Host cell protein LAMP‐2 is the receptor for Trypanosoma cruzi surface molecule gp82 that mediates invasion

Host cell invasion by Trypanosoma cruzi metacyclic trypomastigote (MT) is mediated by MT‐specific surface molecule gp82, which binds to a still unidentified receptor, inducing lysosome spreading and exocytosis required for the parasitophorous vacuole formation. We examined the involvement of the major lysosome membrane‐associated LAMP proteins in MT invasion. First, human epithelial HeLa cells were incubated with MT in the presence of antibody to LAMP‐1 or LAMP‐2. Antibody to LAMP‐2, but not to LAMP‐1, significantly reduced MT invasion. Next, HeLa cells depleted in LAMP‐1 or LAMP‐2 were generated. Cells deficient in LAMP‐2, but not in LAMP‐1, were significantly more resistant to MT invasion than wild‐type controls. The possibility that LAMP‐2 might be the receptor for gp82 was examined by co‐immunoprecipitation assays. Protein A/G magnetic beads cross‐linked with antibody directed to LAMP‐1 or LAMP‐2 were incubated with HeLa cell and MT detergent extracts. Gp82 bound to LAMP‐2 but not to LAMP‐1. Binding of the recombinant gp82 protein to wild‐type and LAMP‐1‐deficient cells, which was dose dependent and saturable, had a similar profile and was much higher as compared with LAMP‐2‐depleted cells. These data indicate that MT invasion is accomplished through recognition of gp82 by its receptor LAMP‐2.     

A Somatic MAP3K3 Mutation Is Associated with Verrucous Venous Malformation

Verrucous venous malformation (VVM), also called “verrucous hemangioma,” is a non-hereditary, congenital, vascular anomaly comprised of aberrant clusters of malformed dermal venule-like channels underlying hyperkeratotic skin. We tested the hypothesis that VVM lesions arise as a consequence of a somatic mutation. We performed whole-exome sequencing (WES) on VVM tissue from six unrelated individuals and looked for somatic mutations affecting the same gene in specimens from multiple persons. We observed mosaicism for a missense mutation ({"type":"entrez-nucleotide","attrs":{"text":"NM_002401.3","term_id":"42794764","term_text":"NM_002401.3"}}NM_002401.3, c.1323C>G; {"type":"entrez-protein","attrs":{"text":"NP_002392","term_id":"42794765","term_text":"NP_002392"}}NP_002392, p.Iso441Met) in mitogen-activated protein kinase kinase kinase 3 (MAP3K3) in three of six individuals. We confirmed the presence of this mutation via droplet digital PCR (ddPCR) in the three subjects and found the mutation in three additional specimens from another four participants. Mutant allele frequencies ranged from 6% to 19% in affected tissue. We did not observe this mutant allele in unaffected tissue or in affected tissue from individuals with other types of vascular anomalies. Studies using global and conditional Map3k3 knockout mice have previously implicated MAP3K3 in vascular development. MAP3K3 dysfunction probably causes VVM in humans.     

Protein Fibrillation Lag Times During Kinetic Inhibition

Protein aggregation is linked to more than 30 human pathologies, including Alzheimer’s and Parkinson’s diseases. Since small oligomers that form at the beginning of the fibrillation process probably are the most toxic elements, therapeutic strategies involving fibril fragmentation could be detrimental. An alternative approach, named kinetic inhibition, aims to prevent fibril formation by using small ligands that stabilize the parent protein. The factors that govern fibrillation lag times during kinetic inhibition are largely unknown, notwithstanding their importance for designing effective long-term therapies. Inhibitor-bound species are not likely to be incorporated into the core of mature fibrils, although their presence could alter the kinetics of the fibrillation process. For instance, inhibitor-bound species may act as capping elements that impair the nucleation process and/or fibril growth. Here, we address this issue by studying the effect of two natural inhibitors on the fibrillation behavior of lysozyme at neutral pH. We analyzed a set of 79 fibrillation curves obtained in lysozyme alone and a set of 37 obtained in the presence of inhibitors. We calculated the concentrations of the relevant species at the beginning of the curves using the inhibitor-binding constants measured under the same experimental conditions. We found that inhibitor-bound protein species do not affect fibrillation onset times, which are mainly determined by the concentration of unbound protein species present in equilibrium. In this system, knowledge of the fibrillation kinetics and inhibitor affinities suffices to predict the effect of kinetic inhibitors on fibrillation lag times. In addition, we developed a new methodology to better estimate fibrillation lag times from experimental curves.     

Molecular findings from influenza A(H1N1)pdm09 detected in patients from a Brazilian equatorial region during the pandemic period

After the World Health Organization officially declared the end of the first pandemic of the XXI century in August 2010, the influenza A(H1N1)pdm09 virus has been disseminated in the human population. In spite of its sustained circulation, very little on phylogenetic data or oseltamivir (OST) resistance is available for the virus in equatorial regions of South America. In order to shed more light on this topic, we analysed the haemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09 positive samples collected during the pandemic period in the Pernambuco (PE), a northeastern Brazilian state. Complete HA sequences were compared and amino acid changes were related to clinical outcome. In addition, the H275Y substitution in NA, associated with OST resistance, was investigated by pyrosequencing. Samples from PE were grouped in phylogenetic clades 6 and 7, being clustered together with sequences from South and Southeast Brazil. The D222N/G HA gene mutation, associated with severity, was found in one deceased patient that was pregnant. Additionally, the HA mutation K308E, which appeared in Brazil in 2010 and was only detected worldwide the following year, was identified in samples from hospitalised cases. The resistance marker H275Y was not identified in samples tested. However, broader studies are needed to establish the real frequency of resistance in this Brazilian region.     

Contextual Fear Conditioning in Maternal Separated Rats: The Amygdala as a Site for Alterations

The first 2 weeks of life are a critical period for neural development in rats. Repeated long-term separation from the dam is considered to be one of the most potent stressors to which rat pups can be exposed, and permanently modifies neurobiological and behavioral parameters. Prolonged periods of maternal separation (MS) usually increase stress reactivity during adulthood, and enhance anxiety-like behavior. The aim of this study was to verify the effects of maternal separation during the neonatal period on memory as well as on biochemical parameters (Na⁺, K⁺-ATPase and antioxidant enzymes activities) in the amygdala of adult rats. Females and male Wistar rats were subjected to repeated maternal separation (incubator at 32 °C, 3 h/day) during postnatal days 1–10. At 60 days of age, the subjects were exposed to a Contextual fear conditioning task. One week after the behavioral task, animals were sacrificed and the amygdala was dissected for evaluation of Na⁺, K⁺-ATPase and antioxidant enzymes activities. Student-t test showed significant MS effect, causing an increase of freezing time in the three exposures to the aversive context in both sexes. Considering biochemical parameters Student-t test showed significant MS effect causing an increase of Na⁺, K⁺-ATPase activity in both sexes. On the other hand, no differences were found among the groups on the antioxidant enzymes activities [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT)] in male rats, but in females, we found a significant MS effect, causing an increase of CAT activity and no differences were found among the groups on SOD and GPx activities. Our results suggest a role of early rearing environment in programming fear learning and memory in adulthood. An early stress experience such as maternal separation may increase activity in the amygdala (as pointed by the increased activity of Na⁺, K⁺-ATPase), affecting behaviors related to fear in adulthood, and this effect could be task-specific.     

Lacunas: a web interface to identify plant knowledge gaps to support informed decision-making

Scientists from megadiverse countries, such as Brazil, face huge challenges in gathering and analyzing information about species richness and abundance. In Brazil, speciesLink is an e-infrastructure that offers free and open access to data from more than 300 biological and data collections. SpeciesLink’s thematic network, INCT-Virtual Herbarium of Plants and Fungi  and the List of Species of the Brazilian Flora, are used as primary data sources to develop Lacunas, an information system with a public web interface that generates detailed reports of the status of plant species occurrence data. Lacunas also integrates information about endemism, conservation status, and collecting efforts over time. Here we describe the motivation and the functionality of this system, showing how it can be useful in detecting under-sampled plant species and geographic areas. We show examples of how knowledge can be extracted from biodiversity primary data using Lacunas. For instance, Lacunas report revealed that 111 angiosperm species (10.3 %), currently considered Data Deficient (DD) in the Official List of Threatened Brazilian Flora, have their distribution well characterized. In addition, the situation of Attalea funifera, a native palm classified as DD, was analyzed in detail, together with other use cases. Information presented in Lacunas reports can thus be used by scientists and policy-makers to help evaluate the status of species occurrence data and prioritize digitization and collecting efforts, as well as some features concerning its conservation status. As Lacunas offers a public online interface, it may also become a valuable tool for helping decision-making processes to become more dynamic and transparent.     

Aspergillus fumigatus calcineurin interacts with a nucleoside diphosphate kinase

The Ca²⁺-calcineurin pathway affects virulence and morphogenesis in filamentous fungi. Here, we identified 37 CalA-interacting proteins that interact with the catalytic subunit of calcineurin (CalA) in Aspergillus fumigatus, including the nucleoside diphosphate kinase (SwoH). The in vivo interaction between CalA and SwoH was validated by bimolecular fluorescence complementation. A. fumigatus swoH is an essential gene. Therefore, a temperature-sensitive conditional mutant strain with a point mutation in the active site, SwoH^V83F, was constructed, which demonstrated reduced growth and increased sensitivity to elevated temperatures. The SwoH^V83F mutation did not cause a loss in virulence in the Galleria mellonella infection model. Taken together these results imply that CalA interacts with SwoH.     

Highly interconnected genes in disease-specific networks are enriched for disease-associated polymorphisms

Background

Complex diseases are associated with altered interactions between thousands of genes. We developed a novel method to identify and prioritize disease genes, which was generally applicable to complex diseases.

Results

We identified modules of highly interconnected genes in disease-specific networks derived from integrating gene-expression and protein interaction data. We examined if those modules were enriched for disease-associated SNPs, and could be used to find novel genes for functional studies. First, we analyzed publicly available gene expression microarray and genome-wide association study (GWAS) data from 13, highly diverse, complex diseases. In each disease, highly interconnected genes formed modules, which were significantly enriched for genes harboring disease-associated SNPs. To test if such modules could be used to find novel genes for functional studies, we repeated the analyses using our own gene expression microarray and GWAS data from seasonal allergic rhinitis. We identified a novel gene, FGF2, whose relevance was supported by functional studies using combined small interfering RNA-mediated knock-down and gene expression microarrays. The modules in the 13 complex diseases analyzed here tended to overlap and were enriched for pathways related to oncological, metabolic and inflammatory diseases. This suggested that this union of the modules would be associated with a general increase in susceptibility for complex diseases. Indeed, we found that this union was enriched with GWAS genes for 145 other complex diseases.

Conclusions

Modules of highly interconnected complex disease genes were enriched for disease-associated SNPs, and could be used to find novel genes for functional studies.     

An in vitro study on spontaneous myometrial contractility in the mare during estrus and diestrus

Uterine smooth muscle specimens were collected from euthanatized mares in estrus and diestrus. Longitudinal and circular specimens were mounted in organ baths and the signals transcribed to a Grass polygraph. After equilibration time and 2 g preload, their physiologic isometric contractility was recorded for a continuous 2.0 h. Area under the curve, frequency and time occupied by contractions were studied. Differences between cycle phases, between muscle layers, and over the recorded time periods were statistically evaluated using linear mixed-effect models. In the mare, physiologic contractility of the uterus decreased significantly over time for all variables evaluated (time as covariate on a continuous scale). For area under the curve, there was a significant effect of muscle layer (longitudinal > circular). For frequency, higher values were recorded in estrus for circular smooth muscle layer, whereas higher values were seen in longitudinal smooth muscle layers during diestrus. In longitudinal layer and in diestrus, more time was occupied by contractions than in circular layer, and in estrus. This study is describing physiologic myometrial motility in the organ bath depending on cycle phase.