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BackgroundEstablishing relationships between transcription factors and target genes is essential for understanding the mechanisms regulating gene expression, which play a fundamental role in plant adaptation to the local environment. Despite the importance of this research area and the tremendous progress in sequencing methods such as ChIP-seq and DAP-seq, we are still far from a complete reconstruction of thecis-regulatory landscape. Only a small number of transcription factors can be assessed from experimental data to identify theircis-regulatory binding sites. This highlights the role thatin silicoapproaches can play to complement experimental data.ResultsWe have developed Plant-PLMview, a web-accessible database for detecting preferentiallycis-regulatory sequences in the gene-proximal regions of 20 plant species. Users of Plant-PLMview can (i) access the proximal regions of controlled genes from 20 plant species, (ii) query their own DNA motifs or access 840cis-regulatory sequences from various plant resources, and (iii) use a tool called PLMdetect to search for preferentially located motifs in the gene-proximal regions of a list of genes. Results are displayed via a web interface with a list of DNA motifs preferentially located in a region near the start or end of genes, the distribution of these motifs and associated annotations. In addition, a graphical map of the preferential locations of the motifs in the 5’ and 3’-proximal regions of genes provides an overview of all motifs and genes examined.ConclusionPlant-PLMview provides the opportunity to study the proximal landscape of gene regulation in 20 plant genomes. The originality of the database lies in its ease of use thanks to the curated data (cis-regulatory sequences and proximal regions of genes) and the possibility to search through PLMdetect in the 5’-gene-proximal region, but also in the 3’-gene-proximal region, which is rarely explored. The web interface provides numerous graphical views that allow the users to get an overview and interpret the results more easily.

The identification of cis-regulatory elements controlling gene expression is an arduous challenge that is being actively explored to discover the key genetic factors responsible for traits of agronomic interest. Here, we have used a de novo and genome-wide approach for preferentially located motif (PLM) detection to investigate the proximal cis-regulatory landscape of Arabidopsis thaliana and Zea mays. We report three groups of PLMs in each gene-proximal region and emphasize conserved PLMs in both species, particularly in the 3’-gene-proximal region. Comparison with resources of transcription factor and microRNA binding sites indicates that 79% of the identified PLMs are unassigned, although some are supported by MNase-defined cistrome occupancy analysis. Enrichment analyses further reveal that unassigned PLMs provide functional predictions distinct from those inferred by transcription factor and microRNA binding sites. Our study provides a comprehensive map of PLMs and points at their potential utility for future characterization of orphan genes in plants.

The grapevine reference genome was published by Jaillon et al. [1]. The sequence for the first version of the genome, called the 8X version, was obtained using a whole genome shotgun strategy and the Sanger sequencing technology and was assembled from reads representing 8X coverage. Soon after, the assembly was improved through the addition of 4X of additional coverage, including more Bacterial Artificial Chromosome end sequences that greatly improved the scaffolding of the sequence contigs [2], [3]. The corresponding scaffolds and raw sequences were deposited in European Molecular Biology Laboratory (EMBL) archives (FN594950-FN597014, 2065 entries, release 102). A new chromosome assembly was also developed, based on an improved version of the maps used for the 8X genome version [2], [3], [4], [5] and was also archived at EMBL (FN597015-FN597047, 33 entries, release 102): it is referenced in the grapevine community as the 12X.v0 version of the grapevine reference genome. The chromosome sequence scaffolding of this version still necessitated improvements as around 9% of the sequence was not anchored to chromosomes (with the corresponding scaffolds stacked in the “Unknown” chromosome) and 3.5% of the sequence could be assigned to a chromosome but without certain placement and orientation within the chromosome (stacked in additional “random” chromosomes)., This work was supported by the French National Institute for Agriculture (INRA, France), the University of Udine and the Institute of Applied Genomics (Italy), the Vlaams Institut voor Biotechnologie and the University of Ghent (Belgium), the Istituto de Ciencas de la Vid et del Vino (Logroño, Spain) and several grants: ANR-Plant-KBBE-2008- GrapeReSeq and ANR-2008-Muscares funded by the French National Research Agency (ANR), Valorizzazione dei Principali Vitigni Autoctoni Italiani e dei loro Terroir (Vigneto, no. COSVIR27129) funded by the Italian Ministry of Agriculture and the COST action FA1106 funded under the European FP7 Research Program

International audience; Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Here, we performed high-resolution copy-number analysis on 125 HCC tumors and whole-exome sequencing on 24 of these tumors. We identified 135 homozygous deletions and 994 somatic mutations of genes with predicted functional consequences. We found new recurrent alterations in four genes (ARID1A, RPS6KA3, NFE2L2 and IRF2) not previously described in HCC. Functional analyses showed tumor suppressor properties for IRF2, whose inactivation, exclusively found in hepatitis B virus (HBV)-related tumors, led to impaired TP53 function. In contrast, inactivation of chromatin remodelers was frequent and predominant in alcohol-related tumors. Moreover, association of mutations in specific genes (RPS6KA3-AXIN1 and NFE2L2-CTNNB1) suggested that Wnt/β-catenin signaling might cooperate in liver carcinogenesis with both oxidative stress metabolism and Ras/mitogen-activated protein kinase (MAPK) pathways. This study provides insight into the somatic mutational landscape in HCC and identifies interactions between mutations in oncogene and tumor suppressor gene mutations related to specific risk factors.

