The control of gene expression in eukaryotic cells occurs both transcriptionally and post-transcriptionally. Although many genes are now known to be regulated at the translational level, in general, the mechanisms are poorly understood. We have previously presented polysomal gradient and array-based evidence that translational control is widespread in a significant number of genes when yeast cells are exposed to a range of stresses. Here we have re-examined these gene sets, considering the role of UTR sequences in the translational responses of these genes using recent large-scale datasets which define 5' and 3' transcriptional ends for many yeast genes. In particular, we highlight the potential role of 5' UTRs and upstream open reading frames (uORFs).We show a highly significant enrichment in specific GO functional classes for genes that are translationally up- and down-regulated under given stresses (e.g. carbohydrate metabolism is up-regulated under amino acid starvation). Cross-referencing these data with the stress response data we show that translationally upregulated genes have longer 5' UTRs, consistent with their role in translational regulation. In the first genome-wide study of uORFs in a set of mapped 5' UTRs, we show that uORFs are rare, being statistically under-represented in UTR sequences. However, they have distinct compositional biases consistent with their putative role in translational control and are more common in genes which are apparently translationally up-regulated.These results demonstrate a central regulatory role for UTR sequences, and 5' UTRs in particular, highlighting the significant role of uORFs in post-transcriptional control in yeast. Yeast uORFs are more highly conserved than has been suggested, lending further weight to their significance as functional elements involved in gene regulation. It also suggests a more complex and novel mechanism of control, whereby uORFs permit genes to escape from a more general attenuation of translation under conditions of stress. However, since uORFs are relatively rare (only ~13% of yeast genes have them) there remain many unanswered questions as to how UTR elements can direct translational control of many hundreds of genes under stress. Much of the focus of post-genome science is now switching from simply cataloguing genes and gene products, to the greater challenge of understanding how they interact and regulate one another. The regulation of gene expression underpins the ability of cells to adapt to environments, deal with stresses, and progress through reproductive and cell cycle changes. Similarly, the increased complexity by which higher eukaryotes can control and manage the levels of their gene products has been suggested to underlie complexity in higher organisms – a compelling argument when the small difference between the numbers of genes in humans and the fly is considered. Yet a detailed understanding of how even the simplest cell controls its gene complement remains elusive, though great strides are being made in the field of systems biology. Much of this research has exploited advances in array-based technologies to characterise how gene expression levels change during the cell cycle or in response to stresses or environmental changes ,2, allowPrevious work from our laboratories has examined the effects that different stresses have on both the transcriptional and translational regulation of genes in the model organism, yeast ,20. Genevia a number of potential post-transcriptional mechanisms [via the nonsense mediated decay pathway (NMD) [YAP1 and YAP2 have been studied in detail, revealing different potential mechanisms for destabilising mRNA post-termination after ribosomal scanning [GCN4 appear to be conserved and are functionally operative.Here we examine the properties of these characterised yeast UTRs in the context of the stress-response microarray data sets ,20, lookchanisms ,6,24-26 ay (NMD) , and mecay (NMD) . Similarscanning ,28, and scanning , concludGCN4, YAP1/2). We show that uORF stop codon readthrough is likely and propose that uORF-mediated changes to reinitiation competency is a common mechanism to regulate gene expression under stress, consistent with that proposed for GCN4. Nevertheless, although uORFs provide some explanations, translational control is apparently both widespread and complex, and other mechanisms must be responsible for the post-transcriptional control of the majority of yeast genes which exploit it.Our results provide further evidence that not only does yeast exert a general, large scale control over its genes at the translational level, but that under stress this effect is controlled by UTR sequence elements. This is the first evidence that suggests UTR elements are specifically responsible for translational control of an experimentally-determined gene set which is differentially expressed under stress. Although rare, we propose that uORFs play a role in this regulation for a significant number of genes, not just restricted to a very limited subset  from the GCapping and 5'SAGE method were combined to create a 5' TSS superset. This resulted in a total of 53,673 individual TSS determinations for 4,149 yeast ORFs which contains considerable redundancy, with many repeated observations. To remove redundancy and maximize coverage, a single TSS was selected for each yeast ORF using a simple selection rule, taking the most abundant TSS position listed, or in the case of ambiguity, the longest UTR. All UTR introns were excised from relevant UTR sequences. The UTR intron co-ordinates were obtained from the Database . Figure Database . The tiln shuffles (where n = length of UTR) for each of the original UTRs to create 100 n different sequences and calculating a mean value for the number of uORFs. There is a significant increase in uORF density for shuffled UTRs compared to the real UTRs . This shows that there are fewer uORFs than would be expected by chance in sequences of identical composition, lending further weight to this observation. This suggests that the knowledge of the true TSS imposes constraints on the number of uORFs found in upstream sequences, and the properties of uORFs should only be considered when formally located within a mapped 5' UTR sequence – and not just in upstream sequences of a single fixed length.The selected 5'UTR sequences for all 4149 ORFs were submitted to a uORF identification procedure. Identified uORFs had to conform to the following rules: start at least 20 nt from the 5' mRNA end to take into account the 5'cap, be minimally 9 nt in length , and not extend more than 100 nt into the principal ORF. Generally, uORFs are relatively rare; a total of 1481 uORFs were identified in 554 UTRs, corresponding to 13% of the yeast ORFs in the dataset. Only 182 of these uORFs extended into the principal ORF. Figure It should also be noted that there is a correlation between 5' UTR length and the number of uORFs present . Despite this, a sizeable fraction of 5' UTR sequences have no uORFs at all, including some of those above 400 nt in length when at least three uORFs are expected by chance  decrease the reinitiation competence of ribosomes to zero . A totalUG-CAI(r), see Methods) has been proposed [UG-CAI(r) score for uORFs is 0.36, lower than the 0.48 for real ORFs. Figure 2 ratio of uORF:ORF AUG-CAI(r) for each uORF-ORF pair on the same transcript and plot these values in a frequency histogram. The true ORFs are generally better adapted since most values are less than zero (mean = -0.26). Interestingly, many genes possess at least one uORF that displays a more adapted start codon than the true ORF, leading to a log AUG-CAI ratio greater than zero. This is the case for 534 uORF start codons corresponding to 52% of all uORF-containing genes. This suggests that the majority of genes containing at least one uORF possess a uAUG at least as adapted to the translational machinery as the true ORF start. However, we found no systematic bias in this gene subset in terms of GO or other functional category (data not shown).A potential general mechanism for uORFs to affect translational efficiency is as a decoy from the principal ORF, causing premature termination, by triggering mRNA decay via NMD, or delaying the ribosomal complex from attaching and initiating translation at the correct AUG start codon. The potential for a uORF to be recognised by the translational machinery depends upon the sequence composition around the AUG, a concept known as leaky scanning where non-optimal sequences may be bypassed. For this reason the AUG codon adaption index  only considers the initiation step. Although this is deemed to be rate-limiting to translation [The Anslation ,32, morenslation . This menslation  and is anslation  and one GCN4 5' UTR, where uORF1 displays the "leaky" UUUxxU motif and positively influences the ability of the ribosome to resume scanning following termination [GCN4.A final feature to be implicated in translational control is the efficiency of translational release coupled to sequence signals immediately downstream of the stop codon ,36. Stopmination . When lo2O2 addition and 2 M H2O2 addition. For each case, changes in both transcriptional and translational expression were characterised using standard array-based technology coupled with polysomal gradient analysis [2O2 additions. The reduction in mapped TSS genes is a result of minor annotation differences between the papers and the Affymetrix GeneChips™.After examining the uORF dataset for features and properties that correlate with the TSS mapped UTRs, we cross-referenced this data with gene sets known to exhibit differential expression as a response to different cellular stresses, particularly at the translational level. The aim was to investigate the reasons for translational changes in gene regulation in terms of the TSS-mapped UTR set and uORFs in particular. This data covered four stress response datasets; amino acid starvation, butanol addition, 0.2 M Hanalysis ,20. In eanalysis ,20. CrosThe TSS filtered stress-response datasets were ranked according to the change in translational state. In the case of amino acid starvation and butanol addition this was characterised as the log ratio of the differential polysomal expression  observed from control (C) to stress (S) conditions (PS/MS:PC/MC). This characterises the translational "shift" in expression in stressed conditions by following the movement of each gene from polysomal to monosomal states. It should be stressed that the nature of these experiments characterise this as a relative change in translational control, since for stresses such as amino acid starvation, there is a general downshift in translation. Given these ratios measure relative changes, the values are not directly comparable between experiments and instead we selected gene lists representing the "extremes" in relative up/down regulation. Where possible, the top and bottom 100 gene log ratios that were above 0.9 and below -0.9, respectively, were used for subsequent analysis; this was possible with all but the butanol addition experiment where only 54 passed this filter (54 up and 54 down). In the subsequent analyses we refer to gene sets as "up" regulated (log ratio > 0.9) and "down" regulated (log ratio < -0.9) where strictly these values characterise differential relative expression states in the stress condition compared to the control state.2O2 are over-represented in stress adaptation and protection GO categories, and down regulated genes are enriched in ion transport and mitochondrial-related GO annotations. For 2 mM H2O2 addition, GO categories for RNA processing and ribosomal biogenesis are over-represented in the up-regulated genes set and oxidoreductase activity is over-represented in the down regulation set. The only stress without significant GO over-representation was butanol addition. This was likely due to the smaller number of genes that satisfied the log ratio cut-off values in this stress condition; butanol stress exerts a general translational effect on only a specific, small number of genes [We next examined whether any of these differentially regulated gene sets were enriched in specific biological functions. This followed on from our previous analyses where functional biases were observed using hand-curated functional assignments. Here, all