Dengue haemorrhagic fever and dengue shock syndrome, the most severe responses to dengue virus (DV) infection, are characterized by plasma leakage (due to increased vascular permeability) and low platelet counts. CLEC5A (C-type lectin domain family 5, member A; also known as myeloid DAP12-associating lectin (MDL-1)) contains a C-type lectin-like fold similar to the natural-killer T-cell C-type lectin domains and associates with a 12-kDa DNAX-activating protein (DAP12) on myeloid cells. Here we show that CLEC5A interacts with the dengue virion directly and thereby brings about DAP12 phosphorylation. The CLEC5A-DV interaction does not result in viral entry but stimulates the release of proinflammatory cytokines. Blockade of CLEC5A-DV interaction suppresses the secretion of proinflammatory cytokines without affecting the release of interferon-alpha, supporting the notion that CLEC5A acts as a signalling receptor for proinflammatory cytokine release. Moreover, anti-CLEC5A monoclonal antibodies inhibit DV-induced plasma leakage, as well as subcutaneous and vital-organ haemorrhaging, and reduce the mortality of DV infection by about 50% in STAT1-deficient mice. Our observation that blockade of CLEC5A-mediated signalling attenuates the production of proinflammatory cytokines by macrophages infected with DV (either alone or complexed with an enhancing antibody) offers a promising strategy for alleviating tissue damage and increasing the survival of patients suffering from dengue haemorrhagic fever and dengue shock syndrome, and possibly even other virus-induced inflammatory diseases.

In response to heat stress, Bacillus subtilis activates the transcription of well over 100 different genes. Many of these genes are members of a general stress response regulon controlled by the secondary sigma factor, B , while others are under control of the HrcA or CtsR heat shock regulators. We have used DNA microarrays to monitor the global transcriptional response to heat shock. We find strong induction of known B -dependent genes with a characteristic rapid induction followed by a return to near prestimulus levels. The HrcA and CtsR regulons are also induced, but with somewhat slower kinetics. Analysis of DNA sequences proximal to newly identified heat-induced genes leads us to propose ϳ70 additional members of the B regulon. We have also identified numerous heat-induced genes that are not members of known heat shock regulons. Notably, we observe very strong induction of arginine biosynthesis and transport operons. Induction of several genes was confirmed by quantitative reverse transcriptase PCR. In addition, the transcriptional responses measured by microarray hybridization compare favorably with the numerous previous studies of heat shock in this organism. Since many different conditions elicit both specific and general stress responses, knowledge of the heat-induced general stress response reported here will be helpful for interpreting future microarray studies of other stress responses.DNA microarray technology provides a powerful tool for the analysis of global transcriptional responses elicited by various physical and chemical stresses. One challenge in this sort of analysis is to distinguish stress-specific responses from more general stress responses. For example, in Bacillus subtilis, many different stresses (including heat shock, osmotic stress, and energy stress) activate the large general stress response controlled by the B transcription factor (20,44,45). While others have used two-dimensional protein gels to classify cellular stress responses (55, 58), DNA-based methods have several advantages: they can be rapidly adapted to new organisms, they provide greater coverage of the genome, and data processing is comparatively easy to automate. Ultimately, it may be possible to integrate both technologies, at least for well-studied model organisms (19,41,56).We have initiated a series of studies to characterize the global transcriptional responses of B. subtilis, a model grampositive microorganism. Here, we document the heat-induced general stress response. Heat shock was chosen for this initial study since it is arguably the best-studied stress response in this organism and includes activation of the large general stress response under the control of B (20, 44). In addition, a subset of antibiotics that inhibit translation have been reported to induce heat shock genes in other organisms (57). It is anticipated that knowledge of transcriptional responses to antimicrobial compounds will be useful for both antibacterial discovery and characterization (47).Analysis of the transcriptional profile of B...

