AR mRNA stability is increased with AR-antagonist resistance Via 3’ UTR variants

Advanced prostate cancer is often treated with AR-antagonists which target the androgen receptor (AR) on which the growth of the tumour depends. Prostate cancer often develops AR-antagonist resistance via a plethora of mechanisms, many of which are as yet unknown, but it is thought that AR upregulation or AR ligand binding site mutations, may be responsible. Here we describe the production of cell lines based on LNCaP and VCaP, with acquired resistance to the clinically relevant AR-antagonists, bicalutamide and enzalutamide.In these resistant cells, we observed, via RNA-seq, that new variants in the 3’UTR of the AR mRNA were detectable and that the levels were increased both with AR-antagonist treatment and with hormonal starvation. Around 20% of AR transcripts showed a 3kb deletion within the 6.7kb 3’UTR sequence. Actinomycin D and luciferase fusion studies indicated that this shorter mRNA variant was inherently more stable in anti-androgen resistant cell lines.Of additional interest was that the AR UTR variant could be detected in the sera of prostate cancer patients in a cohort of serum samples collected from patients of Gleason grades 6-10, with an increasing level correlated to increasing grade.We hypothesise that the shorter AR UTR variant is a survival adaptation to low hormone levels and/or AR-antagonist treatment in these cells, where a more stable mRNA may allow higher levels of AR expression under these conditions

Pyrosequencing of Mytilus galloprovincialis cDNAs: tissue-specific expression patterns.

BACKGROUND: Mytilus species are important in marine ecology and in environmental quality assessment, yet their molecular biology is poorly understood. Molecular aspects of their reproduction, hybridisation between species, mitochondrial inheritance, skewed sex ratios of offspring and adaptation to climatic and pollution factors are priority areas. METHODOLOGY/PRINCIPAL FINDINGS: To start to address this situation, expressed genetic transcripts from M. galloprovincialis were pyrosequenced. Transcripts were isolated from the digestive gland, foot, gill and mantle of both male and female mussels. In total, 175,547 sequences were obtained and for foot and mantle, 90% of the sequences could be assembled into contiguous fragments but this reduced to 75% for the digestive gland and gill. Transcripts relating to protein metabolism and respiration dominated including ribosomal proteins, cytochrome oxidases and NADH dehydrogenase subunits. Tissue specific variation was identified in transcripts associated with mitochondrial energy metabolism, with the digestive gland and gill having the greatest transcript abundance. Using fragment recruitment it was also possible to identify sites of potential small RNAs involved in mitochondrial transcriptional regulation. Sex ratios based on Vitelline Envelop Receptor for Lysin and Vitelline Coat Lysin transcript abundances, indicated that an equal sex distribution was maintained. Taxonomic profiling of the M. galloprovincialis tissues highlighted an abundant microbial flora associated with the digestive gland. Profiling of the tissues for genes involved in intermediary metabolism demonstrated that the gill and digestive gland were more similar to each other than to the other two tissues, and specifically the foot transcriptome was most dissimilar. CONCLUSIONS: Pyrosequencing has provided extensive genomic information for M. galloprovincialis and generated novel observations on expression of different tissues, mitochondria and associated microorganisms. It will also facilitate the much needed production of an oligonucleotide microarray for the organism

Culture dependent and independent analyses of 16S rRNA and ATP citrate lyase genes : a comparison of microbial communities from different black smoker chimneys on the Mid-Atlantic Ridge

Author Posting. © Springer, 2008. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Extremophiles 12 (2008): 627-640, doi:10.1007/s00792-008-0167-5.The bacterial and archaeal communities of three deep-sea hydrothermal vent systems located on the Mid-Atlantic Ridge (MAR; Rainbow, Logatchev and Broken Spur) were investigated using an integrated culture-dependent and independent approach. Comparative molecular phylogenetic analyses, using the 16S rRNA gene and the deduced amino acid sequences of the alpha and beta subunits of the ATP citrate lyase encoding genes were carried out on natural microbial communities, on an enrichment culture obtained from the Broken Spur chimney, and on novel chemolithoautotrophic bacteria and reference strains originally isolated from several different deep-sea vents. Our data showed that the three MAR hydrothermal vent chimneys investigated in this study host very different microbial assemblages. The microbial community of the Rainbow chimney was dominated by thermophilic, autotrophic, hydrogen-oxidizing, sulfur- and nitrate reducing Epsilonproteobacteria related to the genus Caminibacter. The detection of sequences related to sulfur-reducing bacteria and archaea (Archaeoglobus) indicated that thermophilic sulfate reduction might also be occurring at this site. The Logatchev bacterial community included several sequences related to mesophilic sulfur-oxidizing bacteria, while the archaeal component of this chimney was dominated by sequences related to the ANME-2 lineage, suggesting that anaerobic oxidation of methane may be occurring at this site. Comparative analyses of the ATP citrate lyase encoding genes from natural microbial communities suggested that Epsilonproteobacteria were the dominant primary producers using the reverse TCA cycle (rTCA) at Rainbow, while Aquificales of the genera Desulfurobacterium and Persephonella were prevalent in the Broken Spur chimney.This research was supported by NSF grants MCB 04-56676 (C.V.), OCE 03-27353 (C.V.), MCB 04-56689 (S.M.S.), a grant from the New Jersey Agricultural Experiment Station to C.V., and a NIH Ph.D. Training Program in Biotechnology Fellowship (NIH NIGMS 5 T32 GM08339) to J.V. M.H. was supported through a postdoctoral scholarship from the Woods Hole Oceanographic Institution

Reconstruction of Ribosomal RNA Genes from Metagenomic Data

Direct sequencing of environmental DNA (metagenomics) has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes

Korarchaeota Diversity, Biogeography, and Abundance in Yellowstone and Great Basin Hot Springs and Ecological Niche Modeling Based on Machine Learning

Over 100 hot spring sediment samples were collected from 28 sites in 12 areas/regions, while recording as many coincident geochemical properties as feasible (>60 analytes). PCR was used to screen samples for Korarchaeota 16S rRNA genes. Over 500 Korarchaeota 16S rRNA genes were screened by RFLP analysis and 90 were sequenced, resulting in identification of novel Korarchaeota phylotypes and exclusive geographical variants. Korarchaeota diversity was low, as in other terrestrial geothermal systems, suggesting a marine origin for Korarchaeota with subsequent niche-invasion into terrestrial systems. Korarchaeota endemism is consistent with endemism of other terrestrial thermophiles and supports the existence of dispersal barriers. Korarchaeota were found predominantly in >55°C springs at pH 4.7–8.5 at concentrations up to 6.6×106 16S rRNA gene copies g−1 wet sediment. In Yellowstone National Park (YNP), Korarchaeota were most abundant in springs with a pH range of 5.7 to 7.0. High sulfate concentrations suggest these fluids are influenced by contributions from hydrothermal vapors that may be neutralized to some extent by mixing with water from deep geothermal sources or meteoric water. In the Great Basin (GB), Korarchaeota were most abundant at spring sources of pH<7.2 with high particulate C content and high alkalinity, which are likely to be buffered by the carbonic acid system. It is therefore likely that at least two different geological mechanisms in YNP and GB springs create the neutral to mildly acidic pH that is optimal for Korarchaeota. A classification support vector machine (C-SVM) trained on single analytes, two analyte combinations, or vectors from non-metric multidimensional scaling models was able to predict springs as Korarchaeota-optimal or sub-optimal habitats with accuracies up to 95%. To our knowledge, this is the most extensive analysis of the geochemical habitat of any high-level microbial taxon and the first application of a C-SVM to microbial ecology

Gut Bacterial Communities in the Giant Land Snail Achatina fulica and Their Modification by Sugarcane-Based Diet

The invasive land snail Achatina fulica is one of the most damaging agricultural pests worldwide representing a potentially serious threat to natural ecosystems and human health. This species is known to carry parasites and harbors a dense and metabolically active microbial community; however, little is known about its diversity and composition. Here, we assessed for the first time the complexity of bacterial communities occurring in the digestive tracts of field-collected snails (FC) by using culture-independent molecular analysis. Crop and intestinal bacteria in FC were then compared to those from groups of snails that were reared in the laboratory (RL) on a sugarcane-based diet. Most of the sequences recovered were novel and related to those reported for herbivorous gut. Changes in the relative abundance of Bacteroidetes and Firmicutes were observed when the snails were fed a high-sugar diet, suggesting that the snail gut microbiota can influence the energy balance equation. Furthermore, this study represents a first step in gaining a better understanding of land snail gut microbiota and shows that this is a complex holobiont system containing diverse, abundant and active microbial communities

Bacteria associated with the pinewood nematode Bursaphelenchus xylophilus collected in Portugal

Abstract: In this study, we report on the bacterial community associated with the pinewood nematode Bursaphelenchus xylophilus from symptomatic pine wilted trees, as well as from long-term preserved B. xylophilus laboratory collection specimens, emphasizing the close bacteria-nematode associations that may contribute to pine wilt disease development

Like Will to Like: Abundances of Closely Related Species Can Predict Susceptibility to Intestinal Colonization by Pathogenic and Commensal Bacteria

The intestinal ecosystem is formed by a complex, yet highly characteristic microbial community. The parameters defining whether this community permits invasion of a new bacterial species are unclear. In particular, inhibition of enteropathogen infection by the gut microbiota ( = colonization resistance) is poorly understood. To analyze the mechanisms of microbiota-mediated protection from Salmonella enterica induced enterocolitis, we used a mouse infection model and large scale high-throughput pyrosequencing. In contrast to conventional mice (CON), mice with a gut microbiota of low complexity (LCM) were highly susceptible to S. enterica induced colonization and enterocolitis. Colonization resistance was partially restored in LCM-animals by co-housing with conventional mice for 21 days (LCMcon21). 16S rRNA sequence analysis comparing LCM, LCMcon21 and CON gut microbiota revealed that gut microbiota complexity increased upon conventionalization and correlated with increased resistance to S. enterica infection. Comparative microbiota analysis of mice with varying degrees of colonization resistance allowed us to identify intestinal ecosystem characteristics associated with susceptibility to S. enterica infection. Moreover, this system enabled us to gain further insights into the general principles of gut ecosystem invasion by non-pathogenic, commensal bacteria. Mice harboring high commensal E. coli densities were more susceptible to S. enterica induced gut inflammation. Similarly, mice with high titers of Lactobacilli were more efficiently colonized by a commensal Lactobacillus reuteri RR strain after oral inoculation. Upon examination of 16S rRNA sequence data from 9 CON mice we found that closely related phylotypes generally display significantly correlated abundances (co-occurrence), more so than distantly related phylotypes. Thus, in essence, the presence of closely related species can increase the chance of invasion of newly incoming species into the gut ecosystem. We provide evidence that this principle might be of general validity for invasion of bacteria in preformed gut ecosystems. This might be of relevance for human enteropathogen infections as well as therapeutic use of probiotic commensal bacteria