Androgen receptor regulation of the versican gene through an androgen response element in the proximal promoter

Versican, one of the key components of prostatic stroma, plays a central role in tumor initiation and progression. Here, we investigated promoter elements and mechanisms of androgen receptor (AR)-mediated regulation of the versican gene in prostate cancer cells. Using transient transfection assays in prostate cancer LNCaP and cervical cancer HeLa cells engineered to express the AR, we demonstrate that the synthetic androgen R1881 and dihydrotestosterone stimulate expression of a versican promoter-driven luciferase reporter vector (versican-Luc). Further, both basal and androgen-stimulated versican-Luc activities were significantly diminished in LNCaP cells, when AR gene expression was knocked down using a short hairpin RNA. Methylation-protection footprinting analysis revealed an AR-protected element between positions +75 and +102 of the proximal versican promoter, which strongly resembled a consensus steroid receptor element. Electrophoretic mobility shift and supershift assays revealed strong and specific binding of the recombinant AR DNA binding domain to oligonucleotides corresponding to this protected DNA sequence. Site-directed mutagenesis of the steroid receptor element site markedly diminished R1881-stimulated versican-Luc activity. In contrast to the response seen using LNCaP cells, R1881 did not significantly induce versican promoter activity and mRNA levels in AR-positive prostate stromal fibroblasts. Interestingly, overexpression of beta-catenin in the presence of androgen augmented versican promoter activity 10- and 30-fold and enhanced versican mRNA levels 2.8-fold in fibroblasts. In conclusion, we demonstrate that AR transactivates versican expression, which may augment tumor-stromal interactions and may contribute to prostate cancer progression.     

Evaluating the effectiveness of conservation site networks under climate change: accounting for uncertainty

We forecasted potential impacts of climate change on the ability of a network of key sites for bird conservation (Important Bird Areas; IBAs) to provide suitable climate for 370 bird species of current conservation concern in two Asian biodiversity hotspots: the Eastern Himalaya and Lower Mekong. Comparable studies have largely not accounted for uncertainty, which may lead to inappropriate conclusions. We quantified the contribution of four sources of variation (choice of general circulation models, emission scenarios and species distribution modelling methods and variation in species distribution data) to uncertainty in forecasts and tested if our projections were robust to these uncertainties. Declines in the availability of suitable climate within the IBA network by 2100 were forecast as ‘extremely likely’ for 45% of species, whereas increases were projected for only 2%. Thus, we predict almost 24 times as many ‘losers’ as ‘winners’. However, for no species was suitable climate ‘extremely likely’ to be completely lost from the network. Considerable turnover (median = 43%, 95% CI = 35–69%) in species compositions of most IBAs were projected by 2100. Climatic conditions in 47% of IBAs were projected as ‘extremely likely’ to become suitable for fewer priority species. However, no IBA was forecast to become suitable for more species. Variation among General Circulation Models and Species Distribution Models contributed most to uncertainty among forecasts. This uncertainty precluded firm conclusions for 53% of species and IBAs because 95% confidence intervals included projections of no change. Considering this uncertainty, however, allows robust recommendations concerning the remaining species and IBAs. Overall, while the IBA network will continue to sustain bird conservation, climate change will modify which species each site will be suitable for. Thus, adaptive management of the network, including modified site conservation strategies and facilitating species' movement among sites, is critical to ensure effective future conservation.     

Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Gene Expression Analysis in Rice Plants Exposed to Metal Stresses

Environmental pollution by toxic heavy metals may lead to the possible contamination of the rice plant (Oryza sativa L.). Although gene expression analysis through real-time quantitative PCR (RT-qPCR) has increased our knowledge about biological responses to heavy metals, gene network that mediates rice plant responses to heavy metal stress remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving RT-qPCR. In this study, we analyzed the expression stability of eight commonly used housekeeping genes (GAPDH, Actin, eIF-4α, UBQ 5, UBQ 10, UBC, EF-1α and β-TUB) in rice leaves exposed to four kinds of heavy metals (Zn, Cu, Cd and Pb). The expression stability of these genes was determined using geNorm, NormFinder, BestKeeper and RefFinder algorithms. The results showed that UBQ 10 and UBC were the most stable reference genes across all the tested samples. We measured the expression profiles of the heavy metal-inducible gene O. sativa METALLOTHIONEIN2b (OsMT2b) using the two most stable and one least stable reference genes in all samples. The relative expression of OsMT2b varied greatly according to the different reference genes. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in rice plants.     

A gene silencing of V‐ATPase subunit A interferes with survival and development of the tomato leafminer,Tuta absoluta

RNA interference (RNAi) technology is not only considered as a tool to analyze gene function, but it is also potentially considered as a strategy to develop novel biopesticide. In the current study, a double‐stranded RNA specific to v‐ATPase subunit A of the tomato leafminer, Tuta absoluta (Meyrick; Lepidoptera: Gelechiidae), was orally administered. A gradual decrease in the expression of the gene was observed from Day 1 to 3 and resulted in significant larval mortality. These results suggest that v‐ATPases A can be considered as a promising target gene by RNAi technology to be used in the management of the tomato leafminer.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Cellular impact of combinations of endosulfan,atrazine, and chlorpyrifos on human primary hepatocytes and HepaRG cells after short and chronic exposures

Chronic exposure to low doses of pesticides present in the environment is increasingly suspected to cause major health issues to humans. Toxicological evaluations become more complex when the exposure concerns chemical combinations. Atrazine, chlorpyrifos, and endosulfan are pesticides used worldwide in agriculture and are therefore currently found at residual levels in food and the environment, even in countries in which they are now banned. Our study aimed to use Real-Time Cell Impedance Analyzer to investigate changes in phenotypical status of primary human hepatocytes and differentiated HepaRG cells induced by short and chronic exposures to these three chemicals. In contrast to the traditionally used endpoint cytotoxicity test, this technology allows kinetic measurements in real-time throughout the entire experiment. Our data show significantly higher cytotoxic effects of mixtures as compared to individual pesticides and a greater susceptibility of human hepatocytes as compared to HepaRG to short-term exposure (24 h). Repeated exposure over 2 weeks to endosulfan and endosulfan-containing mixture induced HepaRG cell death in a time- and dose-dependent manner. Of the typical genes involved in metabolism and cell-response to xenobiotics, we found an exposure time- and condition-dependent deregulation of the expression of CYP3A4 and UGT1A in HepaRG cells exposed to low doses of pesticides and mixtures. Our data demonstrate the usefulness of real-time cell monitoring in long-term toxicological evaluations of co-exposure to xenobiotics. In addition, they support but at the same time highlight certain limitations in the use of HepaRG cells as the gold standard liver cell model in toxicity studies.