July correction PLoS Biology, volume 2, issue 8In Nicotine's Defensive Function in NatureAnke Steppuhn, Klaus Gase, Bernd Krock, Rayko Halitschke, Ian T. Baldwin10.1371/journal.pbio.0020217DOI: The Academic Editor was erroneously listed as Michael Levine. The Academic Editor for this paper is Joy Bergelson, Chicago University.This correction note may be found online at DOI: 10.1371/journal.pbio.0020382.


Published February 15, 2005PLoS Biology, volume 2, issue 12.In 10.1371/journal.pbio.0020449The art credits were missing from the image accompanying this synopsis. The image caption should read as follows:


PLoS Biology, volume 4, issue 10: DOI: 10.1371/journal.pbio.0040321In The legend to Figure 4 incorrectly identifies the dotted line as representing evolutionary rate and the solid line as representing the size of a morphological character. This identification is reversed in the caption. The dotted line represents the morphological trait's magnitude and the solid is the rate of evolution.


PLoS Biology, volume 5, issue 4: doi: 10.1371/journal.pbio.0050072In On page 0801, in the last sentence of the first paragraph of the Results section, “isoleucine” should be replaced with “threonine.”The corrected version is:“Nonetheless, the overall feature emerging from our analysis is that syntaxins with conserved threonine at the +7 layer appear to be selectively involved in regulated secretion at synapses or neurosecretory cells.”


PLoS Biology, volume 5, issue 3: doi: 10.1371/journal.pbio.0050065In In Figure 5A, the sequence of the subpanels was inadvertently inverted. The correct Figure 5 is shown here.


E. coli reveals that although these viruses don't age, there is a trade-off between mortality and growth rate, which parallels that observed in many other species.A comparison of life-history traits of 16 phages infecting PLoS Biology, volume 4, issue 7: DOI:10.1371/journal.pbio.0040193InIn the abstract, the mortality rate is described as being negatively correlated with multiplication rate, whereas it is positively correlated.


PLoS Biology, volume 4, issue 8: DOI: 10.1371/journal.pbio.0040255In The figure caption is missing a credit. The caption should read as follows: A model based on growth trajectories estimated from fossils provides evidence that dinosaurs were reptiles whose body temperatures increased with body size. (Painting: Skye White).


PLoS Biology, volume 4, issue 5: DOI:10.1371/journal.pbio.0040174InThe caption of the image should be “Transcription foci/factories (in blue) are found within areas of intermingling between Chromosome 3 (in red) and the remaining genome (in green), and may mediate functional interactions between them.”


PLoS Biology, volume 5, issue 5: doi: 10.1371/journal.pbio.0050097In The ninth author's name was incorrectly given as Janet Thonton; it should be Janet Thornton.


A new melanopsin gene, identified in fish, bird, and amphibian genomes, is the true ortholog of the melanopsin gene previously described in mammals. PLoS Biology, volume 4, issue 8: DOI: 10.1371/journal.pbio.0040254In There was an error in the legend for Figure 4. The text describing part A should read “chicken chromosome 6” rather than “chicken chromosome 4.”


PLoS Biology, volume 3, issue 4, DOI: 10.1371/journal.pbio.0030127In The figure credits for Figures 1 and 2 are missing from the PDF versions of the article. For Figure 1, images are courtesy of Langdon Quetin and Robin Ross, researchers at the Marine Science Institute, University of California, Santa Barbara, and funded by the Office of Polar Programs, National Science Foundation. For Figure 2, images are courtesy of Donna Fraser.


PLoS Biology, volume 4, issue 10: doi:10.1371/journal.pbio.0040325In The “female population” part of the x-axis label in Figure 2 has an error: the labels sym and allo are switched in two places. Attached is a PDF of Figure 2 as it should appear. The legend should remain the same.


PLoS Biology, volume 4, issue 2: DOI: 10.1371/journal.pbio.0040031In In the first paragraph of the Materials and Methods subsection “Retroviral infection of INS1 and MIN6 cells,” “pSUPERretro SiRNA-T1 (5′-GCTGCATCCAAGGGCCATG-3′)” should be “pSUPERretro SiRNA-T1 (5′-gatgaagttgacctcctca-3′)”.


PLoS Computational Biology, volume 2, issue 6: DOI:  10.1371/journal.pcbi.0020055In The Figure 5 title and legend should read:Figure 5 Variance of the Natural Logarithm of Size of Memory Lineages Other parameters are the same as in Figure 2B and 2D, and the mean cross-reactivity is kept the same at


PLoS Biology, volume 3, issue 3: DOI: 10.1371/journal.pbio.0030068In In the caption for Figure 5A, the formula for the lognormal fit was given as follows:.The second squaring exponent should have been inside the parentheses, so it reads as follows:.


PLoS Biology, volume 2, issue 11: DOI: 10.1371/journal.pbio.0020363In http://www.microrna.org/ contains the updated information with changes flagged. We thank Dr. Denman for pointing out this error to us.We incorrectly named the products of two genes, gamma actin and betaAPP, as FMRP ligands because of a misreading of Table 1 from [


Correction for:10.1371/journal.pbio.0050163Brennand K, Huangfu D, Melton D (2007) All β cells contribute equally to islet growth and maintenance. PLoS Biol 5(7): e163. doi:The labels in Figure 2B indicating the “Pulse” and “No Pulse” FACS conditions for the Rosa26-rtTA;tetO-H2BGFP cells were switched. Labeled properly, the middle left panel should be titled “No Pulse,” and the bottom left panel should be titled “Pulse.”


Correction for:10.1371/journal.pbio.0050219Steiner CC, Weber JN, Hoekstra HE (2007) Adaptive variation in beach mice produced by two interacting pigmentation genes. PLoS Biol 5(9): e219. doi:Figure S2 was erroneously labeled. The caption should read:Agouti Gene Including the Known Cis-Regulatory and Coding Regions Figure S2. Genomic Structure of the In addition, there was an omission in the acknowledgments. The following sentence should be included:T. Glenn, N. Schable, and C. Hagen produced microsatellite libraries and provided PmBW clone sequences that were funded by National Institutes of Health grant R01Gm069601 (to M. Dewey and T. Glenn).


