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" . 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 . 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 , 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 , 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 , 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 , 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 . 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 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  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 . 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  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 .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:


The identification of the molecular mechanisms of human immunodeficiency virus type 1, HIV-1, transcriptional regulation is required to develop novel inhibitors of viral replication. NF-κB transacting factors strongly enhance the HIV/SIV expression in both epithelial and lymphoid cells. Controversial results have been reported on the requirement of NF-κB factors in distinct cell reservoirs, such as CD4-positive T lymphocytes and monocytes. We have previously shown that IκB-αS32/36A, a proteolysis-resistant inhibitor of NF-κB, potently inhibits the growth of HIV-1 and SIVmac239 in cell cultures and in the SIV macaque model of AIDS. To further extend these observations, we have generated NL(AD8)IκB-αS32/36A, a macrophage-tropic HIV-1 recombinant strain endowed to express IκB-αS32/36A.In this work, we show that infection with NL(AD8)IκB-αS32/36A down-regulated the NF-κB DNA binding activity in cells. NL(AD8)IκB-αS32/36A was also highly attenuated for replication in cultures of human primary monocytes.in vivo.These results point to a major requirement of NF-κB activation for the optimal replication of HIV-1 in monocytes and suggest that agents which interfere with NF-κB activity could counteract HIV-1 infection of monocytes-macrophages  HIV-1 infection is characterized by a long period of clinical latency followed by the development of acquired immunodeficiency syndrome, AIDS. During latency and when viral replication is being controlled in patients treated with antiretroviral therapy, HIV-1 is present in cellular reservoirs and continues to replicate, with each ensuing round of replication giving rise to escape mutants, which further replenish viral reservoirs . This grin vivo by HIV-1 is thought to occur in CD4-positive, CCR5-positive lymphocytes and monocytes. Accordingly, when HIV-1 envelope protein in its oligomerized g160 form contacts the cell surface receptor a signalling cascade is triggered that results in transcriptional activation of specific gene arrays, such as the inflammatory cytokines IL-1 β, IL-6, IL-8, TNF-α, TGF-β; these cytokines, in turn, function to enhance the transcriptional activity of the proviral long terminal repeat (LTR) promoter ATP  using polynucleotide kinase . Equal amounts (5 μg) of cell extracts were incubated in a 20 μl reaction mixture containing 10% glycerol, 60 mM KCl, 1 mM EDTA, 1 mM DTT, and 2 μg of poly [d(G-C)]  for 5 min on ice. One μl of [γ32P]-labelled double-stranded probe  was then added with or without a 100-fold molar excess of competitor oligonucleotide. The reactions were incubated at room temperature for 15 min and run on a 6% acrylamide:bisacrylamide (30:1) gel in 22.5 mM Tris borate, 0.5 mM EDTA. Gels were dried and autoradiographed.Nuclear extracts and gel retardation assays were performed as described previously . Briefly5 cells) were infected with 105 - 106 cpm of RT activity. The cell culture supernatants were collected every two days and replaced with fresh medium. The viral production was measured as RT activity in the culture supernatants as previously described [Human monocytes were isolated from PBMC by elutriation, cultured in RPMI, 10% FCS and GMCSF (20 ng/ml) for 48 hours. Infections were performed with viral stocks measured by reverse-transcriptase (RT) activity . Usuallyescribed . The synNF-κB, nuclear factor kappa BIκB, inhibitor of nuclear factor kappa BIL-1, interleukin-1IL-6, interleukin-6IL-8, interleukin-8TNF-α, tumor necrosis factor alphaTGF-β, transforming growth factor-betacpm, counts per minuteFCS, fetal calf serumGMCSF, granulocyte-macrophage colony-stimulating factorThe author(s) declare that they have no competing interests.CP carried out the analysis of viral growth and DNA band-shift assays. FT was responsible for cell cultures. AP performed the immunoblotting analysis. GF produced the viral plasmids and viral stocks, and performed the artwork of the paper. GS participated in the design of the study and discussion of results. IQ designed this study and edited the manuscript.


Clustering is one of the most commonly used methods for discovering hidden structure in microarray gene expression data. Most current methods for clustering samples are based on distance metrics utilizing all genes. This has the effect of obscuring clustering in samples that may be evident only when looking at a subset of genes, because noise from irrelevant genes dominates the signal from the relevant genes in the distance calculation.We describe an algorithm for automatically detecting clusters of samples that are discernable only in a subset of genes. We use iteration between Minimal Spanning Tree based clustering and feature selection to remove noise genes in a step-wise manner while simultaneously sharpening the clustering.Evaluation of this algorithm on synthetic data shows that it resolves planted clusters with high accuracy in spite of noise and the presence of other clusters. It also shows a low probability of detecting spurious clusters. Testing the algorithm on some well known micro-array data-sets reveals known biological classes as well as novel clusters.© programs for the iterative clustering algorithm are available from The iterative clustering method offers considerable improvement over clustering in all genes. This method can be used to discover partitions and their biological significance can be determined by comparing with clinical correlates and gene annotations. The MATLAB Many diseases, though, are characterized by small numbers of genes that differentiate between different disease states. Giving equal weight to relevant and irrelevant genes will obscure this difference. Figure et al ..2].xs,g~N  for samples s belonging to cluster 1 and xs,g~N  for samples belonging to cluster 2. The rest of the genes are not differentially expressed and are called noise genes and are distributed according to a normal distribution N . If we have k signal genes, each differentially expressed by c between the two clusters, the Euclidean distance between the cluster-means will be .Synthetic data were created by generating normally distributed expression profiles for each gene. Each planted partition is supported by a fraction of the genes  which were differentially expressed between the two clusters. Each signal gene is differentially expressed to the same extent. The expressions were normally distributed; M = 1000 and M = 10000 with varying fraction of signal genes ε and distance between cluster means Dc. Each set of data was analyzed both by the iterative and the hierarchical clustering method (using average linkage). The iterative clustering method was used to obtain the first partition discovered using the Euclidean distance (Pthresh = 99.95). Hierarchical clustering was used to obtain a tree, and the branch at the highest level was split to produce a partition.Sets of synthetic data were generated for number of genes Dc.The match of these two partitions with the true partition was calculated and the detection accuracy was assigned 1 if the match was greater than 75% and 0 otherwise. A logistic regression analysis was used to model the dependence of the probability of detection on the distance between the clusters Pthresh = 99.9. Each sample is a 1000-dimensional vector drawn from a multivariate normal distribution. Thus any clusters detected can be expected to be spurious clusters formed by chance.To estimate the false detection rate, the algorithm was run on synthetic data containing 10 – 100 samples with Pthresh = 99.9) using (1-correlation coefficient) as the distance measure for the MST. For the BRCA data, we also clustered the data using standard hierarchical clustering using centered correlation as the distance metric [For the micro-array data, the iterative method was used to detect the first 10 partitions,  depends only on the distance between the clusters for both hierarchical and iterative clustering.Click here for file


Bone adapts to mechanical strain by rearranging the trabecular geometry and bone density. The common finite element methods used to simulate this adaptation have inconsistencies regarding material properties at each node and are computationally demanding. Here, a volume-based, non-continuum formulation is proposed as an alternative. Adaptive processes corresponding to various external mechanical loading conditions are simulated for the femur.Bone adaptations were modeled for one-legged stance, abduction and adduction. One-legged stance generally results in higher bone densities than the other two loading cases. The femoral head and neck are the regions where densities change most drastically under different loading conditions while the distal area always contains the lowest densities regardless of the loading conditions. In the proposed formulation, the inconsistency of material densities or strain energy densities, which is a common problem to finite element based approaches, is eliminated. The computational task is alleviated through introduction of the quasi-binary connectivity matrix and linearization operations in the Jacobian matrix and is therefore computationally less demanding.The results demonstrated the viability of the proposed formulation to study bone functional adaptation under mechanical loading. Much research effort has been devoted to understanding the functional adaptation of bone under physiological loading ever since the idea of bone functional adaptation was proposed by Wolff more than one hundred years ago -14. VariThe common finite element approach is to take the element densities as the state variables and define elements with either constant or varying densities, then update the material densities for the next step of computation according to the computed strain energy density ,23,26,27One common problem is the inconsistency of material densities on element boundaries . During Some effort has been made trying to solve this problem. For example, a node-based variant of the finite element method was tried with focus on the densities of the nodes rather than densities of the whole elements ,30. The Other previous work has used Voronoi structures  to studyThe long existing problems and the limitations of assuming a continuum drive this new effort to explore the possibility of a non-continuum formulation of bone functional adaptations through nodal analysis in the hope of eliminating the errors present in the previous approaches. In the proposed non-continuum formulation, neighboring nodes are connected by struts that are defined with invariant material densities with respect to time but strut volumes are defined as state variables indicating different configurations of bone structure. The updating of strut volume will depend on the strain energy density in the strut in the previous step. As a result, there is no conflict either in density or in strain energy density. The shift of state variables from bone densities to bone volumes not only eliminates the errors inherent to the density-based finite element approaches but also transforms the continuum formulation to a non-continuum formulation . The advThe proposed volume-based non-continuum formulation shows computational advantages in modeling bone functional adaptations and has much potential for clinical applications in this field.Fig. 3 is used as the maximum value in the present formulation.The nodal density is a percentage relative to its maximum value (0~100%). It is 'converted' based on volume information (BV/TV) for the purpose of easy visual inspection. As suggested by Zhu , the valIn the combined loading case and one-legged stance, the nodal densities are generally high in the proximal area where a considerable number of nodes reach the highest density due to the relatively high load, while in the large distal area, the densities become lower. In the case of abduction, the external load is the smallest out of the three loading cases and the nodal density in this case seldom reaches the maximum. Although the high densities also appear in the proximal area of femoral head, the densities are lower than those of other loading cases. In the large distal area, the node densities generally range at very low levels. In the case of adduction, the highest densities still appear in the proximal area of femoral head, but in the large diaphyseal area, the densities range at very low levels.In summary, one-legged stance and the combined loading case generally result in higher bone densities than the other two loading cases due to higher mechanical loading. The femoral head and neck are the regions where densities change most drastically under different loading conditions while the distal area always contains the lowest densities regardless of the loading conditions.Starting from a uniform material distribution, the program computes the strain energy density, adjusts the strut configurations, and continues on to the next iteration. Fig. A prediction of bone fracture risk has been proposed using stress range levels  from whiA volume-based non-continuum formulation has been developed that describes the adaptation of bone to various mechanical loading situations. In the finite element approach to bone adaptation simulation, the integration of the entries in the coefficient matrix can be a heavy computing task ,23,26,27As mentioned in the introduction, one common problem is the inconsistency of material densities or strain energy densities on element boundaries . Since aAs described above, previous work -33 addreHip fracture is one typical manifestation of osteoporosis, and the results obtained by the simulation indicate that considerable changes of bone structure take place in the regions of femoral head and neck, where the stress level is normally higher than that of distal regions. The variations in stress level as shown in Fig. In the current literature, the time scale for adaptive processes is not very well defined. This general lack of knowledge poses a problem for any experimental proof of concept – while the numbers of strain repetition can be predefined, they must be done within biologically suitable time frames. If a given strain comes too sudden, the bone may break instead of remodel; if the strain is applied over too long a period, it may not be a sufficient stimulus to activate adaptive processes. The lack of well-defined temporal constraints, however, is common. Kim's approach  is very The simulation model presented here may, beyond theoretical calculations, be applied to look at two clinical questions. Firstly, the simulation can be adjusted so that a realistic density distribution is the starting point, and outcomes following certain loading conditions, such as a predefined number of load cycles can then be predicted. Secondly, the program can integrate the measured bone density of a given patient to estimate the fracture risk based on stress level calculations.By eliminating the common inconsistencies at each node, the formulation presented here shows good numerical performance and successfully predicts reasonable bone structure changes under different loading conditions. It is viable to serve as an alternative method apart from the traditional finite element based approached to study bone adaptations. In conclusion, the volume based non-continuum formulation is a new approach to bone adaptation study and has its own advantages. can be obtained though the resultant of the vectorial material components of the struts, as described by equation (1) and (2).In the volume-based non-continuum formulation used here, the trabecular structure is represented by a connected strut system and each strut can take different sizes according to the mechanical loading requirements, that is, strain energy density. The strut representation is shown in Fig. vi is the volume of the i-th strut in the j-th basic unit, Vj is the volume of the j-th basic unit, Rj is the material orientation of the j-th basic unit,  is the orientation of the i-th strut in the j-th basic unit and N is the number of struts in the j-th basic unit.where For the formulation proposed here, the bone structure is decomposed into and represented by a connected network of struts. These struts are a mathematical abstraction of the physical bone structure, from which the BV/TV ratio can be derived. The thickness of a strut is adapted during the bone adaptation process in mimicry of the physiological processes.The bone mass will vary under mechanical loading. In engineering, the general relationship between varying mass, density and volume is described as:, simply vanishes and the mass variation is realized through volume variation under mechanical loading. Based on the density-based adaptation proposed by Zhu X. et al (39), which is stated as:Since the density here is taken as constant regarding time, the second term on the right hand side, the volume-based adaptation can thus be stated as:βi = Ui / ρik, which is a comparative coefficient describing the comparison of a given mechanical stimulus in each sensor cell with the reference value k, and Ui represents the strain energy density for the I-th sensor unit; N is the number of sensor cells and fi(x) is the spatial influence function; B(t) is a remodeling coefficient; α indicates the remodeling power of strain energy density [where  density .With the whole bone represented by a volume-based strut system, the non-continuum formulation can be noted as follows:A is a connectivity matrix describing the connecting relationship between the struts, α is the linearized Jacobian matrix,  is the nodal displacement vector to be solved for and  is the loading vector derived from the external mechanical load. The generalized conjugate residue method is used to solve this formulation [where mulation ,40.Connectivity matrix A is the matrix to show the relationship between connected struts with the entries of 1, -1 or 0. A strut starts from the node with the index corresponding to the entry 1 and ends at the node with the index corresponding to -1. It is further illustrated in Fig. Finally, the different loading conditions to be applied are shown in Fig. The author(s) declare that they have no competing interests.Zhengyuan Wang developed the formulation and partly prepared the manuscript, Adrian Mondry participated in the adaptation controls and partly prepared the manuscript.


To build the complex body plan of higher organisms, thousands of genes must act in a coordinated fashion, becoming active at the right time and in the right place to define structures like head, thorax, and abdomen, or cell types like skin, muscle, and bone. One of the central questions for developmental biologists is how such specific spatiotemporal expression of genes is achieved.The general mechanism of the control of gene expression is well understood: Special proteins, called transcription factors, bind to short stretches of DNA near a gene. By docking to such binding sites, they activate or repress the transcription of the gene into mRNA (which is then translated into protein). Transcription factors often act in a combinatorial fashion—that is, several different factors have to bind in close proximity to each other to achieve a particular transcriptional outcome. As a consequence, their binding sites form clusters, called regulatory elements or modules.Drosophila. Organized in four tiers and acting in combinatorial fashion, the segmentation genes lay out the anterior-posterior axis of the embryo. In a stepwise refinement of expression patterns, they translate broad, overlapping gradients formed by maternally provided transcription factors into a periodic pattern of 14 discrete stripes that prefigure the 14 segments of the larva. The segmentation gene network has long been one of the prime paradigms for studying transcriptional control, and many researchers have worked over the years to experimentally dissect the regulatory interactions within the hierarchy. For some of the most important genes, the regulatory elements driving their expression and the favored binding sites have been identified. Nevertheless, the picture of transcriptional regulation within the segmentation gene network has remained incomplete.In many contexts, the genes that are activated or repressed encode transcription factors themselves, forming a cascade of transcriptional control events. One such transcriptional control hierarchy is the segmentation gene network in the fruitfly Drosophila, the binding sites are typically short and variable, and the search space is large; on the other hand, the fact that sites cluster—where transcription factors work in concert—aids the task.This is where the research reported by Mark Schroeder et al. comes in: With the sequence of entire genomes available, it's possible to use existing binding site information to computationally search the neighborhood of genes for regulatory elements. The difficulty here is that in higher organisms such as To identify regulatory elements, the researchers developed an algorithm, named Ahab, that models the behavior of multiple transcription factors competing for binding sites and fine-tunes the search by detecting clusters of weak sites. Using this approach, Schroeder et al. identified 52 regulatory elements within the segmentation gene network, 32 of them novel. The authors tested a large number of the newly identified modules experimentally by placing them in front of reporter genes that reveal where the modules drive expression within the developing fly. They showed that almost all modules faithfully reproduce the expression pattern of the endogenous gene. To better understand the way segmentation gene modules function, the researchers then systematically analyzed their predicted binding site composition. They correlated the composition of modules with the expression they produce and with the distribution of the transcription factors that bind to them. They were thus able to glean basic composition rules and to derive the mode of action for most of the factors, that is, whether they act as activators or as repressors.Overall, Schroeder et al. show that a computational search can greatly reduce the experimental effort necessary for finding regulatory elements within the genomic sequence. Their study provides an example of how experimental and quantitative methods can be combined to achieve a more global analysis of the regulatory interactions within a transcriptional network.