International audience; Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrPC) into the aggregated misfolded scrapie isoform, named PrPSc. Recent studies on the physiological role of PrPC revealed that this protein has probably multiple functions, notably in cell–cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 50-GACACAAGC CGA-30 was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities.

Immunization with xenogeneic DNA is a promising cancer treatment to overcome tolerance to self-antigens. Heat shock protein 70 (HSP70) is over-expressed in various kinds of tumors and is believed to be involved in tumor progression. This study tested a xenogeneic chicken HSP70 (chHSP70) DNA vaccine in an experimental canine transmissible venereal tumor (CTVT) model. Three vaccination strategies were compared: the first (PE) was designed to evaluate the prophylactic efficacy of chHSP70 DNA vaccination by delivering the vaccine before tumor inoculation in a prime boost setting, the second (T) was designed to evaluate the therapeutic efficacy of the same prime boost vaccine by vaccinating the dogs after tumor inoculation; the third (PT) was similar to the first strategy (PE), with the exception that the electroporation booster injection was replaced with a transdermal needle-free injection. Tumor growth was notably inhibited only in the PE dogs, in which the vaccination program triggered tumor regression significantly sooner than in control dogs (NT). The CD4(+) subpopulation of tumor-infiltrating lymphocytes and canine HSP70 (caHSP70)-specific IFN-γ-secreting lymphocytes were significantly increased during tumor regression in the PE dogs as compared to control dogs, demonstrating that specific tolerance to caHSP70 has been overcome. In contrast, no benefit of the therapeutic strategy (T) could be noticed and the (PT) strategy only led to partial control of tumor growth. In summary, antitumor prophylactic activity was demonstrated using the chHSP70 DNA vaccine including a boost via electroporation. Our data stressed the importance of DNA electroporation as a booster to get the full benefit of DNA vaccination but also of cancer immunotherapy initiation as early as possible. Xenogeneic chHSP70 DNA vaccination including an electroporation boost is a potential vaccine to HSP70-expressing tumors, although further research is still required to better understand true clinical potential.

The pathogenesis of pulmonary fibrosis is linked to oxidative stress, possibly generated by the reactive oxygen species (ROS) generating NADPH oxidase NOX4. Epithelial cell death is a crucial early step in the development of the disease, followed only later by the fibrotic stage. We demonstrate that in lungs of patients with idiopathic lung fibrosis, there is strong expression of NOX4 in hyperplastic alveolar type II cells. Aim: To study a possible causative role of NOX4 in the death of alveolar cells, we have generated NOX4-deficient mice. Results: Three weeks after administration of bleomycin, wild-type (WT) mice developed massive fibrosis, whereas NOX4-deficient mice displayed almost normal lung histology, and only little Smad2 phosphorylation and accumulation of myofibroblasts. However, the protective effects of NOX4 deficiency preceded the fibrotic stage. Indeed, at day 7 after bleomycin, lungs of WT mice showed massive increase in epithelial cell apoptosis and inflammation. In NOX4-deficient mice, no increase in apoptosis was observed, whereas inflammation was comparable to WT. In vitro, NOX4-deficient primary alveolar epithelial cells exposed to transforming growth factor-β1 did not generate ROS and were protected from apoptosis. Acute treatment with the NOX inhibitors also blunted transforming growth factor-β1–induced apoptosis. Conclusion: ROS generation by NOX4 is a key player in epithelial cell death leading to pulmonary fibrosis. Antioxid. Redox Signal. 15, 607–619.

International audience; ABSTRACT The homeostatic self-renewal of the colonic epithelium requires coordinated regulation of the canonical Wnt/β-catenin and Notch signaling pathways to control proliferation and lineage commitment of multipotent stem cells. However, the molecular mechanisms by which the Wnt/β-catenin and Notch1 pathways interplay in controlling cell proliferation and fate in the colon are poorly understood. Here we show that NADPH oxidase 1 (NOX1), a reactive oxygen species (ROS)-producing oxidase that is highly expressed in colonic epithelial cells, is a pivotal determinant of cell proliferation and fate that integrates Wnt/β-catenin and Notch1 signals. NOX1-deficient mice reveal a massive conversion of progenitor cells into postmitotic goblet cells at the cost of colonocytes due to the concerted repression of phosphatidylinositol 3-kinase (PI3K)/AKT/Wnt/β-catenin and Notch1 signaling. This conversion correlates with the following: (i) the redox-dependent activation of the dual phosphatase PTEN, causing the inactivation of the Wnt pathway effector β-catenin, and (ii) the downregulation of Notch1 signaling that provokes derepression of mouse atonal homolog 1 (Math1) expression. We conclude that NOX1 controls the balance between goblet and absorptive cell types in the colon by coordinately modulating PI3K/AKT/Wnt/β-catenin and Notch1 signaling. This finding provides the molecular basis for the role of NOX1 in cell proliferation and postmitotic differentiation.

