Serologic survey of West Nile virus in horses from Central-West,Northeast and Southeast Brazil

Since the emergence of West Nile virus (WNV) in North America in 1999, there have been several reports of WNV activity in Central and South American countries. To detect WNV in Brazil, we performed a serological survey of horses from different regions of Brazil using recombinant peptides from domain III of WNV. Positive samples were validated with the neutralisation test. Our results showed that of 79 ELISA-positive horses, nine expressed WNV-specific neutralising antibodies. Eight of the infected horses were from the state of Mato Grosso do Sul and one was from the state of Paraíba. Our results provide additional evidence for the emergence of WNV in Brazil and for its circulation in multiple regions of the country.     

Changes in biofilm structure during the colonization of chalcopyrite by Acidithiobacillus thiooxidans

Biofilms of Acidithiobacillus thiooxidans were grown on the surface of massive chalcopyrite electrodes (MCE) where different secondary sulfur phases were previously formed by potentiostatic oxidation of MCE at 0.780?≤?E _an?≤?0.965 V (electrooxidized MCE, eMCE). The formation of mainly S⁰ and minor amounts of CuS and S _n ^2? were detected on eMCEs. The eMCEs were incubated with A. thiooxidans cells for 1, 12, 24, 48, and 120 h in order to temporally monitor changes in eMCE's secondary phases, biofilm structure, and extracellular polymeric substance (EPS) composition (lipids, proteins, and polysaccharides) using microscopic, spectroscopic, electrochemical, and biochemical techniques. The results show significant cell attachments with stratified biofilm structure since the first hour of incubation and EPS composition changes, the most important being production after 48–120 h when the highest amount of lipids and proteins were registered. During 120 h, periodic oxidation/formation of S⁰/S _n ^2? was recorded on biooxidized eMCEs, until a stable CuS composition was formed. In contrast, no evidence of CuS formation was observed on the eMCEs of the abiotic control, confirming that CuS formation results from microbial activity. The surface transformation of eMCE induces a structural transformation of the biofilm, evolving directly to a multilayered biofilm with more hydrophobic EPS and proteins after 120 h. Our results suggest that A. thiooxidans responded to the spatial and temporal distribution and chemical reactivity of the S _n ^2?/S⁰/CuS phases throughout 120 h. These results suggested a strong correlation between surface speciation, hydrophobic domains in EPS, and biofilm organization during chalcopyrite biooxidation by A. thiooxidans.     

A comparative protease stability study of synthetic macrocyclic peptides that mimic two endocrine hormones

Peptide therapeutics have traditionally faced many challenges including low bioavailability, poor proteolytic stability and difficult cellular uptake. Conformationally constraining the backbone of a peptide into a macrocyclic ring often ameliorates these problems and allows for the development of a variety of new drugs. Such peptide-based pharmaceuticals can enhance the multi-faceted functionality of peptide side chains, permitting the peptides to bind cellular targets and receptors necessary to impart their role, while protecting them from degrading cellular influences. In the work described here, we developed three cyclic peptides, VP mimic1, VP mimic2 and OT mimic1, which mimic endocrine hormones vasopressin and oxytocin. Making notable changes to the overall structure and composition of the parent hormones, we synthesized the mimics and tested their durability against treatment with three proteases chosen for their specificity: pepsin, alpha-chymotrypsin, and pronase. Vasopressin and oxytocin contain a disulfide linkage leaving them particularly vulnerable to deactivation from the reducing environment inside the cell. Thus, we increased the complexity of our assays by adding reducing agent glutathione to each mixture. Subsequently, we discovered each of our mimics withstood protease treatment with less degradation and/or a slower rate of degradation as compared to both parent hormones and a linear control peptide.     

Using DNA-barcoding for sorting out protist species complexes: A case study of the Nebela tincta–collaris–bohemica group (Amoebozoa; Arcellinida,Hyalospheniidae)

Species identification by means of morphology is often problematic in protists. Nebela tincta–collaris–bohemica (Arcellinida) is a species complex of small to medium-sized (ca.100 μm) testate amoebae common in peat bogs and forest soils. The taxonomic validity of characters used to define species within this group is debated and causes confusion in studies of biogeography, and applications in palaeoecology.We examined the relationship between morphological and genetic diversity within this species complex by combined analyses of light microscopy imaging and Cytochrome Oxidase Subunit 1(COI) sequences obtained from the same individual amoeba cells. Our goals were (1) to clarify the taxonomy and the phylogenetic relationships within this group, and (2) to evaluate if individual genotypes corresponded to specific morphotypes and the extent of phenotypic plasticity.We show here that small variations in test morphology that have been often overlooked by traditional taxonomy correspond to distinct haplotypes. We therefore revise the taxonomy of the group. We redefine Nebela tincta (Leidy) Kosakyan et Lara and N. collaris (Ehrenberg 1848) Kosakyan et Gomaa, change N. tincta var. rotunda Penard to N. rotunda (Penard 1890), describe three new species: N. guttata n. sp. Kosakyan et Lara, N. pechorensis n. sp. Kosakyan et Mitchell, and N. aliciae n. sp. Mitchell et Lara.     

Designing bryophyte surveys for an optimal coverage of diversity gradients

Knowledge on the distribution and abundance of species is plagued by significant taxonomic and geographic biases that influence the analyses on biodiversity patterns. Due to this, standard, easy-to-use methods are needed to design efficient field campaigns that minimize data deficiencies. We evaluate the applicability, usefulness and effectiveness of a survey design protocol based on the Environmental Diversity (ED) criterion under different scenarios, with examples of varying extent of environmental niche, range of spatial distribution and level of previous knowledge. We planned surveys for epiphytic bryophytes growing in three types of forests at NW Iberian Peninsula (dominated by Quercus ilex, Q. faginea and Q. pyrenaica). Knowledge on the distribution and abundance of epiphytic bryophytes in this region presents large gaps and strong geographic biases. Besides, the three forest types differ in their environmental requirements, spatial distribution and level of previous knowledge, providing three working scenarios to test the response of the protocol under different situations. The protocol was implemented as a set of sequential selection rules, starting by an ED-based criterion aiming at maximizing the coverage of climatic and geographic variability; further criteria include an iterative set of qualitative properties: maximizing forest area, conservation status and accessibility. The protocol performed efficiently at different ranges of spatial distribution levels of environmental variability, and degree of previous knowledge and generated an even distribution of sampling points that efficiently covered the diversity of epiphytic bryophytes. The results show that ED protocols are a proficient and time-saving approach to select sampling sites by objective criteria also for groups with high dispersal ability and fragmented landscapes.     

The Tyrosine Kinase Btk Regulates the Macrophage Response to Listeria monocytogenes Infection

In this study we investigated the role of Bruton''s tyrosine kinase (Btk) in the immune response to the Gram-positive intracellular bacterium Listeria monocytogenes (Lm). In response to Lm infection, Btk was activated in bone marrow-derived macrophages (BMMs) and Btk ^−/− BMMs showed enhanced TNF-α, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt) BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of E. coli fragments, there was no difference between wt and Btk ^−/− BMMs in the uptake of Lm upon infection. Moreover, there was no difference in the response to heat-killed Lm between wt and Btk ^−/− BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular Lm. Finally, Btk ^−/− mice displayed enhanced resistance and an increased mean survival time upon Lm infection in comparison to wt mice. This correlated with elevated IFN-γ and IL-12p70 serum levels in Btk ^−/− mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during Lm infection.     

Polymorphisms in DNA-Repair Genes in a Cohort of Prostate Cancer Patients from Different Areas in Spain: Heterogeneity between Populations as a Confounding Factor in Association Studies

Background

Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics.

Objective

To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias.

Design, Setting, and Participants

A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArray® NT Cycler.

Outcome Measurements and Statistical Analysis

Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer.

Results and Limitations

We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics.

Conclusion

Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of SNPs associated with radiation-induced toxicity, especially when extensive meta-analysis with subjects from different countries are carried out.     

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose

Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed.

Materials and Methods

Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR.

Results

Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C.

Conclusion

HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.