This practice guideline was developed to provide recommendations to clinicians in Ontario on the preferred standard radiotherapy fractionation schedule for the treatment of painful bone metastases.A systematic review and meta-analysis was performed and published elsewhere. The Supportive Care Guidelines Group, a multidisciplinary guideline development panel, formulated clinical recommendations based on their interpretation of the evidence. In addition to evidence from clinical trials, the panel also considered patient convenience and ease of administration of palliative radiotherapy. External review of the draft report by Ontario practitioners was obtained through a mailed survey, and final approval was obtained from the Practice Guidelines Coordinating Committee.Meta-analysis did not detect a significant difference in complete or overall pain relief between single treatment and multifraction palliative radiotherapy for bone metastases. Fifty-nine Ontario practitioners responded to the mailed survey (return rate 62%). Forty-two percent also returned written comments. Eighty-three percent of respondents agreed with the interpretation of the evidence and 75% agreed that the report should be approved as a practice guideline. Minor revisions were made based on feedback from the external reviewers and the Practice Guidelines Coordinating Committee. The Practice Guidelines Coordinating Committee approved the final practice guideline report.For adult patients with single or multiple radiographically confirmed bone metastases of any histology corresponding to painful areas in previously non-irradiated areas without pathologic fractures or spinal cord/cauda equine compression, we conclude that:Where the treatment objective is pain relief, a single 8 Gy treatment, prescribed to the appropriate target volume, is recommended as the standard dose-fractionation schedule for the treatment of symptomatic and uncomplicated bone metastases.• Several factors frequently considered in clinical practice when applying this evidence such as the effect of primary histology, anatomical site of treatment, risk of pathological fracture, soft tissue disease and cord compression, use of antiemetics, and the role of retreatment are discussed as qualifying statements.Our systematic review and meta-analysis provided high quality evidence for the key recommendation in this clinical practice guideline. Qualifying statements addressing factors that should be considered when applying this recommendation in clinical practice facilitate its clinical application. The rigorous development and approval process result in a final document that is strongly endorsed by practitioners as a practice guideline. Radiotherapy is a well-recognized, effective modality in the palliative treatment of painful bone metastases. Bone metastases are a common manifestation of distant relapse from many types of malignant tumours, especially from cancers of the lung, breast, and prostate. With the advent of effective systemic therapies and improvements in supportive care, cancer patients are expected to live longer and may suffer from metastatic disease for a considerable length of time. Many patients with bone metastases suffer from compromised mobility and performance status.optimal" with different treatment goals, one could recommend that a "preferred" dose-fractionation is one that provides pain relief without undue toxicity and is least onerous to the patient.The optimal dose-fractionation schedule for the treatment of bone metastases is unclear. Two surveys of Canadian patterns of practice found that various fractionation schedules are employed by radiation oncologists, ranging from a single large-dose fraction (e.g., 8 Gy) to a more prolonged course of 30 Gy/10 fractions over 2 weeks . While dDuring the past decade, significant clinical trial efforts have been devoted to comparing single large-dose radiation 8 Gy to 10 Gy) with multifraction regimens (five to ten fractions) . The two Gy with This provincial guideline was initiated to summarize the evidence and to provide recommendations on the preferred standard radiotherapy fractionation schedule for the treatment of painful bone metastases.This practice guideline was developed by Cancer Care Ontario's Practice Guidelines Initiative (PGI), using the methods of the Practice Guidelines Development Cycle . The praEvidence was selected and summarized by four members of the Supportive Care Guidelines Group (SCGG) and methodologists. Members of the SCGG disclosed potential conflict of interest information, reviewed the analysis of the evidence, and prepared draft recommendations. The SCGG includes palliative care physicians, medical and radiation oncologists, psychiatrists, nurses, psychologists, a chaplain, an anesthetist, a surgeon, methodologists, and administrators. After reviewing the evidence and considering issues of patient convenience and resource utilization, the SCGG reached consensus on draft recommendations. The systematic review and meta-analysis, conducted as the initial step in formulating this practice guideline, has been described elsewhere .External review by Ontario practitioners was obtained through a mailed survey consisting of items that address the quality of the draft practice guideline report and recommendations and whether the recommendations should serve as a practice guideline. The efficacy of the practitioner feedback survey process has been previously described . Final aPractitioner feedback was obtained through a mailed survey of 95 radiation oncologists across Ontario. The survey consisted of items evaluating the methods, results, and interpretive summary used to inform the draft recommendation and whether the draft recommendation should be approved as a practice guideline. Written comments were invited. Follow-up reminders were sent at two weeks (post card) and four weeks (complete package mailed again). The SCGG reviewed the results of the survey.Fifty-nine of the 95 surveys were returned (62% return rate). Key results of the practitioner feedback survey are summarized in Table The practice guideline report was circulated to PGCC members for review and approval. Eleven of the fourteen PGCC members completed and returned ballots. Ten PGCC members approved the practice guideline report as written, and one member approved the guideline and provided suggestions for consideration by the SCGG. Suggestions made were to reword the Target Population section and to clarify the second qualifying statement. The SCGG agreed with the suggestions and modified the guideline accordingly.Standard" refers to what is applicable to the majority of patients, with a preference for patient convenience and ease of administration without compromising treatment efficacy or morbidity. Our meta-analysis of all published randomized trials found no difference in pain relief between single fraction and multifraction treatments [The preferred radiotherapy dose-fractionation schedule for the palliation of uncomplicated painful bone metastases has been a controversial subject -21. The eatments . Based oAvailable evidence does not support the notion that a more durable response can be achieved with higher dose-fractionation .There is a lack of firm evidence relating the fractionation schedule to the prevention of pathologic fracture because no study evaluated the risk of pathologic fracture prior to treatment. Although the pathologic fracture rate was significantly higher after single fraction radiotherapy than after multifraction in the Dutch study , the absIt should be noted that the published studies included a heterogeneous group of patients differing in histologies, performance status, severity of pain, extent of disease, and so forth. The fact that breast, prostate, and lung cancer patients constituted the majority of trial patients implies a greater confidence in reproducing treatment results for these patients in practice. However, the evidence does not provide sufficient materials to allow a recommendation based on treatment outcomes among subgroups of different primary tumours or other patient- and tumour-related factors.The evidence reviewed does not specifically address the results of large-volume (i.e. wide-field, hemibody irradiation), single fraction treatment. Although average treatment volumes were not reported in any of the single fraction trials, a significant proportion of patients did receive treatments to the lumbar spine and pelvis ,11,13Ki. TherefoThe underlying concern for this group of patients is whether single large-dose radiation compromises subsequent tolerance to re-irradiation. Although no untoward late effects were reported by the single fraction studies with follow-up of one year or more ,11, clinWith CT/MRI (magnetic resonance imaging) diagnostic investigations becoming more routinely available, and the introduction of the CT-simulator into many departments, the extent of metastatic disease is likely to be better evaluated than in the past. In cases where lytic disease is associated with a large soft-tissue mass (e.g., in the acetabulum and adjacent pelvic bone), the desired palliative endpoint may be tumour shrinkage as well as pain control. No evidence-based recommendation can be given for this scenario.no clear guideline can be given regarding dose-fractionation of re-irradiation. A new intergroup randomized trial supported by the National Cancer Institute of Canada (NCIC) of single versus multiple fractions for re-irradiation opened in January 2004 and is expected to accrue patients from Canada, the United Kingdom, the Netherlands, and Australia [Re-irradiation may be considered in three scenarios: 1) no pain relief or pain progression after initial radiotherapy, 2) partial response with initial radiotherapy and the hope of achieving further pain reduction with more radiotherapy, and 3) partial or complete response with initial radiotherapy but subsequent recurrence of pain. The response after re-irradiation may be different for each of these scenarios. Two published studies reported response rates to re-irradiation ,27 with ustralia .No increase in acute gastrointestinal morbidity was observed with single fraction treatment compared to multiple fractions. The Canadian study showed significantly fewer vomiting episodes with single fraction treatment after prophylactic ondansetron was used in cases with treatment fields over the abdomen or pelvis [Kirkbride P: Personal communications. 2001]. Antiemetic agents should be considered as a prophylaxis, given that 30% or more of patients experienced vomiting following single or multifraction treatment in the two studies that specifically collected patient-assessed nausea/vomiting data ,10.For patients where the treatment objective is pain relief, a single 8 Gy treatment, prescribed to the appropriate target volume, is recommended as the standard dose-fractionation schedule for the treatment of symptomatic and uncomplicated bone metastases. This recommendation applies to adult patients with single or multiple radiographically confirmed bone metastases of any histology corresponding to painful areas in previously non-irradiated areas without pathologic fractures or spinal cord/cauda equina compression. It does not apply to the management of malignant primary bone tumour. The following qualifying statements are provided to support the application of the recommendation in clinical practice:Standard" refers to what is applicable to the majority of patients, with a preference for patient convenience and ease of administration and without compromising treatment efficacy or morbidity.• "• The recommendation does not apply to lesions previously irradiated, or lesions causing cord compression or pathologic fractures, because such patients were mostly excluded from clinical trials examining fractionation schedules.• Prophylactic antiemetic agents should be considered when a significant proportion of the gastrointestinal tract is in the irradiated volume.• Patients and referring physicians should be advised that repeat irradiation to the treated area may be possible.• There is insufficient evidence at this time to make a dose-fractionation recommendation for other treatment indications, such as long-term disease control for patients with solitary bone metastasis, prevention/treatment of cord compression, prevention/treatment of pathologic fractures, and treatment of soft tissue masses associated with bony disease.This practice guideline incorporates recommendations based on a systematic review, comprehensive consideration of how the evidence may be applied to clinical practice, feedback from Ontario practitioners, and input from the Practice Guidelines Coordinating Committee prior to final approval. It is strongly endorsed by practitioners for whom it was developed.ASTRO, American Society for Therapeutic Radiology and Oncology; CARO, Canadian Association of Radiation Oncologists; CT, computed tomography; Gy, gray(s); met, metastasis(es); MRI, magnetic resonance imaging; NCIC, National Cancer Institute of Canada; PGCC, Practice Guidelines Coordinating Committee; PGI, Practice Guidelines Initiative; RTOG, Radiation Therapy Oncology Group; SCGG, Supportive Care Guidelines Group.The authors declare that they have no competing interests.JW was the lead author responsible for designing and conducting the systematic review of the literature and the meta-analysis that informed the practice guideline, and for drafting and modifying the practice guideline report. JW was a member of the Supportive Care Guidelines Group and a Radiation Oncologist at the Juravinski Cancer Centre during the development of this guideline. RW reviewed all drafts of the guideline report and made major contributions to performing the meta-analysis that informed the practice guideline, and provided extensive input to the guideline as a radiation oncologist and methodologist. RW is co-Chair of the Supportive Care Guidelines Group. NL and MJ coordinated input from members of the SCGG, conducted literature searches, and drafted and edited the guideline report. MJ conducted duplicate data extraction and meta-analysis. NL updated the literature search, incorporated new data, conducted the practitioner feedback survey, and coordinated approval of the guideline by the Practice Guidelines Coordinating Committee. As members of the Supportive Care Guidelines Group, AB and TW provided substantial feedback on the guideline report at several points during its development, from both a radiation oncology and methodology perspective. Members of the SCGG provided feedback on all draft guideline reports.The pre-publication history for this paper can be accessed here:


