Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii

<p>Abstract</p> <p>Background</p> <p><it>Sporothrix schenckii </it>is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in <it>S. schenckii </it>responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in <it>S. schenckii, sscmk1</it>, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle.</p> <p>Results</p> <p>Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of <it>sscmk1 </it>revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of <it>sscmk1 </it>revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 – 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition.</p> <p>Conclusion</p> <p>This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in <it>S. schenckii </it>and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus. The results suggest that the calcium/calmodulin kinases of yeasts are evolutionarily distinct from those in filamentous fungi.</p

Development and validation of a questionnaire to evaluate lifestyle-related behaviors in elementary school children

Background: The SI! Program promotes cardiovascular health through a multilevel school-based intervention on four lifestyle-related components: diet, physical activity, understanding the body and heart, and management of emotions. We report here the development and validation of the KAH (knowledge, attitudes and habits)-questionnaire adapted for elementary school children (6-7 years old) as a tool for the forthcoming evaluation of the SI! Program, where the KAH scoring will be the primary outcome. The efficacy of such an intervention will be based on the improvements in children's KAH towards a healthy lifestyle. Methods: The questionnaire validation process started with a pool of items proposed by the pedagogical team who developed the SI! Program for elementary school. The questionnaire was finalized by decreasing the number of items from 155 to 48 using expert panels and statistical tests on the responses from 384 children (ages 6-7). A team of specialized psychologists administered the questionnaire at schools providing standard directions for the final administration. The internal consistency was assessed using Cronbach's a coefficients. Reliability was measured through the split-half method, and problematic items were detected applying the item response theory. Analysis of variance and Tukey's test of additivity were used for multiple comparisons. Results: The final KAH-questionnaire for elementary school children should be administered to children individually by trained staff. The 48 items-questionnaire is divided evenly between the 4 components of the intervention, with an overall Cronbach's a = 0.791 (a = 0.526 for diet, a = 0.537 for physical activity, a = 0.523 for human body and heart, and a = 0.537 for management of emotions). Conclusions: The KAH-questionnaire is a reliable instrument to assess the efficacy of the SI! Program on instilling healthy lifestyle-related behaviors in elementary school children.This work is supported by the SHE Foundation (Foundation for Science, Health and Education) and the Daniel and Nina Carasso Foundation.S

Non-invasive imaging of atherosclerotic plaque macrophage in a rabbit model with F-18 FDG PET: a histopathological correlation

BACKGROUND: Coronary atherosclerosis and its thrombotic complications are the major cause of mortality and morbidity throughout the industrialized world. Thrombosis on disrupted atherosclerotic plaques plays a key role in the onset of acute coronary syndromes. Macrophages density is one of the most critical compositions of plaque in both plaque vulnerability and thrombogenicity upon rupture. It has been shown that macrophages have a high uptake of (18)F-FDG (FDG). We studied the correlation of FDG uptake with histopathological macrophage accumulation in atherosclerotic plaques in a rabbit model. METHODS: Atherosclerosis was induced in rabbits (n = 6) by a combination of atherogenic diet and balloon denudation of the aorta. PET imaging was performed at baseline and 2 months after atherogenic diet and coregistered with magnetic resonance (MR) imaging. Normal (n = 3) rabbits served as controls. FDG uptake by the thoracic aorta was expressed as concentration (μCi/ml) and the ratio of aortic uptake-to-blood radioactivity. FDG uptake and RAM-11 antibody positive areas were analyzed in descending aorta. RESULTS: Atherosclerotic aortas showed significantly higher uptake of FDG than normal aortas. The correlation of aortic FDG uptake with macrophage areas assessed by histopathology was statistically significant although it was not high (r = 0.48, p < 0.0001). When uptake was expressed as the ratio of aortic uptake-to-blood activity, it correlated better (r = 0.80, p < 0.0001) with the macrophage areas, due to the correction for residual blood FDG activity. CONCLUSION: PET FDG activity correlated with macrophage content within aortic atherosclerosis. This imaging approach might serve as a useful non-invasive imaging technique and potentially permit monitoring of relative changes in inflammation within the atherosclerotic lesion

Human-based approaches to pharmacology and cardiology: an interdisciplinary and intersectorial workshop.

Both biomedical research and clinical practice rely on complex datasets for the physiological and genetic characterization of human hearts in health and disease. Given the complexity and variety of approaches and recordings, there is now growing recognition of the need to embed computational methods in cardiovascular medicine and science for analysis, integration and prediction. This paper describes a Workshop on Computational Cardiovascular Science that created an international, interdisciplinary and inter-sectorial forum to define the next steps for a human-based approach to disease supported by computational methodologies. The main ideas highlighted were (i) a shift towards human-based methodologies, spurred by advances in new in silico, in vivo, in vitro, and ex vivo techniques and the increasing acknowledgement of the limitations of animal models. (ii) Computational approaches complement, expand, bridge, and integrate in vitro, in vivo, and ex vivo experimental and clinical data and methods, and as such they are an integral part of human-based methodologies in pharmacology and medicine. (iii) The effective implementation of multi- and interdisciplinary approaches, teams, and training combining and integrating computational methods with experimental and clinical approaches across academia, industry, and healthcare settings is a priority. (iv) The human-based cross-disciplinary approach requires experts in specific methodologies and domains, who also have the capacity to communicate and collaborate across disciplines and cross-sector environments. (v) This new translational domain for human-based cardiology and pharmacology requires new partnerships supported financially and institutionally across sectors. Institutional, organizational, and social barriers must be identified, understood and overcome in each specific setting

Mortality after coronary artery bypass grafting versus percutaneous coronary intervention with stenting for coronary artery disease: a pooled analysis of individual patient data

Background: Numerous randomised trials have compared coronary artery bypass grafting (CABG) with percutaneous coronary intervention (PCI) for patients with coronary artery disease. However, no studies have been powered to detect a difference in mortality between the revascularisation strategies. Methods: We did a systematic review up to July 19, 2017, to identify randomised clinical trials comparing CABG with PCI using stents. Eligible studies included patients with multivessel or left main coronary artery disease who did not present with acute myocardial infarction, did PCI with stents (bare-metal or drug-eluting), and had more than 1 year of follow-up for all-cause mortality. In a collaborative, pooled analysis of individual patient data from the identified trials, we estimated all-cause mortality up to 5 years using Kaplan-Meier analyses and compared PCI with CABG using a random-effects Cox proportional-hazards model stratified by trial. Consistency of treatment effect was explored in subgroup analyses, with subgroups defined according to baseline clinical and anatomical characteristics. Findings: We included 11 randomised trials involving 11 518 patients selected by heart teams who were assigned to PCI (n=5753) or to CABG (n=5765). 976 patients died over a mean follow-up of 3·8 years (SD 1·4). Mean Synergy between PCI with Taxus and Cardiac Surgery (SYNTAX) score was 26·0 (SD 9·5), with 1798 (22·1%) of 8138 patients having a SYNTAX score of 33 or higher. 5 year all-cause mortality was 11·2% after PCI and 9·2% after CABG (hazard ratio [HR] 1·20, 95% CI 1·06–1·37; p=0·0038). 5 year all-cause mortality was significantly different between the interventions in patients with multivessel disease (11·5% after PCI vs 8·9% after CABG; HR 1·28, 95% CI 1·09–1·49; p=0·0019), including in those with diabetes (15·5% vs 10·0%; 1·48, 1·19–1·84; p=0·0004), but not in those without diabetes (8·7% vs 8·0%; 1·08, 0·86–1·36; p=0·49). SYNTAX score had a significant effect on the difference between the interventions in multivessel disease. 5 year all-cause mortality was similar between the interventions in patients with left main disease (10·7% after PCI vs 10·5% after CABG; 1·07, 0·87–1·33; p=0·52), regardless of diabetes status and SYNTAX score. Interpretation: CABG had a mortality benefit over PCI in patients with multivessel disease, particularly those with diabetes and higher coronary complexity. No benefit for CABG over PCI was seen in patients with left main disease. Longer follow-up is needed to better define mortality differences between the revascularisation strategies

Microbial Diversity of a Brazilian Coastal Region Influenced by an Upwelling System and Anthropogenic Activity

BACKGROUND: Upwelling systems are characterised by an intense primary biomass production in the surface (warmest) water after the outcrop of the bottom (coldest) water, which is rich in nutrients. Although it is known that the microbial assemblage plays an important role in the food chain of marine systems and that the upwelling systems that occur in southwest Brazil drive the complex dynamics of the food chain, little is known about the microbial composition present in this region. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a molecular survey based on SSU rRNA gene from the three domains of the phylogenetic tree of life present in a tropical upwelling region (Arraial do Cabo, Rio de Janeiro, Brazil). The aim was to analyse the horizontal and vertical variations of the microbial composition in two geographically close areas influenced by anthropogenic activity (sewage disposal/port activity) and upwelling phenomena, respectively. A lower estimated diversity of microorganisms of the three domains of the phylogenetic tree of life was found in the water of the area influenced by anthropogenic activity compared to the area influenced by upwelling phenomena. We observed a heterogenic distribution of the relative abundance of taxonomic groups, especially in the Archaea and Eukarya domains. The bacterial community was dominated by Proteobacteria, Cyanobacteria and Bacteroidetes phyla, whereas the microeukaryotic community was dominated by Metazoa, Fungi, Alveolata and Stramenopile. The estimated archaeal diversity was the lowest of the three domains and was dominated by uncharacterised marine Crenarchaeota that were most closely related to Marine Group I. CONCLUSIONS/SIGNIFICANCE: The variety of conditions and the presence of different microbial assemblages indicated that the area of Arraial do Cabo can be used as a model for detailed studies that contemplate the correlation between pollution-indicating parameters and the depletion of microbial diversity in areas close to anthropogenic activity; functional roles and geochemical processes; phylogeny of the uncharacterised diversity; and seasonal variations of the microbial assemblages

CandidaDB: a genome database for Candida albicans pathogenomics

CandidaDB is a database dedicated to the genome of the most prevalent systemic fungal pathogen of humans, Candida albicans. CandidaDB is based on an annotation of the Stanford Genome Technology Center C.albicans genome sequence data by the European Galar Fungail Consortium. CandidaDB Release 2.0 (June 2004) contains information pertaining to Assembly 19 of the genome of C.albicans strain SC5314. The current release contains 6244 annotated entries corresponding to 130 tRNA genes and 5917 protein-coding genes. For these, it provides tentative functional assignments along with numerous pre-run analyses that can assist the researcher in the evaluation of gene function for the purpose of specific or large-scale analysis. CandidaDB is based on GenoList, a generic relational data schema and a World Wide Web interface that has been adapted to the handling of eukaryotic genomes. The interface allows users to browse easily through genome data and retrieve information. CandidaDB also provides more elaborate tools, such as pattern searching, that are tightly connected to the overall browsing system. As the C.albicans genome is diploid and still incompletely assembled, CandidaDB provides tools to browse the genome by individual supercontigs and to examine information about allelic sequences obtained from complementary contigs. CandidaDB is accessible at http://genolist.pasteur.fr/CandidaDB

A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy

Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy. We then selected commonly up-regulated microRNAs for a functional in vivo therapeutic screen using oligonucleotide inhibitors. Argonaute sequencing generated 1.44 billion small RNA reads of which up to 82% were microRNAs, with over 400 unique microRNAs detected per model. Approximately half of the detected microRNAs were dysregulated in each epilepsy model. We prioritized commonly up-regulated microRNAs that were fully conserved in humans and designed custom antisense oligonucleotides for these candidate targets. Antiseizure phenotypes were observed upon knockdown of miR-10a-5p, miR-21a-5p, and miR-142a-5p and electrophysiological analyses indicated broad safety of this approach. Combined inhibition of these three microRNAs reduced spontaneous seizures in epileptic mice. Proteomic data, RNA sequencing, and pathway analysis on predicted and validated targets of these microRNAs implicated derepressed TGF-\u3b2 signaling as a shared seizure-modifying mechanism. Correspondingly, inhibition of TGF-\u3b2 signaling occluded the antiseizure effects of the antagomirs. Together, these results identify shared, dysregulated, and functionally active microRNAs during the pathogenesis of epilepsy which represent therapeutic antiseizure targets

Shape model and spin-state analysis of PHA contact binary (85990) 1999 JV6 from combined radar and optical observations

Context. The potentially hazardous asteroid (85990)1999 JV6 has been a target of previously published thermal-infrared observations and optical photometry. It has been identified as a promising candidate for possible Yarkovsky-O’Keefe-Radzievskii-Paddack (YORP) effect detection. Aims. The YORP effect is a small thermal-radiation torque considered to be a key factor in spin-state evolution of small Solar System bodies. In order to detect YORP on 1999 JV6 we developed a detailed shape model and analysed the spin-state using both optical and radar observations. Methods. For 1999 JV6, we collected optical photometry between 2007 and 2016. Additionally, we obtained radar echo-power spectra and imaging observations with Arecibo and Goldstone planetary radar facilities in 2015, 2016, and 2017. We combined our data with published optical photometry to develop a robust physical model. Results. We determine that the rotation pole resides at negative latitudes in an area with a 5◦ radius close to the south ecliptic pole. The refined sidereal rotation period is 6.536787±0.000007 h. The radar images are best reproduced with a bilobed shape model. Both lobes of 1999 JV6 can be represented as oblate ellipsoids with a smaller, more spherical component resting at the end of a larger, more elongated component. While contact binaries appear to be abundant in the near-Earth population, there are only a few published shape models for asteroids in this particular configuration. By combining the radar-derived shape model with optical light curves we determine a constant-period solution that fits all available data well. Using light-curve data alone we determine an upper limit for YORP of 8.5×10−8 rad day−2. Conclusions. The bifurcated shape of 1999 JV6 might be a result of two ellipsoidal components gently merging with each other, or a deformation of a rubble pile with a weak-tensile-strength core due to spin-up. The physical model of 1999 JV6 presented here will enable future studies of contact binary asteroid formation and evolution

Predictive models for mutations in mismatch repair genes: implication for genetic counseling in developing countries

<p>Abstract</p> <p>Background</p> <p>Lynch syndrome (LS) is the most common form of inherited predisposition to colorectal cancer (CRC), accounting for 2-5% of all CRC. LS is an autosomal dominant disease characterized by mutations in the mismatch repair genes mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), postmeiotic segregation increased 1 (PMS1), post-meiotic segregation increased 2 (PMS2) and mutS homolog 6 (MSH6). Mutation risk prediction models can be incorporated into clinical practice, facilitating the decision-making process and identifying individuals for molecular investigation. This is extremely important in countries with limited economic resources. This study aims to evaluate sensitivity and specificity of five predictive models for germline mutations in repair genes in a sample of individuals with suspected Lynch syndrome.</p> <p>Methods</p> <p>Blood samples from 88 patients were analyzed through sequencing MLH1, MSH2 and MSH6 genes. The probability of detecting a mutation was calculated using the PREMM, Barnetson, MMRpro, Wijnen and Myriad models. To evaluate the sensitivity and specificity of the models, receiver operating characteristic curves were constructed.</p> <p>Results</p> <p>Of the 88 patients included in this analysis, 31 mutations were identified: 16 were found in the MSH2 gene, 15 in the MLH1 gene and no pathogenic mutations were identified in the MSH6 gene. It was observed that the AUC for the PREMM (0.846), Barnetson (0.850), MMRpro (0.821) and Wijnen (0.807) models did not present significant statistical difference. The Myriad model presented lower AUC (0.704) than the four other models evaluated. Considering thresholds of ≥ 5%, the models sensitivity varied between 1 (Myriad) and 0.87 (Wijnen) and specificity ranged from 0 (Myriad) to 0.38 (Barnetson).</p> <p>Conclusions</p> <p>The Barnetson, PREMM, MMRpro and Wijnen models present similar AUC. The AUC of the Myriad model is statistically inferior to the four other models.</p