The most prominent feature of melancholic depression is a near-total loss of the capacity to derive pleasure from activities or other positive stimuli. Additional symptoms can include psychomotor disturbances, anorexia, excessive guilt, and early awakening from sleep. Melancholic patients may exhibit treatment responses and outcomes that differ from those of non-melancholic patients. Pooled data from double-blind, placebo-controlled studies were utilized to compare the efficacy of duloxetine in depressed patients with and without melancholic features.17) total score, HAMD17 subscales , the Clinical Global Impression of Severity (CGI-S) and Patient Global Impression of Improvement (PGI-I) scales, and Visual Analog Scales (VAS) for pain.Efficacy data were pooled from 8 double-blind, placebo-controlled clinical trials of duloxetine. The presence of melancholic features (DSM-IV criteria) was determined using results from the Mini International Neuropsychiatric Interview (MINI). Patients (aged ≥ 18 years) meeting DSM-IV criteria for major depressive disorder (MDD) received duloxetine  or placebo  for up to 9 weeks. Efficacy measures included the 17-item Hamilton Rating Scale for Depression . When analyzed by gender, the magnitude of improvement in efficacy outcomes did not differ significantly between duloxetine-treated male and female melancholic patients. In the two studies that assessed duloxetine 60 mg once-daily dosing, duloxetine-treated melancholic patients had significantly greater improvement compared with placebo on HAMD17 total score, CGI-S, PGI-I, 3 of 4 subscales of the HAMD17, and VAS overall pain severity (p < .01). Estimated probabilities of response and remission were significantly greater for melancholic patients receiving duloxetine 60 mg QD compared with placebo . Duloxetine demonstrated significantly greater improvement in depressive symptom severity, compared with placebo, within both melancholic and non-melancholic cohorts (p ≤ .001 for HAMDIn this analysis of pooled data, the efficacy of duloxetine in patients with melancholic features did not differ significantly from that observed in non-melancholic patients. Diagnostic and Statistical Manual of Mental Disorders, 3rdEdition (DSM-III) to describe a subtype of major depressive disorder (MDD). Although revised in DSM-III-R, the current DSM-IV "With Melancholic Features Specifier" represents a return to the older DSM-III definition. According to DSM-IV criteria, the principal diagnostic feature exhibited by patients with melancholic depression is a loss of pleasure in all, or almost all, activities or a lack of reactivity to usually pleasurable stimuli ,,1 and 2, 1 [4 [5 , and 6 [[4 [5 [6  have beeDiagnostic and Statistical Manual of Mental Disorders, 4thEdition (DSM-IV) [17)[In all studies, patients were 18 years of age or older and met criteria for MDD as defined by the (DSM-IV) . The dia(DSM-IV) , a stand-IV) [17) total sc-IV) [17) score ≥ Either feature 1 or 2 in Criteria A AND three (or more) features from Criteria B must be present to qualify for melancholic features."A. Either of the following, occurring during the most severe period of the current episode.1. Loss of pleasure in all, or almost all, activities;2. Lack of reactivity to usually pleasurable stimuliB. Three (or more) of the following:1. Distinct quality of depressed mood;2. Depression regularly worse in the morning;3. Early morning awakening ;4. Marked psychomotor retardation or agitation;5. Significant anorexia or weight loss;6. Excessive or inappropriate guilt."Patients were excluded for the following reasons: a current and primary Axis I disorder, other than MDD; an Axis II disorder which could interfere with protocol compliance; lack of response of the current depressive episode to two or more adequate courses of antidepressant therapy; serious medical illness; a serious risk of suicide; a history of substance abuse or dependence within the last year, or a positive urine drug screen.Concomitant medications with primarily central nervous system activity were not permitted, with the exception of episodic use of chloral hydrate or zolpidem for insomnia. Chronic use of prescription analgesic medications was not allowed; episodic use was permitted at the discretion of the physician in charge of the study. Use of anti-hypertensive medications was not permitted unless the patient had been on a stable dose for at least 3 months prior to study entry.Efficacy analyses were performed on three sets of data, obtained using the following pooling strategies:(A) "All Studies" – data from all 8 studies were pooled. Placebo: melancholic , non-melancholic . Duloxetine (40–120 mg/d): melancholic , non-melancholic . Duloxetine was compared with placebo in one set of analyses. In a second set of analyses using data from the 6 SSRI-controlled studies, duloxetine was compared with fluoxetine and paroxetine: Placebo: melancholic , non-melancholic . Duloxetine (40–120 mg/d): melancholic , non-melancholic . SSRI: melancholic , non-melancholic ;1, 4, 5, 6,7, and 8) that demonstrated a significant advantage for duloxetine over placebo on the primary efficacy measure. Placebo: melancholic , non-melancholic . Duloxetine (40–120 mg/d): melancholic , non-melancholic ;(B) "Positive Studies" – data from placebo- and duloxetine-treated patients were pooled from the 6 studies (5 and 6) that compared duloxetine 60 mg once-daily with placebo. Placebo: melancholic , non-melancholic . Duloxetine (60 mg/d): melancholic , non-melancholic .(C) "Focus Studies" – data were pooled from the 2 studies , Maier , retardation , and sleep ; the CGI-S scale; and the Patient Global Impression of Improvement (PGI-I) scale [17 total score. Remission was defined as a HAMD17 total score ≤ 7. In Studies 3 – 8, the severity of painful physical symptoms was assessed using Visual Analog Scales (VAS) for pain [In all 8 studies, the primary efficacy outcome was mean change from baseline to endpoint in HAMDI) scale . Responsfor pain .17 total score, CGI-S, and PGI-I were assessed using an analysis of covariance (ANCOVA) with models that included baseline score, treatment, melancholia status (features present Yes/No), investigator, and the treatment-by-melancholia status interaction as independent variables. Hereafter this analysis is referred to as LOCF mean change.Patients with missing melancholia status were not included in the analyses. All other patients with a baseline and at least one postbaseline observation were included in the analyses. Mean changes from baseline to last observation (carried forward) in HAMDThe treatment-by-melancholic status interaction was the main basis upon which differential treatment effects between the subgroups were assessed. Contrasts between duloxetine and placebo within the melancholic and non-melancholic subgroups were used to assess the clinical relevance of treatment effects.17 value, melancholia status, and the two-way and three-way interactions between treatment, visit, and melancholia status. Hereafter this analysis is referred to as MMRM mean change. The categorical longitudinal analyses were similar in concept to the longitudinal mean change analyses, but simplifications were necessary to reduce the computational complexity.Longitudinal mean changes and categorical changes (estimated probabilities) were assessed using a likelihood-based mixed-effects model repeated measures (MMRM) approach. Models for mean changes included treatment, visit, investigator, baseline HAMD17 value, and the treatment-by-visit interaction. A logit link function and a binomial error structure were included to account for the non-linearity of the response and the non-normality of the data, respectively. Hereafter, this analysis is referred to as categorical MMRM. Remission and response rates at last observation were assessed using Fisher's Exact test.The categorical analyses were applied only to patients with melancholic features so that the main effect of melancholic status and its two-way and three-way interactions with visit and treatment could be deleted. Therefore, the model for the categorical analyses included treatment, visit, investigator, baseline HAMD17, CGI-S and PGI-I was applied to all three databases. The focus for the analyses was on all studies and the positive studies with the primary aim of detecting differential effects of duloxetine in patients with and without melancholic features. The MMRM mean change and categorical MMRM analyses were then applied to a wide variety of outcomes from the focus studies in order to gain an in-depth perspective on the efficacy of duloxetine in patients with melancholic features. In addition, LOCF analyses of data from the focus studies were conducted in order to assess robustness of the MMRM results.The LOCF mean change analysis of HAMDBaseline patient demographics are summarized in Table 17 total scores and CGI-S scores were significantly higher (more severe illness) in melancholic patients compared with non-melancholics . There was a marginally significant difference in VAS overall pain severity at baseline , although the clinical relevance of this small difference is questionable.Mean baseline HAMD17 total score, CGI-S and PGI-I (p ≤ .001). Treatment-by-melancholic status interactions were not significant for HAMD17 total score, CGI-S, or PGI-I, in either data set (p > .50 for each comparison). Using pooled data from all studies, the effect size for HAMD17 total score was 0.33 in melancholic patients and 0.32 in non-melancholics. In the six positive studies, the effect size for HAMD17 total score was 0.39 in melancholic patients compared with 0.43 in non-melancholics.Analyses of mean changes from baseline to last observation (LOCF mean change) from all eight studies and the six positive studies are summarized in Table 17 total score, mean change in CGI-S score, and mean endpoint PGI-I score were not statistically significant, indicating that duloxetine's efficacy was similar in male and female melancholic patients  vs. 0.34 .Within the subset of melancholic patients, treatment-by-gender interactions for mean change in HAMDts Table . In pool17 total score were not significant at thresholds of age 55 (p = .777), age 60 (p = .387), or age 65 (p = .540), indicating that the efficacy of duloxetine did not differ between older and younger melancholic patients irrespective of the old/young age threshold.In melancholic patients, treatment-by-age interactions for mean change in HAMD17 total score, mean change in CGI-S score, and mean endpoint PGI-I score were not statistically significant, indicating that the relative efficacy of duloxetine and SSRIs did not differ significantly between melancholic and non-melancholic patients  are summarized in Table 17 total score and HAMD17 Maier subscale compared with those receiving placebo (p < .001). Significant differences from placebo first occurred at Week 1 . On both clinician-rated (CGI-S) and patient-rated (PGI-I) assessments of global improvement, duloxetine-treated melancholic patients had significantly greater mean improvements compared with melancholics receiving placebo (p < .001). Robustness of the MMRM results was confirmed in that significant differences were