Microcebus murinus), the golden brown mouse lemur (M. ravelobensis) and the Goodman's mouse lemur (M. lehilahytsara). The first two species occur sympatrically, the latter lives allopatrically to them.A central question in evolutionary biology is how cryptic species maintain species cohesiveness in an area of sympatry. The coexistence of sympatrically living cryptic species requires the evolution of species-specific signalling and recognition systems. In nocturnal, dispersed living species, specific vocalisations have been suggested to act as an ideal premating isolation mechanism. We studied the structure and perception of male advertisement calls of three nocturnal, dispersed living mouse lemur species, the grey mouse lemur (M. murinus from the field using advertisement calls and alarm whistle calls of all three species. Individuals responded significantly stronger to conspecific than to heterospecific advertisement calls but there were no differences in response behaviour towards statistically similar whistle calls of the three species. Furthermore, sympatric calls evoked weaker interest than allopatric advertisement calls.A multi-parameter sound analysis revealed prominent differences in the frequency contour and in the duration of advertisement calls. To test whether mouse lemurs respond specifically to calls of the different species, we conducted a playback experiment with Our results provide the first evidence for a specific relevance of social calls for speciation in cryptic primates. They furthermore support that specific differences in signalling and recognition systems represent an efficient premating isolation mechanism contributing to species cohesiveness in sympatrically living species. Cryptic species are closely related species which are morphologically similar, but differ genetically ,2. The rA fundamental problem for sympatrically living, cryptic mammalian species is the coordination of reproduction between conspecifics in time and space, especially when individuals of a species forage solitarily. Under these circumstances mating partners do not only have to detect, localise and find each other, they also have to discriminate between conspecifics and remarkably similar heterospecifics. Current evolutionary theory ,9-11 sugThe Malagasy mouse lemurs, small nocturnal primates which inhabit the fine branch niche of forests provide an excellent model to explore the significance of vocal communication for species recognition and discrimination. Mouse lemurs have large mobile ears, exhibit a high auditory sensitivity , are higAt present 15 cryptic species are known which are difficult to distinguish in body characteristics ,27-29. IMicrocebus rufus) and their perception by the grey mouse lemur. These three species are genetically distinct from each other but share a large number of morphological features [We studied the structure of male advertisement calls of the grey, the golden brown and the Goodman's mouse lemur, formerly belonging to the rufous mouse lemur  whereas alarm calls do not . Until nThe present study gives the first account of the relevance of communication calls for species recognition and discrimination in cryptic primates in an area of sympatry combining a call structure analysis and playback experiments. Three questions were addressed:1. To what extent do advertisement calls of sympatric cryptic mouse lemurs differ in structure?2. Do mouse lemurs discriminate between advertisement calls of different species and do they show stronger discrimination between conspecific and sympatric than between conspecific and allopatric calls?3. Do mouse lemurs discriminate between call types of different species which are irrelevant for species recognition in the reproductive context?To answer these questions we recorded male advertisement calls of grey and golden brown mouse lemurs and measured several time and frequency parameters for an interspecific statistical comparison of the sympatrically living species. Per individual, we analysed 3 to 21 (median 5) calls and calculated individual median values for each acoustic parameter. On the basis of these values we conducted a Kruskal-Wallis analysis of variance (ANOVA) to test for species-specificity.In addition we conducted playback experiments with 16 grey mouse lemurs from the field. Six categories of playback stimuli were presented during a single experimental session: conspecific male advertisement calls (referred to as conspecific advertisement), heterospecific male advertisement calls of the golden brown mouse lemur (referred to as sympatric advertisement), heterospecific male advertisement calls of the Goodman's mouse lemur  and male whistle alarm calls ,38 of alThe behavioural responses of the tested individuals were classified into two different response categories: (1) no orientation, not involving any orientation response including no reaction, ear movement, interruption of activity or startle without turning towards the speaker and (2) orientation, including turning towards the speaker and approaching the speaker, sometimes accompanied by antiphonal vocalisation.For statistical comparison of call categories, an individual-based analysis was conducted comparing individual response indices for all call categories of advertisement calls and short whistles, respectively. The individual response index towards a call category was defined by the number of orientation responses divided by all responses of an individual towards stimuli of the respective call category, that isFriedman-ANOVA and Wilcoxon tests with a serial Bonferroni correction procedure  were perThe frequency contour of the harmonically structured advertisement calls from the three species was remarkably different Figure . The gref0 min: H2 = 3.470, p = 0.176; f0 max: H2 = 0.928, p = 0.629; f0 band: H2 = 2.566, p = 0.278; N = 14 for all tests; see Table H2 = 11.623, p = 0.003, N = 14). The calls of the grey mouse lemurs were the longest, those of the Goodman's mouse lemur the shortest and those of the golden brown mouse lemur took an intermediate position.No measured frequency parameter showed any species specificity . In addition, response strength was independent of the presentation number of stimuli . This shows that inter-stimulus intervals were sufficient to avoid any habituation effects owing to the consecutive stimulus presentation design.Neither the sound pressure level nor the signal-to-noise ratio of stimuli had a significant effect on the stimulus response indices .Individual response indices revealed remarkable differences for conspecific, sympatric and allopatric stimuli  and maximum (f0 max) frequency of the fundamental and calculated the bandwidth of the fundamental (f0 band = f0 max - f0 min).Male advertisement calls were recorded in the presence of oestrous females ,60. The Statistics were made using Statistica 6.0 (StatSoft Inc.), the level of significance was 0.05 for all statistical tests.Playback experiments were conducted in the Ankarafantsika National Park , about 110 km south-east of Mahajanga, Madagascar during the dry season from September to October 2000 and from July to October 2001 covering the mating period of the mouse lemurs. They were performed in a part of the dry deciduous forest where the grey and the golden brown mouse lemur occur sympatrically.Sixteen  grey mouse lemurs were subjects of our playback experiments. The experiments were conducted under temporary captivity conditions in the field. A stationary setup under controlled conditions was necessary because mouse lemurs communicate in the ultrasonic range which requires special playback and recording equipment. To test for differences in the perception of sympatric and allopatric calls, we needed animals from the field which were experienced with their sympatric species.The intervention on the individual and population level by the experimental study was reduced to a minimum by the following procedure: we trapped the animals using Sherman Live Traps  by setting them in the late afternoon in trees and bushes . Traps wDue to the fact that mouse lemurs are seasonal breeders , it was For the playback experiment a playback stimulus consisted of one call for the categories conspecific and sympatric advertisement, two calls for the category allopatric advertisement and three calls for the three whistle categories, respectively. By using this setup we accounted for the different duration and repetition rates of male advertisement calls and short whistles from the different species. We used two different advertisement stimuli from each of four conspecific males, two from the Hannover population and two from a Madagascar population. In addition, two different stimuli from each of two sympatric and allopatric males were taken. As whistle stimuli, we used two short whistles each of two males of the grey, two males of the golden brown and one male of the Goodman's mouse lemur.With these stimuli, we produced a playback tape based on the original analogue recordings from the NAGRA tape recorder. To minimise background noise the stimuli were highpass filtered at a frequency of 7 to 15 kHz depending on the minimum frequency of the call. The playback tape was started at a random position using a NAGRA IV-SJ tape recorder , a custom-made amplifier and a speaker (Leaf Tweeter EAS-10Th400A). Stimuli ranged between 70.5 and 83.0 dB sound pressure level at a distance of 1 m , that is, sound pressure levels corresponded to the naturally occurring ranges. The loudspeaker was placed between 0.6 and 0.8 m above the ground at a distance of about 0.5 m from the cage to ensure a sufficient presentation quality of the highly directional ultrasonic calls at any position in the cage.In each playback session seven different stimuli were played back in a random order: two of the category conspecific advertisement (one each of the two different populations), and one stimulus each of the other five categories. To avoid a habituation to playback stimuli, the inter-stimulus interval was kept between 1 and 10 minutes. Each individual took part in one to three playback sessions in which each of them received every stimulus only once.Behavioural responses to playback stimuli were observed at a distance of about 5 m from the observation cage using a headlamp and binoculars and reported to a dictaphone for further analysis. We recorded the behavioural responses belonging to the categories 'orientation' and 'no orientation' within 10 seconds just after the onset of a stimulus. In all cases, response behaviour had finished within this period.Cases were excluded in which animals were not visible to the observer because they went into their bamboo trunk or were hidden by cage enrichment. We were able to analyse 186 responses to playback stimuli. The frequencies of no orientation and orientation responses were determined per stimulus and per individual, respectively. We recorded 5 to 13 (median 8) responses for each stimulus. Each individual contributed between 3 and 20 responses (median 11.5). The behavioural responses were counted for the respective response categories and visualised within each call category.N = 22) with their sound pressure level and their signal-to-noise ratio, respectively. A stimulus response index was defined by the number of orientation responses divided by all responses towards a stimulus. To make sure that the consecutive presentation of playback stimuli resulted in independent responses we conducted a Spearman rank correlation for the response indices with the order of stimulus presentation. The order response index was defined by the order number of the orientation responses divided by all responses for the respective presentation number.We conducted Spearman rank correlations to exclude the effects of stimulus quality by correlating the response indices of all stimuli for which we saw behavioural responses . Therefore, responses of an individual towards conspecific advertisement stimuli were averaged for further analysis.Furthermore, to test for habituation effects we analysed the response strength towards the first and the second stimulus of the two conspecific advertisement stimuli during a given playback session. A chi-square test revealed that the distribution of no orientation and orientation responses did not differ significantly between the first and the second conspecific advertisement stimulus (chi-square-test: χPB participated in the design of the study, conducted the experiments, performed the statistical analysis and prepared the manuscript. SS mentored the study, contributed to the technical design of the study and the preparation of the manuscript. EZ initiated, financed, mentored and contributed to the design of the study and the preparation of the manuscript. All authors read and approved the final manuscript.


