Lymphoma-associated hemophagocytic syndrome (LAHS) occurs in mostly extra nodal non-Hodgkin's lymphoma. LAHS arising from gastrointestinal lymphoma has never been reported. Here we report a case of gastric T-cell lymphoma-associated hemophagocytic syndrome.A 51-year-old woman presented with pain, redness of breasts, fever and hematemesis. Hematological examination revealed anemia. Gastroscopy revealed small bleeding ulcers in the stomach and the computed tomography scan showed liver tumor. She underwent total gastrectomy for gastrointestinal bleeding and the histopathology revealed gastric T-cell lymphoma. She continued to bleed from the anastomosis and died on the 8th postoperative day. Autopsy revealed it to be a LAHS.If Hemophagocytic syndrome (HPS) occurs in lymphoma of the gastrointestinal tract, bleeding from the primary lesion might be uncontrollable. Early diagnosis and appropriate treatment are needed for long-term survival. Hemophagocytic syndrome (HPS) in adults is characterized by reactive and systemic proliferation of benign histiocytes that phagocytose blood cells . It is o4/mm3, hemoglobin 10.3 g/dl (post transfusion), WBC 4,900/mm3, Platelets 51,000/mm3, serum albumin 1.5 g/dl, total bilirubin 0.6 mg/dl, AST 691 IU/l, ALT 187 IU/l, LDH 2976 IU/l, fibrinogen 134 mg/dl, FDP 10 μg/ml, and AT-III 40%. Bleeding from the stomach continued and did not stop with conservative treatment; therefore, two days later the patient underwent total gastrectomy and a partial liver resection. Histopathology of the resected specimen showed it to be a gastric lymphoma  with liver metastasis . There was no generalized lymphadenopathy. On gastroscopic examination multiple small ulcers were observed in the stomach. An abdominal computed tomographic (CT) scan showed liver tumor and a normal spleen. Hematological and biochemical examination at admission showed the following results: RBC 352 × 10in situ hybridization. The final diagnosis was EBV-related T-cell LAHS.On autopsy, malignant lymphoid cell infiltration and hemophagocytosis were observed in the liver, spleen, heart, small bowel, lung, both breasts, kidney, pancreas, uterus, and gastroduodenal lymph nodes Fig. . The bonet al, [et al, [HPS is a clinicopathological entity characterized by systemic proliferation of benign hemophagocytic histiocytes, fever, cytopenia, liver dysfunction, hepatosplenomegaly, and coagulopathy . This syet al,  are wideet al, ,3,4. On  [et al,  has propIn Japan, T-cell LAHS accounts for 48.5% of all adult LAHS . UncontrThe poor prognosis of LAHS, especially T-LAHS, is well known. The median survival time from the diagnosis is reported to be 143 and 69 days respectively in Japan . For LAHIn this case, the initial presentation was mastalgia and hence it took a considerable amount of time to reach a diagnosis. Furthermore, bleeding from the anastomosis continued leading to a rapidly progressive fatal clinical course.In HPS occurring in lymphoma of the gastrointestinal tract uncontrollable bleeding from the primary lesion might occur. Therefore, an earlier diagnosis of HPS should be made by bone marrow aspirates, and appropriate treatments should be started as soon as possible. Surgery if performed, must be performed with utmost caution.LAHS could also occur from lymphoma of the gastrointestinal tract. For long-term survival; early diagnosis and appropriate treatment are needed. Surgery if performed without a proper diagnosis could prove fatal.The author(s) declare that they have no competing interests.RF, FH, TY, RD, KO and KH were gastrointestinal surgeons.MO referred this patient to us.KK and MS performed pathological examination and the autopsy.KS was a member of the intensive care team.TH, YY, HN gave us helpful comments about the manuscript


Due to the low participation in colorectal cancer screening, public preference for colorectal cancer screening modality was determined.A cross-sectional survey was performed of healthy ambulatory adults in a pediatrics primary care office and neighboring church. Overall preference was ranked for each of four colorectal cancer screening modalities: Faecal Occult Blood, Fiberoptic Sigmoidoscopy, Barium Enema and Colonoscopy. Four additional domains of preference also were ranked: suspected discomfort, embarrassment, inconvenience and danger of each exam.80 surveys were analyzed, 57 of which were received from participants who had experienced none of the screening tests. Fecal Occult Blood Testing is significantly preferred over each other screening modality in overall preference and every domain of preference, among all subjects and those who had experienced none of the tests.Efforts to increase public participation in colorectal cancer screening may be more effective if undertaken in the context of public perceptions of screening choices. Screening for colorectal cancer lessens the risk of dying from that disease . KnowledMost publication concerning colorectal cancer screening relates to the choice of screening modality; discussing accuracy, efficacy and cost, since the most inexpensive technique, faecal occult blood testing, is inaccurate in the detection of colorectal neoplasia, though effective in significantly diminishing disease specific mortality , and theHowever the greatest problem related to screening remains the low level of participation by those for whom it is intended: asymptomatic individuals over the age of 50 years with no specific risk factors for colorectal cancer, i.e., no past history of colorectal polyps, cancer, rectal bleeding, colitis, change in bowel habits, iron deficiency anemia, weight loss or a close family member with colorectal cancer. We agree with Dr. Woolf , that stWe have in this report chosen to focus our survey differently and uniquely; first to inform healthy, ambulatory and younger people, and not ailing patients, concerning only the preparation and conduct of each screening test. Secondly, in order to determine how their perceptions of the conduct of each test might affect their participation, participants were then asked to rank not just their overall preference based upon the preparation and conduct of the tests alone, but four other domains of preference for each screening modality: perceived physical discomfort, inconvenience, embarrassment and danger. Test accuracy was not included in the preamble on test performance, first, because we wanted to isolate perceptions of the physical conduct of the screening test, and second, because test accuracy has been part of many of the previous surveys, often presented with considerable bias. Randomized trials of decision aids have also shown that description of a test's ability to detect colorectal cancer has not been successful in increasing participation in screening -17. LastParticipants were a convenience sample of parents or grandparents of children visiting a general pediatrics office , personnel working in that office, or parishioners attending a church social gathering, all aged 18 and over. An introductory letter described the purpose of the survey. This was followed by a brief description of the preparation and performance of each commonly used screening test for colorectal neoplasia: faecal occult blood testing (FOBT), fiberoptic sigmoidoscopy (FS), air contrast barium enema (BE) and total colonoscopy (C). The relative accuracy of each exam was not discussed. Six questions followed. The first asked the participant to rank each test in order of overall preference. The second asked the participant to rank each test according to how much that test might cause physical discomfort, the third, inconvenience; the fourth embarrassment, the fifth, the relative danger of the exam. The sixth question asked participants which of the four tests they had previously experienced, along with their gender and age. No further symptom or medical history was obtained and surveys were only numbered consecutively with no personal identifiers. (see appendix for letter and survey)Based upon a related survey concerning subject preference for tests of colonic inflammation , a samplData were analyzed using SPSS 11.0. Analyses focused on comparisons between ranks assigned each test on preference and the other assessed attributes, and included Friedman's test for ranks (to test the hypothesis that ranks differed for different tests) and the Wilcoxon signed-ranks test (to test the hypothesis that pairs of tests were differently ranked.) We also considered whether those rank orders might differ between participants who have and have not received any of these tests, and how gender and age affected preferences.80 of 84 surveys were available for analysis; twenty nine from men and 51 from women. The mean age of the participants was 38.3 years . Eight subjects had previously had a colonoscopy, five a barium enema, seven a sigmoidoscopy and 17 had stool collected for various reasons. Fifty seven subjects had experienced none of the screening tests. The mean rankings for preference among the entire sample are presented in Table Median scores were determined for each domain for both the whole survey group and the naive subgroup. For each domain and in each group the results were the same, with ranks of 4,3,2 &1 for C, BE, FS and FOBT respectively, 1 being most preferred, except for embarrassment in both groups in which C and BE each had a median rank 3.The results hold up for each gender subgroup in all cases except that men didn't consider FOBT significantly less inconvenient than FS. Age was not significantly correlated with ranking of FOBT  by Spearman's rho. Rho values ranged from -0.10 to +0.16, none significant. Splitting the groups into ages 18–39 (n = 39) and 40+ (n = 40), the results are the same for both groups except that for those over 40, preference for FOBT vs. FS and inconvenience of FOBT vs. FS did not reach significance by two-tailed test (p = .079 and p = .057 respectively)A recent review of colorectal cancer screening stated that, "At present there is no preferred CRC screening strategy". This prpatients, were quite different from the group reported herein. Only one of those reports offered all four of the screening modalities that we did in our study [Among previous surveys there are four randomized controlled trials of the use of decision aids that were designed with the intent of altering participation in screening. Three of these presented choices of screening modality or scenario to both intervention and control groups -17. Thesur study , the othur study ,16. Neveur study . In the ur study  and quesAmong some physicians there is a growing popularity for the use of definitive diagnostic testing as a screening tool, that is, colonoscopy . Though But the public has to want to participate in this program and there is little evidence in this current survey and previous studies, especially those done in primary care settings -18, thatThe author(s) declare that they have no competing interests.RLN conceived of the study, designed the questionnaire and supervised its administration.AS organized the domains of preference and performed the statistical analyses.Both authors participated in the writing of the manuscript.The pre-publication history for this paper can be accessed here:


Current journal requirements forcing clinical trials to be registered  are insu


