Evolutionary biologists have long been interested in understanding the molecular basis for the great diversity in size, shape, and behavior seen in life on earth. Recent attention has focused on the role that gene expression changes play in organismal evolution. Tracing the evolution of gene regulation, however, has proved difficult. This is in large part due to the difficulty in identifying and comparing the regulatory elements that control gene expression in different species.cis-regulatory elements, short sequence motifs embedded in the DNA that flank a gene's coding region. Regulatory proteins bind to specific cis-regulatory sequences, and command the activation or repression of the corresponding gene. The challenge in studying the evolution of cis-regulatory elements lies in identifying those elements in multiple species. Unlike protein sequences, which are typically a few hundred amino acids long and relatively straightforward to identify in related organisms, cis-regulatory elements are often short and can have variations in sequence. This makes it very difficult to distinguish the regulatory elements from the nonfunctional DNA that surrounds them. It is even harder to identify corresponding regulatory elements across species. As the evolutionary distance between species increases, so, too, does the difficulty in identifying corresponding cis-elements in those species.Gene expression depends on PLoS Biology, Audrey Gasch and her colleagues describe a comparative genomics approach that allows them to identify potential cis-regulatory elements in thousands of genes across 14 ascomycete fungi whose diversity represents the effects of several hundred million years of evolution. Ascomycetes are a large class of fungi with extremely diverse morphologies, reproductive strategies, and habitats. A divergence dating back 500 million to 1 billion years ago gave rise to three groups: Archaeascomycetes, Euascomycetes, and Hemiascomycetes. The genome of the brewer's yeast, Saccharomyces cerevisiae, a hemiascomycete, was completely sequenced in 1995, and that of fission yeast, Schizosaccharomyces pombe, an archaeascomycete, in 2002. Since that time, complete genome sequences have been released for more than nine additional hemiascomycetes and three euascomycetes. This gives the authors an opportunity to compare regulatory systems among progressively more distantly related species, on a genomic scale.In this issue of S. cerevisiae have revealed groups of genes whose expression levels vary simultaneously under varying experimental conditions. Such co-regulated genes, the authors reasoned, must harbor common regulatory elements that coordinate their response to experimental triggers. Gasch and colleagues looked for such cis-elements and found 35 groups of co-regulated S. cerevisiae genes with at least one shared cis-element. The authors then argued that co-regulation may reflect selection pressures that also apply to other ascomycetes, and so they identified the equivalent of the 35 co-regulated gene groups in each of the 13 other species. They then looked for shared cis-elements within each group and in each species independently, and compared the regulatory systems across the species.Genome-wide expression studies in the yeast cis-elements first identified in yeast are retained in the equivalent gene groups in other species, in a manner that reflects the species' evolutionary distance from yeast. One cis-element, in a group of co-regulated genes that control the cell cycle, is found all the way from budding yeast to fission yeast, suggesting a selection pressure on the co-regulation of these genes that has withstood greater than 500 million to 1 billion years of evolution.The results of this study show that the majority of cis-elements in each species, suggesting that the regulation of those genes has evolved. In the case of cis-elements found in genes controlling protein degradation, a related element was identified in all of the hemiascomycetes, whereas the euascomycetes appear to have adopted a novel cis-element for this gene group. Interestingly, the hemiascomycete element displays a sequence variation in Candida albicans that is not found in S. cerevisiae. The two species diverged 200 million years ago. Gasch and colleagues showed that the protein that binds to the hemiascomycete element has evolved to have slightly different DNA interactions in the two species, allowing the C. albicans protein to bind the novel sequence found only in the C. albicans genes. This provides evidence for co-evolution between a transcription factor and its target cis-element. Overall, this analysis has uncovered striking cases of conservation and innovation of gene regulatory systems, and therefore provides important insight into the evolutionary forces that have shaped the evolution of gene regulation.In contrast, there were other examples in which the same gene groups contained different putative


Recent studies strongly suggest that due to the limitations and risks of biopsy, as well as the improvement of the diagnostic accuracy of biochemical markers, liver biopsy should no longer be considered mandatory in patients with chronic hepatitis C. In 2001, FibroTest ActiTest (FT-AT), a panel of biochemical markers, was found to have high diagnostic value for fibrosis (FT range 0.00–1.00) and necroinflammatory histological activity (AT range 0.00–1.00). The aim was to summarize the diagnostic value of these tests from the scientific literature; to respond to frequently asked questions by performing original new analyses ; and to develop a system of conversion between the biochemical and biopsy estimates of liver injury.versus other biochemical markers, including hyaluronic acid, the Forns index, and the APRI index. All the comparisons favored FT (P < 0.05). There were no differences between the AUROCs of FT-AT according to genotype or viral load. The AUROCs of FT-AT for consecutive stages of fibrosis and grades of necrosis were the same for both moderate and extreme stages and grades. A conversion table was constructed between the continuous FT-AT values (0.00 to 1.00) and the expected semi-quantitative fibrosis stages (F0 to F4) and necrosis grades (A0 to A3).A total of 16 publications were identified. An integrated database was constructed using 1,570 individual data, to which applied analytical recommendations. The control group consisted of 300 prospectively studied blood donors. For the diagnosis of significant fibrosis by the METAVIR scoring system, the areas under the receiver operating characteristics curves (AUROC) ranged from 0.73 to 0.87. For the diagnosis of significant histological activity, the AUROCs ranged from 0.75 to 0.86. At a cut off of 0.31, the FT negative predictive value for excluding significant fibrosis  was 91%. At a cut off of 0.36, the ActiTest negative predictive value for excluding significant necrosis  was 85%. In three studies there was a direct comparison in the same patients of FT i.e., in case of high risk of error of biochemical tests.Based on these results, the use of the biochemical markers of liver fibrosis (FibroTest) and necrosis (ActiTest) can be recommended as an alternative to liver biopsy for the assessment of liver injury in patients with chronic hepatitis C. In clinical practice, liver biopsy should be recommended only as a second line test,  One of the major clinical problems is how to best evaluate and manage the increasing numbers of patients infected with the hepatitis C virus (HCV) . Liver bAmong the non-invasive alternatives to liver biopsy , severalvs F1) or advanced fibrosis/cirrhosis (F3 vs F4) than at predicting intermediate levels of fibrosis (F1 vs F2)? And 5) what is the conversion between FT-AT results and the corresponding fibrosis stages and necrosis grades?The aim of this study was to summarize the diagnostic value of these tests by an overview of the scientific literature and to respond to the following frequently asked questions by performing original new analyses: 1) what is the range of the FT-AT diagnostic values across the different studies? 2) What are the base evidence comparisons between FT-AT and other published biochemical markers? 3) Are there differences in diagnostic values according to HCV genotype or viral load? 4) Are there differences between the FT-AT diagnostic values according to stages and grades? – In other words, is FT better at predicting no or minimal fibrosis  [In four studies there was a direct comparison in the same patients of FT nic acid , the Fornic acid , the APRnic acid  and the nic acid . All the for FT) .A total of 1,570 subjects were included in the integrated database. Of these, 1,270 were patients with chronic hepatitis C who tested PCR positive before treatment and who had had a liver biopsy and METAVIR staging and grading performed. Of these patients, 453 were from our center ,14, inclThere was no difference between the AUROC of FT-AT for the diagnosis of significant fibrosis (F2F3F4) Figure  and signversus random AUROCs at 0.50, were all significant and similar between these 3 groups in a meta-analysis: mean difference in AUROC = 0.29 (random effect model Chi-square = 549, P < 0.001), including 0.24 for independent, 0.25 for mixed and 0.36 for dependent studies. In the Callewaert et al. [Among the 13 published studies of FT , the AUROCs were not significantly different and ranged from 0.95 to 0.99.The AUROCs between different stage combinations are given in Table versus F3 and F0 versus F4), the AUROCs were not significantly different and ranged from 0.95 to 0.99.The AUROCs between different grade combinations are given in Table th percentile, 0.23) and the median AT value was 0.07 ± 0.004 (95th percentile, 0.26). Among the 1,270 HCV-infected patients, the FT conversion was 0.000 – 0.2100 for F0; 0.2101 – 0.2700 for F0–F1; 0.2701 – 0.3100 for F1; 0.3101 – 0.4800 for F1–F2; 0.4801 – 0.5800 for F2; 0.5801 – 0.7200 for F3; 0.7201 – 0.7400 for F3–F4; and 0.7401 – 1.00 for F4.  was 0.08 ± 0.004  picture of fibrogenic and necrotic events occurring within the liver than hepatic biopsy. The greater accuracies of FT-AT, when assessed with biopsy specimens greater than 15 mm ng error . Several markers ,9,11. ThThe present work allowed frequently asked questions to be answered, the first being whether the diagnostic values of FT-AT had been confirmed in all studies performed to date. A major strength of the studies pertaining to FT-AT is that they were carried out on a large number of patients with chronic hepatitis C, and the results were reproducible in different populations, including patients coinfected with HIV. There was a small variability in the AUROCs, both for the diagnosis of significant fibrosis (0.73 to 0.87) and significant necrosis (0.75 to 0.86).A weakness of this study was that the same group, which developed these tests, performed most of the published studies. However the independent published studies found the same significant diagnostic values than non-independent or multicentre studies. Several recent independent studies confirmed the predictive value of FT-AT ,30.et al. study (n = 323) [The second question concerned the comparison of FT-AT to other tests. In their recent review, Gebo et al.  concluden = 323) , as the n = 323) . The comn = 323)  includedn = 323) .Recently, a study using profiles of serum protein N-glycans found that a profile has a similar AUROC than FT for the diagnosis of compensated cirrhosis. When combined with FT this marker had 100% specificity and 75% sensitivity for the diagnosis of compensated cirrhosis, which is not significantly different from the 92% specificity and 67% sensitivity of the FT . This stHowever FT-AT is the only panel of markers identified by an independent overview , which hThe present analysis of the integrated database demonstrated that the diagnostic value of FT-AT did not depend on HCV genotype or viral load. However, because of the small number of patients included, studies in genotype 4, 5 and 6 would be useful.The present analysis also answered another frequently asked question concerning the predictive values for the intermediate stages of fibrosis. Contrary to the initial hypothesis, the diagnostic values of FT-AT for consecutive stages of fibrosis and grades of necroinflammatory activity were the same for both moderate and extreme stages and grades. Our interpretation is that the same overlap exists between all stages, which is mainly related to the sampling error of the biopsy. It is very reassuring that the medians of FT-AT are linearly associated with stages and grades Figures . The linFinally, the integrated database allowed a simple conversion system to be proposed to clinicians between liver injury as estimated by the FT-AT and that as estimated by liver biopsy Figure . One coni.e., in case of high risk of error of biochemical tests or in transplanted patients. For clinical research, only very high quality liver biopsy (as two samples of 20 mm each) can be considered as a gold standard for validation of new alternatives.Based on these results, the use of the biochemical markers of liver fibrosis (FibroTest) and necrosis (ActiTest) can be recommended as an alternative to liver biopsy for the first line assessment of liver injury in patients with chronic hepatitis C. In clinical practice, liver biopsy should be recommended only as a second line test, We did a search for all publications and communications between February 2001 and March 2004 with the key words "FibroTest" and "ActiTest" in Medline and in the abstract books of hepatology, gastroenterology, internal medicine and infectious diseases annual meetings. Only publications or abstracts concerning FT-AT in chronic hepatitis C were included.For each study we assessed the diagnostic value for the diagnosis of significant fibrosis (bridging fibrosis or stages F2, F3, F4 according to the METAVIR scoring system) and significant necroinflammatory activity (moderate or severe necrosis, grades A2 or A3 according to the METAVIR scoring system) by the area under the receiver operating characteristics curve (AUROC).For several databases it was possible to re-analyze the individual data and we looked at the sensitivity and specificity according to different thresholds (0.10, 0.30, 0.60 and 0.80). When FT-AT was compared to other biochemical tests, we also assessed the corresponding sensitivity and specificity according to several thresholds.We selected studies using direct comparisons of diagnostic values in the same patients. The AUROCs were compared for the diagnosis of significant fibrosis (F2F3F4) and significant necrosis (A2A3).Patients were included in an integrated database if they belonged to a published population of patients with chronic hepatitis C. Liver biopsy was scored using the METAVIR scoring system and FT-AT was assessed using the recommended pre-analytical and analytical procedures ,20. A puUsing the integrated database, we compared the AUROCs of FT-AT for the diagnosis of significant fibrosis (F2F3F4) and significant activity (A2A3) between 4 classes of genotype (1, 2, 3 and the rarer genotypes 4, 5, 6 grouped together). For viral load, only those assessed in the same laboratory were included in the comparison between AUROCs, and the median was used to define low and high viral loads (3,800,000 copies/ml) [Using the integrated database, we compared the diagnostic values according to different stages or grades. We compared the AUROCs for all possible combinations of stages and grades, including combinations with blood donors. This allowed, for example, a comparison to be made of the diagnostic value of FT for discriminating between F1 and F2 after excluding all other stages of the database.In the integrated database, liver biopsies were processed using standard techniques. A pathologist who was unaware of the biochemical markers evaluated fibrosis stage and necrosis grade according to the METAVIR scoring system ,23.Fibrosis was staged on a scale of 0 to 4: F0 = no fibrosis, F1 = portal fibrosis without septa, F2 = few septa, F3 = numerous septa without cirrhosis, F4 = cirrhosis. The grading of activity by the METAVIR system (based on the intensity of necroinflammatory activity, mainly on necrosis) was scored as follows: A0 = no necroinflammatory activity, A1 = mild activity, A2 = moderate activity, A3 = severe activity ,23.We used the previously validated FT-AT ,9,11-21.Corresponding stages and grades were calculated from median scores and 95% confidence intervals were observed in 1,270 patients and 300 healthy blood donors. The AUROC was used as a measure of discrimination, estimated using the empirical (non-parametric) method by DeLong et al. , and werTP and MM conceived the study, performed the statistical analysis, and wrote the manuscript. FIM, BH and DM carried out biochemical analyses. RP, DT, VR, and YB participated in the coordination of the study, and drafted the manuscript. AM participated in the design and coordination of assays in the control group. All authors read and approved the final manuscript.


Little data exist for the effectiveness of communication skills teaching for medical students in non-English speaking countries. We conducted a non-randomized controlled study to examine if a short intensive seminar for Japanese medical students had any impact on communication skills with patients.Throughout the academic year 2001–2002, a total of 105 fifth-year students (18 groups of 5 to 7 students) participated, one group at a time, in a two-day, small group seminar on medical interviewing. Half way through the year, a five-station objective structured clinical examination (OSCE) was conducted for all fifth-year students. We videotaped all the students' interaction with a standardized patient in one OSCE station that was focused on communication skills. Two independent observers rated the videotapes of 50 students who had attended the seminar and 47 who had not. Sixteen core communication skills were measured. Disagreements between raters were resolved by a third observer's rating.There was a statistically significant difference in proportions of students who were judged as 'acceptable' in one particular skill related to understanding patient's perspectives: asking how the illness or problems affected the patient's life, . No differences were observed in the other 15 core communication skills, although there was a trend for improvement in the skill for asking the patient's ideas about the illness or problems (60% vs. 40%, p = .054) and one of the relationship building skills; being attentive and empathic nonverbally (87% vs. 72%, p = .064).The results of this study suggest that a short, intensive small group seminar for Japanese medical students may have had a short-term impact on specific communication skills, pertaining to understanding patient's perspectives. The literature from English-speaking countries indicates that teaching communication skills is effective in improving learners' communication skills with patients . HoweverTeaching communication skills is gaining popularity and proliferating for Japanese health professional students . YoshidaIn many traditional medical schools in Japan, communication skills teaching is limited in time and scope, and isolated from other formal curricula. Thus it is important to know whether such type of training make a difference, at least in the short run. This should also be of interest to educators elsewhere who similarly work in settings where there is not enough formal curricular time for communication skills teaching.The objective of this study was to evaluate the impact of a short, intensive small group seminar, which was based on Western educational principles, on Japanese medical students' communication skills with patients.Medical schools in Japan last six years with the last two years consisting of clerkships. Before the fifth-year, Japanese students typically have few direct interactions with patients. Throughout the academic year 2001–2002, a total of 105 fifth-year students from the Nagoya University School of Medicine rotated through the various clinical services of the Nagoya University Hospital. Students divided themselves into 18 groups of 5 to 7, but the sequential order of rotations is set by the medical school officials.As part of a 1-week clerkship rotation at the Department of General Medicine, students participated in a two-day, small group seminar on the medical interview and communication skills. Typically either or both of two of the authors (KM, NB) facilitated the seminar. Both facilitators had had an experience in learning and teaching the medical interview and communication skills in the United States. The seminar utilized learner-centered, skills-oriented, experiential, and interactive learning methods. To guide the teaching of communication skills, we created a conceptual model for patient-physician communication referring to 3 existing models ,10,11. AIn September 2001, half way through the academic year, a five-station objective structured clinical examination (OSCE) was conducted for all fifth-year students. The primary purpose of the OSCE was to provide trainees with the opportunity to receive feedback on their clinical skills from the faculty in a safe and structured environment. One OSCE station focused on the medical interview. Students engaged in a 5-minute interaction with a standardized patient presenting with cough. A total of 10 fourth-year students were trained to serve as standardized patients in a series of 3 small group sessions, each lasting 60 minutes . During Placed at the mid point of the academic year, the OSCE provided us with the opportunity to evaluate the short-term effectiveness of the small group seminar on students' communication skills. We reviewed the videotapes of 52 students who had attended the seminar  and 53 students who had not at the time of the OSCE (the control group). The group assignment was based on the sequential order of clinical rotations, arbitrarily set by the medical school officials. The time intervals between the seminar and the OSCE ranged from 1 week to 5 months. Students were asked to provide informed consent using a form that had been approved by the Institutional Review Board at the Nagoya University Hospital.establish initial rapport, survey patient's reason(s) for the visit, determine the patient's chief concern, elicit and understand the patient's perspective, manage flow – provide the structure for the interview, and use of relationship building skills). The performance was rated on a 4-point scale labelled as good, satisfactory, insufficient and poor. The skills items were selected for their association with improved patient outcomes. They were derived from evidence-based communication assessment tools  [The interview rating form was created by one of the authors (KM) and includes 16 essential communication skills items. They are grouped into 6 communication tasks that should be accomplished during the initial 5 minutes of an encounter (ication) ,10,11. TTwo staff members (KK, HW) were trained to serve as raters. The tapes were independently reviewed and scored using the students' communication skills rating scale. Ten arbitrarily selected videotapes of students' role-plays of a doctor-patient encounter during the small group seminar were used to ensure accuracy and inter-rater reliability. At the time of the research the raters primarily worked outside the University and did not participate in the teaching seminars. Thus they were blinded to the students' group assignments.From the 105 students who attended the OSCE, 2 did not return the consent form, 5 did not give permission for the video review, for 1 the videotape quality was too poor to be analyzed. Thus, a total of 97 videotapes were available for the analysis.A skill item was considered 'acceptable' if both raters scored the students' performance as 'good' or 'satisfactory.' It was labeled as 'unacceptable' if both raters scored the performance as 'insufficient' or 'poor.' When these two raters disagreed over the judgment about the students' performance (e.g., one rater scored the performance of a skill item as 'acceptable' and the other scored the performance of the same item as 'unacceptable'), a communication educator and researcher (KA) served as the tiebreaker. The overall disagreement rate between the two raters (KK, HW) was 21%. The raters disagreed more often on inviting the patient to tell the story chronologically (41%), actively responding to the patient's concerns and nonverbal cues (34%), and being attentive and empathic nonverbally (31%).We compared baseline characteristics of the two groups using t-tests for a continuous variable (age) and chi-square tests for categorical variables. To evaluate the effect of the educational intervention, the proportion of students with 'acceptable' performance was compared with those whose performance was unacceptable using chi-square tests for all 16 skills items. All statistical analyses were done by JS using Stat View Version 5.0 (SAS Institute Inc. North Carolina).th year and were interested in a future generalist career. For both groups combined, the mean age was 23.5 years and 38 % were women.Student characteristics including gender did not differ between the groups except that more students in the control group engaged in self study as a preparation for the OSCE (p < .05) Table . There wThe proportions of students who were judged to have performed as 'acceptable' for each of the 16 items are shown in Table A short, intensive small group seminar on medical interviewing appeared to have had an impact on some specific skills, pertaining to "eliciting and understanding the patient's perspectives." It did not seemed to have improved the skills associated with the other tasks: establishing initial rapport, surveying the patient's reason(s) for the visit, determining the patient's chief concern, and managing flow – providing the structure for the interview, and the skills for building relationships.There are several strengths of our study. First, this is one of the few empirical, controlled studies from a non-English speaking country. Even though the students were not strictly randomized into intervention and control groups, the assignment occurred arbitrarily by the administration, without regard to students' preferences or interests in medical interviewing. Thus, it is unlikely that the higher scores in the intervention group are attributable to self-selection. Although there was a significant difference between the groups in proportions of students who did a self-study for the OSCE, which might have caused the results