Upright faces are thought to be processed holistically. However, the range of views within which holistic processing occurs is unknown. Recent research by McKone  suggests By necessity, we must be able to generalize across many natural sources of image variation in order to recognize a face, including changes in distance and viewpoint as we move around each other and changes in lighting as we move in and out of different environments. We rarely view faces from directly in front (known as the “full face” or 0° view). Most commonly, our visual experience of faces falls within a range of viewpoints rotated away from 0° by up to 45° to the left or right , above or below , and clockwise or anticlockwise . Within this range of variation in viewpoint, face recognition is remarkably good, even for unfamiliar faces  is the observation that the inversion (180° roll or picture-plane rotation) of faces dramatically impairs recognition compared to upright faces, and that this impairment is disproportionately larger for faces than objects Yin, . BecauseIt is now generally accepted that turning a face upside-down disrupts face-specific holistic processing. For example, if one creates a composite face by aligning the photographs of the top and bottom halves of two different faces, the obligatory holistic processing of the new “whole” face will impair naming accuracy and increase reaction times (RTs) for each half face  in a similar manner. During picture-plane rotations, Rossion and Boremanse  found a To date there has been little investigation of holistic processing in faces rotated in pitch and yaw , configural information appears to be more useful. Second, pitch rotation disrupts configural information to a greater degree than yaw or roll rotation. Compared to rotations about other axes, pitch camera rotations result in a greater foreshortening and occlusion of features as well as a general reduction in the amount of available “face” information. Thus, Favelle et al.’s  findingsMcKone  found evThis study was approved by the University of Wollongong Human Research Ethics Committee, and written consent was obtained from all participants.Participants were 20  volunteer undergraduate students attending the University of Wollongong. The average age of participants was 21.6 years (age range: 18–29 years). All participants had normal or corrected-to-normal vision and none were familiar with the faces used as stimuli.d′, defined below) and RT to correct same matches.Participants made “same/different” judgments of two sequentially presented faces, shown centrally, and both upright or inverted. On each trial, these two faces were typically seen from different views. There were 10 levels of viewpoint  crossed with orientation (upright and inverted), all manipulated within subjects. The dependent measures were matching sensitivity  as well as hair were removed digitally. Faces were illuminated by an ambient light source located directly above the model plus four directional light sources located 1 m in front of the model . The two lights located just above the face were oriented horizontally , whereas the lights located just below the face were oriented 45° below the horizontal. Lighting was held constant across all viewpoints. In addition to a full-face (0°) view, each face was presented from three different viewpoints rotated 15°, 45°, and 75° away from 0° in three different directions: yaw leftAll images were viewed in the center of the computer screen against a white background. The on-screen height of 0° faces was approximately 16 cm with a width of 10 cm, which produced a visual angle of 14.7° × 19.2°. For yaw viewpoints the height of the face image remained constant, however face width increased as the viewpoint was rotated further away from 0°. Face width remained constant for pitch camera rotations, however face height decreased as the viewpoint was rotated further away from 0° (for both pitch-up and pitch-down conditions). The 75° pitch-up camera condition had the shortest image, which was 12 cm high and produced a visual angle of 11° × 9.2°. The rectangular patterned mask used in the experiment subtended a visual area of 18° × 22° and was composed of various elements taken from the stimuli used in the task.www.tarrlab.org) guided the trial sequence. Responses were made via key presses on a keyboard placed in front of the participant.Full color images were presented to participants on a 48 cm flat-screen monitor with a resolution of 1024 × 768 pixels. Trials were run on a Macintosh G4 computer and RSVP experimental software  was presented first followed by a mask, and then a test face  was presented followed by the mask again. Participants were first verbally instructed how to complete the task, with emphasis placed on both speed and accuracy in responding. Written instructions on how to complete the task were also provided on the computer screen. After reading the instructions participants completed 10 practice trials to familiarize them with the task. Stimuli used in the practice trials were different from the stimuli used in the task. Following the practice trials participants were given a chance to ask any questions about the procedure, should they have any, before continuing on with the experiment.Orientation (upright or inverted) was blocked and the order of the blocks counterbalanced. Each block consisted of 180 trials, giving a total of 360 experimental trials. In half of the trials the two faces presented were the same, regardless of viewpoint . In the other half of trials the two faces were different; the different face was randomly selected from the remaining eight faces . Trial type was presented in random order within each block. Participants were given five self-timed rest periods spaced equally throughout each block. The experiment lasted approximately 30 min.Each trial began with a fixation cross displayed for 500 ms. This was followed by the presentation of face 1 for 250 ms. Then the mask was presented for 500 ms. Face 2 was then presented for 250 ms, followed by a second presentation of the mask for 500 ms. Following the second mask the screen remained blank for 2 s or until a response was made by the participant. If a response was not made within this time, the trial ended . The interval between trials was 1 s. Participants were required to respond by pressing clearly labeled “same” and “different” keys on a keyboard depending on whether they judged face 1 and face 2 to be the same face or two different faces.z-scores and then used to calculate d′  and RT (ms) scores were analyzed in a series of two-way (viewpoint × orientation) repeated measures ANOVAs. Pitch viewpoints  were analyzed separately to yaw viewpoints . The alpha level was 0.05. Greenhouse–Geisser corrections were made whenever the assumption of sphericity was violated. Bonferroni adjustments were made where necessary to control for family wise error.Participants’ responses were converted into hit (H) and false alarm (FA) rates, where a hit was a correct “same” response to a face, and a FA was an incorrect “same” response to a “different” face. These H and FA rates were converted into d′ data revealed a main effect of orientation, F = 25.75, p < 0.001, MSE = 30.12, F = 43.07, p < 0.001, MSE = 30.85, F = 3.17, p < 0.01, MSE = 2.19, Post hoc comparisons showed a significant FIE (upright–inverted) at the full-face (0°) view and at pitch viewpoints of 15° and 45° above and below 0°  with no evidence of a FIE at either of the 75° viewpoints (both p > 0.84).As can be seen in Figure F = 4.77, p < 0.05, MSE = 381809.7, F = 8.15, p < 0.001, MSE = 156663.29, F = 0.74, p = 0.56, MSE = 12112.8. Post hoc comparisons showed significantly faster response times to 0° than to either 45° or 75° pitch viewpoints above (both p < 0.01).The pattern of the RT data approximately reflected that of the sensitivity data except at pitch +75°  = 39.76, p < 0.001, MSE = 26.71, F = 15.73, p < 0.001, MSE = 10.28, F = 1.86, p = 0.15. Post hoc comparisons showed significantly higher sensitivity to 0° viewpoints than to any other yaw viewpoint . There was no difference in sensitivity to matching faces between 15°, 45°, and 75° viewpoints .As can be seen in Figure F = 4.8, p < 0.05, MSE = 212161.9, F = 5.98, p < 0.001, MSE = 56426.8, F = 0.16, p = 0.93. Post hoc comparisons showed significantly faster responses to 0° viewpoints than to either 45° or 75° yaw viewpoints (both p < 0.05).The RT data reflects the pattern of the sensitivity data with a FIE evident at all viewpoints . We found that recognition performance was most sensitive for the upright full-face views and declined as these upright faces were rotated away from the full-face view. While sensitivity continued to decline for upright face views following pitch rotations of up to ±75° , no further decline was evident following yaw rotation beyond 45°.As in previous experiments from this lab  the absence of inversion effects for these viewpoints was unlikely to have arisen due to floor effects.Interestingly, inversion impaired face recognition sensitivity  for some viewpoints, but not for others. As Hills et al.  found, rThe advantage of the face inversion manipulation is that the low-level stimulus properties present in an upright face are identical to those in an inverted face. Thus, any differences between the two conditions have to be accounted for by higher-level, probably face-specific, processes. As noted in the Section The current experiment is the first to investigate the holistic processing of pitch rotated face views. While the FIE was observed at pitch ±15° and ±45° viewpoints, the absence of a FIE at pitch ±75° viewpoints suggests that there is a fixed limit to the range of upright face views in which holistic processing occurs. That is, there appears to be a qualitative difference in the way that upright faces are processed at pitch ±75° viewpoints compared to the other pitch and yaw-rotated viewpoints tested in this experiment. Face recognition performance at these viewpoints could be explained in two ways: (i) holistic processing occurs much less efficiently at 75° pitch rotations because we have little to no expertise with these views  and hence cannot be explained in terms of response biasRecently, Richler et al.  have argRossion and his colleagues Rossion,  on the oMore recently, Van Belle et al.  found emFollowing on from this, the current results might be accounted for in the same way. According to this view, all faces  are handled in a similar way by the early visual system. However, it may not be possible to match 75° pitch rotated views of faces to the experience-derived face template/s and so as a result they do not appear to initiate holistic processing. If this is the case, since neither 75° pitch rotated views of faces nor any inverted faces are processed holistically, then there is no basis for the FIE. Importantly, what this suggests is that a visual stimulus simply being categorized as a face, as we assume at least the 75° pitch view from below would, is not enough to trigger holistic processing.Changes in view can result in substantial variations in the visual appearance of a face. Despite this, our results show that holistic processing occurs for a large range of views around the upright full-face view. Specifically, the range of views within which upright faces are processed holistically extends to rotations of 90° in yaw McKone,  and ±45°The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AbstractAchillea millefolium L.) accessions was reported. Cytological analyses on four Achillea millefolium accessions, indicated that two accessions were diploids (2n=2x=18) and two tetraploids (2n=4x=36). Cluster analysis based on chromosomal characteristics and karyotype asymmetry, categorized the four accessions separated into two groups. In terms of the Stebbins’ system, the karyotype of diploid accessions grouped in 2A class. The average value of the total form percentage (TF%) in the group one (diploid accessions) and two (tetraploid accessions) were 40.85 and 41.15, respectively. The group one had the highest mean value for the symmetry index (S%=57.5). Consequently, it can be inferred that diploids belonging to the group one are the earlier evolutionary forms.In this study, a new chromosome number for Iranian yarrow ( Achillea is one of the most recent genera of the Asteraceae family which exists throughout the world  to find any relationship between the karyotype characteristics and asymmetrical index with ploidy levels.he world . Three che world . The aimAchillea millefolium accessions were collected from three provinces in north, west and south of Iran , mean chromosome length (MCL), and mean arm ratio (MAR) were calculated using MICROMEASURE (Version 3.3) Software . Stebbinn = 4x = 36 chromosomes, while Achillea millefolium  that were collected in northern parts of Iran.Am1 and Am2 accessions were diploids (2n= 2x= 18) whereas the two other accessions (Am3 and Am4) showed tetraploid (2n= 4x= 36) level . AccordiAchillea millefolium  in the group one and two were 40.85 and 41.15, respectively. The TF% index has frequently been used to explain karyotype asymmetry  and karyotype asymmetry (TF% and S%)  and agreAchillea millefolium accessions. Cluster analysis indicated that accessions can be classified based on ploidy levels.The results of the present study illustrated a new ploidy level (2n= 2x= 18) in Iranian


