Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways

In addition to being a very well-known ion pump, Na⁺, K⁺-ATPase is a cell–cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na⁺, K⁺-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.     

Comparative morphology of pond,stream and phytotelm‐dwelling tadpoles of the South American Redbelly Toads (Anura: Bufonidae: Melanophryniscus)

We present a comprehensive review of larval morphology in the Neotropical toad genus Melanophryniscus. The taxa studied included 23 species with representatives of recognized phenetic groups and different larval ecomorphological guilds: pond, stream, and phytotelm‐dwelling tadpoles. Their external morphology variation is congruent with current phenetic arrangement based on adult features, but also reflects the habitat where larvae develop. Lotic tadpoles (i.e. M. tumifrons group and M. krauczuki) in general exhibit a more depressed body, a longer tail with lower fins, and larger oral discs than lentic forms (i.e. M. stelzneri group, M. moreirae, M. sanmartini, and M. langonei). Despite their peculiar, confined microhabitat, phytotelm larvae do not diverge markedly from non‐arboreal species. The distinctive features of all species are the presence of a pineal end organ and the placement of the intestinal reversal point at the left of the abdomen in typical larval stages. The buccal cavity and musculoskeletal anatomy are quite conserved between species, yet some characteristics differ from those of other bufonids. The presence of one pair of subhyoid muscles is apparently an exclusive trait of Melanophryniscus among Bufonidae. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 417–441.     

Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.     

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.     

Brazilian Microbiome Project: Revealing the Unexplored Microbial Diversity—Challenges and Prospects

The Brazilian Microbiome Project (BMP) aims to assemble a Brazilian Metagenomic Consortium/Database. At present, many metagenomic projects underway in Brazil are widely known. Our goal in this initiative is to co-ordinate and standardize these together with new projects to come. It is estimated that Brazil hosts approximately 20 % of the entire world’s macroorganism biological diversity. It is 1 of the 17 countries that share nearly 70 % of the world’s catalogued animal and plant species, and is recognized as one of the most megadiverse countries. At the end of 2012, Brazil has joined GBIF (Global Biodiversity Information Facility), as associated member, to improve the access to the Brazilian biodiversity data in a free and open way. This was an important step toward increasing international collaboration and clearly shows the commitment of the Brazilian government in directing national policies toward sustainable development. Despite its importance, the Brazilian microbial diversity is still considered to be largely unknown, and it is clear that to maintain ecosystem dynamics and to sustainably manage land use, it is crucial to understand the biological and functional diversity of the system. This is the first attempt to collect and collate information about Brazilian microbial genetic and functional diversity in a systematic and holistic manner. The success of the BMP depends on a massive collaborative effort of both the Brazilian and international scientific communities, and therefore, we invite all colleagues to participate in this project.     

Solution and high‐pressure NMR studies of the structure,dynamics, and stability of the cross‐reactive allergenic cod parvalbumin Gad m 1

Beta‐parvalbumins from different fish species have been identified as the main elicitors of IgE‐mediated reactions in fish‐allergic individuals. Here, we report for the first time the NMR determination of the structure and dynamics of the major Atlantic cod (Gadus morhua) allergen Gad m 1 and compare them with other known parvalbumins. Although the Gad m 1 structure and accessibility of putative IgE epitopes are similar to parvalbumins in mackerel and carp, the charge distribution at the putative epitopes is different. The determination of the Gad m 1 structure contributes to a better understanding of cross‐reactivity among fish parvalbumins. In addition, the high‐pressure NMR and temperature variation experiments revealed the important contribution of the AB motif and other regions to the protein folding. This structural information could assist the future identification of hot spots for targeted mutations to develop hypoallergenic Ca²⁺‐free forms for potential use in immunotherapy. Proteins 2014; 82:3032–3042. © 2014 Wiley Periodicals, Inc.     

Habitat conversion and galling insect richness in tropical rainforests under mining effect

Human-induced habitat change is the main cause of species loss and can have severe effects on plant communities and the associated herbivore fauna. In this study, we investigated the effects of habitat conversion due to mining on communities of galling insects in areas of tropical rainforest in the Brazilian Amazon. We sampled galling insects in the Floresta Nacional de Saracá Taquera, Pará, Brazil, where forest plateaus are used by the Mineração Rio do Norte Group to extract bauxite. Our results show that human-induced habitat change via mining activities increased the local species richness of galling insects. We also found that after impact there was greater species richness of galling insects closer to the forest edge than in the forest interior. Changes in plant physiology and in the diversity of natural enemies in human-modified habitats, along with the endophagous life-form, might account for the high incidence of galling in human-disturbed habitats. This result highlights the importance of understanding how different insect groups respond to human activities, since such idiosyncrasies might have profound effects on the species’ patterns of ecological interactions and in the outcomes of those interactions.     

Unravelling biodiversity,evolution and threats to conservation in the Sahara‐Sahel

Deserts and arid regions are generally perceived as bare and rather homogeneous areas of low diversity. The Sahara is the largest warm desert in the world and together with the arid Sahel displays high topographical and climatic heterogeneity, and has experienced recent and strong climatic oscillations that have greatly shifted biodiversity distribution and community composition. The large size, remoteness and long‐term political instability of the Sahara‐Sahel, have limited knowledge on its biodiversity. However, over the last decade, there have been an increasing number of published scientific studies based on modern geomatic and molecular tools, and broad sampling of taxa of these regions. This review tracks trends in knowledge about biodiversity patterns, processes and threats across the Sahara‐Sahel, and anticipates needs for biodiversity research and conservation. Recent studies are changing completely the perception of regional biodiversity patterns. Instead of relatively low species diversity with distribution covering most of the region, studies now suggest a high rate of endemism and larger number of species, with much narrower and fragmented ranges, frequently limited to micro‐hotspots of biodiversity. Molecular‐based studies are also unravelling cryptic diversity associated with mountains, which together with recent distribution atlases, allows identifying integrative biogeographic patterns in biodiversity distribution. Mapping of multivariate environmental variation (at 1 km × 1 km resolution) of the region illustrates main biogeographical features of the Sahara‐Sahel and supports recently hypothesised dispersal corridors and refugia. Micro‐scale water‐features present mostly in mountains have been associated with local biodiversity hotspots. However, the distribution of available data on vertebrates highlights current knowledge gaps that still apply to a large proportion of the Sahara‐Sahel. Current research is providing insights into key evolutionary and ecological processes, including causes and timing of radiation and divergence for multiple taxa, and associating the onset of the Sahara with diversification processes for low‐mobility vertebrates. Examples of phylogeographic patterns are showing the importance of allopatric speciation in the Sahara‐Sahel, and this review presents a synthetic overview of the most commonly hypothesised diversification mechanisms. Studies are also stressing that biodiversity is threatened by increasing human activities in the region, including overhunting and natural resources prospection, and in the future by predicted global warming. A representation of areas of conflict, landmines, and natural resources extraction illustrates how human activities and regional insecurity are hampering biodiversity research and conservation. Although there are still numerous knowledge gaps for the optimised conservation of biodiversity in the region, a set of research priorities is provided to identify the framework data needed to support regional conservation planning.     

The importance of the Abn2 calcium cluster in the endo-1,5-arabinanase activity from Bacillus subtilis

Arabinanase is a glycosyl hydrolase that is able to cleave the glycosidic bonds of α-1,5-l-arabinan, releasing arabino-oligosaccharides and l-arabinose. The enzyme has two domains, an N-terminal catalytic domain with a characteristic β-propeller fold and a C-terminal domain whose function is unknown. A calcium ion, located near the catalytic site, serves to stabilize the N-terminal domain, but it has also been proposed to play a key role in the enzyme mechanism. The present work describes the structure of an inactive mutant of the wild-type enzyme (H318Q) and in which the calcium ion has been adventitiously replaced by nickel. These structural studies, together with functional and modelling studies, clearly support the role of the calcium ion in the overall reaction mechanism.     

Confirmatory Factor Analysis of the WHO Violence Against Women Instrument in Pregnant Women: Results from the BRISA Prenatal Cohort

Background

Screening for violence during pregnancy is one of the strategies for the prevention of abuse against women. Since violence is difficult to measure, it is necessary to validate questionnaires that can provide a good measure of the phenomenon. The present study analyzed the psychometric properties of the World Health Organization Violence Against Women (WHO VAW) instrument for the measurement of violence against pregnant women.

Methods

Data from the Brazilian Ribeirão Preto and São Luís birth cohort studies (BRISA) were used. The sample consisted of 1,446 pregnant women from São Luís and 1,378 from Ribeirão Preto, interviewed in 2010 and 2011. Thirteen variables were selected from a self-applied questionnaire. Confirmatory factor analysis was used to investigate whether violence is a uni-or-multidimensional construct consisting of psychological, physical and sexual dimensions. The mean-and-variance-adjusted weighted least squares estimator was used. Models were fitted separately for each city and a third model combining data from the two settings was also tested. Models suggested from modification indices were tested to determine whether changes in the WHO VAW model would produce a better fit.

Results

The unidimensional model did not show good fit (Root mean square error of approximation [RMSEA]  = 0.060, p<0.001 for the combined model). The multidimensional WHO VAW model showed good fit (RMSEA = 0.036, p = 0.999 for the combined model) and standardized factor loadings higher than 0.70, except for the sexual dimension for SL (0.65). The models suggested by the modification indices with cross loadings measuring simultaneously physical and psychological violence showed a significantly better fit compared to the original WHO model (p<0.001 for the difference between the model chi-squares).

Conclusions

Violence is a multidimensional second-order construct consisting of psychological, physical and sexual dimensions. The WHO VAW model and the modified models are suitable for measuring violence against pregnant women.