Recently, several members of a vertebrate protein family containing a six trans-membrane (6TM) domain and involved in apoptosis and cancer , have been identified in Golgi and cytoplasmic membranes. The exact function of these proteins remains unknown.We related this 6TM domain to distant protein families using intermediate sequences and methods of iterative profile sequence similarity search.Here we show for the first time that this 6TM domain is homolog to the 6TM heme binding domain of both the NADPH oxidase (Nox) family and the YedZ family of bacterial oxidoreductases.This finding gives novel insights about the existence of a previously undetected electron transfer system involved in apoptosis and cancer, and suggests further steps in the experimental characterization of these evolutionarily related families. A family of vertebrate proteins containing a six transmembrane domain 6TM) has been recently implicated in apoptosis and cancer. Up to date, this family contains four vertebrate members: STEAP, STAMP1/STEAP2, TIARP and TSAP6/pHyde. STEAP  was the first described member of this family and identified as a prostate-specific cell-surface antigen overexpressed in cancer, located at the cell-cell junction of the secretory epithelium of prostate, and found as well in both colon and bladder cancer cell lines ,4. TIARPTM has beEscherichia coli YedZ protein, which binds a single heme and is involved in electron transfer to the molybdopterin cofactor in YedY, its operon neighbour gene [In this work we present evidence of remote homology of this family to other two families: the mainly eukaryotic Nox and the bacterial YedZ Fig. , both inour gene -18. The To do the sequence analysis of the new domain we took advantage of the possibility of connecting distant protein families via intermediate sequences  and methHaemophilus ducreyi) with an E-value of 0.63. This protein belongs to the large YedZ family of bacterial oxidoreductases. The corresponding YedZ global profile detected the STEAP family  with an E-value of 0.00087. The global profile of STEAP and YedZ detected the Nox family with an E-value of 0.032 and the corresponding Nox global profile localized the YedZ family with an E-value of 0.007 . These searches detected all of the ACRATA domain-containing families after 15 iterations (using a cut-off of E = 0.005 for the inclusion of retrieved sequences in the profile). None of these profile searches retrieved new unrelated sequences, and reciprocal searches produced convergent results. Therefore, we have concluded that ACRATA is a previously undetected, conserved domain that is commonly found in members of the STEAP, YedZ and Nox protein families. The similarity between these proteins was suspected before [To investigate the consistency of our results we performed iterative database searches using the PSI-BLAST program . We usedd before . HoweverThe complete conservation of two histidines in all ACRATA domain containing proteins Fig.  indicateExperimental evidence show that Nox and YedZ families share heme binding capabilities and also involvement in electron transfer chains -13,16. FE. coli in a number of conditions tested . Very differently, changes in the expression patterns of human genes containing the ACRATA domain could be related with apoptosis or cancer [Although the mechanism of action of the ACRATA domain could be the same in all the proteins containing it, its variable cellular role is made conspicuous by the different effects produced by the modifications in the expression of the corresponding genes. For example, the knock-out of the whole YedYZ operon seems not to affect r cancer -14. The r cancer , or in tr cancer ,12.We have described for the first time a 6TM domain present in three protein families : the ACRATA domain. The common functions of the proteins of those families suggest that this domain is involved in electron transfer, mediated by its heme binding capability. We hypothesize that STEAP, STAMP1, TSAP6, and TIARP have this function, and that they form part of electron transfer systems involved in cellular regulation, apoptosis, and cancer. Additional experimental approaches using different members of the ACRATA domain-containing families are required to confirm these hypotheses.STEAP, six-transmembrane epithelial antigen of the prostate;STAMP1, six transmembrane protein of prostate 1;TSAP6, tumor suppressor activated pathway-6;TIARP, Tumor necrosis factor-alpha-induced adipose-related protein;TCTP, Translationally controlled tumor protein;Nox, NADPH oxidase;ROS, reactive oxygen species;TM, Trans-membrane;FAD, flavin adenine dinucleotide;HMM, Hidden Markov Models;The author(s) declare that they have no competing interests.LSP, AMR, and MAA carried out the sequence analysis of the domain.LSP and MAA provided with the initial input of the research.LSP, AMR, AV, CMA, and MAA authored the manuscript.The pre-publication history for this paper can be accessed here:


It is a high priority that health care providers have effective communication skills. It has been well documented that the doctor-patient relationship is central to the delivery of high quality medical care, and it has been shown to affect patient satisfaction, to decrease the use of pain killers, to shorten hospital stays, to improve recovery from surgery and a variety of other biological, psychological and social outcomes. This study sought to quantify the current knowledge of interns in Iran about communication skills.A cross-sectional study using a self-report questionnaire was conducted among interns. Data analysis was based on 223 questionnaires. The internal consistency of the items was 0.8979.Overall, knowledge levels were unsatisfactory. Results indicated that interns had a limited knowledge of communication skills, including identification of communication skills. In addition, there was a significant difference between the mean scores of interns on breaking bad news and sex education. The confidence of males about their communication skills was significantly higher than for females. Analysis of the total scores by age and sex showed that there was a statistically significant main effect for sex and the interaction with age was statistically significant. Free response comments of the interns are also discussed.It is argued that there is a real need for integrating a communication skills course, which is linked to the various different ethnic and religious backgrounds of interns, into Iranian medical curricula. Some recommendations are made and the limitations of the study are discussed. The expectations of the public have been dramatically increased and the majority of them are familiar with their rights in the health care system. As a consequence, it is a high priority that health care providers have effective communication skills. It has been well documented that the doctor-patient relationship is central to the delivery of high quality medical care. It has been shown to affect patient satisfaction, to decrease the use of pain killers, to shorten hospital stays, to improve recovery from surgery and a variety of other biological, psychological and social outcomes -4. Lack However, little is known about the importance of communication skills in the practice and training of doctors in Iran, where the culture differs greatly from that of the West. Sensitivity to religious matters is particularly important in Iranian doctor-patient relationships where Islam is more than a religion; it is a way of life. It controls politics, local laws, behaviour and many other aspects of daily life. It gives guidance in all spheres of human activity from birth to death. Therefore doctors coming into contact with religious patients need to be aware that there are numerous potential barriers to good communication .A major criticism of current medical training in Iran is that communication skills have not been embedded in the curriculum of Iranian medical students, despite the richness and variety of evidence from elsewhere concerning the importance of communication skills. Concerns over poor doctor-patient communication amongst Iranian doctors led to an exploration of the current situation . In thisA quantitative survey was performed at Tehran University of Medical Science (TUMS). A cross-sectional study was conducted using a questionnaire administered to 235 interns. Anonymity was maintained throughout. The subjects received the self-administered questionnaire with a covering letter explaining the project and the subject's rights. 12 subjects did not return the questionnaire and an additional 7 subjects did not give their age and one person did not give his/her sex. Therefore data analysis was based on 223 questionnaires, but covariate-based analysis on fewer. The subjects were asked to complete the questionnaire without referring to source books.The questionnaire consisted of three sections. The first section asked students to give personal details including the demographic items age and gender .The choice of items was based on the communication skills an intern will need. All items were verified and subjected to content validation by three major experts in communication skills. These experts were given copies of the CSKS and the purpose and objectives of the study. They then evaluated the CSKS on an individual basis. Comparisons were made between these evaluations and the authors then made some minor changes within the CSKS. The CSKS had a high internal consistency  is 10 to 100, with 10 indicating low knowledge. Analysis of the total scores produced a mean score of 51.30 [95 per cent confidence interval (CI) 49.05–53.55]. The subjects' performance on the CSKS suggests a knowledge deficit in communication skills. The mean scores for males and females were respectively 53.6 and 48.2 (P = 0.02). The vast majority of interns (78.1%) had not studied a paper on communication skills. When asked whether they had formally attended communication skills courses, 91.4% of interns reported "no". Of the few interns who reported "yes", these interns specified courses such as CPR, injections and semiology . There was a statistically significant main effect for sex  and the interaction effect [F  = 4.06, p = 0.04) did reach statistical significance. However the effect size was small (eta squared = 0.02). The young male interns were more confident than average, while the young female interns were less confident.Free responses included the following comments:Nobody has trained us about communication skills. Our knowledge in respect of communication skills is very poor. Your items show that we are very far behind other countries. Our universities are not as advanced as other universities'.''I feel we are not familiar with the ABC of communication skills'.'A good guide to communication skills needed'.'I feel communication skills would be an excellent course since it gives us an idea of how we can handle bad news'.'Attending doctors are not totally familiar with the aims and use of communication skills in the clinical setting'.'All our courses only focus on biological issues rather than psychosocial issues'There were a number of limitations to this study.1. The CSKS has not been normed for a population of interns.2. Criterion-related validity of the CSKS was not determined, although content validity was established on the instrument.3. Since it is a self-assessed questionnaire, these may be problems with bias, such as prestige bias.The very high response rate (95%) of this questionnaire may have reflected general interest, or may have resulted from the advantages of self-assessment which itself may improve performance. The results on the CSKS show that basic knowledge of interns in Iran about communication skills is limited. Researchers have reported similar findings in other countries which reveal a deficit in the knowledge of doctors about communication skills . The impThe vast majority of research studies have been conducted on the outcome of communication skills in the practice and training of doctors in western countries. Even here, despite doctors trained in communication skills and the advocacy of the use of a patient-oriented approach, some evidence suggests that there are difficulties in practice ,20. HoweThe results of the study show that there were significant differences between males