Aim: In patients with advanced cirrhosis, little is known about the ability of peripheral blood monocytes to spontaneously produce signaling proteins such as cytokines. The aim of this ex vivo study was to evaluate cytokine production under baseline conditions and after stimulation by lipopolysaccharide (LPS), a toll-like receptor (TLR) agonist. Methods: Peripheral blood monocytes were isolated from patients with advanced alcoholic cirrhosis (without ongoing bacterial infections) and normal subjects. Cells were left unstimulated or were stimulated with LPS. The abundance of 24 cytokines was measured using a filter-based, arrayed sandwich enzyme-linked immunosorbent assay (ELISA) in the supernatant of cultured monocytes. Results: Cirrhotic monocytes spontaneously produced six proteins (TNF-α, IL-6, IL-8, MCP-1, RANTES and Gro), whereas normal monocytes produced only small amounts of IL-8 and RANTES. Analyses with the online gene set analysis toolkit WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt) found enrichment for the six proteins in the human gene ontology subcategory (http://www.geneontology.org), Kyoto Encyclopedia of Genes and Genome pathways (http://www.genome.ad.jp/kegg/) and BioCarta pathways (http://www.biocarta.com/genes/index.asp) consistent with a proinflammatory phenotype of cirrhotic monocytes resulting from activated TLR signaling. Interestingly, LPS-elicited TLR engagement further increased the production of the six proteins and did not induce the secretion of any others, in particular the anti-inflammatory cytokine IL-10. LPS-stimulated normal monocytes produced TNF-α, IL-6, IL-8, MCP-1, RANTES, Gro and IL-10. Conclusion: In patients with advanced cirrhosis, peripheral blood monocytes spontaneously produce proinflammatory cytokines, presumably in response to unrestricted TLR signaling.

High-density lipoproteins (HDL) are known to neutralize lipopolysaccharide (LPS). Because patients with cirrhosis have lower HDL levels, this may contribute to LPS-induced ex vivo monocyte overproduction of proinflammatory cytokines. However, the effects of HDL on cytokine production by monocytes from patients with cirrhosis have never been studied. The aim of this study was to determine the effects of HDL on LPS-induced proinflammatory cytokine production in whole blood and isolated monocytes from patients with severe cirrhosis and controls. Plasma levels of HDL and cytokines were determined. The effects of reconstituted HDL (rHDL) on LPS-induced cytokine production in whole blood were assessed by cytokine array and on tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) production in isolated monocytes. Plasma HDL levels were significantly lower in patients with cirrhosis than in controls. Plasma levels of TNF-α and IL-6 were significantly higher in patients with cirrhosis than in controls. Incubation of rHDL with whole blood prevented LPS-induced TNF-α and IL-6 overproduction in patients with cirrhosis. LPS-induced TNF-α production and CD14 expression were significantly more marked in cirrhotic monocytes than in control monocytes, and both decreased significantly after rHDL incubation. LPS-induced down-regulation of scavenger receptor, class B, type I (SR-BI) expression was abolished in cirrhotic monocytes. Conclusions: This study shows that rHDL abolishes the LPS-induced overproduction of proinflammatory cytokines in whole blood from patients with severe cirrhosis. These results were confirmed in isolated monocytes from these patients. This suggests that administration of rHDL might be a useful strategy for the treatment of cirrhosis to limit LPS-induced cytokine overproduction. (HEPATOLOGY 2009;49:175-184.)

Chronic exposure to particulate air pollution is associated with lung function impairment. To determine the molecular mechanism(s) of this phenomenon, we investigated, in an alveolar human epithelial cell line (A549), whether diesel exhaust particles (DEPs), a main component of particulate air pollution, modulates the expression and activity of the matrix metalloprotease (MMP)-1, a collagenase involved in alveolar wall degradation. Interaction of DEPs with cigarette smoke, which also produces structural and functional lung alterations, was also investigated. A noncytotoxic concentration of DEPs induced an increase in MMP-1 mRNA and protein expression and activity in A549 cells without modifying the expression of the MMP inhibitors TIMP-1 and -2. This effect was not potentiated when cells were coexposed to noncytotoxic concentrations of cigarette smoke condensate. DEP-induced MMP-1 was associated with increased ERK 1/2 phosphorylation and upregulation of expression and activity of the NADPH oxidase analog NOX4. Cell transfection with a NOX4 small interfering RNA prevented these phenomena, showing the critical role of a NOX4 ERK 1/2 pathway in DEP-induced MMP-1 expression and activity. Similar results to those observed in A549 cells were obtained in another human lung epithelial cell line, NCI-H292. Furthermore, experiments in mice intratracheally instilled with DEPs confirmed the in vitro findings, showing the induction of NOX4 and MMP-1 protein expression in alveolar epithelial cells. We conclude that alveolar alterations secondary to MMP-1 induction could explain lung function impairment associated with exposure to particulate pollution.