In smooth muscle tissues, the relationship between muscle or cell length and active force can be modulated by altering the cell or tissue length during stimulation. Mechanisms for this mechanical plasticity were investigated by measuring muscle stiffness during isometric contractions in which contractile force was graded by changing stimulus intensity or muscle length. Stiffness was significantly higher in contracted than in resting muscles at comparable forces; however, the relationship between stiffness and force during force development was curvilinear and independent of muscle length and stimulus intensity. This suggests that muscle stiffness during force development reflects properties of cellular components other than cross bridges which contribute to the series elasticity only during activation. During the tonic phase of isometric contraction, muscle stiffness increased while force remained constant. A step decrease in the length of a contracted muscle resulted in a high level of stiffness relative to force during isometric force redevelopment following the length step. We propose that the arrangement of the cytoskeleton can adjust to changes in the conformation of resting smooth muscle cells but that the organization of the cytoskeleton becomes more fixed upon contractile activation and is modulated very slowly during a sustained contraction. This may provide a mechanism for optimizing force development to the physical conformation of the cell at the time of activation.

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Bacillus subtilis exhibits a complex adaptive response to low levels of peroxides. We used global transcriptional profiling to monitor the magnitude and kinetics of changes in the mRNA population after exposure to either hydrogen peroxide (H 2 O 2 ) or tert-butyl peroxide (t-buOOH). The peroxide stimulons could be largely accounted for by three regulons controlled by the PerR, B , and OhrR transcription factors. Three members of the PerR regulon (katA, mrgA, and zosA) were strongly induced by H 2 O 2 and weakly induced by t-buOOH. The remaining members of the PerR regulon were only modestly up-regulated by peroxide treatment. Overall, the magnitude of peroxide induction of PerR regulon genes corresponded well with the extent of derepression in a perR mutant strain. The B regulon was activated by 58 M H 2 O 2 but not by 8 M H 2 O 2 and was strongly activated by either t-buOOH or, in a control experiment, tert-butyl alcohol. Apart from the B regulon there was a single gene, ohrA, that was strongly and rapidly induced by t-buOOH exposure. This gene, controlled by the peroxide-sensing repressor OhrR, was not induced by any of the other conditions tested.

Gefitinib and erlotinib are active in patients with G719X/L861Q/S768I mutations; however, less effective than in those with common mutations.

BackgroundIt is important to select appropriate targeted therapies for subgroups of patients with lung adenocarcinoma who have specific gene alterations.MethodsThis prospective study was a multicenter project conducted in Taiwan for assessment of lung adenocarcinoma genetic tests. Five oncogenic drivers, including EGFR, KRAS, BRAF, HER2 and EML4-ALK fusion mutations, were tested. EGFR, KRAS, BRAF and HER2 mutations were assessed by MALDI-TOF MS (Cohort 1). EML4-ALK translocation was tested by Ventana method in EGFR-wild type patients (Cohort 2).ResultsFrom August 2011 to November 2013, a total of 1772 patients with lung adenocarcinoma were enrolled. In Cohort 1 analysis, EGFR, KRAS, HER2 and BRAF mutations were identified in 987 (55.7%), 93 (5.2%), 36 (2.0%) and 12 (0.7%) patients, respectively. Most of these mutations were mutually exclusive, except for co-mutations in seven patients (3 with EGFR + KRAS, 3 with EGFR + HER2 and 1 with KRAS + BRAF). In Cohort 2 analysis, 29 of 295 EGFR-wild type patients (9.8%) were positive for EML4-ALK translocation. EGFR mutations were more common in female patients and non-smokers and KRAS mutations were more common in male patients and smokers. Gender and smoking status were not correlated significantly with HER2, BRAF and EML4-ALK mutations. EML4-ALK translocation was more common in patients with younger age.ConclusionThis was the first study in Taiwan to explore the incidence of five oncogenic drivers in patients with lung adenocarcinoma and the results could be valuable for physicians in consideration of targeted therapy and inclusion of clinical trials.

BackgroundAngiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma.MethodsSplenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage.Results and conclusionsSequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.