also observed in LOCF mean change analyses.Analyses of mean changes from baseline to Week 9 (MMRM mean change) for melancholic patients in the two focus studies (Studies Mean changes from baseline (MMRM mean change) for VAS overall pain were also assessed. Figure Estimated probabilities  of remission at Week 9 were significantly higher for melancholic patients treated with duloxetine (60 mg QD) compared with placebo . The estimated probability of response among melancholic patients was 74.7% for duloxetine compared with 42.2% for placebo (p < .001). The advantage of duloxetine over placebo in remission and response rates was also significant in LOCF analyses .17 total score, CGI-S, PGI-I). These results suggest that duloxetine's efficacy does not differ substantially between melancholic and non-melancholic patients. Furthermore, within the group of melancholic patients, duloxetine's treatment effects were similar in male and female patients . One exception was noted in the anxiety subscale of the HAMD17. In the two focus studies, duloxetine did not achieve separation from placebo in patients with melancholic features, but demonstrated efficacy in non-melancholic patients. The reason for the disparity in outcomes between melancholic and non-melancholic patients on the HAMD17 anxiety subscale is unclear.The magnitude of duloxetine's advantage over placebo was generally similar in melancholic and non-melancholic patients. Treatment-by-melancholic status interactions were not significant for any of the three assessed efficacy measures (HAMDi.e. the MINI plus DSM-IV criteria) produced a substantial number of false positive results with regard to melancholic features. It is possible that the use of an alternative melancholia screening tool, such as the CORE scale developed by Parker et al [In previous studies, the prevalence of melancholic features in populations of depressed outpatients has ranged from 16% to 53%. The substantial variation in the reported rates of melancholia may be a result of the numerous criteria used to define melancholic features over the past two decades, including DSM-III, DSM-III-R, DSM-IV, and the Newcastle 1 Depression Rating Scale  (N1) . In the er et al , would her et al . In a wier et al , and wheer et al ,52. Althet al reported that the presence of melancholic features was not associated with lower body mass index [There was no significant difference in age between melancholic and non-melancholic patient groups, despite the fact that melancholic features are more common in older patients. Patients with melancholic features had significantly lower mean body weight at baseline compared to non-melancholics, with a between-group difference of 3.6 kg (8.0 lbs). Since one of the DSM-IV criteria for melancholic features is "significant anorexia or weight loss", this result does not appear surprising. However, given that the melancholic cohort contained a significantly greater proportion of females when compared with the non-melancholic group, the lower body weight among melancholics may be an artifact of gender rather than of MDD subtype. For example, in a recent study of 1694 depressed patients, Berlin ss index .17 score almost two points higher  in the melancholic group. Melancholic patients also had significantly higher baseline CGI-S scores when compared to those without melancholic features. Although melancholic patients had a slightly higher overall pain severity at baseline , the difference only approached statistical significance and its clinical relevance is questionable.As expected, melancholic patients exhibited a greater severity of illness at baseline compared with non-melancholics, with a mean baseline HAMD17, CGI-S or PGI-I scores among both placebo- and duloxetine-treated melancholic patients were slightly larger than those of non-melancholic patients, resulting in a very similar drug-placebo difference in both patient groups. In measures of pain severity there was evidence of greater placebo response among non-melancholic patients; however, duloxetine's advantage over placebo was similar in melancholic and non-melancholic patients, since patients without melancholic features exhibited more robust responses to both drug and placebo, when compared with melancholic patients.A frequent finding in placebo-controlled antidepressant studies is that placebo responses among melancholic patients are smaller than those observed in non-melancholic patients -25,54, aThere does not appear to be a clear consensus as to which class of antidepressant medication is most efficacious for depressed patients exhibiting melancholic features. Some studies have demonstrated advantages for TCAs over SSRIs in the treatment of melancholia -32, and 17 scale has only limited capability to assess improvement in melancholic symptoms. Anhedonia, one of the key features of melancholia, is only assessed indirectly as a part of question 7, while psychomotor agitation is only partially addressed by question 9. Furthermore, items such as diurnal variation in mood are not specifically measured by the HAMD17.Results from the present investigation must be considered in light of several limitations. Firstly, this was a post-hoc analysis of pooled clinical trial data. Although subgroup analyses of efficacy assessments stratified by melancholic status were pre-specified in each protocol, the pooling strategies and some of the analyses conducted in the present investigation were not pre-specified. Secondly, while melancholic features were evaluated as part of the MINI at study entry, specific melancholic features were not an entry requirement and randomization was not stratified by melancholic status. Furthermore, as discussed previously in some detail, the MINI is not an ideal tool for assessing the presence of melancholic features. Thirdly, the HAMD17 total score. Therefore, results from other efficacy measures should be viewed as secondary in nature. Together, these limitations necessitate that the results from the present investigation cannot be considered confirmatory. A prospectively designed clinical trial will be required to confirm the results suggested by the current set of analyses.Fourthly, the group of patients receiving the recommended target dose of duloxetine (60 mg QD) could not be compared head-to-head with SSRIs, as these two studies did not feature active comparator treatment groups. Finally, although duloxetine demonstrated significant advantage over placebo on a number of efficacy measures, the individual studies were powered on the basis of the primary outcome – mean change in HAMDIn these analyses of pooled data, the efficacy of duloxetine in patients with melancholic features did not differ significantly from that observed in non-melancholic patients. Additional prospectively designed clinical trials evaluating duloxetine's efficacy in melancholic patients will be required to substantiate the preliminary findings described here.Drs. Mallinckrodt, Watkin, Liu, Wohlreich, and Raskin are employees of Eli Lilly and Company.CHM proposed the data pooling strategies, designed the statistical analyses, and participated in interpretation of data and drafting of the manuscript. MMW, JR, and JGW participated in interpretation of data and drafting of the manuscript. CL performed the statistical analyses. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The relative role of anti apoptotic (i.e. Bcl-2) or pro-apoptotic (e.g. Bax) proteins in tumor progression is still not completely understood.Bcl-2 and Bax transgenes and the viability of theses cell lines was analyzed in vitro and in vivo.The rat glioma cell line A15A5 was stably transfected with human In vitro, the transfected cell lines (huBax A15A5 and huBcl-2 A15A5) exhibited different sensitivities toward apoptotic stimuli. huBax A15A5 cells were more sensitive and huBcl-2 A15A5 cells more resistant to apoptosis than mock-transfected A15A5 cells (pCMV A15A5). However, in vivo, in syngenic rat BDIX, these cell lines behaved differently, as no tumor growth was observed with huBax A15A5 cells while huBcl-2 A15A5 cells formed large tumors. The immune system appeared to be involved in the rejection of huBax A15A5 cells since i) huBax A15A5 cells were tumorogenic in nude mice, ii) an accumulation of CD8+ T-lymphocytes was observed at the site of injection of huBax A15A5 cells and iii) BDIX rats, which had received huBax A15A5 cells developed an immune protection against pCMV A15A5 and huBcl-2 A15A5 cells.We show that the expression of Bax and Bcl-2 controls the sensitivity of the cancer cells toward the immune system. This sensitization is most likely to be due to an increase in immune induced cell death and/or the amplification of an anti tumour immune response Glioblastoma Multiforme (GBM) are the most common and aggressive tumors of the central nervous system (CNS) . Currentin vitro and in vivo in the latter pathway [Proteins of the BCL-2 family play a major role in the control of apoptosis both  pathway . These p pathway . Inhibit pathway  while, o pathway . We have pathway . Note th pathway . One of  pathway .Although the CNS is usually considered an immune privileged site , specifiBax or Bcl-2 in a rat glioma model and analyzed the effects of these transgenes on the in vitro and in vivo growth of these cell lines.We have tested this hypothesis by establishing cell lines, which stably express transgenes encoding either for human ® assay from Promega  as described previously [Unless specified, all reagents used in this study were from Sigma . Monoclonal anti-human Bax antibody (clone 4F11) was from Immunotech  and monoclonal anti-human Bcl-2 antibody (M 0887) was from Dako ; antibodies against rat Bcl-2 or Bax were respectively from Oncogene (Ab5)  and from Pharmingen (13456E) . The fluorogenic peptide Ac-DEVD-AMC was from Bachem  and the lactate dehydrogenase (LDH) activity was measured using the Cytotox 96eviously ,18,21.HelsinkiDeclaration.Experimental research on animals have been conducted according to recommendations of the French National Ethics committee, and are in compliance with the 2 air-humidified atmosphere at 37°C. Plasmids encoding for human Bcl-2 or Bax were subcloned into pRcCMV (Invitrogen) as described by the manufacturer. A15A5 cells were transfected with either 2 μg pCMV vector, pCMV Bcl-2 or pCMV Bax. Plasmid DNA was introduced into 106 cells by electroporation  using 200 V/cm and 250 μF. Transfected cells were selected and cloned in a medium containing neomycin (250 μg/ml) for several weeks before clonal dilution.The rat glioma cell line, A15A5, was obtained from the European Collection of Animal Cell Culture . The cell line was maintained in RPMI-1640  supplemented with 10% heat-inactivated FCS , 100 μg/ml streptomycin, 100 U/ml penicillin and 2 mM L-glutamate in a 5% COin vitro cell proliferation of transfected A15A5 cells.Apoptosis was induced by a short UV-treatment. Both untreated and UV-treated cells were cultured for a further 24 h under serum-free conditions. Cell death was also induced with FasL , doxorubicin , staurosporine , Na-Butyrate  or serum deprivation (d-serum) for 3 days. A MTT assay was used according to the manufacturer's instructions (Promega) to determine 5 cells) or tumors established in rats or mice were homogenized vol./vol. in RIPA buffer . After several passages in a 2 ml glass Dounce homogenizer, the homogenates were centrifuged at 4°C at 13,000 g for 30 min. The resulting supernatants were assayed for protein concentration using the Bradford technique prior to analysis on 15% SDS-PAGE. Western blots were performed as described earlier [A15A5 transfected cells  and were housed under standard conditions in our laboratory. huBax and huBcl-2 as well as pCMV A15A5 cells were injected into the brain of BDIX rats weighing between 200 and 240 g. All animal procedures were performed with approved protocols and in accordance with published recommendations for the proper use and care of laboratory animals. Briefly the rats were anaesthetised with an intraperitoneal injection of pentobarbital (50 mg/kg) and positioned in a stereotactic head frame. Aseptic surgical techniques were used to open the scalp in the midline and to expose the frontal and temporalis bones. A 1.0 mm aperture for implanting tumor cells was drilled through the skull. The stereotaxic position of this injection site was 2.5 mm anterior to the bregma and 2.0 mm to the right of midline. 104 tumor cells were implanted stereotactically at a depth of 3.0 mm into the cerebral parenchyma using a 10 μl Hamilton syringe with a 26-gauge needle. The volume injected was 5 μl and the hole was sealed with sterile bone wax. Alternatively, rats were injected sc with 105 cells into the hindlimbs and tumor growth was monitored every week by measuring the volume of the growing tumors. Swiss nude mice were treated similarly except that 104 cells were injected subcutaneously.Inbred BDIX rats and Swiss sc injections of 3.3 × 104 huBax A15A5 cells 15, 10 and 5 days before injection of pCMV or huBcl-2 A15A5 cells. As a control PBS, A15A5 cell oncolysate, or apoptotic bodies derived from Na-Butyrate (NaB)-treated A15A5 cells as previously described [in vitro by a 10 mM NaB treatment in subconfluent A15A5 cultures. When signs of apoptosis were observed under a microscope  apoptotic bodies were collected, centrifuged and conserved at -80°C prior to use. Typically, 250 μg apoptotic bodies were mixed with 5 mg/ml BCG and injected subcutaneously three times over 15 days. As a control we used oncolysates obtained after several cycles of rapid freezing/ thawing of A15A5 cells and the lysates was injected together with 5 mg/ml BCG to the animals as above. On day 0, 5 days post-treatment, four groups of rats were sc challenged with 105 pCMV or huBcl-2 A15A5 cells. Tumor growth was evaluated daily over 60 days.In order to evaluate the implication of huBax A15A5 cells preventive anti-tumoral treatment, we designed a protocol consisting of three escribed . Briefly2O2 in methanol for 20 min. The sections were incubated overnight at 4°C with anti-rat CD8  diluted 1 in 2 in 1% BSA in PBS, then a secondary antibody coupled to peroxydase was added. The staining was revealed with an AEC substrate.Immunochemical analysis was performed as on 12 μm brain frozen sections. Briefly, sections were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. Endogenous peroxydase activity was inhibited by a treatment with 0,3% H3 pieces, which were incubated in extraction buffer  at room temperature for 45 min and under constant agitation. The cell suspension was filtered through a sterile grid and washed three times with RPMI and maintained overnight in RPMI before analysis. For the phenotypic analysis, monoclonal antibodies obtained from Pharmingen raised against the following molecules were used: CD3 , CD4 , CD8 , CD161 , CMH I (22301 D), OX 41/ CD172 (552297) and OX 62 (555010). Monoclonal antibodies raised against granzyme B  was obtained from Chemicon (France) and Fas (AF 126) from R&D Systems . Cells were incubated with the primary antibodies for 30 min at 4°C and washed twice in PBS + 0.1% BSA. For the intracellular detection of GrB, cells were first fixed in 4% paraformaldehyde for 10 min at room temperature, washed with PBS + 0.1% BSA, then permeabilized with 0.1% saponin then incubated with the anti-GrB antibody for 30 min. The secondary antibody was then added for 30 min at 4°C and the cells washed 3 times with PBS + 0.1% BSA. Cells were analyzed on FACScalibur  using Cell Quest Pro software. A total of 5000 cells were counted in each experiment.Tumors were resected and minced into 1–2 mmBax or Bcl-2 transgenes, were obtained as described in materials and methods. Two different clones were used in each experiment. The expression of the transgenes was monitored by immunoblot analysis using antibodies specific for human Bcl-2 or Bax and, as shown in figure A15A5 cells transfected with the pCMV vector, human To examine the effect of the different transgene expression to cell death, we studied their sensitivity toward different inducers of apoptosis using both drugs such as doxo (20 ng/ml) and STS (20 μM) or treatments such as d-serum, NaB (10 mM) or a short (1 min) UV irradiation. Cell death was monitored and quantified by measuring the activity of caspase 3 (namely the cleavage of the peptide Ac-DEVD-AMC) and the activity of the cytosolic enzyme LDH released into the culture medium . As shown in figure in vitro response to externally added FasL or by transient transfection of pCMV GrB, huBax A15A5 and huBcl-2 A15A5 cells as described previously for human glioma cells [The immune system exerts its anti-tumoral surveillance mainly through cell death induced by CTLs and NK . These cma cells . As showin vitro  into syngenic BDIX rats as described in materials and methods. The survival rates of the different groups of rats were different as the median survival for the group of rats which had received huBcl-2 A15A5 and pCMV A15A5 cells were respectively 10.2 and 15.6 days, a difference, which was highly significant (P = 0.0086) (figure data not shown). Brain sections from rats injected with huBcl-2 or A15A5 cells were histologically examined at the time of their death or after 30 days for rats injected with huBax A15A5 cells. As illustrated in figure To determine the influence of Bax and Bcl-2 on tumoral growth ) figure . On the ) figure . It shouTo gain information about the kinetics of tumoral growth, cells were subcutaneously (s.c.) injected into the hindlimbs of syngenic BDIX rats and the volume of the tumors evaluated every 10 days. As shown in figure 5) were inoculated s.c. into Swiss nude mice . Tumor growth in the mice was measured for a period of 45 days (figure data not shown). This result showed that the growth of the huBax A15A5 was not due to the induction of rat Bcl-2 expression during tumoral growth in the Swiss nude mice.To test the involvement of the innate immunity in the control of proliferation, the different types of transfected cells  class I molecules. Tumoral cells were dissociated and enzymatically treated as described in materials and methods. We observed a significant increase in double positive cells CD3/CD8 in huBax A15A5 tumors (~17%) compared to huBcl-2 (~4%) and pCMV (~6%) tumors  In x figure . Howeverx figure . Howeverx figure . On the The apoptosis index has been found to be of significant prognostic significance in patients with high-grade astrocytomas . Our stuInterestingly, a rat model of colon carcinoma has been described in which some clones gave rise to tumors that constantly expanded in the animal to eventually formed metastasis (Pro) while others progress for several days before complete regression (Reg) . The disOur results show thus for the first time, in the same type of cells, the adverse effects of Bax and Bcl-2 in the antitumoral role of the immune system.Taken together, our data provide evidence that BCL-2 family members control tumor growth through their sensitivity to immune-induced cell death and enhancement of the immunogenicity of tumor cells.The authors declared that they have no competing interests.ic: intra-cerebral; sc: subcutaneous.Bax: Baxα ; CNS: central nervous system; CTL: cytotoxic T lymphocytes; GBM: Glioblastoma Multiforme; Gwenola Bougras carried out the phenotyping characterization of the cell lines and in vivo experiments with tumors, Pierre Francois Cartron the molecular genetic studies, Fabien Gautier carried out the experimental studies with apoptotic bodies and Stephane Martin the intracerebral implantation of the rat glioma cell lines. Marité LeCabellec participated in immunohistochemical characterization of the tumors. Marc Grégoire and Khaled Meflah participated in the coordination of the study. Francois M. Vallette conceived of the study, and participated in its design and coordination and drafted the manuscript.The pre-publication history for this paper can be accessed here:


Wise et al. introduced a rank-based statistical technique for meta-analysis of genome scans, the Genome Scan Meta-Analysis (GSMA) method. Levinson et al. recently described two generalizations of the GSMA statistic: (i) a weighted version of the GSMA statistic, so that different studies could be ascribed different weights for analysis; and (ii) an order statistic approach, reflecting the fact that a GSMA statistic can be computed for each chromosomal region or bin width across the various genome scan studies.We provide an Edgeworth approximation to the null distribution of the weighted GSMA statistic, and, we examine the limiting distribution of the GSMA statistics under the order statistic formulation, and quantify the relevance of the pairwise correlations of the GSMA statistics across different bins on this limiting distribution. We also remark on aggregate criteria and multiple testing for determining significance of GSMA results.Theoretical considerations detailed herein can lead to clarification and simplification of testing criteria for generalizations of the GSMA statistic. Wise, Lanchbury and Lewis  introducn = 120 bins spanning the entire genome, and correspondingly 120 GSMA statistics. Wise  = 0, , andLevinson  proposedand Feng  to derivand Feng , we here.) = φ (z)[1 + c4H4 (z)], where φ(·) is the standard normal density function, , H4 (·) is the 4th degree Hermite-Chebyshev polynomial, H4 (z) = z4 - 6z2 + 3, and the constant c4 given by  (Stuart and Ord ; and, for the remaining order statistics T(110), T(109), ..., T(1), the criterion {PAvgRnk < 0.05 and Pord < 0.05} reduces to {PAvgRnk < 0.05}, equivalently, {Tj) , are less than 1.645 .Levinson  had propPAvgRnk < 0.000417} (0.05 corrected for 120 tests) for evidence that a bin is likely to contain a linked locus or loci. One can improve on this procedure by using Holm's  That is, in this particular instance, the unweighted GSMA statistics with either Bonferroni or Holm  As for the order statistic formulation and the aggregate criteria of Levinson [For practitioners utilizing GSMA statistics, the question arises as to whether the methods proposed here as well as in Koziol and Feng  are mereLevinson ), then tLevinson  or KozioLevinson , and shoLevinson  providesLevinson  and KoziLevinson  will typLevinson  can be bLevinson , we beliJAK conceived, designed and drafted the manuscript. ACF performed the statistical simulation.Both authors read and approved the final manuscript.


Haemophilus influenzae, was published in 1995. Since then, more than 400 microbial genome sequences have been completed or commenced. This massive influx of data provides the opportunity to obtain biological insights through comparative genomics. However few tools are available for this scale of comparative analysis.The first microbial genome sequence, The BLAST Score Ratio (BSR) approach, implemented in a Perl script, classifies all putative peptides within three genomes using a measure of similarity based on the ratio of BLAST scores. The output of the BSR analysis enables global visualization of the degree of proteome similarity between all three genomes. Additional output enables the genomic synteny (conserved gene order) between each genome pair to be assessed. Furthermore, we extend this synteny analysis by overlaying BSR data as a color dimension, enabling visualization of the degree of similarity of the peptides being compared..Combining the degree of similarity, synteny and annotation will allow rapid identification of conserved genomic regions as well as a number of common genomic rearrangements such as insertions, deletions and inversions. The script and example visualizations are available at:  Haemophilus influenzae genome sequence in 1995 [In the decade since the publication of the  in 1995 ; as of OBLAST analysis has become a ubiquitous method of interrogating new sequence data, but there are limitations to using BLAST alone as a discriminating tool. Many other methods and individuals use BLAST output E-values as a criterion for data parsing. While this measure may be efficient, the output is often skewed by both the database used for comparison and the length of the match . Small rSeveral other tools utilize the BLAST algorithms to compare nucleotide or peptide sequences from genome projects. The Wellcome Trust Sanger Institute ACT software  can dispBSR analysis is a departure from traditional genome scale analyses as it overcomes the limitations of BLAST E-values in comparative studies by normalizing the BLAST raw scores. BSR analysis is a tool for the rapid comparison of complete proteomes of any three genomes, and enables a visual evaluation of the overall degree of similarity of these proteomes and their genomic structure.1" and "Query2" respectively. Initiation of the script results in each of the putative peptides in the Reference proteome being compared to all of the other peptides in the Reference and Query proteomes using NCBI BLASTP.We have implemented the BSR algorithm using Perl. The inputs are the predicted proteomes of each of the three genomes under analysis, formatted as multi-FASTA files. An additional file for each of the proteomes is required. This file must contain a unique identifier, matching the FASTA header of the corresponding peptide in the multi-FASTA file, the relative genomic location of the start and stop of the coding region as well as the annotation for each peptide. The user selects one proteome as the "Reference"; the other two proteomes are termed "Query1 and Query2 proteomes with each best BLAST raw score recorded as Query1 and Query2, respectively  output contains an ordered list including the Reference peptide unique identifier, annotation, and Reference BLAST raw score, in addition to the unique identifier of the best hits in the Query proteomes, corresponding BLAST raw scores and the calculated BSR. Additionally, four unique files are generated corresponding to the peptides within the four quadrants delineated in Figure Following calculation of the BSRs, a number of output files are generated, including both text and graphical formats. The text files are tab-delimited for ease of parsing; filenames are derived from the named proteome files used as input into the script. The R_Q1_Q2.txt  between the two genomes , howevere Figure .The Gnuplot, PostScript and xfig outputs allow publication-quality, global visualization of the similarity and synteny of the selected genomes. However these formats do not allow the annotation associated with individual peptides to be viewed interactively. To overcome this limitation, additional XML files for the similarity and synteny plots described above are generated. These files are the input for the freely available GGobi software. GGobi is a data visualization system for viewing high-dimensional data . The tooThe GGobi package also allows the expansion of the BSR approach to include more than three genomes or other additional parameters associated with proteomic or genomic data, enabling interactive, user-driven exploration of these complex datasets. The current BSR implementation uses three genomes as input; however, additional genomes can readily be added as new dimensions simply by repeating the analysis with the same Reference genome and varying the Query genomes. Additional non-BSR dimensions are readily included, such as pI or %GC, or factors such as surface localization or some other feature of the peptides of interest.C. pneumoniae AR39 (GenBank Accession Number AE002161) is more similar to C. caviae GPIC (GenBank Accession Number AE015925) than C. muridarum strain Nigg (GenBank Accession Number AE002160) as 7.3 % of the proteome is shared between only C. caviae GPIC and C. pneumoniae AR39 compared to only 1.6% between C. caviae GPIC and C. muridarum strain Nigg. Finally, Figure C. caviae proteome has no significant hit to any of the peptides in the Query proteomes, although many of these peptides (78.2%) are currently annotated as hypothetical.Genome structure is often altered during the evolution of species . VisualiC. caviae GPIC and C. pneumoniae AR39 reveals that the genomes contain two points of inversion  and greater than 50% amino acid identity. This group of peptides is more readily identified by BSR analysis than BLAST E-value, which is artificially low due to the small peptide size. Additionally, peptides that have a BSR score < 0.4 but a BLAST E-value < 1 × 10-15 correspond 7.8% of the proteome. These represent divergent peptides with an artificially high BLAST E-value score resulting from limited regions of identity. The BSR analysis more accurately classifies these peptides based on the amino acid identity over the entire peptide. As the BSR comparison utilizes a single genome as a reference, the BSR score is calculated using a unidirectional best BLAST hit. However, when the Chlamydial proteomes were compared only one case in over 1000 could be found with a BSR score > 0.4 that was not also a bidirectional best BLAST hit.In the analysis of the Chlamydial proteomes using BSR score and BLAST E-values approximately 1% of peptides examined have a BSR score > 0.4 and BLAST E-value > 1 × 10The BSR approach allows rapid evaluation of the level of conservation of any three proteomes and the degree to which the genome structure between the three genomes is similar. While in this report we discuss the applications of this approach to whole genomes, the analysis has been performed on portions of genomes such as genomic or pathogenicity islands, plasmids and phage to identify peptide similarity and regional structure.More genome sequences are being generated from closely related organisms – a trend which shows no sign of abating. The BSR approach has become a crucial tool in our comparative genomics armamentarium and has been utilized in a number of genomic comparisons, revealing regions of similarity and difference between both closely and distantly related organisms ,15,16.Project name: BSR.plProject homepage: Operating System: Unix and MacOS XProgramming language: PerlOther requirements: Perl Statistics::Descriptive module License: NoneAny restrictions to use by non-academics: NoneBSR – BLAST score ratio; BLAST – basic local alignment search tool.DAR, GSAM and JR conceived and implemented the first versions of BSR and prepared the manuscript. All authors have read and approved the final manuscript.


Ilex (Aquifoliaceae) and discrepancy between plastid and nuclear phylogenies is the rule in this genus. These observations suggest that inter-specific plastid or/and nuclear introgression played an important role in the process of evolution of Ilex. With the aim of a precise understanding of the evolution of this genus, two distantly related sympatric species collected in Tenerife (Canary Islands), I. perado and I. canariensis, were studied in detail. Introgression between these two species was previously never reported. One plastid marker (the atpB-rbcL spacer) and two nuclear markers, the ribosomal internal transcribed spacer (ITS) and the nuclear encoded plastid glutamine synthetase (nepGS) were analyzed for 13 and 27 individuals of I. perado and I. canariensis, respectively.Intra-specific and intra-individual polymorphism is frequently observed in nuclear markers of I. perado, they are highly polymorphic in I. canariensis. The presence of pseudogenes and recombination in ITS sequences of I. canariensis explain this polymorphism. Ancestral sequence polymorphism with incomplete lineage sorting, or past or recent hybridization with an unknown species could explain this polymorphism, not resolved by concerted evolution. However, as already reported for many other plants, past or recent introgression of an alien genotype seem the most probable explanation for such a tremendous polymorphism.The plastid marker is intra-specifically constant and correlated with species identity. On the other hand, whereas the nuclear markers are conserved in I. canariensis, but I. perado is suspected. The introgression would be unilateral, with I. perado as the male donor, and the paternal sequences would be rapidly converted in highly divergent and consequently unidentifiable pseudogenes. At least, this study allows the establishment of precautionary measures when nuclear markers are used in phylogenetic studies of genera having experienced introgression such as the genus Ilex.Data do not allow the determination with certitude of the putative species introgressing  Ilex  using the unique maximum parsimony tree obtained from the ITS matrix of Manen et al.  and these parameters were used to calculate the tree scores of simulated clonal sequences.The minimum number of recombination events RM  was calc to PIST  to calcuThe recombination points of three selected sequences (see results) showing evidence of recombination were calculated by maximum likelihood using LARD  with theITS matrix in NEXUSClick here for file


Schistosoma mansoni and Plasmodium chabaudi develop higher P. chabaudi parasitaemia and had a higher mortality rate. This important observation deserved to be further investigated among human populations.Helminthic infections concomitant with malaria are common in inter-tropical areas. A recent study showed that mice co-infected with S. mansoni, and 175 were not. The incidence rate of malaria attacks was significantly higher among S. mansoni-infected individuals, particularly those carrying the highest worm loads, as compared to uninfected subjects . In contrast, the rate of malaria attacks was lower, without reaching significance, in medium grade S. mansoni infections. Thus, infection by S. mansoni affects susceptibility to malaria, but this can vary according to the intensity of parasite load. The immunological mechanisms underlying this dual effect need to be further explored.Malaria attacks were recorded in 512 children aged 6–15 years living in Richard Toll  among whom 336 were infected by  Schistosoma mansoni and Plasmodium chabaudi co-infected mice develop higher P. chabaudi parasitemia and higher mortality rate [Malaria is prevalent in many parts of tropical Africa, where other concomitant parasitic infections are common. However, little is known about how concurrent infections affect the expression to and/or pathogenesis of each other. A recent study showed that ity rate . This obity rate  and by fity rate .S. mansoni infection [In the Senegal river basin, where malaria is hypoendemic, extensive irrigation programmes have been developed, promoting a spread of nfection . The expThe study took place in Gallo Malick, a district of Richard Toll, which is located near a canal ensuring the irrigation of rice and sugar cane fields. The population was estimated in 1998 to reach 3,685 inhabitants. Annual rainfall amounts to around 250 mm water. The district is approximately 1 km long and 500 m wide. A dispensary is set up in the centre of the district and is thus easily accessible.In September 1998, after obtaining agreement from the parents, two stools examinations were carried out in all children aged between 5 and 15 years. From 25 mg of stools, two slides were prepared, according to the modified Kato-Katz method [Schistosoma haematobium eggs, were performed. Ten ml of urine were filtered through a 12 μ millipore membrane and the eggs on the whole filter surface were counted. A questionnaire was issued to check habits with regard to protection against mosquitoes and usage of anti-malarial drug-prophylaxis. It was also ascertained that none of the included children had been administered anti-S. mansoni treatment (praziquantel) over the past year (September 97 – September 98). In March 99, all children who presented with at least one S. mansoni positive stool examination were treated with praziquantel (40 mg/kg).The follow-up was implemented between September 1998 and April 1999. In January 1999, a further stool examination and a urine filtration for the detection of Any child presenting with a body temperature > 38°C or having a history of fever-in the 24 hours preceding the consultation and with a parasite density ≥ 5000 parasites/μl of blood was defined as suffering from a malaria attack.S. mansoni at least once was defined as S. mansoni positive. If both examinations proved positive, the highest parasitic burden was kept for further analysis. Parasite loads were classified into four categories: A) 1 to 100, B) 101 to 400, C) 401 to 1000, and D) > 1000 eggs per one gram of stools.Any child whose tool tested positive for S. mansoni but presenting other intestinal parasitic infection were excluded from the analysis.In order to exclude possible interactions with other helminthic infections, children from the group not infected by S. mansoni. This is in agreement with the distinct sources of infection, Anopheles larvae breeding in small water collections in gardens, and Schistosoma vectors in rice fields and irrigation canals.When houses where children had a malaria attack and those where the children were carrying schistosomiasis were located on a map, the two pathologies were found to be distributed in a uniform way in the whole district, without evidence of clustering of double infection malaria-S. mansoni categories eggs load and age.Comparison between children presenting at least one malaria attack and children not having presented any malaria attack was performed using a forward logistic regression model using STATA statistical software. The variables included in the model were sex, S. mansoni infections using a lifetime estimate table from the EGRET programme . Any p value lower than 0.05 was considered significant.The period between the beginning of the study and the date of a malaria attack was compared between groups of S. mansoni eggs and 189 were negative for both stool examinations.In September 1998, 525 children underwent two stool examinations. Three-hundred and thirty-six-were found to carry Schistosoma haematobium (2.7%). Among them, 11 were also positive for S. mansoni. The stool examination showed the presence of 31 other intestinal parasitic infections (Ascaris and Trichuris). Patients negative for S. mansoni and carriers of another intestinal (12 subjects) or urinary parasite (2 subjects) were excluded of the analysis.Urine filtration detected only 13 children excreting eggs of S. mansoni (65.7%) and 175 (34.3%) not infected). The prevalence increased as a function of age, reaching 80% in children older than 11 years with > 30% of children presenting with high schistosome loads, i.e. > 1,000 eggs/ 1 g of stools.In total, 511 children were included in the analysis  were attributed to a malaria attack, according to the criteria described. Only 10 children underwent two malaria attacks. In total, 18.9 % (97/511) of the children included in the analysis presented with a malaria attack at least once.S. mansoniior not. However, the malaria attack prevalence was also the same whether or not the children were sleeping under a mosquito bednet.Chemoprophylaxis for children is not recommended by the National Control Programme, but 7.9 % of the parents reported that they were giving a chemoprophylactic treatment to their children. 58.9 % stated they used a mosquito bednet. These proportions were the same in the two groups of children, infected by S. mansoni and 16.6% (29/175) in those non-infected (p = 0.40). The malarial incidence, however, varied depending on the load of eggs. It was high in all groups, but the highest (26.6 %) in those carrying the highest worm loads , except in subjects presenting medium loads (>100 and < 400 eggs/g of stools) where malarial incidence was lower than in S. mansoni negative individuals (9.4 %). Sex and age had no significant influence, which is not surprising since this is a mesoendemic area. Using a logistic regression model and taking the negative group as reference, the difference was significant with the group carrying a high load of eggs (RR = 2.24 (1.2 – 4.2) , the malaria incidence was significantly increased for low S. mansoni egg carriage (1–100) (RR = 2.51 (1.05–6) as well as high S. mansoni egg carriage  RR = 3.12(1.33–7.29).The incidence rate of malaria attacks was 20.2 % (68/336) in the group of children concomitantly infected by Out of 10 children who presented two malaria attacks, nine were infected by S.m.; four presented a schistosome load higher than 1,000 eggs/g of stools, and three > 400 and <1,000.S. mansoni loads and other groups increased over time during the follow-up  or a low load of eggs (1–100), whereas a lower attack rate was observed in children presenting a medium egg load (>100 and <400 eggs/g), although this opposite trend did not reach significance.The analysis of the incidence of the malaria attacks by amount of parasite load in Protection against falciparum malaria has been found to be associated with the preferential production of the cytophilic classes, IgG1 and IgG3, of antibodies , this beS mansoni, but only for those having the highest loads. The opposite effect was found in individuals carrying medium schistosome infections. As immunological studies could not be performed, one can only speculate about this biphasic effect. The production of cytophilic classes against malaria requires that the T-cells helper effect is provided by Th1 type of T-cells, however cytokines produced by T-cells specific for schistosome eggs or worms can influence responses to malaria. It has been reported that, during the first phase of S. mansoni infection, T-cells are stimulated which secrete cytokines belonging, in majority, to the Th1 type. The switch towards Th2 type cytokines occurs later on and is dependent on egg production [S. mansoni eggs.In the present study, this observation has been confirmed with yet another worm infection, oduction . In the Obviously, further studies, particularly of T-cell responses to both malarial and schistosome antigens are required to sort out this issue.S. mansoni infection can increase the susceptibility to malaria in subjects excreting high schistosome egg loads.Helminthic infections are a fact of life in malaria endemic areas and their influence on the course of infection and the epidemiology of malaria is a fascinating though neglected area of research. This study shows that CS, PM, JA, PC, MD were responsible for field and laboratory studies. JYL was responsible for the statistical analysis. PD inspired and designed the study


The discussion about the interplay between theory and intervention has a long history and much has been written and published. The companion perspective papers by Rothman and Jeffery provide alternative points of view on this important and complex question. Their papers are not intended to be critical reviews of the literature. Instead, the aims of the two papers are to delineate the problem, present the different perspectives of the authors on the issue, and suggest constructive steps researchers can take to move the field forward.We hope that these articles generate a lively response from our readers and spur innovative ideas for strategies to cultivate a healthy developmental interplay between theory and intervention in behavioral nutrition and physical activity research.We invite you to consider the intellectual and practical challenges posed by the two authors and perhaps respond with your perspectives, which may be published in the IJBNPA.We look forward to hearing from you soon.Simone A. French, Ph.D.Tony Worsley, Ph.D.Co-Editors, IJBNPA


Multiple studies have linked fungal exposure to asthma, but the link to severe asthma is controversial. We studied the relationship between asthma severity and immediate type hypersensitivity to mold  and non-mold allergens in 181 asthmatic subjects.Aspergillus, Alternaria, Cladosporium, Penicillium and Candida) and 4 other common inhalant allergens .We recruited asthma patients aged 16 to 60 years at a University hospital and a nearby General Practice. Patients were categorized according to the lifetime number of hospital admissions for asthma . All subjects had allergy skin prick tests performed for 5 mold allergens . Multiple mold reactions were also much commoner in the group with multiple admissions . The number of asthma admissions was related to the number and size of positive mold skin allergy tests  and less strongly correlated to the number and size of non-mold allergy tests . Hospital admissions for asthma patients aged 16–40 were commonest during the mold spore season (July to October) whereas admissions of patients aged above 40 peaked in November-February .Skin reactivity to all allergens was commonest in the group with multiple admissions. This trend was strongest for mold allergens and dog allergen and weakest for These findings support previous suggestions that mold sensitization may be associated with severe asthma attacks requiring hospital admission. Alternaria species has been reported to be especially common in patients with life-threatening asthma [Most asthma patients have mild symptoms which are well controlled with anti-inflammatory and bronchodilator therapy but a minority of asthma patients have severe airway inflammation and airflow obstruction requiring multiple hospital admissions. The reasons for these differences in asthma severity are complex and not fully understood . At leasg asthma . Asthma g asthma -11. Moldg asthma -17.Indoor mold exposure might also contribute to asthma severity. Many patients believe that their asthma is aggravated by damp housing, especially if there is visible mold growth . It has These studies prompted us to undertake a study of asthmatic patients in Salford in Greater Manchester (North-West England) to assess whether atopy in general and mold sensitization in particular was associated with increased asthma severity. There are few agreed definitions of severe asthma so we used hospital admissions and treatment category (according to British Thoracic Society Asthma Guidelines) as surrogate markers for asthma severity .This study was undertaken in Hope Hospital, a 900 bedded University Hospital in Salford, Greater Manchester, UK. The study was designed to record the atopic status for mold and non-mold allergens of asthma patients at every level of severity from very mild to very severe . We studied patients with severe asthma during hospital admissions or at subsequent visits to the hospital chest clinic. Patients were recruited opportunistically during hospital admissions or during routine consultations with the Respiratory Nurse Specialist (RNS) in the hospital Chest Clinic. Recruitment was evenly spread over 30 months from January 1996 to June 1998. During this period, the Respiratory Nurse service was under development with only one part-time RNS available to serve a large population of hospital in-patients and ambulatory care patients at chest clinics. Ward-based doctors and nurses referred patients with acute asthma to the RNS on an opportunistic basis based on availability of the part-time RNS and whether the ward teams were aware of the developing RNS service. About 20% of all adult asthma patients admitted during the study period were referred to the RNS and most of these were recruited in the study (provided the RNS had time available to do so). Although recruitment was not systematic (based mainly on availability of RNS service), we were not aware of any potential bias which might have affected the recruitment process and we believe that the recruited patients were typical of adult patients with acute asthma admitted to our hospital. We recruited ambulatory patients  at the same hospital chest clinic and in a single Primary Care practice where 25 patients were recruited during routine consultations with the practice nurse. This recruitment was also opportunistic, based mainly on the availability of time to complete the study protocol during busy clinics. We believe that these patients were typical of patients seen at hospital chest clinics and General Practice asthma clinics in this area. Patients were grouped according to their lifetime history of hospital admissions for asthma . Patients with no hospital admissions were further categorized according to the treatment steps described in the British Thoracic Society guidelines for asthma management; Step 1 requiring only occasional bronchodilator treatment, Step 2 requiring low-doses (<800 mcg per day) of inhaled steroid, Step 3 requiring high doses of inhaled steroids or long acting beta agonists, Step 4 requiring additional therapy such as domiciliary nebulized therapy . We atteAll patients were Caucasians who were lifelong residents of the United Kingdom. Inclusion criteria were a diagnosis of asthma by the patient's doctor, age 16 to 60 and ability to give informed consent. All subjects gave written informed consent prior to partaking in the study which was approved by the Salford Research Ethics Committee. Exclusion criteria included a diagnosis of COPD, non-European ethnic group  and consumption of any antihistamine in the previous 48 hours. All subjects completed a questionnaire concerning respiratory symptoms, smoking status and allergies.Dermatophagoides pteronyssinus, cat, dog, mixed grass pollen, Aspergillus fumigatus, Alternaria alternata, Cladosporium herbarum, Penicillium notatum and Candida albicans.All tests were conducted by one author (LCH) or by one other respiratory nurse specialist using a standardized technique in an open manner. A small drop of allergen was placed on the volar surface of the forearm. Allergens were purchased from Allergopharma , a single batch of each allergen was used throughout the study. We used skin test lancets with a 1 mm tip . The lancet was introduced vertically into the skin through the allergen solution. Allergens studied were: negative control, histamine 0.1%, Aspergillus and a 5 mm reaction to Cladosporium would have a "mold sensitization score" of (6-1)+(5-1) = 9 mm.Weal diameter (if any) was recorded at 15 minutes. If a weal was asymmetrical, the mean of two perpendicular measurements was calculated. Weals less than 3 mm greater than the negative control reaction were regarded as negative in accordance with guidance issued by the European Academy of Allergology . We deviAs a subsidiary study , we also studied the seasonality of asthma admissions in Salford by reviewing the electronic records of 520 asthma admissions under the care of two pulmonary physicians who kept a computerized database at this hospital between 1995 and June 2000 . This covered a wide time-span before, during and after the period of the mold sensitization study; some of the allergy study patients were admitted and recruited during this time but they represented only a small (and random) proportion of the admissions studied for seasonality. Asthma admissions were analyzed by month of admission and divided into 3 four-month "seasons"; March to June ; November to February ; . PatientStatistical analysis was performed using Prism II software . Chi squared test was used to compare the number of positive allergy skin tests between groups of asthma patients. Spearman Correlation Coefficient was used to compare each patients lifetime number of hospital admissions with their "Mold sensitization score" (as described above). Mann Whitney tests were used to compare mean sensitization scores between groups of patients.One hundred eighty-one asthmatic patients were recruited. Their characteristics are given in table Positive skin tests to all allergens were commoner in the group with severe asthma  than patients with milder asthma. Table . Atopic Alternaria) to 41% (Cladosporium) in the severe asthma group compared with 0–10% in the milder asthma groups.Mold sensitization was uncommon in mild asthma but very common in asthma patients with multiple admissions Figure . There wAspergillus and Candida precipitins and specific IgE were not measured in this study. None of these patients had any clinical features suggestive of allergic bronchopulmonary aspergillosis (ABPA) such as pulmonary infiltrates, bronchiectasis or marked eosinophilia. The number of admissions correlated with the number and size of positive skin tests using the scoring system described previously. For mold sensitization, the Spearman Correlation Coefficient was 0.60 (two-tailed p < 0.0001) and for non-mold allergens was 0.34 (two-tailed p = 0.0005). The cumulative "mold sensitization score" and the "non-mold sensitization score" for each group of patients is shown in table Aspergillus, one Penicillium).Of the patients not admitted to hospital, 32 had very mild asthma (BTS Step 1), 25 had mild-moderate asthma (BTS Step 2) and 25 had moderate to severe asthma (BTS Steps 3–4). There was no significant difference in mold or non-mold sensitization between these groups of non-admitted patients with different grades of asthma severity.Our review of asthma admissions to this hospital between 1995 and 2000 identified 520 patients admitted under the care of the two chest physicians who kept a computerized database . There were 173 asthma admissions in the 16–40 age group, these admissions peaked in late summer and fall (figure Alternaria or Cladosporium is a powerful risk factor for severe asthma[D. pteronyssinus) sensitization in our patients with more severe asthma [Although the present study was larger than most previous studies of mold sensitization in severe asthma, it must be regarded as a "pilot study" due to the non-systematic recruitment of asthma patients and the cross-sectional nature of the study. Our data indicate that mold (and dog) sensitization is common in patients with severe asthma requiring multiple hospital admissions in Manchester. The results are consistent with previous evidence that atopy  is related to asthma severity or bronchial hyper-reactivity -6,12-17.re asthma in severre asthma,14. We he asthma ,25. HoweThere has been some debate about the best cut-off point for weal size to define a positive skin-prick test. We accepted the European Academy of Allergology figure of 3 mm greater than the negative control . HoweverA key question is whether severe asthma is actually caused by sensitivity to molds or is simply associated with it. In any case, mold sensitivity will certainly not be the only cause of severe attacks of asthma; upper respiratory tract virus infections and some drugs being two of other well documented causes. The greater degree of mold sensitization in the severe asthma group could simply reflect an extreme example of the generalized increase in atopy amongst this group. However, we believe that mold allergy may be responsible for severe asthma attacks for several reasons.Alternaria-sensitive had their near-fatal asthma episodes in summer and early fall when mold spore levels would be expected to be high [First, the temporal relationship between high environmental spore counts and asthmatic attacks is strong. Airborne spore levels may be up to 1000 times higher than pollen levels . The dat be high .Cladosporium in July, Alternaria and hyaline basidiospores in August, uredospores in September and coloured basidiospores in October. The data from Derby (52 miles south-east of Manchester) are similar[Cladosporium, Alternaria, Penicillium and Aspergillus with maximal levels between June and October [A. alternata and C. herbarium was common and associated with asthma severity – OR of 2.03 for the former, of 3.2 for the latter fungus and 2.34 for both. No such association was found for pollens or cats, although sensitization to house dust mite was slightly more frequent in those with severe asthma (OR 1.61) [Second, the seasonal  peak of asthma admissions occurs when ambient air counts of molds are high. We have documented a late summer-fall peak of asthma admissions involving young adults in Manchester which coincides with the summer-fall peak of asthma deaths in UK patients aged under 35 years ,21. These similar. In addi October . A pan-E October . SensitiOR 1.61) . The preThird, there is evidence that indoor mold exposure may contribute to asthma severity. Many patients report respiratory symptoms in damp and moldy houses and a review of nine population-based studies found that seven reported one or more positive associations between fungal levels and health outcomes . The stuAspergillus in particular is a major respiratory allergen causing the vast majority of cases of allergic bronchopulmonary mycosis [Aspergillus and Candida allergens may share IgE-binding epitopes [Aspergillus and a few other common airborne fungi have and it is likely that sensitization to other fungi will be discovered in the future.Fourth, we know that  mycosis -33. It iepitopes . Howeverepitopes . Few funCandida is present in the gut of most, if not all, humans. The difference may relate to the nature or intensity of exposure to mold allergens or to their ability to become airborne and to gain entry to human airways due to their small size. Also many potent allergenic proteins have been described in Aspergillus and some in other fungi [Aspergillus is inhaled, which is almost hourly. Some fungal antigens are proteases  and as DP1 is also a protease – a similar pathogenic role can be postulated [It is not known why mold allergens should produce more severe airway disease than other common allergens such as house dust mites, cat dander, or grass pollen. Fungi are very common in the environment and er fungi . There istulated ,36.It therefore seems likely that there is a causal relationship between mold allergy and asthma severity for some younger asthma patients. Our seasonal admissions data suggest that up to 6% of adult asthma admissions in Manchester and 19% of asthma admissions in the 16–40 age group may be attributable to mold allergy. These unlucky individuals seem to have more severe asthma than patients with sensitization to other common allergens and an increased risk of fatal or near-fatal asthma or hospital admissions during the mold spore season, especially on days when the local mold spore count is high. The risk may be further increased if the patient lives in a damp, moldy house or attends a damp moldy school ,30,37. ICandida albicans, is a yeast, does not become airborne but does produce hyphae in tissue. Thus it is possible that human asthma due to fungal allergens may have three sources of allergen exposure – outdoor mold spores, indoor mold spores and endogenous fungal growth on body surfaces including the skin and gut [One of the fungi which we investigated and for which antigenic extracts are available,  and gut .Alternaria sensitization in Italian patients with respiratory symptoms ranges from 2% in Northern Italy to 29% in Southern Italy [Fungal sensitization is relatively uncommon in our British asthma patients and in Finnish schoolchildren  comparedrn Italy . Some ofrn Italy . Also furn Italy ,16,17 suThe findings of this study support previous suggestions that mold sensitization may be associated with severe asthma attacks requiring hospital admission.BTS Guidelines = British Thoracic Society Guidelines for Asthma Management, RNS = Respiratory Nurse SpecialistThe author(s) declare that they have no competing interests.ROD originated and co-ordinated the study and contributed to the analysis of the data and preparation of the paper.LCH contributed to the design of the study and was the main clinical investigator. She also contributed to the analysis of the data and preparation of the paper.DWD contributed to the design of the study and contributed to the analysis of the data and preparation of the paper.The pre-publication history for this paper can be accessed here:


Public repositories of microarray data contain an incredible amount of information that is potentially relevant to explore functional relationships among genes by meta-analysis of expression profiles. However, the widespread use of this resource by the scientific community is at the moment limited by the limited availability of effective tools of analysis. We here describe CLOE, a simple cDNA microarray data mining strategy based on meta-analysis of datasets from pairs of species. The method consists in ranking EST probes in the datasets of the two species according to the similarity of their expression profiles with that of two EST probes from orthologous genes, and extracting orthologous EST pairs from a given top interval of the ranked lists. The Gene Ontology annotation of the obtained candidate partners is then analyzed for keywords overrepresentation.We demonstrate the capabilities of the approach by testing its predictive power on three proteomically-defined mammalian protein complexes, in comparison with single and multiple species meta-analysis approaches. Our results show that CLOE can find candidate partners for a greater number of genes, if compared to multiple species co-expression analysis, but retains a comparable specificity even when applied to species as close as mouse and human. On the other hand, it is much more specific than single organisms co-expression analysis, strongly reducing the number of potential candidate partners for a given gene of interest.CLOE represents a simple and effective data mining approach that can be easily used for meta-analysis of cDNA microarray experiments characterized by very heterogeneous coverage. Importantly, it produces for genes of interest an average number of high confidence putative partners that is in the range of standard experimental validation techniques. The availability of genome sequences from several model organisms, including humans, and of high-throughput technologies to study gene function is dramatically changing the approach to biological problems. In particular, the consolidated reductionist gene-by-gene strategy is being replaced by a 'modular approach', in which several genes are studied simultaneously to gather a more comprehensive picture of the many different cellular processes : in liviIt is well recognized that one of the most important and widespread mechanisms used by cells to regulate functional modules is the coordinate transcriptional and/or post-transcriptional modulation of mRNA levels of the interacting genes. Therefore, DNA microarrays represent a fundamental tool to unravel biological complexity on a genome-wide scale. Information concerning the expression of thousands of genes, and also of different transcripts from the same gene, can be obtained in a single experiment, and the relationships among gene expression patterns can be studied systematically . The extBesides being useful to other researchers to confirm the published results, the deposited datasets can be used as a substrate for new analysis, aimed at discovering functional modules by searching for related expression profiles. Recent studies have shown that, if the expression of two or more genes is consistently related throughout many independent microarray datasets, the genes display a significant degree of functional similarity ,8. HowevHomo sapiens, Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans was very effective in identifying new genes involved in core biological functions [Phylogenetic conservation has been recently proposed as a very strong criterion to identify functionally relevant co-expression links among genes . Signifiunctions . AlthougSince regulatory regions diverge much more rapidly than coding sequences ,13, a siAdditionally, when using microarray data to establish significant correlations among gene expression profiles, almost invariably the information obtained with probes covering different gene portions is averaged . Though Coexpression-based Linking of Orthologous ESTs) a new data mining method for the identification of transcripts showing evolutionary conserved co-expression in cDNA microarray datasets. This approach is based on the pairwise comparison of data from two species. The predictive capability of the method was proved by comparing human with mouse data. Our results show that CLOE is a valuable tool for biologists that can be used to identify putative partners for genes of interest and/or to predict some of their functional properties.In this study we describe CLOE   . We then generated the respective ranked lists, subdivided them in 1% rank intervals and analyzed the number of orthologous pairs in corresponding rank intervals. As a control, we performed the same analysis on an equal number of randomly chosen (and hence non-orthologous) human-mouse EST pairs. The analysis was repeated three times with essentially identical results.-94, chi square test). The difference was still very significant in the 2% rank interval, even though with a much higher p value (p = 1.3·10-10), but was not detectable below that threshold. Interestingly, a slight enrichment was also observed in the last rank interval . The latter observation is consistent with the previously noted fact that negative correlations tend to be less common and significant than positive correlations [As shown in Figure Saccharomyces cerevisiae [Drosophila melanogaster [Caenorhabditis elegans [We next investigated the effectiveness of our approach, by comparing it to single and multiple organisms co-expression analysis. To address this point, we analyzed the ability of the three methods to predict known physical and functional interactions among mammalian proteins. Protein-protein complexes have begun to be determined on a genome-wide scale only for revisiae , Drosophnogaster  and Caen elegans , but no  elegans  . On the other hand, the average percentage of CLOE links that correspond to a documented protein-protein interaction was 6.6 %, i.e. approximately 5 times higher than that obtained with the single organism method  ratios.Within these datasets, the number of ESTs representing a given transcription unit, as well as the number of valid experiments for each EST, are highly variable. While this feature would pose serious problems, if one should attempt to average the data of all the probes belonging to the same gene, it may offer extremely valuable information when every EST is considered independently, since each clone explores the expression properties of a particular group of exons, in a particular combination of experimental situations.Non-correlated or anti-correlated expression between two well-represented EST probes belonging to the same gene would strongly suggest that they correspond to alternatively expressed transcripts. For this reason, we decided not to merge the data of probes belonging to the same gene, but to treat separately every EST probe in the datasets.The choice of the most representative EST for the genes of interest represents a particularly critical aspect of our procedure. If interested to a single gene, the more exhaustive solution to this problem would be to generate and evaluate a list of candidate partners for every possible pairwise combination of ESTs probes. However, this would greatly complicate the analysis should one be interested in analyzing the potential partners of many genes. An alternative possibility is to focus, for every Unigene cluster, on the most representative ESTs, i.e. the probes with the highest number of experiments. For these reasons, the decision about what ESTs to analyze is left to the end user.Our implementation of the method can accept as input both a UniGene cluster ID or the results of a BLAST search performed with the sequence of interest against the EST database. In both cases, it retrieves a list of all probes found in the two datasets for the orthologous UniGene clusters.To help the user decide which ESTs to analyze, the program provides basic information about all the EST probes representing the gene of interest in the two datasets. Moreover, to help the user identify the most representative EST probe in each dataset, i.e. the probe with the highest number of experiments, it also provides the number of valid data points for every probe. Finally, to assess the redundancy of the information provided by the different probes, i.e. whether they represent different experiments and display similar/different expression profiles, the program calculates, for every pair of probes belonging to the same UniGene cluster, the number of common experiments and the Pearson correlation coefficient between their expression profiles.r) with every other EST in the respective dataset. The raw r is the normalized for the number of common data points (n) obtained for the analyzed ESTs. This is done by multiplying r for , since the statistical significance of r is a function of the product ·r. Such normalization is particularly important when, as in our case, n has a very wide range of variation. The ESTs are then ranked by decreasing normalized score. Finally, a user-defined top percentage of the two ranked lists is compared to identify those ESTs that are associated to the same InParanoid cluster ID. A non-redundant list of the positive InParanoid clusters, sorted by the average highest rank obtained in the two organisms, represents the main output of the program ;readme.txt (detailed instructions for using our program).MP: development of most of the software, execution of the described analysis.PP: development of the routines used to calculate the normalized Pearson, supervision of the statistical analysis.LS: assessment of the biological significance of results.FD: supervision of the project, manuscript writing.


This article reports a cDNA collection representing more than 2000 new, full-length transcripts from a high-quality cDNA library. A large number of cDNA inserts were sequenced from a high-quality library of chicken bursal lymphocyte cDNAs. Comparisons to public gene databases indicate that the cDNA collection represents more than 2,000 new, full-length transcripts. This resource defines the structure and the coding potential of a large fraction of B-cell specific and housekeeping genes whose function can be analyzed by disruption in the chicken DT40 B-cell line. Large-scale genomic and cDNA sequencing projects have revealed thousands of new genes whose open reading frames (ORFs) are highly conserved during vertebrate evolution, but whose precise cellular functions remain unclear. Although functional analysis by gene disruption is possible after transfection of murine embryonic stem cells and the breeding of knockout mice , these win silico gene structure prediction methods have a high error rate and often do not correctly annotate the intron-exon structure of genes. Only full-length cDNAs unambiguously define the boundaries of the transcription units within whole-genome assemblies and cloned full-length cDNAs are also of immense practical value to complement mutant phenotypes and artificially express the encoded protein [The recent release of the chicken genome sequence  greatly  protein . For the protein ,12. Alth protein  and othe protein , relativHere we describe a project to sequence and characterize a large number of full-length cDNAs from bursal lymphocytes. The corresponding genes are likely to be expressed in DT40 and this should facilitate their analysis by targeted gene modifications. In combination with the recently released cDNAs from other tissues , the burThe overall strategy for producing the greatest possible number of new full-length cDNAs expressed in bursal lymphocytes is outlined in Figure The plasmids corresponding to the remaining 2,796 ESTs were chosen for full insert sequencing by bidirectional primer walks. Once the end of the walks had been reached, the sequences of the full-length cDNA inserts were assembled. From the BLAST search results the most likely methionine start codon was assigned to each sequence. About 15% of the cDNA sequences showed evidence for premature frameshifts in the form of short ORFs and stretches of conserved sequence in a different frame further 3'. If overlapping ESTs were found in the public databases, the cDNA sequences were edited to correct the likely reverse transcription error, otherwise these sequences were discarded.A total of 2,272 high-quality chicken full-length cDNA clones were sequenced and assembled, manually annotated with respect to their likely translation start codon and deposited both at The Bursal Transcript Database website  and in tThe most remarkable feature noted in the analysis of 5' UTRs of the bursal cDNAs is a very high GC content (67%). This supports the observation that the GC content of 5' UTRs is particularly high in warm-blooded species . On the The accurate prediction of the translation start codon remains difficult and in some cases our annotations remain tentative. Sequences surrounding the translation start codons are not random and in mammals match the consensus GCCRCCaugG (where aug is the start codon and R is either A or G) . The mosA detailed analysis shows that the riken1 collection of cDNA sequences contains 4,406 AUG codons upstream of the annotated translation start codons in 2,218 of the bursal cDNAs. Nine hundred one of these alternative start codons were in the same reading frame as the annotated ORF. An in-frame stop codon within the 5' UTR region was present downstream of 501 of these 901 alternative start codons. The total number of ORFs present in 5' UTR regions of riken1 cDNAs was 1,289.We have checked whether the context of the annotated AUG start codons differs from the context of the alternative upstream AUG sequences of the bursal cDNAs. We therefore extracted 10-bp long sequences surrounding the annotated start codons and the alternative upstream AUGs and visualized sequence variability using the sequence logo software  Figure . The annAll full-length cDNAs were compared to the collection of transcripts predicted from the chicken genome sequence by the Ensembl system . The traFigure When compared to available chicken ESTs or cDNAs in the public databases, some of the bursal cDNAs showed significant structural differences most likely due to differential transcript processing. In addition, the bursal cDNA collection has been used to define a large number of intragenic single-nucleotide polymorphisms (SNPs) .All full-length cDNAs were compared to the Pfam database , which sTables There are 22 full-length cDNAs in our collection containing Pfam domains annotated by the GO term 'molecular function unknown'. Experimental information concerning the molecular mechanisms of action is very sparse or nonexistent for proteins sharing these evolutionarily conserved domains. Highly similar human proteins exist for the chicken proteins, an example being the human protein BM02. Taking into account the ease of targeted genome modification and availability of numerous functional assays, the DT40 cell line is an attractive model system to provide first insights into the functions of the evolutionarily conserved domains described above.All the full-length cDNA sequences are stored within the Bursal Transcript database . This dain vitro or for complementation of mutant DT40 phenotypes with the added convenience that the cDNA sequences are not only known, but also available as cloned pieces of fully sequenced DNA.The cDNAs from bursal lymphocytes represent one of the largest full-length cDNA collections in the chicken, comprising about one third of all currently available, experimentally verified transcripts and will be of general interest to researchers using the chicken as an experimental model as well as to the poultry industry. The resource has already been integrated with the chicken genome sequences to build a unigene catalog , to defiLMO1 locus. This sequence was present as a stuffer of the lambda vector used for the library construction and the clones containing it were removed from further analysis. In total, the 5' single-pass sequencing of 14,976 clones yielded 11,116 high-quality ESTs of the riken1 library.The riken1 library was synthesized from mRNA of 2-week-old CB strain bursal lymphocytes using the biotinylated cap trapper method ,31. The BLAST searches against the 'All non-redundant GenBank CDS' database showed that approximately 80% of the 5' EST sequences matched GenBank entries with a score of at least 50. This score threshold was chosen because it allowed us in most cases to align the putative start codon of the query sequence to the EST. These sequences were chosen and clustered  to removSufficient plasmid template for numerous sequencing reactions was prepared from the clones corresponding to the selected ESTs. All plasmids were then sequenced with a primer complementary to a plasmid sequence 3' of the cDNA insertion site. Subsequently, custom-made 20-mer primers based on available sequences were used for sequencing until the 3' and 5' ends of the cDNA inserts were reached. All sequences were processed as described previously , except Gallus gallus ESTs from the public databases. If the short ORF could be corrected by adopting the sequence of an overlapping EST, the cDNA sequence was edited. The type of editing was recorded and the corresponding riken1 clone was annotated as likely to be defective. In total, 293 cDNAs were removed because either a likely artificial frameshift could not be corrected by using sequences of overlapping ESTs or they contained multiple stop codons in all three reading frames or they showed evidence for unspliced introns. All cDNA clones are freely available upon request to the corresponding author and their sequences have been submitted to the EMBL public database (accession numbers AJ719267-AJ721138 and AJ851370-AJ851825).The integrity of the conserved ORF within each assembled cDNA sequence was manually examined by inspecting BLAST search results against the public protein and EST databases. To facilitate this task, a new EstSet module was added to FOUNTAIN . The useThe sequences surrounding the annotated start codons (10 bp upstream and downstream) were exported and submitted for information content visualization by the WebLogo software . Subsequ-6) run on the binary database files downloaded from the National Center for Biotechnology Information (NCBI). Functional classes were assigned according to Pfam to GO mapping provided by the InterPro database. The XML information exchange standard was used to interface the BLAST program outputs with the FOUNTAIN package.The riken1 cDNAs were compared with the collection of predicted chicken transcripts downloaded from the Ensembl ftp site  using th


PLoS Biology, Thomas Bartman et al. use the powerful tools afforded by zebrafish genetics to dissect the early steps of heart valve formation. In the process, they provide evidence for a causal relationship between the early function of the heart and its final structure.The heart starts beating and pumps blood through the body long before it has achieved its mature architecture. In theory, this provides a chance for cardiac function to sculpt cardiac structure, an intriguing possibility for developmental biologists, and one of potentially great clinical import for cardiologists seeking to identify the causes of  cardiac anomalies. In this issue of At the time of its first beat, the vertebrate heart is little more than a tube, lined on its outside by a myocardial cell layer whose contractions (the heartbeats) power blood flow, and on its inside by an endocardial cell layer, an extension of the inner wall of the connecting blood vessels. What it lacks still are valves and septae, the fibrous gates that subdivide the mature heart into atrial and ventricular chambers, and control the directionality of blood flow. These structures derive from the endocardium in a process that begins—shortly after the establishment of blood flow—with the local accumulation of endocardial cells into what are known as endocardial cushions (ECs).cardiofunk (cfk), which was devoid of ECs. Genetic mapping of the cfk mutation revealed a single sequence change in a gene encoding a novel actin molecule that is most closely related to the sarcomeric actins found in sarcomeres, the contracting organelles of muscle cells. The result was surprising because contractions are not a property of endocardial cells. Using RNA detection assays, the authors show that the cfk gene is in fact expressed in the myocardium, rather than in the endocardium. It therefore appears that the inability to form ECs in cfk mutants does not reside in the endocardium per se, but is an indirect consequence of a myocardial anomaly.The zebrafish lends itself well to large-scale genetic screens, and powerful genomic tools are now available to efficiently identify the gene affected by any mutation. The authors have used genetic screens to identify several mutations that affect early cardiac function or morphology. Heart anomalies are easy to detect in zebrafish, and can be examined in real time and in live specimens because the embryos develop outside the mother and are fully transparent. Using a fluorescent molecular marker highly expressed in the ECs, the authors narrowed in on mutations that result in valve defects, and identified a mutant they named cfk mutation introduces a single amino acid change in the actin protein, and through detailed biochemical analyses, the authors show that the mutant actin is impaired in its ability to assemble into fibers in vitro. What might be the consequence in vivo? The authors note that cfk mutants display abnormal heart contractions prior to the development of their EC defect. Support for the notion that myocardial contractions are required for EC formation comes from the examination of silent-heart (sih) mutants. sih mutants, which lack a heatbeat, have been shown to harbor a mutation in troponin T, one of the motors of actin contractions; the authors find that sih mutants also fail to develop ECs. The mechanisms linking myocardial contractions and cushion formation remain unclear. Blood flow may be a trigger, though the authors find that ECs can develop even in the presence of pharmacological compounds that abolish it. The characterization of additional mutants should help answer this question.The Valve or septal defects represent 40% of cardiac anomalies in humans. Bartman and colleagues suggest that, by analogy with zebrafish, some may result from congenital defects affecting very early myocardial function. Their work thus opens new avenues for the early detection of human cardiac malfunctions and malformations.