of the 8 gene lists were submitted to GoStat  in orderof genes . Relatedof genes -43. For Our data suggest that a significant proportion of the yeast proteome appears to be, in part, regulated at the translational level, particularly under stress conditions. Given this observation, we wished to examine the transcript sequences of the genes involved to try and explain the differential regulation in terms of known control mechanisms. Namely, these were uORF-based attenuation of translation, known functional 5' UTR motifs, and the propensity of 5'UTRs to form secondary structures, all of which have been proposed as general translational control mechanisms ,44. Alth2O2 addition experiments. These data suggest that they also play an important, but different, role to 5'UTRs in regulating translation [2O2 addition with no significant length differences found for 5'UTR lengths, although there is a significant difference for 3'UTR lengths which are shorter in up-regulated genes , the C2 codon must code for the same amino acid  and C1 and C2 must be in-frame in the aligned species. The average number of "conserved" species was then calculated for phastCons scores in the range 0.99 to 1.We considered a dataset of 61 "ultra-conserved" uORFs in more detail which had phastCons score > 0.99 and were completely conserved in at least 1 other species  which has a highly conserved uORF at position -281 with respect to the start codon, and is down regulated in low peroxide concentrations.We also examined the correspondence between these 61 conserved uORFs and up/down regulation in our stress response gene sets. Unfortunately, only 4 of these genes appeared in the stress response data sets and it is therefore difficult to draw any general conclusions. However, one notable gene is the mitochondrial alcohol dehydrogenase UG CAI index), their overall codon bias (tAI), and importantly, their preferred stop codon contexts. The decreased translational adaption of uORFs has also been noted for those associated with NMD [In this study we have examined the role of upstream sequence elements in yeast UTRs, considering whether they play a concerted role in regulation of gene expression under stress conditions. Specifically, we considered available data sets of full length mapped yeast transcripts to define a superset of mapped transcriptional start sites (TSSs) representing over 70% of yeast protein coding genes. This is the most comprehensive survey of yeast uORFs using known 5' UTR sequence, and the first time this data has been considered in light of known translationally regulated genes under stress conditions ,18. The with NMD  and is cwith NMD . Intereswith NMD .via some motif or element contained within either the 5' or 3' UTR. However, here we demonstrate this for a variety of stresses for the first time, and importantly, demonstrate that this is a statistically significant trend. It offers a simple approach to select genes which are more likely to be translationally regulated on the basis of the UTR size and contents. Interestingly, the recent tiling array study of the yeast genome also defined 5' UTRs for a subset of the gene set [Regarding differential regulation at the translational level, a significant trend is observed across most of the stresses examined here. Genes which are observed to undergo relative translational up-regulation under stress have longer UTRs. This observation seems self-evident when considered at face value – namely, that any gene which can be regulated at the translational level must have a mechanism to support this, and this should be gene set  and thesgene set . This prgene set .Pinpointing the precise nature of the elements conferring translational regulatory properties is rather more challenging. Our data suggests that uORFs play a significant role mediating gene expression during stress responses, as they are over-represented in translationally up-regulated genes, particularly under 0.2 mM peroxide stress. However, this trend is not so striking as the UTR length correlation, and must in part be a result of this; longer UTRs are more likely to have a uORF.It should also be noted that the over-representation in up-regulated genes is difficult to reconcile with the "standard" uORF mechanism where they are generally expected to down-regulate gene expression at the translational level. This suggests that they are either acting in a novel way, that the complex "GCN4"-type mechanism is more widespread, or that other UTR elements than uORFs are responsible. Regardless, given their relative scarcity it is clear that there is still much to learn about UTR and uORF function.Other authors have focused on conservation as a strong predictor of functional significance ,28,46,47It has been reported that secondary structure in 5' UTRs mediates translational control of gene expression on a genomic scale in yeast . We re-eIn summary, the results presented here demonstrate convincing evidence that 5'UTR sequence has a major role to play in the regulation of gene expression, particularly under stress conditions and at the translational level. This effect appears to be widespread, affecting large numbers of different yeast genes under different conditions. Yeast has evolved a variety of mechanisms to effect these changes, including upstream open reading frames which are over-represented in translationally up-regulated genes.S. cerevisiae open reading frame (ORF) chromosomal co-ordinate information was obtained from the SGD website via:  and all S. cerevisiae sequences were obtained from All . The two gene sets were merged by ORF name selecting a single TSS for each ORF. For each ORF with multiple mapped TSSs, the occurrence of each TSS was counted. If the modal TSS for each ORF had a count of greater than two it was taken as the representative site for that gene, otherwise the longest 5'UTR was selected, the most distal from the ATG start codon. The 5'UTRs sequences were then taken from the ATG start codon up to the chosen TSS. A complete list of uORFs and 5' UTRs is given in Additional File Transcription start site (TSS) information was obtained from published studies, for 5' UTRs from Zhang and Dietrich , and fro using accession numbers E-MEXP-323 (amino acid starvation), E-MEXP-324 (butanol addition) and E-MEXP-526 (0.2 mM and 2 mM H2O2 additions). Translational up- and down-regulation under stress for each gene was calculated as previously described [2O2 stress experiments PS/MS:PC/MC was not used as the sole measure of translational state, due to oxidative stress inhibiting ribosomal transit [Microarray data pertaining to the stress response gene sets were obtained from ArrayExpress escribed ,20 usingUG-CAI is then calculated for each candidate AUG context by calculating the geometric mean of these look-up values (multiplying the 9 weights together and taking the 9th root). This mean value is then normalised, dividing by the maximum theoretical value, to give an AUG-CAI(r) value between 0 and 1.A previously published method was implemented  taking t). This estimates codon weights from gene copy numbers of the tRNA isoacceptors for a given codon, essentially reflecting the relative ease with which a gene can be translated based on the availability of the necessary tRNAs for that codon. A look-up table of codon adaptation weights were calculated for S. cerevisiae genes were and subsequently used to calculate a geometric mean for a given open reading frame as described in ref [The translation adaption (tAI) index implements a similar method, originally developed by dos Reis and colleagues ,34 (avaid in ref . using yeast SGD gene names of the respective gene sets as input.Over-representation statistics of Gene Ontology categories within gene-sets were performed using GOStat  at  usinUpstream open reading frames (uORFs) were identified in 5'UTRs and up to 100 nucleotides into the real open reading frame (ORF). We defined uORFs from the relative nucleotide positions and coding frame of all start and stop codons with respect to the ATG start of the true ORF. All in-frame uORFs were collected from this set, taking the longer of any subsets that contained that same stop codon position. All identified uORFs were discarded if they were either less than three codons in length (including the stop codon) or within 20 nt of the 5'end, as the latter are not considered to have an effect on translation .Predicted secondary structure with 5'UTR sequences was calculated using the Randfold method . All 5'US. cerevisiae, using the PhastCons [S. cerevisiae genomic alignments against S. paradoxus, S. Mikitae, S. kudriavzevii, S. bayanus, S. castelli and S. kluyveri where obtained using the Table Browser at UCSC [S. cerevisiae. The average phastCons scores were calculated for all 3,877 uORFs across the all positions from start to stop, inclusive. An average "background" phastCons score was also calculated for each uORF from the 5'UTR sequence containing it, and the uORF scores converted to Z-scores. This provides an additional measure of whether a uORF is conserved over and above the level of conservation observed generally in each UTR, In addition, multiZ genomic alignment fragments were also obtained from the UCSC browser for 90 highly conserved uORFs with a mean phastCons scores >0.99. The aligned species sequences were analysed for in-frame start and stop codons that corresponded to those of the S. cerevisiae uORFs.Conservation of individual base positions in each UTR was calculated using related fungal genomic sequences aligned to hastCons  algorith at UCSC , and indSJH, MA, GP, CG conceived and designed the study, with help from all authors. CL carried out all the computational analyses on UTRs, gene function and uORFs, under the guidance of SJH with the following exceptions; RP carried out an initial analysis on upstreamAUGs/uORFs in fixed upstream segments, JNS carried out the processing of microarray data and generation of candidate gene lists. JS carried out all the experimental microarray work. CL and SJH wrote initial drafts of the manuscript, all authors offered comments and have read and approved the final manuscript, edited by SJH.Comparison of UTR lengths in two datasets. Comparison of 5' and 3' UTR lengths from the dataset generated for this study with that of David et al's tiling arrat study [at study .Click here for fileObserved and expected frequencies of uORFs in mapped 5' UTRs. This file contains a figure showing the distribution of observed and expected frequencies of uORFs in 5'UTRsClick here for fileObserved reading frame distribution of uORFs in mapped 5' UTR. Figure showing the relative frequency of uORF open reading frames with respect to the main ORF reading frame. For those uORFs wholly contained within the 5' UTR there is clearly no bias towards any frame suggesting that there are no likely missannotations of the true ORF start or independent ORFs.Click here for fileSummary statistics for long uORFs greater than 35 amino acids in length. This Excel format file file contains statistics for individual uORFs (one per line) whose length exceeds 35 amino acids. For each uORF, the ORF, ORF length, 5' UTR length, frame and start position are indicated, as well as the total number of uORFs contained in the given 5' UTR.Click here for fileGoStat over-representation statistics for translationally controlled gene subsets under H2O2 stress. This Figure shows similar over-representation statistics as shown in Figure Click here for fileUTR length distributions for different gene sets under stress conditions using tiling array definitions of TSS. Length distributions equivalent to those shown in Figure Click here for fileFull Statistics for conserved 61 uORF sub set. This spreadsheet provides the full data for conserved uORFs, listing the parent gene, position of uORF in the UTR, PhastCons score, conservation Z-score, and number of aligned species in which a uORF is directly conserved.Click here for fileAnalyses of 5' UTR secondary structures using RanFold. Tabulated statistics of Randfold calculations are shown for the 5 most likely secondary structure containing UTRs along with potential secondary structure diagrams.Click here for fileComplete statistics for all uORFs and UTRs used in the study. This details all the uORFs from all UTRs used in this study, providing locations, parent genes, and sequence information, as well as the details on all the 5' UTRs used, and their start sites with respect to each named yeast gene.Click here for file


The feasibility of evaluating an objective grading of cervical intraneoplasia lesions (CIN) is attempted using an automatic computerized system able to measure several valuable parameters with special reference to epithelium differentiation.4 groups of 10 images each were selected at random from 68 consensus images coming from 80 archival cervical biopsies, normal (n = 10), CIN 1 (n = 10), CIN 2 (n = 10), CIN 3 (n = 10). Representative images of lesions were captured from the microscopic slides and were analyzed using mathematical morphology, with special reference toVoronoï tessellation and Delaunay triangulation. Epithelium surface, nuclear and cytoplasm area, triangle edge and area, total and upper nuclear index were precisely measured in each lesion, and discriminant coefficients were calculated therewith. A dilation/erosion coefficient was automatically defined using triangle edge length and nuclear radius in order to measure the epithelium ratio of differentiation. A histogram ratio was also automatically established between total nuclei and upper nuclei on top of differentiated epithelium. With the latter two ratios added to the nucleo-cytoplasmic ratio, a cervical score able to classify CIN is proposed.There is a quasi-linear increase of mean cervical score values between normal epithelium and CIN 3: (27) for normal epithelium, (51) for CIN 1, (78) for CIN 2 and (100) for CIN 3, with significant differences (P < 0.05).Our results highlight the possibility of applying a cervical score for the automatic grading of CIN lesions and thereby assisting the pathologist for improvement of grading. The automatic measure of epithelium differentiation ratio appears to be a new interesting parameter in computerized image analysis of cervical lesions. Cervical intraepithelial neoplasia (CIN) has aroused a lot of interest with regard to classification. The world-wide classification, proposed by Richart , is baseSeveral attempts have already been made to automatically and objectively grade CIN using image analysis. Based on mathematical morphology Keenan et al.  focused Archival cervical biopsies came from 80 patients (age 25 - 43 years) undergoing punch biopsy during colposcopy, after detection of cytological abnormalities in cervical smear. Cervical tissues, which were treated with formaldehyde-acetic acid fixative and embedded in paraffin, were cut into 2.5-μm serial sections. Sections were deparaffined and stained automatically  with a modified Weigert stain [MJ], listed on table 2 real surface on the microscopic slide, so that 1 pixel corresponded to 0.22 × 10-6 μm2.Microscopic slides were observed with a Zeiss Axioskop microscope  with a ×25 magnification (Zeiss Plan-Neofluar 25/0.8 lens) under standard illumination (same voltage and diaphragm aperture). For each biopsy, at least 1 image was recorded. In some samples, where different grades of lesion were observed on the same biopsy, a microscopic field of each grade was recorded. The images were recorded with a black and white CCD camera , they were digitized  by an electronic video card  and recorded in bitmap format. Each image had a 286,976 pixel area, corresponding to a 0.066 mmA total of 86 microscopic fields, corresponding to normal and cervical lesions of different grades, were submitted to 4 gynaeco-pathologists for classification, one of them being the first author [MJ]. Images were submitted to the 4 pathologists independently, and the same images were viewed again by each one 3 months later, independently of the first vision. The pathologists were asked to grade the images in 4 groups . An intra- and inter-observer consensus was established with 68 images, among which 10 images were selected at random in each group.Micromorph 1.4 version image analysis system  was used for image processing. This software was developed for the use of mathematical morphology and contains nearly all the tools of this image analysis procedure . Some feEach gray image was manually processed with Photoshop 5.5 in order to select the original epithelial tissue, thereby eliminating artifacts and stroma. The basal lamina and the surface of the epithelium were delineated, before running image analysis in the region of interest (ROI), delimited in that way. The cleared image was then treated by the image analysis system for segmentation of nuclei. After increasing light and contrast, the image was submitted to thresholding, followed by proper segmentation. A 1 pixel border was added to the final image and the non-tissue areas were filled by superposition of the original image with the segmented one. With such a procedure, the nuclei cutting the image edge were suppressed, allowing a precise measurement of ROI and average nuclear areas.Once the segmentation was properly achieved, an automatic algorithm provided several measurement parameters. A Voronoi tessellation was done delimiting the zone of influence on nuclei. This procedure gave an approximation of the cytoplasmic area, considering that squamous epithelium is made of joint cells. The geodesic center of each zone of influence was then used to proceed towards a Delaunay triangulation, with subsequent retrieval of the mean edge length of the triangles. The procedure is represented on figure The epithelial differentiation, representative of the CIN definition, has been introduced by Richart  who arbiThe dilation/erosion coefficient was established from the mean value of the triangle edge and the mean nuclear area measured in the 4 groups and defined by dividing the mean triangle edge by 1/4th of the mean radius of the nucleus, which was achieved automatically by the vision machine. Some other mathematical tools, such as hole closing and cleaning, were applied in order to afford a properly designed image for measurements. The final result is represented on figure By superimposition of the binary segmented nuclei on the original grey level image and inversion of the resulting image, it was possible to measure the histogram corresponding to all nuclei. The same procedure was applied to the non-differentiated epithelium giving the nuclei histogram value in the upper part of the epithelium. This is represented on figure The following parameters were automatically measured by the vision machine on the resulting images:1 - area of the selected epithelium (A)2 - mean area of the nuclei (N)3 - mean area of the zone of influence (V)4 - epithelial differentiation (D)5 - total nuclei histogram (Hst)6 - upper nuclei histogram (HstU).From these 6 parameters, 3 ratios were inferred:- nucleo-cytoplasmic ratio (N/V) = ratio of nucleus area to cytoplasmic area;- epithelial differentiation ratio (DR) = ratio of differentiated epithelium area (D) to total epithelium area (A);- upper nuclei histogram ratio (HstR) = ratio of upper nuclei histogram (HstU) to total nuclei histogram (Hst).The 3 discriminant ratios were used to derive a mathematical score, in order to get a near to 100 value for high grade lesions (CIN 3).The same calculation was applied for all groups of images.vs CIN1, CIN 1 vs CIN 2, CIN 2 vs CIN 3) have been made by t-test, with Welch correction when unequal variance was assumed. The levels of significance are P < 0.05. GraphPad Prism 5.02  was the statistical software used.Distributions have been found normal, except for 2 out of 40 data . Therefore comparisons between 2 successive groups  decreases without statistical significance;- mean nucleus area (N) increases significantly from normal to CIN 1 and remains further at the same level for high grade lesions;- mean cytoplasm area (V) decreases from normal up to CIN 3;- mean epithelium differentiation (D) increases significantly from normal up to CIN 3;- total nucleus histogram (Hst) increases significantly from normal up to CIN 3;- upper nucleus histogram (Hst U) increases significantly from normal to CIN 1, then without significant difference between CIN1 and CIN 2, and with a significant decrease between CIN 2 and CIN 3 due to a drastic diminution of non differentiated epithelium in CIN 3 .Most of these results confirm the general impression when observing a cervical biopsy under the microscope. Of interest are the ratios calculated from data on table - nucleo-cytoplasmic ratio (N/V) increases significantly from normal up to CIN 3;- epithelium differentiation ratio (DR) increases significantly from normal up to CIN 3, and this means that the mathematical procedure is in accordance with pathology. Measurements have been made on the nuclei and did not take into account the cytoplasm, thereby resulting into ratios which do not reach 100%, as expected. This is due to the fact that epithelium differentiation was measured with nuclei area and did not take into account cytoplasm area, although present, but in a small amount on the upper side of the basal part of epithelium;- upper nuclei histogram ratio (Hst R) decreases with increasing epithelium differentiation from normal up to CIN 1, with a drastic fall for CIN 3 due to the low number of nuclei in the upper part.Finally, the cervical score, which represents a calculation from the preceding ratios, displays a quasi-linear and significant progression from normal up to CIN 3 lesions with no overlapping between the 4 groups.The 3 preceding ratios and the cervical score are represented on figure A vision machine is able to measure a nearly unlimited number of parameters when analyzing an image, but we completely agree with Keenan  who poinThe purpose of this work was to limit the number of parameters by retaining only those values which are as close as possible to the parameters taken into account when a pathologist analyses and grades a cervical biopsy. With a specific algorithm we were able to record automatically 6 reliable parameters and to establish a cervical score for grading objectively a cervical biopsy. We took into account the original classification proposed by Richart  which isImage captures were done with a ×25 objective which suits perfectly to our optical system, allowing maximum vision of total epithelium height, especially in the normal group. The application of contrast staining appeared to be a valuable procedure in the segmentation process. Segmentation is a major problem in image analysis, as the nuclei are often superimposed, particularly in high grade lesions and cancer. Up to now there is no valid solution, because the manually segmenting of clusters is time consuming and subject to errors, particularly in high grade lesions and cancer. After several attempts to achieve a fully automatic process, it appeared that a manual or semi-automatic procedure was necessary to reach better segmentation before the automatic measurement of the pertinent parameters. This is a general problem in image analysis and represents an important issue for the standardization of image quality, as pointed out by Kayser ,15.Voronoï tessellation is based on nuclei segmentation and determines on each nucleus the zone of influence, which is a mathematical morphology item that suits well with biological observation, because the nucleus exerts an "influence" on the surrounding cytoplasm. Therefore, we can relate the zone of influence area to cytoplasm area. As proposed by Serra , the Delaunay triangulation was then drawn from the geodesic center of each zone of influence, this procedure allowing a more accurate triangulation because it takes into account the nearest neighbors and eliminates false relation of 2 cells being far away from each other, when there is convexity in the epithelium for example.The Delaunay triangulation measures the mean triangle edge length which was used to define the dilation/erosion coefficient. By combining this value with the mean value of nuclei area it became possible to automatically measure differentiated epithelium. An alternative for the measurement the dilation/erosion coefficient consist in the application of the minimum spanning tree technique giving rise to the minimum distance between the nuclei's nearest neighbors . A speciper se, the intensity of the nucleic staining, which is taken into account in routine microscopy, can bring up an interesting data. When establishing the histogram of all nuclei on one end and the upper nuclei on the other end, the histogram ratio gives an adequate appreciation of nucleic chromaticism, independent of specimen thickness and staining intensity.Cell maturation in the upper part of the differentiated epithelium represents an important factor in the grading, especially in cases of koïlocytosis. Our staining procedure is based on iron-haematoxylin treatment. By increasing the nucleus contrast with our modified technique, although haematoxylin is not stochiometric Nucleo-cytoplasmic ratio is an important data from the spatial point of view. In normal epithelium near the surface, the cytoplasm is very large and the nucleus becomes smaller during normal maturation. As the histological section is 2.5 μm thick, only a few nuclei are present with their surrounding cytoplasm, leading to a false estimation of nucleo-cytoplasmic ratios. Data are nevertheless comparable because they are objectively done by the machine and are reproducible.We did not consider mitoses which is an important parameter in the classification. This can be achieved when images are captured with a ×1000 magnification ,18, but The use of mathematical morphology makes our algorithm transposable to any system based on that image analysis system, after proper calibration of the optical system specific to each user. The staining procedure, the microscopic field illumination and the automatic measures with our system were standard and gave reproducible results, these parameters being considered as very important .The cervical score we proposed presents a quasi-linear progression from normal to high grade lesions and fits closely to Richart's classification. The problem however is to determine an adequate cut-off value for the decision to treat or not to treat a patient.per se, the selection of the optical field to be analyzed remaining operator dependent. Nevertheless it can help the pathologist to accurately establish the most valuable diagnosis. Although some artefacts may occur, precision seems fairly good, which is hardly the case when grading is done by routine observation on microscopic slides, as inter and intra variability is a limiting factor. The present investigation should be considered as a contribution to total morphologic analysis of any cervical lesion by measuring objective parameters, with special reference to epithelial differentiation.Though it may not always be comfortable to accept that a machine can be able to grade a CIN, we hope that our contribution to the proposal for a reliable scoring will help pathologists to deliver objective diagnoses, further more in experimental studies where objectivity and quantification are requested. So far, we consider that the vision machine is not able to produce a diagnosis Using Voronoï tessellation and Delaunay triangulation on an image analysis system we were able to demonstrate that it is possible to grade automatically and precisely a cervical intraneoplasia lesion. This was achieved using a modified Weigert dye enhancing nuclear contrast and we were able to propose a cervical scoring. Our contribution to this difficult approach, compared to previous published results, was to add the automatic measurement of the pathologic epithelium differentiation. Our algorithms developed for this specific application can be adapted to any vision machine using mathematical morphology. More information can be made available by immuno-histochemistry combined with image analysis. We are looking forward pursuing further developments to this preliminary work in order to help pathologists to become more and more accurate and objective in their grading of cervical intraneoplasia.The authors declare that they have no competing interests.MJ initialized the study and drafted the paper. RA was involved in developping the specific algorithms and LD was involved in the statistical aspect and reviewing the manuscript. All authors read and approved the final manuscript.


Persistent pulmonary hypertension of the newborn (PPHN) is a severe condition that determines a profound brain hypoxia. Inhaled nitric oxide was approved for the treatment of PPHN since the end of the 1990s. The debate upon the long term outcome of these children is still open. Our aim was to investigate the incidence of minor long-term neurodevelopmental problems in a cohort of children affected by severe PPHN.All neonates with severe PPHN treated with inhaled nitric oxide in our facility between 01.01.02 and 31.12.07 were seen in a follow up visit and evaluated with a neurodevelopmental scale, according to their age at the time of observation.in the study period 31 children were diagnosed with severe PPHN. 29 survived. 27 accepted to come for follow-up. Mean age: 41 months (range 12 - 70 months).26% of the evaluated children had some behavioural problems, while 22% had some language disturbances.This is the first neurodevelopmental follow-up of neonates with PPHN in which children older than 36 months have been evaluated.There is an unexpected high incidence of minor neurological deficits, mainly regarding the fields of language and behaviour. These deficits seem to be related to the severity of illness rather than to the treatment. Language and behaviour are considered "higher functions" in humans and their integrity can be better defined in older children. Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome that results from failure of the pulmonary circulation to adapt to extrauterine life-3.There is a persistently elevated pulmonary vascular resistance, associated with a right-to-left shunting through the foramen ovale and/or ductus arteriosus.Although PPHN is commonly associated with a parenchymal lung disease, about 20% of the cases are classified as idiopathic,5.Hypoxia is one of the major causes of PPHN.The clinical presentation of PPHN might be the result of different pathophysiological processes, but the diagnosis is anyway based on three fundamental features:- severe hypoxia- right-to-left shunting, proven by echocardiography- absence of a cyanotic congenital heart disease2 exchanges.PPHN is to be suspected in case of very poor oxygenation and particularly when hypoxia is relatively too deep compared to the severity of pulmonary parenchymal disease or to the effectiveness of COThe severe instability of these patients make them susceptible to respiratory and cardiac complications but also to neurological sequelae, considering the effects of pressure impairment on cerebral blood flow and the immaturity and fragility of the newborn's central nervous system.To our knowledge, relatively few studies have tried to evaluate the long-term neurodevelopmental outcome of newborns affected by PPHN, often leading to discordant results. Previous works reported a 10% to 20% rate of long-term neurodevelopmental sequelae in infants with PPHN-9.We know that neonates who needed prolonged mechanical ventilation are at risk for minor neurological deficits: hypo-oxygenation as a matter of fact plays a relevant role in the pathogenesis of brain damage, especially in terms of cortical organization, associative cortical areas or neurotransmitters system. This condition affects the normal development of intellectual skills and behaviour, so that cognitive outcome could be particularly compromised in case of severe neonatal hypoxia.Our study had two main objectives:1. To carry out a long-term neurodevelopmental follow-up of the children with PPHN treated with nitric oxide in the Neonatology Division of Udine  over a period of 6 years.2. To revise retrospectively these cases considering enrolment criteria to treatment with nitric oxide (NO), nitric oxide dosage, clinical response and short-term outcome.st of January 2002 to the 31st of December 2007, 31 neonates were admitted in the Neonatal Intensive Care Unit of The S. Maria della Misericordia Hospital in Udine with a diagnosis of severe PPHN confirmed by echocardiographic findings and were treated with inhaled nitric oxide (iNO).From the 1We retrospectively selected from the database all children with a proven diagnosis of PPHN with echocardiographic evidence of pulmonary hypertension who had been treated with iNO. Patients who did not undergo echocardiography were excluded, as were those who did not receive iNO. The excluded patients are not reported in this study.Incidence of PPHN was about 1:1000 born alive.ad hoc database was created to retrospectively collect all these data and analyze them. We also analyzed ventilation parameters such as mean airways pressure (MAP), inspired oxygen fraction (FiO2), haemogas-analysis values , oxygenation index (OI), alveolar-arterial oxygen gradient (AaDO2) and arterial blood pressure values.Data recorded from the neonatal period included sex, gestational age, weight, APGAR score, admitting diagnosis. An All these parameters were measured before starting iNO therapy, at any significant variation of mechanical ventilation or any relevant change in clinical conditions, until discontinuation of iNO and/or resolution of PPHN.All enrolled children presented severe hypoxic respiratory failure and were treated with conventional mechanical ventilation or high frequency oscillatory ventilation .Inotropic agents (dopamine and/or dobutamine) were administered for blood pressure support and sedation with fentanyl was performed too-12.Patients were eligible for iNO therapy if they had PPHN clinical features and there was echocardiographic evidence of indirect signs of pulmonary hypertension.In our facility, we usually perform cardiac ultrasound before starting iNO because we can perform it by ourselves in the NICU. In the 31 considered patients, cardiac ultrasound was always performed before starting iNO.An initial dose of 20 ppm iNO was used, according to previous Italian and international trials,13-21.Responses to iNO were classified into two groups: early response was defined as a 25% OI reduction occurring within 6 hours after iNO initiation; late response as OI reduction within 6-24 hours. Infants who did not show a 25% OI reduction after 24 hours were considered unresponsive-17,22.All infants who survived were offered participation in our study and they were invited to come for a clinical and neurodevelopmental evaluation.Informed consent was given by parents of each infant.Some of the inborn patients had also been seen before in our conventional follow-up programme (every third month).Outborn patients were supposed to have been seen in the centre closest to their home, but some of them had had no follow-up at all.In the follow-up visit, we interviewed parents on growth and development, intercurrent illnesses, respiratory problems, rehospitalization.Growth parameters were measured: weight, height and cranial circumference were plotted on the reference curves. A complNeurodevelopmental examination was based on Bayley Scales of Infant Development for chilOlder patients were tested with Wechsler Preschool and Primary Scale of Intelligence WPPSI.The final scores are classified as extremely low if they are < 70; borderline between 70 and 89; in the average range between 90 and 109; higher scores are considered superior or very superior (> 130). One child with cerebral palsy was tested using the Griffith score.Both for Bayley Scales and WPPSI, the scores of the mental/verbal area (MDI) and those concerning the performance/motor area (PDI) were considered separately, so that total score was not falsely decreased by a specific deficit in a single area.Data analysis was performed in the Epidemiology Institute of the University of Udine. Comparison analyses were carried out with the SAS software.Frequencies of the neurodevelopmental scores, underlying diseases, gestational age and of any ventilation parameter were calculated.Some variables have been categorized.2) analysis and Fisher test.Statistical significance of the differences between groups was evaluated through chi-square , pneumothorax (5/31), severe asphyxia (5/31), respiratory distress syndrome (3/31), sepsis (3/31), adenomatoid cystic pulmonary malformation (2/31), placental disruption (2/31), idiopathic PPHN (4/31).The minor cardiovascular defects that we found were not directly responsible for the clinical manifestations of PPHN. They included only small defects of interventricular and interatrial septum and irrelevant patent ductus arteriosus. None of these was hemodinamically significant. In these cases PPHN was considered idiopathic, given that there was not another major cause.Major congenital cyanotic heart diseases were excluded from the study.We had no cases of diaphragmatic hernia.Male sex was sharply prevalent: 25/31 were male (80.6%); 6/31 female (19.4%).Median gestational age was 38 weeks (range 26 - 41 weeks).The range of gestational age seems to be wide, but really premature children were very few; actually only 5 children were born at less than 34 weeks. One was 26 weeks and one was 27 weeks; the other three were older than 30 weeks. None of them showed any major complication of prematurity  that could be responsible for the long-term outcome. All of them had negative cerebral ultrasound and none of them had any major neurological deficit.Median weight at birth was 3.200 gr (range 1.100 - 4.640 gr) and 29 out of 31 newborns had an adequate for gestational age weight (AGA); the two remaining children were considered large for gestational age with weight > 97° percentile.APGAR score was < 6 at 5 minutes of life in 6 cases.All infants were treated with iNO. Nitric oxide administration was started at the post-natal mean age of 1.4 days, according to literature,16,27,2816 patients (51.6%) of the studying population received conventional mechanical ventilation; the remaining 15/31 received high frequency oscillatory ventilation (HFOV). In our facility, we usually started HFOV in the patients in whom PPHN was associated to a particularly severe parenchymal pulmonary disease.2O (range 5 - 23).Average initial MAP was 11.8 cmH2 was 100% in all cases and we attempted optimization of ventilation before starting iNO treatment. Mean oxygenation index was 28.36, while median OI was 27 (range 76.5 - 5).FiO15 infants (48.4%) showed an early response to treatment, while 14 (45.2%) had a late response. 2 neonates (6.4%) were unresponsive and their oxygenation index worsened even after starting inhaled nitric oxide.No statistical significant differences were found between early and late responders in term of initial OI, type of ventilation, duration of iNO treatment, iNO dosage or any other recorded parameter.Response was not even statistically dependent on gestational age or underlying pathology. Table 2 infants died before discharge (6.4%), but their death was not directly related to PPHN itself: one child was severely compromised by septic shock; the other one was affected by congenital haemochromatosis and presented a multi-organ failure confirmed by post-mortem examination.29 children survived, yet only 27 accepted to come for follow-up.At the follow up visit, auxologic parameters were included between 10° and 90° percentile for all children.No significant respiratory pathologies were found.Mean age at the time of the neurodevelopmental evaluation was 41 months (range 12 - 70). This is the first follow-up study of children with PPHN in children older than 36 months.The wide age range at the time of observation is due to the long period considered for the study (from 01.01.02 to 31.12.07). Anyway, only nine patients were younger than 36 months at the time of neurodevelopmental examination, while 18 children out of 31 were 36 months or older.14/27 children (51.8%) have been evaluated with the Bayley Scales and theiWe found a pathologic score (< 70) in the mental/verbal area in 5 out of 25 cases (20%).The incidence of a performance/motor score < 70 is 8%. Table Despite the numeric score, in the complete sample of 27, 6 children turned out to have deficits in the linguistic area (22%).7 (26%) patients, moreover, showed "minor" behavioural problems even tough they did not have a pathologic MDI score.Electrophysiological tests have been performed in 17 out of 25 of our children, but none of them showed significant abnormalities.All children underwent cerebral ultrasound in the neonatal period. It turned out negative in 22 cases; mild persistent flare was reported in 7 cases. Only two children had IVH of III° or IV° grade: one of these is the one with cerebral palsy (CP); the other is one of the two who died before discharge.7 cases underwent a cerebral magnetic resonance, but it resulted negative except for the child with CP.Statistical analysis could not prove any significant association between the neurodevelopmental score and the underlying pathology determining PPHN. The neurodevelopmental outcome does not statistically correlate with the early or late response to iNO treatment.In the present medical setting, treating pathologies and healing the single patient is not any longer enough.We need to verify the effectiveness of our practice.The result to be evaluated is not only the short-term outcome and the survival of our patients, but also the long-term impact on population and quality of life.Actually, we found an excellent short-term outcome.The raw mortality rate is 6.4% (2/31). It seems to be lower than what presented in literature,32-39, yIn the current clinical practice in our NICU, the criteria to start iNO treatment are based on clinical conditions of the patients with severe hypoxemia, unresponsive to mechanical ventilation with inspired oxygen fraction of 100% and with echocardiographic evidence of PPHN.OI was calculated ex-post just for this study. So we found that iNO was given to children with a very wide range of OI (5 - 76.5) and 17 cases had an OI lower than 25, which is the accepted cut-off for iNO initiation,16,17,40Even tough an earlier initiation of iNO is not associated with lower mortality, it might be related to a minor rate of illness progression to more severe stages,39.As we consider long-term outcome, the great majority of works on children with PPHN try to demonstrate whether there is a difference in the incidence of neurological deficits in those treated with iNO as opposed to those who received other therapies-38,40-43The Nitric Oxide Study Group in 2000 published data that didMinor disabilities were not evaluated in either of these studies.In his follow-up, Lipkin analyzedHaemodynamic instability during PPHN might cause pressure and metabolic impairment that may alter the auto-regulation of the cerebral blood flow of the newborn,44.In our study, we start from the hypothesis that PPHN is a severe pathology and infants who underwent prolonged invasive ventilation should have a neurodevelopmental follow-up aiming especially at the search for minor deficits.15 of the 27 children (56%) who accepted to come to our follow-up had not had any previous evaluation of the psychomotor development. This might be related to the good general conditions of these patients after discharge from NICU, but also to the fact that many of them (67.7%) came from areas that refer to other hospitals.In literature, the reported incidence of neurodevelopmental deficits in infants with PPHN is 10% to 20%-8. We foThe incidence of language disturbances seems to be very high and they are more evident in the group of children who were tested with the WPPSI test.Published data are based almost exclusively on the Bayley Scales performed on children with a mean age of 20-24 months.At this age language evaluation is limited and a language delay or alteration might be evident only later on. Our study is the first neurodevelopmental follow-up for infants with PPHN, in which children older than 36 months have been evaluated. We found both expressive and receptive linguistic deficits, that did not seem to be related to any auditory impairment.Neuroradiological examination did not show any major abnormality of the cerebral parenchyma, and cerebral ultrasound that had been performed in all children in the neonatal period was normal in the children who had language or behavioural problems at follow up visits.In human beings, language is a higher function and not just a motor or sensitive function regulated by a single functional brain area.Studies on post-traumatic aphasia demonstrate that there is no direct relation between lesions in the classical linguistic areas (Broca and Wernike) and the development of the corresponding forms of aphasia.The ability to comprehend and produce language is based on an interwoven constellation of skills influenced by exogenous factors  and by endogenous factors such as the interactions among neural systems. The anatomo-functional subdivision of cortical areas then does not follow established developmental trajectories, but is driven by the sensimotor experience of the individual.PPHN can cause a diffuse and multifocal hypoxic brain damage that does not determine the development of major neurological deficits such as cerebral palsy, but it might involve and compromise the connection network among different cortical areas.This kind of damage may involve more subtle clinical features that become evident in older children who are required more elaborated performances, such as linguistic expression or the maintenance of attention while performing a specific task.A correct follow-up programme is then important to allow early identification of these problems. Moreover, it might prevent a negative evolution by starting a specific treatment as soon as possible.The therapeutic alliance with the family established in the follow-up programme may also be helpful to limit or correct behavioural disturbances that were proven to be relevant in these children.Follow-up must continue to pre-school and school age, so that specific deficits regarding language, calculation skills and/or abstract thinking may be found out.We already knew that PPHN is a very severe condition associated with a high mortality rate, but also with the risk of long-term neurodevelopmental sequelae. We also knew that inhaled nitric oxide is an effective treatment to reduce mortality and the need for ECMO.Thanks to the results achieved through this study, we can say that it might be reasonable to start iNO therapy on the base of clinical criteria even in children with OI < 25. This attitude might not reduce total mortality rate, but it could be associated with a lower rate of illness' severity progression and thus with a better long-term outcome.Anyway, specific trials to evaluate this topic are not yet available.Regarding long-term outcome:- we did not find a significant incidence of respiratory problems;- neurodevelopmental tests showed a high prevalence of minor deficits, especially in the linguistic area. These children are asymptomatic for major neurologic disabilities and so they can be lost in normal follow-up programmes;- children with PPHN are exposed to hypoxemia and therefore they need to be monitored in a strict follow-up programme for psychomotor development, possibly up to school age.Having a better knowledge of the long-term neurodevelopmental deficits in children who were admitted in NICU, we get an improved awareness on the effects of pathologies and their treatment.Up to now, more researches are certainly needed to better comprehend the neuro-physiological alterations underlying the minor deficits of these children.PPHN: Persistent pulmonary hypertension of the newborn; NO (iNO): Nitric oxide ; RDS: Respiratory distress syndrome; PaO2: Arterial partial oxygen pressure; PaCO2: Arterial partial CO2 pressure; SatO2: arterial oxygen saturation; HFOV: High frequency oscillatory ventilation; FiO2: Inspired oxygen fraction; MAP: Mean airway pressure; ECMO: Extracorporeal membrane oxygenation; AaDO2: Alveolar-arterial oxygen gradient; OI: Oxygenation index; NINOS: Neonatal inhaled nitric oxide study group; AGA: Adequate for gestational age; MDI: Motor developmental index; PDI: Performance developmental index; NICU: Neonatal intensive care unit.The authors declare that they have no competing interests.Not required. This is a retrospective study. All neonates had been treated according to the therapeutic protocol for PPHN already approved by our Ethical Committee. Informed consent for neurodevelopmental evaluation was given by parents of each child.AB, AJ, RF and FM designed the study protocol; AB, AJ and RF were directly involved in the clinical management of the patients. AB, AJ and RF carried out the data collection and carried out the analysis and interpretation of the data. AB, AJ and FM drafted the article. All authors read and approved the final manuscript. AB and AJ are guarantors of the paper.None.


Hydrozoa and Anthozoa classes, and from copepods of the Pontellidae family, as well as non-fluorescent proteins from Anthozoa. Recently, an mRNA encoding a fluorescent GFP-like protein AmphiGFP, related to GFP from Pontellidae, has been isolated from the lancelet Branchiostoma floridae, a cephalochordate .Members of the green fluorescent protein (GFP) family share sequence similarity and the 11-stranded β-barrel fold. Fluorescence or bright coloration, observed in many members of this family, is enabled by the intrinsic properties of the polypeptide chain itself, without the requirement for cofactors. Amino acid sequence of fluorescent proteins can be altered by genetic engineering to produce variants with different spectral properties, suitable for direct visualization of molecular and cellular processes. Naturally occurring GFP-like proteins include fluorescent proteins from cnidarians of the Branchiostoma floridae encodes at least 12 GFP-like proteins. The evidence for expression of six of these genes can be found in the EST databases. Phylogenetic analysis suggests that a gene encoding a GFP-like protein was present in the common ancestor of Cnidaria and Bilateria. We synthesized and expressed two of the lancelet GFP-like proteins in mammalian cells and in bacteria. One protein, which we called LanFP1, exhibits bright green fluorescence in both systems. The other protein, LanFP2, is identical to AmphiGFP in amino acid sequence and is moderately fluorescent. Live imaging of the adult animals revealed bright green fluorescence at the anterior end and in the basal region of the oral cirri, as well as weaker green signals throughout the body of the animal. In addition, red fluorescence was observed in oral cirri, extending to the tips.We report that the nearly-completely sequenced genome of Metazoa. Their evolutionary history includes losses in several metazoan lineages and expansion in cephalochordates that resulted in the largest repertoire of GFP-like proteins known thus far in a single organism. Lancelet expresses several of its GFP-like proteins, which appear to have distinct spectral properties and perhaps diverse functions.GFP-like proteins may have been present in the primitive This article was reviewed by Shamil Sunyaev, Mikhail Matz (nominated by I. King Jordan) and L. Aravind. Among the fluorescent proteins that have been developed as reporters, members of the GFP family, with the green fluorescent protein of hydroid Aequorea victoria as its founding member 19]. The Sequence similarity searches were performed using the gapped BLAST and PSI-BLAST family of programs . MultiplB. floridana EST libraries as two of the proteins supported by the largest number of EST. Prior to the release of the lancelet genome assembly, there were six nucleotide positions in the LanFP1 coding sequence showing variations between the ESTs in the database, and we used the most frequently conserved nucleotide for these positions. The sequences were subsequently verified by comparing them to the genome assembly. To optimize the codons for expressing in mouse and other mammalian systems, we reverse translated the LanFP1 and LanFP2 into DNA using a standard mouse codon set. For ease of cloning, we included HindIII and BamHI restriction sites at 5' and 3' ends of both sequences. The gene was synthesized by Bioclone, Inc. . The synthesized gene was cloned into Sigma's p3XFlag-Myc-CMV plasmid HindIII and BamHI sites, which contained a 5' Flag and a 3' Myc tags plus a stop codon on the vector backbone. The sequence for optimized sequences can be found in Additional File LanFP1 and LanFP2 coding sequences have been initially assembled from the Human embryonic kidney (HEK) 293 cells were cultured in the minimum essential medium (MEM) (Invitrogen) supplemented with 5% fetal bovine serum (FBS) and 2 mM glutamine and were maintained at 37°C in a humidified environment of 5% CO2. The cells were plated on 25 mm round coverslips coated with poly-D Lysine (Sigma) 24 to 48 hours and transfected with DNA plasmids when cells reached 70–80% confluence. Plasmid DNA used for transfection was obtained using the HiSpeed maxi-prep kit (Qiagen) and repurified by sodium acetate and ethanol precipitation. 2 μg of plasmid DNA was mixed with 12 μg of Nupherin  in 300 μl of MEM containing no FBS or antibiotics for 15 min and then combined with 300 μl of MEM containing 6 μl of LipofectAMINE 2000 (Invitrogen) for another 15 min at room temperature. The culture medium was replaced with 600 μl of transfection medium containing the LipofectAMINE-Nupherin-DNA complex. After incubating for 0.5 to 1 hour, the transfection medium was replaced with 2 ml of culture medium. Cell imaging occurred 24–72 hours post transfection.BL-21 Ecoli cells were transformed with LanFP1 or LanFP2 subcloned into pRSET-B bacteria expression vector  in frame with 6 histidine tags. Overnight cultures were grown to OD 0.4 prior to induction. Bacterial cultures were induced by the addition 1 mM IPTG, grown to OD 0.8, lysed and the His-tagged protein was extracted using a Ni-agarose beads solution (Qiagen cat no. 30210). Concentration of purified protein was established using a BCA assay  and fluorescence correlation spectroscopy.GFP: Green fluorescent protein.The authors have a financial interest in patent applications which cover some of the work described in the paper.The order of authors is alphabetical. AM, JS and CRY designed research and wrote the manuscript. All authors performed research, analyzed the data, read and approved the final manuscript.B. floridae. The authors expressed two proteins and detected fluorescent signals of different brightness and wavelengths. Phylogenetic analysis that included the analysis of intron position conservation suggested an early metazoan origin of GFPs followed by a massive parallel loss in multiple lineages. The manuscript also discusses the potential evolutionary relationship between structurally similar G2 domains and GFPs.This manuscript describes a discovery of a family of GFP-like proteins in the newly sequenced genome of a lancelet in vivo fluorescence of lancelets. Still, this paper contains substantially more material than the short note by Deheyn et al. First, the whole family of GFP-like proteins in one genome was surveyed. Second, two of the 12 detected genes have been heterologously expressed and spectroscopically characterized. Third, and this is the most intriguing information for me, red fluorescence has been observed in the lancelet. All these are valuable contributions to our knowledge of diversity and evolution of GFP-like proteins.Unfortunately, the novelty edge has been taken away from this work by the prior note by Deheyn et al. of green fluorescent proteins in three species of lancelets . Deheyn et al already discussed the phylogenetic relationships of one of the lancelet GFPs, including its relationships with G2 domain proteins, and described I regret, however, that the authors did not go as far as to express and characterize all 12 family members found in the lancelet genome. This would have been a logically complete study, and the first one to describe the full complement of GFP-like proteins from an organism.Authors' response: We are doing it now with all B. floridae family members and, for good measure, with the homologs from sea anemone Nematostella vectensis that have been revealed in the genome project but, as far as we know, were not characterized before.In addition to possible identification of the red fluorescent protein, such study might have turned up interesting patterns related to evolution and/or deterioration of gene functions in multigene families. In this regard, it is really intriguing that the two characterized proteins differ so much in their spectral properties. Whereas LanFP1 is a rather generic GFP, LanFP2 is dimmer because of its low quantum yield but instead possesses impressively high molar extinction coefficient, on par with anthozoan purple-blue chromoproteins that are known for their striking color appearance. I would mention in the discussion that this difference in spectral properties may be the result of functional sub-specialization that maintained both genes in the lancelet genome after duplication. Also, given its very high molar extinction, I would expect that LanGFP2 protein should look quite colorful (not necessarily green!) in solution. Is that so?Authors' response: Yes! LanFP2 solution is yellow-brown.I am rather surprised that the authors doubt the homology of G2 domains and GFPs. In addition to the fold similarity and same collection of the secondary structure elements, these proteins also have the elements in identical  order within their primary structure. Importantly, this order does not have to be conserved for the protein to work, as has been demonstrated by successful circular permutation of several GFP-like proteins. I believe therefore that in this case, complete identity of the fold including the composition and order of secondary structure elements constitutes a far stronger evidence of homology than any structure-oblivious algorithm may provide.Authors' response: Unfortunately, there is no accepted quantitative model of structural similarity that can be used for testing evolutionary hypotheses in the absence of sequence signal – similar number and similar topology of strands is certainly something worth knowing about, but this qualitative argument cannot (yet) be translated in a hard number, the way the "structure-oblivious" sequence similarities can be. On the other hand, we have nothing against the suggestion that nidogen G2 domain and GFP share a common ancestor. In fact, the relevant paragraph in the Conclusions section includes evolutionary scenarios that presume the existence of just such an ancestor, on the basis of exactly the evidence that the Reviewer highlights. Note also related comments by L. Aravind.I also have a few minor questions, listed below.1. How was the fluorescence measured in live animals?Authors' response: The fluorescence was measured using a LSM-510 confocal microscope. The animals were immobilized with a muscle inhibitor and images collected using 488 nm laser excitation and spectral imaging with either a 20× or 40× objective (see Methods for more detail).2. What were the excitation wavelengths for "green" and "red spectra", Fig. Authors' response: The excitation wavelength was 488 nm with an argon laser for both green and red spectra. The red spectrum can also be excited with a 561 nm laser. The two-photon excitation scan indicates the green fluorescence is produced when excited with 720 to 1020 nm light. The red fluorescence was not apparent until using two-photon excitation above 980 nm light.3. The "red spectra" does not look like any of the GFP-like proteins known thus far. What can you say about it?Authors' response: The spectrum does not provide any information about the potential source of the red fluorescence, i.e., even whether it is a protein or a small molecule. Note, however, the claims in a very recent patent (ref. 14) about proteins from a related species of Branchiostoma.4. I can't help noticing that the "green spectra" are different in Fig. Authors' response: They are identical. We hope that reformatted figure makes this clearer.5. I'd like to see excitation spectra for the expressed proteins .7. Please make the phylogenetic tree one of main figures. I would also appreciate if the tree was reformatted into a more conventional form.Authors' response: We feel that the tree is too trivial to add much to discussion , but we now provide, as Additional File 8. The role of fluorescence in attracting food is an interesting proposition, but why it is important particularly during twilight? I don't see the connection.Authors' response: Phytoplankton and zooplankton exhibit phototaxis, and different parts of spectra may be most effective at different times of day and at different depths in attracting food from the surface. We agree that there is no particular evidence that would compel one to focus on twilight, so we deleted "during twilight".B. floridae) and experimental characterization of a few of them. The point of note is the proposal that that GFP was present in the common ancestor of Bilateralia and Cnidaria and repeatedly lost in various animal lineages. Despite the extremely sporadic distribution of these proteins, the phylogenetic analysis and the argument of conserved intron positions support this scenario. Yet, it might be useful to check environmental metagenomic sequences to address the possibility of independent lateral transfer from some unknown source into the few animal lineages in which GFPs are found.The paper by Baumann et al describes the recovery of several new GFP related proteins in amphioxus  do make it a distinctive possibility.Authors' response: We agree that this is a possibility worth discussing, and we amended the text accordingly. Note, however, that we have not seen any G2 domains in complete proteome of sea anemone Nematostella or in the available sequences of planarian Schmidtea or any other primitive metazoan clade. Thus, if the common 11-stranded barrel ancestor of G2 domain and of the present-day GFPs was not itself part of the GFP family, it either had to spawn GFP and G2 domains on separate occasions, or to split into GFP and G2 followed by at least one, or possibly many losses, of G2 (in Anthozoa and perhaps in Plathelminthes). Better sequence sampling of sponges, coelenterates and flatworms would help to sort out these possibilities.Minor points: -Pg 10 "At this level of significance, GFPs did not stand out from the background of other β-barrel proteins." What were the other β barrel proteins detected by HHpred and their p-values? Was a reciprocal search done using the GFP profiles as queries against a profile database lacking GFPs?Authors' response: This paragraph was a bit too convoluted in the initial version. Current HHPred server (release 2.2.1) does not report GFP-G2 similarity if the complete database is used as the search space. The same negative results are obtained with the stand-alone version 1.5.0 whether or not the selfsame models are removed from the database. The matches between G2 and GFP can be only observed when we search the subset of HMMs built upon proteins with the known three-dimensional structures. In that case, the G2 model finds the best GFP model with p-value 0.0017, and other GFP-related models have p-values from 0.002 to 0.005. An apparently unrelated β-barrel family, specified by At1g79260 protein from Arabidopsis thaliana, is seen with p-value 0.039, though the alignment is only partial and reciprocal search does not detect the G2 domain. G2 model also finds bacterial semi-barrel LolB in searches against the complete database . GFP does not detect G2 even under these forced conditions. All these weak similarities, however, only reinforce our main argument, i.e., that we still do not have a robust statistical validation of the GFP-G2 monophyly at the sequence level. Thus, interested as we are in discussing scenarios that assume a common ancestor, we can do it only hypothetically. We have modified the Conclusions section with this in mind.What is the evidence that the red fluorescence comes from a GFP like protein and not some other fluorochrome?Authors' response: This question was also raised by M. Matz, see our response .Phylogenetic tree of GFP-like proteins. The image of the neighbour-joining tree inferred from the alignment of GFP-like proteins.Click here for fileSpectral properties of LanFP2 and LanFP2. The table summarizing absorbance, emission, extension and quantum yield for several GFP-like proteinsClick here for fileB. floridae fluorescent proteins, with codon usage optimized for expression in murine cellsMurinized sequences of LanFP1 and LanFP2. Nucleotide sequences encoding two Click here for fileB. floridae fluorescent proteins compared with several other spectra of GFP-like proteinsNormalized absorbance spectra of LanFP1 and LanFP2. Image of spectra of two Click here for fileSource file of the tree shown in Additional file 1. Newick-formatted trees, one of which contains branch length information, and the other is the consensus of 100 trees built on the basis of the bootstrap replicates of the first tree, contains the information on the bootstrap support for each partition in the first tree but lacking the branch length information.Click here for file


FMDV virus has been increasingly recognised as the most economically severe animal virus with a remarkable degree of antigenic diversity. Using an integrative evolutionary and computational approach we have compelling evidence for heterogeneity in the selection forces shaping the evolution of the seven different FMDV serotypes. Our results show that positive Darwinian selection has governed the evolution of the major antigenic regions of serotypes A, Asia1, O, SAT1 and SAT2, but not C or SAT3. Co-evolution between sites from antigenic regions under positive selection pinpoints their functional communication to generate immune-escape mutants while maintaining their ability to recognise the host-cell receptors. Neural network and functional divergence analyses strongly point to selection shifts between the different serotypes. Our results suggest that, unlike African FMDV serotypes, serotypes with wide geographical distribution have accumulated compensatory mutations as a strategy to ameliorate the effect of slightly deleterious mutations fixed by genetic drift. This strategy may have provided the virus by a flexibility to generate immune-escape mutants and yet recognise host-cell receptors. African serotypes presented no evidence for compensatory mutations. Our results support heterogeneous selective constraints affecting the different serotypes. This points to the possible accelerated rates of evolution diverging serotypes sharing geographical locations as to ameliorate the competition for the host. Foot-and-Mouth Disease Virus (FMDV), a single-stranded RNA virus, belongs to the Picornaviridae family . It has Syncerus caffer)  1–3, Asia-1, A, O and C). It has an enormous wide spectrum of infection reflecting its genetic and antigenic diversity. O is the most predominant of the serotypes. Type A possesses greater geographical spread than any of the other serotypes. It is also the most antigenically diverse of the Eurasian serotypes with a large amount of variants in Asia, Africa and South America . To date caffer) . They ar caffer) .A number of evolutionary and population parameters are believed to be responsible for the emergence and dispersal of the different virus serotypes. These constraints can be broadly classed as genetic, for example the ability of the virus to avoid the host immune response, or ecological such as host mobility and population density. Elucidating the mechanisms utilised by viruses that subsequently drive the evolution and infectivity would critically aid in our understanding of viral epidemiology and disease prevention. Recently, Carrillo et al.  have perIt has been long recognised that the VP1 gene differs in about 30–50% between serotypes . We postA dataset comprising 665 FMDV VP1 sequences were downloaded from Genebank. This includes all serotypes and only comprises sequences with length greater than 400 nucleotides to account for most of the antigenic regions within the VP1 protein. Alignments were obtained for VP1 amino acid sequences for each individual serotype using the T-coffee program . We thenα) of the gamma distribution for the amino acid sequence alignment using the program AAml from the PAML package version 3.14  importance of particular amino acid sites in the VP1 protein we used two approaches. The first approach was based on the collection of the functional information available for this protein. However, most of the amino acid sites in the molecule have not been tested for their functional importance since studies have been biased towards testing antigenic peptide regions. We hence conducted a neural-network analysis implemented in the program CONSEQ . We useddS) to the number of replacements per non-synonymous site (dN) (ω = 1), replacements have been removed by purifying selection (ω < 1), or mutations have been fixed by adaptive evolution (ω > 1). Furthermore, since dS values are neutrally fixed (except when constraints operate at the RNA level in these sites or when synonymous sites are saturated) they are proportional to the time since the sequences compared have diverged. Consequently, the normalisation of dN values by dS makes ω independent of time. It has been shown, however, that ω is a poor indicator of the action of adaptive evolution  . The ratvolution . The ratdN and dS using a number of simulated data sets. We used 1000 simulated data sets in our analysis obtained using the program EVOLVER from the PAML (ω transition-to-transversion rates ratio and codon frequency table generated under the Goldman and Yang model (dN and dS by the method of Li (ω > 1) but also accelerated rates of amino acid substitutions without the restriction of ω > 1 (that can be due to either adaptive Darwinian evolution or to the fixation of slightly deleterious mutations by genetic drift), saturations of synonymous sites  and hot spots (where both dS and dN are significantly high but where ω < 1).We have used Kimura-based models to measure the intensity of selection between and within serotypes using the set of 665 sequences. To detect selective constraints we used a sliding-window based approach  implementhe PAML  package,ng model . These pod of Li . The sigNormally, in phylogenetic tree inference and detection of selective constraints, fast evolving sites and slow evolving sites are considered to remain fast and slow sites, respectively, throughout the entire evolutionary history , something called the homogeneous gamma model or rates-across-site (RAS) model . If the θ), which is equivalent to the anti-correlation in the evolution of the two group of sequences (e.g. FMDV serotypes) being compared. It estimates the posterior Bayesian probability for an amino acid site to belong to the group of sites responsible for functional divergence. These sites share the property of being highly constant in one of the sequence groups and variable in the other, supporting the gain/loss of a functional/structural constraint in one of the groups.A non-homogeneous gamma model of evolution can be statistically described by the functional divergence type I . BecauseAmongst FMDV antigenic sites in VP1 protein, Site A is involved in conferring the virus its ability to escape from the immune pressure and to recognise host cell receptors . Importai, s′j), of occurrence of symbol i at position s and j at position s′. The MIC values generated range between 0, indicating independent evolution, and a positive value whose magnitude depends on the amount of covariation. We only included in the analysis those variable positions in the alignment that were parsimony-informative (i.e. they contain at least two types of amino acids and at least two of them occur with a minimum frequency of two). The statistical power of the test is dependent on the number of sequences as well as the amount of variability at each amino acid site. In this case, both parameters were secured since the number of sequences was high and the variability as well. The significance of the MIC values was assessed by randomisation of pairs of sites in the alignment, calculation of their MIC values and comparison of the real MIC values with the randomly generated distribution of MIC values. One million permutations were conducted to test MIC value significance.We tested intra-molecular coevolution in each one of the serotypes by applying a method based on the information theoretic quantity called mutual information to measure the dependence of mutations in VP1 , except C and SAT3, presented evidence of positive Darwinian selection in the VP1 protein . We haveThe comparison of the five serotypes under positive selection highlights diverse patterns of selective constraints at the molecular and phylogenetic levels. Interestingly, all those serotypes under positive selection comprise a similar region around N131- R149 that is located in the immunodominant site A within the G-H loop. However only serotypes A and O, the wider geographically spread FMDV serotypes, have the full RGD motif under positive selection. Moreover, serotypes SAT1 and 2 present evidence of adaptive evolution at overlapping VP1 protein regions. In fact, all of the regions detected in SAT1 to be under adaptive evolution were also found in SAT2 to be positively selected . InteresIf we were to classify these serotypes in terms of percentage of codon sites under positive selection the following is observed: SAT2 > ASIA 1 > A > SAT 1 > O. The difference from SAT 2 to type O represents a 10-fold increase in the percentage of sites under adaptive evolution.For each individual serotype we applied the neural-network analysis to detect functional/structural sites implemented in the program CONSEQ. This analysis also enabled us to know whether sites under adaptive evolution had any functional/structural role. In the seven serotypes, the RGD motif from the VP1 protein was identified as functionally important, except in the case of Asia1 and A serotypes where only the G144 and D145 were identified as important.χ62 = 14.034, P = 0.029). Similarly, the proportion of structurally important sites was also significantly different among serotypes . Comparison of the number of sites under functional and structural constraints between serotypes uncovers an interesting pattern that suggests certain correlation between the selective constraints acting on VP1 and the functional/structural selection shifts among serotypes. In fact, those serotypes that have not undergone adaptive evolution present the greatest number of functionally and structurally important regions . The percentage of sites under functional constraints as detected by CONSEQ were not an artefact of the amino acid variability produced by adaptive evolution since all positively selected regions were detected to be functionally/structurally important. Thus, high variability due to positive selection was not leading to an underestimation of functional/structural important sites.The level of functional important sites varied significantly between serotypes . For exaFMDV serotypes were compared to each other and regions under functional divergence type I determined. Only amino acid sites showing posterior Bayesian probabilities greater than 0.95 were considered as having been subjected to functional divergence. In all the pairwise comparisons between serotypes, most of the sites involved in functional divergence were found in the carboxy-terminal half of the molecule, affecting antigenic sites A, C and D.t19 = 2.538, P = 0.02; t7 = 3.789, P = 0.007; Functional divergence type I between serotypes followed a trend in agreement with the geographical pattern observed in the analysis of coevolution and functional/structural sites distribution. Functional divergence affected a significantly greater number of sites when the serotypes compared belonged to different-continent locations  in SAT2, 83% in A, 63% in Asia1, 33% in SAT1 and 21% in O . ResultsThere was no evidence for compensatory mutations in African serotypes. In these serotypes, sites under adaptive evolution were generally included in different coevolution groups . No singAll four antigenic regions presented evidence of co-evolution between each other. Interestingly, the BC loop presented the highest percentage of co-evolving sites with antigenic sites A, C and D compared to other VP1 regions. A trend emerges that shows that serotype A has the greatest number of co-evolving sites in antigenic regions closely followed by C, then types O, SAT2, ASIA1, SAT1 and SAT3.Previous works have attempted to answer the question of whether the appearance of a new serotype is subjected to a change in evolutionary constraints. ω values due to the purifying selection acting on the RNA molecule. However, this would affect all the regions in the protein and thus selective constraints would not be concentrated in few biologically important protein regions.Adaptive evolution in FMDV has been previously detected in natural isolates and has been associated with the emergence of immune-escape and persistent mutants . BecauseOn examination of the sites under adaptive evolution we find that G-H loop that ranges from amino acids 131–149 have undergone positive selection in all the serotypes. Within SAT serotypes we find Site 3 and antigenic region C under positive selection. Most of the regions under positive selection in SAT serotypes are not found in non-African serotypes, sparking speculation that there is a difference in the selective pressures governing the SAT types compared to non-African serotypes. On the other hand, the difference in the percentage of sites that have undergone positive selection is very noticeable, with SAT2 having a significantly greater percentage of sites undergoing selection compared to SAT1. This coincides with the fact that SAT2 is widely distributed in the African continent whereas SAT1 is confined to specific African regions, yet both serotypes overlap in some African territories. A plausible explanation for this correlation is that SAT2 has accumulated amino acid replacements in its VP1 protein that permitted the generation of immune-escape mutants and the emergence of variants in SAT1-SAT2 overlapping geographical areas as to minimize the competition by utilizing different niches in these areas (adaptive radiation). If this hypothesis were true, we would expect greater accelerated rates of evolution in the VP1 protein from serotypes in overlapping geographical areas compared to the same serotypes in non-overlapping areas. Examination of the Poisson-corrected amino acid distances between serotypes in overlapping regions reveals faster evolutionary rates for data sets of sequences in overlapping geographical regions for all the serotypes . This faAmino acid sites detected to be under adaptive evolution in FMDV serotypes are mostly located in antigenic regions known to interact with both, integrin receptors  and neutExamination of the three-dimensional localisation of coevolving sites suggests fixation of mutations with compensatory effects in VP1 from serotypes with a wide geographical distribution. In fact, in these serotypes we identified fast-evolving sites co-evolving with positively selected sites. These accelerated sites are in contact with functionally important sites as highlighted in the three-dimensional structures. Mutations at these sites may have important negative effects on the functional regions that must be compensated by advantageous mutations at adjacent sites. Both types of mutations together would have neutral effects on the protein’s function. In addition, for those accelerated sites affecting important regions embedded in complex local structures, such as the RGD motif, several advantageous mutations at nearby sites would be needed to compensate for the deleterious effect of accelerated sites. This would explain the significant number of positively selected sites belonging to the same co-evolution group in the case of these serotypes. Conversely, those viruses with a distribution confined to the African continent showed no indication of compensatory mutations.This pattern of coevolution may have probably been produced by fixation of slightly deleterious mutations in the widely distributed serotypes  subjected to population bottlenecks due to the high host geographical mobility. Caution is however stressed in interpreting these results since high host population densities may ameliorate the effects of genetic drift caused by host mobility. Conversely, SAT serotypes are confined to specific areas of the African continent probably infecting persistently the host  through improving their ability to generate highly competitor immune-escape mutants. In addition, compensatory mutations and the co-evolution among antigenic sites, as demonstrated in this work, would permit fixing immune-escape mutations probably maintaining the ability to recognise host-cell receptors in these serotypes. This is in agreement with previous studies suggesting the co-evolution of antigenicity and receptor usage . FurtherOur analysis supports that FMDV serotypes geographically distant have diverged functionally/structurally during their independent evolution. In contrast to the African serotypes that show a geographical confinement, serotypes in the Asian continent present evidence for inter-serotype contact due to their lower functional divergence. This has been probably produced due to the greater mobility of the virus or the host that could eventually lead to bottlenecks on the viral population.In this manuscript we give details about the complex dynamics of evolution of an economically important virus. Variability in RNA viruses caused by high errors rates during virus replication seems to be important for the survival of virus populations under changing environmental conditions. It has been recently shown that low fidelity increases virus pathogenesis and that selective pressures are operating at the population level . The maiSampling limitations may indeed play a factor in the interpretation of these results. In order to provide accurate reflections of the disease, there is need for complete temporal, spatial coverage and full recording of all disease events. This study attempts to maximise the geographic distribution and range of sampling times for sequences in order to obtain a random and true representation of the worldwide situation rather than single episodes or epizootics. Indeed it is inevitable that the current sequences sampled here are only a subset of those actually in nature but nevertheless they aid in our understanding of the disease. In any case, this is to our knowledge the most comprehensive study done on the evolutionary constraints on FMDV serotypes.