Background/Aims Endoplasmic reticulum (ER)-related unfolded protein response (UPR) is mediated by PKR-like ER kinase (PERK), ATF6 and IRE1. PERK phosphorylates eukaryotic translation initiation factor-2α (eIF2α) to attenuate protein synthesis, including in NF-κB-dependent antiapoptotic proteins. We hypothesized that an altered UPR in the liver may sensitize cirrhotic livers to LPS-induced, TNFα-mediated apoptosis. Thus, we examined in vivo UPR and NF-κB activity in livers from cirrhotic and normal LPS-challenged rats. Methods Livers were harvested in rats that did or did not receive LPS. Results Under baseline conditions, no UPR was found in normal livers while PERK/eIF2α and ATF6 pathways were activated in cirrhotic livers. After LPS, in normal livers, the PERK/eIF2α pathway was transiently activated. ATF6 and IRE1 were activated. In cirrhotic livers, the PERK/eIF2α pathway remained elevated. ATF6 and IRE1 pathways were altered. LPS-induced, NF-κB-dependent antiapoptotic proteins increased in normal livers whereas their expression was blunted at the posttranscriptional level in cirrhotic livers. Conclusions Cirrhotic livers exhibit partial UPR activation in the basal state and full UPR, although altered, after LPS challenge. Sustained eIF2α phosphorylation, a hallmark of cirrhotic liver UPR, is associated with a lack of LPS-induced accumulation of NF-κB-dependent antiapoptotic proteins which may sensitize cirrhotic livers to LPS/TNFα-mediated apoptosis.

International audience; The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole-genome shotgun (WGS) approach, without the use of costly and time-consuming methods, such as fosmid or BAC clone-based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS-FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired-end and mate-pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired-end reads and contaminants detected, resulting in a total of 17,910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.

publié Epub 2014 Nov 11; CATdb (http://urgv.evry.inra.fr/CATdb) is a database providing a public access to a large collection of transcriptomic data, mainly for Arabidopsis but also for other plants. This resource has the rare advantage to contain several thousands of microarray experiments obtained with the same technical protocol and analyzed by the same statistical pipelines. In this paper, we present GEM2Net, a new module of CATdb that takes advantage of this homogeneous dataset to mine co-expression units and decipher Arabidopsis gene functions. GEM2Net explores 387 stress conditions organized into 18 biotic and abiotic stress categories. For each one, a model-based clustering is applied on expression differences to identify clusters of co-expressed genes. To characterize functions associated with these clusters, various resources are analyzed and integrated: Gene Ontology, subcellular localization of proteins, Hormone Families, Transcription Factor Families and a refined stress-related gene list associated to publications. Exploiting protein-protein interactions and transcription factors-targets interactions enables to display gene networks. GEM2Net presents the analysis of the 18 stress categories, in which 17,264 genes are involved and organized within 681 co-expression clusters. The meta-data analyses were stored and organized to compose a dynamic Web resource.

La production des formes reactives de l’oxygene par la NADPH oxydase phagocytaire a ete longtemps consideree comme restreinte aux phagocytes professionnels. Recemment, six homologues de la sous-unite catalytique de la NADPH oxydase phagocytaire (gp91phox ou Nox-2) ont ete identifies et appartiennent a la famille des Nox : Nox-1, Nox-3, Nox-4, Nox-5, Duox-1 et Duox-2. Ces enzymes ont la capacite de transporter des electrons a travers la membrane plasmique et produisent de l’anion superoxyde, luimeme precurseur des autres formes reactives de l’oxygene. Cependant, la distribution tissulaire et les mecanismes d’activation des membres de la famille Nox sont tres differents, suggerant qu’ils exercent des fonctions physiologiques distinctes dans des mecanismes aussi varies que la defense de l’hote, la croissance, la differenciation et l’apoptose. La modulation de la fonction des differentes Nox peut etre a l’origine d’une grande variete d’evenements physiopathologiques.

The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b(558), the cytosol factors p47(phox), p67(phox), p40(phox), and the small GTPase Rac2, which translocate to the membrane to assemble the active complex following neutrophil activation. Interleukin-8 (IL-8) does not activate NADPH oxidase, but potentiates the oxidative burst induced by stimuli such as formyl-methionyl-leucyl-phenylalanine (fMLP) via a priming mechanism. The effect of IL-8 on the components of NADPH oxidase during the priming process has never been investigated in human neutrophils. Here we showed that within 3 min, IL-8 treatment enhanced the Btk- and ERK1/2-dependent phosphorylation of p47(phox), as well as the recruitment of flavocytochrome b(558), p47(phox), and Rac2 into cholesterol-enriched detergent-resistant microdomains (or lipid rafts). Conversely, IL-8 treatment lasting 15 min failed to recruit flavocytochrome b(558), p47(phox), or Rac2, but did enhance the Btk- and p38 MAPK-dependent phosphorylation and the translocation of p67(phox) into detergent-resistant microdomains. Moreover, methyl-beta-cyclodextrin, which disrupts lipid rafts, inhibited IL-8-induced priming in response to fMLP. Our findings indicate that IL-8-induced priming of the oxidative burst in response to fMLP involves a sequential assembly of the NADPH oxidase components in the lipid rafts of neutrophils.

Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS).