Recent analysis of the yeast gene network shows that most genes have few inputs, indicating that enumerative gene reconstruction methods are both useful and computationally feasible. A simple enumerative reconstruction method based on a discrete dynamical system model is used to study how microarray experiments involving modulated global perturbations can be designed to obtain reasonably accurate reconstructions. The method is tested on artificial gene networks with biologically realistic in/out degree characteristics.It was found that a relatively small number of perturbations significantly improve inference accuracy, particularly for low-order inputs of one or two genes. The perturbations themselves should alter the expression level of approximately 50–60% of the genes in the network.Time-series obtained from perturbations are a common form of expression data. This study illustrates how gene networks can be significantly reconstructed from such time-series while requiring only a relatively small number of calibrated perturbations, even for large networks, thus reducing experimental costs. This process is expected to lead to novel classes of drug based on a network approach to cellular dynamics.Recent technological advances have led to an explosive growth in high-throughput genomic and proteomic data such as DNA microarrays. The rapid growth in available data has led in turn to a need for novel quantitive methods for analysis. As a consequence of this need, the reconstruction of gene network architectures from DNA microarray expression data has become a major goal in the field of systems biology. An increased understanding of the network architectures and their respective dynamics will enable novel approaches to disease treatments by allowing us, for example, to identify drug targets Frequently, the gene expression data itself is derived from perturbation experiments such as stress conditions, temperature shifts, and chemical treatments; for example, the widely used yeast cell-cycle datasets of Cho  and SpelO(log(n)) experiments (input/output pairs) might be required for n genes, but that to infer these relationships requires the use of computationally costly enumeration methods.A range of computational and mathematical techniques have been adopted in the effort to find a successful gene network reconstruction technique. Reconstruction methods often have to negotiate a tradeoff between intensive (often intractable) computations, and having to perform a large number of costly experiments. Certain progress can be achieved by making simplifications, such as imposing a limit on the number of inputs to each gene, or making steady state assumptions about the system ,4. Some n ~ 100), and still tractable for large networks (n ~ 1000), though this would require some parallelisation. The global perturbations themselves are simulated by changing the state of each gene at random. A perturbation intensity measure q, defines the probability that each gene will change state (see Methods).In this paper, we propose to explore the issue of how perturbation microarray experiments might be designed, and to suggest how such experiments might be optimised so as to maximize inference capability. Logical gene network models have previously been used to investigate gene network robustness , perturbsensitivity  was measured against P, the number of additional perturbations. Figure P).A discrete dynamical model was used to generate time series data from random networks (see Methods). To measure the effect of adding perturbations on inference ability, inference i which has one or more negative inputs, as well as having default value OFF. Since the discrete dynamics for this gene will be the same as if it had no inputs at all (i.e. zero gene expression for t > 0), the presence of the inputs is impossible to infer. This underestimation effect is clear in table Yi|, see Methods) with the actual solution sizes (i.e. the indegree distribution), and shows that the method is only able to produce roughly half the number of one and two input solution sets that actually exist.It should be noted that the algorithm tends to underestimate the number of inputs a gene may have. This is to be expected in genes for which dynamics cannot be informative: for example, consider a gene P can be explained at least partially, in the following way. Since the time series are discrete, many of the genes may have identical behaviour over time despite having different inputs (i.e. si(t) = sj(t) for two different genes i and j). If we define a "concatenated" time series vector  for gene i, and then map each gene i onto Si, we obtain a many-to-one mapping. As we increase the number of perturbations, we might expect the number of distinct time series also to increase. We define a simple measure to quantify this mapping, M = n'/N where n' is the number of distinct vectors Si, and N is the number of genes. The maximum value of M = 1 indicates that the mapping of genes to time series is one-to-one, whereas lower values indicate degenerate mappings. The manner in which M increases with the number of perturbations is shown in figure The increase in sensitivity with N, and the perturbation intensity parameter q .The experiments described above were repeated to consider variations in two other parameters: the network size P* required to reach a given high accuracy criterion was measured for different values of the network size N. The high accuracy criterion was defined as average sensitivity = 0.95 for one-input solution sets . To find P*, we first find the number of perturbations P+, such that average sensitivity P+ ≥ 0.95, and average sensitivity (P+ - 1) < 0.95. If average sensitivity P+ > 0.95, we use simple linear interpolation to find the  value of P* between P+ and (P+ - 1) for which average sensitivity = 0.95.To consider the first case, the minimum number of perturbations P* are shown in figure log(N) against P* (logarithms used are base 10). A least squares best fit gives P* ≃ 1.75 log(N) + 7.02, which, for N = 1000, gives P* ≃ 12.26. In order to obtain a measure of variance for P*, we would need to calculate P*-equivalent values for many individual networks separately, then consolidate these values to obtain the relevant statistics. However, because it was only feasible to consider medium-sized networks (20 ≤ N ≤ 70), and for any such network we often find only a small number of one-input solution sets, such statistics were found to be unreliable.The resulting values for q. Figure q, and figure q < 0.6. Together with a corresponding minimisation of the standard deviation in this interval (though it still remains fairly high in absolute terms), these results suggest that perturbation intensity should be close to this range to optimise inference accuracy.The second case (varying perturbation intensity) suggests an optimal range for P, and b) perturbation intensity, q.A recent analysis of the yeast genetic network has shown that 93% of genes are regulated by between 1 and 4 genes . This suN. These relatively modest experimental requirements would presumably imply lower experimental costs. The results also suggest that the perturbations should be calibrated (by changing stress intensity, for example), so as to alter the expression levels of approximately half the genes in each experiment. Generating perturbations which alter the expression level of half the genes at random may be difficult to achieve in practice, though experiments can be designed to come as close to this goal as possible. Even in the absence of optimal perturbations, we hope the simulation approach described here will still serve as a useful tool for planning experiments.The inference method itself is most useful for low order inputs, with inference accuracy maximized for predicted single input genes. More accurate methods have been proposed, though these generally require a much larger number of experiments ,15. MethN genes, the state of each gene si (i = 1, .., N) is represented by the binary values 0(OFF) and 1(ON). Additionally, each gene is assigned a default ON/OFF state θi ∈ {0, 1}. The gene interactions are described by an (N × N) matrix C, composed of elements Cij ∈ {-1, 0, +1}, representing the positive(+1), zero(0) or negative(-1) influence of gene j on gene i. State transitions are calculated as follows:For a system of ith gene at the next timestep, si(t + 1), is therefore determined by the balance of positive versus negative inputs which are ON at the previous timestep t. If the balance is positive, then ui(t) > 0 and the next state will be 1(ON). Similarly, if the balance is negative, then ui(t) < 0 and the next state will be 0(OFF). If ui(t) = 0 , then the default value θi determines the next state. This default value needs to be given a priori, and for the purpose of this study will be random.The state of the s(t) and the default vector θ, the problem is to find the necessary model parameters (C) which will reproduce these dynamics. Specifically, a system initialised at s(0) should reproduce the given dynamics s(t) for t > 0. Note that multiple s(t) expression patterns may be defined, which will be denoted as sr(t) for r = 0, .., P, corresponding to time series with different initial states sr(0). Our problem is to find at least one interaction matrix that will reproduce all given dynamics sr(t). The problem of finding an appropriate matrix C may be broken up into N sub-problems, since in this system, each gene i may be solved independently from the others. More precisely, the inputs to gene i (i.e. Ci, the ith row of C), can be found independently of the other genes. This reduces the search space from  down to O(N3N). Each input zi to gene i is represented as an ordered pair (j, g), j ∈ {1, .., N}, g ∈ {± 1}, indicating an input from gene j of sign g. A solution y(i) for gene i is a set of K inputs  (with y(i) = φ if K = 0). For K inputs there are  solutions to evaluate. Starting with K = 0 (no inputs), we progress up to a maximum of K = 4, exhaustively evaluating all possible solutions for each K. However, making a parsimony assumption, if solutions are found for some Ks < 4, the method no longer evaluates for K >Ks. Note that the method does not stop as soon as a solution is found, but evaluates all possible solutions for Ks. The failure rate (percentage of genes for which no solution was found for K ≤ 4) never exceeded 3% of the genes in any single network for which reconstruction was attempted.Assuming we are given the state dynamics s0(t) is generated by setting s0(0) = θ. The other time series sr(t), r > 0 are obtained from initial conditions which are perturbations of θ, and correspond to standard experiments such as stress conditions, or chemical treatments. Since, experimental perturbations can usually be modulated in intensity (for example, a temperature shift), this was represented using modulated artificial perturbations. Perturbed initial states sr(0) were generated by randomly changing each state s0(0) with probability q. This means that, on average, there will be qN random state differences between each perturbed initial state sr(0), and θ.The control time series y1(i), y2(i), ... are found for gene i, these are consolidated into a solution set, Yi = ∪l{yl(i)}. Note that some information about the solutions has been lost using this approach. For example, a solution set  obtained from a single two-input (K = 2) solution: , may be equal to another solution set  resulting from two single-input (K = 1) solutions:  with  and .Assuming one or more solutions sensitivity and specificity. Here, accuracy was measured using sensitivity, defined as true positives / . The relatively large number of true negatives, makes specificity, defined as true negatives / , an uninformative statistic. Here, true positives are members of the solution set Yi which are also true inputs , and false negatives are those true inputs which are not members of the solution set Yi.However, this consolidation is convenient in that the solution set is easily compared with the known network structures using standard accuracy measures such as N ≤ 70). Inferences on R random networks (each with N genes), will produce approximately RN sensitivity measurements (slightly fewer due to the nonzero failure rate).Accuracy statistics were gathered from inferences performed on a large number of medium-sized random networks (20 ≤ k follows pk ~ Cineβk -with β ~ 0.45, whereas the distribution for outdegree follows pk ~ Coutkτ-, with τ ~ 1 (Cin, Cout constants) [k is given by:It appears to be the case in gene networks that indegree follows an exponential distribution, whereas outdegree appears to follow a scale-free distribution. More specifically, for the yeast network, the probability distribution for indegree nstants) . Here, anstants)  were crenstants) . The exppk = (1 - eβ-)eβk-,β = 0.45 is a constant. Similarly, the power law distribution  for outpk = Ckτ -eγk-,C, γ, and τ = 1 are constants. Since the algorithm begins by generating in/out-degree pairs for each node, we require equal means for both indegree (<kin >) and outdegree (<kout >). Following [where ollowing , we obtaβ is given, we obtain a value <kin > = 1.76, and fit the free parameter γ = 0.436 to obtain <kout > = <kin > Since the resulting networks are unweighted, non-zero weights (Cij ∈ {-1, +1}) are assigned at random with probability 0.5, as in [Since 5, as in . It shouTM devised and implemented the experiments and drafted the manuscript. RS and AP supervised the project. All authors read and approved the final manuscript.


Medical training is increasingly occurring in the ambulatory setting for final year medical students and residents. This study looks to identify if gender, school, level of training, or speciality affects learner's  preferred site characteristics and preceptor behaviours for learning in the ambulatory setting.All final year medical students and residents at the five medical schools in Ontario (N = 3471) were surveyed about the site characteristics and preceptor behaviours most enhancing their learning in the ambulatory setting. Preferred site characteristics and preceptor behaviours were rank ordered. Factor analysis grouped the site characteristics and preceptor behaviours into themes which were then correlated with gender, school, level of training, and speciality.Having an adequate number and variety of patients while being supervised by enthusiastic preceptors who give feedback and are willing to discuss their reasoning processes and delegate responsibility are site characteristics and preceptor behaviours valued by almost all learners. Some teaching strategies recently suggested to improve efficiency in the ambulatory teaching setting, such as structuring the interview for the student and teaching and reviewing the case in front of the patient, were found not to be valued by learners. There was a striking degree of similarity in what was valued by all learners but there were also some educationally significant differences, particularly between learners at different levels and in different specialities. Key findings between the different levels include preceptor interaction being most important for medical students as opposed to residents who most value issues pertaining to patient logistics. Learning resources are less valued early and late in training. Teaching and having the case reviewed in front of the patient becomes increasingly less valued as learners advance in their training. As one approaches the end of ones' training office management instruction becomes increasingly valued. Differences between specialities pertain most to the type of practice residents will ultimately end up in .Preceptors need to be aware of, and make efforts to provide, teaching strategies such as feedback and discussing clinical reasoning, that learners have identified as being helpful for learning. If strategies identified as not being valued for learning, such as teaching in front of the patient, must continue it will be important to explore the barriers they present to learning. Although what all learners want from their preceptors and clinic settings to enhance their learning is remarkably similar, being aware of the educationally significant differences, particularly for learners at different levels and in different specialities, will enhance teaching in the ambulatory setting. Since learning is best done contextually ,2 it is An article by Kernan  outlinedWe surveyed all final year medical students and residents in Ontario about the site characteristics and preceptor behaviours that they find most enhance learning in the ambulatory setting and determined if these were related to demographic factors, level of training or residency program. Implications for teaching in the ambulatory setting are explored based on these results.All medical students (n = 532) and residents (n = 2939) at the five medical schools in Ontario were surveyed using a four part questionnaire which collected information on demographics, preferred site characteristics, preferred preceptor behaviours, and approaches to learning and perceptions of learning climate. Questions for the site characteristics and preceptor behaviours included previously validated questions -15 and qStudents rated 24 site characteristics and 38 preceptor behaviours on a Likert scale from 1 (very important for learning) to 5  or D . Within each section they indicated the five most important and 5 most unimportant or detrimental items for learning. A section for general comments was included at the end of the survey. The survey was piloted with a group of Queen's University residents and final year medical students checking for ambiguity and content. Ethical approval was granted by the Queen's University General Research Ethics Board. To ensure privacy for their students schools requested that the questionnaires be addressed by their own undergraduate and postgraduate offices. Coded questionnaires were thus sent with student's names and bulk mailed to the undergraduate and postgraduate medical schools who then addressed and forwarded the questionnaires to their final year medical students and residents. Entry into a draw for a Personal Digital Assistant or equivalent monetary prize was offered for completed surveys. Non-responders were identified by a lack of a returned coded questionnaire. Two subsequent mailings were sent to the non-respondents through their schools' undergraduate or postgraduate office. In addition an email reminder was sent to everyone between the second and third mailing.Data were analyzed using SPSS for Windows, version 11.0 . A systeSurvey response was 48% 1642/3430). Of these 44 had not worked in an ambulatory setting and so were eliminated from further analysis (N = 1598). The demographics of the five medical schools are listed in Table 642/3430.The rank ordering of site characteristics and preceptor behaviours, including missing data and number judging an item to not only be unhelpful but detrimental for learning are shown in Tables Six factors, accounting for 55 % of the variance for the 24 site characteristics, and 7 factors, accounting for 54% of the variance for the 38 preceptor behaviours, were identified Tables . Labels Logistic regression analysis of the independent variables revealed striking similarities, but some significant differences, in valued site characteristics and preceptor behaviours for male and female students and those in different schools, at different levels of training and in different residencies Figures ,5,6,7,8.Male and female residents rank ordered all site characteristics and preceptor behaviours identically. Women ranked all factors, with the exception of teaching in the patient's presence, higher than men, usually significantly so Figures ,2.Across schools the rank ordering of factors was identical with the exception of Toronto and Ottawa ranking office management instruction higher than the other schools. Toronto valued six factors significantly more than the other schools, Queen's and Western ranked three and two items respectively significantly less than the other schools Figures ,4.Across levels the only difference in rank ordering was clerks ranking preceptor interaction as the most important site characteristic whereas all other groups ranked patient logistics as most important. Those at the beginning and end of their training valued having learning resources available less than all other levels. Clerks were most different from all the other levels in what they valued or did not value (indicated by the number of * and # for this group) Figures ,6. SubanThe ambulatory teaching site characteristics most valued by clerks and residents are having an adequate number and variety of patients while being supervised by enthusiastic and available preceptors. These characteristics have been identified before and are well summarized by Bowen and Irby . Little Computer resources were more valued than books, likely reflecting a generation of learners who are comfortable accessing electronic information. Proximity of the clinic to university campus was unimportant. In contrast to other studies -24 we ford year medical students to also value these preceptor behaviours[Valued preceptor behaviours identified in this study are feedback by enthusiastic, open preceptors who are willing to discuss their reasoning processes and delegate responsibility. Recent studies report 3ehaviours,27. Leskehaviours suggest We have confirmed the value of feedback found in most studies ,28-31  most valuing office management and health care system interaction instruction.Strengths of this study are the large multi-institutional sample size (n = 1642) encompassing students at multiple levels in all specialties. The response rate (48%) limits the external validity of the result. A confounding factor within the level of training data set may be the variability in residency lengths as suggested by the subanalysis of the PGY2 data. Rather than years from graduation from medical school what seems to influence valued site characteristics and preceptor behaviours more are years from independent practice."Software"  is valued more than "hardware" (clinic set-up, learning resources). All learners value the above preceptor behaviours; most do not value, and a significant number consider detrimental, having the structure of the patient encounter dictated to them and having the patient present during review and teaching. Future work is needed to explain why learners do not value these practices. Learners at all levels and in all specialties are strikingly similar in what they value from their preceptors and clinic sites for their learning. There are some differences between levels and residencies however that require consideration when teaching these different groups. Educationally significant differences within levels include preceptor interaction being paramount for medical students; patient logistics (adequate number and variety of patients seen independently and in follow-up) being second. The reverse is true for residents. Proportioning time accordingly deserves attention. The more senior the learner the more being taught or having the case reviewed in the patients' presence is not valued. Sensitivity to the patient-learner relationship is required if these practices are utilized but particularly so for more senior learners. Finally relevance not surprisingly dictates importance. Office management instruction is valued by those at the end of their training and those primarily in office-based specialties. Similarly office-based specialties appreciate instruction in health care system interaction. This study identifies preceptor behaviours and site characteristics valued by medical students and residents for their learning in the ambulatory setting. Further studies are needed to determine the effect of providing these valued site characteristics and preceptor behaviours on learning outcomes.None declared.KS conceived of the study, prepared the manuscript and participated in the conceptual planning and design of the study and data interpretation. JK, DD, and MG participated in the conceptual planning and design of the study, statistical analysis and data interpretation and manuscript revision. SV and RB contributed to the design of the study and manuscript revision. CK contributed to the design of the study. All authors read and approved the final manuscript. RS participated in the statistical analysis.The pre-publication history for this paper can be accessed here:


Plasmodium chabaudi, we experimentally tested whether immune pressure promotes the evolution of more virulent pathogens by evolving parasite lines in immunized and nonimmunized (“naïve”) mice using serial passage. We found that parasite lines evolved in immunized mice became more virulent to both naïve and immune mice than lines evolved in naïve mice. When these evolved lines were transmitted through mosquitoes, there was a general reduction in virulence across all lines. However, the immune-selected lines remained more virulent to naïve mice than the naïve-selected lines, though not to immunized mice. Thus, immune selection accelerated the rate of virulence evolution, rendering parasites more dangerous to naïve hosts. These results argue for further consideration of the evolutionary consequences for pathogen virulence of vaccination.Evolutionary models predict that host immunity will shape the evolution of parasite virulence. While some assumptions of these models have been tested, the actual evolutionary outcome of immune selection on virulence has not. Using the mouse malaria model,  Immunisation against malaria can promote the evolution of more virulent pathogens in mice, making non-immunised mice more vulnerable -- a result with important implications for the development of malaria vaccines Genetic variation in pathogen virulence (harm to the host) has been found whenever it has been looked for. A considerable body of theory, based on the transmission consequences of virulence, has been developed to predict how natural selection will act on this genetic variation and how it will shape virulence levels in natural populations of disease-causing organisms . For insAs well as altering between-host selection pressures on virulence, host immunity can alter the nature of inhost selection. Different directions of virulence evolution are expected depending on the details of inhost competition among parasites e.g., . UnfortuOne barrier to testing theoretical models of virulence evolution is that the models typically predict the outcome at evolutionary and epidemiological equilibrium. New equi-libria may or may not take a long time to reach, but will in any case depend on the dynamics of the host population and the environmental conditions under which transmission occurs: this means that experimental evolution to new equilibria will be hard to study in the laboratory for medically relevant pathogens. However, the short-term consequences for virulence evolution, which are at least as important to public health policy as the long-term consequences, may be more tractable. This is especially true for diseases for which animal models are available.Plasmodium chabaudi, in laboratory mice. We evolved multiple lines of P. chabaudi in immunized and naïve mice by repeated serial passage of blood-stage parasites  starting from two different starting populations. After 20 passages, the lines had evolved sufficiently to make comparisons between the immune-selected lines (I-lines) and naïve-selected lines (N-lines) for virulence and transmissibility.In this study, we begin the empirical effort to determine the likely direction of immune-mediated virulence evolution by performing experimental evolution of the rodent malaria parasite, We found that both the I-lines and N-lines evolved to become more virulent than their ancestral populations, but the I-lines became even more virulent than the N-lines A. This hp > 0.10 by 2-tailed Fisher's Exact test, zero mortality in immunized mice), and all but one of the deaths occurred after the day of transfer. The most likely explanation is that immunity generated more intense selection by killing a greater proportion of the parasite population up until the point of transfer  for (i) parasitemia on day 4, (ii) parasitemia on day 6 or 7, (iii) the increase in parasitemia from day 4 to day 6 or 7, and (iv) maximum parasitemia, with one exception: maximum parasitemia was significantly higher in I-lines than N-lines derived from unadapted ancestors when measured in immunized mice, and this only in one of the two replicate experiments (23% versus 6.9% parasitemia, p < 0.001). Thus, there is little evidence to suggest that the increased virulence was due to a higher asexual multiplication rate  in those parasites that successfully made it into the syringe. Our data demonstrate that immunity acts as a powerful and upward inhost selective force on virulence, but the precise mechanism awaits further study.But why would selection favor more virulent parasites? Our previous studies have consistently shown that peak parasite densities in the acute phase are positively correlated to the level of virulence that they generate . We therp > 0.05). Gametocyte densities are a good predictor of transmission probability in P. chabaudi and other Plasmodium species . Alternatively, virulence reduction may be due to the deterministic forces of selection against virulent variants that have lost or reduced the ability to transmit through mosquitoes : the potAny of these mechanisms could explain the loss of virulence during mosquito transmission, but none are sufficient to explain why the I-lines were more virulent than the N-lines in naïve mice both before and after mosquito transmission. Thus, more than one distinct underlying mechanism probably explains the virulence differences observed here, such as differences in intrinsic virulence properties and differences in levels of antigenic diversity within the lines. Identifying the mechanisms, any links between them, and their relative roles in determining parasite survival in naïve versus immunized hosts are of key importance in understanding virulence evolution and immunoepidemiology of malaria in the field.P. falciparum at least , most had no replication of lines within selection treatment, and some used just a single selection step. Nevertheless, some tentative conclusions may be drawn. Comparisons of selected and ancestral parasites have been made after three different forms of immune manipulation: (i) down-regulation of immunity by removal of the spleen prior to infection, (ii) up-regulation of immunity by transfer of immune serum at the beginning of infection, and (iii) up-regulation of immunity by infection, sometimes with subcurative drug treatment in order to establish a chronic infection. In the first two, parasites were selected from the primary wave of parasitemia, as in our experiment, whereas in the third, selected parasites were isolated from relapses much later in the infection (40–150 d PI). Parasite lines passaged through splenectomized hosts often lose the ability to bind to host endothelial cells (cytoadherence) in the microvasculature of the deep tissues and therefore the ability to avoid being passaged through the spleen , the priat least , this coat least . Importaat least . If our However, the second form of immune selection—passage of acute-phase parasites from hosts injected with antiserum at the beginning of the infection—yielded parasites with lower virulence to naïve mice than their ancestors in one study , althougOur data demonstrate that host immunity can increase the potency of inhost selection for higher virulence in malaria. Whether our results generalize to other immunization protocols, parasite clones, parasite species, host genotypes, repeated mosquito passage, and so on requires extensive further experimentation. But, coupled with the malaria parasite's famous ability to rapidly adapt to novel conditions in the laboratory (see above) and to variant-specific vaccine pressure  and drug5 parasites from a donor mouse that had been infected 7 d previously. Day 7 PI is during the period of rapid population growth, and is about 2 d prior to peak parasitemia, after which population size rapidly declines , five parasite lines (“sublines”) from each ancestral line were repeatedly passaged in mice  that were naïve to malaria infection (N-lines), and five from each ancestral line were passaged in immunized mice (I-lines, see below), forming 20 lines (“sublines”) in total. Passages involved the syringe transfer to a fresh mouse of 0.1 ml of diluted blood containing 5 × 10ines see . Parasit4 parasites of a different clone (denoted ER), followed by drug cure with 10 mg/kg of mefloquine for 4 d starting on day 5 PI. Naïve mice were injected with parasite-free media but were not drug treated. Re-infection took place on average 3 wk after the end of drug treatment (range 1.5–5 wk): as the half-life of mefloquine in mice is reported to be 18 h —one avirulent (CW-0) and one virulent (CW-A). CW-0 had been cloned by serial dilution from an isolate obtained from its natural host, the thicket rat, passages . Prior tAnopheles stephensi mosquitoes by allowing 50–100 mosquitoes aged 2–5 d to take a blood meal for 20–30 min on an anaesthetized gametocytemic mouse that had been inoculated 6–10 d previously, i.e., prior to the peak of infection. Then, 11–12 d later these mosquitoes—typically 10–20 of them infected as assessed by random surveys of oocyst prevalence—were allowed to feed back onto anaesthetized naïve mice. After 7–10 d, the blood from these sporozoite-infected mice was harvested and stored in liquid nitrogen. These aliquots were used to initiate blood infections in naïve mice that were then used as donors of asexual parasites to mice involved in the posttransmission experiments. As the lines were transmitted through mosquitoes noncontemporaneously, and involved typically one mouse per subline, comparisons among the lines for infectivity to mosquitoes were not made during these transmission exercises.All the lines, including the ancestral lines, were transmitted once through After 18 passages, the pretransmission lines were evaluated in two replicate experimental blocks in naïve (generations 19 and 21) and immunized mice (generations 20 and 22). Ancestral lines were only evaluated in generations 21 and 22. This set of trials was denoted the “pretransmission experiments.” In a separate set of experiments, the “posttransmission experiments,” the mosquito-transmitted lines were compared with each other, as well as with the nontransmitted ancestral lines in two replicate experimental blocks in both naïve (generations 23 and 24) and immunized mice (generations 25 and 26). In both these experiments, across both blocks, ten mice were used for each of the four selection groups (two per subline), and five mice were used per ancestral line. Red blood cell density was measured every 1 or 2 d until day 18 PI by flow cytometry (Coulter Electronics, Luton, United Kingdom), and the minimum density reached was taken as a measure of virulence. Liveweight of the mouse was also recorded every 1–2 d. During the pretransmission experiments (generations 19–22), parasitemia and gametocytemia  were evaluated from Giemsa-stained thin blood smears every 2 d from day 4 PI until day 18 PI, and then four more times until day 43 PI. Total lifetime transmission potential was measured as the average gametocytemia throughout the infection from day 4 to day 18 PI.Statistical analyses were performed separately for the pretransmission and posttransmission experiments as these were carried out at different times. The virulence measure used for the final analysis was minimum red blood cell density, though other measures of virulence were also analyzed (unpublished data). Since selection treatment was replicated on sublines, thus making subline the independent experimental unit, the means of mice within sublines were first calculated. These were then analyzed for the effects of immune environment on selection response by fitting a linear model to these data with factors for selection line , ancestral population (CW-0, CW-A), and an interaction between these two factors. Thus, statistical tests of differences between the selection lines and other factors in the model were made using t-tests, with the variance for subline means as the residual. An alternative model fitted to data on individual mice (rather than means of sublines) that incorporated subline as a random effect was found to be unsatisfactory because in some treatment groups, the model did not converge and estimates of the subline variance were highly variable between groups. To determine the effects of mosquito transmission on the line differences in virulence, a further analysis was performed on the combined data from the pretransmission and posttransmission experiments fitting a fixed effect factor of line-within-experiment in the statistical model (seven levels—three lines for the pretransmission experiment and four for the posttransmission experiment). These analyses were carried out separately for each of the four immune-treatment-by-ancestral-line groups. Since the pretransmission ancestral line was included in both the pretransmission and posttransmission experiments, the effect of mosquito transmission (and its standard error) on the N-lines and I-lines, which was not measured directly (i.e., in a single experiment), could be estimated by reference to this line. For example, the effect of mosquito transmission in the N-lines was estimated from the difference between the N-lines and their pretransmission ancestral line in the pretransmission experiment minus the analogous contrast in the posttransmission experiment. This was done using the method of linear contrasts provided for in the SAS GLM procedure (SAS 1990). The effect of mosquito transmission on the difference between the I-lines and N-lines was similarly calculated but without reference to the pretransmission ancestral line. The effect of mosquito transmission on the ancestral lines was estimated from the direct comparison available from only the posttransmission experiment data.


Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 × C57BL/6) model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE. Several strains of gene-targeted mice develop systemic lupus erythematosus (SLE). Analysis of these strains demonstrates that the genetic background profoundly influences the development of autoimmunity Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterised by the production of autoantibodies (auto-Abs) against a wide spectrum of self-antigens, mainly from subcellular compartments, especially the cell nucleus. Genetic predisposition is an important contributor to susceptibility to SLE in both humans and animals . Genes ilpr/lpr strain, which carries the lpr mutation of the FAS receptor gene; and the BXSB strain, which carries the Y chromosome autoimmune accelerator (Yaa) gene ; the MRL/Mp aa) gene . Extensiaa) gene .lpr mutation, none of the genetic contributions to disease in the three well-documented murine SLE strains have yet been fully resolved at the molecular or protein level. Thus, targeted genetic disruption of candidate genes encoding proteins of the immune system has been extensively used to examine their role in immune regulation. However, the most surprising result of this powerful approach has been the high frequency with which such mutations have been associated with an autoimmune phenotype. In this regard, it is of note that hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, are spontaneously predisposed to development of humoral autoimmunity with low levels of glomerulonephritis (Apcs). The Apcs-deficient mice (Apcs−/−) were chosen as an example of a gene-targeted strain previously reported to develop a lupus-like disease on the hybrid genetic background (129 × C57BL/6); autoimmunity in Apcs−/− mice persists even after backcrossing the mutated gene onto C57BL/6  and the severity of the renal pathology were not different between the two experimental groups. In view of the possibility of an association between the fixed 129-derived segment flanking the mutated Apcs gene and the autoimmune traits observed, the genome-wide linkage analysis of the two experimental cohorts was carried out separately.To investigate the genetic basis of the lupus-like disease observed in the (129 × C57BL/6) hybrid mice, we generated two large cohorts of (129 × C57BL/6)F2 animals, one carrying a mutation in the Apcs−/− mice as this region was of fixed 129 origin. Chromosomes where linkages were present only in one of the two cohorts are shown in Mice were genotyped with 143 microsatellite markers distributed throughout the autosomes such that 98% of the genes were within 20 cM of an informative marker. A summary of the genome-wide linkage analysis for each of the disease traits measured is shown in Apcs gene, anti-dsDNA or anti-ssDNA Ab production was also linked to other segments on Chromosome 1, indicating a more complex genetic contribution from the 129 mouse strain.The quantitative trait linkage (QTL) analysis identified several intervals on Chromosome 1 with linkage to disease serological markers, and these regions were all derived from the 129 mouse strain see  and 5. IApcs−/− mice were caused by a gene(s) within the fixed 129 region surrounding the mutated Apcs gene, rather than caused by the mutated Apcs gene itself. We compared the levels of these auto-Abs between all (129 × C57BL/6)F2.Apcs−/− mice and a group of 33 wild-type mice that were selected for being homozygous 129 in the region of Chromosome 1 between microsatellites D1Mit105 and D1Mit 223 (80–106 cM) . The C57BL/6 strain has previously been reported to be one of the murine strains defined as low Apcs producers (Apcs coding region in both strains failed to identify any coding sequence polymorphisms in the Apcs gene (data not shown), indicating that a structural variant of the protein is unlikely to be the explanation for our findings. This is consistent with a previous report by Apcs coding sequence differences amongst several autoimmune and nonautoimmune murine strains.Guided by these observations, we investigated whether the increased levels of ANA and anti-chromatin Ab observed in the –106 cM) A–6D. In roducers . In addiIn addition to the 129-derived segments, in both cohorts the C57BL/6 strain contributed to the autoimmune traits with one major susceptibility locus on Chromosome 3. A genomic region between D3Mit40 and D3Mit13, with an estimated peak at position approximately 51 cM, showed a significant linkage to ANA production and weaker linkages to anti-ssDNA and anti-chromatin production see . The higp = 0.0013), and one on Chromosome 17 between D17Mit100 (11.7 cM) and D17Mit216 (29.4 cM) from the C57BL/6 strain (LOD 1.3, p = 0.049 and LOD 1.67, p = 0.021 in the wild-type and Apcs−/− mice, respectively). Histological evidence of glomerulonephritis was only found in approximately 20% of the mice in each cohort, which reduces the power of the QTL analysis for this disease trait.In contrast to these strong associations with disease serological markers, the QTL analysis identified only two potential linkages to glomerulonephritis: one in the wild-type mice on Chromosome 7 across a 10 cM region between D7Mit246 (15 cM) and D7Mit145 (26.5 cM) of 129 origin (LOD 2.86, We generated a C57BL/6 congenic line carrying the telomeric region of Chromosome 1 from the 129 mouse strain, in order to dissect the complex polygenic disease phenotype of the (C57BL/6 × 129/Sv)F2 hybrid strain into its individual genetic components. The 129 interval was backcrossed seven times onto C57BL/6, and at each generation the presence or absence of the Chromosome 1 interval was determined with several microsatellite markers. Each backcrossed generation was screened with more than three markers per chromosome to facilitate the removal of unselected 129 genomic regions. At the end of the backcrossing, the 129-derived Chromosome 1 interval in the congenic mice extended from microsatellite marker D1Mit105 to D1Mit223 (80–106 cM), which encompasses the most important 129 interval identified by the linkage studies in the (C57BL/6 × 129/Sv)F2 mice.Apcs−/− mice backcrossed onto C57BL/6 for ten generations (C57BL/6.Apcs−/−) and C57BL/6 controls, were monitored for the presence of lupus. In the C57BL/6.Apcs−/− mice, the 129 genome around the Apcs locus was mapped as a stretch of approximately 17 cM, positioned from 87.9 cM (D1Mit15) to 105 cM (D1Mit17). Thus, the congenic line carried a similar 129 region (80–106 cM) to the one present in the C57BL/6.Apcs−/− mice (87.9–105 cM). At 1 y of age, all animals were sacrificed, the auto-Abs assessed, and the renal histology examined. The results of this analysis are shown in Apcs−/− compared to the wild-type C57BL/6 controls. However, the C57BL/6.129(D1Mit105–223) animals also expressed high levels of auto-Abs, and these titres were not different from those detected in the matched congenic mice containing a null mutation of the Apcs gene. These results clearly demonstrated that epistatic interactions between 129 loci on Chromosome 1 and C57BL/6 genes were sufficient to mediate the loss of tolerance to nuclear autoantigens. However, in contrast to the serological data, the histological assessment of the kidneys showed evidence of markedly increased glomerulonephritis in the C57BL/6.Apcs−/− compared to both control groups , together with sex-matched l groups , suggestApcs gene, we demonstrated that serum amyloid P component deficiency influences the severity of glomerulonephritis, but is not the prime mover in the induction of anti-nuclear autoimmunity, contrary to our own original interpretation of our data  hybrid strain, the most common genetic background in gene-targeted mice. Although spontaneous autoimmunity has not been documented in either of the pure 129 or C57BL/6 strains, a spontaneous lupus-like phenotype has been described in (129 × C57BL/6) hybrid strains , suggestThis was confirmed by the mapping study reported here. We showed that there are multiple genetic loci contributing to the disease and these are derived from both 129 and C57BL/6 mice. We demonstrated that a 129-derived segment of Chromosome 1 was strongly linked to the expression of auto-Abs. This region is probably capable of causing the initiation of a humoral autoimmune response to nuclear antigens; however, this response does not occur in the absence of C57BL/6 genes. In support of this, we identified a C57BL/6 segment on Chromosome 3, which may interact with the 129 genes on Chromosome 1 to mediate the loss of tolerance. Interestingly, although the C57BL/6 SLE-susceptibility region on Chromosome 3 is novel, disease-modifying alleles derived from C57BL/10 and C57BL/6 strains have been mapped to a portion of Chromosome 3 close to the region identified in this study . FurtherApcs−/− mice with Chromosome 1 genetically matched controls.Taken together our results suggest a complex genetic contribution from the (129 × C57BL/6) hybrid background genome, with both enhancing as well as inhibitory loci from the 129 mouse, in addition to genes promoting autoimmunity from the C57BL/6 mice. The impact that these interacting loci may have on the lupus-like disease present in several gene-targeted animals was further assessed by comparing Sle1 F2.Apcs−/− mice. A possible explanation for this discrepancy may lie in the fact that the Apcs−/− mice analysed in the original study were generated from a limited number of founders and that this may have caused a nonrandom inheritance of the loci from the parental strains. Furthermore, the whole-genome analysis identified the 129 region surrounding the Apcs gene as the main locus contributing to the development of ANA and anti-chromatin Ab. In agreement with this, when we carried out a selective comparison between the (129 × C57BL/6)F2.Apcs−/− mice and Chromosome 1 genetically matched controls, we failed to detect any significant difference in the levels of these two auto-Abs. These findings, taken together, indicated that the phenotype associated with Apcs deficiency was caused by the presence of unaltered 129 genes from the telomeric region of Chromosome 1 operating in the C57BL/6 genomic background. Strong supportive evidence for this was provided by the analysis of the C57BL/6 mice congenic for this 129 region.The in vitro  and alsoin vitro . We haveephritis . On the Apcs gene to each disease trait. Using this approach, we demonstrated that the 129 interval on distal Chromosome 1, when transferred onto the C57BL/6 genome, a combination commonly created by backcrossing onto C57BL/6 a mutated gene located in that chromosomal region, was sufficient to mediate the production of auto-Abs. In this context, it is of note that several strains of mice with targeted mutations of genes encoded in this region have been reported to express a lupus-like illness, including mice lacking FcγRIIB F2 and 158 (129 × C57BL/6)F2.Apcs−/− female mice were produced and monitored for 1 y. Wild-type congenic C57BL/6.129(D1Mit105–223) mice were generated by backcrossing the 129 interval between microsatellites D1Mit105 and D1Mit223 (80 cM to 106 cM) onto the C57BL/6 strain. Inherited 129 regions were mapped with microsatellite markers polymorphic between 129 and C57BL/6 mice (see below). After seven generations of backcrossing, siblings were intercrossed to generate C57BL/6.129(D1Mit105–223) congenic mice homozygous for the 129 Chromosome 1 interval. Inside 129 markers at positions 81.6 cM (D1Mit159) and 105 cM (D1Mit17), respectively, and an outside C57BL/6 marker at position 74.3cM (D1Mit159) were used to further define the interval. In the C57BL/6.Apcs−/− mice (backcrossed onto C57BL/6 for ten generations), the 129 genome around the Apcs locus was mapped as a segment from 87.9 cM (D1Mit15) to 105 cM (D1Mit17). In this analysis, the inside 129 markers were at positions 93 cM (D1Mit36) and 99.7 cM (D1Mit115) and the outside C57BL/6 markers at positions 81.6 cM (D1Mit159) and 106 cM (D1Mit223). Along with 28 C57BL/6.Apcs−/−mice and 30 C57BL/6 wild-type animals, 26 C57BL/6.129(D1Mit105–223) female mice −/−were followed up to 1 y of age. Animals were maintained in specific pathogen-free conditions. All animal procedures were in accordance with institutional guidelines.All the mice were females. Wild-type C57BL/6 and 129/Sv (129S6, according to the revised nomenclature) were bred and maintained in the animal care facility at Imperial College, London, United Kingdom. (129 × C57BL/6)F1 mice were generated by intercrossing the two wild-type strains and (129 × C57BL/6)F2 mice by interbreeding the (129 × C57BL/6)F1 mice. The reported , and theCrithidia luciliae as previously described (lpr/lpr mouse), which was assigned a value of 100. For interplate comparison, serial dilutions of a positive control serum sample were included on each plate. Apcs levels were assessed by ELISA using sheep anti-mouse Apcs and rabbit anti-mouse Apcs Abs . Samples were screened at a 1:3,000 dilution, and the results were expressed in milligrams per liters, referring to a standard curve derived from an acute-phase serum with a known concentration of Apcs . Apcs−/− mouse serum was included as a negative control.Sera, collected at 6 and 12 mo of age, were assayed for the presence of auto-Abs. Levels of IgG ANA were sought by indirect immunofluorescence using Hep-2 cells, and anti-dsDNA Abs were detected by indirect immunofluorescence on escribed . Serum sescribed . SamplesAll the mice, except the few that died before the end of the experiment, were sacrificed at 1 y of age, and kidney portions were fixed in Bouin's solution and paraffin embedded, and sections were stained with periodic acid–Schiff reagent. Glomerular histology was graded in a blinded fashion as follows: grade 0, normal; grade 1, hypercellularity involving greater than 50% of the glomerular tuft in 25%–50% of glomeruli; grade 2, hypercellularity involving greater than 50% of the glomerular tuft in 50%–75% of glomeruli; grade 3, glomerular hypercellularity in greater than 75% of glomeruli or crescents in greater than 25% of glomeruli.TM version 3.0 for Windows . Non-parametric tests were applied throughout, with differences being considered significant for p values < 0.05. The Mann–Whitney test was used for comparison of two groups, whilst for analysis of three groups the Kruskal–Wallis test with Dunn's multiple comparison test was used.Non-parametric data are expressed as median with range of values in parentheses. All statistics were calculated using GraphPad Prism2 and 0.4 μM primers. Microsatellite markers were screened for size polymorphisms between 129 and C57BL/6 mice. Only primers with differences detectable on ethidium bromide-stained agarose gels or on SDS-polyacrylamide gels were used.Genotyping was carried out by PCR of genomic DNA using 143 polymorphic markers (list available on request) distributed throughout all 19 autosomes. PCRs were performed using standard reagents containing 1.5 mM MgClftp://mcbio.med.buffalo.edu/pub/MapMgr/) was used, and the two experimental groups were examined independently. Only data from mice at 12 mo of age were analysed. Log transformations of auto-Abs levels resulted in more normalised distribution and were used in QTL mapping. LOD thresholds for suggestive and significant linkages were determined by using a cohort- and trait-specific permutation test (1,000 permutations). The average threshold for suggestive, significant, and highly significant linkages were LOD ≥ 2.1 (p ≤ 7.8 × 10−3), LOD ≥ 3.6 (p ≤ 2.4 × 10−4), and LOD ≥ 5 (p ≤ 1 × 10−5), respectively (The QTL program MAPMANAGER.QTL (ectively .http://www.ncbi.nlm.nih.gov/LocusLink/) ID numbers for the genes and gene products discussed in this paper are Apcs (LocusLink ID 20219), CD35/CD21 (LocusLink ID 12902), CD55 (LocusLink ID 13136), the FAS receptor gene (LocusLink ID 14102), and FcγRII (LocusLink ID 14130).The LocusLink (


Systematic assessment of recent health research output from India, and its relation with the estimated disease burden, is not available. This information would help understand the areas in health research that need improvement in India to enhance the health of India's population.The health research output from India during 2002, which was accessible in the public domain, was assessed by searching PubMed and other internet health literature databases, and was related to the disease burden suggested by the Global Burden of Disease Study. The main outcome measures were number of health papers with abstracts in basic, clinical and public health sciences; quality-adjusted research output based on the impact factors of journals in which the papers were published; classification of papers in disease/condition categories and comparison of research output with the estimated disease burden in each category. Comparison of the health papers from India during 2002 included in PubMed was done with those from Australia during one quarter of 2002.Of the 4876 health papers from India in 2002 in PubMed, 48.4%, 47.1% and 4.4% were in basic, clinical and public health sciences, respectively. Of the 4495 papers based on original research, only 3.3% were in public health. Quality-adjusted original research output was highest for non-communicable diseases . Of the total quality-adjusted original research output, the proportions in injuries (0.7%), cardiovascular diseases (3.6%), respiratory infections (0.2%), diarrhoeal diseases (1.9%), perinatal conditions (0.4%), childhood cluster diseases (0.5%), unipolar major depression (0%), and HIV/AIDS (1.5%) were substantially lower than their proportional contribution to the disease burden in India. Human resources, health policy, health economics, and impact assessment of interventions were particularly poorly represented in public health research. The Australia-India ratio for quality-adjusted health research output per unit gross domestic product was 20 and for public health research output was 31.Good-quality public health research output from India is grossly inadequate, and strategic planning to improve it is necessary if substantial enhancement of population health were to be made possible. There is inordinately low relative research output in several diseases/conditions that cause major disease burden in India. India suffers a large proportion of the disease burden of the world, which has been estimated to be more than its 16.8% share of the world's population . One of Health research output was defined as tangible research information related to human health that was readily accessible in the public domain. PubMed ,4 , twelve academic institutions of India involved with public health, and sixteen publishing houses, were searched to locate research on public health reported in the public domain from India in 2002.affiliation option in PubMed for all journals as several papers in Australian journals mentioned only city and state but not Australia in the author affiliation.For comparison of the Indian health research output with a developed country, a PubMed search was also done for papers published from Australia during the April–June 2002 quarter, using "Australia" OR "names/abbreviations of the states and territories of Australia " in the Data were entered in an MS Access database and analysed using SPSS software.Of the 5718 papers with abstracts located on PubMed that were published from India in 2002, 842 (14.7%) papers were considered as non-health papers as they were on pure botany, chemistry, physics or zoology that could not be related to human health, and the other 4876 were health papers. The distribution of the types of research and the types of papers for the health papers is shown in Table Table Overall, the diseases/conditions that were substantially underrepresented in the relative proportion of quality-adjusted original research output as compared with their contribution to the disease burden were injuries, cardiovascular disease, respiratory infections, diarrhoeal diseases, perinatal conditions, childhood cluster diseases (including measles and tetanus), unipolar major depression, and HIV/AIDS Table .As the research output was least in public health, a brief description follows to understand this deficiency better. Figure Of the total 4876 health papers from India in PubMed for 2002, 1300 (26.7%) were published in Indian journals, but these papers accounted for only 1.5% of the total impact factor of all health papers from India due to the very low impact factors of Indian journals. Among the public health papers 44.4% were published in Indian journals, for clinical sciences papers this was 39.7%, whereas this proportion was much smaller for basic sciences (12.4%).The highest proportion of quality-adjusted basic research output was by university departments, institutions affiliated with the Council of Scientific and Industrial Research, and technical institutions; the predominant proportion of clinical research was by medical colleges / hospitals; and public health research by medical colleges / hospitals, government departments , and institutions affiliated with the Indian Council of Medical Research Table . The NatSearch of websites of major academic institutions in India, international agencies, and publishing houses revealed that substantial original public health research output that was accessible in the public domain was not readily available from these sources. Among the major academic institutions in India involved with public health research, only one was found to have a few reports on health research accessible on its website  and anotIn the April-June quarter of 2002, 1905 health papers published from Australia were located on PubMed, of which 722 (37.9%) were in basic sciences, 954 (50.1%) in clinical sciences, and 229 (12%) in public health. Taking into account the population and total gross domestic product (GDP) adjusted for purchasing power parity (PPP) of Australia and India [The data presented in this paper suggest that the health research output from India is not commensurate with the magnitude and distribution of disease burden. The research output in public health is particularly meagre, which is a major concern as public health sciences are a necessary tool to facilitate improvement in population health. Within this low research output, several diseases/conditions contributing substantially to the disease burden and several major areas of public health importance have relatively less representation. Without dynamic, relevant, good quality and adequate original research in the various aspects of public health it is difficult to imagine how the sub-optimal health status of the Indian population would improve on rhetoric or theoretical concepts alone ,15.In this paper we used impact factors for journals as a measure of the quality of papers published in those journals. Although impact factors are not without their limitations, they still offer a tangible, and perhaps the best available, option to compare the quality of publications in journals .We explored several sources where information about health research output from India could be available in the public domain, as the utilisation of research findings is facilitated most if they are readily accessible in the public domain. However, we did not find any source that would add substantially to the information available in the PubMed database. Indeed, there are more Indian health journals than are included in PubMed, but their quality in general is not as high as those included in PubMed with none of them having an impact factor above zero. Non-inclusion in our analysis of the papers published in these journals, therefore, did not bias our assessment of quality-adjusted research output based on impact factors. The relative low quality and impact factor of a large proportion of Indian journals has been discussed previously ,17. PubMWe used the disease burden in India as estimated by the Global Burden of Disease Study . AlthougThere has been a previous attempt to assess the health research output from India using the Science Citation Index of 1981–85 and relating the number of papers published in journals of various medical/health specialities with the perceived areas of major disease burden . Howeverdemand, supply and environment issues would have to be addressed:Since public health sciences seem to be the weakest link in improving health in India currently, it is imperative that a strategic framework for developing original public health research in India be evolved. To do so, the Demand. Among the multitude of factors that influence the demand for relevant public health research, the role of policy makers and senior health academics is of particular importance. This is seriously sub-optimal in India at present. Political compulsions push many policy makers into short-term gains instead of investments in comprehensive research for long-term benefits. Although there has recently been an increasing trend in India towards commissioned research by government and international agencies in some aspects of public health, this by itself is not enough to boost comprehensive public health research in India, and the reports of such studies are many times not available in the public domain which reduces the chance of their widespread utilisation. Many senior health academics in India continue to disregard public health research as a less-respectful cousin of basic and clinical research. Systematic efforts are needed to demonstrate to these groups the linkages between all aspects of health research , and the linkages between public health research and improvements in population health, in order to boost the demand for relevant and good-quality public health research in India.• Supply. Enhancing the output of public health research will require effort on various fronts. Establishing schools of public health and other institutions to train quality scientists in public health is a priority, as India has a surprisingly few number of institutions that can provide proper training in public health research. Another area that needs quick attention is to make public health exposure in medical and paramedical colleges more practical to encourage hands-on investigative thinking, as currently it is so theoretical that it rarely inspires enthusiasm in young professionals towards public health research. Setting higher standards for the research dissertations currently required for post-graduate degrees in preventive and social medicine would also encourage better quality and practically relevant public health research. It is also necessary to systematically develop performance-based opportunities to public health research scholars for career enhancement. Another element that would help develop public health research capacity in India is evolving mechanisms to encourage contribution to this effort by the many Indian public health researchers living abroad.• Environment. A conducive environment is necessary for the demand and supply of public health research to function optimally. Efforts are needed to develop this by attempting to develop broad-based coalitions, that include health care providers, civil society and non-governmental sector, for-profit private sector and industry, and national and international agencies providing financial support, which would understand and support the need for vibrant public health research as a vital element of societal development. This is a necessary element that has so far received scant attention, which must be addressed if sustainable development of public health research to improve population health is to become possible in India. An environment of good-quality and comprehensive public health research in India would also infuse the much-needed originality in teaching public health sciences and their practical application to the local context.• Evolving such frameworks would require building up a critical momentum for this effort through perseverance and wisdom. One such opportunity is provided by the recent initiative of the Indian Ministry of Health and Family Welfare to develop more effective institutes of public health in India, with relevant public health research and its utilisation an important key to improving population health .The recent attention towards revitalising the academic aspects of health care / medicine through evidence  and evid• Publications from India in PubMed were 11 times less in public health than those in basic sciences and in clinical sciences in 2002.• Injuries, cardiovascular diseases, respiratory infections, diarrhoeal diseases, perinatal conditions, childhood cluster diseases, unipolar major depression, and HIV/AIDS had substantially less proportion of quality-adjusted original research output in India as compared with their contribution to the disease burden.• India produced 20 times less quality-adjusted health research output than Australia per unit gross domestic product adjusted for purchasing power parity, and this ratio for public health research output was even higher at 31 times.• Good-quality public health research output from India is grossly inadequate, and strategic planning to improve it is necessary if substantial enhancement of population health were to be made possible.The author(s) declare that they have no competing interests.LD conceived the idea of this study, guided the design, data collection and analysis, and wrote the initial draft of this paper. YSS contributed to the design, data collection and analysis. MNJ contributed to data collection and analysis. VSUB contributed to data management and analysis. RD contributed to the idea of this study, design and data analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Phyllodes tumors (cystosarcoma phyllodes) are uncommon lesions in the female breast. Rarely, the occurrence of carcinoma within a phyllodes tumor has been reported in the literature, but has never been associated with lymph node metastases.in-situ (DCIS) and invasive carcinoma of no special type, located entirely within the phyllodes tumor. Subsequent axillary lymph node dissection revealed metastatic carcinoma in four lymph nodes.A 26-year-old woman presented with a firm, mobile, non-tender mass in the left breast and palpable lymph nodes in the left axilla. The excised lesion appeared well circumscribed and lobulated, with variable fleshy and firm areas. Microscopic examination showed a circumscribed fibroepithelial lesion with a well developed leaf-like architecture, in keeping with a benign phyllodes tumor. The epithelial component showed extensive high grade ductal carcinoma Although rare, phyllodes tumors may harbor DCIS and invasive carcinoma, with potential for lymph node metastasis. Phyllodes tumors constitute less than 1% of breast tumors and 2–3% of fibroepithelial breast tumors . Metastain-situ and invasive carcinoma occurring within a benign phyllodes tumor in a 26-year-old woman, which, to our knowledge, is the youngest presenting age for this combination tumor. We also report the first case of this combination tumor with documented lymph node metastases. Our case highlights the importance of assessing phyllodes tumors for concurrent carcinomatous involvement, as this may affect both the need for axillary lymph node examination and subsequent treatment options.We describe a case of A 26-year-old woman presented with a 1 month history of a palpable mass in her left breast. Her mother died at the age of 31 years of breast cancer. Examination revealed a 2 cm firm and mobile mass in the lower outer quadrant of the left breast. The left axilla contained two enlarged palpable lymph nodes.Mammography showed a 2 cm oval density that was homogenous with sharp margins. Ultrasound characterized the mass as solid but with a complex internal architecture. Fine needle aspiration of the mass was performed. It was positive for carcinoma, showing a very cellular sample with many malignant epithelial cells in cohesive groups, as well as lying singly in a background of necrosis and inflammation. The surgeon subsequently performed an excisional biopsy.in-situ, with multiple foci of invasive mammary carcinoma  chemotherapy and underwent further local excision to achieve wider margins; pathological examination was negative for residual tumor. She subsequently received local radiation and is currently undergoing tamoxifen therapy. The patient is presently alive and disease free, nearly three years after presentation.in-situ and invasive lobular and ductal  carcinoma, tubular carcinoma and squamous carcinoma [The epithelial component of phyllodes tumors  may show a range of metaplastic (apocrine, squamous) and proliferative changes ,6. Howevarcinoma -7,9-13. arcinoma .The prognosis for cases of carcinoma within phyllodes tumors is generally favorable, with no deaths yet reported ,10. The in situor invasive carcinoma.Invasive carcinoma occurring within a phyllodes tumor is rare; occurrence of axillary lymph node metastasis is even rarer. Besides the stromal component, the epithelial component of phyllodes tumors should also be examined in detail, as it may harbor an The authors declare that they have no competing interests.JRP prepared the manuscript.CA and ABT were involved with initial diagnosis of the pathologic specimen and were involved with manuscript editing.FO provided consultation for the final diagnostic interpretation and was involved with manuscript editing.JR provided patient history, obtained patient consent and helped with manuscript editing.All authors read and approved the final manuscript.


TMPRSS3 are associated with nonsyndromic recessive deafness (DFNB8/B10). TMPRSS3 encodes a predicted secreted serine protease, although the deduced amino acid sequence has no signal peptide. In this study, we searched for mutant alleles of TMPRSS3 in families from Pakistan and Newfoundland with recessive deafness co-segregating with DFNB8/B10 linked haplotypes and also more thoroughly characterized the genomic structure of TMPRSS3.Mutant alleles of TMPRSS3. The structure of TMPRSS3 was characterized bioinformatically and experimentally by sequencing novel cDNA clones of TMPRSS3.We enrolled families segregating recessive hearing loss from Pakistan and Newfoundland. Microsatellite markers flanking the TMPRSS3 locus were used for linkage analysis. DNA samples from participating individuals were sequenced for TMPRSS3 in four Pakistani families with recessive, nonsyndromic congenital deafness. We also identified two recessive mutations, one of which is novel, of TMPRSS3 segregating in a six-generation extended family from Newfoundland. The spectrum of TMPRSS3 mutations is reviewed in the context of a genotype-phenotype correlation. Our study also revealed a longer isoform of TMPRSS3 with a hitherto unidentified exon encoding a signal peptide, which is expressed in several tissues.We identified mutations in TMPRSS3 contribute to hearing loss in many communities worldwide and account for 1.8% (8 of 449) of Pakistani families segregating congenital deafness as an autosomal recessive trait. The newly identified TMPRSS3 isoform e will be helpful in the functional characterization of the full length protein.Mutations of  Genetic analysis of inherited deafness is a powerful tool for discovering the molecular mechanisms that control the development, function and maintenance of the auditory system. Linkage analysis of deafness segregating in large families has resulted in the mapping of more than 55 non-overlapping loci and the discovery of over 30 genes that are essential for hearing . Given tTMPRSS3 gene, spanning approximately 24 kb on chromosome 21, contains thirteen reported exons [TMPRSS3 a, b, c and d), encoding predicted polypeptides of 454, 327, 327 and 344 amino acids, respectively [TMPRSS3e, which encodes 538 amino acid residues, including a signal peptide, and is expressed in many human tissues.DFNB8/B10, an autosomal recessive deafness locus, was independently mapped in two consanguineous families from Palestine and Pakistan ,3. HaploTMPRSS3 mutations in four out of a total of 159 Pakistani families segregating profound congenital recessive deafness [TMPRSS3 mutations to recessive deafness in Pakistan, we screened an additional 290 Pakistani families for linkage to the DFNB8/B10 locus. We also screened TMPRSS3 for mutations in a large kindred from Newfoundland, Canada segregating hearing loss linked to markers for DFNB8/B10 and surprisingly found two different mutant alleles.We previously reported pathogenic Institutional review board approvals (OH93-N-016 and OH95-DC-N-050) were obtained for this study from the National Institutes of Health, USA, the Centre of Excellence in Molecular Biology, Lahore, Pakistan and the Newfoundland and Labrador Medical Genetics Program, Health Sciences Centre, St. John's. Participating individuals gave written informed consent. Medical histories indicated that all four Pakistani families segregate congenital, profound, non-syndromic sensorineural hearing loss. Pure tone air and bone conduction audiometry was performed on affected and unaffected individuals of the family from Newfoundland. Pneumatic otoscopy was used to confirm or deny cases in which conductive hearing loss was suspect. All testing was done in non-sound-attenuated exam room. Prior to testing, a biologic calibration was done in the room used for testing with a listener having normal hearing thresholds (LS). Possible effects of ambient noise on thresholds were then taken into account and only those frequencies unaffected were included. DNA was extracted from venous blood samples from the participating individuals.TMPRSS3 mutations in families segregating DFNB8/B10, the sequence of each exon of TMPRSS3 was evaluated in two affected subjects from each family as described previously [DNA samples were PCR amplified using fluorescently labeled primers surrounding microsatellite repeats at known DFNB loci and analyzed on an ABI 377 DNA sequencer. Genotypes were determined using Genescan and Genotyper software (PE Biosystems). To detect eviously .TMPRSS3a exon 1. 5' RACE PCR using human retina Marathon-Ready cDNA as template (Clontech) with primers 5'CAGACCAATGGCCAGTGCTAATATC3' and the AP1 primer was performed using the following thermal cycling conditions: 94°C for 1 min, 25 cycles of 94°C for 30 s and 72°C for 5 min. Five microliters of amplified PCR product was used for the second round of amplification with nested primer 5'TTTTCAAATCATCAAGGCCAAAAAG3' and the AP2 primer. Five microliters of each sample were analyzed on a 1.2% agarose/ethidium bromide gel, extracted using a QIAquick gel extraction kit (Qiagen) and cloned into E. coli using pGEM®-T Easy vector (Promega). DNA was purified from minipreps using QIAquick miniprep kit (Qiagen) and inserts were sequenced using T7 and SP6 primers. To amplify the full length TMPRSS3e isoform, 5'ATGGTGAGTAAAATGGGTGTGAGGA3' and 5'CTTGGAAGTAGAAAGGGTGGGTTTG3' primers and LA-Taq polymerase (Pan Vera) were used as recommended by the manufacturers.Sequence analysis of the subcloned PCR product amplified from human retina GETLarge full length cDNA (Genemed Biotechnologies) using primers 5'GGGTTGCTTCAAATGGCTTACTAGATCC3' and 5'CATTTTCCCCCATGGTGACTATTTCAG revealed an additional 385 bp of transcribed sequence downstream of TMPRSS3e was evaluated using a Human Tissue cDNA panel (Clontech). TMPRSS3e cDNA was amplified with primers 5'CCAGAAATGGTGAGTAAAATGG3' and 5'AGCAACAGCATCTGCATCTGGT3' using 20 pg of each cDNA as template following the manufacturer's recommended protocol (Clontech). PCR amplified products were sub-cloned into pGEM-T Easy vector (Promega) and sequenced.Expression of In four out of 290 newly investigated Pakistani families, nonsyndromic congenital deafness was found to co-segregate with DFNB8/B10 linked haplotypes  of the mRNA . The amplicon generated using a forward primer from the reported non-coding exon 1 and a reverse primer from the protein coding region of exon 2 was larger than the expected size product based on the sequence of TMPRSS3 isoform a [TMPRSS3e  in which deaf subjects were homozygous for the markers spanning the DFNB8/B10 region with the simulated maximal lod score of 3.8 . HoweverTMPRSS3 . One pos TMPRSS3 . To probsoform a . After ssoform a . We furtTMPRSS3e transcript encodes a putative polypeptide of 538 amino acids, including 84 previously unknown amino acids at the amino terminus  does not meet Kozak consensus criteria, there are two reasons for believing that this is the translation initiation site. First, there are stop codons in all three reading frames upstream of this site. Secondly, the 252 bp open reading frame between ATG 705 and ATG 957 Figure , the pres Figure  and 3C. s Figure  and 3B. s Figure  and 3B. s Figure . ThroughTMPRSS3 is the only protease reported thus far to be involved in nonsyndromic deafness [TMPRSS3 segregating in a large family from Newfoundland and in additional Pakistani families with nonsyndromic, recessive deafness. The Newfoundland family has at least two different mutated genes associated with hearing impairment. The majority of the affected individuals in this family are homozygous for 207delC  and IV:12  show an identical chromosome 21 haplotype. Similarly the individuals VI:1 (affected) and VI:2  share identical haplotypes for the TMPRSS3 locus. Hence, it is unlikely that the deafness of individuals IV:10 and VI:1 is due to cryptic TMPRSS3 mutations  are segregating in the Pakistani population [We have analyzed a total of 449 Pakistani families [ and thispulation ,9,10.TMPRSS3, six are present in exons common to all isoforms, including isoform "e" reported herein. Of the isoforms of this serine protease, TMPRSS3e has the longest open reading frame and is the only isoform of this gene with a predicted signal sequence at the amino terminus. The identification of 84 additional amino acids, including a predicted signal sequence, may aid functional assays to determine the role of TMPRSS3 in cochlear development and function. TMPRSS3 isoform a was identified with a forward primer  of TMPRSS3 have not been reported in the auditory system, TMPRSS3 is thought to regulate the activity of the epithelial amiloride sensitive sodium channel (ENaC) in vitro, which was suggested to control critical signaling pathway(s) in the inner ear and may have a role in the maintenance of the low sodium concentration of endolymph [ cochlea . Althougelopment ,13. Althndolymph . Howeverndolymph , suggestTMPRSS3 mutations account for hearing loss in 1.8% (8 of 449) of Pakistani families segregating deafness as an autosomal recessive trait. We also identified two recessive mutations of TMPRSS3 segregating in a six-generation extended family from Newfoundland. Our study also revealed a longer isoform of TMPRSS3 with an exon encoding a signal peptide, which should help in the functional dissection of this secreted serine protease.TMPRSS3; Transmembrane Protease, Serine 3 [OMIM entry 605511].DFNB8; Deafness, childhood-onset neurosensory autosomal recessive 8 [OMIM entry 601072]DFNB10; Deafness, congenital neurosensory, autosomal recessive 10 [OMIM entry 605316]The authors declare that they have no competing interests.TMPRSS3 isoform e and prepared the manuscript. XCL analyzed all linkage data from family B from Newfoundland and identified DFNB8/B10 segregation in this family. SDP performed DNA sequencing to determine mutant alleles of TMPRSS3 and confirmed linkage in Pakistani families. SR, KR and ZA enrolled families in Pakistan. TLY re-sampled some members of family B from Newfoundland, confirmed genotypes and sequenced normal controls from Newfoundland. SL participated in the clinical assessment of Newfoundland deaf family members. KD sequenced normal controls from Newfoundland. LM was the genetic counsellor involved in family B enrolment from Newfoundland. BP provided considerable technical assistance. LIS performed hearing tests on Newfoundland family members. EI identified Newfoundland families with inherited deafness. SR was responsible for the enrolment of 449 families from Pakistan. TBF was in charge of oversight, editing and analyses. RJM and ERW trained investigators and were involved in the experimental design and genetic analyses of all families. All authors contributed to and edited the manuscript.ZMA performed DNA sequencing, characterized the novel The pre-publication history for this paper can be accessed here: