Identification of novel TMPRSS2:ERG mechanisms in prostate cancer metastasis: involvement of MMP9 and PLXNA2.

Prostate cancer (PCa) is one of the major public health problems in Western countries. Recently, the TMPRSS2:ERG gene fusion, which results in the aberrant expression of the transcription factor ERG, has been shown to be the most common gene rearrangement in PCa. Previous studies have determined the contributions of this fusion in PCa disease initiation and/or progression in vitro and in vivo. In this study on TMPRSS2:ERG regulation in PCa, we used an androgen receptor and TMPRSS2:ERG fusion double-negative PCa cell model: PC3c. In three cell clones with different TMPRSS2:ERG expression levels, ectopic expression of the fusion resulted in significant induction of cell migration and invasion in a dose-dependent manner. In agreement with this phenotype, high-throughput microarray analysis revealed that a set of genes, functionally associated with cell motility and invasiveness, were deregulated in a dose-dependent manner in TMPRSS2:ERG-expressing cells. Importantly, we identified increased MMP9 (Metalloproteinase 9) and PLXNA2 (Plexin A2) expression in TMPRSS2:ERG-positive PCa samples, and their expression levels were significantly correlated with ERG expression in a PCa cohort. In line with these findings, there was evidence that TMPRSS2:ERG directly and positively regulates MMP9 and PLXNA2 expression in PC3c cells. Moreover, PLXNA2 upregulation contributed to TMPRSS2:ERG-mediated enhancements of PC3c cell migration and invasion. Furthermore, and importantly, PLXNA2 expression was upregulated in metastatic PCa tumors compared with localized primary PCa tumors. This study provides novel insights into the role of the TMPRSS2:ERG fusion in PCa metastasis.

Airway structural cells regulate TLR5-mediated mucosal adjuvant activity.

Antigen-presenting cell (APC) activation is enhanced by vaccine adjuvants. Most vaccines are based on the assumption that adjuvant activity of Toll-like receptor (TLR) agonists depends on direct, functional activation of APCs. Here, we sought to establish whether TLR stimulation in non-hematopoietic cells contributes to flagellin's mucosal adjuvant activity. Nasal administration of flagellin enhanced T-cell-mediated immunity, and systemic and secretory antibody responses to coadministered antigens in a TLR5-dependent manner. Mucosal adjuvant activity was not affected by either abrogation of TLR5 signaling in hematopoietic cells or the presence of flagellin-specific, circulating neutralizing antibodies. We found that flagellin is rapidly degraded in conducting airways, does not translocate into lung parenchyma and stimulates an early immune response, suggesting that TLR5 signaling is regionalized. The flagellin-specific early response of lung was regulated by radioresistant cells expressing TLR5 (particularly the airway epithelial cells). Flagellin stimulated the epithelial production of a small set of mediators that included the chemokine CCL20, which is known to promote APC recruitment in mucosal tissues. Our data suggest that (i) the adjuvant activity of TLR agonists in mucosal vaccination may require TLR stimulation of structural cells and (ii) harnessing the effect of adjuvants on epithelial cells can improve mucosal vaccines.

Transcriptome analysis in pea allows to distinguish chilling and acclimation mechanisms.

In order to distinguish chilling and freezing tolerance mechanisms in pea, responses to cold exposure were compared between the freezing tolerant line Champagne and the sensitive line Terese. Global gene expression was considered in the two lines and associated with morphological, histological and biochemical approaches. The chilling tolerance in both lines was related to responses of the CBF, COR and LEA genes belonging to the CBF regulon, with greater earliness of expression in the Champagne genotype. The freezing tolerance, only observed in Champagne, was associated with acclimation processes such as cellular osmotic stabilization, photosynthesis modifications, antioxidants production, modifications in hormone metabolism, cell wall composition and dynamics.

Transcriptomic and genetic studies identify IL-33 as a candidate gene for Alzheimer's disease.

The only recognized genetic determinant of the common forms of Alzheimer's disease (AD) is the epsilon 4 allele of the apolipoprotein E gene (APOE). To identify new candidate genes, we recently performed transcriptomic analysis of 2741 genes in chromosomal regions of interest using brain tissue of AD cases and controls. From 82 differentially expressed genes, 1156 polymorphisms were genotyped in two independent discovery subsamples (n=945). Seventeen genes exhibited at least one polymorphism associated with AD risk, and following correction for multiple testing, we retained the interleukin (IL)-33 gene. We first confirmed that the IL-33 expression was decreased in the brain of AD cases compared with that of controls. Further genetic analysis led us to select three polymorphisms within this gene, which we analyzed in three independent case-control studies. These polymorphisms and a resulting protective haplotype were systematically associated with AD risk in non-APOE epsilon 4 carriers. Using a large prospective study, these associations were also detected when analyzing the prevalent and incident AD cases together or the incident AD cases alone. These polymorphisms were also associated with less cerebral amyloid angiopathy (CAA) in the brain of non-APOE epsilon 4 AD cases. Immunohistochemistry experiments finally indicated that the IL-33 expression was consistently restricted to vascular capillaries in the brain. Moreover, IL-33 overexpression in cellular models led to a specific decrease in secretion of the A beta(40) peptides, the main CAA component. In conclusion, our data suggest that genetic variants in IL-33 gene may be associated with a decrease in AD risk potentially in modulating CAA formation.

Evidence for induction of the ornithine transcarbamylase expression in Alzheimer's disease.

To more rapidly identify candidate genes located within chromosomal regions of interest defined by genome scan studies in Alzheimer's disease (AD), we have developed a customized microarray containing all the ORFs (n=2741) located within nine of these regions. Levels of gene expression were assessed in total RNA from brain tissue of 12 controls and 12 AD patients. Of all genes showing differential expression, we focused on the ornithine transcarbamylase (OTC) gene on Xp21.1., a key enzyme of the urea cycle which we found to be expressed in AD brains but not in controls, as confirmed by RT-PCR. We also detected mRNA expression of all the other urea cycle enzymes in AD brains. Immunochemistry experiments revealed that the OTC expression was strictly restricted to vascular endothelial cells in brain. Furthermore, OTC activity was 880% increased in the CSF of probable AD cases compared with controls. We analysed the association of the OTC -389 G/A and -241 A/G promoter polymorphisms with the risk of developing AD. We observed that rare haplotypes may be associated with the risk of AD through a possible modulation of the methylation of the OTC promoter. In conclusion, our results suggest the involvement of a new pathway in AD brains involving the urea cycle.

Structure-based mechanism of ligand binding for periplasmic solute-binding protein of the Bug family.

Bug proteins form a large family of periplasmic solute-binding proteins well represented in beta-proteobacteria. They adopt a characteristic Venus flytrap fold with two globular domains bisected by a ligand-binding cleft. The structures of two liganded Bug proteins have revealed that the family is specific for carboxylated solutes, with a characteristic mode of binding involving two highly conserved beta strand-beta turn-alpha helix motifs originating from each domain. These two motifs form hydrogen bonds with a carboxylate group of the ligand, both directly and via conserved water molecules, and have thus been termed the carboxylate pincers. In both crystallized Bug proteins, the ligands were found enclosed between the two domains and inaccessible to solvent, suggesting an inter-domain hinge-bending motion upon ligand binding. We report here the first structures of an open, unliganded Bug protein and of the same protein with a citrate ion bound in the open cavity. One of the ligand carboxylate groups is bound to one half of the carboxylate pincers by the beta strand-beta turn-alpha helix motif from domain 1, and the citrate ion forms several additional interactions with domain 1. The ligand is accessible to solvent and has very few contacts with domain 2. In this open, liganded structure, the second part of the carboxylate pincers originating from domain 2 is not stabilized by ligand binding, and a loop replaces the beta turn. In the unliganded structure, both motifs of the carboxylate pincers are highly mobile, and neither of the two beta turns is formed. Thus, ligand recognition is performed by domain 1, with the carboxylate group serving as an initial anchoring point. Stabilization of the closed conformation requires proper interactions to be established with domain 2, and thus domain 2 discriminates between productively and non-productively bound ligands.

In vitro expression and analysis of secreted fowlpox virus CC chemokine-like proteins Fpv060, Fpv061, Fpv116 and Fpv121.

The four CC chemokine-like proteins (Fpv060, Fpv061, Fpv116 and Fpv121) of fowlpox virus (FWPV) were over-expressed as His-tagged versions from a T7 promoter/EMCV IRES construct in vitro, by coupled transcription/translation, or in cell culture, by co-infection with two recombinant FWPVs (one expressing the chemokine-like protein and one expressing T7 RNA polymerase). All, except Fpv116, appeared to be glycosylated in the presence of microsomal membranes in vitro. In culture, all were secreted (even though secretion of Fpv061 was not predicted). Secreted forms of Fpv060 and Fpv121 were the most abundant forms of those two proteins. Glycosidase analysis of cellular and secreted forms confirmed that Fpv060, Fpv061 and Fpv121 were N-glycosylated and that the most abundant, cellular form of Fpv061 had been glycosylated but remained Endo H-sensitive (retained in the endoplasmic reticulum or Golgi). N-terminal sequence analysis of His-tagged Fpv060 and Fpv121 showed that they were processed at the predicted signal cleavage sites. Fpv060- and Fpv061-specific antipeptide sera allowed confirmation that the expression, processing and secretion of the native proteins were as determined for the His-tagged proteins. Isolation of knock-out mutants showed that all four proteins were non-essential for replication in tissue culture.

Detection of somatic phages, infectious enteroviruses and enterovirus genomes as indicators of human enteric viral pollution in surface water.

In the present study, we aimed to determine whether the concentrations of somatic coliphages, infectious enteroviruses or the detection of enterovirus genomes were associated with the detection of human pathogenic viruses in surface water. Four French rivers were sampled monthly or semimonthly for the quantitative detection of somatic coliphages, infectious enteroviruses and the qualitative RT-PCR detection of enterovirus, hepatitis A virus, Norwalk I viruses, Norwalk II viruses, astrovirus and rotavirus genomes over 12 months. All the 68 water samples tested were positive for the quantitative detection of somatic coliphages (range of concentrations: 4 x 10(2) to 1.6 x10(5) FUl(-1)). Infectious enteroviruses were isolated by a cell culture system in only two (3%) of the 68 concentrated water samples tested, whereas enterovirus genomes were detectable in 60 (88%) of the same samples. A positive RT-PCR detection of the genome of hepatitis A virus, Norwalk-like virus genogroup II, astrovirus, rotavirus and Norwalk-like virus genogroup I was demonstrated, respectively, in 1.5% (1/68), 1.5% (1/68), 3% (2/68), 0% and 0% of the 68 concentrated water samples tested. All of these four water samples were positive for the detection of enterovirus genomes, whereas only one of them was positive for the isolation of enteroviruses on cell culture. Moreover, the genomic detection of human pathogenic viruses appeared not to be statistically associated with the concentration levels of somatic coliphages in the 68 concentrated water samples tested (Wilcoxon rank test; P=0.14). Taken together, our findings indicate that the quantitative detection of somatic coliphages and the isolation of enteroviruses on cell culture are not suitable parameters for the control of the viral contamination in surface water, whereas the detection of enterovirus genomes may be useful for predicting the presence of waterborne viruses.

Relevance and limitations of public databases for microarray design: a critical approach to gene predictions.

In conjunction with the completion of the human genome sequence, microarray technology offers a complementary strategy to traditional methodologies used to search for genetic determinants involved in multifactorial diseases such as Alzheimer's disease. In order to gain benefits from this strategy, we have designed home-made microarrays to compare the expression of all ORFs located within loci of interest defined by genome scanning in Alzheimer family studies. Two approaches were selected using either probes amplified by PCR from a cDNA bank or specific oligonucleotides. Here, we report the challenging task of validating, prioritising and selecting the best ORFs derived from the genome sequence. The initial inventory from the NCBI website allowed us to select 5849 ORF's within nine loci. Half of them resulted from prediction models using the GenomeScan software. However, our data have shown that predicted ORFs may not be representative of exonic sequences, or even real genes. These observations have led us to exclude these ORFs from our study, decreasing their number from 5849 to 2748. Microarrays may be only 'snapshots' of our current knowledge of the human genome.

Differential modulation of Bordetella pertussis virulence genes as evidenced by DNA microarray analysis.

The production of most factors involved in Bordetella pertussis virulence is controlled by a two-component regulatory system termed BvgA/S. In the Bvg+ phase virulence-activated genes (vags) are expressed, and virulence-repressed genes (vrgs) are down-regulated. The expression of these genes can also be modulated by MgSO(4) or nicotinic acid. In this study we used microarrays to analyse the influence of BvgA/S or modulation on the expression of nearly 200 selected genes. With the exception of one vrg, all previously known vags and vrgs were correctly assigned as such, and the microarray analyses identified several new vags and vrgs, including genes coding for putative autotransporters, two-component systems, extracellular sigma factors, the adenylate cyclase accessory genes cyaBDE, and two genes coding for components of a type III secretion system. For most of the new vrgs and vags the results of the microarray analyses were confirmed by RT-PCR analysis and/or lacZfusions. The degree of regulation and modulation varied between genes, and showed a continuum from strongly BvgA/S-activated genes to strongly BvgA/S-repressed genes. The microarray analyses also led to the identification of a subset of vags and vrgs that are differentially regulated and modulated by MgSO(4) or nicotinic acid, indicating that these genes may be targets for multiple regulatory circuits. For example, the expression of bilA, a gene predicted to encode an intimin-like protein, was found to be activated by BvgA/S and up-modulated by nicotinic acid. Furthermore, surprisingly, in the strain analysed here, which produces only type 2 fimbriae, the fim3 gene was identified as a vrg, while fim2 was confirmed to be a vag.

Enteroviruses can persist with or without active viral replication in cardiac tissue of patients with end-stage ischemic or dilated cardiomyopathy.

To investigate enterovirus replication versus persistence in end-stage cardiac diseases, endomyocardial biopsies from explanted hearts of 70 patients with idiopathic dilated cardiomyopathy (IDCM), 64 patients with chronic coronary disease (CCD), and 45 donors of healthy hearts (controls) were examined by reverse transcriptase-polymerase chain reaction for genomic and antigenomic enterovirus RNA and by VP1 antigen immunohistochemistry. Enterovirus genome was detected in 25 of 70 patients with IDCM and in 21 of 64 patients with CCDs (35.7 vs. 32.8%, respectively; P=.12). Of the 46 patients positive for genomic RNA, only 3 exhibited antigenomic RNA and VP1 antigen that demonstrated active viral replication, whereas 43 had latent infection characterized by the absence of antigenomic RNA associated with or not with VP1 antigen expression. No viral component was detected in control subjects. The findings demonstrate that a small percentage of patients with end-stage chronic cardiac diseases had active enterovirus replication in their myocardium.