In eukaryotic cells, DNA is packaged into chromatin and this compact storage in the nucleus promotes transcriptional repression of genes. Chromatin remodeling complexes such as the SWI/SNF complex are involved in making DNA accessible to transcription factors and thereby are implicated in the regulation of gene expression. Mutations and altered expression of chromatin remodeling complex genes have been described in cancer cells. Indeed, non-small cell lung cancer cell lines have been shown to harbor mutations in SMARCA4 (BRG1), a member of the SWI/SNF complex, but evidence has been less clear in primary tumors. Recently, inactivating mutations in AT-rich interaction domain 2 (ARID2) were found in liver cancer related to HCV infection and in melanoma. Here, we explored, using a genome-wide strategy and subsequent sequencing of targeted genes, whether chromatin remodeling is implicated in primary lung adenocarcinoma. Two genes were individualized from the genome screening as homozygously deleted in a set of samples: JARID2 and ARID2. Subsequent analysis of the entire coding sequences showed that ARID2 loss-of-function mutations were found in 5% of nonsmall cell lung cancers, thereby constituting one of the most frequently mutated genes in this cancer type after TP53, KRAS, EGFR, CDKN2A and STK11.

Background In the contexts of genomics, post-genomics and systems biology approaches, data integration presents a major concern. Databases provide crucial solutions: they store, organize and allow information to be queried, they enhance the visibility of newly produced data by comparing them with previously published results, and facilitate the exploration and development of both existing hypotheses and new ideas. Results The FLAGdb++ information system was developed with the aim of using whole plant genomes as physical references in order to gather and merge available genomic data from in silico or experimental approaches. Available through a JAVA application, original interfaces and tools assist the functional study of plant genes by considering them in their specific context: chromosome, gene family, orthology group, co-expression cluster and functional network. FLAGdb++ is mainly dedicated to the exploration of large gene groups in order to decipher functional connections, to highlight shared or specific structural or functional features, and to facilitate translational tasks between plant species (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa and Vitis vinifera). Conclusion Combining original data with the output of experts and graphical displays that differ from classical plant genome browsers, FLAGdb++ presents a powerful complementary tool for exploring plant genomes and exploiting structural and functional resources, without the need for computer programming knowledge. First launched in 2002, a 15th version of FLAGdb++ is now available and comprises four model plant genomes and over eight million genomic features.

International audience; In the context of the sequencing project for the grapevine reference genome sequence, two genetic maps were completed with SSR markers in order to align the sequence contigs of the Vv12X.0 version of the genome assembly along the chromosomes. For this purpose, two full-sib families, 'Syrah' x 'Grenache' (SxG, 192 individuals) and 'Chardonnay' x 'Bianca' (CxB, 358 individuals) were respectively scored with 223 and 401 SSR. These two maps, supplemented with former data, allowed anchoring over 90% of the reference genome sequence. SNP markers were then defined from 37 anchored but not oriented and 44 not oriented and not anchored scaffolds of sequence. So far 110 new markers have been mapped, allowing anchoring 31 not yet anchored scaffolds (similar to 9 Mb) and orienting 35 scaffolds (similar to 15.5 Mb). In parallel, the recombination rate along the chromosomes is under study, in relation with sequence features (gene/repeat density, %GC, distance to the centromere/telomere). Finally, we are developing a cytogenetic map in Vitis vinifera using BAC-FISH. All these approaches will be combined to study the structure of the Vitis vinifera genome, including the factors affecting recombination rates and the dynamics of the genome evolution.

In hepatocytes, the accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and the unfolded protein response (UPR), mediated by the ER-resident stress sensors ATF-6, IRE1, and PERK. UPR-responsive genes are involved in the fate of ER-stressed cells. Cells carrying hepatitis C virus (HCV) subgenomic replicons exhibit in vitro ER stress and suggest that HCV inhibits the UPR. Since in vivo ER homeostasis is unknown in livers with chronic HCV infection, we investigated ER stress and the UPR in liver samples from untreated patients with chronic hepatitis C (CHC), in comparison with normal livers. Electron microscopy, western blotting, and real-time RT-PCR were used in liver biopsy specimens. Electron microscopy identified features showing ER stress in hepatocyte samples from patients with CHC; however, 'ER-stressed' hepatocytes were found in clusters (3-5 cells) that were scattered in the liver parenchyma. Western blot analysis confirmed the existence of hepatic ER stress by showing activation of the three ER stress sensors ATF-6, IRE1, and PERK in CHC. Real-time RT-PCR showed no significant induction of UPR-responsive genes in CHC. In contrast, genes involved in the control of diffuse processes such as liver proliferation, inflammation, and apoptosis were significantly induced in CHC. In conclusion, livers from patients with untreated CHC exhibit in vivo hepatocyte ER stress and activation of the three UPR sensors without apparent induction of UPR-responsive genes. This lack of gene induction may be explained by the inhibiting action of HCV per se (as suggested by in vitro studies) and/or by our finding of the localized nature of hepatocyte ER stress.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Background: Unlike in tomato, little is known about the genetic and molecular control of fleshy fruit development of perennial fruit trees like grapevine (Vitis vinifera L.). Here we present the study of the sequence polymorphism in a 1 Mb grapevine genome region at the top of chromosome 18 carrying the fleshless berry mutation (flb) in order, first to identify SNP markers closely linked to the gene and second to search for possible signatures of domestication. Results: In total, 62 regions (17 SSR, 3 SNP, 1 CAPS and 41 re-sequenced gene fragments) were scanned for polymorphism along a 3.4 Mb interval (85,127-3,506,060 bp) at the top of the chromosome 18, in both V. vinifera cv. Chardonnay and a genotype carrying the flb mutation, V. vinifera cv. Ugni Blanc mutant. A nearly complete homozygosity in Ugni Blanc (wild and mutant forms) and an expected high level of heterozygosity in Chardonnay were revealed. Experiments using qPCR and BAC FISH confirmed the observed homozygosity. Under the assumption that flb could be one of the genes involved into the domestication syndrome of grapevine, we sequenced 69 gene fragments, spread over the flb region, representing 48,874 bp in a highly diverse set of cultivated and wild V. vinifera genotypes, to identify possible signatures of domestication in the cultivated V. vinifera compartment. We identified eight gene fragments presenting a significant deviation from neutrality of the Tajima's D parameter in the cultivated pool. One of these also showed higher nucleotide diversity in the wild compartments than in the cultivated compartments. In addition, SNPs significantly associated to berry weight variation were identified in the flb region. Conclusions: We observed the occurrence of a large homozygous region in a non-repetitive region of the grapevine otherwise highly-heterozygous genome and propose a hypothesis for its formation. We demonstrated the feasibility to apply BAC FISH on the very small grapevine chromosomes and provided a specific probe for the identification of chromosome 18 on a cytogenetic map. We evidenced genes showing putative signatures of selection and SNPs significantly associated with berry weight variation in the flb region. In addition, we provided to the community 554 SNPs at the top of chromosome 18 for the development of a genotyping chip for future fine mapping of the flb gene in a F2 population when available.