and females with regard to their reported knowledge of the main communication skills. Women were less confident of their skills. The deficit may partly be an artefact of an inadvertent prestige bias of the male students. The deficit is particularly notable in sex education. There are three possible explanations for this. Firstly, in general, Iranian female interns are very shy to ask patients about sexual issues. Therefore they may feel that sex education skills have no implications for their practice and hence pay less attention to sex education training. Secondly, it may be a systematic error in female respondents, i.e. they may be shy to discuss their knowledge about sex education skills rather than lack knowledge. Thirdly, in the past, sex education was regarded as a taboo in Iran and was not available in schools, especially for girls . This peThe results on the CSKS suggest that there are areas of weakness in the communication skills confidence of interns, particularly in breaking bad news. While it is well recognised that delivering bad news is a difficult task that requires skills and sensitivity , both feThere is a significant difference between the mean score of the interns on breaking bad news. The female interns have reported lower confidence than the male interns. The deficit could be an inadvertent prestige bias of the male students. However, to our knowledge, there is no evidence that underpin such finding. Although Orlander et al's work  demonstrGiven the poor levels of confidence about communication skills, particularly sex education skills, revealed in this study, it is concluded that educational programmes are necessary. In sex education skills training, given the complex interplay of cultural and religious beliefs in Iran, particular attention must be paid to multicultural and religious issues. Therefore, further work is needed on gender education and stereotypes in sex education; learning styles; the 'hidden curriculum'; and how far medical schools make organisational and administrative arrangements on the basis of gender and the implication for female and male interns.The enthusiastic response to the questionnaires may suggest that medicine is accepting the need for developing communication skills within the medical curriculum. Medical education in Iran must respond to this challenge.Finally, our findings may be somewhat limited in generalisability because they are derived from only one medical school in Iran. Self-assessment data may suffer from biases such as prestige bias. Despite these caveats, the authors believe the data to be an accurate reflection of current practice in Iran, based on the Iranian authors training experiences, and consistency with previous accounts.Whilst the approach to this research has been shaped by a government-recognised health need, the authors recognise the need for, and welcome, further examination of these findings from multiple perspectives, especially with regards to ethnicity and social issues. Since not enough attention has been focused on individuals as makers of health as a service rather than customers of health care services, it is strongly recommended, therefore, that medical students be trained in the context of psychosocial issues that may influence health behaviour, as has been indicated by one of the participants. It is particularly important that this type of approach be incorporated into the curricula of medical training. This may assist in transferring from the disease-oriented to the patient-oriented approach and ultimately lead to patients understanding more and taking greater responsibility for their own health.The author(s) declare that they have no competing interests.MT and ST carried out the conception, design, initial analysis and interpretation of the data. MT drafted the paper. ODL was involved in revising the draft critically, revising the statistical analysis and gave final approval of the version to be published. AAZ contributed to the collection of data and the reviewing of the manuscript.The pre-publication history for this paper can be accessed here:


Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000–40,000. Only 2%–3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.We report improved whole-genome shotgun sequences for the genomes of  indica and japonica rice reveals that duplication of genes and genomic regions has played a major part in the evolution of grass genomesComparative genome sequencing of  This draft was acquired by a whole-genome shotgun (WGS) method /39.6 = 40,216. Similarly, we get 37,794 for Syngenta japonica and 37,581 for IRGSP japonica. If unique genes are truly expressed at lower levels than shared genes, this procedure might underestimate the gene count. One should thus interpret these numbers as lower bounds.Even after removing likely TEs, two particular subclasses warrant caution, as they contain a higher than normal rate of erroneous predictions, which is reflected in a reduced rate of confirmation by ESTs. Overall, we used 200,648 ESTs from indica that are not found in either japonica assembly is 1,064. Conversely, Syngenta japonica has 1,517 predicted genes that are not in indica . As a fraction of the totals, 2.2% and 3.3% of indica and japonica genes, respectively, are unique to the subspecies, which is plausibly comparable to the amount of sequence that might still be missing. There is little difference in gene content between indica and japonica, but major differences are seen in the intergenic regions. Only 260 Mb (72%) of the mapped sequences can be aligned. This remains true no matter how much we relax the alignment parameters, and despite the fact that we had 34,190 “anchor points”  and a decrease in gene density. A more detailed view is given by the pullout figures of indica and Syngenta japonica.Arabidopsis, the number and timing of these duplication events is still unknown [Duplication of individual genes, chromosomal segments, or even entire genomes is an important source of raw materials for gene genesis . In the  unknown ,57,58,59 unknown ,61,62 be unknown  and an a unknown . Here, windica.We accept that every class of duplication is present in the same genome, and we thus explicitly assign, to every homolog pair, a status as to the class of duplication from which it came. For the sake of discussion, we define three classes: segmental duplication of multiple genes along a chromosome, tandem duplication of individual genes, and a category called background duplications to encompass everything else that cannot be so easily classified. In this conception, a WGD is a collection of segmental duplications that cover a majority of the genome, all of which date back to a common time in evolutionary history. All three rice assemblies give the same result, so we show only Beijing −7. Exons in the same order and orientation are linked together, and success is declared if these linked exons can account for 50% of the original protein sequence. This technique has the advantage that the homolog need not be a cDNA or a predicted gene (as neither dataset is likely to be complete). In fact, the homolog might even be a remnant of an ancient duplication that is no longer a functional gene. Complications are found at two extremes. Many cDNAs have no homologs, but many others have too many homologs. In particular, 24.5% of WH genes have no homologs in rice, whereas 64.4% of NH genes have no homologs in rice. Because NH genes are dispersed throughout the genome, sandwiched between WH genes, we cannot adopt a strict colinearity rule in our search for duplicated segments. There would be too many exceptions. Conversely, when there is at least one homolog in rice, the mean (median) number of homologs per cDNA is 40 (5). Rather than deal with the complexities of this situation, we focus first on the cDNAs with one and only one homolog. This reduces the background duplication noise and allows us to identify trend lines indicative of segmental and tandem gene duplications. We can then add back those cDNAs with more than one homolog that we had rejected earlier by using our newly defined trend lines to constrain the choices.Unlike previous analyses, we avoid predicted genes. Instead, we define a homolog pair to be a single nr-KOME cDNA and one of its potentially many homologs within rice. These homologs are defined by translating the cDNA's coding sequence into protein and searching the rice genome in all six reading frames for putative exons, with TBlastN at E-values of 10The above procedure leaves us with 2,271 homolog pairs (or cDNAs). We adopt a graphical approach, because in the presence of massive background noise, trend lines are often easier to identify by eye than by software. −9 substitutions per silent site per year [One can date the duplications by computing the number of substitutions per silent site (Ks). Multiple substitution corrections are done within K-Estimator . To imprper year . Most ofper year  dates th−9 evolutionary rate . However, all we really want to know is whether the duplication event occurred before or after the origin of the grasses, 55–70 Mya [The molecular clock can also vary between genes and between taxa ,67. Evid5–70 Mya . To this5–70 Mya . Almost 5–70 Mya . HoweverIf we accept that a WGD occurred before the divergence of maize–rice, and that a duplication in Chromosomes 11 and 12 occurred afterward, we might then expect to find two levels of duplication in this region of rice. We thus extended our analysis to consider cDNAs that map to as many as four loci. No indications of such a multilevel duplication could be found. Undaunted, we decided to try another approach and analyzed the maize–rice synteny, starting from the maize genetic map . The resGiven how so much of the rice genome is covered by segmental duplications, and the fact that all but one of our 18 segment pairs date back to the same time, give or take a standard deviation of 14.5%, the simplest interpretation is that a WGD did occur and that it happened before the origin of the grasses. However, it is equally clear that other classes of duplications are also present, and these are worth investigating too.Arabidopsis genome paper, we find that 16.5% of the rice genome is tandemly duplicated, compared to 16.2% of the Arabidopsis genome. Note, however, that the Ks distribution for tandemly duplicated genes in Arabidopsis is highly unusual, in the sense that it does not exhibit the big peak at zero Ks that is seen in virtually every other plant genome [Tandem duplications are represented by the trend along the diagonal, Y = X, that is observed in all chromosomes see . Segmentt genome .In addition to segmental and tandem duplications, there is a third and last class of duplications that looks like background noise in our figures. The number of homolog pairs is 1,351 and 32,384 before and after higher-order homologs, respectively, although with no trend line to constrain the choice of homologs, that second number is almost certainly an overestimate, since only 4,212 cDNAs are involved. Surprisingly few of these higher-order homologs are the result of processed pseudogenes, as the number of cases in which a multiexon cDNA pairs with a single-exon TBlastN homolog is 9.8%. To demonstrate how overwhelmingly these higher-order homologs contribute to the background noise, Tandem and segmental duplications show markedly different Ka/Ks distributions, a popular test for evolutionary selection, where Ka and Ks refer to the fraction of nonsynonymous and synonymous sites, respectively, that are changed within a homolog pair . Ka/Ks iOur WGD is in good agreement with the results of Paterson et al. , but we Arabidopsis was the only sequenced plant genome. When two rice genomes were first published in draft format, the comparative analyses that could be done were hindered by a lack of long-range contiguity. Now, there are three plant genomes  with multimegabase contiguity. In our analyses, we strived to maintain methodological consistency. To assess the accuracy of our assemblies, we first compared IRGSP japonica to Syngenta japonica, so that polymorphic differences would not be a confounding factor. To compare gene content in the three rice assemblies, we annotated them all with the same procedures. Our conclusion is that, even if the WGS method does fall just slightly short of the clone-by-clone method in terms of accuracy and completeness, it comes remarkably close. This is why all the genome-sequencing projects now being funded by the National Human Genome Research Institute (in the United States) are being done with WGS methods (http://www.genome.gov/11007951). Rice is also now one of the few organisms with the luxury of having a complete genome sequence for two important subspecies. Comparisons of indica and japonica reveal strikingly little difference in the gene content, but there are massive intergenic differences. This vindicates our strategy to focus on genic sequences, because if the intergenic sequences are so unstable even between indica and japonica, they are highly unlikely to be functional.Until recently, Our analysis of the duplication history in rice resolves a simmering dispute and, at the same time, raises some intriguing questions. We find evidence for an ancient WGD, a recent segmental duplication, and massive ongoing individual gene duplications. This last phenomenon can explain certain unexpected findings. Sequencing of orthologous loci between grass genomes has identified many smaller-scale rearrangements that were not seen in the original map-based studies. Many of these exceptions to synteny are due to tandem duplications ,77,78, windica variety represented by93–11, but also the maternal strain of the LYP9 superhybrid, PA64s, which has a complex breeding history incorporating genetic material from indica, japonica, and javanica—all of the major subspecies of cultivated rice. Work on PA64s is continuing at our Beijing center. For the research community, we will be providing DNA microarrays to facilitate the systematic studies of gene expression in different tissues and developmental stages, and under different physiological and environmental conditions. We will develop molecular markers for mapping causative genes in mutant lines and marker-assisted breeding. This publication, and the associated data release, is also a fitting way to celebrate the end of 2004, which the General Assembly of the United Nations declared to be the International Year of Rice (http://www.fao.org/rice2004).Looking toward the future, we would point out that the Chinese Superhybrid Rice Genome Project was designed to include not only a major subspecies of rice, namely, the japonica cDNAs with complete open reading frames. These cDNAs were aligned to Syngenta japonica, and when two alignments overlapped by at least 100 bp, the smaller cDNA was removed. A small number of clones could not be aligned—not even partially—to any of our three rice assemblies . Removing these as nonrice contaminants gave a set of 19,079 nonredundant cDNAs that we call nr-KOME. Because the sequence quality is so high, we could use the longest open reading frame for the overwhelming majority of these cDNAs, without having to correct for sequencing errors. Minor corrections are applied to 2.5% of these cDNAs, following the methods first developed for GenScan [The initial Knowledge-Based Oryza Molecular-Biological Encyclopedia dataset  had 28,4 GenScan .The basic procedure for converting sequence reads into contigs and scaffolds was described in our original publication on RePS , our WGShttp://www.repeatmasker.org). However, the degradation makes it trivial to distinguish between two copies of an ancient TE, so these do not cause assembly problems. It is also relatively easy to distinguish between gene duplicates, because their introns and flanking intergenic regions are under fewer evolutionary constraints than their exons. Even for recent TEs and gene duplicates, assembly problems can be avoided, because RePS computes the copy number for every 20-mer in the WGS assembly, and it will refuse to join anything that might be ambiguous. Indeed, the only way a misassembly can occur is if there is a low copy MDR and its copy number is underestimated by RePS. All of our tests show that, although this can happen, it is a rare event.In a WGS assembly, the problems arise from the MDRs, which are not equivalent to the biologically defined repeats. For example, TEs qualify as biologically defined repeats, and they can be recognized, even after many millions of years of degradation, by specialized programs like RepeatMasker . This is preferred to any of the BLAST alignment tools, which, although they are faster, occasionally miss subtle details. To discriminate between polymorphisms and sequencing errors, we use the error probability p attached to every base, and given as Q = −10 × log(p). Following the rules established in the early days of large-scale polymorphism discovery [Q > 23 at the SNP site and Q > 15 for the two flanking 5-bp regions. Experience has taught us that higher thresholds  are required for the indels. For comparison, an independent analysis [The sequence alignments for nts” see . We partiscovery , we use analysis  reportedSaccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Helicobacter pylori analyses [Bioverse annotations in this article are dated July 2003 (FGENESH) and November 2002 (nr-KOME). Two proteins are said to interact if they are similar to two other proteins that are known to interact. Our criterion is that the product of the similarity measures (percentage identity) must exceed 0.15. For example, two proteins with 45% and 30% identity to two other proteins that are experimentally determined to interact would be rejected, as their score is 0.45 × 0.30 = 0.135. The reliability of this approach, especially for transfer of interaction data between organisms, has been demonstrated in analyses . As an eanalyses . A generArabidopsis, we use the methods described in the Arabidopsis genome paper and analyze predicted genes with BlastP at E-values of 10−20.We defined a homolog pair as a single nr-KOME cDNA and its TBlastN homolog, but occasionally that TBlastN homolog will overlap with another cDNA. To avoid double counting, we keep only the larger of these two cDNAs. Segmental duplications identified by visual inspection must have at least five homolog pairs, with no more than 5 Mb between adjacent homolog pairs. We approximate the trend line with a second- or third-order polynomial, and to capture what our eyes indicate should be captured, we accept homolog pairs within a 500-kb radius of this polynomial. Slightly different definitions are used for tandem duplications, depending on application. For Ks, we allow two TBlastN homologs to count as a homolog pair and accept homolog pairs within a 50-kb radius of the diagonal, although the mean (median) center-to-center distance is 6.8 kb (4.7 kb). To compare tandem duplications in rice and To determine the maize–rice synteny, we began with 1,063 maize genetic markers  and searFigure S1japonica and IRGSP japonica.Similar results are seen with the other two assemblies, Syngenta (1 MB EPS).Click here for additional data file.Figure S2We count discrepancies where the markers are found (A) on different chromosomes and (B) in different locations on the same chromosome.(458 KB ZIP).Click here for additional data file.Figure S3Genomic size refers to the unspliced transcript, with introns, but constrained to the region from the start to stop codons. CDS size refers to the spliced transcript, without introns. Predictions are assessed with FP and FN rates, where per-aa (per amino acid) refers to the fact that we check whether the reading frame is correct.(351 KB ZIP).Click here for additional data file.Figure S4gypsy/copia TE), where most of the 20-mer copy numbers exceed ten. Lower copy number regions are tolerated up to a “maximum gap size,” which defaults to 150 bp. Regions lying between two adjacent intergenic repeat clusters are taken to be gene islands.Intergenic repeat clusters are regions of size larger than 1.5 kb .Click here for additional data file.Figure S5gypsy and copia) and a decrease in gene density. This is not an artifact of the fact that WGS assemblies underrepresent larger TEs, as much the same effect is observed when we use IRGSP japonica instead (data not shown).Near the centromeres, there is an increase in TE density .Click here for additional data file.Figure S6Arabidopsis. TEs are decomposed into classes I, II, and III. Correspondence between indica and japonica is indicated by drawing a connecting line between the 5′ ends of the nr-KOME cDNAs that clearly align to both assemblies.We depict all the genetic markers, nr-KOME cDNAs, FGENESH gene predictions, and transposable elements identified by RepeatMasker. Genes are depicted as WH (colored blue) or NH (colored red) based on their similarity to (9.6 MB ZIP).Click here for additional data file.Figure S7(507 KB ZIP).Click here for additional data file.Figure S8x-axis shows a specific chromosome for one genome, and the y-axis shows all chromosomes for a second genome, with the chromosome numbers color-coded as per the legend. We show here 12 panels for rice.Each point indicates the genomic positions for a maize genetic marker and its highest confidence match in rice. The (311 KB ZIP).Click here for additional data file.Figure S9x-axis shows a specific chromosome for one genome, and the y-axis shows all chromosomes for a second genome, with the chromosome numbers color-coded as per the legend. We show here ten panels for maize.Each point indicates the genomic positions for a maize genetic marker and its highest confidence match in rice. The (288 KB ZIP).Click here for additional data file.Table S1−2 or better. Effective coverage is based on the depth of reads in contigs over 5 kb in size, ignoring regions with 20-mer repeats. Clone insert sizes are specified in terms of tenth and 90th percentiles.Read length is the number of Q20 bases with an error rate of 10(16 KB XLS).Click here for additional data file.Table S2indica–japonica polymorphisms, the sequence is divided into four nonoverlapping categories: unassembled, assembled-but-unmapped, mapped-but-unaligned, and aligned .As a result of our efforts to identify (28 KB XLS).Click here for additional data file.Table S3japonica sequence. This supports the idea that the WGS method can sometimes identify genes that are not well represented in the BAC clone libraries.For each region of the Venn diagram, we use BLAT to align the predicted gene to the other assembly (or assemblies) where the gene is supposedly missing. The objective is to determine whether it is the sequence that is missing, or whether the discrepancy is due to the errors in the ab initio predictions. What we find is a bit of both. However, fragmented sequence assemblies are not a problem. If the gene is found at all, it is usually found in one piece. What is striking is that predicted genes that are unique to the two WGS assemblies do tend to be genuinely missing from IRGSP (17 KB XLS).Click here for additional data file.Table S4Arabidopsis and Beijing indica. The second table compares predicted genes from Beijing indica with nr-KOME cDNAs.One table compares predicted genes from (169 KB XLS).Click here for additional data file.http://rise.genomics.org.cn [indica  and Syngenta japonica .The DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank (BGI-RIS s.org.cn ) projectThe idea that TEs are often mistakenly annotated as genes was also suggested in a recent paper by Bennetzen et al. .


Single-pass, partial sequencing of complementary DNA (cDNA) libraries generates thousands of chromatograms that are processed into high quality expressed sequence tags (ESTs), and then assembled into contigs representative of putative genes. Usually, to be of value, ESTs and contigs must be associated with meaningful annotations, and made available to end-users.A web application, Expressed Sequence Tag Information Management and Annotation (ESTIMA), has been created to meet the EST annotation and data management requirements of multiple high-throughput EST sequencing projects. It is anchored on individual ESTs and organized around different properties of ESTs including chromatograms, base-calling quality scores, structure of assembled transcripts, and multiple sources of comparison to infer functional annotation, Gene Ontology associations, and cDNA library information. ESTIMA consists of a relational database schema and a set of interactive query interfaces. These are integrated with a suite of web-based tools that allow a user to query and retrieve information. Further, query results are interconnected among the various EST properties. ESTIMA has several unique features. Users may run their own EST processing pipeline, search against preferred reference genomes, and use any clustering and assembly algorithm. The ESTIMA database schema is very flexible and accepts output from any EST processing and assembly pipeline.Apis mellifera), cattle (Bos taurus), songbird (Taeniopygia guttata), corn rootworm (Diabrotica vergifera), catfish , and apple . The entire resource may be downloaded and used as is, or readily adapted to fit the unique needs of other cDNA sequencing projects.ESTIMA has been used for the management of EST projects of many species, including honeybee (. The entity-relationship (E-R) diagrams and the programs used to generate the Oracle database tables are also available. We have also provided detailed installation instructions and a tutorial at the same website. Presently the chromatograms, EST databases and their annotations have been made available for cattle and honeybee brain EST projects. Non-academic users need to contact the W.M. Keck Center for Functional and Comparative Genomics, University of Illinois at Urbana-Champaign, Urbana, IL, for licensing information.The scripts used to create the ESTIMA interface are freely available to academic users in an archived format from  Expressed sequence tag (EST) collections represent partial descriptions of the transcribed portions of genomes. They are generated from single-pass cDNA library sequencing that is often carried out by small or mid-size sequencing centers and research groups. The research may be aimed at transcriptome expression analysis using microarray, RT-PCR, or hybridization techniques. An increasing number of research centers are involved in sequencing multiple cDNA libraries. This is manifest by the exponential growth in the number of ESTs deposited to dbEST at NCBI , and as of the May 21, 2004 update, that number is at over 21.2 million ESTs from 686 species. Small sequencing centers are faced with searching for cost-effective and convenient tools for EST management and querying, visualization, public and private user access, and functional classification.EST processing pipelines that transform raw chromatograms into high-quality filtered sequences, and software to cluster and assemble them into contigs are convenient to implement, and various tools are now available to researchers  software that provides a database schema for management of raw and annotated ESTs, and is coupled with a suite of custom web-based tools that facilitate searching various aspects of ESTs and contigs, visualization, pairwise searching by BLAST , and funTo form an association between a GO term and an EST, an organism that has already been annotated with the GO terms is selected. The ESTs are searched against this reference organism's annotated sequences, and the resulting alignments are used to ascribe putative function. The ESTIMA database schema provides users the flexibility to use any reference database that has GO term association, and any number of external databases, such as NCBI's non-redundant (nr) database or EMBL's Swissprot collections, to annotate ESTs and their contigs. This flexibility in the schema is critical given the increasing number of sequenced genomes, and specialized reference databases that researchers can download and integrate with existing annotations. Other database schemas that house EST project and analysis data have been reported ,12. But The inputs to ESTIMA are the chromatograms, raw and processed ESTs, clusters and assembled EST contigs from any source, reference database annotations of ESTs, and contigs. All of these get loaded into the tables generated from the ESTIMA database schema and loading scripts. The raw and processed data, with their annotations, are made available to users through the ESTIMA query interfaces, for exporting, visualization, and further research.ESTIMA is composed of three major components: an ODBC-compliant database, loading scripts and a web application. Figure The heart of the system is a pair of database schemas. Figure Term_Seq_Count contains the pre-calculated counts of ESTs associated with each term, and all the terms' child terms. Pre-calculation saves time at execution, and is handled by a Perl script that is rerun each time the GO tables are updated.The Gene_Association and Annotation table contains information that links the ESTs to the reference genomes. The Gene_Association table has one record for each reference gene that is associated with an EST or contig. The reference gene is then related to a GO term in the Term table.GENOME also contains the Blast_All table that holds information regarding the association between the ESTs and any project-specific DNA sequences. The ESTs are searched against sequences of interest, such as NCBI's nr, and the results are parsed and loaded into the Blast_All table.In addition to the common data, each project has its own schema that contains information such as DNA sequences, contig assemblies and paths to the chromatogram files. The PROJECT schema is centered on the Project table that contains a simple link between the other PROJECT tables and the GENOME schema. This organization has proven useful when the contents of the PROJECT schema are to be transferred directly from a sequencing facility to a Laboratory Information Management System (LIMS).Each project may have several different contig assemblies deriving from different assembly techniques. The assemblies are all housed in the Assembly_Project, Contig, Consensus_Link and Singlet tables. Each assembly has a single ID that is used by the web application to determine which is "live". After a new assembly is completed and loaded, it can be taken live by simply changing a single number in the web applications configuration file.Finally, the sequence data itself is stored in the DNA_Sequence table, while related GenBank accession numbers are stored in their own table. Although accession numbers are not required by the web application, if they are available, then the web application will accept them where a sequence ID would be used.The second major component of the ESTIMA system is a series of Perl and JAVA applications used to parse and load data into the database. Because each project will have its own needs, the loading system was not automated. Instead, several dozen separate scripts and applications have been developed to allow researchers to manipulate and analyze large data sets using standard analysis applications.The third component of ESTIMA is the web-application that queries and reports the EST information to the end-users.The ESTIMA web application is organized around seven points of entry into the system – the start screen and six query applications. Each query application interfaces with the databases, and allow users to query raw and annotated ESTs and contigs. ESTIMA supports password-protection, multiple-projects, and multiple libraries within a project. This is achieved with an XML configuration file with project-specific information that is called by the various applications when project-related information is needed. Figure The start screen provides a convenient point of reference to the system. The page contains information about each library  in the project. All of the elements of the start page are contained within the configuration file.The GO Browser Figure  allows aESTs can be accessed directly from the sequence ID interface. More commonly, results from queries on annotations or contigs are dynamically linked to the EST sequence information. From these screens users can access chromatograms, and both raw and filtered sequences.Information about the association between the ESTs and the reference genome sequences can be accessed through these pages.ESTIMA provides a Blast User Interface that allows FASTA-formatted sequences to be searched against the EST libraries. The libraries that are available for BLAST  can be dThe Annotation page is an optional page, the presence or absence of which is controlled from a configuration file. This page allows additional BLAST-derived annotations, beyond GO annotations, to be displayed and queried in a number of different ways. The web application creates an interface that allows users to query the Blast_All table directly. For example, songbird ESTs  were seaThe Contig Viewer provides an image of the assembled contigs showing the relationship of the contig with the ESTs it contains as members. It provides the contig's consensus sequence, and links to each of the member ESTs.The web application maintains a common look and feel to all the pages within a project. This is implemented by having a single block of HTML stored within a configuration file. As each page is rendered, the same HTML is called to generate the header block of the page. Each project has its own HTML, so the look of each project's web site can be customized.The inter-connectivity of the different ESTIMA modules allows researchers to engage in a "free form" exploration of the data. Users can query the GO Browser, for example, to find a contig associated with a term of interest, drill down to see the structure of the contig, and then, if desired, drill down to get specific information on each EST in the contig. Just as easily, the users could take a sequence of interest from their research, and similarity-search it against the assembled ESTs. Then they could drill down on an EST that aligned with their sequence, and see the additional information about the sequence, including any annotations.ESTIMA allows users to view the actual chromatograms of the ESTs. Chromatogram files are stored in a file system visible to the web server, while file paths are stored in the database. When a user requests a chromatogram, Phred ,16 is caESTIMA is unlinked from the backend EST processing pipeline, clustering, and assembly of ESTs. It serves as a stand-alone web application that allows users to store, access, research, and visualize the raw and annotated ESTs and contigs, including GO annotation. The output from a sequencing center's EST processing pipeline  gets loaded into databases, and serves as input to ESTIMA. (The W.M. Keck Center will gladly share the source code to its pipeline with any interested academic institution).Additionally, researchers may choose to use any reference genome to annotate their sequences. ESTIMA comes packaged with a flexible database schema that supports the linking of sequences to GO terms, and other user-supplied sequences. The flexibility of the ESTIMA database schema becomes more relevant with the increasing number of sequenced genomes. ESTIMA provides a flexible, password-protected, multi-project environment to researchers. It facilitates analysis of an unlimited number of ESTs and contigs linked to GO and non-GO annotations, and the download of annotated sequences related to any GO term. ESTIMA comes with an implementation of a GO browser that allows users to view the entire child term tree for any term, conveniently from a single query interface.ESTIMA is designed to be a stand-alone application. Each installation of the web application has all system dependent information in its own configuration files, including the information needed to connect to the databases. We maintain two instances of the interface, a development and a production copy. As new projects are introduced, they can be tested, and any interface modifications that are needed can be perfected on the development machine, concurrent with the creation of the production version of the databases. The production database can be examined using the development web application and any errors corrected before taking the system "live" to the production version.ESTIMA is designed to accept new projects. As new EST sequencing projects are finished, they can be easily added to an existing ESTIMA installation. A new schema is created for the new project and the loading scripts are used to populate the database. All that is required to activate the web application is the addition of a block of XML code to the configuration file, and a connection string and some HTML to the system configuration file. XML tags within a configuration file control project- specific issues such as whether the data is password protected, or which BLAST databases can be accessed from the Blast User Interface.Drosophila genome, and based on these similarities the sequences were tentatively assigned one or more molecular functions and biological processes. Likewise, BOVEST, the cattle EST database [There are three public ESTIMA projects currently administered through the W.M. Keck Center for Comparative and Functional Genomics at the University of Illinois. The bee-ESTdb  is a resdatabase , containdatabase . The sondatabase  that werThe presence of multiple projects in ESTIMA allows for efficient cross-tissue or cross-species homology searches. For example, a mouse brain EST may be used to interrogate the honeybee brain or songbird brain library to test the hypothesis that the gene is expressed as a brain-specific transcript. Thus, a mouse brain transcript, NCBI accession number BM875176, similar to human tubulin alpha-1 chain protein may be used to do a TBLASTX against the honeybee brain assembled ESTs from the BlastUI interface in ESTIMA. The BLAST results retrieve a significant hit, Contig2466, to the honeybee brain database (Figure ESTIMA projects, as compared to other public web-applications such as TIGR gene indices , allow aESTIMA is a full-featured web-application and database, designed to simplify exploring and sharing EST libraries and databases. It can be easily adapted to a wide variety of system configurations, and back-end database engines. Our installation of ESTIMA easily supports three public projects, with five different EST libraries, and additionally a growing number of private projects.. Under 'Downloadable Software' section of the web page, detailed installation instructions and a user manual have been included as well. ESTIMA is still in active development. New features are constantly being developed to meet the changing needs of the research projects that use it. Further, new projects are being added to our ESTIMA installation. The system has been written to facilitate its own change, and as such, researchers should find it approachable with a good working knowledge of Perl, SQL, and HTML.ESTIMA is available free to academic users at ESTIMA requires Perl and a database. All communication with the database is handled through Perl DBI, which is extensible to any ODBC compliant database. The use of Perl DBI and ODBC allow the databases to reside on separate servers from the web interface. Although many databases may be used, in practice, there are several complex joins in the code that could result in slow performance on large EST sets unless a well-optimized database was selected.ESTIMA requires certain additional Perl modules, specifically BIO, CGI, DBI, and GD. Bioperl, the BIO module  is used We have been using ESTIMA throughout its development. Our schemas are housed in an Oracle 8I database on a Silicon Graphics Origin 2000 16 × 250 MHz machine running IRIX 6.5.20. The web application, including the user requested BLAST jobs are run on a Sun Microsystems SunFire 280 R with dual 700 SPARK V9 CPUs.GG and LL developed the database schemas. CGK, LL and LR developed the initial web application prototype. HAL managed project development and contributed to design concepts. RL modified the web application and optimized the code. All authors read and approved the manuscript.


Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector – target cell contact and adsorption periods were studied. MOI between 0–32 was assessed on commonly used cell lines as well as a new cell line.Lentiviral vectors encoding a marker gene were packaged and supernatants concentrated. The number of viable vector particles was determined by We demonstrated that the resulting values of lentiviral vector titre varied with changes of conditions in the transduction process, including inoculum volume of the vector, the type and number of target cells, vector stability and the length of period of the vector adsorption to target cells. Vector inoculum and the number of target cells determine the frequencies of gene transfer event, although not proportionally. Vector exposure time to target cells also influenced transduction results. Varying these parameters resulted in a greater than 50-fold differences in the vector titre from the same vector stock. Commonly used cell lines in vector titration were less sensitive to lentiviral vector-mediated gene transfer than a new cell line, FRL 19. Within 0–32 of MOI used transducing four different cell lines, the higher the MOI applied, the higher the efficiency of gene transfer obtained.in vitro vector titration and resulted in vastly different values from the same vector stock, thus complicating the use of MOI for predicting gene transfer events. Commonly used target cell lines underestimated vector titre. However, within a certain range of MOI, it is possible that, if strictly controlled conditions are observed in the vector titration process, including the use of a sensitive cell line, such as FRL 19 for vector titration, lentivector-mediated gene transfer events could be predicted.Several variables in the transduction process affected in  Multiplicity of infection (MOI) is a parameter that has been commonly used to predict viral infectivity in a population of target cells. With wild type viruses, an "infectious unit" refers to the smallest amount of virus capable of producing an infection in a susceptible cell. The titre of the original suspension is defined as the number of infectious units per unit volume of the preparation . In the in vitro and in vivo gene therapy studies [in vitro and in vivo including hematopoietic stem cells [Recently, lentivirus-based gene transfer vectors have been developed and have shown considerable promise for gene therapy research. It is evident that this vector system has several distinct advantages, and rapidly emerges as the vector of choice for  studies ,4. Most  studies -10. Seveem cells ,11, termem cells , retinalem cells , muscle,em cells  and dendem cells .Other lentivectors, such as those based on the feline immunodeficiency virus (FIV) , equine et al (1999) found that the ratio of cells transduced with the VSV-G-pseudotyped retroviral vectors based on MoMLV correlated with the result predicted from a Poisson distribution [in vitro transduction process, and how the variables affect vector titre determination and MOI usage.Just as with Moloney murine leukaemia virus (MoMLV) based retroviral vectors, many variables could theoretically affect the measurement of infectivity of lentiviral vector particles, such as target cell type, number, cycle, other modulators of cell membrane ingredients, the time needed for vector uptake and vector viability/susceptibility, half life during the transduction process or even Brownian motion in which the vector makes way to the target cell . In addiet al 199 found ribution ,19. Howeribution . The ratin vitro vector titration process, including the number of target cells being transduced, total number of viral vector particles, inoculum volumes (well beyond the depth of relevance to diffusion), vector decay and the period of vector adsorption (and thus vector decay). We also examined the use of various MOIs on several commonly used cell lines and tried to establish the relationship of MOI with the efficiency of gene transfer.In this study, we characterised factors that influenced the -7 M of insulin, and 10-7 M of dexamethasone in a 5% CO2 incubator. All other cells were maintained at 37°C in DMEM containing 2 mM glutamine, 10% Fetal Calf Serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin, similarly in a 5% CO2 incubator. 293, 293T and NIH3T3 were maintained in DMEM containing 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomcycin at 37°C similarly in a 5% CO2 incubator. Cells were seeded at 5 × 105 on 10 cm or 7.5 × 105 on 15 cm plate and were at 70 – 80% confluence at the time of transfection or transduction.Cell lines used in this study were a fetal rat liver carcinoma cell line, FRL 19; a human embryonic kidney cell line, 293 and its derivative, 293T; and a murine embryonic fibroblast cell line, NIH 3T3. FRL 19 was maintained at 37°C in Ham and Dulbecco's modified Eagle's medium  containing 2 mM glutamine, 4% Fetal Calf Serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1 μg of fungizone per ml (Ham and DMEM), 104 precipitation method as we previously reported [2 in 1/10-TE buffer  in 0.5 ml before an equal volume of 2× HBS  was added and mixed by gently bubbling air through the mixture for 1 min. The solution was then added drop-wise onto the cells (100 μl per 1 ml of culture media). The cell cultures were rinsed with PBS and given fresh media within 10–12 hr after initiating transfection. The medium was harvested 48 hr post-transfection, centrifuged at low speed to remove cell debris and filtered through a 0.45 μm filter. The supernatant was stored at 4°C no more than 24 hr before it was used for transduction.Replication-defective retroviral particles were generated by transient co-transfection of 293T cells with the three plasmids , using a CaPOreported . BrieflyThis was performed as reported previously ,21. Brie5 cells and transduced with serially diluted and concentrated viral vector stocks 16–18 hours after seeding when cells were about 70% confluent. For each transduction, 8 μg/mL of polybrene (Sigma) was included in the transducing inoculum. Forty-eight hours after transduction, EGFP positive fluorescent cells were counted using epifluorescent microscope  with the fluorescein isothiocyanate (FITC) excitation-emission filter set at 470 nm. The viral vector titre was determined as the average number of EGFP positive cells per 20 1-mm2 fields multiplied by a factor to account for dilution of the viral stock as well as plate size and thus total cell number. Alternatively, 48 hours after transduction, cells were harvested, resuspended and sent for FACs analyse at a local FACS facility .This was performed as we previously reported . Briefly3, 3 × 104 or 1 × 105 per well were seeded in triplicate in 24-well plates. Transduction was performed with the same stock of viral supernatant using volumes of 100 μl, 300 μl and 1 ml for 2 hours in the presence of 10 μg/ml polybrene. After the incubation period the cells were washed with fresh growth medium twice and allowed to grow for 2 days before the cells were trypsinised and fixed with 2% formaldehyde + 0.2% glutaraldehyde in PBS. EGFP positive and total cell numbers were counted with a haemocytometer using epi-fluorescence microscopy.293T cells at 1 × 103Fe(CN)6, 5 mM K4Fe(CN)6.3H2O, 2 mM MgSO4, and 1 mg/ml X-gal in PBS. Blue cells or colonies were counted as positive for gene transfer.293T cells were grown in 24-well plates to approximately 70% confluence. The cells were incubated with 500 μl of pHR' CMVLacZ supernatant for 10 min, 30 min, 1 hr, 2 hr, 4 hr and 17 hours. After the indicated incubation period the viral supernatant was removed and replaced with fresh media. Forty-eight hours post-transduction, cells were stained to check for the presence of LacZ with the following solution: 5 mM KCell-free viral vector-containing supernatant was incubated at 37°C for 30 min, 2 hr, 4 hr, or 24 hr prior to being used as the transducing medium (500 μl), with experimental samples in triplicate. 293T cell at 70% confluent cultures were exposed to the transducing media for 2 hours, after which the inoculum was removed and the cultures replenished with fresh media. Forty-eight hr post-transduction, cells were stained to check for the presence of LacZ with the X-gal solution. Blue cells/colonies were counted in 3 fields and the average used as the titre at that time point.5 cells/plate. Transduction was performed with viral vector stocks at a MOI of 2, 4, 8, 16 and 32 in the presence of 10 μg/ml polybrene (Sigma). Transduced cells were passaged every three days and EGFP positive cells sorted at a local FACS facility .293T cells were plated in a 10 cm plate at 1 × 10Flow cytometry analysis was performed to evaluate the expression of lentivirus vector-mediated gene transfer. Cells were washed with PBS, and then fixed with 1% paraformaldehyde before the analysis. Samples were analysed on a FACScan flow cytometry in QIMR.3 to 1 × 105 cells/ml. The results suggest the higher inoculum volume of the vector the more opportunity for a viral vector to reach a given target cell.Figure 2) of the cell culture, the depth of the fluid varied from 0.5 mm (with a volume of 0.1 ml) to 1.5 mm for the 0.3 ml volume, and a depth of 5.0 mm for 1 ml of the vector preparation. All of these depths were well beyond the diffusion limit of relevance to the adsorption of 95% of a retrovirus preparation. This was because the rate decreased with the square of the depth, equating to 0.16 mm for a 2.5 hours adsorption period [However, the results contradicted the data from an amphotropic MoMLV viral vector-mediated gene transfer where it was found that by keeping the virus vector concentration constant while the inoculum volume varied, the infectivity remained the same . This di3 and 3 × 104 there was only an average of 9.17-fold increase in total number of transduced cells . For a further 3.3-fold increase in cell number exposed in the same area, there was only a further 2.3 fold increase in total number of transduced cells. Thus, overall for a 100-fold increase in cell numbers (from 1 × 103 – 1 × 105) exposed to vectors there was only a 21.3-fold increase in total number of transduced cells. Interestingly, the increase of the number of positively transduced cells was not proportional to the increase of the vector inoculum volume. The increase in the number of transduced cells was proportionally less than the increase in inoculum volume, e.g. a 10-fold increase in inoculum volume resulted in only a 3.7 to 4.7-fold increase in the number of positively transduced cells.Similarly, vector titre was also affected by the number of target cells used in the vector titration process. A very significant increase in vector titre was noticed with increasing the cell numbers, but the increase was also not proportional. For a 30-fold increase in target cell number between 1 × 10The length of period of vector adsorption to target cells was shown to alter the transduction efficiency significantly. As the incubation period increased so did the number of transduced cells Figure . At 4 ho(1/2)) of the vector system used here, we pre-incubated the inoculum for increasing periods of time before applying aliquots to the target cell monolayer for vector titre determination. The length of time for which the viral supernatant harvest was left at 37°C (in a cell-free environment) prior to use, noticeably affected the value of the vector titre  and t(1/2) = ln(2)/kd to the data, {where VA is the concentration of active virus at time t, VAO is the initial concentration of active virus, and Kd is the virus decay rate constant}, the half-life of the vector was in the 8–9 hours range. This is the first time that lentivector stability has been examined. This estimation was twice as long as that for wild-type HIV [To estimate the half time  of th2 TU/mL to 1.2 × 104 TU/mL for the same viral suspension, more than a 50 fold difference. Likewise, because MOI is based on vector titre (MOI = titre × TD volume / number of cells), the use of MOI was thus affected.Lentiviral vector titre  was calculated using the number of TU/ml times the dilution factor of the vector stock, divided by the volume of vector used in the transduction. As shown in the above results, the number of positively transduced cells changed when the transduction conditions varied. Therefore, the vector titre was affected by inoculum volume, vector stability and target cell numbers. If vector titres were to be calculated using the existing formula that was developed based on retroviral vector-mediated gene transfer, i.e. EGFP-positive cells (TU) ÷ volume of vector inoculum (ml), the titre of the original vector suspension would result in absurdly different figures . Medium was changed every day, with cells harvested 72 hrs after transduction, washed twice with PBS, and then examined by FACS analysis. Figure 4a shows that transduction efficiency of lentivectors was higher on the FRL-19 cell line than the other three cell lines. Transduction efficiency was 67.4% in FRL-19 cells, 33.1% in 293T cells, 23.1% in NIH 3T3 cells, and 8.7% in 293 cells at a MOI of 32. Generally, it was the higher the MOI, the higher the transduction efficiency  played such an important role in the determination of transduction efficiency. It has been demonstrated that above the cell's surface in MoMLV based retroviral vector mediated gene transfer, a fluid layer of 0.1–1 mm thick remained stationary, and this layer is seen to be the major source of origin of the transducing elements. The large effects seen with non-agitated cultures in the present series of experiments with lentiviral vectors indicated some fundamental differences in the processes of the transduction pathways of MoMLV based retroviral vectors and lentivirally derived vectors. During the transduction process, the rate of collision between the virions and the surface of the target cells could be predicted from Brownian theory even when the viral suspension was being shaken continuously . This ap5 cells positive for a given marker gene expression, then the titre of the vector stock would be 1 × 106 TU/ml. When this vector stock is further used to transduce a new cell line, MOI is then determined by simply dividing the number of viral vector units added (ml added × TU/ml) by the number of target cells added (ml added × cells/ml). The average number of viral vector particles per cell in a transduction experiment could be less than 0.1 or more than 1000 depending upon how the experiment is designed. However, recent research showed that if MOI is too low, one may not get enough gene transfer and transgene expression [The viral stocks of most lentiviral vectors are generally produced from a 293 or 293T cell lines and the titre calculated by determining the number of foci (effect of the marker gene expression) produced in the cell line . For exapression . If MOI pression .1, slows drastically during S phase, and picks up the pace again as cells exit mitosis and re-enters G1[Employing MOI from 0–32, we demonstrated that efficient transduction of four different cell lines  resulted in a near liner relationship of MOI to transduction efficiency, the higher the MOI, the higher the transduction efficiency. This was somewhat surprising and contradicted traditional MoMLV based vectors, which showed an obvious plateau when the MOI was increased to about 3 . This isenters G1, which senters G1. Theoretenters G1 and thisIn the real world of gene transfer experiments, transduction conditions will be optimised to achieve the maximum efficiency. Generally, a high MOI is needed for satisfactory levels of gene transfer. Ideally, with a MOI of 2, every single cell might be expected to experience an average of two gene transfer events in a given transduction experiment, but probabilistic considerations of viral and vector-cell interactions ensure that this does not occur (i.e. only 67% of the cells would be "infected"). As seen in the current data, however, the efficiencies of transduction are very much less than the theoretical outcomes. Our study with lentiviral vector convincingly showed that the higher the MOI, the higher the efficiency of gene transfer and the level of gene expression. However, experiments employing MOI even greater than 1000 have still resulted in less than 100% of cells transduced ,28,29 inin vitro vector titration process will cause proportional changes in the magnitude of the transduction efficiency. It is thus evident that MOI is not applicable as a simply manipulable quantity in most gene therapy uses of the lentiviral vector system. Since clinical applications are an important outcome of gene transfer manipulations, and ultimately this may be done by in vivo delivery, the awesome task of evaluating the efficiency of transduction via this route will require considerable ingenuity. If MOI for lentiviral vector transduction has to be used for rigorous comparisons of data, then the specific experimental conditions for vector titration, with using the most sensitive cell lines, such as FRL 19, must be strictly observed for infectivity outcomes to be predictable.MOI is only a useful term for predicting transduction efficiency under very carefully defined experimental conditions. The assumption is not valid that changes in any one of the variables shown to be important in the CAEV, caprine arthritis/encephalitis virus; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; EIAV, equine infectious anaemia virus; FCS, Fetal Calf Serum; FITC, fluorescein isothiocyanate ; FIV, the feline immunodeficiency virus; HIV, Human Immunodeficiency Virus; JDV, Jembrana disease virus; MOI, Multiplicity of Infection; MoMLV, Moloney murine leukaemia virus; PC, phosphatidylcholine; PS, phosphatidylserine; VPC, vector-producing cell; VSV-G, Vesicular Stomatitis Virus G glycoprotein;None declared.in vitro transduction; GC provided some in BZ and HJ's work; M West provided advice on analysis of the data and manuscript writing; M Wei helped with the design and day to day supervision of all the experiments, assisted with analysing the data and prepared and prove read the manuscript.BZ performed the use of MOI to predict gene transfer events in the four cell lines; PM performed titration of lentiviral vectors; HJ performed the statistics and Table


The influence of ageing on the density of the functional entities of the papillae containing nutritive capillaries, here in terms as the papillary index, and the effect of topically applied vitamin C were investigated by confocal laser scanning microscopy (CLSM) in vivo.The age dependency of the papillary index was determined by CLSM on 3 different age groups. Additionally, we determined the effect of a topical cream containing 3% vitamin C against the vehicle alone using daily applications for four months on the volar forearm of 33 women.There were significant decreases in the papillary index showing a clear dependency on age. Topical vitamin C resulted in a significant increase of the density of dermal papillae from 4 weeks onward compared to its vehicle. Reproducibility was determined in repeated studies.Vitamin C has the potential to enhance the density of dermal papillae, perhaps through the mechanism of angiogenesis. Topical vitamin C may have therapeutical effects for partial corrections of the regressive structural changes associated with the aging process. A consistent feature of aged and photoaged skin is the flattening of the epidermal-dermal junction, evidenced in histological sections as a loss of rete ridges and the disappearance of papillary projections. The depth of interdigitation of the retepegs and the dermal papillae decreases with age . Using cIn extremely aged skin, papillae virtually disappear and the junction with the atrophic epidermis is a straight line versus undulations in younger skin. The papillary dermis also thins along with a loss of capillaries. Grove described that the corneocytes in aged skin become larger as a result of decreased epidermal turn-over . SimilarUnlike for example in mice, in humans like in primates in general and in guinea pigs vitamin C must be supplied exogenously in the diet. Diets deficient in vitamin C cause the multiple clinical signs of scurvy.Stones evolutionary treatise showed that a genetic mutation resulted in the loss of the ability of humans and some other animal species to synthesize vitamin C . The maiRetinoids, too, are known to improve the clinical appearance of photoaged skin and to promote the downgrowth of rete ridges, restoring the undulating dermo-epidermal interface . A functThe height of the epidermal-dermal junction like it is measured as a parameter in conventional histology, can be determined by confocal microscopy only in a very inaccurate manner because of the horizontal orientation of the images. The aim of this study is first to establish a CLSM measurable parameter comparable to the height of the epidermal-dermal interface. It should be sensitive to find age-associated changes of the dermal-epidermal interdigitation. Second to examine the effects of a topical vitamin C containing moisturizing cream on the developed CLSM parameters. We used a dose of 3% vitamin C as it shows a good stability and a good release of the active ingredient in a special emulsion system .In the first experiment (I), three groups of volunteers of different ages were compared measuring the density of the functional entities consisting of a dermal papilla and the nutritive capillary (papillary index). In a second experiment (II) a topically applied cream containing 3% vitamin C and its excipient were tested on the volar forearms of 33 volunteers. The effect of vitamin C cream was compared to excipient and to untreated control sites, respectively. (III) This experiment was repeated with a slightly different vehicle and for a two month treatment period only.The Vivascope 1000  is a commercially available confocal laser scanning microscope, which allows to examine human skin in vivo non-invasively. The system uses a Laser source with a wavelength of 830 nm, an illumination power up to 20 mW on the object and water immersion. In all three studies two different fields of view were used to obtain the papillary index (640 μm × 480 μm), and the projection areas of cells in the granular layer (230 μm × 88 μm). The lateral resolution of the instrument is 0.4 μm, the vertical resolution is about 1.9 μm.In confocal images of the forearm the epidermal-dermal interface shows dark round areas, the dermal papillae, surrounded by bright circles of basal cells, containing melanin granules and therefore reflecting strongly. Capillary loops are located in the centre of dermal papillae as black holes, often showing bright erythrocytes flowing through the capillary. The density of dermal papillae was evaluated by counting the papillae containing a capillary loop. 20 fields of view were investigated on each test site.gran) was evaluated by saving images of the most apical plane of the epidermis that still showed living dark nucleated cells. Although in the same image you can have virtual sections of different cell layers, the cells belonging to this layer right underneath the stratum corneum can be identified by the characteristic morphologic features the cells display. The size of cells was analysed using the image analysis program image tool. At least 20 cells in at least three different images were analysed by trained examiners.The size of cells in the upper granular layer (A(I) In the first study three groups of female volunteers were studied: twelve, ages 18 to 25, eleven, ages 40 to 50 and twelve, ages 65 to 80 years. Informed consent was obtained. Images were obtained on the volar side of the right forearm during the winter months.(II) In a second experiment a cream containing 3% vitamin C was compared to its vehicle on a group of initially 36 volunteers. 33 volunteers finally participated using it twice daily. Applications for four months to mid-volar forearms of healthy female postmenopausal subjects, ages 45 to 67 years (mean 55.3 years), phototype II and III. One area was left untreated. The double-blind study was conducted in winter. Informed consent was obtained. Evaluations were obtained at baseline and each month for four months, with one and two months follow-ups after stopping the treatment.(III) To determine reproducibility a second study was conducted over an eight weeks treatment period with a slightly different vehicle. The parameter of interest was the papillary index. Informed consent was obtained. Measurements were made after 20 minutes of acclimatisation at 21°C and 50% relative humidity.Morphometric results are expressed in box-whisker-plots as the median +/- the quartile. Statistical analysis used the Student t-test for normally distributed data or the U-Test (Mann-Whitney) for the age comparison study. The Wilcoxon test was used for analysing the effect of the vitamin C studies. Statistical significance was established for the 95% confidence level (p < 0.05).2, was significantly the highest of the three age groups. The middle aged group showed a significantly higher index, median 29.6 papillae per mm2, than the oldest group, median 24.7 papillae per mm2. The decrease of the papillary index was more pronounced between the younger group (18 to 25 years) and the middle-aged group (40 to 50 years) than between this group and the old group (65 to 80 years) (fig. (I) The papillary index in the young group, with a median of 54.3 papillae per mmIn the elderly group there were often large segments of flat epidermal-dermal junctions with a complete loss of papillae. In these regions the microvasculature consisted of horizontally orientated vessels having noticeably larger diameters than in young skin, probably venules rather than capillaries. A few curled remnants were occasionally found just beneath the atrophic epidermis.The vessel walls could not be resolved into endothelium, pericytes or smooth muscle cells by confocal examination, owing to low reflectiveness.(II) From four weeks onwards ascorbic acid treated areas showed a significantly higher density of capillary containing papillae than untreated and vehicle treated sites fig. .2 for the untreated and from median 22.7 to 30.3 papillae per mm2 for the vehicle treated sites) over the treatment period as well.Statistically significant differences were also observed in the vitamin C treated sites from four weeks onward in comparison to the beginning of the study. The untreated as well as the vehicle treated site showed a slight, but significant increase  The increase of the density of the papillae under influence of vitamin C was reproduced by repetition of the treatment schedules, ruling out the likelihood of changes due to chance.The decrease of the height of the epidermal-dermal junction is a well described histological finding in aged and photoaged skin ,24. We cVitamin C has been reported to improve the clinical appearance of photoaged skin and to enhance the synthesis of composite elastin fibres and of collagen . Our datIn aged skin, the cells in the apical granular layer show larger projection areas than in the skin of younger individuals  similar Another implication of the increase in papillae is that new blood vessels are formed during the treatment with vitamin C. The newly formed blood vessels show a normal anatomical structure in confocal microscopical examination and are apparently integrated in a healthy vascular architecture. In the confocal images there are no signs of pathologic changes of the vasculature like, for example, increased diameter, parallel orientation or clew of vessels in a papillae. No perivascular infiltrations of lymphocytes could be observed during the treatment period.The mechanism, by which Vitamin C restores dermal papillae is unknown.These are early results which strongly suggest that topical vitamin C may have important anti-aging effects in correcting the structural and functional losses associated with skin aging.The studies were funded by the Beiersdorf AG, Germany. Parts of the results presented in the manuscript are subject of patents pending by Beiersdorf AG.The pre-publication history for this paper can be accessed here: