidenced by using chicken dt40 cells deficient in znt5 , znt6 , and znt7 ( 49 ) . znt7 is homologous to znt5 in cation efflux domains ( pfam01545 ) , but it fails to form hetero - oligomeric complexes with znt6 ( unpublished data ) , instead , it forms homo - oligomeric complexes ( 49 ) . in vertebrates , these two different zinc transport complexes , znt5/znt6 hetero - oligomeric complexes and znt7 homo - oligomeric complexes , operate to activate zinc - requiring enzymes like alkaline phosphatases that are synthesized and activated by binding with zinc in the secretory pathway ( 49 ) . moreover , since msc2 and zrg17 are involved in the unfolded protein response ( upr ) because the mutant yeast strains lacking neither or either of the genes are defective in the er - associated degradation ( erad ) and show the increased upr under low - zinc conditions 52 . in fact , zinc deficiency can up - regulate the upr in mammalian cells ( 54 ) . cdf1 , a nematode znt1 orthologue , positively regulates the ras - raf - mek - erk signal transduction by promoting zinc efflux and reducing the concentration of cytosolic zinc 55 . , 56 .. cdf1 binds to raf-1 and promotes the biological and enzymatic activity of raf-1 ( 57 ) . this interaction occurs between the intracellular c - terminal tail of cdf1 and the n - terminal regulatory portion of raf-1 . , 57 .. as the binding of znt1 to raf-1 is inhibited by zinc ( 57 ) , it is plausible that znt1 lowers the cytosolic zinc , which promotes its binding to raf-1 and facilitates raf-1 activation . however , it has not been elucidated whether the mammalian ras - mediated signaling pathway is fine - tuned by znt1 in physiological condition . the divergent cdf protein in nematode , sur7 , which is probably localized to the er , also positively regulates ras signaling through modulating the activity of kinase suppressor of ras ( ksr ; ref . in fact , the toc-1 protein ( zc395.3 ) that shows homology to znt6 is reported to be involved in ras signaling ( 51 ) . the toc-1 protein seems to have essential functions of supplying zinc to proteins in the secretory pathway by forming hetero - oligomeric complexes with the znt5 orthologue protein ( y105e8a.3 ) , because the znt7 gene is not found in the nematode genome sequence ( table 2 ) . the putative hetero - oligomeric complexes may have important functions in ras signaling in nematode . to date , ten cdf proteins designated as znt ( zn transporter ) proteins of human or murine origin have been molecularly characterized or identified 7 . , 10 . , 42 .. cdf transporters are divided into three subgroups , cdf subfamilies i , ii , and iii , based on their sequence similarities ( 5 ) . most eukaryotic members are assigned to subfamilies ii and iii ( 5 ) but znt9/huel and its homologous proteins are classified into cdf subfamily i ( figure 1 ) , which contains mostly prokaryotic members from both eubacterial and archeael sources ( 5 ) . there are sequence similarities among znt2 , znt3 , znt4 , and znt8 , between znt1 and znt10 , and between znt5 and znt7 ( figure 1 ) , which suggests that these closely related proteins have similar functions in the cells . cdf transporters have the same predicted membrane topology of six membrane - spanning domains with both n- and c - terminal ends thought to reside intracellularly and a cytoplasmic his - rich loop between membrane - spanning domains iv and v , although znt5 and its homologous proteins have a long n - terminal portion with extra membrane - spanning domains ( 43 ) . in znt6 and its orthologues , the his - rich loop is not rich in histidine residues but retains serine residues instead ( 44 ) . in znt10 and its orthologues , the loop lacks histidine residues but bears a long loop rich in serine and basic amino acid residues ( 42 ) . the his - rich loop is thought to function as a metal - binding site and is shown to have essential functions ( 45 ) . znt9/huel has a cation efflux domain ( pfam01545 ) ; therefore , it has been assigned to the cdf family 7 . , 10 .. however , znt9/huel and its homologous proteins have significant homology to the dna - binding domain and the nuclear receptor interaction motif ( 46 ) . furthermore , znt9/huel is predominantly localized to the cytoplasm and translocates to the nucleus in a cell cycle - dependent manner ( 46 ) . the cdf proteins found in the human , mouse , chicken , zebrafish , fruit fly , nematode , and yeast genome sequences are arranged in table 2 according to the similarity to human znt proteins as in table 1 . compared with zip proteins , cdf proteins in the indicated organisms are arranged to each human znt member except for znt3 . znt3 is specifically expressed in the brain , which suggests that znt3 has important neural functions in mammals ( 47 ) . since the zinc transported by znt3 into the synaptic vesicles is implicated in -amyloid plaque formation ( 48 ) , the expression level of znt3 may be an important factor in the incidence of alzheimer s disease . the sequences of znt5 and znt6 are found simultaneously in all organisms except for fruit fly ( table 2 ) , which is consistent with their characteristic to form hetero - oligomeric complexes ( 49 ) . in fruit fly , the znt7 homologous protein is found ( table 2 ) . as znt5 and znt7 have similar functions in the secretory pathway ( see below and ref . 45 ) , the expression of either znt5 ( with znt6 ) or znt7 would be sufficient in fruit fly , nematode , and yeast . mft1 and mft2 , which were identified as mitochondrial iron transporters in yeast ( 50 ) , and sur7 , which is the nematode cdf protein involved in ras signaling ( 51 ) , are not classified into human members because of low homology and different subcellular localization and functions ( table 2 ) . nematode has five more cdf proteins that fail to show similarity to human members ( table 2 ) . further investigation is needed to identify their functions and the relationship between these unclassified cdf proteins and mammalian members . like the zip proteins , interesting cdf functions have been found in the model organisms . in yeast , msc2 and zrg17 form hetero - oligomeric complexes and have essential functions to maintain homeostasis in the er by transporting zinc into the er ( 52 ) . they are counterpart proteins of znt5 and znt6 , although msc2 is homologous to znt5 only in the c - terminal portion including six membrane - spanning domains 43 . , 53 . the hetero - oligomeric formation of znt5 and znt6 has been evidenced by using chicken dt40 cells deficient in znt5 , znt6 , and znt7 ( 49 ) . znt7 is homologous to znt5 in cation efflux domains ( pfam01545 ) , but it fails to form hetero - oligomeric complexes with znt6 ( unpublished data ) , instead , it forms homo - oligomeric complexes ( 49 ) . in vertebrates , these two different zinc transport complexes , znt5/znt6 hetero - oligomeric complexes and znt7 homo - oligomeric complexes , operate to activate zinc - requiring enzymes like alkaline phosphatases that are synthesized and activated by binding with zinc in the secretory pathway ( 49 ) . moreover , since msc2 and zrg17 are involved in the unfolded protein response ( upr ) because the mutant yeast strains lacking neither or either of the genes are defective in the er - associated degradation ( erad ) and show the increased upr under low - zinc conditions 52 . in fact , zinc deficiency can up - regulate the upr in mammalian cells ( 54 ) . cdf1 , a nematode znt1 orthologue , positively regulates the ras - raf - mek - erk signal transduction by promoting zinc efflux and reducing the concentration of cytosolic zinc 55 . , 56 .. cdf1 binds to raf-1 and promotes the biological and enzymatic activity of raf-1 ( 57 ) . this interaction occurs between the intracellular c - terminal tail of cdf1 and the n - terminal regulatory portion of raf-1 . , 57 .. as the binding of znt1 to raf-1 is inhibited by zinc ( 57 ) , it is plausible that znt1 lowers the cytosolic zinc , which promotes its binding to raf-1 and facilitates raf-1 activation . however , it has not been elucidated whether the mammalian ras - mediated signaling pathway is fine - tuned by znt1 in physiological condition . the divergent cdf protein in nematode , sur7 , which is probably localized to the er , also positively regulates ras signaling through modulating the activity of kinase suppressor of ras ( ksr ; ref . in fact , the toc-1 protein ( zc395.3 ) that shows homology to znt6 is reported to be involved in ras signaling ( 51 ) . the toc-1 protein seems to have essential functions of supplying zinc to proteins in the secretory pathway by forming hetero - oligomeric complexes with the znt5 orthologue protein ( y105e8a.3 ) , because the znt7 gene is not found in the nematode genome sequence ( table 2 ) . the putative hetero - oligomeric complexes may have important functions in ras signaling in nematode . various roles of zip or cdf transporters have been clarified , but further studies are needed to fully elucidate their physiological functions . a comprehensive comparison of similarities and differences in the functions and regulations in transcription , translation , trafficking , and turnover of homologous proteins of zip and cdf among mammals and other organisms should help elucidate the true role of each transporter in zinc homeostasis . by elucidating which of the redundant transporters is the principal or the backup , and identifying which transporter forms homo - oligomeric complexes or hetero - oligomeric complexes to express zinc transport activity , we should be able to ultimately solve the intriguing question why living organisms including humans need so many zinc transporters to survive .

zip ( zrt / irt - like protein ) and cdf ( cation diffusion facilitator ) are two large metal transporter families mainly transporting zinc into and out of the cytosol . several zip and cdf transporters have been characterized in mammals and various model organisms , such as yeast , nematode , fruit fly , and zebrafish , and many candidate genes have been identified by genome projects . unexpected functions of zip and cdf transporters have been recently reported in some model organisms , leading to major advances in our understanding of the functions of mammalian counterparts . here , we review the recent information on the sequence similarity and functional relationship among eukaryotic zip and cdf transporters obtained from the representative model organisms .

Introduction ZIP Transporters Arrangement of ZIP proteins found in the genome sequences of the representative model organisms Interesting characteristics of ZIP transporters obtained from the model organisms CDF Transporters Arrangement of CDF proteins found in the genome sequences of the representative model organisms Interesting characteristics of CDF transporters obtained from the model organisms Conclusion

lymphocytic plasmacytic enteritis ( lpe ) , a chronic enteropathie , is the most frequently described type of inflammatory bowel disease in dogs . lpe is recognized as one of the most common causes of chronic vomiting and diarrhoea . the name of this disorder refers to the population of inflammatory cells present in the lamina propria of the small bowel . despite being an important disease , the exact aetiology remains unclear but appears to involve an exaggerated reaction of the mucosal immune system against the environment ( bacteria and food antigens ) in a susceptible host . some investigators have recently focused on the effect disturbing the homeostasis between the immune system and luminal antigens in the intestinal microenvironment . similarly , several reports have evaluated the role of cytokines , subpopulations of leukocytes , lymphocyte apoptosis , toll - like receptors , nuclear factor kappa - beta or intestinal microbial communities . diagnosing inflammatory bowel disease is based on ruling out diseases that may cause intestinal inflammation along with histological evidence of inflammatory infiltration into the intestinal mucosa . marked advances in endoscopic equipment combined with the advantages of flexible endoscopy make upper gastrointestinal endoscopy the procedure of choice for obtaining intestinal biopsies to diagnose enteropathies in dogs and cats . information about macroscopic parameters of gastric and duodenal mucosa evaluated during endoscopy can vary depending on who performs the technique . recently , the gastrointestinal standardization group sponsored by world small animal veterinary association ( wsava ) proposed a set of guidelines for endoscopic examinations in order to obtain information about gastrointestinal endoscopic findings that could be universally used . moreover , the following should also be evaluated in the duodenum : distensibility of the lumen , hyperemia / vascularity , edema , discoloration , friability , texture , haemorrhage , erosion / ulcer , lacteal dilation , and contents ( mucus , bile , or food ) . distended lacteals , defined as many expanded white villi in the duodenum , is an endoscopic finding strongly indicative of intestinal lymphangiectasia ( il ) . these scattered white spots have been described as having a snowflake - like or rice grain - like appearance . information about the presence of these white spots and their significance is limited . as far as we know , this finding has been reported in one dog with il and lpe and in eight dogs with ultrasonographic intestinal hyperechoic mucosal striations . the aim of this retrospective study was to evaluate the significance of white spots in the duodenal mucosa of dogs with lpe . we analysed endoscopy examination data that were compiled between january 2000 and december 2008 at the complutense university of madrid veterinary medical teaching hospital ( spain ) . all the dogs were cared for according to protocols approved by the animal experimentation committee of the complutense university of madrid ( spain ) . the dogs in our study were diagnosed with lpe with white spots in the mucosa of the duodenum found by endoscopy ( n = 22 ) . control dogs consisted of animals randomly selected during the review of the endoscopic data that had lpe but no white spots ( n = 28 ) . the following information was obtained from the medical records of all dogs : signalment , clinical signs , and physical examination findings and laboratory results at the time of presentation . the severity of the clinical signs was assessed using two previously described activity indexes : the canine inflammatory bowel disease activity index ( cibdai ) and canine chronic enteropathy clinical activity index ( ccecai ) . the cibdai is the sum of the score of six different clinical signs including attitude / activity , appetite , vomiting , stool consistency , stool frequency , and weight loss . the recently introduced ccecai , which is based on the previously established cibdai , also includes the scoring of serum - albumin concentration , peripheral edema and ascites , and severity of pruritus . the minimum information obtained for each dog included a complete anamnesis , complete blood count and serum biochemical profile ; fecal examination for three consecutive days for cestodes , nematodes , and protozoa ( direct smear with saline solution , direct smear with methiolate iodine formaldehyde solution and zinc - sulfate flotation , or the telemann technique ) ; fecal chymotrypsin , and serum trypsin - like immunoreactivity . persistent gastrointestinal signs ( > 3 weeks in duration ) in combination with normal results from a thorough diagnostic evaluation and absence of a response to diet modification ( prescription dry diets for gastrointestinal disease with low concentration of lipid ) were noted in these dogs . gastroduodenoscopy was performed in all dogs in order to obtain gastric and duodenal mucosal biopsy specimens . videoendoscopes of variable lengths and diameters were used according to the size of the dog for endoscopic exploration . dogs were denied access to food 24 h prior to endoscopy and to water 12 h before the examination . an average of five to six images showing representative findings ( through the descending duodenum ) were routinely captured by a video printer . for all animals , photographs of the duodenum with gross white spots were reviewed in order to assess the density of the spots ( 1 = mild , 2 = moderate , 3 = severe ) . this evaluation method was chosen by the authors after taking into account the scores for other endoscopic parameters proposed by the wsava gastrointestinal standardization group . the density of the white spots was graded by one of the authors ( fr ) . information regarding the animals ' medical history , clinical signs , laboratory results , or histopathologic descriptions was not available to the clinician when evaluating the spots . all duodenal mucosal biopsy specimens were taken using flexible , through - the - endoscope , pinch biopsy forceps with smooth - edged oval cups . biopsy specimens were fixed by immersion in neutral - buffered 10% formalin , embedded in paraffin wax , cut into 5 m - thick sections , and stained with hematoxylin and eosin , masson trichromic , and periodic acid - schiff reagent . lpe was diagnosed in each dog based on information from the diagnostic evaluation combined with a histopathologic finding of lymphocyte and plasma cell infiltration into the lamina propria of the duodenum . a complete histopathological evaluation of all biopsies was performed according to the histopathologic criteria recently proposed by the wsava gastrointestinal standardization group for diagnosing gastrointestinal inflammation in dogs and cats . specifically , the findings for cases of lacteal dilation were scored from 0 to 3 as follows : 0 = normal , when the central lacteal represented up to 25% of the villous lamina propria width on the longitudinal section ; 1 = mild dilation , when this width represents up to approximately 50% ; 2 = moderate dilation , when this width represented up to 75% ; and 3 = marked dilation , when central lacteal represented up to 100% of the villous lamina propria . statistical analysis was performed using commercially available software ( ibm spss statistics 19 ; spss , usa ) . the clinical and histological findings of the two lpe groups ( with and without white spots ) were compared . data were analysed using student 's t - tests or willcoxon signed - rank tests depending on the distribution of the variables . correlations between the density of the white spots in the duodenum , serum proteins , serum albumin , activity index , and histological grading were analysed in lpe group with white spots using spearman 's test . a chi - square test was used to compare percentages ; a fisher exact test was used when needed . the lpe group with white spots in the duodenum included 22 animals : 17 males and 5 females . the median age at the time of endoscopy was 5.2 years ( range , 2~10 years ) . six animals were mixed breeds along with 16 dogs that were 11 different pure breeds . the lpe group without white spots in the duodenum included 28 animals with 18 males and 10 females . the median age at the time of endoscopy was 5 years ( range , 2~9 years ) . statistical significant differences were not found between both groups regarding age , sex and breed distribution . decreased serum protein concentrations ( 5.6 g / dl ) were found in 31.8% ( 7/22 ) of the lpe dogs with white spots and in 7.1% ( 2/28 ) of the lpe dogs without white spots . hypoproteinemia was significantly more frequent in dogs with white spots compared to ones without ( p = 0.02 ) . mean serum protein concentrations were 5.68 g / dl ( range , 2.4~8.4 g / dl ) in lpe dogs with white spots and 6.58 g / dl ( range , 4.6~7.6 g / dl ) in lpe dogs without . serum protein concentration was significantly lower in white spots lpe dogs related to the other group ( p = 0.038 ) . mean serum albumin concentrations were 2.84 g / dl ( range , 1.0~4.3 g / dl ) in lpe dogs with white spots and 3.29 g / dl ( range , 2.3~4 g / dl ) in ones without . serum albumin concentrations were significantly lower in the lpe dogs with spots compared to the dogs without ( p = 0.039 ) . no significant differences in activity indices were observed between the two groups according to ccecai ( white spots group : mean , 4.54 ; range , 0~15 ; group without spots : mean 5.53 ; range , 2~14 ) . cibdai scores were significantly lower ( p = 0.016 ) in the white spots group ( mean , 3.77 ; range , 0~11 ) compared to the group without white spots ( mean , 5.21 ; range , 2~9 ) . lacteal dilation histological scores were significantly higher ( p = 0.027 ) in lpe dogs with white spots ( mean , 0.45 ; range , 0~2 ) compared to dogs without white spots ( mean , 0.12 ; range , 0~1 ) . no statistically significant differences were found between the two groups when analyzing the other histological parameters . the density of the white spots in the duodenal mucosa was graded as mild in 14 dogs ( 64% ; fig . hypoproteinemia was found in 21% of the dogs ( 3/14 ) with mild white spot density , in 25% ( 1/4 ) with moderate density , and in 75% ( 3/4 ) with severe density . no statistically significant correlation was found between density of white spots and serum protein or albumin concentrations . likewise , no statistically significant correlation was found between the density of the white spots and either disease activity indices ( cibdai , p = 0.894 ; ccecai , p = 0.076 ) . however , the results showed a statistically significant correlation between density of the white spots and lymphatic dilatation histological scores ( p = 0.023 ; = 0.481 ; fig . to date , the present study is the first that compares two populations of lpe dogs , with or without white spots in the duodenal mucosa . the characteristics of both populations were similar in terms of age , gender , and breed distribution . until now , the appearance of many white spots on endoscopy strongly suggested intestinal il . to our knowledge , this feature has not been routinely described in dogs with lpe . according to our results , the presence of hypoproteinemia in lpe dogs is significantly higher when white spots are present in the duodenal mucosa than when they are not . previous studies reported a higher prevalence of hypoproteniemia in these lpe dogs compared to our study , ranging from 24~63% . it is unclear why the lpe dogs in the present study had a lower prevalence of hypoproteinemia compared to previous studies . although not statistically significant , correlation between serum protein or albumin levels and the density of the white spots was found . significant lower serum protein and albumin concentrations in lpe dogs with white spots was probably related to the presence of these spots in the duodenal mucosa . a recent study was the first to describe a significant association between lacteal dilation and hypoalbuminemia . an increase in plasma proteins leakage from the intestine can be the result of several causes via one of two main mechanisms : 1 ) mucosal injury with or without erosions or ulcerations , and 2 ) increased lymphatic pressure in the gut due to different factors . in our dogs , both mechanisms could be implicated . rupture of intestinal villous lacteals that appeared as " rice - grain " spots in duodenum in our study indicated lymph leakage into the intestinal lumen with other components such as chylomicrons , lymphocytes , and proteins . although some lymph constituents can be digested and reabsorbed at more distal sites in the intestine , the presence of lacteals dilation , inflammation , or oedema in the mucosa can limit intestinal absorptive capacity , resulting in a net loss of lymph . it was surprising that cibdai scores was significantly lower in the group with white spots compared to the group without . in this study , hypoalbuminemia has been reported to be a sign of poor prognosis which is why this group of dogs was expected to be in a poor clinical condition . after reviewing the activity indices of this group , we hypothesised that a possible reason for the relatively low cibdai scores might be that these animals presented only one clinical sign like vomiting or severe abdominal pain that is not noted in this activity index . up to now , the presence of scattered white spots in the duodenal mucosa seems to correspond to lacteals that are dilated and filled with chyle . the results from our study support this hypothesis based on the significant correlation found between the histological grade of lacteals distension and density of the white spots observed during endoscopy exploration . in cases of il ( congenital or acquired ) , the intestinal villous lacteals can dilate , become more fragile , and rupture easily when pressure in the mesenteric or intestinal lymph vessels increases independently of the disease . this explanation could also explain the presence of white spots in cases of lpe because distended lacteals are frequently found in the presence of this disease . in animals with intestinal diseases such as inflammatory lpe the results obtained from this study suggest that the appearance of white spots in the duodenal mucosa of dogs is not a finding exclusive to il . low serum protein and albumin concentrations are probably a result of these white spots . in our study , the density of the white spots in the duodenum significantly correlated with the histological lacteal dilation scores . further studies are needed in order to evaluate the clinical significance of these white spots in dogs with lpe , especially in terms of prognosis .

distended lacteals , described as expanded white villi in duodenum , are strongly indicative of primary intestinal lymphangiectasia . in the present study , we evaluated the significance of white spots present in the duodenal mucosa of dogs with lymphocytic plasmacytic enteritis ( lpe ) . fifty dogs with lpe were included in this study , and white spots were detected in the duodenal mucosa in 22 dogs during endoscopy . hypoproteinemia was more frequent in dogs with white spots than in dogs without spots ( p = 0.02 ) . serum protein and albumin concentration were significantly lower in lpe dogs with white spots ( p = 0.038 ) compared to lpe dogs without white spots ( p = 0.039 ) . there was a significant correlation between white spots density and lymphatic dilatation histological scores ( p = 0.023 ; = 0.481 ) . these results suggest that the presence of white spots in the duodenal mucosa of dogs is not a finding exclusive for intestinal lymphangiectasia . low serum protein and albumin concentrations together with lymphatic dilatation seem to be related to the presence of white spots in the duodenal mucosa of lpe dogs .

Introduction Materials and Methods Results Discussion

the united states maintains a national network of hospitals and clinics , using a single - payer , single - employer model , dedicated to providing care to former members of the united states armed forces . collectively , this network is known as the veterans health administration ( vha ) , which is a subsidiary of the cabinet - level department of veterans affairs . in these respects , the vha bears some resemblance to the british national health service and other nationalised healthcare systems in social democracies . it is common in these systems to provide performance measures , such as the national health service 's 4-hour rule , as a large - scale means of standardised care . given that long emergency department ( ed ) stays are known to be associated with increased risks of adverse events,1 the vha holds a national performance measure that no more than 10% of patients should spend > 6 h in the ed from arrival to disposition ( discharge , transfer or admission ) . moreover , waiting times are a major reason for patients leaving without being seen ( lwbs ) by a healthcare provider2 3 and , likewise , improvements in waiting times are associated with decreases in lwbs.4 5 the preponderance of evidence suggests that patients who leave without being seen are similar with regard to illness and acuity to patients who remain for care and , therefore , may have similar risk of adverse events after leaving.6 7 case reports of front - end interventions to improve ed length of stay ( los ) and lwbs have demonstrated success.8 however , the generalisability of such interventions remains in question . most interventions to reduce ed los involve costly process interventions for example , changes in staffing , facilities , computer or laboratory systems , and so on . moreover , successful interventions generally require buy - in from clinical staff ; as such , a failed intervention may diminish staff morale and might lower the likelihood of success for future interventions . a rapid , accurate , low - cost , low - risk means of testing and evaluating such interventions would be highly advantageous . discrete event simulation ( des ) is a technology that facilitates analysis of non - linear interactions between variables and their intermediary agents and is therefore highly suited for the complex , dynamic system of ed patient flow . such models are useful in guiding process interventions in a low - cost , minimal - risk manner . however , published reports of des in the ed are essentially case reports ; it is not clear how changes in metrics predicted by des compare quantitatively with real - world interventions . computer simulation of clinical ed patient flow was first proposed in the economics literature as early as 1975,9 but was not described in the medical literature until 1989,10 without further mention until coats and michalis introduced a proof - of - concept model in 2001.11 since that time , des has been described as a means of analysing patient flow,1214 predicting demand for services and addressing the related problems of crowding,1517 inpatient boarding18 19 and evaluating various other interventions in patients requiring emergency services.20 21 however , the authors know of no published studies reporting the comparison of real - world implementation with the results predicted a priori by simulation in the ed . this study represents an attempt to assess reduction in the daily mean los , and the proportion of patients with los > 6 h , by employing a triage intervention consisting of adding a physician and mid - level provider ( currently a nurse practitioner , but potentially also a physician 's assistant ) in triage , consolidating fast track into triage and discharging low - acuity patients directly from triage whenever possible . fast track is a separate , dedicated portion of the ed designed to handle lower - acuity patients . because of the restricted nature of this cohort , it is generally possible to maintain a higher patient to nurse ratio and have the primary provider as a mid - level provider ( in the case of st . acuity is defined according to the emergency severity index ( esi ) , which sorts patients by both severity and expected resources needed to complete the visit . esi 1 represents patients in need of resuscitation , esi 2 represents patients in dire emergent condition , and esi 35 represent patients in urgent to non - urgent condition and requiring higher to lower numbers of resources to treat . esi levels and procedures are defined in the agency for healthcare research and quality 's emergency severity index implementation handbook.22 the intervention was devised by ara - r based on personal communication and collaboration with other veterans administration ed directors . additionally , we aim to determine how accurately changes in clinically useful throughput metrics are predicted by the des model , by comparing simulation - based prediction with the results of a real - world trial of the proposed intervention . the design of the study was to develop and validate a des of the ed , and to use this tool to predict the effectiveness of the proposed change to triage services , followed by a real - world implementation of the strategy . the pre - intervention triage system is denoted below as the control , and the post - intervention triage system is denoted as the test. the computer simulation was first modelled and then validated against the real - world control ; then it was used to predict the test . then , upon generation of a favourable prediction , the triage intervention was adopted in the real - world ed . finally , post - hoc analysis of the result from the real - world test was compared with the simulated prediction , to determine precisely how accurate the simulation was in predicting the consequences of adopting the intervention . thus , there are four total instantiations of the study : computer simulation before and after and the real - world system , before and after . level 3 trauma centres are characterised by having less than full specialist availability , but full resources for resuscitation , and icu . the ed includes 14 patient beds , of which two are dedicated for mental health patients , and receives approximately 20 000 patient visits annually . the study intervention was approved by hospital administration and declared exempt by the institutional review board of the st . the intervention was implemented in the real - world ed on 19 september 2010 for a trial period of 1 month , until 18 october 2010 . comparison data were drawn from 19 september 2009 to 30 october 2009 , to be used for pre - intervention validation data , which are , respectively , the trial period and the test period. with the exception of ara - r , providers were unaware that intervention results would be compared with the simulated prediction . prior to the intervention under study , data were collected from daily aggregated patient encounter sheets for all patients presenting for care between 19 september 2009 and 30 october 2009hereafter referred to as the control period. during this trial period , patients presenting to the ed between 08:00 and 16:00 were seen first by a pre - triage nurse responsible for directing patients in urgent or emergent need directly to the ed treatment area ; others , with less urgent needs , were directed to resources outside of the ed ( eg , pharmacy , primary care or other clinics ) . outside of these hours , the remaining patients were then directed to a triage room , where they were interviewed and assessed by a dedicated triage nurse . as a result of this evaluation , the triage nurse assigned an esi and directed the patient to the main ed ( esi 3 or sometimes 4 ) , fast track ( esi 4 and 5 ) or discharged the patient directly from triage ( a small subset of esi 5 ) . those with esi 1 and 2 were directed to the ed treatment area , effectively bypassing triage . whether a patient with esi 4 is directed to the ed or to fast track similarly , whether a patient with esi 5 was dispositioned from triage or sent to fast track was determined by the triage nurse ( figure 1 , ) . process flowchart for emergency department ( ed ) patients , from arrival to disposition pre - intervention conditions . decision points are depicted as diamonds ; percentages indicate the percentage of patients assigned to each pathway , based on staff discretion or emergency severity index ( esi ) ( in the real ed ) or random assignment ( in the discrete event simulation model ) . the main ed treatment area was then reserved for care of patients of moderate to high acuity ( esi 13 ) and included standard ed treatment , including laboratories , medications , full radiological services and other services typical of a level 3 trauma centre ( figure 3 ) . this area was staffed by four attending physicians , in addition to the lone mid - level provider dedicated to the fast track area . following the intervention , patients were seen first by the pre - triage nurse from 08:00 to 16:00 and presented directly to triage at other times , in a similar manner . patients determined to be esi 1 or 2 at this stage are brought directly to an ed bed , bypassing the full triage protocol . patients continuing on to triage were registered and then divided between two providers , both present at triage : a physician , who evaluates higher - acuity patients ( ie , esi 3 ) , and a mid - level provider , who evaluates lower - acuity patients ( esi 4 and 5 ) . these providers communicate as necessary and may reassign patients to one another during this triage process . all esi 4 and esi 5 patients are treated and discharged from the triage area , rather than being assigned to a separate fast track. any patients requiring services that can not be provided by providers at triage are assigned ed beds , and may be reassigned a lower esi . as many esi 3 patients as possible ( ie , all those who can safely and comfortably wait in the waiting room rather than an ed bed ) were also evaluated entirely from the triage area , rather than being assigned to a separate fast track. the remaining high - acuity patients were transferred promptly to ed treatment beds for further care ( figure 2 ) . as a result of this change , fast track was eliminated as a separate treatment path and consolidated with triage , as was the mid - level provider formerly assigned to those patients . decision points are depicted as diamonds ; percentages indicate the percentage of patients assigned to each pathway , based on staff discretion or emergency severity index ( esi ) ( in the real ed ) or random assignment ( in the discrete event simulation model ) . md , attending physician ; mlp , mid - level provider ( nurse practitioner ) ; rn , nursing staff assigned to the triage area . further care in the ed treatment area was performed as before ( figure 3 ) , with the exception that only three attending physicians were assigned to this area . common elements of patient flow ( ed treatment bed ) , pre- and post - intervention conditions . as described above , a computer simulation was developed to model the ed as it existed prior to any intervention ( control ) . then , the simulation was updated with the proposed intervention ( test ) , to determine the consequences of the proposed changes . the triage processes were mapped by study investigators , in flowcharts representing both pre - intervention ( figure 1 ) and post - intervention ( figure 2 ) conditions , as well as the internal ed processes common to each of them ( figure 3 ) . probability frequencies for decision trees were calculated based on data collected from actual patient visits during the test period . process turnaround times for each step in the model were abstracted from daily ed status reports in the case of processes estimable from those ( admission / discharge rates , esi distributions , arrival rates ) , and for smaller processes ( provider time with patient , nurse time with patient ) estimated from interviews with experienced staff members responsible for those processes . simulations of this event sequence were created as des models using commercially available software for this purpose ( anylogic professional 6.4 ) . two des models were created , prior to actual intervention in the real world , one simulating the pre - intervention flowcharts ( figures 1 and 3 ) and the other simulating the post - intervention flowcharts ( figures 2 and 3 ) . simulations were constructed according to a standard four - step process : the system was decomposed into its constituent elements : entities ( patients ) ; resources ( physicians , nurses , etc . ) ; locations ( exam rooms , triage rooms , etc . ) ; and path networks ( hallways ) . the system was integrated into the anylogic 6.4 architecture , detailing how entities consume resources at locations , and then proceed to the next location according to the flowcharts . proportional likelihoods of following particular paths at decision trees were assigned according to probability frequencies described above . the system underwent face validation with members of the ed staff , who confirmed that processes were accurately mimicked by the simulation , and was then validated against a test data set as follows : the pre - intervention simulation was run for a 6-week period , then repeated for a total of 10 instantiations . the post - intervention simulation was also run for ten 6-week runs and the data were averaged similarly . similar data from the real - world ed were collected both prior to and pursuant to implementation of the change in triage process . comparisons between mean daily los were performed with unpaired , two - tailed student t tests ; comparisons between % 6-hour los were performed with two - proportion z tests . the design of the study was to develop and validate a des of the ed , and to use this tool to predict the effectiveness of the proposed change to triage services , followed by a real - world implementation of the strategy . the pre - intervention triage system is denoted below as the control , and the post - intervention triage system is denoted as the test. the computer simulation was first modelled and then validated against the real - world control ; then it was used to predict the test . then , upon generation of a favourable prediction , the triage intervention was adopted in the real - world ed . finally , post - hoc analysis of the result from the real - world test was compared with the simulated prediction , to determine precisely how accurate the simulation was in predicting the consequences of adopting the intervention . thus , there are four total instantiations of the study : computer simulation before and after and the real - world system , before and after . level 3 trauma centres are characterised by having less than full specialist availability , but full resources for resuscitation , and icu . the ed includes 14 patient beds , of which two are dedicated for mental health patients , and receives approximately 20 000 patient visits annually . the study intervention was approved by hospital administration and declared exempt by the institutional review board of the st . the intervention was implemented in the real - world ed on 19 september 2010 for a trial period of 1 month , until 18 october 2010 . comparison data were drawn from 19 september 2009 to 30 october 2009 , to be used for pre - intervention validation data , which are , respectively , the trial period and the test period. with the exception of ara - r , providers were unaware that intervention results would be compared with the simulated prediction . prior to the intervention under study , data were collected from daily aggregated patient encounter sheets for all patients presenting for care between 19 september 2009 and 30 october 2009hereafter referred to as the control period. during this trial period , patients presenting to the ed between 08:00 and 16:00 were seen first by a pre - triage nurse responsible for directing patients in urgent or emergent need directly to the ed treatment area ; others , with less urgent needs , were directed to resources outside of the ed ( eg , pharmacy , primary care or other clinics ) . outside of these hours , the remaining patients were then directed to a triage room , where they were interviewed and assessed by a dedicated triage nurse . as a result of this evaluation , the triage nurse assigned an esi and directed the patient to the main ed ( esi 3 or sometimes 4 ) , fast track ( esi 4 and 5 ) or discharged the patient directly from triage ( a small subset of esi 5 ) . those with esi 1 and 2 were directed to the ed treatment area , effectively bypassing triage . whether a patient with esi 4 is directed to the ed or to fast track similarly , whether a patient with esi 5 was dispositioned from triage or sent to fast track was determined by the triage nurse ( figure 1 , ) . process flowchart for emergency department ( ed ) patients , from arrival to disposition pre - intervention conditions . decision points are depicted as diamonds ; percentages indicate the percentage of patients assigned to each pathway , based on staff discretion or emergency severity index ( esi ) ( in the real ed ) or random assignment ( in the discrete event simulation model ) . the main ed treatment area was then reserved for care of patients of moderate to high acuity ( esi 13 ) and included standard ed treatment , including laboratories , medications , full radiological services and other services typical of a level 3 trauma centre ( figure 3 ) . this area was staffed by four attending physicians , in addition to the lone mid - level provider dedicated to the fast track area . following the intervention , patients were seen first by the pre - triage nurse from 08:00 to 16:00 and presented directly to triage at other times , in a similar manner . patients determined to be esi 1 or 2 at this stage are brought directly to an ed bed , bypassing the full triage protocol . patients continuing on to triage were registered and then divided between two providers , both present at triage : a physician , who evaluates higher - acuity patients ( ie , esi 3 ) , and a mid - level provider , who evaluates lower - acuity patients ( esi 4 and 5 ) . these providers communicate as necessary and may reassign patients to one another during this triage process . all esi 4 and esi 5 patients are treated and discharged from the triage area , rather than being assigned to a separate fast track. any patients requiring services that can not be provided by providers at triage are assigned ed beds , and may be reassigned a lower esi . as many esi 3 patients as possible ( ie , all those who can safely and comfortably wait in the waiting room rather than an ed bed ) were also evaluated entirely from the triage area , rather than being assigned to a separate fast track. the remaining high - acuity patients were transferred promptly to ed treatment beds for further care ( figure 2 ) . as a result of this change , fast track was eliminated as a separate treatment path and consolidated with triage , as was the mid - level provider formerly assigned to those patients . decision points are depicted as diamonds ; percentages indicate the percentage of patients assigned to each pathway , based on staff discretion or emergency severity index ( esi ) ( in the real ed ) or random assignment ( in the discrete event simulation model ) . md , attending physician ; mlp , mid - level provider ( nurse practitioner ) ; rn , nursing staff assigned to the triage area . further care in the ed treatment area was performed as before ( figure 3 ) , with the exception that only three attending physicians were assigned to this area . common elements of patient flow ( ed treatment bed ) , pre- and post - intervention conditions . as described above , a computer simulation was developed to model the ed as it existed prior to any intervention ( control ) . then , the simulation was updated with the proposed intervention ( test ) , to determine the consequences of the proposed changes . the triage processes were mapped by study investigators , in flowcharts representing both pre - intervention ( figure 1 ) and post - intervention ( figure 2 ) conditions , as well as the internal ed processes common to each of them ( figure 3 ) . probability frequencies for decision trees were calculated based on data collected from actual patient visits during the test period . process turnaround times for each step in the model were abstracted from daily ed status reports in the case of processes estimable from those ( admission / discharge rates , esi distributions , arrival rates ) , and for smaller processes ( provider time with patient , nurse time with patient ) estimated from interviews with experienced staff members responsible for those processes . simulations of this event sequence were created as des models using commercially available software for this purpose ( anylogic professional 6.4 ) . two des models were created , prior to actual intervention in the real world , one simulating the pre - intervention flowcharts ( figures 1 and 3 ) and the other simulating the post - intervention flowcharts ( figures 2 and 3 ) . simulations were constructed according to a standard four - step process : the system was decomposed into its constituent elements : entities ( patients ) ; resources ( physicians , nurses , etc . ) ; locations ( exam rooms , triage rooms , etc . ) ; and path networks ( hallways ) . the system was integrated into the anylogic 6.4 architecture , detailing how entities consume resources at locations , and then proceed to the next location according to the flowcharts . proportional likelihoods of following particular paths at decision trees were assigned according to probability frequencies described above . the system underwent face validation with members of the ed staff , who confirmed that processes were accurately mimicked by the simulation , and was then validated against a test data set as follows : the pre - intervention simulation was run for a 6-week period , then repeated for a total of 10 instantiations . the post - intervention simulation was also run for ten 6-week runs and the data were averaged similarly . similar data from the real - world ed were collected both prior to and pursuant to implementation of the change in triage process . comparisons between mean daily los were performed with unpaired , two - tailed student t tests ; comparisons between % 6-hour los were performed with two - proportion z tests . during the pre - intervention period , the real - world ed saw 2194 patient visits over a 6-week period . daily mean throughput time ( arrival to disposition ) was 247 min ( sd 39.8 ) , with 437 ( 19.9% ) visits taking > 6 h. the pre - intervention simulation run saw 2178 patient visits over a similar time period . simulated mean throughput time was 249 min ( sd 39.7 ) , with 413 ( 19.0% ) visits taking > 6 h. there was no statistically significant difference between mean los ( p=0.694 ) or % 6-hour los ( p=0.909 ) , between the real - world situation and the simulation . the post - intervention des model reported 2154 patient visits over a 6-week period , with a daily mean throughput time of 200 min ( sd 19.0 ) . of these , 282 visits took > 6 h ( 13.1% ) , for a relative reduction of 31.1% compared with the pre - intervention simulation . during the post - intervention period , mean throughput time was significantly different between pre- and post - intervention samples ( p<0.0001 ) . it should be noted that the decrease in patients is due to the real - world intervention trial period being 30 days , rather than the 42 days of the other periods , and not a decrease in daily census . during the post - intervention period , there were 243 ( 14.3% ) visits that took > 6 h , which represents a relative reduction of 28.2% compared with the pre - intervention , real - world ed ( p=0.045 ) . five hundred and seventy - seven patients with acuity esi 4 or esi 5 were discharged directly from triage under the new flow model , rather than being sent to there was no statistically significant difference between the post - intervention states in the simulated and real - world mean los ( p=0.499 ) and % 6-hour los ( p=0.880 ) . all mean daily los values were verified to fit normal distributions using stat::fit ( geer mountain software corporation , south kent , connecticut , usa ) . the reassignment of a physician and nurse practitioner to triage , coupled with the consolidation of fast track into triage , appears to have been effective in reducing the ed patient turnaround time in two ways . first , provider coverage allowed for a large percentage of patients to be discharged directly from triage . because provider coverage was accessible earlier in the emergency visit process , some treatment decisions could be made earlier in the patient encounter , resulting in shorter stays for some patients . this was critical in decreasing the number of visits that took > 6 h ; previously , some patients would experience prolonged delays , while higher - acuity patients were treated first . second , by reducing the number of patients requiring beds in the ed , more resources were available for those patients who did require such beds . it has been suggested , but not easily demonstrated , that provider coverage in triage23 and discharge from triage24 have the potential to improve the ed throughput . des was instrumental in predicting the consequences of such interventions prior to implementation , and such predictions were confirmed to be highly accurate , when compared with the actual ed . des holds promise as a low - cost , low - risk method for evaluating clinical process changes in the ed . while numerous case studies exist demonstrating qualitatively that hypotheses generated by des models can yield effective interventions , it is unclear how accurate these models are in this regard and , therefore , how trustworthy such models are in ranking the effectiveness of such process changes . this study shows that , in our institution , a des model was highly accurate in predicting the decrease in mean los and the percentage of patients with los > 6 h. while des can not provide a single optimised state of a department , it is extremely versatile at testing hypotheses related to process change . this study demonstrates the potential for des in testing and accurately predicting the results from a complex intervention involving changes in staffing ( adding a physician and mid - level provider to triage ) , upstream process ( changing the underlying criteria by which patients will be seen in triage ) and downstream process ( changing the treatment location of patients following triage ) . des has a long history of use in medical systems for the analysis and prediction of changes to system dynamics and policy . however , outcomes of implementations based upon simulated predictions are rarely reported in the medical literature . the simulation was able , in this case , to provide valuable insight into the likely outcome of the proposed triage intervention , and the intervention was adopted in large part due to the simulated results . one perceived strength of the simulation was its capacity for graphical representation of the ed , thereby allowing stakeholders unfamiliar with simulation methods ( ie , ed administration ) to demonstrate the viability of the intervention . the outcome of the intervention was to significantly improve system performance and was consistent with the predicted results . our validation methods that is , comparing los and % 6-hour los data between the des model and the real - world data represents an additional degree of robustness compared with los validation techniques which are seen in the literature.25 accurately capturing the % 6-hour los provides a reflection of the high end of the distribution of patient times , rather than restricting the validation to aggregated averages over time . as with any simulation , it is impossible to capture every potential occurrence in the ed ; therefore , rather than attempting to incorporate every possible variation in care , the flow is designed to represent the general practice of emergency care in the st . it should be noted that these results are based on limited real - world sample sizes . the reference data set represents only a 6-week period , during which there were no extraordinary or unexpected demands on the ed facility or seasonal variation in patient arrivals . we could not necessarily guarantee such a high degree of predictability over a longer , or more heterogeneous , period of time . individual process times ( eg , patient time with a physician in an exam room ) were estimated based on interviews with ed staff and modelled with triangular distributions . nevertheless , the similarity of results in the real - world and simulated data suggests that this bias was insignificant to the present study . it is possible that this bias introduces a similar systematic error in pre- and post - intervention simulations and , therefore , may cancel out when comparing between pre- and post - interventional conditions . protected health information , such as patient identity and demographics ( age , sex , ethnicity and gender ) , was not available . as such , it was not possible to assess the effects of patient characteristics on throughput data . the accuracy of the des model before and after intervention suggests that patient characteristics other than illness severity ( eg , esi ) are unlikely to influence results . the duration of the simulated period , as well as pre- and post - intervention control period , was determined as a quality assurance project . as a result , there was a discrepancy between the duration of the predictions made in simulation and the real - world trial period . the authors recommend that simulation - tested interventions be implemented under the same conditions as those simulated , and for the same length of time . louis vamc , we designed , simulated and adopted an intervention consisting of adding a physician and mid - level provider in triage , absorbing fast track into triage and discharging low - acuity patients directly from triage whenever possible . this resulted in a mean reduction in los by 15% , with a 28.2% reduction in patients with los > 6 h. our des models were effective in quantitatively predicting these results . further work is suggested to demonstrate ( 1 ) the applicability of such changes in patient flow in other eds and ( 2 ) that des is similarly accurate in predicting the results of other process changes , in this and other eds . future work is indicated to employ des in the capacity of identification of crowding causes and the testing of proposed interventions designed to ameliorate those factors .

objective(1 ) to determine the effects of adding a provider in triage on average length of stay ( los ) and proportion of patients with > 6 h los . ( 2 ) to assess the accuracy of computer simulation in predicting the magnitude of such effects on these metrics.methodsa group - level quasi - experimental trial comparing the st . louis veterans affairs medical center emergency department ( 1 ) before intervention , ( 2 ) after institution of provider in triage , and discrete event simulation ( des ) models of similar ( 3 ) before and ( 4 ) after conditions . the outcome measures were daily mean los and percentage of patients with los > 6 h.resultsthe des - modelled intervention predicted a decrease in the % 6-hour los from 19.0% to 13.1% , and a drop in the daily mean los from 249 to 200 min ( p<0.0001 ) . following ( actual ) intervention , the number of patients with los > 6 h decreased from 19.9% to 14.3% ( p<0.0001 ) , with the daily mean los decreasing from 247 to 210 min ( p<0.0001).conclusionphysician and mid - level provider coverage at triage significantly reduced emergency department los in this setting . des accurately predicted the magnitude of this effect . these results suggest further work in the generalisability of triage providers and in the utility of des for predicting quantitative effects of process changes .

Background Methods Design Setting Control condition (pre-intervention) Test condition (post-intervention) Simulations Data analysis Results Discussion Limitations Conclusions

health - related professionals are at high risk of job burnout which will in turn lead to effects on health services provision . the present study was conducted to define job burnout and its association with personal characteristics among the midwives working in isfahan , iran . this descriptive correlational study was performed on 193 midwives working in health centers and hospitals in isfahan . the data was collected by a researcher - made personal characteristics questionnaire as well as maslach burnout inventory . in the present study , the highest frequencies of job burnout dimensions were for the low levels of emotional exhaustion ( 58% ) and depersonalization ( 65.8% ) , and high levels of personal performance ( 58% ) . there was a significant inverse association between age and depersonalization ( p = 0.02 ) . however , no significant relations were found between job burnout dimensions and variables of marital status , number of children , level of education , and residential status . although the results of this research showed a low prevalence of job burnout among midwives , the stressful nature of midwifery as a profession necessitates educational intervent . job burnout syndrome , as a recent professional issue , is considered as the main feature of occupational stress , and a delayed reaction to chronic stressors in the work place . this psycho - cognitive syndrome comprises three dimensions of emotional exhaustion and mental power discharge , depersonalization ( negative reaction accompanied with ignoring the clients ) , and the decline of personal performance ( losing capability and success at work ) . job burnout results in numerous inappropriate mental and psychical effects such as depression , reduction of job satisfaction , criticizing the clients and losing empathy , decline of professional function , increase of family and interpersonal problems , irregularity in patient care and eventually , absence from work . it also imposes a 4% waste of work hours to organizations and thus brings about financial loss by wasting millions of dollars each year . previous research has showed health professions ( such as midwifery ) to be at the highest risk of occupational complications , such as job burnout , compared to other occupations . individuals are believed to experience different levels of job burnout based on personal and occupational factors . some studies have evaluated the association between personal characteristics and job burnout and reported controversial findings . for instance , talaei et al . found a significant association between job burnout subscales and variables such as age and education . however , alparslan and doganer demonstrated that age , marital status and number of children had no effects on job burnout . likewise , toubaei and sahraeian concluded that job burnout was not significantly associated with age . in another study , bahri bynabaj et al . reported that although there was not a significant association between job burnout and the variable of age , marital status and level of education were significantly related with job burnout . some other researchers have believed personal factors not to be able to influence job burnout . therefore , considering the existing controversies and the high prevalence of job burnout in modern societies resulting in leaving jobs , reduction of working forces and their outcome in economy and production of the country , further studies seem necessary . on the other hand , the midwives are important members of health providing team whose high quality health services play a key role in health indexes such as life expectancy and maternal mortality . in addition , having a crucial responsibility , midwives can be predisposed to job burnout that consequently causes diminished quality of patient care and low quality and quantity of health services . the present study was thus conducted to define job burnout and its association with personal factors ( age , marital status , number of children , level of education , and residential status ) amongst midwives in order to reduce job burnout and to promote the quality of care they provide . this descriptive correlational study included all midwives working in health centers , and university and private hospitals in isfahan ( iran ) as well as isfahan charity center in 2011 . the inclusion criteria were having at least one year of working experience , no history of mental and psychotic problems and not being exposed to acute stressful events . in order to collect data , subjects filled out a researcher - made personal factor questionnaire and maslach burnout inventory ( mbi ) . mbi includes 22 questions on three components of emotional exhaustion , personal performance , and depersonalization . the personal factor questionnaire was based on a seven - item likert scale in which scores of 06 were respectively associated with never , several times a year , once a month , several times a month , once a week , several times a week , and every day . after scoring the questions for each subject , total scores of each dimension was summed up and categorized as low , moderate , or high . in order to do so , high , moderate , and low scores were respectively considered as over 30 , 1829 , and 17 in emotional exhaustion , over 12 , 611 , and 6 in depersonalization , and over 40 , 3439 , and 33 in personal performance . the scores of subscales were not addable since in two subscales of emotional exhaustion and depersonalization , high scores showed job burnout but in the subscale of personal performance , lower scores indicated job burnout . the employed questionnaire has been frequently confirmed by reliability of over 90% by iranian researchers . the collected data was analyzed by descriptive ( mean , standard deviation ( sd ) , and frequency distribution ) and inferential statistical tests ( pearson s correlation test , spearman test , analysis of variance , and independent t- test ) in spss . most subjects were married , had two children , had a bachelor s degree , and owned a house . low levels of emotional exhaustion ( 58% ) and depersonalization ( 65.8% ) , and high levels of personal performance ( 58% ) were the most frequent levels of job burnout dimensions ( table 1 ) . mean values of job burnout in dimensions of emotional exhaustion , depersonalization , and personal performance were 15.3 ( 9.9 ) , 5.4 ( 4.8 ) , and 39.7 ( 6.3 ) , respectively . mean values in dimensions of emotional exhaustion , depersonalization and personal performance were respectively 13.58 ( 9.92 ) , 4.74 ( 4.44 ) , and 40.28 ( 6.39 ) in health centers . the corresponding values in the hospitals were 16.79 ( 9.82 ) , 6.00 ( 5.17 ) , and 39.25 ( 6.34 ) . independent t - test showed a significant association between workplace location and dimensions of emotional exhaustion ( t = 2.25 ; p = 0.01 ) and depersonalization ( t = 1.79 ; p = 0.03 ) . however , personal performance and workplace location were not significantly related ( t = 1.12 ; p = 0.13 ) . in addition , mean values of emotional exhaustion and depersonalization in the age group of 22 - 32 years were higher than other age groups ( 16.41 ( 10.13 ) vs. 6.50 ( 5.31 ) ) . mean value of personal performance was equal to 38.77 ( 6.8 ) in this age group which was in a lower level compared to other subjects . distribution of subscale levels of burnout in subjects pearson s correlation test revealed a significant inverse association between age and depersonalization . however , no significant associations were found between age and dimensions of emotional exhaustion and personal performance . analysis of variance showed no significant associations between marital status and subscales of emotional exhaustion , depersonalization and personal performance . in addition , no significant relations between number of children and emotional exhaustion , depersonalization , and personal performance were indicated by pearson s correlation test . moreover , educational level and emotional exhaustion , depersonalization and personal performance were not found to be significantly related according to spearman correlation test . finally , independent t - test showed no significant associations between residential status and the three dimensions of job burnout ( table 2 ) . job burnout values stratified based on the three dimensions of burnout and demographic factors ( values are presented as mean and standard deviation ) the findings of this study showed the highest frequencies of job burnout dimensions to be for low levels of emotional exhaustion and depersonalization , and high levels of personal performance , respectively . therefore , a high percentage of the subjects in the present study seemed not to have experienced job burnout . khaghani zadeh reported the highest frequency of the three component dimensions of job burnout amongst nurses as low levels of emotional exhaustion ( 53% ) , depersonalization ( 48% ) , as well as personal performance ( 45% ) . indicated the highest frequency for low levels of emotional exhaustion ( 70% ) and personal performance ( 54% ) , and high levels of depersonalization ( 94.7% ) amongst physicians . sotodeh asl and bakhtiari concluded that the highest frequencies were for moderate emotional exhaustion ( 93.2% ) , low personal performance ( 93.1% ) , and high depersonalization ( 94.1% ) amongst nurses and midwives . however , their study generally considered nurses and midwives and did not evaluate different types of the professions . therefore , job burnout was less common amongst the studied midwives in the present study than the physicians taee et al . studied . alparslan and doganer reported the mean values of job burnout dimensions as 17.41 , 5.03 , and 20.38 for emotional exhaustion , depersonalization , and personal performance , respectively . on the other hand , momeni et al . published 41.38 , 9.28 , and 29.31 as the mean values of emotional exhaustion , depersonalization , and personal performance , respectively . mean values of the three dimensions of burnout in the present study were thus lower than the abovementioned studies . since depersonalization is defined as the negative reaction and ignoring the clients , lower scores of depersonalization in the midwives in the present study can possibly be due to their appropriate interpersonal communication . it should be indicated that personal performance is felt when individuals can play a role in their related organizations , show their abilities , and attain positive attitudes about themselves and their clients . in this case , they can have a better judgment about their job , attain more authority , and cope with their duties while feeling self - confident . thus , high personal performance among the studied subjects may reflect their positive attitude toward their profession and their high self - confidence . on the other hand , high social class or other successful life backgrounds among the studied subjects seem to have helped them cope with their occupational stressors , and to have lowered their emotional exhaustion . . believed a higher social class or successful backgrounds to be effective on diminishing emotional exhaustion . the present study found higher mean values of emotional exhaustion and depersonalization , and lower values of personal performance among midwives working in hospitals compared to those in health centers . therefore , midwives working in hospitals seem to suffer more from job burnout possibly due to the type and intensity of occupational stress they experience at work . moreover , a significant inverse association was observed between age and depersonalization , while emotional exhaustion and personal performance were not significantly related with age . on the other hand , the mean value of depersonalization was the highest in the age group of 22 - 32 . while momeni et al . suggested various age groups of nurses to obtain different scores of depersonalization bargellini et al . ahmadi and sheikh alizadeh and sotodeh asl and bakhtiari rejected any significant association between age and job burnout . based on the relation between depersonalization and age in the present study , some other elements such as working conditions and obtained experiences at work may also be effective in this regard . . believed that individuals get more prepared to cope with stressful situations at higher ages , and consequently experience less job burnout . it can be thus concluded that 22 - 33 year - old midwives suffered more from job burnout as a result of their higher mean emotional exhaustion and depersonalization , and their lower personal performance compared to other age groups . this issue may be due to the fact that younger individuals are more susceptible to job burnout . not being significantly correlated with job burnout dimensions , marital status seems not to be an appropriate predictor for job burnout , i.e. individuals with different marital statuses would experience the same level of job burnout . likewise , gonzalez and bernard and bartly did not suggest a significant association between emotional exhaustion and marital status among academic members . consistent with the findings of alparslan and doganer , yaman and soler , and gullap et al . , the present study did not reveal a significant association between job burnout subscales and number of children . in addition , the present study showed that despite the higher mean values of job burnout dimensions among the employees with a bachelor s degree , the relations were not significant . similar results were previously published by alparslan and doganer , yaman and soler , mollao lu et al , and aziz nejad and hosseini . in addition , analysis of variance showed no significant associations between the three dimensions of job burnout and residential status , which was also reported by aziz nejad and hosseini . job burnout was lower among the midwives attending the present study compared to the abovementioned researches . however , the prevalence of job burnout was higher among midwives working in hospitals compared to the health centers . in addition , considering the higher mean values of depersonalization in the age group of 22 - 32 years in the present study , younger midwives need to receive more attention . finally , the education authorities are suggested to hold related educational workshops to prevent and amend job burnout and ultimately enhance the quality of care .

background : health - related professionals are at high risk of job burnout which will in turn lead to effects on health services provision . the present study was conducted to define job burnout and its association with personal characteristics among the midwives working in isfahan , iran.materials and methods : this descriptive correlational study was performed on 193 midwives working in health centers and hospitals in isfahan . the participants were selected through cluster random sampling . the data was collected by a researcher - made personal characteristics questionnaire as well as maslach burnout inventory . the data was analyzed by descriptive and inferential statistical tests in spss.findings:in the present study , the highest frequencies of job burnout dimensions were for the low levels of emotional exhaustion ( 58% ) and depersonalization ( 65.8% ) , and high levels of personal performance ( 58% ) . there was a significant inverse association between age and depersonalization ( p = 0.02 ) . however , no significant relations were found between job burnout dimensions and variables of marital status , number of children , level of education , and residential status.conclusions:although the results of this research showed a low prevalence of job burnout among midwives , the stressful nature of midwifery as a profession necessitates educational intervent .

Background: Materials and Methods: Findings: Conclusions: I M F D