The incidence of obesity and non-esterified (‘free’) fatty acid-associated metabolic disorders such as the metabolic syndrome and diabetes is increasing dramatically in most countries. Although the pathogenesis of these metabolic disorders is complex, there is emerging evidence that ROS (reactive oxygen species) are critically involved in the aberrant signalling and tissue damage observed in this context. Indeed, it is now widely accepted that ROS not only play an important role in physiology, but also contribute to cell and tissue dysfunction. Inappropriate ROS generation may contribute to tissue dysfunction in two ways: (i) dysregulation of redox-sensitive signalling pathways, and (ii) oxidative damage to biological structures (DNA, proteins, lipids, etc.). An important source of ROS is the NOX family of NADPH oxidases. Several NOX isoforms are expressed in the liver and pancreatic β-cells. There is now evidence that inappropriate activation of NOX enzymes may damage the liver and pancreatic β-cells. In the context of the metabolic syndrome, the emerging epidemic of non-alcoholic steatohepatitis is thought to be NOX/ROS-dependent and of particular medical relevance. NOX/ROS-dependent β-cell damage is thought to be involved in glucolipotoxicity and thereby leads to progression from the metabolic syndrome to Type 2 diabetes. Thus understanding the role of NOX enzymes in liver and β-cell damage should lead to an increased understanding of pathomechanisms in the metabolic syndrome and diabetes and may identify useful targets for novel therapeutic strategies.

Background Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed. Results The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome. Conclusion Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance loci in grapevine.

UTILLdb is a database of phenotypic and sequence information on mutant genes from a reference Pisum sativum EMS-mutant population., The systematic characterization of gene functions in species recalcitrant to Agrobacterium-based transformation, like Pisum sativum, remains a challenge. To develop a high throughput forward and reverse genetics tool in pea, we have constructed a reference ethylmethane sulfonate mutant population and developed a database, UTILLdb, that contains phenotypic as well as sequence information on mutant genes. UTILLdb can be searched online for TILLING alleles, through the BLAST tool, or for phenotypic information about mutants by keywords.

Reactive oxygen species (ROS) generated by the NADPH oxidases are conventionally thought to be cytotoxic and mutagenic and at high levels induce an oxidative stress response. The phagocyte NADPH oxidase catalyzes the NADPH-dependent reduction of molecular oxygen to generate superoxide O2-., which can dismute to generate ROS species. Together, these ROS participate in host defence by killing or damaging invading microbes. Flavocytochrome b558 is the catalytic core of the phagocyte NADPH oxidase and consists of a large glycoprotein gp91phox or Nox-2 and a small protein p22phox. The other components of the NADPH oxidase are cytosolic proteins, namely p67phox, p47phox, p40phox and Rac. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections. Evidence is rapidly accumulating that low level of ROS were produced by NADPH oxidase homologs in non-phagocytic cells. To date, six human homologs (Nox-1, Nox-3, Nox-4, Nox-5, Duox-1 and Duox-2) have been recently identified in a variety of non-phagocytic cells. The identification of Nox-1 was quickly followed by the cloning of Nox-3, Nox-4, and Nox-5. In parallel, two very large members of the Nox family were discovered, namely Duox-1 and Duox-2, initially also referred to as thyroid oxidases. The physiological functions of Nox-dependent ROS generation are in progress and still require detailed characterization. Activation mechanisms and tissue distribution of the different members of the Nox family are very different, suggesting distinct physiological functions. Nox family enzymes are likely to be involved in a variety of physiological events including cell proliferation, host defence, differentiation, apoptosis, senescence and activation of growth-related signaling pathways. An increase and a decrease in the function of Nox enzymes can contribute to a wide range of pathological processes.

The search for effective chemopreventive compounds is a major challenge facing research into preventing the progression of cancer cells. The naturally occurring polyphenol antioxidants look very promising, but their mechanism of action still remains poorly understood. Here, we show that 2-(3,4-dihydroxyphenyl)ethanol (DPE), a phenol antioxidant derived from olive oil, induces growth arrest and apoptosis in human colon carcinoma HT-29 cells. The mechanisms involve prolonged stress of the endoplasmic reticulum (ER) leading to the activation of the two main branches of the unfolded protein response (UPR), including the Ire1/XBP-1/GRP78/Bip and PERK/eIF2alpha arms. DPE treatment led to overexpression of the pro-apoptotic factor CHOP/GADD153 and persistent activation of the Jun-NH2-terminal kinase/activator protein-1 signaling pathway. DPE concomitantly modulated the extracellular signal-regulated kinase 1/2 and Akt/PKB pro-survival factors by altering their phosphorylation status as well as inhibiting tumor necrosis factor-alpha-induced nuclear factor-kappaB activation by inactivating the phosphorylation of nuclear factor inhibitor-kappaB kinase. These findings prompted us to investigate the possible involvement of phosphatases in DPE-mediated action. Using phosphatase inhibitors and RNA interference to silence the Ser/Thr phosphatase 2A (PP2A) prevented DPE-induced cell death. These findings demonstrate that DPE specifically activates PP2A, which plays a key initiating role in various pathways that lead to apoptosis in colon cancer cells.

Using flow cytometry, we observed that interleukin-18 (IL-18) primed human neutrophils (PMNs) in whole blood to produce superoxide anion (O 2 ° − ) in response to N-formyl peptide (fMLP) stimulation, whereas IL-18 alone had no significant effect. In contrast to tumor necrosis factor alpha (TNF-α), which is a cytokine known to strongly prime O 2 ° − production, IL-18 did not induce either p47 phox phosphorylation or its translocation from the cytosol to the plasma membrane. However, IL-18 increased PMN degranulation, as shown by increased levels of cytochrome b558 and CD11b expression at the PMN surface. Moreover, addition of IL-18 to whole blood for 45 min reduced the ability of PMNs to bind to fMLP, suggesting endocytosis of fMLP receptors, as visualized by confocal microscopy. 2,3-Butanedione 2-monoxime, which inhibits endosomal recycling of plasma membrane components back to the cell surface, concomitantly accentuated the diminution of fMLP binding at the PMN surface and increased IL-18 priming of O 2 ° − production by PMNs in response to fMLP. This suggests that fMLP receptor endocytosis could account, at least in part, for the priming of O 2 ° − production. In addition, genistein, a tyrosine kinase inhibitor, and SB203580, a p38 mitogen-activated protein kinase (p38MAPK) inhibitor, completely reversed the decreased level of fMLP binding and increased the level of CD11b expression after IL-18 treatment. Flow cytometric analysis of intact PMNs in whole blood showed that IL-18 increased p38MAPK phosphorylation and tyrosine phosphorylation. In particular, IL-18 induced phosphorylation of focal adhesion kinase (p125 FAK ), which has been implicated in cytoskeleton reorganization. Taken together, our findings suggest several mechanisms that are likely to regulate cytokine-induced priming of the oxidative burst in PMNs in their blood environment.

The mechanisms involved in the cytotoxic action of oxysterols in the pathogenesis of atherosclerosis still remain poorly understood. Among the major oxysterols present in oxidized low-density lipoprotein, we show here that 7-ketocholesterol (7-Kchol) induces oxidative stress and/or apoptotic events in human aortic smooth muscle cells (SMCs). This specific effect of 7-Kchol is mediated by a robust upregulation (threefold from the basal level) of Nox-4, a reactive oxygen species (ROS)-generating NAD(P)H oxidase homologue. This effect was highlighted by silencing Nox-4 expression with a specific small interfering RNA, which significantly reduced the 7-Kchol-induced production of ROS and abolished apoptotic events. Furthermore, the 7-Kchol activating pathway included an early triggering of endoplasmic reticulum stress, as assessed by transient intracellular Ca(2+) oscillations, and the induction of the expression of the cell death effector CHOP and of GRP78/Bip chaperone via the activation of IRE-1, all hallmarks of the unfolded protein response (UPR). We also showed that 7-Kchol activated the IRE-1/Jun-NH(2)-terminal kinase (JNK)/AP-1 signaling pathway to promote Nox-4 expression. Silencing of IRE-1 and JNK inhibition downregulated Nox-4 expression and subsequently prevented the UPR-dependent cell death induced by 7-Kchol. These findings demonstrate that Nox-4 plays a key role in 7-Kchol-induced SMC death, which is consistent with the hypothesis that Nox-4/oxysterols are involved in the pathogenesis of atherosclerosis.

Genetic studies were performed in a French series of hepatocellular carcinomas. New oncogenes (NFE2L2) and tumor suppressor genes (IRF2, ARID1A and RPS6K3) were found to be recurrently altered. Moreover, a genotoxic signature was identified, raising the possible implication of a genotoxic exposure in the etiology of HCC, which remains to be characterized.

Determination of left-right axis is a precocious embryonic event, and all phenotypic anomalies resulting from disruption of the normal lateralization process are collectively referred to as the lateralization defect. A transgenic mouse with lateralization defect and hepatic, kidney, and pancreatic anomalies has resulted from disruption of the inv gene by insertion of a transgene. The human ortholog is thus a good candidate for lateralization defect in humans, in particular in cases with associated hepatic anomalies. Here, we have identified, mapped, and characterized the INV human gene and screened a series of heterotaxic patients (with or without biliary anomalies) for mutation in this gene. In a German family of Turkish origin, we have found that all available affected and unaffected individuals are heterozygous for a mutation in the splicing donor site of intron 12 in the INV gene resulting in two different aberrant splicing isoforms. This can be explained either by a randomization of lateralization defects or, as suggested earlier, di- or trigenic inheritance, although we have been unable to detect, in this family, a mutation in genes known to be involved in the human lateralization defect ( LEFTY1, LEFTY2, ACVR2B, NODAL, ZIC3, and CFC1). In contrast to the mouse, the affected individuals have no biliary anomalies, and the absence of mutation in a series of seven cases with lateralization defect and biliary anomalies demonstrates that INV is not frequently involved in such a phenotype in humans.

P003 Selective inhibition of eIF2alpha dephosphorylation prevents colitis and shows therapeutic potential in a new model of ulcerative colitis X. Treton1 *, E. Pedruzzi1, C. Guichard2, M. Vallee1, S. Sedghi1, Y. Ladeiro1, N. Montcuquet3, J.N. Freund4, I. Van Seuningen5, F. Barreau1, J.P. Hugot1, D. Cazals-Hatem6, F. Daniel1, E. OgierDenis1, Y. Bouhnik7. 1Paris 7 University, Inserm U773, Paris, 2Univ. Paris-Diderot Sorbonne Paris-Cite, INSERM U674, 3Univ. Paris-Diderot Sorbonne Paris-Cite, INSERM U989, 4Inserm U 682, Strasbourg, 5INSERM UMR837, Lille, 6Hopital Beaujon, APHP, Pathology, Clichy, 7Hopital Beaujon, APHP, Gastroenterology, Clichy, France

The genetic alterations involved in the development lung adenocarcinoma (ADC) have been widely characterized allowing the identification of the most frequent oncogenes or tumor suppressor genes linked to this malignancy. Most studies have focus on proliferation pathway, anti-apoptotic factors or cell cycle alterations. Here we show that alterations in chromatin remodeling complexes are frequent in lung ADC. Chromatin structure plays a key role in the regulation of tissue-specific gene expression during development and differentiation therefore it is not surprising that proteins implicated in remodeling complexes play a role in cancer development. For example, mutations have been found in genes belonging to the SWI/SNF complex in different tumors. In lung cancer BRG1 knockout was shown to potentiate tumor development and mutations were recently identified in tumors by the use of a very sensitive method: massive parallel DNA pyrosequencing that suggests the presence of the alteration in cell subclones. In the course of identifying novel chromatin remodeling complex genes implicated in lung ADC development we analyzed a series of 82 lung adenocarcinomas by SNP array and looked for the presence of homozygous deletions on the entire genome. Three regions of homozygous deletions in known tumor suppressor genes CDKN2A, FHIT and PTEN and in JARID2, which is involved in chromatin remodeling, were found in more than 2 samples. Another ARID-domain containing gene; ARID2 was found deleted in one sample and we therefore decided to screen both ARID-domain genes for mutations by direct sequencing on cryopreserved tumor tissues. One missense somatic mutation was found in JARID2 and 6/82 tumors had ARID2 alterations predicted to inactivate the protein in at least 4 out of 6 samples. Indeed 3 mutations were frameshift or non-sense, one specimen had a homozygous deletion and 4 tumors demonstrated loss of heterozygosity associated to the mutation. Our results indicate that ARID2 inactivation is a common feature of lung cancer development and provide new insight on the impact of chromatin remodeling alteration in lung ADC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5121. doi:1538-7445.AM2012-5121

BACKGROUND: Ulcerative Colitis (UC) and Crohn's Disease (CD) are two chronic Inflammatory Bowel Diseases (IBD) affecting the intestinal mucosa. Current understanding of IBD pathogenesis points out the interplay of genetic events and environmental cues in the dysregulated immune response. We hypothesized that dysregulated microRNA (miRNA) expression may contribute to IBD pathogenesis. miRNAs are small, non-coding RNAs which prevent protein synthesis through translational suppression or mRNAs degradation, and regulate several physiological processes. METHODOLOGY/FINDINGS: Expression of mature miRNAs was studied by Q-PCR in inactive colonic mucosa of patients with UC (8), CD (8) and expressed relative to that observed in healthy controls (10). Only miRNAs with highly altered expression (>5 or 100 -fold and 0.05-0.19 -fold for over- and under- expression, respectively; 0.001