SHR using quantitative real-time PCR. Kidney cortex, but not liver or heart Mt-gene expression was decreased ~2–5 fold in 12 of 13 protein encoding genes of HT BN/SHR-mtSHR. Kidney cortex but not liver mRNA expression of the nuclear transcription factors Tfam, NRF1, NRF2 and Pgc1α were also decreased in HT BN/SHR-mtSHR. Kidney cortical tissue of HT BN/SHR-mtSHR exhibited lower cytochrome oxidase histochemical staining, indicating a reduction in renal oxidative phosphorylation but not in liver or heart. These results support the hypothesis that renal cortex of rats with SHR mitochondrial genome has specifically altered renal expression of genes encoding mitochondrial proteins. This kidney-specific coordinated reduction of mitochondrial and nuclear oxidative metabolism genes may be associated with heritable hypertension in SHR.Mitochondrial (Mt) dysfunction contributes to the pathophysiology of renal function and promotes cardiovascular disease such as hypertension. We hypothesize that renal Mt-genes derived from female spontaneously hypertensive rats (SHR) that exhibit hypertension have reduced expression specific to kidney cortex. After breeding a female Okamoto-Aoki SHR (SAP = 188mmHg) with Brown Norway (BN) males (SAP = 100 and 104 mmHg), hypertensive female progeny were backcrossed with founder BN for 5 consecutive generations in order to maintain the SHR mitochondrial genome in offspring that contain over increasing BN nuclear genome. Mt-protein coding genes  and nuclear transcription factors mediating Mt-gene transcription were evaluated in kidney, heart and liver of normotensive (NT: n = 20) vs. hypertensive (HT: n = 20) BN/SHR-mt Hypertension constitutes a primary and significant factor in the development of cardiovascular disease. Despite major gains in the long-term treatment of hypertension, cardiovascular disease remains the number one cause of death and disability in developed countries. Primary or essential hypertension is regarded as a multi-factorial disease, influenced by both genetic inheritance and environmental conditions that influence gene expression. The genetic basis of hypertension has been focused primarily on inheritance and expression of nuclear genes (nDNA) , despitelle gene (GenBank accession no. NC_001807) and hypertension. The genetic association of mtDNA variants and tRNA mutations [Mitochondrial dysfunction has been implicated in a wide variety of genetic disorders , 5, and utations  to type utations . Taken tSHR) [The impact of the kidney on long-term blood pressure regulation is well known. It has been hypothesized that the “set-point” for the long-term control of BP resides in the kidney –15. In tSHR) . HyperteAll experiments were carried out in strict accordance with the recommendations provided in the AAALAC Guide to the Care and Use of Laboratory Animals and the National Institutes of Health. All protocols in this study were specifically approved by the University of Kentucky Institutional Animal Care and Use Committee (IACUC #000649L2003). All surgeries were performed following complete anesthesia of individual animals. Tissue collection for these studies also was conducted under full anesthesia. At the time of any change in protocol (minor or major) amendments were submitted to the IACUC and approved prior to conducting any individual experiment. Since this was a long term breeding study, the original IACUC approval served for a significant time period. All individual studies for this protocol approval were reevaluated at 3 year intervals as required by AAALAC standards. A “conplastic” colony using phenotypic selection was employed. The development and phenotypic characterization have been described in detail elsewhere . The AokAfter repeated blood pressure recordings that assured consistent determination of arterial pressure, rats not scheduled for rebreeding were euthanized with an overdose of sodium pentobarbital (60 mg/kg i.p), decapitated, and organ tissues were rapidly frozen in acetone super-cooled on dry ice and stored for later analysis.SHR rats  were utilized for the current study  was evaluated in parents and offspring beginning no earlier than 10–12 weeks of age. Phenotypes were assigned as either normotensive (NT: SAP ≤ 124mmHg), borderline hypertensive (BHT: 125 ≤ SAP ≤149 mmHg) or hypertensive  as described above. Total RNA was extracted with Trizol reagent  and purified using RNeasy minicolumns  according to the manufacturer’s protocol. Possible genomic DNA was digested with DNase I . Concentration and purity of all RNA samples was determined by the Nanodrop ND-1000 spectrophotometer . Extracted RNA was reverse-transcribed into complementary DNA (cDNA) using qScript cDNA Supermix  in a total volume of 20μl using a MyCyler Thermal Cycler .Renal cortex, liver and left ventricular cardiac tissue were selected from age and sex-matched HT and NT BN/SHR-mtmt-ND1- Rn03296764_s1, mt-ND2- Rn03296765_s1, mt-ND3- Rn03296825_s1, mt-ND4- Rn03296781_s1, mt-ND4L- Rn03296792_s1, mt-ND5- Rn03296799_s1, mt-ND6- Rn03296815_s1, mt-CO1- Rn03296721_s1, mt-CO2- Rn03296737_s1, mt-CO3- Rn03296820_s1, mt-CYB- Rn03296746_s1, mt-ATP6- Rn03296710_s1, mt-ATP8- Rn03296716_s1, NRF1- Rn01455958_m1, NRF2a- Rn01767215_m1, NRF2b-Rn01514289_g1, Pgc1α-Rn00598552_m1, Tfam-Rn00580051_m1, Cyc1-Rn01504159_g1, Cox6c-Rn00820983_gH, GAPDH- Rn01775763_g1). Primers and probes were verified as operating at similar efficiencies. Target gene and endogenous control amplicons were labeled with either FAM or VIC. The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression were measured in all samples to normalize gene expression for sample-to-sample differences in RNA input, RNA quality and reverse transcription efficiency. Each sample was analyzed in triplicate, and the expression was calculated according to the 2−ΔΔCt method [Quantitative Real-Time PCR was performed on a StepOnePlus Real-time PCR system . Real-time quantitative PCR amplifications were performed in triplicate on a 96-well plate. Pre-designed TaqMan primers and hydrolysis probes for all genes of interest were purchased from Applied Biosystems  . Total mCytochrome oxidase (CO) activity was determined in fresh frozen sections (20μm) in kidney, liver and heart tissue, as described previously . BrieflyU Test comparisons. The 0.05 level of probability was utilized as the minimum criterion of significance. All statistical analyses were performed using GraphPad Prism 4.0 .Blood pressures and citrate synthase activity among animals were initially analyzed by 1-way analysis of variance (ANOVA) followed by post-hoc comparisons using the Bonferroni t-test. Tissue mRNA expression levels were analyzed using Mann-Whitney SHR, including five complex I, one complex III, three complex IV and both subunits of ATP synthase. Additionally, the nuclear trans-factors Tfam, NRF1, NRF2a, NRF2b and Pgc1a were all downregulated in the kidney, but not elsewhere, of HT BN/SHR-mtSHR.Multiple mtDNA encoded genes of the mitochondrial respiratory chain were significantly reduced in renal, but not liver or cardiac tissue of HT BN/SHR-mtSHR. mt-ND1 was reduced ~3.7 fold in HT BN/SHR-mtSHR (P = 0.0317). mt-ND3 was reduced ~2.6 fold in HT SHR/BN-mtSHR (P = 0.0077). mt-ND4 was reduced ~10.8 fold in HT SHR/BN-mtSHR (P = 0.0221). mt-ND4L was reduced ~7.7 fold in HT SHR/BN-mtSHR (P = 0.05). mt-ND5 was reduced ~2.7 fold in HT SHR/BN-mtSHR (P = 0.00167). mt-ND6 was reduced ~1.7 fold in HT SHR/BN-mtSHR (P = 0.0822) (mt-ND2 was not different between the two phenotypes (P = 0.5994).The renal expression of five of the seven mt-encoded genes of complex I were significantly reduced in hypertensive versus normotensive BN/SHR-mt 0.0822) . mt-ND2 mt-CYB) was significantly reduced in hypertensive versus NT BN/SHR-mtSHR. mt-CYB was reduced ~5 fold in HT BN/SHR-mtSHR (P = 0.0103)  .SHR. mt-CO1 was reduced ~3.2 fold in HT BN/SHR-mtSHR (P = 0.0164), mt-CO2 was reduced ~3.6 fold in HT BN/SHR-mtSHR (P = 0.0063), and mt-CO3 was reduced 4.1 fold in HT BN/SHR-mtSHR (P = 0.0085) (All three mt-encoded genes of complex IV were significantly reduced in hypertensive versus normotensive BN/SHR-mt 0.0085) .mt-ATP6 was reduced ~2.3 fold in HT BN/SHR—mtSHR (P<0.05), while mt-ATP8 was reduced ~3.1 fold in HT BN/SHR-mtSHR (P = 0.0255)  .SHR as described above. mt-CYB, mt-CO2 and mt-ND1, mt-ATP6 were shown to be not different (P>0.05) between the HT and NT BN/SHR-mtSHR liver or heart tissues. This is in contrast to renal tissue wherein each of these genes exhibited reduced expression in HT vs. NT animals  in the kidney, but not the liver or heart (P>0.05) in HT BN/SHR-mtSHR  in NT (n = 6) and HT (n = 6) BN/SHR-mtHR-mtSHR .PGC-1α regulates NRF-dependent transcription, increases expression of both mitochondrial and nuclear encoded genes of oxidative phosphorylation and induces mitochondrial biogenesis. HT BN/SHR-mtSHR exhibited ~2.5 fold reduction in PGC-1α mRNA in kidney tissue compared with NT BN/SHR-mtSHR (P = 0.0098) ( 0.0098) .Tfam, whose expression is regulated by NRF1. All three NRFs were reduced in the kidney, but not liver of HT compared to NT BN/SHR-mtSHR. Renal NRF1 mRNA expression was reduced ~1.8 fold in HT BN/SHR-mtSHR compared with NT BN/SHR-mtSHR (P = 0.0307) (NRF2a mRNA expression was reduced ~2.3 fold in HT BN/SHR-mtSHR compared with NT BN/SHR-mtSHR (P = 0.0083) (NRF2b mRNA expression was reduced ~1.9 fold in HT BN/SHR-mtSHR compared with NT BN/SHR-mtSHR (P = 0.0377)  . Renal N 0.0083) . Renal N 0.0377) .Tfam is a key activator of mammalian mitochondrial transcription. Kidney, but not liver exhibited reduced Tfam mRNA expression ~2.5 fold in HT BN/SHR-mtSHR compared with NT BN/SHR-mtSHR (P = 0.0281) ( 0.0281) .CYC or Cox6c between NT (n = 10) and HT (n = 10) BN/SHR-mtSHR (P>0.05)  .SHR  and HT (n = 10) BN/SHR-mtSHR . There wSHR compared with NT BN/SHR-mtSHR , also exhibited reduced expression in kidney cortex, but not liver, compared with NT BN/SHR-mtSHR  subunits and the potential for disease has been previously evaluated , 30. Lopet al.  reported [et al.  reportedet al. [SHR compared with NT BN/SHR-mtSHR. It is possible that mitochondrial activity is elevated in renal proximal tubules of very young SHR prior to the onset of hypertension. As the development of hypertension progresses in the maturing SHR, renal mitochondrial gene expression may then decline as a compensatory mechanism to, for instance, decrease sodium transport along various segments of the nephron. This is plausible, as altered renal sodium handling and renal function are a hallmark of hypertension. Ongoing and future studies are being conducted to address this possible relationship and mechanism specific to the SHR.Data presented here poses the important and critical question: Does the reduction of renal mitochondrial gene expression and function contribute significantly to the etiology of hypertension? Or does hypertension cause the downregulation of mitochondrial gene transcription and function? Recently, Lee et al.  postulattrans-factors that regulate them. As shown in SHR, hence, it appears that mitochondrial function was decreased in the kidneys of HT BN/SHR-mtSHR. Altered renal function has been well recognized as a key factor in the development and maintenance of hypertension [et al. [Agtr1a gene resulted in the reduced age-related cardio-renal complications, improved mitochondrial biogenesis, and increased longevity in mice. Treatment with antioxidants, mitochondrial superoxide dismutase mimetics, and AT1r blockers decreased vascular O2.- production and attenuated development of hypertension in SHR [SHR exhibit elevated AT1r mRNA (Agtr1a) expression compared to NT BN/SHR-mtSHR, while the renal and systemic expression of renin, angiotensinogen, and angiotensin-converting enzymes were not different [SHR also had elevated AT1r protein compared with NT BN/SHR-mtSHR, and this increase was positively correlated with elevated systolic BP [et al. [1r blockade with Losartan. These data support the hypothesis that hypertensive phenotype derived from the SHR is driving the reduced mitochondrial gene expression, which may lead to decreased OXPHOS.One of the more interesting aspects of this study is a potential ETC dysfunction driven by transcript differences in the kidneys, but not in other tissues, of mt-genes and the nuclear rtension , 33–38.  [et al.  Deletionn in SHR , 41. In ifferent . Renal ctolic BP . De Cava [et al.  demonstrPGC-1α plays a central role in regulating mitochondrial content and function within cells, because of its ability to co-activate and augment several promoters of nuclear-encoded mitochondrial genes, as well as regulating mitochondrial transcription via the NRF-Tfam pathway [PGC-1α regulates NRF-dependent transcription, increases expression of both mitochondrial and nuclear encoded genes of oxidative phosphorylation and induces mitochondrial biogenesis [PGC-1α in a tissue specific manner in brown fat, muscle and liver [PGC-1α and its effectors is unknown. Results from this study show a coordinated reduction of the kidney-specific expression of nuclear and mitochondrial genes vital to OXPHOS in hypertensive BN/SHR-mtSHR, which may play a significant role in the development and maintenance of hypertension  nor cytochrome c oxidase subunit Vic (Cox6c) were differentially expressed in kidney cortex of HT and NT BN/SHR-mtSHR .None of the 7 mitochondrial encoded genes of complex 1 exhibited gene expression differences (P>0.05) between the HT and NT BN/SHR-mt(TIF)Click here for additional data file.S2 FigA: Superoxide dismutase 1 (SOD1) nor B: superoxide dismutase 2 (SOD2) were different (P>0.05) between NT and HT BN/SHR-mtSHR .Neither renal (TIF)Click here for additional data file.


DNA extraction from clinical samples is commonly achieved with a silica solid phase extraction column in the presence of a chaotrope. Versions of these protocols have been adapted for point of care (POC) diagnostic devices in miniaturized platforms, but commercial kits require a high amount of input DNA. Thus, when the input clinical sample contains less than 1 μg of total DNA, the target-specific DNA recovery from most of these protocols is low without supplementing the sample with exogenous carrier DNA. In fact, many clinical samples used in the development of POC diagnostics often exhibit target DNA concentrations as low as 3 ng/mL. With the broader goal of improving the yield and efficiency of nucleic acid-based POC devices for dilute samples, we investigated both DNA adsorption and recovery from silica particles by using 1 pg– 1 μg of DNA with a set of adsorption and elution buffers ranging in pH and chaotropic presence. In terms of adsorption, we found that low pH and the presence of chaotropic guanidinium thiocyanate (GuSCN) enhanced DNA-silica adsorption. When eluting with a standard low-salt, high-pH buffer, > 70% of DNA was unrecoverable, except when DNA was initially adsorbed with 5 M GuSCN at pH 5.2. Unrecovered DNA was either not initially adsorbed or irreversibly bound on the silica surface. Recovery was improved when eluting with 95°C formamide and 1 M NaOH, which suggested that DNA-silica-chaotrope interactions are dominated by hydrophobic interactions and hydrogen bonding. While heated formamide and NaOH are non-ideal elution buffers for practical POC devices, the salient results are important for engineering a set of optimized reagents that could maximize nucleic acid recovery from a microfluidic DNA-silica-chaotrope system. Furthermore, an a posteriori Tukey test showed that DNA recovery using 1 M NaOH and 95°C formamide were significantly different than recoveries using buffer EB, water, and 50 mM Tris-Cl (pH 8.5) at α = 0.01. In addition, there was a significant difference in DNA recovery using 1 M NaOH versus 95° formamide at α = 0.01.The elution curve data is shown in The DNA loss data suggests that DNA adsorption on silica is mainly dependent on pH. DNA has an inherently greater affinity for the silica surface in acidic environments. However, the addition of the chaotrope (GuSCN) increased the surface affinity for DNA at pH 3 and 5.2. We expected this result because the isoelectric points of DNA and silica are 5 and 1.5–3.6 respectively. At pH 3, we did not expect electrostatics to interfere with the chaotropic mechanism that increased DNA adsorption, resulting in the highest surface affinity. Both the DNA and the silica surface become more negative at pH 5.2, and the corresponding increase in electrostatic repulsion resulted in more DNA loss. At pH 8, the loss was maximized. It has been theorized that chaotropes increase DNA-silica affinity by dehydrating the surface and promoting hydrogen bonding between the DNA and silica surface . Our DNAFocusing on events downstream of the DNA adsorption process, our elution results suggest that an increase in DNA adsorption capacity at low pH does not necessarily leads to a higher DNA recovery rate. Instead, the best DNA recovery results were obtained by taking advantage of a weaker DNA-silica-chaotrope adsorption complex facilitated at the intermediate pH of 5.2. Buffer EB, the gold-standard, was not able to disrupt the DNA-silica-chaotrope complex created at pH 3 since DNA recovery did not exceed 10.1%. Conversely, when the DNA-silica-chaotrope complex was formed at pH 8, weak DNA adsorption events resulted in massive 48.8% DNA loss prior to the EB elution steps. Thus, a DNA-silica-chaotrope adsorption event at pH 5.2 represents an optimum situation where: (A) The initial DNA loss due to a weaker solid-phase complex is offset by (B) An ease in complex dissociation with buffer EB at pH 8.5, thus resulting in a multi-factor local maximum in total DNA recovery.Diving deeper into 2 in the presence of a chaotrope, depending on the pH of the adsorption solution [2. In this regime when using GuSCN, no appreciable DNA was adsorbed at pH 8 and no appreciable DNA will be eluted when adsorbed at pH 3. This is in contrast to other experimental designs in literature that have demonstrated recovery of significant amounts of DNA after adsorbing at pH 4 or 8, with the caveat that the amount of unrecoverable DNA within the system was not addressed. To elaborate, these studies were conducted using large concentrations of input DNA, such that the net unrecoverable amount of DNA was negligible compared to the net recovered [The experiments were conducted such that DNA, rather than the available surface, was the limiting factor for adsorption. Existing literature has shown that the adsorption capacity for DNA per unit surface of silica is 240–800 μg/msolution ,19,26. Oecovered ,26. ThesOur results suggest that the standard method for eluting DNA using buffer EB from silica following chaotrope-mediated adsorption may not be ideal for low concentration of DNA. Only two conditions allowed for the recovery of more than 30% of input DNA with buffer EB. Based on these results, DNA recovery depends on 1) the concentration of DNA in the adsorption solution, 2) the adsorption solution pH, 3) the presence of a chaotrope, and 4) the elution buffer. It also depends on the ratio of input DNA to the available surface area of silica when comparing to previous results. This however was controlled for in these experiments. Thus, designing a device to use this technology requires not only an understanding of the DNA-silica-chaotrope interaction, but the dynamic range of characteristics associated with clinical samples and how sample preparation steps affect these characteristics. Our data suggests that we can increase the range of initial sample conditions for which this technology can be implemented.We developed a protocol to assess the adsorption and elution of DNA from silica particles while emulating POC DNA extraction conditions for samples containing ≤ 1 μg DNA. Controlling for presence of a chaotrope, DNA adsorption increased with increasing acidity of the aqueous adsorption solution. Adsorption was further enhanced by adding 5 M GuSCN to the adsorption solution. Elutions exceeded 30% DNA recovery only when DNA was adsorbed at pH 5.2 with 5 M GuSCN for inputs of ≥ 100 ng net DNA. Adsorption at pH 3 resulted in DNA-silica-chaotrope complex that was too strong for buffer EB to disrupt effectively, while adsorption at pH 8 was not strong enough for DNA to adsorb sufficiently. Due to the poor recover of DNA with buffer EB at pH 3, we attempted to disrupt DNA-silica-chaotrope complex by varying the eluting solution. Hot formamide and 1 M NaOH resulted in increased recovery of 27.5% and 71.9% respectively, leading to the conclusion that the interaction was dominated by hydrophobic interactions and hydrogen bonding. However, neither of these buffers is practical for POC use. The optimal adsorption condition for POC use studied here was achieved using pH 3 with 5 M GuSCN resulting in up to 99.998% DNA adsorbed, while the optimal DNA recovery with buffer EB was 53.5% DNA using pH 5.2 with 5 M GuSCN. As our knowledge of these interactions improve, so will the range of clinical applications for this technology.


N- and O-linked glycosylations that define the individual properties of extracellular and membrane-associated proteins. In this study, we utilized different computational tools to perform in silico based genome-wide mapping of 1,117 human proteins and unravel the contribution of both penultimate and vicinal amino acids for the asparagine-based, site-specific N-glycosylation. Our results correlate the non-canonical involvement of charge and polarity environment of classified amino acids  in the N-glycosylation process, as validated by NetNGlyc predictions, and 130 literature-reported human proteins. From our results, particular charge and polarity combinations of non-polar aliphatic, acidic, basic, and aromatic polar side chain environment of both penultimate and vicinal amino acids were found to promote the N-glycosylation process. However, the alteration in side-chain charge and polarity environment of genetic variants, particularly in the vicinity of Asn-containing epitope, may induce constitutive glycosylation  of membrane proteins causing constitutive proliferation and triggering epithelial-to-mesenchymal transition. The current genome-wide mapping of 1,117 proteins  was used to explore charge- and polarity-based mechanistic constraints in N-glycosylation, and discuss alterations of the neoplastic phenotype that can be ascribed to N-glycosylation at preferred and non-preferred sites.The structural and functional diversity of the human proteome is mediated by  O- and N- linked sugar moieties (N-glycosylation) and hydroxyl groups of serine and threonine (O-glycosylation) generates a large number of glycoforms that are credited for the modulation of diverse cellular functions , the oligosaccharyl transferase (OT) mediates the co-translational transfer of a lipid-linked tetradecasaccharide (GlcNAc2-Man9-Glc3) from a dolichol phosphate to an asparagine included in a NXS/T sequon. The selective recognition by OT of the consensus sequence (NXS/T) has enabled investigation of the structural requirements for N-glycosylation. The rapid increase of substrate data for protein N-glycosylation has led to the development of different databases and prediction tools: dbPTMs, UniProt, NetNGlyc and MAPRes  (Proteins that undergo cations) , 12–15.N-glycosylated to perform key biological functions (N-glycosylation (preferred and non-preferred motifs) is needed to explore the biological relationships between sequence, structure, and function of glycoproteins. MAPRes is a valuable tool to define the significantly preferred and non-preferred amino acids in the vicinity of a N-glycosylation site by resorting to the association rule mining technique (N-glycosylation (N+) and non-N-glycosylation (N−) sites on the basis of potential score and consensus sequences within the target protein  in 1,117 human proteins in which the majority (96.5%) of N + sites is followed by the canonical motif of NXS/TY. According to our MAPRes analysis for general protein sequence analyses, Val at +1, Ser/Thr at 2, Leu/Val at 3 and Leu at −5 positions were found significantly preferred residues to mediate the glycosylation of Asn residues in the human proteome. After classifying amino acids’ charge and polarity according to properties of their side-chain R-groups, significant preference for N-glycosylation was found for non-polar, uncharged R-groups  at position 1, polar R-group (Met/Thr/Ser/Cys/Asn/Gln: L) at position 2, polar, negatively charged acidic R-groups  at position 3/5/−4 and aromatic amino acids: Phe/Trp/Tyr: A, at position 3/−5/−1. Furthermore, we validated the MAPRes-predicted preferred association pattern for the wider N-glycosylation sequence contexts by using the NetNGlyc 1.0 server and 130 literature-reported UniProt proteins, and provided further evidence that charge and polarity of O amino acids  at position 1, A amino acids (Phe/Trp/Tyr) at positions −6, −5, −2, −1,1,3, and 10, P-amino acids (Lys/Arg/His) at positions −9, −3, 9, 10, and N amino acids (Asp/Glu) at positions −4/3/5, in combination with L amino acids at position 2, is likely to generate significantly preferable environments for the N-glycosylation of human proteins. Any change in charge and polarity environments may, therefore, result in aberrant N-glycosylation on both preferred and non-preferred sites.In this study, we have identified 2,909 N-glycosylated (N+) and non-N-glycosylated (N−) residues by using MAPRes. Then, preferred amino acids and association patterns were determined on the basis of polarity and charge of the surrounding amino acids of N+ and N− residues. Next, association patterns mined by MAPRes were validated with the NetNGlyc 1.0 server (see text footnote 1) for 15 biologically important proteins. The second level of validation was performed by using 130 literature-reported human proteins with 438 N-glycosylated motifs from the UniProt database.As a first step, preferred amino acids and association patterns were found around N-glycosylated proteins were downloaded from dbPTM, version 3.0,N-glycosylation included 16,915 entries in total and provided all required information for MAPRes analysis except for the protein primary sequences (from UniProt), which were added manually in the final dataset. All entries other than human proteins were removed. In the final dataset, only 1,117 proteins and 2,909 asparagine residues were found modified.The primary sequences for The data cleaning of the final dataset was essential for estimating modified residues on vicinal amino acids. Several types of errors that can be generated during the compilation of the dataset such as incorrect position of the modified residues, presence of non-standard characters in the sequence, incorrect sequence length and repetitions of entries. These inconsistencies were identified and removed by utilizing the Data Inconsistency and Duplication Check module of MAPRes.N-glycosylation proteins (Table To the environments of the N+ (positive) and N− (negative), the negative sites were annotated in the final dataset. There were in total 31,052 negative sites (non-modified Asn) in the final dataset of human The new version of MAPRes can mine association patterns for neighboring amino acids of modified residues on the basis of their specific biophysical and biochemical properties. In this study, the association patterns mined on the basis of polarity and charge of the amino acids were distributed into five different groups Table . SpecifiN-glycosylated and non-N-glycosylated datasets. MAPRes supplied the charge and polarity information to estimate the significantly preferred amino acids and association rule mining for neighboring amino acids for all datasets (Table N-glycosylated “N+” and other is non-N-glycosylated “N−”) and two for classified/encoded sequence of proteins (N-glycosylated and non-N-glycosylated) for which MAPRes mined association rules  found by NetNGlyc from the selected 15 proteins. The rest of the 642 Asn were considered to be N-residues. Peptides of 21 amino acids were generated for both N+ and N− residues and patterns mined by MAPRes for N-glycosylated and non-N-glycosylated, and found in both datasets. The confirmatory percentage for association patterns in the peptide dataset was found to be 94% for the N-glycosylated dataset and 59% for the non-glycosylated dataset  and sequence-based statistical analysis of N-glycosylated motifs validated the association rules of MAPRes. From the general dataset, around 99% of N-glycosylated motifs were found in the NXS/TY sequon . From classified datasets, among 438 residues, 99% were L amino acids at position 2 , and 30% of total were O-amino acids at position 1, in combination with L amino acids at position 2, to validate the <O,1><L,2> rule. Therefore, a strong correlation was observed between polar and non-polar R-groups in the NXS/TY sequon. The presence of O amino acids  at position 1 and of L amino acids at position 2 were characteristic for N-glycosylation  at 5, −4, and 3 positions. In addition to O and N amino acids, A amino acids also showed preference with at −2, 10, 3, −1, and −5 positions, in combination with L amino acids at 2 , N73 (NYDL), N128 (NKTG), N175 (NMSM), N196 (NGSC), N352 (NATN), N361 (NCTS), N413 (NRTD), N444 (NITS), N528 (NVSR), N568 (NITC), and N603 (NNTL)], localized in the extracellular EGFR , another key regulator of neoplastic cells, the extracellular N-glycosylation sites “N558 (NSTY), N570 (NGSP), N622 (NTSP), N637 (NWTI)” were modeled to validate the MAPRes association pattern  found by NetNGlyc from the selected 15 proteins. The rest of the 642 Asn were considered to be N-residues. From the second level of validation, we selected the 438 experimentally defined glycopeptides from 130 reviewed proteins and found validation of MAPRes rules in all 438 glycoepitopes. We further modeled two cell membrane proteins, epidermal growth factor receptor (EGFR), and E-cad, because these proteins are heavily decorated with N-glycans and involved in cancers as a result of aberrant or excessive glycosylation  inhibitor erlotinib, a frequently utilized drug to downregulate EGFR activation supporting constitutive proliferation of non-small cell lung cancer (N-glycosylation (a modification of the extracellular portion of EGFR) on the intracellular TK enzyme strongly suggests that the allosteric organization of EGFR TK is dependent on extracellular N-glycosylation events and that EGFR functions are indeed linked to the N-glycosylation status of EGFR.In the first level of validation, the association patterns mined by MAPRes for sylation , 44, 45.d breast –48. Actug cancer . This efN-glycosylation sites  in its extracellular domain were reported in the UniProt database  achieves the conformation favoring N-glycosylation of N56 -based structural modification of E-cad N-glycosylation in epithelial-to-mesenchymal transitions and susceptibility to colorectal cancer (N-glycosylation sites “N558 (NSTY), N570 (NGSP), N622 (NTSP), N637 (NWTI)” , and N637 (as Asn633) were found aberrantly decorated with complex-type high-mannose N-glycans , and critical for regulating the biological functions of E-cad in cancer ” , supportn cancer , 34. ForN-glycosylation site, favor the normal N-glycosylation process. However, the mutational changes at N-glycosylation site and/or vicinal preferred sites may impact the oncogenicity of proteins  and reinforces the neoplastic phenotype. Thereby, the genetic alterations in many transmembrane receptors and adhesion proteins are important to determine the success or failure of glycosylation in these transmembrane proteins. This is particularly critical in neoplastic cells where disease-associated risks need to be assessed for many transmembrane oncogenic TKs.Glycans remodel the protein backbone by reducing its conformational freedom and the loss of configurational entropy upon folding. To unravel the phenomenon of N-glycosylation. Moreover, remote parts of the protein chain rich in aromatic amino acids (A) support protein folding and promote the glycan–protein hydrophobic and nucleophilic interactions at positions 3/−5/−1  and O amino acids at position 1  was found significantly preferred for 1 Tables , in the 1 Tables . The N aThis publication is dedicated to the memory of Professor Dr. Nasir-ud-Din (1937–2016), founder and chairman of Institute of Molecular Sciences and Bioinformatics and Fellow of Pakistan Academy of Sciences.MM designed the basic research theme, and contributed for data collection, analysis and validation. He also contributed significantly in manuscript writing. ZI defined and designed the research theme, methods, and generated various results. WQ contributed to generating results and participated in the improvement of the results interpretations. DH provided suggestions for improving the structure of the basic theme of the research and writing of the manuscript. He also helped to improve the scope of the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Endobronchial and endotracheal metastases from extra-pulmonary solid tumors are rare.Patients and methods: We reported the case of a patient diagnosed with endobronchial and endotracheal metastases from rectal adenocarcinoma.Case report: Patient P.G., 62 years old, was diagnosed with a rectal tumor in 2011, for which, a surgical intervention was performed . Afterwards, she underwent adjuvant chemotherapy and concomitant radiochemotherapy.In September 2013, the chest CT showed 2 nodules for which, an incomplete surgical resection was done and which were histopathologically diagnosed as metastases from rectal cancer. The patient continued the treatment with chemotherapy associated with Bevacizumab and after 6 months only Bevacizumab for maintenance.In June 2015, the chest CT pointed out a nodule in the right upper lobe and the bronchoscopy highlighted a 4-5 mm lesion at the level of the right primary bronchus, whose biopsy proved the rectal origin. Afterwards, another surgical intervention was performed. Unfortunately, the postoperative chest CT revealed an intratracheal tissue mass (11/ 7mm) and multiple metastases in the right lung. The bronchoscopy showed 2 endotracheal lesions, out of which one was biopsied . Despite the fact that chemotherapy was continued, other endobronchial lesions appeared. All of them were removed and the patient started radiotherapy on the tracheal area. Afterwards, she refused to continue chemotherapy. The last bronchoscopy highlighted one endobronchial and two endotracheal secondary malignant lesions.Conclusion: Endobronchial and endotracheal metastases must be taken into consideration in all the patients with a history of extra-pulmonary cancer.Abbreviations: CT = computed tomography, MRI = magnetic resonance imaging, IMRT = intensity-modulated radiotherapy, ESMO = European Society for Medical Oncology, NCCN = National Comprehensive Cancer Network, iv = intravenous, PET – CT = Positron Emission Tomography – Computed Tomography Endobronchial and endotracheal metastasis secondary to extra-pulmonary solid tumors or to primary lung tumors are rare, but can be life-threatening [1]. The most frequent solid primary non pulmonary tumors which determine the appearance of endobronchial/ endotracheal metastasis are breast cancer, colorectal cancer, renal cell carcinoma and malignant melanoma .Among the different localizations of the tracheobronchial tree, the trachea represents an extremely rare site for metastasis from solid extra-pulmonary cancers, being involved in only 5% of the cases , have occurred in the evolution of a rectal adenocarcinoma. Usually, they appear late after the diagnosis of a primary tumor, at a median of 50 months, being associated with other metastases . Our patient presented tracheobronchial tree metastases after 43 months from the diagnosis of a rectal cancer.The particularity of this case was represented, firstly, by the fact that endobronchial and endotracheal metastases, which are a rare condition . Through histopathology, it is very important to make the differential diagnosis with a primary lung cancer, because the treatment and prognosis differ [5-7]. As in the presented case, the symptoms that can appear in this condition can be life-threatening and are characteristic for an upper airway obstruction: cough, hemoptysis, dyspnea and stridor .In this association with rectal cancer it is possible that they are underdiagnosed because bronchoscopy is the most important test through which the physician establishes the diagnosis of certitude, but it is not seldom used between the investigations that are done in a rectal cancer . It can relief the symptoms which are due to the upper airway obstruction and improve the quality of life . It is represented by surgery, chemotherapy, external beam radiotherapy, cryotherapy, and brachytherapy [4]. Regarding the treatment of endobronchial and endotracheal metastasis, usually it is palliative . The goals of neoadjuvant radiochemotherapy are to increase local control, pathologic complete response and the rate of sphincter preservation; in addition, it is less toxic than administered postoperative [9]. Maybe it would have been better for this patient, whose tumor was cT3 on the MRI performed in August 2011, to receive neoadjuvant radiochemotherapy and according to the postoperative pathological result, adjuvant treatment.Neoadjuvant treatment (radiotherapy +/ - chemotherapy) represents the standard for locally advanced rectal cancers tumor (T3-4 N0 or Tany N+) [The tracheobronchial tree should be taken into consideration as a metastatic site for primary solid tumors localized extrapulmonary.Source of foundingThis work received financial support through the project entitled “CERO – Career profile: Romanian Researcher”, grant number POSDRU/159/1.5/S/135760, co-financed by the European Social Fund for Sectoral Operational Programme Human Resources Development 2007-2013.DisclosuresAuthors declare that there is no conflict of interest regarding the publication of this paper.


Enterococcus faecalis in a chemostat of our own design with limiting nutrient in the inflow set near saturation constant at three dilution rates . The highest dilution rate was near the critical rate calculated by the model. The one-day total biofilm buildup was 21× larger and its estimated growth rate 2.4× higher at highest dilution rate than at the lowest one. This increased biofilm formation with increased dilution rates is in agreement with previously published data on pure and mixed continuous flow cultures.We revisited the mathematical model of the chemostat and examined consequences of considerably decreasing the concentration of limiting nutrient in the inflow for the growth of both the planktonic and biofilm cells in the chemostat tank (fermenter). The model predicts a substantially lower steady-state biomass of planktonic cells in response to decreasing inflowing nutrient concentration. Contrarily, the steady-state concentration of nutrient inside the fermenter is expected to remain the same, as long as the inflowing concentration does not fall below its value. This allows the biofilm cells to grow at a rate regulated only by the exchange rate of the medium (dilution rate). We maintained a strain of  A large amount of both experimental data and mathematical description of biofilm buildup has accumulated in the past , yet litA chemostat as a physical instrument allows bacterial populations to grow at a constant rate for an indefinite period of time. As a mathematical model, the chemostat is simple, realistic, and thoroughly tested against the physical instrument by several generations of researchers .Even though the chemostat model itself does not include a biofilm component, it calculates a “common currency” for both plankton and biofilm, i.e., concentration of substrate limiting the growth of the strain in question. As soon as a cell attaches to a submerged surface , the current concentration of substrate ceases to be the limiting factor for its survival which creates a competitive advantage for attached cells over the cells still in suspension. The difference between the two types of cells is in prerequisites for surviving in the system: The suspended cells will be washed from the tank unless they divide at a rate dictated by its dilution rate. The biofilm cells can adjust to lower concentration of substrate by lowering their growth rate without punishment, their challenge, however, is to stay attached. Adhesion is a key trait that biofilm cells need to succeed in competition. It is accomplished using attachment factors and extracellular polymers. The production of extracellular matrix varies among strains and may result in eliminating the less producing cells from the systems by sloughing .The mathematical model of the chemostat devised by A part of the following analysis reiterates the structure of the theory of the chemostat and a reader not fully familiar with the topic is recommended to read more comprehensive texts such as by μmax), its saturation constant (Ks), and a biomass yield coefficient , all for a given limiting substrate.The dynamic budget of the chemostat model is determined by three constants: the bacterial strain’s maximum growth rate (μ) from the increment of the natural logarithm of the biomass (X) plotted against the increment of time (t).For probing the applicability of the chemostat model to biofilm growth rates, we need to presume that the growth kinetics of both planktonic and biofilm cells of a given microbial species in relation to their nutrients (substrates) is similar, despite the differences in gene expression between the two phenotypes . We willS): The maximum specific growth rate μmax and the saturation constant Ks . The growth rate at a given limiting nutrient concentration is approximated by the function:Equations (1) and (2) are sufficient to characterize growth in a closed system (static culture) with high initial nutrient concentrations to support it. In order to describe the growth of populations under changing nutrient conditions, we must use kinetic parameters to characterize the relationship between growth rates, and the availability of nutrients. Experimental data typically follow the hyperbolic Monod’s function , a saturX, in the chemostat, substrate-dependent growth μ(S) increases the biomass, while dilution at rate D (volumetric flow rate per volume of the container) decreases the biomass, leading to the rate equation:For the planktonic biomass, μ(S) – D = 0; both values have the same physical dimension (h-1)]. The steady state value of the planktonic biomass, , depends on how efficiently the substrate can be converted into cells, and this is typically characterized with a strain-specific empirical yield coefficient, Y: = Y (Sr), where  is the steady-state limiting substrate concentration and Sr is the in-flowing (“reservoir”) concentration of the limiting substrate.When they reach a steady-state concentration, the cells in the chemostat will have adjusted their substrate-controlled growth rate so that it matches the dilution rate  and after 15 min carefully rinsed with PBS for the same period in another petri dish. The surface treated with the fluorescent probe was then covered with a coverslip and the edges sealed with nail polish to prevent evaporation. The bottom side of the slide was cleaned with 95% ethanol.The slide was transferred to a confocal laser scanning microscope  and processed promptly. Scanning was performed in inverted position (coverslip at the bottom) with a 63× oil objective. Green fluorescence of Syto 9 was excited with a laser wavelength of 488 nm , while tImages of individual bacteria acquired with CLSM using DNA probes are often poorly defined for the purposes of quantification with “classical” image analysis, as the cell size nears the resolution limits of optical microscopy including CLSM . GrowingE. faecalis (ATCC 29212) for estimating initial rates of exponential growth at serial dilutions of its common culture medium, Trypticase Soy Broth (TSB). The initial concentrations of the medium were divided in half in each consecutive dilution. When we plotted the highest growth rate reached in the respective batch culture against dilutions of the original complex medium, the graph looked like the classical Monod’s saturation curve. Even though the original function had been designed for a single limiting substrate, it allowed estimating the concentration of sterile medium just sufficient to support population of bacteria growing at a given rate  values. This way of estimating the above parameters is not regarded as the most accurate . To apply the model, we used two constants, μmax (1.07 h-1) and Ks (0.019) derived from our E. faecalis static cultures. The third constant, the yield coefficient, was set as 40%, Y = 0.4 based on typical literature data for bacteria. Lastly, the fourth parameter  had to be dramatically adjusted upward  so that the model did not produce negative values at high settings of dilution rate, D ( (D), suspended cell biomass , and its production ( =  ⋅ D) covering the range of dilution rates equivalent to 0 – μmax. The arbitrary units of substrate and biomass could be easily converted to physical units, such as dry weight, carbon, etc.We exploited the potential of the  rate, D . The modmax and the planktonic cells are no longer able to grow quickly enough to compensate for the dilution rate; the level of available substrate rises, until it eventually reaches a physically constrained maximum of Sr; and the production of planktonic biomass increases to a maximum where the effects of increased dilution rate and decreased biomass are optimized, before falling as the planktonic biomass begins to drop.As the plot  indicateKs ) concentration of substrates in the inflow (rS ), we applied the same model at 1/16 of the full TSB medium (rS = 0.06). These conditions generated positive values of cell biomass in planktonic cells, (D), only forIn order to address the effect of low (near D < 0.8 ( (D) accompanying extremely low values of  (D) and , when the dilution rate (D) approached the highest value (0.8 h-1). However, comparing the same scale of  (D) concentrations in both graphs shows that that the calculated values were identical. In D that were implemented in the subsequent E. faecalis chemostat experiments. Corresponding values are marked in the graph on the limiting nutrient  (D) – green line . Three different dilution rates (D) were used to establish steady-state cultures: 0.09, 0.28, and 0.81 h-1. In each run, a sterile biofilm slide was immersed in the chemostat culture for 24 h. Before the slide was inserted in the culture, the flow was kept constant for a period of at least 9 residence times  for inoculation and sampling of biofilms. All additional operations were performed in a sterile biosafety cabinet. Back-flow contamination was prevented by a protective stream of sterile gas. The constancy and homogeneity of nutrient supply to the fermenter was improved by instant dispersion of medium by gas bubbles. A precision pump allowed control of a precise and stable dilution rate. Lastly, cell quantification was carried out by the combination of CLSM and 3-dimensional image analysis allowing adjustment of the algorithm to image condition.rS value used in the chemostat culture lies near the junction of the two branches of the curve. Therefore, the steady-state substrate values [ (D) ≤rS] were located on a steep part of the function, where small changes in nutrient concentration (S) seem to be correlated with large changes of specific growth rate (μ) and dilution rate . Thus we re-calculated the “starting” number of cells per cm2 from the one-day estimates for the assay reversing equation (2)  was much slower than the growth rate of plankton biomass .As opposed to the assay with the middle and highest dilution rate, in the run at  minimal . We prestion (2) . We can o assays . Based o-1h-1  with highest P had the lowest proportion of dead cells (0.8%). Therefore, the noticeable lack of significant difference between cell density at D = 0.28 h-1 and 0.81 h-1 could also be caused by a higher rate of attachment at D = 0.28 h-1 due to a higher production of plankton. It has been previously demonstrated , represents the biomass that can be “harvested” , its physical unit being g L-1h-1 . Using t0.81 h-1 . The midnstrated  that thenstrated .Apparently, the tools used in this study allowed only a rough comparison between the mathematical and physical model. Even over standardized time, the biofilm biomass increase was not a direct measure of its growth rate or production. On the other hand, we have not yet fully utilized the versatility of our chemostat design. If employed in multiple tanks, it would enable researchers to dissect the plankton/biofilm interplay owing to its capability to transfer independently the biofilm-covered slide and/or the entire tank contents (plankton) to a new, sterile tank in real time. It would allow us using markers  and third assay . As the exposure would be shortened roughly in proportion to the plankton population doubling time  than at faster dilution rates . E. faecalis developed a 21-fold denser biofilm in response to a ninefold increase in the chemostat dilution rate  after evaluating a series of batch cultures and applying the function of the saturation curve (S). According to the mathematical chemostat model (rS ). However, there is a restriction of the S value by the fact that S ≤rS at all times. We used this rule for setting the limits for calculation of variables in continuous culture fed by medium with limiting nutrient concentration. Using the Monod’s function, we estimated the highest  dilution rate still allowing a steady-state planktonic growth.We established the on curve . Then, wat model , this vaμmax, hence, the equations held only for a range of dilution rates (D) lower than its critical value. In our physical continuous culture, there was a trend for an increased rate of biofilm formation over that range. Since the calculated steady-state substrate concentration (S) was the only variable that increased with D at rates approaching its critical value, the rate of biofilm formation may be predominantly driven by the growth rate of attached cells due to increased ambient substrate concentration (S). Thus, the biofilm formation is controlled by the dilution rate (D) through the growth rate (μ) of the planktonic component. Higher planktonic growth rates resulted in increased biofilm growth while the planktonic biomass decreases.We fed the system with a concentration of nutrients supporting a growth rate lower than E. faecalis cultures predicted the maximum planktonic biomass production when D approached the critical value (either due to availability of substrate in our “nutrient restricted” chemostat or approaching limits of the strain’s capacity for growth – μmax). This could also provide insight into the contribution of the planktonic biomass to the biofilm buildup. The calculated “production” variable , D = 50 h-1), and D between 65 and 260 h-1). While, due to  plug flow in the flow cells, the actual dilution rates at any point would be even higher, the calculated values of D are far beyond the highest known growth rates for bacteria. Research of these systems underscores the understanding that in addition to the biological behavior of biofilms as a result of nutrient concentration and fluid flow, their physical properties determine their mechanical response to the flow  organisms including humans.Even though a certain range of shear stress values has been found beneficial for building the biofilm architecture, a wider scale of shear stress spans from high values causing detachment, i.e., destruction of biofilms to low values, where no influence, detrimental, or beneficial could be observed. Both the Although the two parameters are positively correlated with the rate of flow, their effective values depend on a set of additional conditions, such as a container shape and its dimensions, fluid viscosity, etc. Yet, we suggest that in the same hydraulic space, two separate boundary values of flow rate can be distinguished that control the biofilm development. Dilution rate appears to enhance the rate of biofilm formation up to a certain value, beyond which the acceleration of biofilm growth does not happen. Similarly, in the opposite way, the shear stress seems to restrict the biofilm formation down to a value, beyond which its influence is negligible.Thus, for biofilm microorganisms, there may be three zones of flow involvement in their biofilm building capability:(a)-1.Slow flow domain of positive dilution rate influence that we addressed in this study, limited to dilution rates slower than 4 h(b)Intermediate flow domain where dilution rate or shear stress fluctuations have virtually no effect.(c)-1 and shear stress alone selects the organisms capable of attachment to the substratum under the given conditions. Within this domain values around 0.01 Pa allow for biofilm buildup, whereas the shear stress 1 Pa and higher generally causes biofilm destruction.Fast flow domain where dilution rates are substantially higher than 4 hvice versa.The intermediate domain, by definition, represents the conditions where the biofilm buildup is maximized. Hence, those conditions should be met, where biofilms may be beneficial and avoided wherever biofilm formation  is undesirable. The behavior and interactions of biofilm organisms reported in the flow cell studies are derived from the fast-flow domain, thus they do not necessarily characterize the same organisms growing under conditions of the slow-flow domain, and For the proposed boundary between the slow and intermediate domain, the implication of results of the present study is that the borderline value of flow/dilution rate is further controlled by the concentration of limiting nutrients.E. faecalis can be achieved with a more data intensive study, our results demonstrated that in a defined range of the fluid exchange rate, the chemostat theory is a good predictor of the biofilm buildup.Experimental data describing the positive correlation between biofilm formation and dilution rate have been previously published referring to both clonal  and mixeML contributed to the conception and study design; acquisition, analysis, data interpretation, and application of the mathematical model; and performed the experiments and wrote the manuscript. DC contributed to the study materials, analysis tools and reagents, and wrote the manuscript. DM contributed to the application of the mathematical model and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.