Abdominal surgical site infections are among the most common complications of inpatient admissions and have serious consequences for outcomes and costs. Different risk factors may be involved, including age, sex, nutrition and immunity, prophylactic antibiotics, operation type and duration, type of shaving, and secondary infections. This study aimed to determine the risk factors affecting abdominal surgical site infections and their incidence at Imam Khomeini, a major referral teaching hospital in Iran.Patients (n = 802) who had undergone abdominal surgery were studied and the relationships among variables were analyzed by Student's t and Chi-square tests. The subjects were followed for 30 days and by a 20-item questionnaire. Data were collected through pre- and post-operative examinations and telephone follow-ups.Of the 802 patients, 139 suffered from SSI (17.4%). In 40.8% of the cases, the wound was dirty infected. The average age for the patients was 46.7 years. The operations were elective in 75.7% of the cases and 24.7% were urgent. The average duration of the operation was 2.24 hours, the average duration of pre-operative hospital stay 4.31 days and the average length of (pre- and post-operation) hospital stay 11.2 days. Three quarters of the cases were shaved 12 hours before the operation. The increased operation time, increased bed stay, electivity of the operation, septicity of the wound, type of incision, the administration of prophylactic antibiotic, type of operation, background disease, and the increased time lapse between shaving and operation all significantly associated with SSI with a p-value less than 0.001.In view of the high rate of SSI reported here (17.4% compared with the 14% quoted in literature), this study suggests that by reducing the average operation time to less than 2 hours, the average preoperative stay to 4 days and the overall stay to less than 11 days, and approximating the timing of shaving to the operation and substituting cefazolin for cefaluthin when prophylactic antibiotic is to be administered, the SSI may be reduced to a more acceptable level. SSIs are among the most common hospital acquired infections comprising 14–16 percent of inpatient infections. Such inClean wounds (1.5–3.7%); clean-contaminated wounds 3–4%); contaminated wounds (8.5%); dirty-infected wounds (28–40%); in laparoscopy (10%) umbilical hernia (2–5%); in the cancer of the colon without taking antimicrobial drugs (30–60%); or with antibiotic and proper intestine wash (10%); in colostomy (above 50%); in colon perforation (20%); in stomach cancer and surgery (20%); in hernionite (50%); in adult appendectomy (10–20%); in children's appendicitis (2–5%); in aged appendicitis and in pregnant women (10–50%); and in AIDS victims (above 50%); in liver abscess (20%); in hydatid cyst (2–5%); in acute and chronic cholecystectomy without stones (10%); in acute septic cholangitis (10–20%); in laparoscopic cholecystectomy (2–5%); and in splenectomy (2–5%) [%; contamSSI is identified with redness, inflammation, heat, pain, a temperature of 38°C, and septic drainage from the surgical site during the 30 days following operation. SeveralWhile we could not find any significant correlation between sex and SSI rate, age proved to be an important factor; the rate of wound infection for 15 to 24-year-old patients was only10% but increased significantly for those over 65 years of age. The extent of SSI was doubled for obese patients. The duration of surgical operation also proved to be a significant factor: only 3% of operations lasting 30 minutes or less led to infection, while for operations lasting more than 6 hours this rate leapt to 18%. SSI rate increased with longer durations of preoperative bed stay, but preoperative showers with a disinfecting soap such as chlorhexidine or Betadine decreased the cutaneous bacterial load. According to Kruise, the rate of infection was reduced to 1.3% among patients who showered with disinfecting soap containing hexachlorophene. In those who showered with ordinary soap the corresponding figure was 2.1%, and for patients who did not shower at all it increased to 2.3%. However, another study on 5536 subjects showed no decrease in SSI in patients who showered with chlorhexidine preoperatively; the rate was 4% [In this study, 802 patients in a teaching hospital during the 15 months from April 2002 to July 2003 underwent abdominal surgery and were studied for SSI and the factors affecting it. Initially, 884 patients were recruited for the study but 82 were excluded on the basis of the following criteria: deficient medical records; patients operated at a different hospital and subsequently transferred to Imam's Hospital: or patients deceased during the operation or within the following 30 days.The dependent variable in this study was abdominal surgical site infection, defined as redness, swelling, pain, temperature above 38°C, during the 30 days after operation. The independent variables were: age, sex, site operated, body mass index, time of shaving the site of incision, administration of prophylactic antibiotics, type of surgical operation, duration of operation, duration of preoperative bed stay; preoperative shower, type of shaving, and accompanying conditions.The data were collected through a 20-item questionnaire. The stages for data collection and information completion were as follows: identification of patients; preoperative interview; postoperative interview; record completion; weekly examinations and telephone follow-ups for 30 days following operation; and pre-discharge examinations. The collected data were analyzed by the SPSS 10 package, using Student's t-test for continuous variables and chi-square test.for categorical variables.To the best of our ability, this study was conducted with due attention to research ethics.however, problems met in the follow-ups the patient's discharge imposed limitations on the studyOf the 802 patients studied who had undergone abdominal surgery 139 (17.4%) suffered from SSI as defined in Table The body mass index for 73 patients (9.1%) was above 30, indicating obesity. Over half the patients (403 cases) suffered from accompanying conditions such as diabetes, high arterial blood pressure, kidney or liver failure, malignancy, febrile condition, cardiac disorders, thyroid disorders, blood disease, chronic obstructive pulmonary disease, convulsion, hyperlipidemia, or immunological disorders; or had previously undergone surgical operations. The rest were free of accompanying conditions Table .We found no significant correlations between SSI incidence and sex or preoperative shower. However, correlations with duration of operation, duration of preoperative bed stay, electivity of surgery, lengthening of preoperative shaving time, increasing age, wound infection, site of surgery, type of incision, accompanying disorders, and type of prophylactic antibiotic administered before operation were all significant at p < 0.001. Although differences in SSI rates were not significantly related to BMI, a trend was apparent: SSI rate was higher with low and high BMI.The following prophylactic antibiotics were used: ampicillin, gentamicine, cephalothin, metronidazole, ceftriaxone and cefazoline table .For the 802 participants in this study, the SSI reported was 17.4%, which is well above the 14–16% reported in other studies [SSI is not correlated with sex , in agreThe findings supported the literature by showing that administration of prophylactic antibiotic half an hour before the operation would bring about the best results and the lowest SSI . This waThe literature shows that with the duration of above 2 hours, the risk of SSI increases . The aveThe time of shaving when it approaches the operation and if done by clippers, reduces the SSI risk. In this study the two times: one hour before surgical operations and 12 hours before that were contrasted which supported previous findings with p = 0.001. This is one area where we can lower the risk by approximating the time of shaving as much as possible to that of operation.Other such factors quoted in the literature as the conditions of the operating theatre, personal hygiene, acompanying diseases, immunological disorders, smoking, techniques of surgery, the surgeon's expertise, duration of surgical scrub, preoperative skin preparation, poor hemostasis, failure to obliterate dead space, tissue trauma, and inadequate sterilization of instruments, which were not included in this study might be considered as confounding factors.Considering the relatively higher rate of SSI in this study (17.4% compared with the 14% quoted in the literature), especially in such cases as stomach cancers and umbilical hernia, where the rate is considerablly higher, we should carefully reconsider the whole operation procedure. In general, we should do our best to reduce the average operation duration to less than 2 hours and the average preoperative bed stay to less than 4 days. Thus, the present average of 11.2 days for the total bed stay would be reduced to less than 11 days. The time of shaving should approximate the operation time as much as possible. Finally, when the administration of prophylactic antibiotics is required, cefazoline is recommended to substitute cephalothin.Razavy: main researcher Ibrahimpour: Sabouri Kashani: (report, edit)Jafarian: (counsult)The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


Engaging practicing physicians in educational strategies that reinforce guideline adoption and improve the quality of healthcare may be difficult. Push technologies such as email offer new opportunities to engage physicians in online educational reinforcing strategies. The objectives are to investigate 1) the effectiveness of email announcements in engaging recruited community-based primary care physicians in an online guideline reinforcement strategy designed to promote Chlamydia screening, 2) the characteristics of physicians who respond to email announcements, as well as 3) how quickly and when they respond to email announcements.Over a 45-week period, 445 recruited physicians received up to 33 email contacts announcing and reminding them of an online women's health guideline reinforcing CME activity. Participation was defined as physician log-on at least once to the website. Data were analyzed to determine participation, to compare characteristics of participants with recruited physicians who did not participate, and to determine at what point and when participants logged on.Of 445 recruited physicians with accurate email addresses, 47.2% logged on and completed at least one module. There were no significant differences by age, race, or specialty between participants and non-participants. Female physicians, US medical graduates and MDs had higher participation rates than male physicians, international medical graduates and DOs. Physicians with higher baseline screening rates were significantly more likely to log on to the course. The first 10 emails were the most effective in engaging community-based physicians to complete the intervention. Physicians were more likely to log on in the afternoon and evening and on Monday or Thursday.Email course reminders may enhance recruitment of physicians to interventions designed to reinforce guideline adoption; physicians' response to email reminders may vary by gender, degree, and country of medical training. Repetition of email communications contributes to physician online participation. Many clinical practice guidelines have been disseminated through print publications and the Internet to improve the quality of healthcare. Complex physician behavior-change interventions have been created and applied to clinical practice guidelines to determine if the guidelines are being practiced. Research indicates that the adoption of clinical practice guidelines continues to lag behind their production and dissemination . Adoptioformation; they require minimal effort on the part of the recipient, which greatly supports their adoption [Proceedings of the American Medical Informatics Association's 2002 Annual Symposium, screen savers have been shown to be effective as a reminder system in engaging physicians in bioterrorism CME activity, although the use of email has exceeded all other Internet applications [Over the past ten years, accessibility to educational opportunities has increased with advancements in technology, particularly the Internet. The Internet offers a delivery system for educational interventions and research activities that are more convenient to the physician learner than traditional live large group lectures and seminars . The Intadoption . Email wadoption . Other fications .Flanagan and colleagues demonstrated promise for email as a means of engaging physicians in online activities designed to reinforce guideline use. They found physician response rate to email solicitation to be 50% over a 14-month study period . Beyond This study is a subanalysis of data from a randomized controlled trial (RCT), described in detail elsewhere . The goaThe study was conducted between February 1, 2002 and December 31, 2002. The intervention included a series of four modules, feedback of performance data and a quality improvement toolbox. The education modules were based upon adult learning and behavior change theory . Our driThe subanalysis of this trial focuses on the use of email reminders to engage physicians in the online intervention. Physician recruitment occurred from November of 2001 to January of 2002. Following recruitment, the intervention was initiated in February of 2002 via email broadcast to recruited physicians. During 2002, four separate educational modules were offered. Each module was introduced with a series of email announcements followed by email reminders that contained tailored subject line (i.e. Dear Dr. John Smith) and text message containing the URL that would connect the physician directly to the educational module. The first three modules, emphasized: (1) young sexually active women are at high risk for asymptomatic infection that may lead to future serious health consequences; (2) newer urine-based screening allows diagnosis without a pelvic examination; and (3) infection may be effectively treated with a one-dose antibiotic. The fourth module reviewed previously introduced concepts. A total of 33 announcements and reminders were sent to recruited physicians between February 1, 2002 and December 31, 2002, which represents one reminder overall for each 1.5 weeks of the study duration. Initially, we were concerned that frequent email reminders would be considered intrusive. After a trial period, we found that we could increase the frequency of reminders without difficulty, thus fewer email reminders were sent during the initial phases of the study.For the subanalysis of this trial, the principal outcome measure is participation, defined as physician log-on at least once to the website. Data was collected electronically when physicians logged on to the intervention. In addition, several evaluation questions were used at the conclusion of the online educational activities to explore CME preferences. Descriptive analyses of patient and physician demographics are used to compare baseline characteristics of patients and physicians in the email participants versus the recruited non-participants. Statistical significance is determined with tests appropriate to the distribution of the data . Two-tailed tests are used for all analyses.Four hundred eighty physicians were recruited to participate, representing 380 physician offices. Of the 463 recruited physicians, 445 were successfully contacted by email using the addresses they had furnished at the time of recruitment. Of these 445 recruited physicians, 210 (47.2%) physicians from 190 offices logged on to at least one of the educational modules. Of the 210 physicians who logged on at least once during the 45-week study period, one hundred twenty-four (59%) returned again to log-on for Module 2, eighty-seven (41%) logged on for Module 3, and forty-four (21%) logged on to Module 4.Two hundred and ten physicians of 445 logged on at least once to the website, leaving 235 physicians as non-participants. Figure The participant characteristics n = 210) were compared with those of recruited but non-participating physicians (n = 235)  and national meetings (50.0%). MDs were more likely to prefer web-based CME (37.0%) to local (35.1%) or national (16.6%) meetings. Female physicians reported a preference for web-based CME (50.0%), whereas male physicians preferred local (35.0%) and national (27.5%) meetings over web-based CME (25.0%).The results of this study indicate that email reminders may be useful in engaging nearly half of a group of practicing primary care physicians recruited to participate in an online women's health educational series. This data is consistent with McMahon et al.'s findings in comparing the use of email, fax and mail, finding that email reminders were more useful to increase response rates . The stuFindings from follow-up evaluation question data indicate that CME providers interested in targeting specific groups of physicians may benefit from using alternative methods of CME recruitment and delivery. CME providers targeting DOs may want to explore ways to engage DOs in web-based learning activities or consider focusing activities that target DOs to local or national meetings. Providers of CME who are interested in engaging male physicians and graduates of U.S. medical schools may find email reminders useful, but they may also want to explore additional methods of recruitment.th reminder. Based on our experience, it would seem reasonable that providers and researchers with limited resources consider focusing their announcements/reminders on the first 3–10 encounters. Data from time of log-on underscores the advantages of asynchronous online interventions for busy clinicians. Traditional "live" online symposia scheduled for the middle of the day might appeal to the physicians who logged on between 10 AM and 2 PM (10:00–14:00), but data from this intervention suggest that many physicians have more available time later in the day for online educational activity. The investigators findings of Monday being a frequent day for log-on was unexpected, but may offer an opportunity for future study designs to include weekend email broadcasting rather than a Thursday broadcast.Persistent email reminders did increase physicians' response rates to online education, but response rate decreased with the number of reminders. The first three reminders produced the largest responses, with decline after the 10The topic of the online educational activity may influence response to email announcements and reminders. While baseline Chlamydia screening rates were relatively low in both groups, the significant difference between the groups may indicate that those who are likely to perform better according to clinical practice guidelines are more likely to participate in online educational strategies that reinforce their use. Or the higher screening rates may be associated with a higher degree of interest in the overall topic area of women's health.Also related to the topic addressed in this online study, previous studies of preventive practices of female physicians have indicated they are more likely than males to promote preventive practices  and screComparisons of characteristics of participant physicians and non-participant recruited physicians may be useful to those designing online recruitment and engagement strategies for future studies. Those using email reminders to communicate with physician populations including large numbers of DOs, however, may benefit from considering blended methods of CME recruitment and delivery. Using various methods of reaching providers, may also enhance DO participation. CME providers targeting DOs may want to explore additional ways to engage DOs in web-based learning activities or consider focusing activities that target DOs at local or national meetings.Physicians' online clinical information seeking and engagement in online education continues to grow . ResearcReminding physicians often via email about online educational opportunities appears to increase engagement in a community-based primary care physician audience. The early and consistent implementation of this push technology may increase physicians' utilization of interventions designed to improve practice.MA participated in the statistical analysis and drafted the manuscript. BCC participated in the statistical analysis and drafting of the manuscript. LC participated in all phases of the project. TW participated in the draft of the manuscript. CS participated in the design and coordination of the study and the drafting of the manuscript. MNR participated in the coordination of the study. NWW participated in the design and implementation of the study. JJA conceived of the study and participated in all phases of the study. All authors read and approved the final manuscript.The authors declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


ELA2), causing increased intracellular concentration of this serine protease, inhibits neutrophil differentiation in severe congenital neutropenia (SCN). Since the clinical symptoms of agranulocytosis and SCN are similar, we hypothesized that it may origin from a common genetic variation in ELA2 or that sulphasalazine may affect human neutrophil elastase activity and protein expression.Drug-induced agranulocytosis, a severe side effect marked by a deficit or absolute lack of granulocytic white blood cells, is a rare side-effect of the anti-inflammatory drug sulphasalazine. Mutations in the human neutrophil elastase gene (ELA2 in DNA from 36 patients who had suffered from sulphasalazine-induced agranulocytosis, and compared them with 72 patients treated with sulphasalazine without blood reactions. We also performed in vitro studies of the blood cell lines HL60 and U937 after sulphasalazine exposure with respect to cell survival index, neutrophil elastase protein expression and activity.We screened for genetic differences in ELA2, which previously have been reported to be associated with SCN, was found in this material. Protein expression of human neutrophil elastase in lymphoma U937 cells was not affected by treatment with concentrations equivalent to therapeutic doses. Cell survival of lymphoma U937 and promyelocytic leukemia HL-60 cells was not affected in this concentration range, but exhibited a decreased proliferative capacity with higher sulphasalazine concentrations. Interestingly the promyelocytic cells were more sensitive to sulphasalazine than the lymphoma cell line.None of the mutations in ELA2 mutations do, however, not seem to be involved in the etilogy of sulphasalazine-induced agranulocytosis. Why sulphasalazine is more toxic to promyelocytes than to lymphocytes remains to be explained.Neutrophil elastase expression and  Sulphasalazine (SA) has anti-inflammatory, immunosuppressive and antibiotic actions, and is a component in the therapy of Crohn's disease, ulcerative colitis and rheumatoid arthritis. Bacterial enzymes in the colon split sulphasalazine into sulphapyridine and 5-aminosalicylic acid before it is absorbed. Sulphapyridine acts as a sulphonamide antibiotic, whereas 5-aminosalicylic acid is believed to be the anti-inflammatory metabolite. Common side/toxic effects are vomiting, skin rash and headache. The incidence of the hematological adverse effects associated with sulphasalazine is generally low, but the reactions can be severe and sometimes fatal. The risk of sulphasalazine-induced agranulocytosis, i.e. profoundly depressed circulating neutrophils is highest within the first three months of sulphasalazine-treatment, with a fatality rate of 6.5 % . ClinicaELA2, encoding human neutrophil elastase, have been identified in a majority of the cases with CN and two-thirds of the cases with SCN [Severe congenital neutropenia (SCN) and cyclic neutropenia (CN) occur both as inherited and as sporadic diseases. SCN has a constant low neutrophil number if left untreated, whereas CN manifests with cyclic oscillations of neutrophil number with a 21-day cycle. Recently, diverse heterozygous mutations in ELA2, one third which of had experienced sulphasalazine-induced agranulocytosis. We, furthermore, tested for cytotoxic doses of sulphasalazine, and studied protein expression of human neutrophil elastase in sulphasalazine-treated blood cell lines.In this study, we hypothesized that sulphasalazine-induced agranulocytosis, with clinical symptoms similar to congenital neutropenia, may arise from genetic variation in the human neutrophil elastase gene. We genotyped 108 sulphasalazine-treated patients for Patients were treated with sulphasalazine  for inflammatory joint diseases and inflammatory bowel disease. The cases with sulphasalazine-induced agranulocytosis were originally collected through the Swedish Medical Products Agency's register of adverse side effects . The conELA2 were amplified by PCR using primer pairs listed in Table ELA2.Genomic DNA was extracted from whole blood using standard techniques. Fragments covering exons 2–5 of The lymphoma cell line U937 and the promyelocytic cell line HL-60 (American tissue culture collection) were cultured in Dulbeccos modified Eagles Medium, DMEM (Sigma) supplemented with 10 % fetal bovine serum , L-glutamine and penicillin-streptomycin (Sigma).6) of U937 cells were grown in 75 cm2 dishes in complete medium containing 0, 125 and 250 μM sulphasalazine for 24 h. For protein isolation, cells were washed in PBS and lysed in buffer containing 1% Triton X-100, 50 mM Tris-HCl pH 8.0 and protease inhibitor cocktail (Sigma) and were kept on ice for 30 min. Lysates were centrifuged for 10 min at 10 000 × g, and protein concentration was determined using BioRad protein assay. Criterion precast gels (BioRad, Sweden) were used to perform SDS-page with 20 μg protein loaded per well. After gel transfer to a nitrocellulose membrane, the membranes were blocked over night in 5 % dry milk in TBS-Tween. Primary antibody against human neutrophil elastase  was diluted 1:1000 in 5 % dry milk in TBS-T. After 2 h incubation, and four sets of washing, a secondary antibody was added (1:5000) and blots were developed using ECL (ECL Western blotting system, Amersham, Sweden). Western blot analysis of human neutrophil elastase expression was performed twice.Equal numbers (8 × 102, 0.1 % Triton X-100. After homogenization, 300 μl of 1.4 M NaCl in 0.1 % Triton X-100 was added and samples were centrifuged at 15 000 × g, for 15 min at 4°C. The supernatants were transferred to new tubes and assayed for elastase activity using Suc-Ala-Ala-Ala-pNA (Sigma) as a substrate. For each assay we took 25 μl sample, mixed with 100 μl buffer containing 100 mM Tris-HCl pH 8.5, 1 M NaCl, 500 mM MgCl2 and 0.1 % Triton X-100. To this, 50 μl of substrate was added, to a final concentration 1 μM. After 30 min of incubation in room temperature, absorbance was read at 405 nm and the concentration was calculated from a standard curve of elastase (Sigma).Cells (HL-60 and U937) treated with 0, 125, 250 and 500 μM of for 24 h were lysed with 100 μl of buffer containing 100 mM Tris-HCl pH 7.4, 1 mM MgCl50).For the cell viability assay, we used a fluorometric microculture cytotoxicity assay (FMCA) previously described by Larsson et al . Briefly2-test with one degree of freedom, using Minitab 14. A p-value less than 0.05 was denoted with (*), p < 0.01 with (**) and was considered as statistically significant.Two-tailed Student's t-test was used to compare subject characteristics and results from cell culture between cases and controls. Frequencies of subject characteristics male versus females was tested with Chip = 0.023). The white blood cell count (WBC) before sulphasalazine-treatment did not differ between cases and controls, nor did the dose of sulphasalazine. Bone marrow aspirates had been taken from 10 patients (cases). In all samples, the myelopoesis was seriously reduced and a maturation arrest at the promyelocyte-myelocyte stage of neutrophilic differentiation was seen.The characteristics of the subjects are presented in Table ELA2 was found in this material, although we found a silent single nucleotide polymorphism, called S173 [None of the previously reported mutations in led S173  that corled S173 . No corr50) of sulphasalazine was approximately 370 μM for HL-60 cells and 820 μM for U937 cells.By western blot analysis, we analyzed neutrophil elastase protein expression in U937 cells. No difference in human neutrophil elastase expression was detected after treatment with 125 and 250 μM sulphasalazine Figure , but at ELA2) are implicated as the primary abnormality [ELA2, which, however, was equally represented among cases and controls.Idiosyncratic drug-induced agranulocytosis can be due to several different mechanisms of action, including immunological, toxic and genetic ,8. Toxicormality . The focELA2 act in a dominant manner, interfering with sub-cellular trafficking of neutrophil elastase, and leading to an accumulation of neutrophil elastase in the cytosol [ELA2 may disrupt the AP3β-recognition site [GFI1, a transcriptional repressor of ELA2, causes over-expression of neutrophil elastase in mice, thus, making them neutropenic [Heterozygous mutations in  cytosol . For nor cytosol . When G- cytosol . In theo cytosol , and mution site . Furthertropenic .50 values of 370 μM and 820 μM, respectively. Human neutrophil elastase expression in lymphoma U937 cells did not differ after sulphasalazine at 125 and 250 μM, indicating that human neutrophil elastase production is not affected by sulphasalazine at subtoxic levels.During maintenance therapy with sulphasalazine, trough serum sulpha concentration is on average approximately 100 μM at the Department of Clinical chemistry and pharmacology, Uppsala University hospital. To avoid toxic effects, trough serum concentration of sulpha should stay below 600 μM . Our in ELA2 mutations were found, and therapeutic concentrations of sulphasalazine did not increase the expression of human neutrophil elastase. High concentrations of sulphasalazine were toxic to white blood cells in vitro; however, there is no evidence that this toxicity is mediated through human neutrophil elastase. Promyelocytic cells were more sensitive to sulphasalazine than lymphoma cells, and the reason for this difference may also explain sulphasalazine-induced agranulocytosis.In conclusion, neutrophil elastase does not appear to be involved in the etiology of sulphasalazine-induced agranulocytosis. No causative The author(s) declare that they have no competing interests.AJ carried out the molecular genetic studies, participated in the sequence alignment, drafted the manuscript and carried out the in vitro experiments. MW participated in the design of the study and performed the statistical analysis. HM conceived the study, and AJ, MW and HM participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The management of acute myocardial infarction (AMI) has improved over the last 50 years with the more frequent use of effective medicines and procedures. The clinical benefit of the speciality of the attending physician is less clear. The United Kingdom National Service Framework for coronary heart disease (CHD) suggested that patients with CHD are likely to benefit from cardiological supervision. We set out to assess the effect of access to cardiologists on survival among AMI patients admitted in two UK hospitals.The study was conducted in a university hospital and a district general hospital in England. Information was obtained on age, sex, ethnicity, Carstairs socioeconomic deprivation category derived from postcode of residence, comorbidity, distance from hospital and medication from all patients admitted with acute myocardial infarction in two acute trusts between July 1999 and June 2000. Record linkage to subsequent Hospital Episode Statistics and Registrar General's death records provided follow up information on procedures and mortality up to eighteen months after admission. Cox proportional hazard models were used to investigate the main hypothesis controlling for confounding. The main outcome measure was 18-month survival after myocardial infarction.Access to a cardiologist was univariately associated with improved survival (hazard ratio 0.16, 95% CI 0.10 to 0.25). This effect remained after controlling for the effect of patient characteristics (hazard ratio 0.22, 95% CI 0.14 to 0.25). The effect disappeared after controlling for access to effective medication (hazard ratio 0.70, 95% CI 0.33 to 1.46).Access to a cardiologist is associated with better survival compared to no access to a cardiologist among a cohort of patients already admitted with AMI. This effect is mainly due to the more frequent use of effective medicines by the group referred to cardiologists. Hospitals may improve survival by improving access to effective medicines and by coordinating care between cardiologists and general physicians. The management of acute myocardial infarction (AMI) has improved over the last 50 years with the more frequent use of effective medicines and procedures. The clinical benefit of the speciality of the attending physician is less clear. The effect of the speciality of the attending physician on mortality in AMI has been studied mainly in the United States (US) with conflicting results arising from the studies -6. The eThe National Service Framework (NSF) for coronary heart disease (CHD) published in March 2000, suggested that patients with CHD are likely to benefit from cardiological supervision. To provide this level of care, all acute hospitals will eventually need a minimum of two cardiologists . A recenThere is a need to establish whether the involvement of a cardiologist in the management of AMI patients affects the quality and outcome of care, and if so to identify ways to improve the outcome of care for patients unable to gain access to a cardiologist. This study aims to assess the effect of access to cardiologists on survival among AMI patients accounting for access to effective investigation, medication, procedures and the underlying condition of the patient at presentation.A retrospective cohort design was used.st July 1999 and 31st June 2000 with a discharge diagnosis of AMI were included in the analysis. A diagnosis of AMI was based on evidence of raised cardiac enzymes and/or other indicators of myocardial necrosis and on a physician's judgement of ECG changes indicative of AMI.All patients admitted to two hospitals in Eastern England between 1Hospital Episode Statistics were used to extract records of all patients admitted with the ICD10 diagnostic codes for Acute Myocardial Infarction (I21, I22, I23) in any diagnostic field. The records were restructured to produce one record per patient using the new NHS number. For records with missing NHS number, a unique identifier was created using date of birth, sex and post code. The unique identifier was also used as a check for the records with NHS numbers. Records of all Finished Consultant Episodes with OPCS 4 codes of either Coronary Artery Bypass Grafting (K40–46) or Percutaneous Transluminal Coronary Angiography (K49–50) were extracted. The process was repeated for angiography (K63 – 65). Record linkage with subsequent Hospital Episode Statistics and Registrar General's records provided follow up information on procedures and death up to eighteen months after the index AMI.All cases were reviewed by examining the case notes. Detailed information on variables summarised below was obtained.List of variables extracted from patients' case notes• Hospital where patient was first admitted• Whether the patient was seen by a cardiologist or not, determined by evidence of a cardiology review in a patients case note• The average distance from residence to hospital first seen was determined by mapping the distance in miles using the RAC™ website distance calculator• A history of comorbidity – defined as the presence of diabetes, metastatic malignancy, cerebrovascular accident (CVA), asthma, chronic obstructive airway disease, renal impairment, endocrine disorder, chronic infection, dementia• Physical impairment with poor mobility• Procedures – exercise testing, angiography, CABG, PTCA• Appropriate medication – β blockers, aspirin, ACE inhibitors, thrombolysis table • A history of Smoking Previous MI Hypertension• Impaired left ventricular function based on echocardiography result or angiography• Severity of AMI using the number of vessels affected (vessels were considered affected if a lesion of 50% or more was noted in the angiography report)• Demographic factors: age, sex, ethnicity, socioeconomic status and Carstairs socio-economic deprivation category derived from postcode of residence2 test. Continuous variables were tested using the Student t test comparing patients seen by cardiologists and those seen by other physicians.Baseline patient characteristics were tabulated. Categorical variables were tested for statistical significance using the χThe relative odds of drug and procedure use by speciality of physician were calculated. Specific variables included the use of thrombolytic therapy, β blockers, aspirin, ACE inhibitors among clinically appropriate groups, and the use of exercise testing, coronary angiography, angioplasty and coronary artery bypass grafting (CABG).Cox proportional hazards models were used to investigate the effect on survival of seeing a cardiologist. Multivariate models were fitted to control for the effect of patient and hospital characteristics. Controlled factors included: age, sex, comorbidity, hospital, distance from patients residence to hospital and Carstairs socio-economic deprivation category in the first instance. The effect of angiography, revascularisation and the use of effective medicines (aspirin and/or β blockers and/or thrombolysis – eligibility criteria listed below) were subsequently introduced into the model to investigate whether these affected outcome. The factors were included in the model either because they are known to confound the association between type of physician and survival or are known to be associated with survival and were not comparable between the two groups.Eligibility criteria for appropriate use of effective medicines .Thrombolytic therapy• No warfarin therapy on admission• ST-segment elevation on initial ECG• Less than 12 hours since onset of chest pain• Systolic BP less than 180 mmHg and diastolic BP less than 110 mmHg at presentation• No severe CVA, gastrointestinal disease or chronic liver diseaseAspirin• No haemorrhagic complication• No severe CVA, gastrointestinal disease or chronic liver disease or renal failure• No warfarin therapy on admissionβ blockers• No chronic lung disease• No cardiogenic shock, hypotension, complete heart block or decompensated heart failureThe characteristics of the two hospitals are summarised figure The main outcome was eighteen-month survival defined as time between date of AMI and death.Statistical analysis was carried out using Stata (Version 6.0).In general for Cox regression analysis, the number of events i.e. deaths (and similarly the number of non-events) per variable modelled should be at least 10 .Ethical approval was obtained from the two local research ethics committees of the hospitals involved in the study. Permission from the director of research and Caldicott guardians of data in each hospital was also obtained.Medical records were obtained for 94% of eligible patients (476 of 506 patients). Four patients did not have enzyme changes of myocardial infarction or were clear cases of miscoding. The mean age of the cohort was 69.3 years (SD 13.2) and 34.5% were female. The patients with missing records did not differ from those with records in terms of age or sex using information from routine data . There were no significant differences in the proportion of data missing between the two hospitals including those for eligibility to effective medications and co morbidities.Patients in our sample seen by a cardiologist were younger, with a higher proportion of males to females. They were more likely to have a history of CHD prior to myocardial infarction, but less likely to have a history of congestive heart failure or hypertension Table . PatientThe likelihood of receiving effective medication (thrombolytic therapy, β blockers, aspirin) was higher among patients seen by a cardiologist compared with those not seen by a cardiologist. Furthermore, patients seen by a cardiologist were more likely to have undergone exercise testing, angiography and revascularisation procedures Table . The useUnivariate analysis indicated that patients seen by a cardiologist had better survival (hazard ratio 0.16, 95% CI 0.10 to 0.25).The survival of patients seen by a cardiologist was still better than that for those not seen by a cardiologist (hazard ratio 0.22, 95% CI 0.14 to 0.25) after controlling for the confounding effect of pre-existing comorbidity, age, sex, hospital first seen, deprivation and distance Table . The effThe effect of seeing a cardiologist on survival became non-significant once access to angiography, revascularisation procedures and effective medicines (aspirin and/or β blockers and/or thrombolysis) had been adjusted for. The hazard ratio was 0.70 95% CI 0.33 – 1.46). The fully adjusted model indicated that the most important factor affecting survival was access to effective medication. . The usAll cases diagnosed by the managing clinician as AMI with ECG changes and found to have enzyme changes indicating myocardial necrosis were included in the analysis after the case notes were reviewed. This may have involved some misclassification due to misdiagnosis, but this is likely to occur at random, affecting only a small number of cases, and can only underestimate the effect of each factor.Limitations of this study include potential for bias in the allocation of patients to treatments and physicians (ie cardiologist/non cardiologist) that were not measured. Another possible limitation is bias arising from medical case notes which were unobtainable. Records from routine data were used to examine whether those with missing records differed in any systematic way from those with available records; it was found that they did not differ in age or sex. The study did not use any criteria for judging clinical appropriateness of the procedures used. Incomplete recording of information in the case notes hampered the determination of appropriateness for effective medication. However there was no difference in the proportion of missing data between the two hospitals.Since doctors may selectively be referring younger patients with lower comorbidity to a cardiologist, we controlled for age and comorbidity in the analysis. However, residual confounding from age and comorbidity could still account for some of the observed difference in survival.All patients admitted to the Coronary Care Unit (CCU) in one of the hospitals are seen by a cardiologist and have a higher chance of accessing thrombolysis, angiography and revascularisation. A cardiologist also sees the majority of patients admitted to the CCU in the second hospital. The protocol for the management of AMI in both hospitals stipulates a cardiology review of all AMI patients. Access to a cardiologist may be a proxy measure of access to effective treatment and it may not be the trigger for effective treatment. These limitations mean that the findings should be interpreted with a degree of caution. In addition our inability to use formal appropriateness criteria limits the interpretation of findings.Despite these limitations the results indicate that, in the short term, acute trusts can improve survival of patients by increasing the use of effective medicines among general physicians.We observed better survival among patients seen by a cardiologist compared with patients with no access to a cardiologist, among a cohort of patients already admitted with AMI. This effect was entirely explained by the more frequent use of effective medicines by cardiologists in the multivariate analysis. About eight hospitals in the UK Royal College of Physicians survey have no cardiologist and another 30% have a single cardiologist . It willNone declaredAll authors contributed in writing the paper, in addition, IA collected the data and conducted the analysis, DK conceived the idea, BA provided advice on statistical analysis, JP provided input in the conduct of the study, PW contributed in refining the research question and discussing the findings of paper.The pre-publication history for this paper can be accessed here:


Since 3D structural data of the LV is now obtainable using standard transesophageal echocardiography (TEE) devices intraoperatively, the present study describes a method to transfer this data into a commercially available FEM analysis system: ABAQUS© Software was used for semi-automatic endocardial border detection, reconstruction, and volume-rendering of the clinical 3D echocardiographic data. A newly developed software program MVCP FemCoGen©, written in Delphi, reformats the TomTec file structures in five patients for use in ABAQUS and allows visualization of regional deformation of the LV.In this prospective study TomTec LV Analysis TEEThis study demonstrates that a fully automated importation of 3D TEE data into FEM modeling is feasible and can be efficiently accomplished in the operating room.For complete intraoperative 3D LV finite element analysis, three input elements are necessary: 1. time-gaited, reality-based structural information, 2. continuous LV pressure and 3. instantaneous tissue elastance. The first of these elements is now available using the methods presented herein. Intraoperative TEE is currently available in most cardiac surgical operating rooms. In some centers, intraoperative 3D echocardiography is used to evaluate geometry and to plan surgical interventions prior to LV remodeling surgery. However, quantitation of LV geometry is limited to rather imprecise measures such as ejection fraction. Thus the cardiac surgeon has no sophisticated, immediate, quantitative analysis of the preoperative 3D LV geometry. Intraoperative quantitative analysis of the dynamic behavior of the LV might provide optimal information upon which to base precise patient-specific planning of the surgical intervention, as well as to assess the adequacy of the completed surgical repair.Because the LV cannot be realistically described by a symmetric mathematical model, the modern approach consists of using a FEM mesh which approximates LV geometry  or wholeInitial attempts at FEM in the heart have been carried out with 3D segmentation and tracking using sophisticated and expensive cardiac MRI , but untFEM modeling of 3D intraoperative echo data provides an excellent tool for incorporating material properties, volumetric data and boundary pressures to more accurately record and then to simulate LV dynamic performance. Accurate simulation will be the foundation of surgical planning. The limitation until now in applying FEM intraoperatively has been the technical complexity of this technique. The purpose of this study is to take the first step towards introducing FEM into the operating room environment. The goal is to facilitate transfer of geometric data from 3D ultrasound data set into FEM.-6 m-3 kg-1 at a respiratory rate sufficient to maintain end-tidal CO2 levels between ~4.4*103 m kg-1 s-1 and ~5.1*103 m kg-1s-1.After obtaining institutional review board approval, LV images from clinical TEE data sets were obtained in five patients via the midesophageal window using a Philips 5500/7500 or and Acuson Sequoia ultrasound system. After induction of general anesthesia and airway protection, the esophagus was intubated using an omniplane TEE probe. 3D TEE data sets of the LV structures including mitral annulus and leaflets, chordae tendinae, papillary muscles and ventricular wall were obtained using the automated Philips/Acuson acquisition protocol at 10° increment. Images were gaited for both beat-to-beat variability and respiratory motion. In order to facilitate acquisition in the shortest possible timeframe, ventilation was modified to provide a tidal volume of 5*10© software module [All images were stripped of patient identifiers. For LV geometry reconstruction, the TomTec LV-Analysis TEEe module  was emple module . It alsoThe first landmark was set in the middle of the mitral valve at the level of its annulus. Care was taken to avoid having the mitral valve cusps cross this landmark. Two additional landmarks were placed in the middle of the aortic valve at the level of its annulus and at the endocardial level of the LV apex. With this landmarking procedure, a time-resolved LV geometric analysis with 18 models per heart cycle was obtained see Fig. .The rendered LV geometry resulting from the TomTec analysis tool was transferred to an ABAQUS input file using software written in Delphi. In this program, the TomTec file structure was reformatted to an ABAQUS system (version 6.3) input file based on standard ABAQUS FEM elements.© cardiac simulator [ABAQUS creates a time series of LV model files and requires continuous intraventricular pressure and tissue elastance parameters to process the model. For this analysis LV pressure was modeled using wave forms obtained from Columbia University's HeartSimimulator . A singlThe time required for each step in this process was recorded for each patient data set.© [th image out of a set of 18 images per heart cycle) are displayed and can be visualized using ABAQUS viewer options. Each mesh element can be analyzed separately. This is shown in Fig. Both, the Philips Sonos 5500/7500 or the Acuson Sequoia ultrasound systems required less than 10 minutes acquistion time per patient. The application of the TomTec LV analysis algorithms with manual placement of the necessary landmarks took approximately 7 minutes per patient. In five patient data sets conversion from TomTec data to the FEM model was carried out in less than a minute for a heart cycle using the conversion tool MVCP FemCoGenThe general intention of this study was to demonstrate the feasibility of transporting individual patient's LV geometry data into a FEM model. Standard laptop computer technology was utilized to accomplish the transfer from common TEE-machines (Philips Sonos 5500/7500 and Acuson Sequoia).© package and ABAQUS FEM system, plus the recently developed MVCP FemCoGen©. Accomplishing this transfer will form the foundation for intraoperative surgical planning and quantitative outcome assessment of valvular and LV reconstructive surgery.The software running on the laptop was the commercially available TomTec LV Analysis TEEThe scope of this study was to produce a prototype in which the feasibility of the method could be assessed. In a fully operational system, we could postulate clinical applications such as enhanced/automated wall motion abnormality detection, assessment of regional relaxation which encompases the entire ventricle, assessment and guidance of ventricular remodeling operations, and serial assessment of recovery of regional wall function post myocardial stunning.FEM meshes have been used for approximately 30 years  in the aThe total time required for acquisition to a completed FEM model was approximately 24 minutes and can be accomplished during the time period when the patient is being prepared for cardiopulmonary bypass . Thus the feasilbility in terms of duration is clearly demonstrated.© Software is under review by the US FDA, but despite of lack of validation TEE is the only practical technology in the cardiac operating room for the forseeable future. Ultrasound tissue Doppler technologies may be developed in the future to allow automation of the registration process.In terms of procedure accuracy, reproducibility and duration, the primary limitation is the dependence of the TomTec software on manual entries of the three registration landmarks. This requirement is iterative. Manual entries must be done for multiple frames within the TEE data sets. Inter- and intraobserver variability is a general problem for ultrasonic imaging. The validation of the TomTec border detection has not been published. TomTec LV Analysis TEEA limitation of the present study is that it is focused on the deployment of the transfer method. The entire process will require extensive validation. The validation strategy will most likely involve comparision with preoperative cardiac MRI as well as comparison with bypass and post bypass tissue geometry in the same patients. Creating models from MRI based data sets analogous to the TomTec LV analysis and transfering these models to ABAQUS might lead to a new validation strategy which is not been possible up to now. The tool for modeling presented here facilitates vector-subtraction analysis for different points within the cardiac cycle. Quantification is therefore immediately available for both global and regional wall motion, shape and volume analysis. The future use of such instantaneous analysis has a number of potential applications for LV function assessment and surgical planning. This technology could enable a comprehensive automated regional wall motion analysis. A significant challenge in the evaluation and management of patients with coronary artery disease is determining the viability of myocardium. A biomechanical FEM of the LV myocardium can be imported to evaluate dynamic mechanical properties of regions of the myocardium. This approach could provide the basis for a new index of regional myocardial viability.For complete intraoperative 3D LV finite element analysis, three input elements are necessary: 1. time-gaited, reality-based structural information, 2. continuous LV pressure and 3. instantaneous tissue elastance. The first of these elements is now available using the methods presented herein. The later two parameters will be required for robust modeling and analysis. Pressure data will be easily available in the cardiac operating room. Strategies for computing elastance are presently under development. With all three parameters, it will be possible to begin to develop the computational strategies which will allow virtual procedures to be performed utilizing 3D display technology and a haptic-feedback robotic "instruments". Whether this new intraoperative information will be useful in assessing the effectiveness of surgical interventions such as LV remodeling remains to be studied.FEM analysis has not been feasible for LV in the intraoperative setting. The major roadblock was the complexity and the practicality of transfer of structural 3D data to a FEM analysis program. This study describes a method to rapidly transfer 3D structural data from the TEE device into a FEM analysis program. Once mesured pressure and calculated elastance are added to the model, near real-time dynamic stress-strain information in the operating room will be achievable.®, NSN did the data acquisition and the medical part. Both authors read and approved the final manuscript.JFV did the technical part implementing the FEM model in ABAQUS


Our analysis suggests that miRNA genes, which are about 1% of all human genes, regulate protein production for 10% or more of all human genes.MicroRNAs (miRNAs) interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. The specific function of most mammalian miRNAs is unknown. We have predicted target sites on the 3′ untranslated regions of human gene transcripts for all currently known 218 mammalian miRNAs to facilitate focused experiments. We report about 2,000 human genes with miRNA target sites conserved in mammals and about 250 human genes conserved as targets between mammals and fish. The prediction algorithm optimizes sequence complementarity using position-specific rules and relies on strict requirements of interspecies conservation. Experimental support for the validity of the method comes from known targets and from strong enrichment of predicted targets in mRNAs associated with the fragile X mental retardation protein in mammals. This is consistent with the hypothesis that miRNAs act as sequence-specific adaptors in the interaction of ribonuclear particles with translationally regulated messages. Overrepresented groups of targets include mRNAs coding for transcription factors, components of the miRNA machinery, and other proteins involved in translational regulation, as well as components of the ubiquitin machinery, representing novel feedback loops in gene regulation. Detailed information about target genes, target processes, and open-source software for target prediction (miRanda) is available at  This computational analysis provides evidence that as many as 10% of human genes are targets for regulation by small RNA molecules called microRNAs In the past three years, several hundred novel genes encoding transcripts containing short double-stranded RNA hairpins, named microRNAs (miRNAs), were identified in plants and animals . More relin-4   . While cThe following rules for matches between miRNA and target messages have been deduced from a range of experiments. (1) Asymmetry: experimentally verified miRNA target sites indicate that the 5′ end of the miRNA tends to have more bases complementary to the target than its 3′ end. Loopouts in either the mRNA or the miRNA between positions 9 and 14 of the miRNA have been observed or deduced . Recent lin-41, a 27-nucleotide (nt) intervening sequence between two consecutive let-7 sites is necessary for its regulation (C. briggsae, incorporation of a corresponding rule is premature.Given the overlap between the siRNA and miRNA pathways, it is reasonable to assume that rules of regulation in the siRNA pathway will partly apply to miRNA target recognition . Lately,gulation . BecausemiRNAs are transcribed as longer precursors, termed pre-miRNAs , sometimOne of the unwound strands is subsequently incorporated into a ribonuclear particle (RNP) complex, RNA-induced silencing complex (RISC) . Every RAmong the prime candidates for miRNA control are the genes that are posttranscriptionally regulated. The mRNA-binding protein fragile X mental retardation protein (FMRP) is involved in the regulation of local protein synthesis  and bindDrosophila homolog of FMRP (FXR) and the Vasa intronic gene were identified as components of RISC , mouse (Mus musculus), and rat (Rattus norvegicus) genomes for potential target sites. The scanning algorithm was based on sequence complementarity between the mature miRNA and the target site, binding energy of the miRNA–target duplex, and evolutionary conservation of the target site sequence and target position in aligned UTRs of homologous genes. We identified as conserved across mammals a total of 2,273 target genes with more than one target site at 90% conservation of target site sequence (Tables (Danio rerio) and fugu (Fugu rubripes) fish genomes for potential targets using known and predicted miRNAs  from the human e Tables  and S3 ad miRNAs  and S5 aIn addition to the analysis of 3′ UTRs, we also scanned all protein-coding regions for high-scoring miRNA target sites. For convenience, these results are reported both as hits in cDNAs  nonuniform distribution of the number of sequence-complementary target sites for different miRNAs; (2) 5′–3′ asymmetry  We compared predicted target numbers from real and shuffled miRNA sequences and estimated the rate of false-positive predictions. (3) We assessed the enrichment of miRNA targets in mRNAs that are known cargoes of FMRP, an RNA-binding protein known to be involved in translational regulation.Only a small number of target sites of target genes regulated by miRNAs have been experimentally verified, so we sought direct and indirect evidence to help validate or invalidate the proposed set of mammalian targets. (1) We compared predicted targets with experimentally verified targets in mammals, let-7 and lin-4 miRNAs in Drosophila to develop the target prediction method and check for consistency (C. elegans (let-7 in the UTR of lin-41 down to three elements, two target sites, and a 27-nt intervening sequence (a possible binding site for another factor). The surviving two target sites have high alignment scores, S = 115 and S = 110, while the other four sites are below threshold  and passes the conservation filter.We previously used known miRNA sites for the sistency . More re elegans  has narrhreshold , fully cD. melanogaster have identified six new miRNA–target gene pairs: miR-7 targets the notch signaling genes HLHm3, HLHm4, and hairy, and miR-2b targets the genes reaper, grim, and sickle , first discovered in worm , let-7b , and miR-351 (1 site). Another known lin-4 and let-7 target in C. elegans is lin-41. The human homolog of lin-41  and another closely related gene (encoding Tripartite motif protein 2) are predicted as high-ranking targets of let-7 and miR-125 (the human homolog of lin-4)  known target sites and target genes at reasonably high rank. However, given the small number of experimentally verified miRNA–target pairs, additional validation tests are desirable, such as statistical tests using randomization of miRNA sequences to estimate false positives.As a computational control of the validity of the prediction method, one can perform a statistical test that attempts to estimate the probability that a predicted site is incorrect. Here, a “false positive” is a predicted target site of a real miRNA on a real mRNA that has passed all relevant thresholds but is incorrect in that it is not biologically meaningful. The statement “not biologically meaningful” is rarely clearly defined, but can reasonably be taken to mean that no functionally effective miRNA:mRNA interaction occurs under conditions of co-expression at physiological concentration, where “functionally effective” is defined in terms of detectable changes of phenotypic attributes.T > c,T where cT is a fixed threshold that may be, in general, different for each miRNA. We chose to estimate the background distribution using shuffled miRNAs obtained by swapping randomly selected pairs of bases of each given miRNA 1,000 times, keeping the nucleotide composition constant. The shuffled miRNA sequences were scanned against human, mouse, and rat 3′ UTR sequences exactly as for the prediction procedure for real miRNA sequences. In the procedure, a miRNA:mRNA match site is predicted to be a target site if it passes three thresholds, S > cS for match score, |ΔG| > |ΔcG| for free energy of duplex formation, and C > cC for conservation, where C reflects a binary evaluation of orthology of mRNAs, similarity of position of the site on the mRNA, and a threshold percentage of conserved residues in the two mRNA target sites. Finally, the predicted target sites for a set of shuffled miRNAs are counted and then averaged over a total of ten randomized runs. The percentage of false positives for target transcripts with more than two, three, and four sites is 39%, 30%, and 24%, respectively, using a non-permissive conservation threshold of 100% for target site sequences  the background distribution of scores for biologically non-meaningful miRNA target sites and then deriving the probability that a non-meaningful target site passes all score thresholds, i.e., for a single aggregate score, that the incorrect site has a score equences . In addi(S) and energy (ΔG) calculated by the miRanda algorithm or may be due to different polynucleotide compositions of shuffled miRNAs compared to real miRNAs. Even if this difference represents a real effect, by far the most predictive criterion for accurate target detection is conservation of target sites across species, and not alignment scores or energies  should be similar to the distribution of incorrect (non-meaningful) hits from real trials . The difference between these two distributions is difficult to compute in principle, as very few validated correct predictions are known at present. For human sequences, without any conservation filter, we obtained a total of 2,538,431 predicted target sites for real miRNAs, and, for shuffled miRNAs, on average, 2,033,701 (± 82,172) target sites—a difference of 20%. This difference may be indicative of a biological signal in the raw score Dicer, and the mammalian homologs of AGO1 and AGO2 –associated (SAPAP4), a neuron-specific protein, is regulated by many miRNAs highly expressed in rat brain primary cortical neurons .In summary, the three validation approaches  suggest that the current version of the miRanda algorithm, in spite of clear limitations, can predict true miRNA targets at reasonable accuracy, provided that (1) the targets are detected as conserved and (2) the gene contains more than one miRNA target site or a single high-scoring site (S > 110) have an estimated false-positive rate comparable to that of genes with multiple sites and thus also are good candidates for experimental verification. Some of the genes with single sites may contain additional sites that we cannot detect for a number of reasons, including truncated UTRs. A significant subset of the total number of single-site target genes (7%) has near complementary single sites. These near complementary sites may indicate cleavage, for which additional sites may not be necessary. The targets listed in let-7b), but the distribution is nonuniform  terms see  and domaHomeobox domain, 5.3-fold; KH domain, 4.0-fold; and Guanine-nucleotide dissociation stimulator CDC25 domain, 3.4-fold . The semaphorins and plexins (semaphorin receptors) are involved in axon guidance, angiogenesis, cell migration, the immune system, and the adult nervous system (let-7c, miR-125b, miR-153, miR-103, miR-323, miR-326, and miR-337). The plexins dimerize with Neuropilin (NP1) to signal the Semaphorin ligand attachment; neuropilin is a predicted high-ranking target of let-7g and miR-130, both brain-expressed miRNAs. A significant proportion of ephrin receptors (seven out of ten genes) and ephrin ligands (five out of seven genes) are predicted targets. The family of ephrins is involved in boundary formation, cell migration, axon guidance, synapse formation, and angiogenesis, and the ephrin ligand, EphA2, which contains a conserved cytoplasmic polyadenylation element (CPE) motif, is considered to be under translational regulation in axon growth cones , miR-208 in “transcription factor” (6-fold), and miR-7, which lies in the intron of the hnRNPk (an RNA-binding protein) gene, in “RNA binding proteins.” Neuronal differentiation of embryonic carcinoma cells by retinoic acid in both mice and humans is coupled to induction of let-7b, miR-30, miR-98, miR-103, and miR-135  cloned from rat neuronal polyribosomes have a large number of neuronal target genes and share many targets, e.g., miR-352 and miR-327 target 5HT-2c, and miR-340, -328, -326, -331, and -333 potentially target beta-catenin, which is implicated in various stages of neural differentiation.Do specific miRNAs target particular functional groups? We analyzed each miRNA individually for GO term and domain enrichment . The tar miR-135 , and themiR-181a and miR-142–3p are key components of a molecular circuitry that modulates hematopoietic lineage (miR-181a causes a 2-fold increase in the cells of the B cell lymphoid lineage. Some of our high-ranking targets for miR-181a may provide clues for the mechanism of this effect. Germ cell nuclear factor GCNF (NR6A1) (the second-highest-ranked target for miR-181a) is expressed in the thymus and bone marrow. miR-181a itself is encoded on the antisense strand of an intron of GCNF. We also predict that the gene Bcl11b, known to affect B cell growth, is a target of miR-181a, ranking third, as well as Lim/homeobox protein LHX9, recently found expressed in developing thymus (Two highly expressed miRNAs in the thymus,  lineage . Ectopicg thymus .KH and one RRG) that bind messages. The specific binding motifs for FMRP on messages are incompletely known, but are thought to include G-quartet patterns and/or U-rich sequences  is an FMRP-bound protein that is translationally regulated. The ing FMRP . In addiAPP mRNA . We predt region , and ano(mir-9, miR-340, and miR-135b), APP-binding family member 2 (let-7 and miR-218), and APP-binding family 2 (miR-188 and miR-206) were also predicted targets, some of which had near exact target site matches. In summary, the APP gene appears to be subject to translational regulation by the combinatorial control of a number of different miRNAs.Other APP-interacting proteins, APP-binding family B member 1 miR-125, miR-135, miR-320, and miR-327, all of which are either exclusively expressed in brain or enriched in brain tissue (miR-125b are found copurified with polyribosomes in rat neurons in (PSD95 has one reported G-quartet in its 3′ UTR at position 648 (PSD95. One of the miRNA (miR-125) target sites overlaps with the G-quartets, raising the possibility that miRNAs directly compete with FMRP to bind the message in this location. Likewise, NAP-22, which has three miRNA target sites , SAP90, and CamKII are predicted miRNA targets (see mGluR, the protein product of which is an agonist that induces the rapid translation of PSD95 (Other gets see , as wellof PSD95  and threof PSD95 . These r. The fact that miRNAs target transcripts for FMRP and FMRP-binding proteins suggests another negative feedback loop in which high levels of these proteins inhibit their own production (depending, of course, on the concentration of miRNAs and mRNAs)  . The genDicer appears to be controlled by let-7 and miR-15b; Ago-1 by let-7 and miR-29b/c; Ago-2 by miR-138; Ago-3 by miR-138, miR-25, and miR-103; and Ago-4 by miR-27a/b. Dicer and two of the Argonautes also are predicted to be targets in both zebrafish and fugu. The let-7 sites on the 3′ UTR of Dicer and Ago-1  are expressed fairly widely, while others (e.g., miR-138 and miR-25) have lower and more restricted expression.Our data suggest that the RNAi–miRNA machinery itself is under miRNA regulation; for example nd Ago-1  will accrebellum . The tar message . Ago-3 iElavl1 (known to regulate its own mRNA), Gap-43, c-fos, PN-1, Krox-24, CD51, CF2R, CTCF, NF-M, GLUT-1, c-myc, and N-cadherin (The highly conserved RNA-binding proteins, ELAV-like proteins (HuR, HuB, HuC, and HuD), contain three RNA-recognition motifs, which bind AU-rich elements in 3′ UTRs of a subset of target mRNAs . These Acadherin . Three ocadherin . This isCPEB2 is the highest-ranking gene of all transcripts. The orthologs to CPEB1 in fish and fly (known as orb in D. melanogaster) are also predicted as targets. CPEB is an RNA-binding protein first shown to activate translationally dormant mRNAs by regulating cytoplasmic polyadenylation in Xenopus oocytes , a member of the Argonaute family  known in mammals as miRNA targets ranked within the top 170 target genes with 6–20 sites in their UTRs . Indeed, oocytes . It also oocytes  and dend oocytes  and faci oocytes . CPEB bi oocytes , thereby oocytes . In addind CPEB3 . Second,nogaster . The CPEe family  that is HuB, HuR, Eif-4 gamma, DAZ associated protein 2, VAMP-2 , Presynaptic protein SAP102, and brain-derived neurotrophic factor precursor. Taken together these data suggest that the CPEB genes, the known CPEB-regulated genes, and the predicted CPEB-regulated genes are strong miRNA target candidates and provide rich ground for experimentation.Among the predicted miRNA targets, 115 genes also contained CPE motifs, which were conserved in at least two mammals in the same positions in the UTRs and are therefore candidates for CPEB regulation . Our premiR-15 and miR-16 are located within a 30-kb region at Chromosome 13q14, a region deleted in 50% of B cell chronic lymphocytic leukemias, 50% of mantle cell lymphomas, 16%–40% of multiple myelomas, and 60% of prostate cancers  CNOT7, a gene expressed in colorectal cell lines and primary tumors ((miR-15a); (2) LASS2, a tumor metastasis suppressor ((miR-15b); (3) ING4, a homolog of the tumor suppressor p33 ING1b, which stimulates cell cycle arrest, repair, and apoptosis ((miR-143); (4) Gab1, encoding multivalent Grb2-associated docking protein, which is involved in cell proliferation and survival ((miR-155); and (5) COL3A1, a gene up-regulated in advanced carcinoma ((miR-145).Our method predicted cancer-specific (by annotation) gene targets of y tumors   and Hox-A5, both known to be dysregulated at the level of protein expression in colon cancers (miR-16 and the target genes (indicating possible cleavage). Both of these target genes are also targets for another cancer related miRNA, miR-15.ntegrity . The fir cancers . Both of.Roughly 55 miRNAs have identical mature sequences in fugu and mammals, and 80 have very similar sequences in the two species; additional fish miRNA sequences can be predicted with confidence based on sequence similarity. It is therefore reasonable to expect that the targets of these probably functionally homologous miRNAs are orthologous genes in the different species. To follow up on this hypothesis, we assessed conservation of mammalian miRNA–target pairs between the 2,273 mammalian and 1,578 fish miRNA targets (with more than one target site per UTR). The analysis yielded 240 target genes conserved between mammals and fish. The number 240 is probably an underestimate because of several factors, including: (1) unfinished annotation of genomes, particularly rat and fugu; (2) ambiguity in assigning orthologs; and (3) lack of UTR informationHox genes are conserved as targets, including the miR-196 targets, Hox-A4:miR-34a, Hox-C9:let-7b (near prefect complementary match), and Hox-B5:miR-27b. Examples from the notch signaling pathway include miR-30:hairy enhancer of split 1 (Hes1) and miR-152:noggin.The full set of conserved target genes between fish and mammals indicates a wide functional range of conserved targets . Many Hohox genes and axon guidance genes as common targets between vertebrates and flies, e.g., capicua and sex combs reduced (one of the vertebrate homologs of Hox-A5). The hox gene cluster in Drosophila contains high-ranking predicted targets  and before application of conservation filters. The statistical assessment of the influence of conservation filters in coding regions would have raised complicated issues, as nearly two-thirds of nucleotides in coding regions are conserved between mammalian genomes to preserve amino acid sequences. Interestingly, we found, on average, 11 pre-conservation target sites per 1 million nucleotides in coding regions, versus 15 such target sites per 1 million nucleotides in UTRs. This is consistent with a stronger “raw” prediction signal in UTRs and may indicate a lower number of biologically relevant target sites in coding regions in mammals, consistent with early experimental findings.To investigate this issue we computed the average density of target sites for high-scoring targets  and also has two conserved “normal” sites in the UTR. Similarly, miR-198 has a site in the coding region, as well as conserved sites in the UTR region, of a sodium and chloride GABA transporter (Ensembl ID ENSG00000157103). However, we see no trend for miRNAs that have conserved sites in UTRs to have additional sites in the coding region; rather, stronger target sites for a given miRNA tend to be confined either to the UTR or the coding region and are rarely in both.As a guide to experimentation, we report all sites in coding regions with an alignment score above 110 for miRNAs of length up to 20 nt and an alignment score above 130 for miRNAs longer than 20 nt (scores depend on the length). These cutoff scores approximately correspond to a 75% complementary match between miRNA and target, leaving open the question of how many match pairs are needed to lead to translational inhibition in coding regions, by any mechanism. We identified 942 genes that contained such sites in their coding regions. Strikingly, there was only one site with a perfect match, and this was for the imprinted e strand . Of the S >120), and these target genes may be cleaved rather than translationally repressed as in the case of miR-196 and Hox-B8. For example miR-298, an embryonic-stem-cell-specific miRNA  has a near match with LIMK-1, which is known to be involved in synapse formation and function. miR-129, expressed in mouse cerebellum, has a near perfect complementary match with Musashi-1, which is an RNA-binding gene essential for neural development, regulated in the cerebellum, and up-regulated in medulloblastoma  or position on the mRNA. Using shuffled miRNA sequences, with the constraint that shuffled controls match real miRNAs in relevant sequence properties, the false-positive rate of predictions was estimated to be 50% for target genes conserved between mouse and human, 31% for target genes conserved in human, mouse, and rat, and 22% for target genes identified in fugu as well as mammals. As a final result, Lewis et al. reported 400 conserved target genes for the 79 miRNAs. Among these targets, 107 genes were reported as conserved in the fish fugu.let-7b miRNA on the UTR of the human homolog of worm lin-28. The target site sequence was engineered into a Luciferase reporter, followed by sequence variation of the target site and test of an initial set of 15 predictions in the same reporter assay. Using shuffled miRNA sequences, and applying the same rules and parameters, the false-positive rate of predictions was estimated to be 50% for targets conserved between human and mouse. As a final result, Kiriakidou et al. reported 5,031 human targets, with 222 reported as conserved in the mouse.In the second method, Kiriakidou et al. used 94 miRNAs in human and mouse, seeking targets in a dataset of 13,000 UTRs conserved in mouse and human (from Ensembl, date not given). The algorithm used a 38-nt sliding mRNA window and calculation of miRNA–mRNA duplex free energies, keeping duplexes with energies below −20 kcal/mol. The duplexes were further filtered using a set of requirements regarding matches and loop lengths in certain positions, as derived and extrapolated from experimental tests involving a predicted target site for In the third method (this work), we used 218 mammalian miRNAs and 29,785 transcripts derived from Ensembl  and, as Direct comparison of the three prediction methods is complicated by the fact that the noticeable differences between the target lists of the three methods are due to the aggregate effects of datasets, algorithm, including selection rules, use of conservation, and cutoffs. The following characteristics of the three methods underlie these differences and should be taken into consideration when choosing targets for experimentation. (1) As to UTR datasets, Lewis et al., with the earliest published report, used a smaller set of UTRs, with some likelihood of false positives as a result of UTR extension. The UTR sets used in this work, the third in terms of publication date, are the most comprehensive and plausibly the most reliable (as of February 2004). (2) As to miRNA datasets, there was an increase from 79 for Lewis et al. to 94 for Kiriakidou et al. to 218 miRNAs used in this work. (3) As to the cooperativity of binding, the scoring system of Lewis et al. evaluated cooperativity of multiple target sites by the same miRNA on a target gene, but disregarded multiple target sites from different miRNAs on one gene; that of Kiriakidou et al. focused on single sites; and that of this work gave high scores to multiple hits on a target gene, no matter whether these hits involved the same miRNA or different miRNAs. These tendencies are not exclusive where scores involve functions of several real numbers, with cutoffs applied to the aggregate score; e.g., our method also allows strong single target sites. (4) As to assessment of false positives using statistical methods based on shuffling, the comparison of percentages is inconclusive, as the statistics of the background distribution of true negatives is not well known. It appears certain, however, from both Lewis et al. and this work, that statistical confidence increases with the extent of conservation among increasingly distant species. (5) As to validation experiments, each of the methods used a different type and set, with mixed overall conclusions. On the reassuring side, there was direct validation of some of the predicted target sites of Lewis et al. and of Kiriakidou et al. using reporter constructs in cell lines. We found some agreement between the sites validated in this way and our predicted targets (details in S > 90) or one target site with a higher threshold (S > 110), counting only sites with up to one G:U pair in residues 2–8 counting from the 5′ end of the miRNA.The predicted targets are useful in practice for the design of experiments as they increase the efficiency of validation experiments by focusing on target lists significantly enhanced in likely targets, compared to random. It is plausible that targets near the top of lists are the most likely to lead to successful experiments. Task-specific filtering of target lists for particular planned experiments is recommended, especially with respect to cooperativity of binding (more than one site for one or more miRNAs on one gene transcript) and coincidence of expression, as new data on expression patterns of miRNAs and mRNAs in different tissues become available. For example, a recommended conservative approach to the design of experiments would use all available expression information and restrict the predicted target genes to those with two or more target sites at normal threshold  of all mammalian genes have more than one miRNA target site in their 3′ UTRs, with 1,314 being stronger candidates with more than two target sites. This could well be an underestimate of the total number of genes subject to miRNA regulation, as we have used a conservative conservation filter. On the other hand, not all predicted miRNA–mRNA pairs would have a biological consequence unless both miRNA and mRNA are expressed at the same time in the same cell and at sufficient concentration. The human genome has about 250 miRNA genes, compared to about 35,000 protein genes. Thus, the the determination that about 1% of genes (miRNAs) control the expression of more than 10% of genes is a reasonable first order estimate. It is currently not known if any miRNAs control the expression of miRNA genes, i.e., the progression from miRNA transcript to mature miRNA.As many miRNA sequences are detectably conserved across large evolutionary distances, they must be subject to strong functional constraints. These constraints are unlikely to come from single-site interactions with the target, as experimentally validated animal miRNAs rarely have perfectly matched target sites. Plausibly, the evolution of miRNAs is constrained by functional interactions with multiple targets. As a consequence, any compensatory mutation in the miRNA in response to mutations in a target site would be disruptive to the miRNA's interaction with other target sites. Co-evolution of the miRNA sequence and all of its target sequences is therefore a rare event. With these assumptions, the constraints on the local mRNA sequence of individual target sites are weaker than those on the miRNA sequence. We were therefore surprised to observe a substantial number of cases (28.6% of the 2,273 targets) with 100% conservation of target site sequence and with the target sites being within ten nucleotides of each other on the globally aligned UTRs of orthologous genes between mammals.Lacking more detailed knowledge of miRNA evolution, we draw two operational conclusions. (1) Conservation of target site sequence and position is a practical information filter for predicted target sites, reducing the rate of false positives. (2) It is very likely that new miRNAs have continuously appeared in evolution  at some (lin-4 and let-7) seemed to indicate. The distribution of predicted targets reflects more complicated combinatorics, both in terms of target multiplicity (more than one target per miRNA) and signal integration (more than one miRNA per target gene).Regulation by miRNAs is obviously not as simple as one miRNA–one target gene, as perhaps the early examples let-7b, with an average of 7.1 targets per miRNA. It is difficult to describe in detail, beyond the examples discussed in this text and beyond the annotation of target genes in lin-4 (The distribution of the number of target genes (and target sites) per miRNA is highly nonuniform, ranging from zero for seven miRNAs to 268 for in lin-4 .The number of miRNA target sites per gene is also nonuniform, with a mean of 2.4. Although we do list target genes with single miRNA sites, there is increasing evidence that, in general, two or more sites are needed in the context of repression of translation. Although the details of these distributions see  depend oWe conclude that multiplicity of targets and cooperative signal integration on target genes are key features of the control of translation by miRNAs. Neither multiplicity nor cooperativity is a novel feature in the regulation of gene expression. Indeed, regulation by transcription factors appears to be characterized, at least in eukaryotes, by analogous one-to-many and many-to-one relations between regulating factor and regulated genes . We are,The role of a few animal miRNAs as posttranscriptional regulators of gene expression and, in particular, as inhibitors of translation is well established. However, the molecular mechanism of action is not well understood. Posttranscriptional control of protein levels can be achieved, for example, by cleaving the mRNA, by preventing RNP transport to ribosomes, by stalling or otherwise inhibiting translation on ribosomes, or by facilitating the formation of protein complexes near ribosomes that degrade nascent polypeptide chains. What do our results imply regarding the mechanism of action?miR-196 and miR-138; see Hox-B8, cleavage has been experimentally shown in mammalian cells  details of strand bias as deduced from siRNA experiments , (2) conAlthough the predicted targets are subject to error  and the prediction rules in need of improvement, several general principles of gene regulation by miRNAs are emerging. (1) Except in cases where a highly complementary match causes cleavage of the target message, miRNAs appear to act cooperatively, requiring two or more target sites per message, for either one or several different miRNAs. (2) Most miRNAs are involved in the translational regulation of several target genes, which in some cases are grouped into functional categories. (3) miRNAs carried in the context of RNPs appear to be sequence-specific adaptors guiding RNPs to particular target sequences. miRNA regulation of cellular messages may therefore range from a switch-like behavior (e.g., cleavage of mRNA message) to a subtle modulation of protein dosage in a cell through low-level translational repression .These aspects of miRNA regulation complicate the design of experiments aiming at testing target predictions, or, more generally, at discovering biologically meaningful targets. Straightforward experiments that test one target site for one miRNA on one UTR will not be able to disentangle the effects of multiplicity or cooperativity. Tests for multiple sites on one UTR for one miRNA capture aspects of cooperativity , but stiThe results of this genome-wide prediction for mammals and fish are meant to be a guide to experiments that will in time elucidate the genetic control network of regulators of transcription, translation/maturation, and degradation of gene products, including miRNAs.Mature human and mouse miRNA sequences were obtained from the RFAM miRNA registry . To coveMature miRNA sequences for zebrafish and fugu were predicted starting from known human and mouse miRNA precursor sequences . Each prThe Ensembl database  served aOrthology mappings between genes from different genomes were obtained using “orthologue tables” from the EnsMart  feature i, j in the alignment scoring matrix, the gap-elongation parameter was used only if the extension to i, j of a given stretch of gaps ending at positions i–1, j or j–1, i (but not of stretches of gaps ending at i–k, j or j, i–k for k > 1) resulted in a higher score than the addition of a nucleotide–nucleotide match at positions i, j. Removal of this restriction with the availability of more computing power would result in a moderate increase in average loop length, but the advantages of this would probably be superceded by overall refinement of target prediction rules. Importantly, complementarity scores at the first eleven positions, counting from the miRNA 5′ end, were multiplied by a scaling factor of 2.0, so as to approximately reflect the experimentally observed 5′–3′ asymmetry; for example, G:C and A:T base pairs contributed +10 to the match score in these positions. The value of the scaling factor at each position is an adjustable parameter subject to optimization as more experimental information becomes available. Because of the ongoing discussion about the rules for target prediction, target genes  that contained target sites with more than one G:U wobble in the 5′ end are flagged in the S > 90 and ΔG < −17 kcal/mol, where S is the sum of single-residue-pair match scores over the alignment trace and ΔG is the free energy of duplex formation from a completely dissociated state, calculated using the Vienna package as in The miRanda algorithm  via a shared (or homologous) miRNA. We required that the positions of pairs of target sites in two species fall within ±10 residues in the aligned 3′ UTRs. Conserved target sites with sequence identity of 90% or more (human versus mouse or rat) and 70% or more (zebrafish versus fugu) were selected as candidate miRNA target sites and stored in a MySQL database. Using human as the reference species, we predicted 10,572 conserved target sites (conserved in either mouse or rat) in 4,463 human transcripts, of which 2,307 transcripts of 2,273 genes contained more than one target site. Similarly, using zebrafish as a reference species, we predicted 7,057 conserved target sites (conserved in fugu) in 4,820 zebrafish transcripts.To focus on the strongest predictions, conserved target sites for each miRNA were sorted according to alignment score, with free energy as the secondary sort criterion. In cases where multiple miRNAs targeted the same site on a transcript (or within 25 nt of a site), only the highest scoring, lowest energy miRNA was reported for that site.class,F using the log-odds ratio of the fraction of annotated target genes with the same class 1):,.Here, For each random experiment all miRNAs were shuffled by randomly swapping two bases of a miRNA 1,000 times. These shuffled sequences were then searched against human, mouse, and rat 3′ UTR sequences in the same way described for the main analysis, including analysis of conservation of target site sequence and position in orthologous 3′ UTRs. A total of ten randomized experiments were performed. Counts were averaged across all experiments, and the standard deviation and other statistical measures were calculated.We compiled a list of 464 gene identifiers of FMRP-associated mRNAs from five different publications . Among tWe predicted CPE motifs in human, mouse, and rat UTRs. We used a search pattern using four criteria: (1) presence of the CPE motif UUUUAU, (2) presence of the hexanucleotide AAUAAA, (3) the CPE and the hexanucleotide motif being within 100 nucleotides of each other, and (4) the conservation of these motifs and the positions of the motifs in the mouse ortholog .Figure S1(347 KB PDF).Click here for additional data file.Table S1(25 KB XLS).Click here for additional data file.Table S2(8.0 MB XLS).Click here for additional data file.Table S3(17.0 MB XLS).Click here for additional data file.Table S4(5.6 MB XLS).Click here for additional data file.Table S5(9.8 MB XLS).Click here for additional data file.Table S6(601 KB XLS).Click here for additional data file.Table S7(512 KB XLS).Click here for additional data file.Table S8(23 KB XLS).Click here for additional data file.Table S9(678 KB XLS).Click here for additional data file.Table S10(357 KB XLS).Click here for additional data file.Table S11(529 KB XLS).Click here for additional data file.Table S12(621 KB XLS).Click here for additional data file.Table S13(68 KB XLS).Click here for additional data file.