of no difference in most of the skills, the other characteristics such as age and gender were similarly distributed Table . Second,Our study also has weaknesses that should be addressed. First, our teaching method was based on the research findings in Western world, and this is based on the untested assumption that these findings are equally valid in Japan. There is evidence that patient-physician communication patterns in Japan are different from those in the West. Previous research by Ohtaki and colleagues compared patient-physician communication patterns in Japan and the USA . It inclOne can hypothesize the reasons why the intervention appeared to make a difference to some communication skills competencies but not to others. One could speculate that the competencies that were not influenced by the intervention were either very easy in general or too difficult to acquire in such a short teaching session. For example, the skills for establishing initial rapport (greet patient and obtain the patient's name, introduce self and clarify roles) and skills for determining the patient's chief concern (ask closed-ended questions that are non-leading and one at a time, define the concern completely) may be already present from the outset or so easy to acquire that a self-study just before the OSCE would make no differences in scores between the groups regardless of the intervention. On the other hand, the skills for surveying the patient's reason(s) for the visit, which requires being open at the beginning of the interview, may be too difficult for students to demonstrate, with or without the intervention. In particular, only 9% in the intervention group and 6% in the control group demonstrated an acceptable performance for the skills of allowing patients to complete their opening statements. These very low scores may also indicate that during small group sessions, we did not emphasize enough the importance of not interrupting patients at the beginning of the interview. Another explanation is that 'content' skills (i.e., what we communicate) are easier for students to acquire than 'process' skills (i.e., how we communicate). Kurtz at al. noted that the skills for understanding patient's perspectives, which our intervention made a difference, are actually 'content' skills, not 'process' skills . One couThe results of this study suggest that a short, intensive small group seminar for Japanese medical students may have had an impact on specific communication skills, namely, skills for exploring how the illness or problem affected the patient's life, asking the patient about ideas concerning the illness or problem, and being attentive and empathic nonverbally at least in the short term. Further studies should be done to confirm this preliminary finding and to clarify the skills for which educational interventions could make a difference.The author(s) declare that they have no competing interests.KM contributed to the conception and design of the study, design and implementation of the educational intervention, interpretation of the data, and drafting of the manuscript.KK and KW contributed to the collection of the data and reviewing of the manuscript.KA contributed to the collection of the data and reviewing of the manuscript.JS contributed to the conception and design of the study, analysis and interpretation of the data and reviewing of the manuscript.NB contributed to the conception and design of the study, design and implementation of the educational intervention, interpretation of the data, and reviewing of the manuscript.The pre-publication history for this paper can be accessed here:


One estimate suggests that by 2010 more than 30% of a physician's time will be spent using information technology tools. The aim of this study is to assess the information and communication technologies (ICT) skills of medical students in Tanzania. We also report a pilot intervention of peer mentoring training in ICT by medical students from the UK tutoring students in Tanzania.Design: Cross sectional study and pilot intervention study. Participants: Fourth year medical students (n = 92) attending Muhimbili University College of Health Sciences, Dar es Salaam, Tanzania. Main outcome measures: Self-reported assessment of competence on ICT-related topics and ability to perform specific ICT tasks. Further information related to frequency of computer use (hours per week), years of computer use, reasons for use and access to computers. Skills at specific tasks were reassessed for 12 students following 4 to 6 hours of peer mentoring training.The highest levels of competence in generic ICT areas were for email, Internet and file management. For other skills such as word processing most respondents reported low levels of competence. The abilities to perform specific ICT skills were low – less than 60% of the participants were able to perform the core specific skills assessed. A period of approximately 5 hours of peer mentoring training produced an approximate doubling of competence scores for these skills.Our study has found a low level of ability to use ICT facilities among medical students in a leading university in sub-Saharan Africa. A pilot scheme utilising UK elective students to tutor basic skills showed potential. Attention is required to develop interventions that can improve ICT skills, as well as computer access, in order to bridge the digital divide. Developments in information and communication technology occur at an astonishing rate. The World Wide Web (WWW) doubled in size during the first 6 months of 2000 and by 2005 the number of Internet users is likely to pass the one billion mark . This haIn a world afflicted by poverty, debt and HIV, why has the digital divide continued to trouble so many academics and development policymakers? The basic concern is that the spread of information and communication technologies in developed countries is leaving the rest of the world behind.The development of online databases allows medical professionals throughout the developed world immediate access to hundreds of e-journals at the touch of a button, a striking contrast to the plight of many of their colleagues in developing countries who are forced to trawl empty libraries. Highlighting one of the greatest tragedies of the digital divide, it threatens the very communities that could benefit the most from the developments in ICT.Many programmes have concentrated on increasing the number and spread of telephones and computers . Other sDuring January and February 2003 we studied ICT skills of medical students at Muhimbili University College of Health Sciences (MUCHS), University of Dar es Salaam, in Dar es Salaam, Tanzania. We also report a pilot intervention of peer mentoring training in ICT by medical students during the elective period.MUCHS is the largest medical school in east Africa and the only public medical school in Tanzania. A lack of resources has resulted in the university library being filled with out-dated textbooks. The majority of its graduates go on to serve rural communities throughout Tanzania and its neighbouring countries, often as the only qualified doctor serving populations of over 100,000.Access to computer facilities is a key problem. The University of Dar es Salaam is aiming for a ratio of one computer for every ten students. One-hundred-and-twenty computers would therefore be required for the 1200 MUCHS students. Help from international donors has allowed MUCHS to secure the presence of 40 computers, but only 25 are fully functional at any one time. Of these, less than half were connected to the Internet or loaded with basic software, bringing the real ratio of students to adequately equipped computers to around 100:1. Most MUCHS students found commercial Internet cafes too expensive to use on a regular basis. The cost of one hour at an Internet café can often be as high as $1, an important limiting factor considering that over 50% of an estimated 36 million people live in extreme poverty, surviving on less than US $1 per day .j.murphy@chime.ucl.ac.uk at the Centre for Health Informatics and Multiprofessional Education (CHIME, ) in London, UK, to assess ICT skills amongst first year medical students (MD1s) attending University College London (UCL). The questionnaires were distributed to all MD4 students by Tanzanian student representatives, to be filled in independently, and were then collected by the representatives.The abilities and attitudes of the fourth year MUCHS medical students (MD4s) towards ICT was assessed using Questionnaire 1 see , an adapThe questionnaire addressed different ICT-related variables associated with generic skills Figure  and specInformation related to frequency of computer use (hours per week) and years of computer use was also gathered. Access to computers at home or at educational facilities, last time of computer use, reasons for use and resources for reference in students' medical studies was also evaluated.The data were analysed using SPSS v.11 to calculate frequencies and percentages. Pearson Rho coefficient was used to look at the correlation between variables, with logarithmic transformation where necessary. Comparisons of frequencies before and after tutoring were performed using Wilcoxon's test. Data are shown as mean (± SD) for normally distributed data and median (interquartile [IQ] range) for skewed data.Ninety two (72.7%) of 120 Tanzanian MD4s completed the questionnaires. 76% of them did not have a computer at home and 74% never use a computer as part of any course either at school or university. Only 48 students (52%) felt that they understood the basic terminology and concepts of computing.The mean (± SD) of the generic and specific ICT scores were 11.1 (± 7.6) (out of a maximum score of 33) and 7.7(± 4.1) (out of a maximum score of 16) respectively. The two scores were significantly correlated, r = 0.81, p < 0.001.The highest levels of competence, assessed using the generic ICT score, were for email, Internet and file management . The median years of computer use was 3 (2–5) years.The main reasons for using a computer during the last year was to communicate by email (75%), Internet navigation (33%), learning purposes (27%), and to prepare reports (22%). Only 21 students (23%) had ever consulted an electronic journal, and nearly 70% did not use any electronic resource. The participants unanimously agreed that medical students should receive training in the use of computers.During our time in Tanzania we piloted an ICT peer mentoring training scheme aimed at the fourth year Tanzanian medical students whose skills had been assessed. We utilized tutorials that had been developed by CHIME for underskilled UCL medical students . Topics The willingness of the Tanzanian students to participate was identified as vital to the success of the scheme, since one of our main concerns was that we would we be seen as a neo-colonial force trying to impose our values. This was not the case: the response from the students was overwhelmingly positive. The majority of those who returned the questionnaires wished to receive peer mentoring training.Four tutorials (1.0–1.5 hours) were conducted utilising MUCHS computers. The course covered computing skills such as file management, word processing, spreadsheet use, Internet access and email.Competence levels were compared for 12 students before and after 4 to 6 hours of peer mentoring training using Questionnaire 2 see . The genWe have assessed ICT competence of a representative sample of 4th year medical students in a Tanzanian medical school and have found substantial limitations in computing skills. A mean generic score of 11.1 would be equivalent to approximately 1 point (very basic) in each of the 11 skills studied. The corresponding score among first year UK medical students in 2002 was 18.5. Initially at UCL, students with an overall score of less than 10 are considered to have low skills and are offered peer mentoring training. Using this criterion, around 50% of the Tanzanian students would fall into the low skills category compared with 9% of first year UCL medical students in 2002. Scores amongst UCL medical students have been rising in recent years .The mean of the specific ICT score was 7.7, in comparison with 11.9 for first year UCL medical students. . The strong correlation between generic and specific scores suggests that the students were good at judging their own ability, thus decreasing a potential bias from self-assessment in our results.Two studies from Nigeria show that there is poor knowledge of computer use. Ajuwon  reports The Internet was the most common application utilised in our sample in Tanzania, especially for email communications (75%). This is concordant with the reports from Nigeria where 76.4% of first year clinical and nursing students in Ibadan  and 58% Around half of the Tanzanian students were able to print a document, cut and paste information from one application to another and word process an essay, letter or their CV. These results, clearly addressed through items in the specific ICT score, show us that even though the students are able to communicate by email and use the Internet, they cannot perform some basic skills necessary for working with files. The lack of confidence in use of packages such as PowerPoint and Excel was also