Systemic lupus erythematosus (SLE) is an autoimmune disorder affecting multiple organ systems. It is characterized by the presence of autoantibodies reactive against various self-antigens. Susceptibility to SLE is found to be associated with many major histocompatibility complex (MHC) and non-MHC genes, one of which is APO-1/Fas gene, which is present on chromosome 10 in humans. The APO-1/Fas promoter contains consensus sequences for binding of several transcription factors that affect the intensity of Fas expression in cells. The mutations in the APO-1/Fas promoter are associated with risk and severity in various autoimmune diseases and other malignancies. The APO-1/Fas receptor is expressed by many cell types. Two forms of APO-1/Fas protein that are involved in regulation of apoptosis have been identified. Fas receptor-mediated apoptosis plays a physiological and pathological role in killing of infected cell targets. In this review, we have focused on APO-1/Fas gene structure, promoter variants and its association with SLE and other autoimmune diseases. Functional aspects of Fas receptor in apoptosis are also discussed. Origin of the disease is multigenic and involves different sets of genes in different individuals. There is a familial aspect to lupus, suggesting its inheritance, but it is not inherited in a typical Mendelian manner. Diagnosis of SLE is largely based on the clinical manifestations and autoantibody (anti-nuclear antibody [ANA] and anti-dsDNA) detection. It is treatable mainly with corticosteroids and immunosuppressants, but currently no cure is available.t-lymphoct-cells after presentation by APCs and production of autoantibodies. Immune complexes of autoantigen/autoantibody bind to the basement membrane of different organs, especially the skin and kidney, where these induce inflammation and may cause lupus nephritis, the most serious manifestation of SLE. Antibodies against dsDNA, Ro62, Ro50, La and anionic phospholipids occur commonly in SLE patients. Mutation in APO-1/Fas has been associated with the loss of regulation of B-lymphocytes, which predisposes to SLE.[Normally, apoptotic cells are removed by macrophages or other professional phagocytes. This regulated removal of unwanted cells by apoptosis helps in tolerance induction for autoantigens. Defects in induction of apoptosis and removal of apoptotic cells have been reported to be involved in SLE development. Deranged clearance of apoptotic cell debris leads to the release of nuclear autoantigens. These autoantigens become clustered in apoptotic blebs that are restricted to sites of free radical generation in apoptotic cells for oxidative modification. A potential source of these antigenic components is abundant generation of nucleosomes (dsDNA), which are fragments of chromatin released in cells undergoing apoptosis. These autoantigens when exposed on the surface of dying cells trigger an autoimmune response by autoreactive s to SLE.-30t-cell leukemia,[Gene upregulation precedes apoptosis. APO-1/Fas expression on cells can be induced by cytokines like IFN-γ and TNF-α. Increaseleukemia, sarcoidoleukemia, hepatitileukemia, and hepaleukemia, infectioleukemia,39 The leleukemia,35 Patienleukemia, silicosileukemia, and non-leukemia, also havMultiple factors contribute to the development of an autoimmune disease. These can be immunological, genetic, hormonal and environmental. Immunogenetic studies on autoimmune diseases have been reported from all over the world in different ethnic populations. Some polymorphisms are common risk factors for autoimmune disease development, e.g. TNF-308A variant of TNF gene at 6p21.3 for SLE, RA and primary SS and some are disease-specific, like CCR5 gene at 3p21.31 and IL1B at 2q13 for SLE. SusceptiThere is a need to explore genetic association in the diverse populations of India. Population studies revealed that each population holds a mutational pool in which most mutations or polymorphisms have undetectable effects on an individual, but, in combination with other alleles, it may promote or prevent from autoimmune diseases. Lack of Protein profiling using proteomic techniques like immobilized antibody array, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry is high throughput and does not rely on the predetermined notions of disease pathogenesis.46 It has


Problem-based Learning (PBL) has been suggested as a key educational method of knowledge acquisition to improve medical education. We sought to evaluate the differences in medical school education between graduates from PBL-based and conventional curricula and to what extent these curricula fit job requirements.Graduates from all German medical schools who graduated between 1996 and 2002 were eligible for this study. Graduates self-assessed nine competencies as required at their day-to-day work and as taught in medical school on a 6-point Likert scale. Results were compared between graduates from a PBL-based curriculum (University Witten/Herdecke) and conventional curricula.Three schools were excluded because of low response rates. Baseline demographics between graduates of the PBL-based curriculum  and the conventional curricula  were similar. No major differences were observed regarding job requirements with priorities for "Independent learning/working" and "Practical medical skills". All competencies were rated to be better taught in PBL-based curriculum compared to the conventional curricula , except for "Medical knowledge" and "Research competence". Comparing competencies required at work and taught in medical school, PBL was associated with benefits in "Interdisciplinary thinking" (Δ + 0.88), "Independent learning/working" (Δ + 0.57), "Psycho-social competence" (Δ + 0.56), "Teamwork" (Δ + 0.39) and "Problem-solving skills" (Δ + 0.36), whereas "Research competence" (Δ - 1.23) and "Business competence" (Δ - 1.44) in the PBL-based curriculum needed improvement.Among medical graduates in Germany, PBL demonstrated benefits with regard to competencies which were highly required in the job of physicians. Research and business competence deserve closer attention in future curricular development. The framework of medical education has always been a controversial subject. In 1899, Sir William Osler claimed that the complex and growing structure of medical knowledge was associated with difficulties in imparting the necessary skill set for a well-trained physician. Osler concluded that one way to overcome this issue may be to allow more time for independent study  for categorical variables. Outcomes of interest were the self-assessed competencies as reported by graduates of German medical schools. Graduates were stratified by whether their medical curriculum was PBL-based (University Witten/Herdecke) or conventional . To ensure generalizabilty, universities were excluded if less than 50 of their graduates responded.Differences in baseline demographics between the two stratified cohorts were determined using chi-square and two-tailed t-tests for categorical and continuous variables, respectively. Fisher's exact test was used if subgroups included less than ten subjects.To determine whether medical school education sufficiently covers the job requirements of physicians, we calculated the mean difference (Δ) between the ratings of competencies as required at the day-to-day work and as taught in medical school by subtracting those from each other. This comparison demonstrates whether an education is perceived to cover job requirements Figure . It alsoTo express effect size, Hedges' g was computed for all significant differences between graduates from the PBL-based curriculum and from the conventional curricula regarding the research questions "Required competencies at the day-to-day work", and "Competencies as taught in medical school" . A HedgeAll analyses were performed using SPSS  and SAS . A p-value < 0.05 was considered to indicate statistical significance.Overall, more than 35,000 graduates of all 37 German medical schools were contacted. Among these, three universities were excluded from the analysis due to low response rates . Thus, completed questionnaires of 4,821 graduates of 34 universities formed the study sample (14%). Of these, 101 were graduates of the PBL-based medical school curriculum and 4,720 were graduates of conventional medical curricula. The response rate was significantly higher in the group of PBL-graduates .Baseline demographics of the two groups are detailed in Table Overall, graduates of both curricula rated the job requirements similarly. The most required competencies at their day-to-day work were "Independent learning/working", "Practical medical skills", and "Psycho-social competence"  and moreover, the PBL-based provided a significant educational surplus in five competencies, especially in "Interdisciplinary thinking" and "Independent learning/working"  offered a PBL-based curriculum during 1996 and 2002, there may be differences with respect to realization of the PBL framework which may limit external validity of the observed findings. Even so our data was collected 2004/05, it has still relevant for the development of medical education systems, especially while the major part of medical schools is still based on a conventional curriculum. In addition, PBL-based curriculum should be developed further under the consideration of the identified weaknesses. Finally, our assessment of competencies required at day-to-day work as well the competencies as taught in medical school was based on self-perception. Some studies suggest that self-perception can differ from objective measures ,29. In tGiven the limitations of the present analysis, further research to confirm and extend our findings is warranted. It may be of additional value to approach and survey employers or professional colleagues about their perceived skills of PBL and conventional medical graduates. Moreover, to address the limitation of low response rates, it may be more feasible to use personal interviews rather than postal or online questionnaires. Finally, more medical schools should be included which offer PBL-based curricula today. Thus longitudinal follow-up studies and objective assessment of competencies are necessary to fully evaluate the impact of PBL on medical education.In summary, this large survey among medical graduates demonstrates similar self-perceived job requirements between graduates from PBL-based and conventional curricula. Nevertheless, the PBL curriculum is associated with a strong positive effect on all key competencies, which are highly required in the job of physicians. However, research and business competencies deserve closer attention in future curricular development.CHE: Centre for Higher Education Development; PBL: problem-based learning; SD: standard deviation.The authors CLS, HD, JD and OP are medical students at the University Witten/Herdecke. The authors MB and MRF are employees of the University Witten/Herdecke.Authors CLS, HD, JD, OP, GF, and MB all contributed to the design of the study, data analysis, and drafting of the manuscript. Authors FB and MRF contributed to data analysis and interpretation, and critical revision of the manuscript. All authors have read and provided approval to the manuscript as submitted.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6920/10/1/prepub


Osteoarthritis of the hip is successfully treated by total hip arthroplasty with metal-on-polyethylene articulation. Polyethylene wear debris can however lead to osteolysis, aseptic loosening and failure of the implant. Large head metal-on-metal total hip arthroplasty may overcome polyethylene wear induced prosthetic failure, but can increase systemic cobalt and chromium ion concentrations. The objective of this study is to compare two cementless total hip arthroplasties: a conventional 28 mm metal-on-polyethylene articulation and a large head metal-on-metal articulation. We hypothesize that the latter arthroplasties show less bone density loss and higher serum metal ion concentrations. We expect equal functional scores, greater range of motion, fewer dislocations, fewer periprosthetic radiolucencies and increased prosthetic survival with the metal-on-metal articulation.A randomized controlled trial will be conducted. Patients to be included suffer from non-inflammatory degenerative joint disease of the hip, are aged between 18 and 80 and are admitted for primary cementless unilateral total hip arthroplasty. Patients in the metal-on-metal group will receive a cementless titanium alloy acetabular component with a cobalt-chromium liner and a cobalt-chromium femoral head varying from 38 to 60 mm. Patients in the metal-on-polyethylene group will receive a cementless titanium alloy acetabular component with a polyethylene liner and a 28 mm cobalt-chromium femoral head. We will assess acetabular bone mineral density by dual energy x-ray absorptiometry (DEXA), serum ion concentrations of cobalt, chromium and titanium, self reported functional status (Oxford hip score), physician reported functional status and range of motion (Harris hip score), number of dislocations and prosthetic survival. Measurements will take place preoperatively, perioperatively, and postoperatively .Superior results of large head metal-on-metal total hip arthroplasty over conventional hip arthroplasty have been put forward by experts, case series and the industry, but to our knowledge there is no randomized controlled evidence.This randomized controlled study has been designed to test whether large head metal-on-metal cementless total hip arthroplasty leads to less periprosthetic bone density loss and higher serum metal ion concentrations compared to 28 mm metal-on-polyethylene cementless total hip arthroplasty.Netherlands Trial Registry NTR1399 Painful osteoarthritis of the hip can be successfully treated by total hip arthroplasty (THA). Conventional total hip prostheses consist of a 28 mm metal head and a polyethylene cup. Polyethylene wear debris can however lead to osteolysis, bone loss, aseptic loosening and eventually failure of the implant, especially in high demand young patients . Metal-oThe objective of this study is to conduct a randomized controlled trial to compare two cementless total hip arthroplasties: a conventional 28 mm metal-on-polyethylene articulation and a metal-on-metal large head articulation. We hypothesize that the large head metal-on-metal arthroplasties show less bone mineral density loss and higher serum metal ion concentrations (primary outcome parameters). We expect equal functional scores, greater range of motion, less dislocations, fewer periprosthetic radiolucencies and increased prosthetic survival with the MM articulation (secondary outcome parameters). The present paper reports on the design of the study.A randomized controlled trial will be conducted and concealed allocation will be used to allocate patients to either metal-on-polyethylene or metal-on-metal cementless total hip arthroplasty. The randomization procedure is based on sequentially numbered opaque sealed envelopes, produced by an external institution not involved in the selection, clinical care and evaluation of the patients. The study design, procedures and informed consent are approved by the local Medical Ethical Committee (registration number 2005–42). The trial is registered in the Netherlands Trial Registry (NTR1399). Guidelines of the Consort Statement are followed .The study will be conducted at the Department of Orthopaedic Surgery of the Martini Hospital, which is a large teaching hospital in the city of Groningen, the Netherlands. Patients to be included suffer from non-inflammatory degenerative joint disease of the hip including osteoarthritis, avascular necrosis and traumatic arthritis, are aged between 18 and 80 and are admitted for primary cementless unilateral THA. Patients with active infection, revision arthroplasty, marked bone loss, and unwillingness or inability to follow instruction are excluded. Participation in the study is voluntary and informed consent is required. The inclusion period is planned from September 2006 to September 2009.Patients in the metal-on-metal group will receive a metal-on-metal articulation total hip arthroplasty, a cementless plasma sprayed porous coated titanium alloy acetabular component with a cobalt-chromium liner  and a cobalt-chromium femoral head with a carbon concentration between 0.20% and 0.30%. The radial clearances of the articulations vary between 17.5 and 150 micrometers. The head sizes vary from 38 to 60 mm, depending on the shell sizes which range from 44 to 66 mm. The geometry of the patient determines the largest possible shell size and head size to be implanted.®, Biomet) with a polyethylene liner  and a 28 mm cobalt-chromium femoral head with a carbon concentration between 0.20% and 0.30%. In both the MM and MP groups the same cementless femoral component is used: a proximally plasma sprayed porous coated titanium alloy (Ti6Al4V) stem .Patients in the metal-on-polyethylene group will receive a metal-on-polyethylene total hip arthroplasty, a cementless plasma sprayed porous coated titanium alloy acetabular component , self reported functional status, physician reported functional status, range of motion, number of dislocations, radiographic evaluation and prosthetic survival (secondary outcome parameters). Measurements will take place preoperatively, perioperatively, and postoperatively .Bone mineral density (BMD) measurements will be performed using a dual energy x-ray absorptiometry (DEXA) scanner  in order to calculate bone density changes around the acetabular component. Four horizontal regions of interest (ROI) are defined, as suggested by Wilkinson . In addiSerum ion concentrations for cobalt, chromium and titanium will be determined by venous blood sampling. Cobalt and titanium concentrations are analyzed by inductively coupled plasma mass spectrometry  and chromium is measured by grafite furnace atomic absorption spectrometry with Zeeman correction . The patients' sera may also be used to assess cytokine levels and effects of these ions on osteoblast cells.Surgical approach, surgical time and intra-operative blood loss are recorded. Perioperative complications will be registered, including hip dislocations.The validated Oxford self-rating questionnaire will be used to assess self reported functional status . The valDuring every follow-up visit standard supine anteroposterior (AP) pelvic hip radiographs (with 115% magnification) will be taken. The AP radiographs at 6 weeks will serve as baseline, and will be compared to the X-rays 5 years and 10 years postoperatively. Radiographs are reviewed for presence of femoral radiolucent lines and scored according to the 7 zones described by Gruen et al. . Peri-ac2 with a standard deviation of 0.4, 41 patients are needed in each group . Based on previous work with cemented THA we expect a drop-out rate of 10%, but we also expect conversion to cemented cups if adequate cementless fixation fails. We therefore aim to include 50 patients in each group. Comparable studies also used 50 patients in each group [It is our hypothesis that large head metal-on-metal arthroplasties will show less bone mineral density loss and higher serum metal ion concentrations compared to the conventional 28 mm metal-on-polyethylene articulations. In order to detect a least clinical relevant difference in bone mineral density (BMD) of 0.25 g/cmch group ,16. In och group .The Statistical Package for the Social Sciences version 14.0 for Windows (SPSS Inc.) will be used. Group comparisons are based on intention-to-treat analysis. Non-parametric tests are used for comparisons of means within groups (Wilcoxon's Signed Ranks Test) and between groups (Mann-Whitney Test) if our expectation of a skewed distribution of Oxford and Harris Hip scores postoperatively is confirmed. Chi-square (Fisher's Exact) tests are employed for analyses of categorical variables. Cumulative implant survival is calculated by Kaplan-Meier time series (Mantel-Cox log rank test). A two-sided p-value of < 0.05 is assumed to be significant.Superior results of large head metal-on-metal total hip arthroplasty over conventional hip arthroplasty have been put forward by experts, case series and the industry, but to our knowledge there is no randomized controlled evidence. This study will compare both arthroplasties. Periprosthetic bone density loss will be the main focus. In the short term we will be able to determine whether large head articulations increase clinical range of motion and reduce the number of dislocations. Furthermore, the trial will provide insight in short-term and long-term serum metal ion levels. A related research project will focus on the effects of metal ions on human osteoblast cells in vitro. This is important since the long-term risks of systemic metal ion exposure are unknown. Major pitfalls in orthopaedic surgery research have been the absence of a control group and the lack of randomization. This study overcomes both these drawbacks.This randomized controlled study has been designed to test whether large head metal-on-metal cementless total hip arthroplasty leads to less periprosthetic bone density loss and higher serum metal ion concentrations compared to 28 mm metal-on-polyethylene cementless total hip arthroplasty.Biomet Netherlands financed the bone densitometry measurements and the serum metal ion analysis. The authors did not receive any reimbursements, fees or salary for performing the study.WPZ designed the study and the data collection protocols, included and reviewed part of the patients, coordinated the trial, wrote the manuscript, and will analyze the data. NB included part of the patients and coordinated the trial. JJAMVR designed the study, included, operated and reviewed part of the patients. All authors read, edited and approved the final manuscript.The pre-publication history for this paper can be accessed here:


To the Editor: Toscana virus (TOSV) causes acute meningitis and meningoencephalitis in Mediterranean countries , and varicella-zoster virus (VZV), were negative in CSF specimens taken at admission and 1 month later. TOSV was detected by cell culture and nested RT-PCR in both samples (Patient 2, a 54-year-old man, was admitted to a regional hospital in Granada Province in November 2007. He was confused and agitated, and he reported having fever and headache 2 days before. On admission, he was receiving treatment with corticosteroids for Crohn disease. Analysis of the CSF specimen showed lymphocytic pleocytosis, a normal glucose level, and increased protein levels. Results of PCR for HSV, VZV, and enterovirus were negative. TOSV was detected in the CSF sample by cell culture and real-time RT-PCR (,The most relevant common signs observed in patients 1 and 2 were the ischemic complications. Few cases of complicated encephalitis with sequelae caused by TOSV have been described (Rickettsia conorii were negative. Specific anti-TOSV IgM was detected. The infection was self-limited, and no signs of neurologic involvement were associated with the rash. This was the only case of anti-TOSV IgM detection in 358 serum specimens analyzed from patients with nonneurologic syndromes. Although this finding is not conclusive, it suggests that TOSV infection might be involved occasionally in other mild syndromes. Two other cases of TOSV infection without neurologic involvement have been reported elsewhere: febrile erythema in Italy (Patient 3, a 41-year-old woman, sought treatment for exanthema at her health care center in July 2004. Test results for IgM antibodies against rubella, parvovirus B19, and The unusual manifestations of TOSV infection reported here occurred in persons from rural areas within Granada Province, where seroprevalence rates have been shown to be higher than in urban areas (


Rheumatoid arthritis (RA) is associated with altered metabolism leading to muscle wasting. In the general population, cigarette smoking is known to affect body composition by reducing fat and inhibiting muscle synthesis. Even though smoking has been implicated in the pathophysiology and progression of RA, its possible effects on body composition of such patients have not been studied. This cross-sectional study aimed to identify potential associations of smoking with body weight and composition of RA patients.A total of 392 patients  with RA were assessed for body mass index (BMI), body fat (BF), fat-free mass (FFM), and waist circumference. Erythrocyte sedimentation rate, C-reactive protein, Disease Activity Score-28, and Health Assessment Questionnaire score were used to assess disease activity and severity. Smoking habit  and intensity (pack-years) were also noted.P < 0.001), BF (P < 0.05), and waist circumference (P < 0.05). Pack-years were inversely correlated with BF , and heavy smokers exhibited a significantly lower FFM (P < 0.05) compared with all other participants.Current smokers had a significantly lower BMI compared with ex-smokers  and never-smokers . Similarly, the BF of current smokers was lower compared with that of ex-smokers  and never-smokers . FFM did not differ between groups. Finally, current smokers had a significantly smaller waist circumference compared with ex-smokers only . Following adjustments for age, disease duration, and HAQ score, smoking remained a significant predictor for BMI (Within the limitations of a cross-sectional study, it appears that cigarette smoking associates with reduced BMI and BF in patients with RA and heavy smoking associates with lower muscle mass. Smoking cessation appears to associate with increased BMI, BF, and waist circumference in these patients. These results should be confirmed in prospective studies. Given the numerous adverse effects of smoking on general health and RA, patients should be actively advised against it. However, smoking cessation regimes in RA may need to include more general lifestyle counselling, particularly about weight control. Rheumatoid arthritis (RA), the commonest inflammatory arthritis, is associated with altered metabolism . CompareCigarette smoking is an important risk factor for several diseases . It is aWe have recently demonstrated that smoking further increases REE in RA  and thisConsecutive patients attending routine rheumatology clinics at the Dudley Group of Hospitals NHS Trust, UK, were invited to participate. All applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research. The study had local research ethics committee and research and development directorate approvals, and all volunteers provided informed consent. A total of 400 volunteers  with RA (1987 revised American College of Rheumatology criteria ) were asAll volunteers were subjected to the same data collection procedures overseen by the same trained investigators. Standing height was measured to the nearest 0.5 cm on a Seca 214 Road Rod portable stadiometer . Body weight and composition  were assessed using a Tanita BC- 418 MA Segmental Body Composition Analyzer . After initial manual entry of their demographic details, participants stood barefooted on the analyzer and held the handgrips provided until the apparatus printed the results. BMI was calculated on the basis of measured height and weight in kilograms per square metre. Waist circumference was also measured. Contemporary disease activity was assessed by the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and the Disease Activity Score-28 (DAS28)  was usedData were analysed using the Statistical Package for Social Sciences version 15.0 . A preliminary evaluation of the variables using a Kolmogorov-Smirnov test of normality revealed that none of them required transformation to reach normality. Mean ± standard deviation was calculated for all variables. Differences in BMI, BF, and FFM between smoking groups are presented as mean differences with 95% confidence intervals (CIs).According to their smoking status, patients were grouped into never-smokers, current smokers, and ex-smokers. Analysis of variance (ANOVA) assessed differences in demographic characteristics, BMI, and body composition between groups for each gender. Analysis of covariance (ANCOVA) was employed to determine whether the differences observed were attributed to smoking status or other confounding factors .In the current smoker and ex-smoker groups, further associations between pack-years with BMI and body composition were examined. Thereafter, patients in these groups were divided into quartiles according to pack-years. ANOVA was employed to assess differences in the measured variables between these subgroups. ANCOVA was used to correct for any confounding factors.P value of less than 0.05.Thereafter, patients were grouped according to (a) RA-specific BMI  and (b) Table 2,387 = 8; P < 0.001), BF , and waist circumference . Smoking also emerged as a significant predictor of FFM , but inclusion of BMI as a covariate eliminated the effect of smoking on FFM (P > 0.05).In ANCOVA with gender and smoking as factors and age, DAS28, HAQ score, and disease duration as covariates, smoking was a significant and independent predictor for BMI  in the current smoker and the ex-smoker groups. This remained significant after adjustment for gender, age, DAS28, HAQ score, and disease duration . Following pack-year grouping into quartiles (pack-group), ANOVA did not reveal any differences for BMI or body composition among the current and ex-smoker pack-groups. However, an ANCOVA model with gender and pack-group as factors and age and weight as covariates  revealed a significant effect of pack-group on FFM , with heavy smokers exhibiting the lowest values. Mean (95% CI) values of this variable in the pack-year subgroups appear in Figure There was a significant negative correlation between pack-years and BF (P < 0.05) in the prevalence of overweight and obesity among smoking groups, with obesity being more prevalent in ex-smokers (50%) followed by never-smokers (39%) and current smokers (30%). Similarly, ex-smokers had a significantly (P < 0.05) higher prevalence of increased waist circumference (69%) compared with never-smokers (60%) and current smokers (49%). However, FFM did not differ between groups (P > 0.05) (Figure Following BMI and BF grouping, chi-square analyses showed significant differences  Figure .To our knowledge, this is the first study to identify significant associations between smoking, body weight, and body composition of RA patients: current smokers had a significantly lower BMI and BF compared with never-smokers. Both BMI and BF were significantly increased in ex-smokers, whereas very heavy smoking appeared to associate with reduced FFM. The study has several potential limitations. These are all cross-sectional associations, and although they can serve for hypothesis generation, they do not provide definitive evidence for causality or directionality: longitudinal studies are required for this. In addition, body composition was assessed by bioelectrical impedance. This method has been validated -23 and iOur observations for BMI are consistent with those in the general population. Both male and female smokers tend to have decreased BMI compared with their non-smoking counterparts ,31. In cIn patients with RA, smoking has been shown to elevate REE ; howeverInterestingly, the lower BMI of current smokers in the present study seems to be due to decreased BF rather than FFM. A possible mechanism by which smoking may affect fat metabolism is through a reduction in neuropeptide Y. This molecule not only stimulates food intake, but also promotes white fat lipid storage and decreases brown fat thermogenesis , so its In predominantly healthy people who are from the general population and who do not have wasting muscle disease, smoking of any intensity has been implicated in muscle wasting  by impaiOverall, this study suggests that, in RA, smoking associates with reduced body mass and fatness without inducing further muscle loss, except in very heavy smokers; in contrast, smoking cessation associates with increased body mass and fatness. This should not be interpreted as favouring what is a very unhealthy habit. Smoking cessation, even if it occurs in mid-life, reduces most of the later risk of death from tobacco . HoweverWithin the limitations of this study, it is concluded that RA smokers have a lower BMI and BF than RA non-smokers, while heavy smokers also have a reduced FFM. A history of smoking cessation appears to associate with increases in BMI, BF, and waist circumference. Nevertheless, given the numerous adverse effects of smoking on health, smokers with RA should be actively advised against it, but smoking cessation programs should include wider lifestyle counselling for weight control, also focusing on increased physical activity and a healthy diet.ANCOVA = analysis of covariance; ANOVA = analysis of variance; BF = body fat; BMI = body mass index; CI = confidence interval; CRP = C-reactive protein; DAS28 = Disease Activity Score-28; ESR = erythrocyte sedimentation rate; FFM = fat-free mass; HAQ = Health Assessment Questionnaire; RA = rheumatoid arthritis; REE = resting energy expenditure.The authors declare that they have no competing interests.AS-K participated in patient recruitment, data collection and analysis, and the drafting of the manuscript. GSM participated in patient recruitment and in data collection and analysis. VFP and KMJD participated in patient recruitment, rheumatological clinical assessments, and application of diagnostic/classification criteria. AMN provided expert statistical advice and supervision and participated in the review of the manuscript. AZJ participated in the inception and development of protocol and in the review of the manuscript and served as PhD program supervisor. MK provided advice on protocol development and body composition assessments and participated in the review of the manuscript. YK participated in the inception and development of protocol and served as PhD program supervisor. GDK participated in the inception and development of protocol, patient recruitment, clinical assessments, and analytical approach, provided supervision in the drafting of the manuscript, and served as PhD program supervisor and study guarantor.


Approximately 5% of patients with an acute coronary syndrome are discharged from the emergency room with an erroneous diagnosis of non-cardiac chest pain. Highly accurate non-invasive stress imaging is valuable for assessment of low-risk chest pain patients to prevent these errors. Adenosine stress cardiovascular magnetic resonance (AS-CMR) is an imaging modality with increasing application. The goal of this study was to evaluate the negative prognostic value of AS-CMR among low-risk acute chest pain patients.We studied 103 patients, mean 56.7 ± 12.3 years of age, with chest pain and no electrocardiographic evidence of ischemia and negative cardiac biomarkers of necrosis, who were admitted to the Cardiac Decision Unit of our institution. All patients underwent AS-CMR. A negative AS-CMR was defined as absence of all the following: regional wall motion abnormalities at rest; perfusion defects during stress (adenosine) and rest; and myocardial scar on late gadolinium enhancement images. The patients were followed for a mean of 277 (range 161-462) days. The primary end point was defined as the combination of cardiac death, nonfatal acute myocardial infarction, re-hospitalization for chest pain, obstructive coronary artery disease (>50% coronary stenosis on invasive angiography) and coronary revascularization.In 14 patients (13.6%), AS-CMR was positive. The remaining 89 patients (86.4%), who had negative AS-CMR, were discharged. No patient with negative AS-CMR reached the primary end-point during follow-up. The negative predictive value of AS-CMR was 100%.AS-CMR holds promise as a useful tool to rule out significant coronary artery disease in patients with low-risk chest pain. Patients with negative AS-CMR have an excellent short and mid-term prognosis. Acute chest pain is one of the most common reasons for presentation to the emergency department . The maiFrom July 2007 through May 2008, we enrolled 103 patients who presented to the emergency department with low-risk acute chest pain. This was a single center, retrospective study. All patients were examined clinically and cardiovascular risk factors were assessed, including tobacco use, diabetes mellitus, hypertension, hypercholesterolemia, family history of coronary artery disease (CAD) and history of previous CAD. Previous CAD was defined as previous myocardial infarction, CAD on cardiac catheterization, previous percutaneous coronary intervention or previous coronary artery bypass grafting. All cardiovascular medications were recorded. Low-risk acute chest pain was specified as no persistent ischemic-type chest pain with no objective evidence of myocardial ischemia, based on the negative results of serial serum cardiac biomarkers of necrosis  as well as normal or inconclusive electrocardiograms . The exclusion criteria were standard contraindications for magnetic resonance imaging, such as internal pacemaker or defibrillator, cerebral aneurysm clips or metal in the eye; contraindications for adenosine administration, such as bronchospasm, high degree atrioventricular block, known allergic reaction to the medicine; pulmonary embolism; acute neurological disease; high clinical suspicion of acute aortic syndrome; and New York Heart Association class IV congestive heart failure symptoms.All patients underwent AS-CMR within 24 hours of their presentation to the emergency department. This study had institutional review board approval from Emory University.The AS-CMR protocol was carried out on a 1.5T Siemens Avanto system . The patient was positioned feet-first supine, with a 6-channel flexible body phased array coil anteriorly and a 24-element spine array for posterior signal reception. Three-lead ECG was applied to the chest with waveform monitoring throughout the study. The entire imaging protocol was completed in approximately 45 minutes  sequence. Subsequently, breath-hold steady-state free precession (SSFP) cine acquisitions were acquired in the vertical long-axis, horizontal long-axis, basal short-axis, true four-chamber, and left ventricular outflow track orientations. Each orientation was prescribed using the preceding acquisition. First-pass perfusion (both at stress and rest) was performed with a saturation-prepared turbo flash acquisition. Three slices were oriented in the short-axis plane , prescribed from the cine four-chamber view. A test scan was performed with 5 dynamic images to confirm absence of artifacts and proper visualization of the LV myocardium and blood pool. The sequence was copied and 60 dynamics were implemented for perfusion imaging. Adenosine was administered intravenously at 140 μg/kg/min. Four minutes into the infusion, the stress perfusion imaging was performed, with 0.05 mmol/kg gadobenate dimeglumine (MultiHance), Bracco Diagnostics Inc., Princeton, NJ. The perfusion pulse sequence was initiated 3 seconds prior to gadolinium infusion, followed by a breath-hold command, and lasted for approximately 1 min. Adenosine infusion was stopped after the acquisition. After a brief period, during which optional images, such as flow sequences, were obtained, rest perfusion was performed with a second dose of 0.05 mmol/kg MultiHance, using the same acquisition method as was used for stress perfusion imaging. An additional 0.1 mmol/kg of MultiHance contrast was administered following rest perfusion for late gadolinium enhancement (LGE) imaging. Multi-slice SSFP cine short-axis imaging was subsequently performed. Acquisition extended from the atrial side of the ventricular valve plane to just beyond the apex. Approximately seven minutes after the final contrast dose, a multi-phase inversion-recovery SSFP scan was acquired in the mid short-axis plane, in order to determine the optimal inversion time (TI) to null normal myocardium for LGE imaging. The time that corresponded to the image with sufficiently nulled myocardium was entered as the TI into the subsequent inversion recovery segmented turbo flash sequence used for LGE imaging. Acquisition was performed in the short-axis, and covered the same anatomic range as the SSFP cine short-axis stack. In addition, single slices were acquired in the vertical long-axis, four chamber and left ventricular outflow tract views to correspond to the cine acquisitions. The TI was increased as needed as time passed.Left ventricular ejection fraction was calculated off-line from end-diastolic and end-systolic endocardial tracings of the multi-slice SSFP cine short-axis images using computer-assisted planimetry. A negative AS-CMR was defined as the absence of all of the following: resting wall motion abnormalities on cine images, perfusion defects during adenosine infusion, or LGE in a coronary disease-type pattern . A positive AS-CMR had one or more of these features. A myocardial perfusion defect was reported if a segment was definitely darker than surrounding myocardium and persisted more than three images beyond initial peak enhancement of the segment which appeared most normal. LGE images were displayed with a gray scale to optimally show normal myocardium as dark and the regions of LGE or fat as bright. The AS-CMRs were interpreted qualitatively by consensus of two Level III CMR trained cardiologists or radiologists.The follow-up was completed in October 2008. The primary end point was defined as the combination of cardiac death, nonfatal myocardial infarction, re-hospitalization for unstable angina, obstructive CAD (defined as at least one coronary lesion of >50% stenosis on invasive coronary angiography) and coronary revascularization. Cardiovascular events during follow-up were identified for these patients by reviewing their medical data from our comprehensive institutional electronic records for clinic visits, new emergency department visits or hospital admissions. Myocardial infarction was defined based on the 2007 ESC/ACCF/AHA/WHF expert consensus document for the universal definition of myocardial infarction . CardiacContinuous variables are presented as mean ± SD or median (range). Qualitative variables are presented as absolute and relative frequencies. All analysis used a two tailed p value with a significance level of < 0.05. Statistical analyses were performed using the SAS statistical package version 9 .During the 13 months of enrollment, 103 patients, 56.7 ± 12.3 years of age, with low-risk chest pain were included as study patients. Patients' characteristics, demographic data and medications at baseline are listed in Table A total of 14 patients (13.6% of the study population) had abnormal findings on AS-CMR. Six of these 14 patients (5.8%) had resting regional wall motion abnormalities, ten patients (9.7%) had perfusion defects during stress, and five patients (4.9%) had evidence of scar on LGE images. Six of the patients with an abnormal AS-CMR had a previously known history of CAD. Four patients refused further evaluation. From the remaining ten, five were advised to undergo coronary angiography, and all of these had obstructive CAD (one received percutaneous coronary intervention). Another four of the ten had known coronary anatomy from recent coronary angiography, and one with only a small area of ischemia was managed conservatively without undergoing coronary angiography. All patients received standard pharmacological treatment for secondary prevention of CAD. During the follow-up period, the conservatively-managed patient with a small area of ischemia presented again with chest pain and was admitted; coronary angiography was performed revealing luminal irregularities with no need for interventional treatment. Another two patients with known CAD and scar in the original AS-CMR were readmitted because of chest pain and underwent coronary angiography, with no need for revascularization. There were no readmissions for the patients who refused coronary angiography.The remaining 89 patients (86.4% of the study population) had negative AS-CMRs. Seven of them had a known history of CAD. All patients were discharged from the emergency department with risk modification counseling and/or pharmacological treatment. During the period of follow-up a total of 9 patients presented again to the emergency department, all of them with atypical chest pain. Evaluation of chest pain with serial electrocardiograms, serial cardiac biomarkers of necrosis and non-invasive imaging studies did not reveal any case of acute coronary syndrome and all patients were discharged from the emergency department, without being admitted. The negative predictive value of AS-CMR was 100%.The present study has shown that a negative AS-CMR performed within 24 hours of patients presenting to the emergency department with low-risk chest pain, as indicated by negative serial electrocardiograms and cardiac biomarkers of necrosis, is associated with an excellent prognosis.Approximately 5% of patients with an acute coronary syndrome are discharged from the emergency room with an erroneous diagnosis of non-cardiac chest pain . These pThe present study extends the findings of previous studies examining the predictive value of stress perfusion CMR -15. JahnAlso in the non-acute clinical setting, Pilz et al.  have shoThe high negative predictive value of AS-CMR found in the present study in patients presenting to the emergency department with low-risk chest pain concurs with findings from a prior study by Ingkanisorn et al , in whicChoosing the appropriate non-invasive evaluation for acute chest pain involves considering test characteristics, as well as the risks inherent to the test itself. High sensitivity and negative predictive value, as demonstrated for AS-CMR in the present study, are essential. For example, while several studies have shown that patients presenting to the emergency department with low-risk chest pain can safely undergo exercise testing within 6 to 12 hours, sensitivity of exercise treadmill alone for detection of CAD may be as low as 29% . Adding Stress echocardiography is an attractive alternative for the evaluation of acute chest pain as it avoids exposure to radiation or nephrotoxic contrast agents, and includes an evaluation of cardiac structure and function similar to AS-CMR. However, the sensitivity of dobutamine stress echocardiography (DSE) with regional wall motion analaysis (WMA) for detection of significant CAD has been questioned, and its risks of induced arrhythmia and acute infarction are appreciable. Tsutsui et al.  suggest In addition to its high diagnostic accuracy and negative predictive value, AS-CMR is a safe and well tolerated procedure with only minimal risk to the patient. Due to the extremely short half-life of adenosine any adverse reactions caused by its infusion are rapidly ended with discontinuation of the drug. In the present study, there were no adverse effects associated with AS-CMR. Gadobenate dimeglumine carries a very low risk of nephrotoxicity, except in patients with end stage renal disease . The imaAS-CMR has specific advantages over other imaging modalities in addition to the avoidance of radiation, iodinated contrast, and dobutamine with its known risks. Unlike other imaging modalities, body habitus does not affect AS-CMR image quality nor the propensity for false positive results. In addition, alternative etiologies of chest pain including aortic dissection, pericarditis, and myocarditis may be accurately identified on AS-CMR. Furthermore, gold standard quantification of ejection fraction as well as assessment of valvular function are inherent to the exam. Finally, AS-CMR images can be read immediately and clinical decisions made quickly, as there is no post-processing required. This may reduce average length of stay in chest pain units, increase diagnostic efficiency, and allow practitioners to care for more patients.The results of our study may have specific implications for women. Although usually underrepresented in studies of acute coronary syndromes, females were the majority of our population (63%). This may have partly accounted for the low incidence of adverse events in our study population and for the excellent negative predictive value of AS-CMR. Other forms of stress testing encounter specific obstacles among women, including low specificity for exercise testing and lower diagnostic accuracy for nuclear perfusion imaging due to the presence of breast artifact . FurtherThe excellent negative predictive value of AS-CMR in our study population applied to patients both with and without a known history of CAD, although few patients had known CAD . According to current recommendations, patients with known CAD and low-risk chest pain can be observed in a chest pain unit and discharged if they have a negative stress test . AS-CMR Our study has several limitations. First, this was a single-center study with small sample size resulting in the absence of a large number of hard cardiac events such as myocardial infarction or cardiac death. Secondly, we did not compare AS-CMR with other non-invasive or invasive imaging modalities. Third, follow-up was by review of medical records rather than directly contacting the patients. Furthermore, we calculated the negative but not the positive predictive value of AS-CMR. We believe that the intensive preventive management strategies in our positive AS-CMR patients could have accounted for a reduction in their risk; thus, the positive predictive value of AS-CMR would have been underestimated. Furthermore, a high negative predictive value is the most valuable characteristic for stress imaging in this clinical setting.Our study suggests that AS-CMR has high negative predictive value and can be used as a non-invasive stress imaging modality to safely discharge low-risk chest pain patients from the emergency department. Additional multicenter, large-scale studies could provide further insights into this area.The authors declare that they have no competing interests.SL conceived of the study and helped draft the manuscript. DSM helped gather data and helped draft the manuscript. MJ edited and revised the manuscript. JNO provided critical input on study design, and technical support for image acquisition. NA assisted with data analysis and statistics. AVA helped design the study and helped draft the manuscript. EZ assisted with statistical analysis and study design. EV assisted with study design. AES helped conceive the study and gather the data. All authors read and approved of the final manuscript.


Animal mitochondrial genomes are potential models for molecular evolution and markers for phylogenetic and population studies. Previous research has shown interesting features in hymenopteran mitochondrial genomes. Here, we conducted a comparative study of mitochondrial genomes of the family Braconidae, one of the largest families of Hymenoptera, and assessed the utility of mitochondrial genomic data for phylogenetic inference at three different hierarchical levels, i.e., Braconidae, Hymenoptera, and Holometabola.Cotesia vestalis, in which 13 protein-coding genes and 14 tRNA genes changed positions or/and directions through three kinds of gene rearrangement events. Remote inversion is posited to be the result of two independent recombination events. Evolutionary rates were lower in species of the cyclostome group than those of noncyclostomes. Phylogenetic analyses based on complete mitochondrial genomes and secondary structure of rrnS supported a sister-group relationship between Aphidiinae and cyclostomes. Many well accepted relationships within Hymenoptera, such as paraphyly of Symphyta and Evaniomorpha, a sister-group relationship between Orussoidea and Apocrita, and monophyly of Proctotrupomorpha, Ichneumonoidea and Aculeata were robustly confirmed. New hypotheses, such as a sister-group relationship between Evanioidea and Aculeata, were generated. Among holometabolous insects, Hymenoptera was shown to be the sister to all other orders. Mecoptera was recovered as the sister-group of Diptera. Neuropterida , and a sister-group relationship with (Diptera + Mecoptera) were supported across all analyses.Seven mitochondrial genomes from seven subfamilies of Braconidae were sequenced. Three of the four sequenced A+T-rich regions are shown to be inverted. Furthermore, all species showed reversal of strand asymmetry, suggesting that inversion of the A+T-rich region might be a synapomorphy of the Braconidae. Gene rearrangement events occurred in all braconid species, but gene rearrangement rates were not taxonomically correlated. Most rearranged genes were tRNAs, except those of Our comparative studies indicate that mitochondrial genomes are a useful phylogenetic tool at the ordinal level within Holometabola, at the superfamily within Hymenoptera and at the subfamily level within Braconidae. Variation at all of these hierarchical levels suggests that the utility of mitochondrial genomes is likely to be a valuable tool for systematics in other groups of arthropods. Most animal mitochondrial genomes are about 16 Kb in size and contain 37 genes: 13 protein-coding genes, 22 transfer RNA genes (tRNA) and two ribosomal RNA genes (rRNA)  a [TA(A)S. agrili, D. longicaudata, C. vestalis and M. camphoraphilus. Elements presumed to be involved in genome replication and transcription were found in all sequenced A+T-rich regions except that of M. camphoraphilus  species, in which at least a large segment including six protein-coding genes and three tRNAs are inverted [C. vestalis and the three Nasonia species indicate that large-scale protein-coding gene rearrangement are probably independent events in Hymenoptera.Gene rearrangement events occurred in all seven species of Braconidae Figure . All reaangement ,21, howeinverted . Gene recox1 to trnH, which was successfully sequenced in all seven species; gene rearrangement rates were unequal among them. Gene arrangement pattern was conserved in A. gifuensis and P. flava, and derived in the other five species. Of the two microgastroid species P. flava and C. vestalis [We compared gene rearrangement rates among braconid mitochondrial genomes in the region from vestalis ,41,51, ttrnI and trnM were inverted and translocated, forming the arrangement pattern trnI(-) - trnM(-) - A+T-rich region - trnQ in S. agrili. D. longicaudata and C. vestalis, and trnM(-) - trnI(-) - A+T-rich region - trnQ in M. camphoraphilus.Gene rearrangement events comprise three classes, i.e., translocation, local inversion (inverted but remaining in the position), and shuffling with remote inversion (translocated and inverted) . All detS. agrili, D. longicaudata and C. vestalis, trnH was inverted and translocated (remote inversion) to the junction of cox2 and atp8. The arrangement pattern, trnD(-) - trnH - trnK or trnH(-) - trnD - trnK, has been found in many subfamilies of Braconidae. It has been reported that both the inversion of trnD and the remote inversion of trnH are independent evolutionary events in this family [Jarra phorocantha and Heterospilus sp., the ancestral arrangement pattern of trnK - trnD occurs [S. agrili occurred after the origin of the subfamily.In the three species s family . In the trnY in C. vestalis is the only locally inverted gene in these seven braconid species, except for the region from trnE to cob in C. vestalis. The duplication/random loss model, and the intramitochondrial genome recombination and duplication/nonrandom loss model are possible mechanisms to explain translocation. Of these, intramitochondrial genome recombination is presumed to be more common. Shuffling is thought to be the result of duplication/random loss. The tRNA clusters trnW - trnC - trnY and trnK - trnD are frequently shuffled regions. trnD was not only shuffled but also inverted. This may have been the result of two independent events caused by separate mechanisms.Mechanisms for the three classes of gene rearrangement events in hymenopteran mitochondrial genomes are widely discussed ,21. IntetrnH, trnI and trnM). However, remote inversion could not be deduced in previous studies due to incompleteness of sequence data [S. agrili, an inverted pseudo-trnH sequence is located between nad4 and nad5, the ancestral position of trnH  [In d Figure : (1) Earonoidea) . Since ionoidea) , recombinad3 and nad5 in their ancestral position are heavily rearranged. And the most apparently conserved order of this tRNA cluster is trnA - trnR - trnN, which indicates that the derived relative positions of these tRNA genes are most likely the result of translocations of trnF and trnE and shuffling of trnS2. Nevertheless, trnH may have been inverted and translocated to the junction of cox2 and atp8 before the large-scale inversion event. The most possible mechanisms of gene translocation and shuffling are recombination, rather than tandem duplication followed by deletion (TDRL), which might change the remnant order of tRNA and the neighbouring protein-coding genes.(2) tRNA translocations. The six tRNA genes between nad4 - nad5, trnT - nad4l, cox3 - trnG - nad3 and cob - nad6 - trnP, were translocated after the large-scale inversion at the junction of atp6 - trnS1. Although tRNA gene rearrangement is more frequent than that of protein-coding genes, translocation of three gene boundaries is more parsimonious than tRNA gene rearrangement. Recombination is also favoured in these small-scale translocation events, because neither pseudo-genes nor large intergenic spacers are present in the boundaries of these rearranged genes, which are the intermediate state of the TDRL model [(3) Small-scale translocations. Four gene boundaries, i.e., RL model .Braconidae mitochondrial genomes have high A+T content, a characteristic typical of other hymenopterans, with values from 82.4% to 87.2%.Strand asymmetry  are usually reflected by AT skew, as expressed by (A-T)/(A+T), and GC skew, as expressed by (G-C)/(G+C) . StrandsHassanin et al. (2005) suggested that strand asymmetry is best reflected in the GC skew. Hence, all braconid species in the present study show reversal of strand asymmetry, as in some other arthropods ,58-61, fInversion of replication origin located in the A+T-rich region would lead to reversal of strand asymmetry ,59,66, wD. semiclausum or Enicospilus sp. (Hymenoptera: Ichneumonidae) as reference sequences , the rate of synonymous substitutions (Ks) and the ratio of the rate of non-synonymous substitutions to the rate of synonymous substitutions (Ka/Ks) were calculated for each braconid mitochondrial genome using s Figure . SpeciesS. agrili (subfamily Doryctinae) and D. longicaudata (subfamily Opiinae) were recovered in all analyses employing different inference methods and data. This is consistent with the widely accepted group, the cyclostomes. A. gifuensis (subfamily Aphidiinae) are firmly placed as the sister-group to the cyclostomes. Four species C. vestalis (Microgastrinae), P. flava (Cheloninae), M. camphoraphilus (Macrocentrinae) and M. pulchricornis (Euphorinae) constitute the other major group, the noncyclostomes. However, the internal relationships among noncyclostome subfamilies were not well resolved. In previous studies, Microgastrinae and Cheloninae were generally recovered in a clade referred to as microgastroids, while Macrocentrinae and Euphorinae form part of the clade, helconoids [M. camphoraphilus or P. flava were excluded from the Bayes analyses, both microgastroids and helconoids were recovered with improved support values  is supported across all analyses, forming the sister-group to (Diptera + Mecoptera), differing from the presently preferred sister-group relationship of Neuropterida and Coleoptera .In the previous analysis using complete mitochondrial genome sequence data, the relationships among holometabolous orders were not well resolved ,68. HereNasonia vitripennis among three Nasonia species is supported, which is congruent with the analyses based on nuclear gene sequence data and phylogeny of Wolbachia bacteria that they host [Within Hymenoptera, three species of Symphyta representing three superfamilies and three families and 17 species of Apocrita representing seven superfamilies and eight families were used in analysis. Symphyta was shown to be paraphyletic; Apocrita was monophyletic with Orussoidea as its sister-group. Two major clades were recovered within Apocrita: Stephanoidea + Proctotrupomorpha, and Ichneumonoidea + (Evanioidea + Aculeata). Proctotrupomorpha , Ichneumonoidea (Ichneumonidae + Braconidae) and Aculeata ((Eumeninae + Vespinae) + Apoidea) in this study) were strongly supported in all analysis. Evaniomorpha proposed in  was not hey host ,70.Phylogenetic relationships among Hymenoptera at the superfamily level remain controversial . Many weCyphon + (Elateroidea + (Chaetosoma + (Tribolium+ (Priasilpha + (Anoplophora + Crioceris))))). The intraordinal relationships of Lepidoptera are also unstable among datasets and methods. BI analyses supported early divergent of Rhopalocera (Lycaenidae + Pieridae in this study) most ML and MP analyses supported that of Tortricidae (Tortricoidea). Monophyly of Bombycoidea (Saturniidae + (Sphingidae + Bombycidae) in this study) was firmly supported in most analysis. The position of Lepidoptera and its internal relationships is likely negatively affected that the small sample of all taxa all of which were restricted to the clade Apoditrysia. We will not discuss the relationships among Coleoptera and Lepidoptera further since phylogenetic relationships are not robustly supported. However, our results might be of service to future researchers.Within Diptera, well established relationships were recovered in nearly all of the analyses. Suborder relationships within Coleoptera were less stable. Adephaga and Myxophaga were recovered as sister group and then sister to Polyphaga. However, Archostemata was either recovered as sister-group to Polyphaga, or to Adephaga. Monophyly of the suborder Polyphaga is recovered in most analyses with internal relationships of In this study, we reported seven complete or nearly complete mitochondrial genomes representing seven subfamilies of Braconidae. Four sequenced A+T-rich regions were shown to be inverted, as reported for species of Philopteridae (Phthiraptera) and Aleyrodidae (Hemiptera). Reversal of strand asymmetry was found in all seven sequenced mitochondrial genomes, which is correlated with the inversion of the A+T-rich region, indicating that inversion of A+T-rich region might be a ground-plan feature of braconid mitochondrial genomes. Mitochondrial gene rearrangement rates differed markedly among Braconidae, revealing that gene rearrangement might be more diverse than that previously reported ,21,22,29Noncyclostome species have a higher evolutionary rate in mitochondrial genome sequences than those of cyclostome species. Those species with high gene rearrangement rates also have high nucleotide sequence evolutionary rates.Phylogenetic analysis using complete mitochondrial genomes sequences recovered most of the well corroborated phylogenetic relationships among major lineages of Braconidae, depending on the method and data matrix used. Cyclostomes and noncyclostomes were recovered in all analysis, and Aphidiinae was firmly recovered as be a sister-group to the cyclostomes.Within Hymenoptera, many well accepted relationships, such as the paraphyly of Symphyta and Evaniomorpha, the sister-group relationship between Orussoidea and Apocrita, and the taxa Proctotrupomorpha, Ichneumonoidea and Aculeata, were recovered with high support values. New views among major groups in Hymenoptera are suggested, such as the sister-group relationship between Evaniidae and Aculeata. Within Diptera, relationships were very stable across analysis, but not stable among suborders of Coleoptera and major lineages of Lepidoptera.Relationships among holometabolous orders were improved with the increase of sampling based on complete mitochondrial genome sequences and the recently suggested basal position of Hymenoptera was supported in most analysis.In conclusion, complete mitochondrial genome data have obvious potential to infer phylogenetic relationships at the subfamily, family, ordinal levels.For each species, one male adult or a leg was homogenized in liquid nitrogen and total genomic DNA was extracted using the DNeasy tissue kit  following manufacturer protocols.A range of universal insect mitochondrial primers and hymenopteran mitochondrial primers modified from universal insect mitochondrial primers were used ,57,80,81http://rna.ucsc.edu/rnacenter/xrna/xrna.html).Protein-coding and rRNA genes were initially identified using BLAST searches in GenBank and subsequently by alignment with genes of other insects. Protein-coding genes were translated using invertebrate mitochondrial genetic code. The tRNA searches were carried out with tRNAscan-SE search server . The parD. semiclausum or Enicospilus sp. as reference sequences.The software packages DnaSP 4.0  was used to compute the number of synonymous substitutions per synonymous site (Ks) and the number of nonsynonymous substitutions per nonsynonymous site (Ka) for each braconid mitochondrial genomes, using that of D. semiclausum and Enicospilus sp., both Ichneumonidae, the family commonly accepted as the sister-group of the Braconidae, were employed as outgroups.For construction of the phylogenetic relationships among Braconidae, seven species with mitochondrial genomes sequenced in this study were used, representing seven subfamilies of Braconidae. S. agrili representing the cyclostomes and C. vestalis representing the noncyclostomes were used. Twelve species in nine families of the Lepidoptera, 14 species in 14 families of Coleoptera and 14 species in nine families of Diptera were included. Additionally, three species of Neuroptera, three species of Megaloptera and one species of Mecoptera were used to improve the sampling of orders among Holometabola. Hydaropsis longirostris and Triatoma dimidiata (Hemiptera) were used as outgroup taxa.For construction of phylogenetic relationships among holometabolous insects, 69 species with complete or nearly complete mitochondrial genome sequences were used , nucleotide sequences of first and second codon positions (Pos12), all codon positions (Pos123), first and second codon positions, and RY-coded (purines coded by R and pyrimidines coded by Y) third codon position (Pos12RY3) of all protein-coding genes. Gene Phylogenetic analyses were performed using Maximum Parsimony (MP) with PAUP* 4.0b10 , MaximumConceived and designed the experiments: SJW and MS. Performed the experiments: SJW. Analyzed the data: SJW and MS. Wrote the paper: SJW. Intellectual contributions during the design and implementation of this study, and during the writing of the manuscript: XXC, MJS and CVA. Provided funding in support of this study: XXC and MJS. All authors read and approved the final manuscript.Phylogenetic trees constructed in this studyClick here for fileSpecies used in phylogenetic reconstruction of HolometabolaClick here for file


Since 2003, the Global Fund has supported the scale-up of HIV/AIDS, tuberculosis and malaria control in low- and middle-income countries. This paper presents and discusses a methodology for estimating the lives saved through selected service deliveries reported to the Global Fund.Global Fund-supported programs reported, by end-2007, 1.4 million HIV-infected persons on antiretroviral treatment (ARV), 3.3 million new smear-positive tuberculosis cases detected in DOTS  programs, and 46 million insecticide-treated mosquito nets (ITNs) delivered. We estimated the corresponding lives saved using adaptations of existing epidemiological estimation models.falciparum malaria were estimated to have achieved protection from malaria for 26 million of child-years at risk cumulatively, resulting in 130,000  under-5 deaths prevented.By end-2007, an estimated 681,000 lives  were saved and 1,097,000  life-years gained by ARV. DOTS treatment would have saved 1.63 million lives (1.09 - 2.17 million) when compared against no treatment, or 408,000 lives  when compared against non-DOTS treatment. ITN distributions in countries with stable endemic These results illustrate the scale of mortality effects that supported programs may have achieved in recent years, despite margins of uncertainty and covering only selected intervention components. Evidence-based evaluation of disease impact of the programs supported by the Global Fund with international and in-country partners must be strengthened using population-level data on intervention coverage and demographic outcomes, information on quality of services, and trends in disease burdens recorded in national health information systems. Only in some five years, the Global Fund to Fight AIDS, Tuberculosis and Malaria  has become a major player in international health development. In 2005, the Global Fund provided 21% of international funding for HIV/AIDS programs in low- and middle-income countries, 67% for tuberculosis (TB), and 64% for malaria[For evaluation of disease impact, Global Fund recipients collect epidemiological data on relevant changes in mortality and morbidity. Given the nature of the diseases and interventions and data collection mechanisms, it may take several years before impact becomes detectable. For example, reductions in new HIV infections are inferred retrospectively from prevalence trends over preceding years. WHO and UNAIDS regularly publish regional-level HIV incidence and prevalence estimates, using country surveillance and survey data that often require two years to be released -7. Similinterim approach, we estimated the scale of lives saved, by summarizing deaths averted from key service delivery results reported by recipient programs. Interventions considered are antiretroviral treatment (ARV), Directly Observed TB Treatment, short-course (DOTS) and insecticide-treated mosquito nets (ITN). Estimates of their efficacies against mortality are available[In view of these data limitations, it is still early for the impact of Global Fund support to be fully measured. As an available,12-17. Tand below).Lives saved and, for ARV, life-years gained, were estimated using mortality estimation models and assumptions used by UNAIDS, WHO Stop TB and Roll Back Malaria. Models were adapted to use as main input service delivery results available from recipient countries Table and belowhttp://www.theglobalfund.org, as the basis for performance-based funding[Supported programs regularly report progress against targets on standardized indicatorshttp://wwd funding. Reported funding,21  that the UNAIDS Reference Group on Estimates, Modelling, and Projections[ARV programs routinely report to the Global Fund  on people ojections recommenojections, becauseSpectrum uses demographic data and estimates of country HIV prevalence, incidence, mortality and ARV coverage rates over time, to estiThese projections yielded average ratios of 'life-years gained per patient-year of ARV', and 'lives saved per patient on ARV', which varied among countries and over calendar years according to varying stages of disease that patients initiated ARV, indirect demographic effects (restored fertility etc.), and dynamic effects of ARV, through reduced viral load, on reducing HIV transmission. To subsequently obtain mortality effect estimates, the regionally averaged ratios of 'life-years gained per patient-year of ARV' over the period 2003-2007, and of 'lives saved per patient on ARV' at end-2007 were applied to regionally summed ARV numbers reported by Global Fund recipients at end-2007.We estimated TB mortality based on numbers of new smear-positive case detections in supported DOTS programs and TB cFor DOTS, two comparison scenarios were considered. The simplest comparison, consistent with the 'no intervention' counterfactual scenario for ARV and ITNs, was against no treatment. The alternative is against non-DOTS treatment, which is commonly used in TB impact evaluations,27, as DCase fatality rates for DOTS, non-DOTS and no treatment scenarios were available from WHO, stratified by HIV and smear status. These country-specific rates were combined with country-specific proportions of DOTS-treated smear-positive TB cases that are HIV-positive, for the year 2006.falciparum malaria, effective use of ITNs results in averting 5.5 under-five deaths per 1000 children protected per year[falciparum malaria occurs of a similar endemicity[falciparum malaria and in places with non-stable falciparum malaria was not estimated, due to limited evidence of intervention effectiveness and lack of reliable estimates of populations at risk[In areas of sub-Saharan Africa with stable endemic  per year  were calculated applying the delta method to the cFor service delivery results, on-site verification had revealed that in 2005-6 83% of country reports were within a 20% margin, without systematic bias in either direction (over-reporting or under-reporting)[For other parameters, errors were (conservatively) assumed to be systematic rather than country-specific. When summing lives saved across the 3 diseases, errors in disease-specific estimates were taken as independent.As of end-2007, 1.44 million people were reported to be on ARV, in 91 supported HIV programs Table . The resSupported TB programs in 68 countries reported a cumulative 3.3 million smear-positive TB case detections by end-2007 Table . Out of falciparum malaria; 231,000 in Papua New Guinea, and 7.4 million in lower-endemic areas  up to end-2007  by December 2007. Alternatively, when using non-DOTS treatment as comparison (instead of no treatment), lives saved sum to 1.22 million .The presented estimates of lives saved and life-years gained by Global Fund-supported ARV, DOTS and ITNs indicate the magnitude of mortality reductions achieved by recipient programs. While not being an actual measurement or formal evaluation of program impact, the estimations allow the Global Fund to gauge progress as service delivery reports from programs accumulate.The estimates do not reflect the whole spectrum of interventions supported. Notably, HIV infections averted by prevention services will in future save lives from AIDS, but cannot readily be estimated from routine delivery reports. Effective HIV prevention often results from a multi-sectoral package of services-36, and For malaria, artemisinin-based combination treatments (ACTs) probably have important mortality effects. By December 2007, Global Fund-supported programs had delivered over 24 million ACT courses, across 49 countries. The effectiveness of ACTs against mortality has not been established through randomized trials and will depend on the timeliness of and compliance with treatment; proportions of ACTs for true malaria episodes ; possible effects of ACTs on preventing repeat episodes due to recrudescent infection; and the comparison scenario (no treatment or ineffective mono-therapy). The presented model furthermore excluded indoor residual spraying (IRS). As more knowledge becomes available, ACT and IRS should be included in lives saved estimation.The gap-filling nature of Global Fund financing makes it difficult to define a specific contribution as distinct from other donors and (pre-)existing in-country efforts. Some programs, particularly HIV and TB, report program-wide results covering all contributions from multiple domestic and international funding sources to the Global Fund, which promotes program supports rather than a project approach. Lives saved result from collective efforts of in-country programs, to which the Global Fund contributes alongside other partners. For malaria, on the other hand, total service deliveries covering all partners' contributions considerably outnumber results reported to the Global Fund: e.g. in 2006 alone, 63 million ITNs were produced worldwide. For ARVSeveral limitations and caveats concerning input data and assumptions must be acknowledged. Errors may occur at any stages. Misclassification of smear positivity for TB is one example. While service delivery numbers may be imprecise and performance-based funding may provide incentives for over-reporting, the errors are not systematically in one direction, so that aggregated portfolio results should roughly reflect what was delivered. In realThe presented methodology was designed exclusively to produce a portfolio-aggregate indication of global-level health gain, although we used certain country-specific input assumptions and presented estimates by regional break-down. Country-specific evaluations would, in contrast, require more refined methods and inputs.The largest uncertainties in these estimates lie in the epidemiological models and their basic (cross-country) assumptions. The 2007 UNAIDS model for estimating HIV-related deaths may be pFor TB, lives saved depend critically on the comparison scenario Figure . While aOur estimate of lives saved by Global Fund-supported ITNs depends critically on assumptions to convert ITN deliveries into child-years of protection. The assumption that each ITN distributed provides an average 1.1 child-year of protection (see Methods) is simplistic and probably conservative, because: (i) some 38% of ITNs distributions reported by supported programs were long-lasting insecticidal nets (LLINs) that remain effective throughout their lifetime of typically 3-4 years; (ii) evfalciparum malaria, for given levels of coverage, in all recent malaria impact models[There is reasonable consensus about the efficacy of ITNs against mortality in children exposed to endemic ct models, includict models. Malariact models contribuThe presented uncertainty ranges, particularly for ARV and DOTS, may be minimum intervals, which do not capture the complex chain of factors that intervene between service delivery counts, population coverage and impact, such as quality of services including (mis-)diagnosis, drug quality, patient counselling and support to promote patient retention and treatment adherence. Periodic evaluation of quality of services must therefore be added to program progress tracking and performance evaluation.Longer-term, improved approaches to measuring disease impact are essential in low- and middle-income countries. To enable this and to evaluate its institutional performance and partners context, the Global Fund recommends that programs spend 5-10% of grant budgets on strengthening monitoring and evaluation systems as well as supporting vital registration. Furthermore, the Global Fund Board has commissioned an independent Five-Year Evaluation including impact evaluations reinforcing quality data collection and analysis in 20 countries.The presented estimates indicate the scale of mortality effects that Global Fund-supported programs may have achieved over recent years. While the numbers carry great margins of uncertainty and cover only selected program components, they reflect the best information available to date. Evidence-based evaluation of the impact of HIV/AIDS, TB and malaria programs supported by the Global Fund and other partners is ongoing and must be further strengthened to maximize health gain. Great improvements will be made by triangulating service delivery reports with population-level data on intervention coverage and demographic outcomes, information on quality of services, and trends in health facility disease burdens recorded in national health information and management systems.No particular funding was provided to develop this manuscript. The authors declare that they have no competing interests.RK, EK and DLB designed the study, led the analyses, and developed the manuscript. CD and CW oversaw the TB estimation and contributed to interpretation and writing. EK implemented the malaria estimation, with advise from RS and BN who also contributed to interpretation and writing. RL and JMGC contributed to estimating and interpreting the effects of ARV and to writing. JC and BS conceptualized and contributed to interpretation and writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/109/prepubExample calculation of lives saved by ITNs. (a) basic calculation; (b) additional step to correct for 'over 100% child coverage' in selected countries.Click here for file


The epidermal growth factor receptor (EGFR/HER1) and its downstream signaling events are important for regulating cell growth and behavior in many epithelial tumors types. In breast cancer, the role of EGFR is complex and appears to vary relative to important clinical features including estrogen receptor (ER) status. To investigate EGFR-signaling using a genomics approach, several breast basal-like and luminal epithelial cell lines were examined for sensitivity to EGFR inhibitors. An EGFR-associated gene expression signature was identified in the basal-like SUM102 cell line and was used to classify a diverse set of sporadic breast tumors.In vitro, breast basal-like cell lines were more sensitive to EGFR inhibitors compared to luminal cell lines. The basal-like tumor derived lines were also the most sensitive to carboplatin, which acted synergistically with cetuximab. An EGFR-associated signature was developed in vitro, evaluated on 241 primary breast tumors; three distinct clusters of genes were evident in vivo, two of which were predictive of poor patient outcomes. These EGFR-associated poor prognostic signatures were highly expressed in almost all basal-like tumors and many of the HER2+/ER- and Luminal B tumors.In vivo, the EGFR-signatures were of prognostic value, were associated with tumor subtype, and were uniquely associated with the high expression of distinct EGFR-RAS-MEK pathway genes.These results suggest that breast basal-like cell lines are sensitive to EGFR inhibitors and carboplatin, and this combination may also be synergistic.  The epidermal growth factor receptor EGFR/HER1) is a member of the human epidermal growth factor receptor (HER) family of transmembrane receptor tyrosine kinases that is linked to growth control, cell adhesion, mobility, and apoptosis  is a mem-5. Two oHigh expression of EGFR has been reported in a variety of epithelial tumors , leadingin vivo has been partially attributed to activation of this pathway downstream of EGFR, or ineffective methods of identifying those tumors that show an EGFR-dependent signature. EGF independent activation of the EGFR-pathway via the PI3K/AKT pathway may occur through either loss of PTEN or mutation/activation of PI3K, both of which have been linked to gefitinib resistance ; CI: Combination Index; SAM: Significance Analysis of Microarrays; HMEC: human mammary epithelial cell; FDR: false discovery rate; GO: gene ontology; RFS: relapse-free survival; OS: overall survival; HR: hazard ratio; 95% CI: 95% confidence interval; ICC: Intraclass correlation; PgR: progesterone receptor.KAH performed the cell line experiments, cell line and tumor data analysis, drafted the paper and helped with the design of the study. VJW, CF, MAT assisted with data analysis. CIS made initial observations of EGFR dependency of SUM102 cells and assisted with the discussion. Tumor sample collection, clinical data acquisition and interpretations were accomplished by LAC, LRS, TRH, and PSB. XH performed tumor RNA preparation and microarray experiments for tumor samples. CMP was the Principal Investigator, instigated and designed the study, and helped draft the paper.Gefitinib and carboplatin combinations in breast cancer-derived cell lines. Cells were treated for 72 h with constant ratios of the IC50 doses for both gefitinib and carboplatin. Combination Index (CI) values below one are synergistic, equal to one are additive, and greater than one are antagonistic.Click here for fileFull cluster diagram for the gene expression patterns of SUM102 cells treated with gefitinib or cetuximab.Click here for filein vivo EGFR-activation profiles clustered on the UNC tumor data set.Full cluster diagram for the Click here for fileGenes from Cluster #1–3. Genes identified from the 500 SUM102 genes clustered on the UNC tumor dataset.Click here for fileClinical data associated with each tumor sample.Click here for file


Multiplex ligation-dependent probe amplification (MLPA) is an efficient and reliable technique for gene dosage analysis. Currently MLPA can be conducted on two platforms: traditional electrophoresis-based, and FlexMAP bead-coupled. Since its introduction in 2002, MLPA has been rapidly adopted in both clinical and research situations. However, MLPA probe design is a time consuming process requiring many steps that address multiple criteria. There exist only one or two commercial software packages for traditional electrophoresis-based MLPA probe design. To our knowledge, no software is yet available that performs bead-coupled MLPA probe design.We have developed H-MAPD, a web-based tool that automates the generation and selection of probes for human genomic MLPA. The software performs physical-chemical property tests using UNAFold software, and uniqueness tests using the UCSC genome browser. H-MAPD supports both traditional electrophoresis-based assays, as well as FlexMAP bead-coupled MLPA.H-MAPD greatly reduces the efforts for human genomic MLPA probe design. The software is written in Perl-CGI, hosted on a Linux server, and is freely available to non-commercial users. Multiplex ligation-dependent probe amplification (MLPA) has proven to be an efficient and reliable technique for gene dosage analysis. This technique was first introduced in 2002 , and hasCompared to other gene dosage detection methods, including, for example, Southern blot analysis and FISH (fluorescent in situ hybridization), MLPA is high throughput, requires only small amounts of starting DNA (cf. Southern blotting), does not require cells for chromosome spreads (as in FISH) and can be used to target any genomic sequences for copy number analysis, irrespective of their size or proximity to each other. Quantitative PCR (qPCR) is a potential alternative, but its use in a multiplex assay is limited by the spectral overlap of the fluorescent dyes used , and detHowever, while kits covering multiple regions/genes of interest are available from MRC-Holland, for those regions not yet commercially available as kits, there are significant challenges in designing custom assays for MLPA. In the original description, MLPA probe sets were generated by cloning the target specific sequence into a family of M13 derived vectors that already contained the variable length fragments. With improvements in oligonucleotide synthesis, completely synthetic probe sets have been successfully used for MLPA reactions, obviating the need for the implementation of the M13 method [H-MAPD accepts one or more nucleotide sequences in FASTA format. Due to technical limitations, electrophoresis-based MLPA supports up to 45 assays per reaction (per fluorescent reporter), while bead-coupled MLPA currently supports up to 100 assays per reaction. The software sets a maximum limit of 50 and 100 sequences for electrophoresis-based and bead-coupled MLPA, respectively. To prevent server overloading, aggregate sequence lengths up to 100,000 bases are allowed . Two platforms are provided, depending on the available technology: one for traditional electrophoresis-based and one for FlexMap bead-coupled MLPA.For the electrophoresis-based MLPA platform, the probe length is increased by 4 bases for each input sequence, starting with the minimum length of ligation product specified by the user. If the user chooses to use stuffer sequences  is calculated by subtracting the length of primers, stuffers or bead tag sequence from the total probe length. A series of hybridizing sequences are generated by walking along the input sequence at 1 base steps, and extracting fragments of the desired length. The hybridizing sequences are split in the middle: the left hybridizing sequence (LHS) becomes the 3' end of the LPO and the right hybridizing sequence (RHS) becomes the 5' end of the RPO.MRC-Holland has the most experience in MLPA probe design. H-MAPD adopted most of the criteria described in MRC-Holland's probe design guidelines . The worProbe sets that meet all physical-chemical criteria  are subject to uniqueness screening. Homology search is performed for LPO and RPO using our local partial mirror of the UCSC BLAT server ,7. SinceProbe sets passing all criteria will be sorted by their scores and returned online . Depending on the size of the input sequence, results will be returned in minutes or hours.Our software applies to human genomic MLPA probe design only but will be generalized in the future to deal with other genomes. In addition, H-MAPD is currently only for the design of MLPA copy number assays, but may be generalized in the future to deal with the design of MLPA assays for other applications, such as SNP detection and methylation changes. For multiple input sequences, the stuffer and bead tag modifications will be applied to the input sequences sequentially. For example, in bead-coupled MLPA, the first bead tag will be inserted into the first input sequence and so on.The software takes into consideration most factors predicted to affect MLPA probe performance. However, there are likely to be unexpected factors that might cause problems. These factors include secondary structure of genomic DNA at the hybridization site, the presence of un-described SNP(s) at the hybridization site, sequence errors in the reference genome assembly, etc. The software performs sequence homology searches using BLAT due to its speed and excellent programming interface. Another popular sequence search tool is BLAST. Because they are implemented using different algorithms, BLAT and BLAST may not return identical results. The authors strongly recommend that users verify H-MAPD results with BLAST for independent validation.Two platforms are available for MLPA assays. The electrophoresis-based platform requires the use of capillary electrophoresis systems which are available in most institutions; however long oligonucleotides (> 150 nt) in high quality are difficult to synthesize currently. The bead-coupled platform works with half probes (LPO/RPO) less than 100 nucleotides in length, but requires the use of a Luminex system. For the electrophoresis-based platform, probe length increase is achieved either by inserting stuffer sequences between the PCR primer and hybridizing sequences, or by extending the length of hybridizing sequences. By using stuffer sequences, hybridizing sequences have identical length and therefore have a more unified Tm. Since the hybridizing sequences are short, the use of stuffer provides the potential for detecting mutations/polymorphisms that are in close proximity to each other. However, designing stuffer sequences takes time. The stuffer sequences in additional file It is a challenge to design MLPA probes that can distinguish closely related sequences in the genome. For highly similar but non-identical sequences, H-MAPD will treat them as non-specific matches and is likely to fail the probe set. One can design MLPA probes for highly related sequences by allowing multiple perfect matches to amplify the common identical fragments. Of course the result represents multiple targets, and can not be used to distinguish the highly related but different sequences. A practical solution, as indicated in the MRC-Holland probe design guidelines , is to fH-MAPD is a web-based MLPA probe design program implemented in Perl-CGI, and hosted on a Linux server. With the increasing application of MLPA in biomedical research and the lack of free probe design software, we hope that H-MAPD will become a valuable tool for automatic MLPA probe generation and selection.. Commercial users should contact the authors. Since the software utilizes the UCSC genome browser and UNAFold, commercial users also need to obtain a license for those programs.H-MAPD is freely available to non-commercial users at URL MLPA: multiplex ligation-dependent probe amplification; FISH: fluorescent in situ hybridization; UCSC: University of California at Santa Cruz; SNP: single nucleotide polymorphism; LPO: left probe oligo; RPO: right probe oligo; LHS: left hybridizing sequence; RHS: right hybridizing sequence; Tm: melting temperature; ΔG: minimum free energy change; qPCR: quantitative polymerase chain reaction; nt: nucleotide.JZ implemented the software and web interface, defined score calculation equations, and drafted the manuscript. EH initiated the project, specified the probe design requirements, and modified the manuscript.Diagram of electrophoresis-base MLPA. Two sequence-tagged half probes are annealed to adjacent sites on the genomic target sequence and ligated using a thermostable DNA ligase. The ligated probes are subsequently amplified with universal primers (one of which is fluorescently labeled) and quantified using electrophoresis. By inserting different-sized stuffer sequence between hybridizing sequence and primer sequence, or by extending the length of the hybridizing sequences, each product has a distinct size, which allows for identification by electrophoresis. The default left and right primers used in H-MAPD are GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC, respectively. These are the same primers included in the commercial MRC-Holland MLPA kits.Click here for fileDiagram of bead-coupled MLPA. Similar to electrophoresis-based MLPA, except that the stuffer sequence is replaced by a fixed-length bead tag. Identification of distinct sequences is based on association with a distinct bead. Bead tags can be inserted either between the left primer and the LHS or between the RHS and the right primer. The default left and right primers used in H-MAPD are GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC, respectively. These are the same primers included in the commercial MRC-Holland MLPA kits.Click here for fileStuffer sequences. The stuffer sequences (used in electrophoresis-based MPLA) are from different locations of the Lambda genomic sequence with minor modifications. To ensure that the union of primer and stuffer sequence itself does not fail any of the criteria, firstly all (default left primer GGGTTCCCTAAGGGTTGGA + left stuffer) sequences and (right stuffer + default right primer TCTAGATTGGATCTTGCTGGCAC) sequences were verified to be free of secondary structure at 60°C and 0.35 M Sodium concentration; secondly the maximum Tm of primer and stuffer union sequences to the human genome is verified to be less than 55°C; thirdly no self or inter-probe annealing is detected for all (left primer + left stuffer) and (right stuffer + right primer) sequences.Click here for fileFlexMAP bead tag sequences. FlexMAP bead tag sequences (used in bead-coupled MPLA) are commercially available. ΔG and maximum Tm to the human genome was calculated for (default left primer GGGTTCCCTAAGGGTTGGA + tag) and (tag + default right primer TCTAGATTGGATCTTGCTGGCAC) union sequences. Some of the tag sequences are not suitable for certain MLPA assays. For example, the tag corresponding to bead 062 or bead 071, when attached to the right primer, has a secondary structure that is significant (ΔG = -1.538 and -1.514 respectively).Click here for fileComparison of Tm calculated by RAW and UNAFold. Melting temperatures of reference sequences mentioned in the MRC-Holland MLPA probe design guidelines, were calculated using two different software, RAW and UNAFold (version 3.5), at 0.35 M Sodium concentration. Tm calculated by RAW is on average 9.1°C higher than that calculated by UNAFold with a standard deviation of 2.8°C.Click here for file