noticeable in the reported score and in the reported reasons for using the computer.The median duration of computer use was 3 years, meaning that most doctors in training had their first contact with a computer at university. Access is one of the main issues when one considers the fact that the current student/computer ratio at MUCHS is 100:1, compared to 35:1 in Portugal, 9:1 in the UK and 5:1 in Norway . SeventyThese findings have substantial implications for addressing the digital divide in this population. These range from a marked limitation of knowledge of basic packages to the possibility of making incorrect assumptions that students who are able to communicate electronically are automatically able to perform basic tasks such as word processing. Graduates recruited by foreign institutions to pursue postgraduate training would face additional difficulties in their learning experience due to their problems with ICT. Also important is the fact that several academic institutions are now looking to expand their teaching programmes using online courses. Again, course organisers could erroneously enrol participants with a lack of basic ICT skills, based solely on the ability to communicate by email.The peer mentoring training piloted in the present study shows promising results, achieving a doubling of competence scores. This programme ,19, whicOur study has found a low level of ability to use ICT facilities among medical students in one university in sub-Saharan Africa. These findings reinforce the idea that more is needed to bridge the digital divide than simply increasing the number of computers. A pilot scheme utilizing visitors from the developed world to tutor basic skills showed potential, but it would be naïve to think that volunteers alone can bridge the gap. Some would argue that increasing the number and distribution of computers will eventually result in an improved skills base. Our experiences would support this. Tanzanian medical students are keen to learn new skills and are aware of the implications of being left behind in the technological revolution. The main concern of such a scenario is that without direct intervention the time required to attain these skills increases, to the further detriment of some of the world's most vulnerable societies.None declared.JCC, RM, MS, EJWY and JJM took part on the initial planning of the project. JCC, RM, MS and EJWY did the field work and the peer mentoring training to Tanzanian medical students. PA and JJM did the data input and analysis. MS, JJM and JCC wrote the initial version of the paper. All the authors contributed to subsequent versions of the paper.The pre-publication history for this paper can be accessed here:Questionnaire 1 Instrument used to assess baseline ICT skills amongst MUCHS medical students. ©Jeannette MurphyClick here for fileQuestionnaire 2 Instrument used to assess skills after the ICT peer mentoring training amongst MUCHS medical students. © Jeannette MurphyClick here for file


Cellular processes require the interaction of many proteins across several cellular compartments. Determining the collective network of such interactions is an important aspect of understanding the role and regulation of individual proteins. The Gene Ontology (GO) is used by model organism databases and other bioinformatics resources to provide functional annotation of proteins. The annotation process provides a mechanism to document the binding of one protein with another. We have constructed protein interaction networks for mouse proteins utilizing the information encoded in the GO annotations. The work reported here presents a methodology for integrating and visualizing information on protein-protein interactions.GO annotation at Mouse Genome Informatics (MGI) captures 1318 curated, documented interactions. These include 129 binary interactions and 125 interaction involving three or more gene products. Three networks involve over 30 partners, the largest involving 109 proteins. Several tools are available at MGI to visualize and analyze these data.Curators at the MGI database annotate protein-protein interaction data from experimental reports from the literature. Integration of these data with the other types of data curated at MGI places protein binding data into the larger context of mouse biology and facilitates the generation of new biological hypotheses based on physical interactions among gene products. Cellular processes require the interaction of many proteins across several cellular compartments. Interactions can range in stability from persistent, such as between members of a stable complex, to transient, such as binding while being phosphorylated. Determining the collective network of such interactions should provide insight into which processes the individual members participate, and how they may be regulated.Understanding protein interaction networks requires two steps. First, the interacting proteins must be identified, usually through some experimental methods. Secondly, the significance of the interaction networks needs to be assessed. Recently, there has been a focus on devising large scale screening methods to collect data on interacting proteins -3. AdditIn addition to the large screening efforts, a significant amount of specific protein-protein interaction data has been reported in the literature over the years. Quite often, these studies report on only a few interacting proteins. It is difficult to place these isolated, yet specific reports in the larger biological context and interconnect them with other data. Recently, there have been efforts to extract such literature-based interaction information using text mining , or combe.g. [Model Organism Databases (MODs) have been collecting diverse types of data about the genes and proteins from their respective organisms since the early 1990s e.g. -13). The. Thee.g.The Mouse Genome Informatics system (MGI) is the MOD for the laboratory mouse . MGI intThe Gene Ontology Consortium provides the biological community a structured vocabulary with which to enable consistent functional annotation of genes and gene products. . Guidelimurine protein gene product is investigated. Many of the detailed annotations have been added on a paper-by-paper basis using the MGI literature collection that contains primary experimental information about mouse genes from over 90,000 references. The accumulation and use of these papers in annotation has been, for the most part, undirected. However, the structure of the GO and the relationships among terms allow grouping of the gene products that share common annotations. Such strategies may reveal hitherto unsuspected relationships between these proteins.In the course of over six years, curators at MGI have made 79690 annotations to 15231 gene products using 3742 GO terms (All database statistics used in this paper are from the MGI release as of 7/30/04). The curation policy focuses on experiments in which the "Protein binding" (GO:0005515), as used by the GO in the Molecular Function ontology, is defined as "interacting selectively with any protein or protein complex" . This teAger. In the case of Ager . A curator at MGI has captured this information in an MGI GO annotation for Ager. For completeness, a curator also annotated the gene product of Hmgb1 with "protein binding" with an IPI to SPTR:Q62151, the protein product of Ager, using the same reference. In this case, these are the only "protein binding" annotations for either of these proteins. These annotations represent an experimentally tested interaction of two proteins.An example of GO annotation that includes "protein-binding" is shown for the gene product of e et al.  have demHcph (hemopoietic cell phosphatase), has been shown to bind both the protein product of Jak2 (Janus kinase 2)   and Klrnase 2) . JAK2 nnase 2) , but alnase 2) (, which inase 2) . KLRB1Bnase 2) , which nase 2)  of the gene product or the process it is involved in . A simple example is seen in the case of TIPIN (timeless interacting protein) Figure . It has ila gene . Howeverogenesis , and thunificant . These iTimeless .Cellular location may also be inferred from protein interactions. SOCS1  has "kinase inhibitor activity" (GO:0019210) and has been implemented in the "cytokine and chemokine mediated signaling pathway" (GO:0019221), and the JAK-STAT cascade (GO:0007259). However, its cellular location has not been documented in the available mouse literature. Analysis of the SOCS1 protein using predictive software such as Psort ) and SubThree networks involve over 30 partners, the largest involving 109 proteins Figure . Can we For the set of 109 proteins shown in figure Tools such as GO_Term_Finder  and its Finally, twelve of the proteins displayed in Figure The networks suggested by the collection of annotations to this GO term involve interactions that are more or less stable under experimental conditions. A gene product is shown to have protein binding activity by a variety of direct assays such as yeast two-hybrid screening , co-immuThe Mouse Genome Informatics system integrates not only data used for GO annotation, but also data on a variety of aspects of mouse biology including embryonic gene expression, alleles and their phenotypes, and chromosome location. The integration of these datasets allows for complex queries, such as "list all genes expressed in the liver at Tyler Stage 15, located on chromosome 12, annotated to "protein binding" AND "nucleus". The integration of protein-protein network visualization into such queries can aide in determining the significance of more complex interaction networks. By combining the above query with our graphical tools, it is possible to get a graphical view of all protein interaction networks in the nucleus of a 9.5 dpc mouse embryo. As annotation progresses and becomes more complete, these types of queries will become more and more informative.During the generation of the interaction sets, it was found that programs such as Graphviz, could easily visualize missing annotations based on the interaction of two proteins. When information about a protein comes from different sources, a curator that is curating a single reference may not necessarily record all of the information implied by a physical interaction, such as cellular location in the example above. Views such as Graphviz can help curators to spot missing data and they may at some point be useful in themselves to display annotations.Drosophila, and C. elegans interaction sets [MGI curators aggressively adopted the use of the "with" field when annotating to "protein binding" during the early stages of annotation efforts at the database. Similar networks may also be mined from the GO data sets available from the other model organism databases participating in the GO. Recently, Lehner and Fraser used GO annotation to analyze a human interaction set predicted from orthology to yeast, ion sets . The GO ion sets  and HPRDion sets  to guideWe have demonstrated that functional annotations curated via GO hierarchies can be used to obtain a summary set from independent annotations to "protein-binding" to form protein-protein interaction networks. The members of these protein-protein interaction sets can be further examined for additional shared GO annotations. Integration of these data with the other types of data curated at MGI places protein binding data into the larger context of mouse biology and will aid in the discovery of new biological knowledge based on physical interactions among gene products.Gene annotations for protein binding interactions are made by manual inspection of published literature. In every case, experimental evidence is supplied in the manuscript to support the interaction that is reported. Annotation of genes to other GO terms is made by a variety of methods including the conservative translation of functional information contained in SwissProt protein records, conservative inference from InterPro domains, and manual curation of the published literature.Data was obtained from the Mouse Genome Informatics system by use of custom SQL queries to collect all markers that had been annotated to "protein binding" or its children using the IPI evidence code. The protein sequence identifier in the "inferred from field" was matched to the appropriate gene in the database. The final output consisted of a two-column file with column 1 being the first protein, and column 2 the protein it binds. This formed the basic data set that was passed to Graphviz  for disp.The two column lists were also used as the basis for data files listing all unique genes in each network. These were then used for input files for GO_Slim Tool  and GO_TGraphViz on the Macintosh OS X platform is a product of Pixelglow . GraphViHJD conceived of and designed this study and implemented the graphical displays using Graphviz and analyzed the significance of the results. He has also been involved in contributing to the GO annotations. CH devised the Perl scripts used to parse out individual interaction sets and removal of redundancies. DPH helped to draft the manuscript and contributed to the annotations. JAB has been involved in the overall design of the GO project at MGI and of gene annotations in MGI overall, and has added critical revisions for important intellectual content to this paper.


Hepatitis C (HCV), hepatitis B (HBV), the human immunodeficiency viruses (HIV), and other viruses that replicate via RNA intermediaries, cause an enormous burden of disease and premature death worldwide. These viruses circulate within infected hosts as vast populations of closely related, but genetically diverse, molecules known as "quasispecies". The mechanism(s) by which this extreme genetic and antigenic diversity is stably maintained are unclear, but are fundamental to understanding viral persistence and pathobiology. The persistence of HCV, an RNA virus, is especially problematic and HCV stability, maintained despite rapid genomic mutation, is highly paradoxical. This paper presents the hypothesis, and evidence, that viruses capable of persistent infection autoregulate replication and the likely mechanism mediating autoregulation – Replicative Homeostasis – is described. Replicative homeostasis causes formation of stable, but highly reactive, equilibria that drive quasispecies expansion and generates escape mutation. Replicative homeostasis explains both viral kinetics and the enigma of RNA quasispecies stability and provides a rational, mechanistic basis for all observed viral behaviours and host responses. More importantly, this paradigm has specific therapeutic implication and defines, precisely, new approaches to antiviral therapy. Replicative homeostasis may also modulate cellular gene expression. Hepatitis C (HCV), HBV and HIV are major causes of premature death and morbidity globally. These infections are frequently life-long; Hepatitis viruses may result in progressive injury to the liver and cirrhosis, and death from liver failure, or hepatocellular carcinoma, while HIV causes progressive immune depletion and death from the acquired immunodeficiency syndrome (AIDS). Together, these infections cause millions of premature deaths annually, predominantly in "developing" countries. Other viruses replicating via RNA intermediaries cause similar morbidity among domestic and wild animal populations. While education, public health measures and vaccination (for HBV) have resulted in significant progress in disease control, therapy of established viral infection remains unsatisfactory.pol), enzymes that lack either fidelity or proofreading function [pol infidelity that introduces errors at ~1 × 10-5 mutations / base RNA synthesised.RNA viruses and retroviruses replicate, at least in part, by RNA polymerases (RNAfunction . During function  due to Rt = [RNA](t-1)ek, where [RNA]t is virus concentration at time (t) and k a growth constant. However, because of RNApol infidelity, wild-type (wt) virus will accumulate at [RNAwt]t = [RNAwt](t-1)•(1-ρ)•K1 and variant forms (mt) at [RNAmt]t ≈ ([RNAwt](t-1)•ρ + [RNAmt](t-1))•K1, where ρ is the probability of mutation during replication and K1 = ek. Therefore, while wild-type virus predominates early, replication (and intracellular accumulation) of variant virus and viral proteins will accelerate (in a ratio of ([RNAwt](t-1)•ρ + [RNAmt](t-1))/ [RNAwt](t-1)•(1-ρ) compared to wild type) and variant viral RNAs will rapidly predominate  represent the immune forces favouring viral clearance and Ve(t) viral forces promoting quasispecies expansion pressures at time (t).2. Let Ic required to clear virus are proportional to viral concentration [V], that is; Ve ∝ [V] (or Ve = ke [V] where ke is some constant), so that Ic required to clear one viral particle Ic(1) is less than that Ic required to clear 10 viral particles Ic(10).3. Assume immune pressures Ic(b or c) ≈ Ve(b or c).     Eq.14. At equilibrium ] ⇒ [Ve(b or c)], then immune clearance pressures must exceed viral expansion pressures at that time i.e. Ic(a) > Ve(a).     Eq.25. If I2–3 [V(a)] ≈ [V(b or c)]• 102–3, and Ic(b or c)= Ve(b or c) then immune clearance pressures at time A exceed those at time (B or C) by102–3 Ic(a) >Ic(b or c)• 102–3. That is, immune pressures fall by 102–3 between time A and B or C,  by 102–3 over days between time A and B or C?i) Why, and by what mechanism, would immune forces, or any other host defense mechanisms, fall by 10cause reduced viral replication. Evidence that prior HCV infection does not confer protective immunity against either heterologous HCV infection in chimpanzee [There is, of course, no evidence immune pressures fall, and very considerable evidence both antibody and adaptive T cell responses are increasing when viral replication is falling ,12. Thesimpanzee or eitherimpanzee  or heterimpanzee  human re10 molecules / day [11 molecules/day resulting in an average viral load of 1010 molecules/person [2–3 times the basal rate [Chronic viral persistence raises other issues; At steady state n), where (Mμ) is mutation rate and (n) genome size. Hepatitis C (a ~9200 bp RNA virus) RNApol introduces mutation at 10-5 substitutions/base, ρ≈0.912. However, for multiple (θ) replications cycles, ρ = (1-(1-Mμ)n)θ. After 20 replication cycles, occurring in <7 days in most patients [o≈7.5 × 10-22, meaning effective loss of sequence information, an outcome that should cause quasispecies extinction [pol infidelity are incorrect  or b) that innate viral mechanism(s) control RNApol fidelity and mediate selective replication of consensus sequence genomes. Thus, rates of viral mutation are tightly constrained by the necessity to retain sequence information. On the other hand, overly faithful template replication will restrict antigenic diversity, rendering virus susceptible to immune destruction and unresponsive to ongoing cellular changes. The necessity to retain sequence information by adequate replicative fidelity, and the later requirements (in terms of replicase ⇒ RNApol evolution) of viruses to access cells via evolving cell receptors and evade host defence mechanisms, has placed constraints on replicase (RNApol) function that dictate polymerase fidelity must be tightly, and dynamically, controlled  and mutation frequency (that is, polymerase fidelity) must be closely regulated Figure .pol to alter processivity and fidelity.That viruses capable of chronic persistence auto-regulate replication and mutation rates by replicative homeostasis. Replicative homeostasis results when RNA polymerase end-translation products (envelope and contiguously encoded accessory proteins) interact with RNASubstantial clinical and in-vitro evidence, including the kinetic paradox indicate viruses auto-regulate. During successful antiviral treatment levels of virus fall sharply ,52,53,57pol output depends on the functionality of protein(s) translated from any RNA synthesized by RNApol. For viruses, and their polymerase, evolutionary survival – i.e. whether the polymerase, and its viral shell, avoids immune surveillance, gains access to cells, and replicates to infect other hosts – is a function of the properties that the sequence, topological variability and structural integrity of envelope proteins impart. RNA polymerase is responsive to and is influenced by accessory proteins that induce conformational changes to alter both processivity and fidelity [Most cellular enzymes are under some form of kinetic control, usually by product inhibition. While simple negative-feedback product inhibition is sufficient to control enzyme reaction velocity and the rate of product synthesis, it is inadequate to ensure the functional quality of any complex molecules – including proteins – synthesised. The functionality of RNAfidelity ,31, reprpol attributes, fidelity and processivity, be dynamically modifiable, and controllable. These linked functional requirements imply a dynamic nexus between the functional output of RNApol (i.e. envelope proteins) and that polymerase.Evolutionary stability requires adaptability to changing environmental circumstances. For viruses, an ability to modulate replication and mutation rates dynamically in response to cellular changes is essential. Viruses intrinsically capable of adaptation to environmental changes, including variations in host density, and evolving cell receptor polymorphisms, immune and other host responses, among other variables, will enjoy a competitive advantage over viruses lacking innate responsiveness. Contrariwise, self-replicating molecules, including viruses, that lack innate adaptability, for whom replication is contingent upon a chance confluence of appropriate cellular conditions – including permissive cell receptors, absence of cell defences and so on – are highly vulnerable to extinction by both adverse environmental changes and competition for scarce intracellular resources by molecules capable of adaptation. For viruses, this adaptability requires antigenic and structural diversity be controlled and, in turn, that means the two critical RNAPol); ii) that these Env :RNAPol interactions alter both polymerase processivity and fidelity; iii) that wild-type (consensus sequence) Envwt :RNAPol complexes cause more rapid, less faithful RNA replication than variant (variant) Envmt :RNAPol complexes. There is solid evidence for each requirements of replicative homeostasis.Systems capable of homeostatic regulation (auto-regulation) have the following characteristics: i) an efferent arm that effects changes in response to perturbations of an equilibrium; ii) an afferent arm that measures the systems response to those changes; iii) mechanism(s) by which i) and ii) communicate. The mechanism of viral autoregulation – Replicative Homesostasis – described here requires: i) that viral envelope (Env) proteins interact with viral RNA polymerases (RNAPol processivity and fidelity.A large body of literature, for many viruses, establishes an important relationship between envelope and polymerase proteins and documents that Env proteins influence both RNAFirst, for HIV, overwhelming evidence suggests HIV polymerases properties, and those of related retroviruses – for example, simian immunodeficiency virus (SIV) and the feline immunodeficiency virus (FIV) – are influenced by Env proteins  antibodies, whereas failure to produce anti-E2 was associated with viral clearance [highly paradoxical result difficult to rationalize unless E2 proteins are important for sustained HCV replication, as we argued previously [Second, for HCV, many separate observations document HCV replication and polymerase functionality is dependent on envelope proteins: i) HCV viral genotypes are defined by sequences of either envelope or polymerase regions ,73,74 anlearance , intuitieviously . vi) Fineviously ,67 an obeviously .envelope genes may change envelope protein conformation and therefore alter normal envelope/polymerase interactions, thus altering processivity and fidelity of the replication complex. This latter interpretation is convincingly supported by data demonstrating that abnormal polymerase function of HBV envelope variants is reversed by co-transfection of Hep G2 cells with clones expressing wild-type envelope sequences [Third, HBV envelope and polymerase protein genes have overlapping open reading frames and significant alterations in envelope and polymerase gene and protein sequences cannot, therefore, occur independently, a genetic nexus again implying an important functional relationship. Mutations in envelope sequences occurring spontaneously  followinequences  and is fequences .ic ≈ 107–8 M-1, inhibition Ki ≈ 109 M-1s-1) [Fourth, studies of the coliphage Qβ demonstrate phage coat proteins bind to genomic RNA to strong M-1s-1)  RNA repl M-1s-1) . This in M-1s-1) , suggest M-1s-1) . An impr M-1s-1) , brome m M-1s-1)  and othe M-1s-1) . Thus, tpol in a series of feedback epicycles linking RNApol function, RNA replication and protein synthesis , are inevitable outcomes that result from equilibria reached because of these interactions  and variant (Mt) envelope proteins on RNAs Figure , 5. Intrpol allows determination of viral mutation rates. Envelope proteins, as opposed to other viral products, are the obvious products to examine for functional variability, and must form part of the afferent arm necessary to "sense" perturbations in the viral equilibrium. While other viral products could be "sensed" to gauge effective viral replication, only functional measurement of envelope protein concentration and topological variability simultaneous measures both the rate of viral replication and envelope functions – properties determined by envelope structure and antigenic diversity – essential for viral survival; immune escape and cell access. Furthermore, envelope and polymerase proteins are typically coded at transcriptionally opposite ends of the viral genome; replication contingent upon a dynamic nexus between envelope and polymerase proteins is, therefore, a functional check of the integrity of the entire viral genome. Importantly, this facet of replicative homeostasis is a direct mechanism of Darwinian selection operating at a molecular level, that ensurs preferential selection and replication of "fit" viral genomes, and maintenance of genotypes (species).Viral polymerases are clearly the effector mechanism – the efferent arm – that determines rate of viral RNA replication and mutation. The afferent arm needs to measure both the rate of viral replication and degree of viral mutation. Intracellular envelope concentrations are a direct function of effective viral replication, while competition between wild-type and variant envelope proteins for interaction with RNAfunctionally with any mutation affecting envelope sequences  to reset replicative equilibrium (by changing replication rate or mutation frequency or both), stable equilibria will still result providing the sum effect of variant proteins encoded within the envelope ORF is to decrease RNApol processivity (v) and mutation (Mu) frequency relative to wild-type protein polymerase interactions.Viruses, notably HIV, produce many accessory proteins (such as HIV Nef, gag, rev and HBeAg) that affect viral replication and mutation rate. However, these proteins are encoded within envelope open reading frames (ORFs) or are contiguous with them and are likely to alter s Figure . While tWt] or reducing intracellular [Envmt] should accelerate viral replication and mutation. In fact, observations relevant to every aspect of this hypothesis have been reported in a variety of systems and circumstances. All outcomes are completely consistent with those predicted by replicative homeostasis. Replicative homeostasis predicts, for example, HCV E2 proteins derived from genotype 1 HCV sequences would reduce HCV replication when administered to patients with heterologous HCV infection (genotypes 2,3 or 4, for example) and studies examining heterologous envelope proteins as direct RNApol inhibitors are underway.This hypothesis is simply tested. Manoeuvres that increase intracellular concentrations of variant envelope proteins or decrease wild-type envelope proteins should inhibit viral replication and reduce mutation rates. Conversely, manoeuvres increasing intracellular [EnvReplicative homeostasis immediately resolves the paradox RNA viral quasispecies stability and explains how these viruses persist and, thereby, cause disease. Replicative homeostasis also explains the initial decline of viral replication, resolving the kinetic paradox, rationalizing the dynamics of chronic viral infection and other enigmatic and unresolved viral behaviours. Most importantly, replicative homeostasis implies a general approach to antiviral therapy.Wt: RNApol interactions that, in turn, increase polymerase processivity but reduce fidelity accelerating synthesis of variant viral RNAs and, consequently, increased translation of antigenically diverse proteins, reactively driving quasispecies expansion and generating the extreme antigenic diversity of RNA quasispecies. Alternatively, in the absence of immunological recognition, variant envelope / polymerase interactions predominate, restricting viral replication and mutation, thus maintaining basal output of consensus viral sequences, thus maintaining genotype. Immune escape and maximal cell tropism are inevitable consequences of the potential antigenic diversity generated by RNA replication mediated by the reactive equilibria of replicative homeostasis.The equilibria formed by replicative homeostasis are responsive to disturbance of envelope concentrations ensuring viral mutation is neither random nor passive but highly reactive to external influence: Sustained reduction of viral envelope (by immune or other mechanisms) would favour high affinity Env20 (about 1026) possible conformations, greatly exceeding the ~1010 antibody [5–7 viral equivalents / ml they are not likely to be any more effective at 108–11 eq/ ml.Potential viral antigenic diversity is numerical superior to any immune response; Theoretically, a small envelope protein of 20 amino acids could assume 20antibody  or CTL rpol inhibition would cause rapidly progressive or fulminant disease , while inadequate replication or generation of diversity will result in viral clearance  will alter extracellular concentrations of Env proteins, thus changing intracellular envelope concentrations once extracellular /intracellular Env concentrations equilibrate. Therefore, antibodies to heterologous envelope proteins – developing, for example, during immunization against other viruses or heterotypic co-infection – will reduce relative intracellular concentrations of variant envelope, favouring RNApol:EnvWt interactions, thus enhancing replication and increasing mutation rates, a prediction confirmed in practice [Perturbations of relative intracellular wild-type and variant envelope concentrations alter RNApractice ,56. Contpractice  – would practice , Dengue practice , Murray practice , Ebola [practice  Coxsackipractice  and othepractice  or tetanpractice  and Dengpractice  co-infecpractice , are alspractice ; persistpractice ; long-tepractice ; spontanpractice   transmission, a prerequisite for viral survival on an evolutionary time scale Figure . A subtlpol properties  and modulate mutations introduced into the RNA templates RNApol synthesises, a subtle form of "quality control" is exerted over protein synthesis [Although proposed specifically to explain RNA viral quasispecies stability, replicative homeostasis is, fundamentally, a mechanism that regulates RNA transcription and modulates protein expression. If proteins (i.e. phenotype) modulate RNAynthesis . This meynthesis , a reproynthesis  and, by pol transcription by DNA viruses, cellular micro-organisms , and eukaryotic cells, subtly modulating cell-surface protein expression, via replicative homeostasis, to mediate immune escape, control cell division and differentiation, or other functions would not be surprising.Finally, accessory proteins that alter the processivity and fidelity of both DNA-dependent RNA polymerases  and DNA-


Severe acute respiratory syndrome (SARS) emerged in later February 2003, as a new epidemic form of life-threatening infection caused by a novel coronavirus. However, the immune-pathogenesis of SARS is poorly understood. To understand the host response to this pathogen, we investigated the gene expression profiles of peripheral blood mononuclear cells (PBMCs) derived from SARS patients, and compared with healthy controls.The number of differentially expressed genes was found to be 186 under stringent filtering criteria of microarray data analysis. Several genes were highly up-regulated in patients with SARS, such as, the genes coding for Lactoferrin, S100A9 and Lipocalin 2. The real-time PCR method verified the results of the gene array analysis and showed that those genes that were up-regulated as determined by microarray analysis were also found to be comparatively up-regulated by real-time PCR analysis.This differential gene expression profiling of PBMCs from patients with SARS strongly suggests that the response of SARS affected patients seems to be mainly an innate inflammatory response, rather than a specific immune response against a viral infection, as we observed a complete lack of cytokine genes usually triggered during a viral infection. Our study shows for the first time how the immune system responds to the SARS infection, and opens new possibilities for designing new diagnostics and treatments for this new life-threatening disease. Severe acute respiratory syndrome (SARS) emerged in 2003, as a new epidemic form of life-threatening infection . As of SDuring a viral infection, most cell types in the body respond by secreting high levels of type 1 interferons (IFN-α and IFN-β) . IFN-α/βTo study the differential immune-gene expression patterns induced by SARS coronavirus, PBMCs from patients with SARS and normal subjects were examined using microarray technology. We shooed to use PBMCs as these cells are more easily obtained from patients compared to other infected tissues, and the SARS-CoV has recently been shown to infect PBMCs ,10. The P < 0.01) was performed and significant genes were selected. Cluster analysis was performed to find distinct groups of genes that were significantly changed. The criteria used for cluster analysis is different from filter criteria used for differentially expressed genes. 248 genes were selected for Hierarchical clustering  but share a common three-dimensional structure ,22. Tissvia remodeling of the actin component of the membrane [There is also up-regulation of expression of genes, such us, Bactericidal Permeability Increasing Protein (BPI) and Carcino Embryonic Antigen related Cell adhesion Molecule 8 (CEACAM 8 or CD66b). BPI is released from activated neutrophils and is an endogenous antibiotic, which rapidly kills Gram negative bacteria by high affinity binding to the LPS component of the cell wall . CEACAM membrane . Up-regumembrane .There is moderate up-regulation of the kappa light chain of the Nuclear Factor NFκB 1A) and the B-cell lymphoma 3-encoded protein (BCL3) , or genes involved in complement mediated cytolysis, suggesting that the immune response against the SARS-CoV may be different from other viral infections, or that the virus may be using a unusual strategy to evade the host immune system and cause the pathogenesis and mortality.This differential gene expression profiling of PBMCs from patients with SARS, strongly suggests that the response of SARS affected patients seems to be mainly an innate inflammatory response, rather than a specific immune response against a viral infection. There is no significant level of up-regulation of MHC-I genes or major cytokines, including IFNs, or complement mediated cytolysis, suggesting that the immune response against the SARS-CoV may be different from other viral infections or that the virus may be using a different strategy to evade the host immune system and cause the pathogenesis and mortality. The severity of the disease does not seem to change the genetic profile of the PBMCs: 5 of our patients were in intensive care, whereas the other 5 patients did not require intensive care, at the time the blood was taken. However, no substantial gene-expression differences were observed that could correlate to disease severity. Moreover, we confirmed  that cytokines usually associated with viral infections (such as interferons and other cytokines) were not detected in the SARS samples, but were triggered in another viral infection (influenza patients). We also showed that genes triggered in the SARS patients were not triggered in the influenza patients.Our study shows, for the first time, how the immune system responds to the SARS infection and opens new possibilities for designing new diagnostics and treatments for this new life-threatening disease.260 nm.Our study included ten adult patients (S1-S10) who were diagnosed with SARS according to the World Health Organization (WHO) SARS criteria and admitted to the Tan Tock Seng Hospital, Singapore, for treatment and four additional normal human subjects (C1-C4). Our protocol was approved by the Tan Tock Seng Hospital Research Ethics committee. Peripheral blood was collected by vein-puncture of the brachial vein; mononuclear cells (PBMC) were prepared using histopaque (Sigma- Aldrich, St. Louis, MO) according to manufacturer's recommendations. Sample preparation and processing procedures were carried out as described in the Affymetrix Gene Chip Expression Analysis Manual (Affymetrix Inc., Santa Clara, CA). Briefly, total RNA was extracted from PBMCs, using Trizole method (Invitrogen, Carlsbad, CA) and further purified using RNeasy columns according to manufacturer's instructions . Integrity of total RNA was confirmed by formamide gel electrophoresis and quantification was carried out by measuring the Ain vitro transcription (IVT) using T7 RNA polymerase at 37°C for 5-6 hr as per manufacturers' instructions (ENZO labeling kit, Ambion, USA). Labeled cRNA obtained after IVT was purified using RNeasy columns (Qiagen). Purified cRNA was fragmented using fragmentation buffer (40 mmol/L Tris acetate, pH 8.1, 100 mmol/L Potassium acetate, 30 mmol/L Magnesium acetate) at 94°C for 35 min.5 μg of total RNA was used to synthesize double stranded cDNA using T7-(dT24) oligonucleotide primer and Superscript reverse transcriptase (Invitrogen). The resultant cDNA was purified by phenol:chloroform extraction and ethanol precipitation in presence of 7.5 M ammonium acetate. 1 μg of purified cDNA was subsequently used to synthesize biotin labeled cRNA by Fragmented cRNA (10 to11μg/ probe array) was used to hybridize to human focus array (HG-Focus Array) at 45°C for 16 hr with constant rotation of 60 rpm in a Gene chip hybridization oven 640 (Affymetrix). The chips were washed and stained using Gene chip fluidics Station 400 (Affymetrix). Staining was performed using streptavidine phycoerythrin conjugate (SAPE, Molecular Probes, Eugene, OR), followed by the addition of biotinylated antibody to streptavidine (Vector Laboratories, CA), and finally with streptavidine phycoerythrin conjugate. Probe arrays were scanned using Agilent Gene Array Scanner Series US 74900593 (Agilent technologies, USA).P value for each gene. Any gene for which this P value was < 0.01 was considered to be differentially expressed. We selected 186 genes for further analysis that met the following criteria:An absolute expression analysis was performed using Microarray Suite Software 5.0 (Affymetrix) and relative mRNA expression levels were expressed as plus or minus fold changes compared to normal controls. Each chip was scaled to an overall intensity of 500 to correct for minor differences in overall chip hybridization intensity, and to allow comparison between chips. The data from ~8700 genes was imported in to MicroDB 3.0 and Data Mining Tool 3.0 (Affymetrix) for further analysis. Pearson Correlation coefficient (r) was used to ensure the reproducibility of the data using signal from normal samples. T-test was performed to identify genes that were differentially expressed in SARS patients over normal samples. The statistical significance of the differential expression of any gene was assessed by computing (i) Changes in expression of at least 2 fold higher or lower comparing the normal(ii) Signal >500P < 0.01 and(iii) Detection (iv) Genes which met the above criteria in at least 30% of samples.Finally, data representing 10 SARS patients fold changes over control samples were averaged..Genes were annotated according to biological process using the Gene ontology: tool for the unification of biology from the Gene Ontology Consortium . The comUnweighted average linkage Hierarchical clustering was applied for samples using the 'Genesis' software . Genes w® instrument (Roche) as follows: reverse transcription at 61°C for 20 min, initial denaturation at 95°C for 2 min; amplification for 45–65 cycles of denaturation (95°C, 5s, ramp rate 2°C /s), annealing  and extension (72°C, product length [bp]/25 s, ramp rate 2°C /s). A single online fluorescence reading for each sample was taken at the end of extension step. Quantitative results were expressed by identification of the second derivative maximum points, which marked the cycles where the second derivatives of the fluorescence signal curves are at maximum. These points were expressed as fractional cycle numbers. Then, these cycle numbers were plotted against the logarithm of the concentrations of serially 2-fold diluted standard samples to obtain a standard curve. The concentrations of unknown samples were calculated by extrapolation from this standard curve. Positive sample specificity was confirmed by determining the melting curve .Real-time PCR was performed for ten genes, namely: Lactoferrin (Primers: 5' tcg tcc tgc tgt tcc tcg ggg 3' and 5' tcc agc ggt cct gcg aag gcc 3'); Lipocalin (Primers: 5' aag ccc ctg ctc ctg gcc atc agc 3' and 5' cga cct gat gct gta tgc cac gtg 3'); S100P (Primers: 5' cat gat cat aga cgt ctt ttc 3' and 5' aca cga tga act cac tga agt 3'); TLR2 (Primers: 5' gta tct gca agg gca gct cag gat 3' and 5' ttc ctc aag gaa ggt aag tcc agc 3'); FCGR3A (Primers: 5' ctc cgg ata tct ttg gtg act 3' and 5' tgc aga gca gtg ttc ttc cag 3'); IFNA (Primers: 5'atg gcc ttg acc ttt gct tt 3' and 5'tgg aag att tcc tca tag c 3'); IFNB (Primers: 5'atg acc aac aag tgt ctc ctc caa a 3' and 5' ttc ttc cag gac tgt ctt ca 3'); IL-12 p40 (Primers: 5'atg tgt cac cag cag ttg gtc atc 3' and 5'ctg aat gtc aaa tca gta ct 3'); TNFA (Primers: 5'gag tga caa gcc tgt agc cca tgt tgt agc 3' and 5'gca atg atc cc a aag tag acc tgc cca gac 3'); GAPDH (Primers: 5'acc aca gtc cat gcc atc ac 3' and 5'tcc acc acc ctg ttg ctg ta 3'). For amplicon detection, the Light Cycler RNA Master SYBR Green Kit (Roche) was used as described by the manufacturer. PCRs were performed in a LightCyclerRR carried out the sample preparation, hybridization and drafted the manuscript. MJ carried out statistical analysis, data filtration, and data deposition and helped with manuscript preparation. LYH, HHC and DT provided the blood samples and the clinical comments for the study. BPL advised on study design and development. AJM devised and coordinated the study, revised and finalized the manuscript. All authors read and approved the final manuscript.


Escherichia coli bacterium confronts oxidative stress, an enzyme that stands at a central point in the amino acid supply line for protein synthesis is rapidly and reversibly inactivated.Among the many stresses faced by a cell, one of the most serious is exposure to oxidizing agents. An invading organism, for example, must defend itself against the oxidative assault mounted by a host's immune system. Since oxidation can rapidly destroy many types of molecules, cells have developed multiple means of protecting against it. Rapid mobilization of these defenses requires diversion of resources and temporary suspension of many normal cellular functions, including protein synthesis. In a new study, Elise Hondorp and Rowena Matthews show that when the E. coli through the action of the enzyme cobalamin-independent methionine synthase (MetE), which makes up between one and five percent of all protein in the cell. Thus, by turning off MetE in the face of oxidative stress, protein synthesis can be slowed or stopped, freeing cellular resources to be used elsewhere.Of the twenty amino acids that make up proteins, methionine plays a special role. It is the first amino acid added to every polypeptide chain, and without it, protein synthesis grinds quickly to a halt. Methionine is formed in E. coli, MetE is acutely vulnerable to oxidation under a variety of conditions. These results are in accord with a companion study by Leichert and Jakob, also in PLoS Biology, showing that MetE is one of the proteins most sensitive to oxidative damage. When the active site of MetE is stressed by an oxidant, Hondorp and Matthews show, it is temporarily blocked by the attachment of a glutathione subunit. Glutathione is a small molecule that includes a reactive sulfur atom. During “glutathionylation” of MetE, a sulfur on an amino acid of the enzyme is oxidized and links up with a sulfur on glutathione. This study shows that glutathionylation occurs only on a specific amino acid (cysteine 645), which recent structural work indicates sits at the entrance to the active site.Hondorp and Matthews show that in Attachment of the bulky glutathione subunit to this cysteine would be expected to block the active site, thus shutting down enzymatic activity. The results indicate that glutathionylation does indeed prevent activity of the enzyme, and furthermore, causes the enzyme to change its three-dimensional form. As the oxidative challenge abates, glutathionylation may be reversed, and the normal activity of the enzyme restored. Thus, glutathionylation of MetE may also serve to protect the active site from permanent oxidative damage. While glutathionylation is a common strategy in eukaryotes, MetE is so far one of the few proteins in bacteria known to be affected in this way.E. coli. Methionine is a precursor for the signaling molecule AI-2, which is released extracellularly and appears to serve as a key indicator of colony health and density. This information enables neighboring cells to better respond to changing and potentially hostile environments. Thus, the glutathionylation and inactivation of MetE may provide a simple mechanism by which a bacterium and its neighbors attempt to deal with oxidative stress.Shutting down MetE and limiting methionine production may play another important role, namely, communicating the bacterium's metabolic state to other nearby


Carcinoma of urinary bladder is one of the leading causes of death in India. Successful treatment of bladder cancer depends on the early detection & specific diagnostic approaches. In the present study, microsatellite instability (MSI) has been evaluated as a prognostic marker in patients with superficial urinary bladder cancer in lower urinary tract for determining risk of recurrence.A total of 44 patients with bladder tumors diagnosed with Transitional Cell Carcinomas [TCC] from lower urinary tract were selected for the study. Tumors were staged and graded according to AJCC-UICC (1997) classification and patients were followed with cystoscopy as per the protocol. Polymerase chain reaction (PCR) was done to amplify microsatellite sequences at mononucleotide BAT – 26, BAT – 40, TGFβ RII, IGFIIR, hMSH3, BAX and dinucleotide D2S123, D9S283, D9S1851 and D18S58 loci in blood (control) and tumor DNA. PCR products were separated on 8% denaturing polyacrylamide gel and visualized by autoradiography.MSI was observed in 72.7% of tumors at BAT – 26, BAT – 40, D2S123, D9S283, D9S1851 and D18S58 loci. Good association of MSI was seen with tumor stage and grade. MSI – High (instability at > 30% of loci) was frequently observed in high stage (40.6%) and high grade (59.4%) tumors. Of 24 tumors of Ta-T1 stage with different grades, 11 (9/18 high grade and 2/6 low grade tumors) recurred in the mean duration of 36 months. MSI positivity was significantly high in patients who had one or more recurrences (p = 0.02 for high grade and 0.04 for low grade tumors).MSI may be an independent prognostic marker for assessing risk of recurrence in superficial tumors irrespective of the grade. Further studies on progression would help in stratifying the patients of T1G3 for early cystectomy vs bladder preservation protocol. Amongst the genitourinary cancer, carcinoma of the urinary bladder is one of the leading causes of death in Indian population. Transitional cell carcinoma (TCC) is the commonest histopathological variant where stage and grade are the two important prognostic factors to know the clinical behavior of these tumors. Superficial tumors with different grades behave differently e.g. tumors with high grade recur early and progress to invasive bladder cancer very soon. This behavior of same stage of the tumor but with varied grades is attributed to genetic alterations.Bladder cancer manifesting from superficial to aggressive muscle invasive tumors undergoes a sequence of genetic alterations. Primary chromosomal aberrations are associated with tumor development while secondary chromosomal abnormalities lead to progression to a more advanced stage. A frequent loss of heterozygosity (LOH) on chromosomes 4, 5, 8, 9, 11 and 17 is considered a major event in the carcinogenesis of bladder cancer .Defects in mismatch repair (MMR) genes result in replication errors and genetic instability. Faulty mismatch repair, generally observed as somatic variation in size of microsatellites (short tandem repeat sequences in genome) is referred as microsatellite instability (MSI) . MSI andA successful treatment of bladder cancer depends on early detection and more specific diagnostic approaches. Preneoplastic changes of the bladder epithelium or superficial tumors as an early event precede invasive bladder carcinomas. Though the higher grade and stage portends a worst prognosis, superficial tumors of same stage and grade have different outcome in different patients. Due to limited utility of these prognosticators in patients with superficial bladder tumor, there is a need to analyze new molecular parameters in predicting the prognosis and risk of recurrence. The following study is based on MSI analysis in tumor tissues to evaluate its utility as a marker for early detection of recurrent bladder carcinomas from lower urinary tract and thus help in deciding treatment modalities.Total of 44 patients with male & female ratio of (42:2) of TCC with a mean age of 62 years were included for the study after the approval from ethical committee. All the patients selected for the study were not having any familial cancer syndrome or had previous history of cancer to the best of our knowledge.All the tumors were resected transurethrally from the lower urinary tract. Part of superficial tissue specimen obtained after transurethral resection of bladder tumor (TURBT) was collected immediately in liquid nitrogen. Matched control sample  from all patients was collected in 200 μl of 0.5 M EDTA. The tumor stage and grade was assigned according to the TNM classification by American Joint Committee on Cancer (AJCC-UICC, 1997) . Tumors Superficial tumor tissue specimens of histologically confirmed bladder tumors and peripheral blood (frozen) of the same patient were processed for DNA isolation using phenol-chloroform extraction method .2, 0.25 units of Taq polymerase (MSI, Fermentas), 100 ng DNA and 2 μCi [α-32P] dCTP (specific activity: 4000 Ci/mM) (BRIT, India) in a volume of 25 μl. PCR conditions involved an initial denaturation at 95°C for 3 min followed by 30 cycles (95°C for 1 min, 50°C to 60°C for 2 min and 72°C for 3 min) and a final extension at 72°C for 8 min. PCR products were mixed with equal volume of formamide loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% Xylene cyanol), denatured for 5 min at 95°C and loaded onto 8% polyacrylamide gel containing 7 M urea. Gels were run at 55 W for 2 hours, transferred onto a Whatman sheet followed by an exposure to X-ray film (Kodak) for desired time and then developed.Table The tumor was designated unstable if its PCR product had altered band pattern when compared to alleles in corresponding matched blood DNA . Out of Statistical tests, including, 2 × 2 contingency table, Fisher's exact probability test (one or two tailed), Karl Pearson's correlation test were applied to assess the relation between the microsatellite instability in tumors and clinicopathological parameters. A student t test was applied to compare the number of genomic alterations between tumors of different grades and stages.A panel of ten microsatellite markers situated on chromosomes 2, 3, 5, 6, 9, 18 and 19 were screened to look for microsatellite instability in superficial tumor tissues and compared with blood DNA. Alterations were detected in 32 of 44 patients (72.7%).Out of the six mononucleotide microsatellite markers analyzed, only BAT – 26 and BAT – 40 in 17.7% and 24.4% of the cases could demonstrate changes respectively while TGFβ RII, IGFIIR, hMSH3 and BAX were microsatellite stable (MSS). BAT-26 & BAT-40 exhibited borderline changes in tumor DNA as compared to control DNA. We also sequenced the tumor & normal PCR product for these microsatellites to prove the change. The dinucleotide markers- D2S123, D9S283, D9S1851 and D18S58 exhibited altered electrophoretic migration pattern in 15.5%, 40%, 31.1% and 17.8% bladder tumors respectively. The most frequent microsatellite alteration was detected on the markers of chromosome 9 (D9S283 followed by D9S1851)  and ≥ G2 grade tumors (Fisher's exact probability test, two tailed, p = 0.08) was observed Table .Total genomic alterations were analyzed among different stages and grades. Changes were comparable among (24/44) T1-Ta and (20/44) T2-T3 tumors that included 35 (13 insertions, 6 deletions, 10 LOH and 6 BA) and 31 (10 insertions, 8 deletions & LOH and 5 BA) respectively (p > 0.05). High grade (G2-G3) (37/44) carcinomas encompassed 63 alterations (24 insertions, 14 deletions, 15 LOH and 10 BA) and low grade (G1) (7/44) bladder tumors had only 5 (1 each deletion & BA and 3 LOH) (p = 0.01)Recurrence of tumors (mean duration of 36 months) was correlated with MSI in 24 superficial Ta-T1) tumors in patients who turned up for regular follow up  and invasive bladder carcinomas (T2-T3) suggesting the role of MSI in progression of bladder cancer as well. However, strong association of MSI – H with T2-T3 and G2-G3 was observed. MSI at ≥ 30% of loci has been found in 59.4% (19/32) of TCC bladder, which is not in accordance with reported earlier ,12. A goWAF/C1P1, Rb, c-erb B-2 are reported to be associated with tumor recurrence and progression but little is known to address MSI [Bladder cancer presents as superficial tumor in 75% of the patients, which can easily be removed by transurethral resection (TUR). Around 60–80 % of these treated patients develop recurrence in due course of time. Out of them, 15% progress to higher grade and stage. With so much potential for recurrence, patients need to be followed up with cystoscopy at regular intervals. Although many new tumor markers have been proposed but all have limitations with respect to execution and interpretation in predicting the recurrence of bladder tumors . Among tress MSI . MSI anaMSI has been observed to play important role in evolution, initiation and progression in bladder tumors. Patients with high grade superficial disease are reported to have higher incidence of MSI. Also high frequency of MSI in superficial tumors showing recurrence irrespective of grade may provide an indication for more radical approach to improve the survival.The author(s) declare that they have no competing interests.MV carried out the molecular genetic studies, participated in analyzing the data & drafted the manuscript. AM provided the clinical material & information, helped in analyzing the data & designing the manuscript. RDM helped in manuscript drafting. BM participated in its designing of the study & manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: