Modélisation des bi-grappes et sélection des variables pour des données de grande dimension : application aux données d’expression génétique

Les simulations ont été implémentées avec le programme Java.

The nuclear pore complex and its transporters : from virus-host interactors to subverting the innate antiviral immunity

Les virus ont besoin d’interagir avec des facteurs cellulaires pour se répliquer et se propager dans les cellules d’hôtes. Une étude de l'interactome des protéines du virus d'hépatite C (VHC) par Germain et al. (2014) a permis d'élucider de nouvelles interactions virus-hôte. L'étude a également démontré que la majorité des facteurs de l'hôte n'avaient pas d'effet sur la réplication du virus. Ces travaux suggèrent que la majorité des protéines ont un rôle dans d'autres processus cellulaires tel que la réponse innée antivirale et ciblées pas le virus dans des mécanismes d'évasion immune. Pour tester cette hypothèse, 132 interactant virus-hôtes ont été sélectionnés et évalués par silençage génique dans un criblage d'ARNi sur la production interferon-beta (IFNB1). Nous avons ainsi observé que les réductions de l'expression de 53 interactants virus-hôte modulent la réponse antivirale innée. Une étude dans les termes de gène d'ontologie (GO) démontre un enrichissement de ces protéines au transport nucléocytoplasmique et au complexe du pore nucléaire. De plus, les gènes associés avec ces termes (CSE1L, KPNB1, RAN, TNPO1 et XPO1) ont été caractérisé comme des interactant de la protéine NS3/4A par Germain et al. (2014), et comme des régulateurs positives de la réponse innée antivirale. Comme le VHC se réplique dans le cytoplasme, nous proposons que ces interactions à des protéines associées avec le noyau confèrent un avantage de réplication et bénéficient au virus en interférant avec des processus cellulaire tel que la réponse innée. Cette réponse innée antivirale requiert la translocation nucléaire des facteurs transcriptionnelles IRF3 et NF-κB p65 pour la production des IFNs de type I. Un essai de microscopie a été développé afin d'évaluer l’effet du silençage de 60 gènes exprimant des protéines associés au complexe du pore nucléaire et au transport nucléocytoplasmique sur la translocation d’IRF3 et NF-κB p65 par un criblage ARNi lors d’une cinétique d'infection virale. En conclusion, l’étude démontre qu’il y a plusieurs protéines qui sont impliqués dans le transport de ces facteurs transcriptionnelles pendant une infection virale et peut affecter la production IFNB1 à différents niveaux de la réponse d'immunité antivirale. L'étude aussi suggère que l'effet de ces facteurs de transport sur la réponse innée est peut être un mécanisme d'évasion par des virus comme VHC.

Prediction and functional analysis of native disorder in proteins from the Three Kingdoms of Life

An automatic method for recognizing natively disordered regions from amino acid sequence is described and benchmarked against predictors that were assessed at the latest critical assessment of techniques for protein structure prediction (CASP) experiment. The method attains a Wilcoxon score of 90.0, which represents a statistically significant improvement on the methods evaluated on the same targets at CASP. The classifier, DISOPRED2, was used to estimate the frequency of native disorder in several representative genomes from the three kingdoms of life. Putative, long (>30 residue) disordered segments are found to occur in 2.0% of archaean, 4.2% of eubacterial and 33.0% of eukaryotic proteins. The function of proteins with long predicted regions of disorder was investigated using the gene ontology annotations supplied with the Saccharomyces genome database. The analysis of the yeast proteome suggests that proteins containing disorder are often located in the cell nucleus and are involved in the regulation of transcription and cell signalling. The results also indicate that native disorder is associated with the molecular functions of kinase activity and nucleic acid binding.

Environmentally-acquired bacteria influence microbial diversity and natural innate immune responses at gut surfaces

Background: Early microbial colonization of the gut reduces the incidence of infectious, inflammatory and autoimmune diseases. Recent population studies reveal that childhood hygiene is a significant risk factor for development of inflammatory bowel disease, thereby reinforcing the hygiene hypothesis and the potential importance of microbial colonization during early life. The extent to which early-life environment impacts on microbial diversity of the adult gut and subsequent immune processes has not been comprehensively investigated thus far. We addressed this important question using the pig as a model to evaluate the impact of early-life environment on microbe/host gut interactions during development. Results: Genetically-related piglets were housed in either indoor or outdoor environments or in experimental isolators. Analysis of over 3,000 16S rRNA sequences revealed major differences in mucosa-adherent microbial diversity in the ileum of adult pigs attributable to differences in earlylife environment. Pigs housed in a natural outdoor environment showed a dominance of Firmicutes, in particular Lactobacillus, whereas animals housed in a hygienic indoor environment had reduced Lactobacillus and higher numbers of potentially pathogenic phylotypes. Our analysis revealed a strong negative correlation between the abundance of Firmicutes and pathogenic bacterial populations in the gut. These differences were exaggerated in animals housed in experimental isolators. Affymetrix microarray technology and Real-time Polymerase Chain Reaction revealed significant gut-specific gene responses also related to early-life environment. Significantly, indoorhoused pigs displayed increased expression of Type 1 interferon genes, Major Histocompatibility Complex class I and several chemokines. Gene Ontology and pathway analysis further confirmed these results.

Comparative genomics suggests that an ancestral polyploidy event leads to enhanced root nodule symbiosis in the Papilionoideae

Root nodule symbiosis (RNS) is one of the most efficient biological systems for nitrogen fixation and it occurs in 90% of genera in the Papilionoideae, the largest subfamily of legumes. Most papilionoid species show evidence of a polyploidy event occurred approximately 58 million years ago. Although polyploidy is considered to be an important evolutionary force in plants, the role of this papilionoid polyploidy event, especially its association with RNS, is not understood. In this study, we explored this role using an integrated comparative genomic approach and conducted gene expression comparisons and gene ontology enrichment analyses. The results show the following: (1) approximately a quarter of the papilionoid-polyploidy-derived duplicate genes are retained; (2) there is a striking divergence in the level of expression of gene duplicate pairs derived from the polyploidy event; and (3) the retained duplicates are frequently involved in the processes crucial for RNS establishment, such as symbiotic signalling, nodule organogenesis, rhizobial infection and nutrient exchange and transport. Thus, we conclude that the papilionoid polyploidy event might have further refined RNS and induced a more robust and enhanced symbiotic system. This conclusion partly explains the widespread occurrence of the Papilionoideae.

Genes driving potato tuber initiation and growth: identification based on transcriptional changes using the POCI array

The increasing amount of available expressed gene sequence data makes whole-transcriptome analysis of certain crop species possible. Potato currently has the second largest number of publicly available expressed sequence tag (EST) sequences among the Solanaceae. Most of these ESTs, plus other proprietary sequences, were combined and used to generate a unigene assembly. The set of 246,182 sequences produced 46,345 unigenes, which were used to design a 44K 60-mer oligo array (Potato Oligo Chip Initiative: POCI). In this study, we attempt to identify genes controlling and driving the process of tuber initiation and growth by implementing large-scale transcriptional changes using the newly developed POCI array. Major gene expression profiles could be identified exhibiting differential expression at key developmental stages. These profiles were associated with functional roles in cell division and growth. A subset of genes involved in the regulation of the cell cycle, based on their Gene Ontology classification, exhibit a clear transient upregulation at tuber onset indicating increased cell division during these stages. The POCI array allows the study of potato gene expression on a much broader level than previously possible and will greatly enhance analysis of transcriptional control mechanisms in a wide range of potato research areas. POCI sequence and annotation data are publicly available through the POCI database (http://pgrc.ipk-gatersleben.de/poci).

Metaproteomics analysis reveals the adaptation process for the chicken gut microbiota

The animal gastrointestinal tract houses a large microbial community, the gut microbiota, that confers many benefits to its host, such as protection from pathogens and provision of essential metabolites. Metagenomic approaches have defined the chicken fecal microbiota in other studies, but here, we wished to assess the correlation between the metagenome and the bacterial proteome in order to better understand the healthy chicken gut microbiota. Here, we performed high-throughput sequencing of 16S rRNA gene amplicons and metaproteomics analysis of fecal samples to determine microbial gut composition and protein expression. 16 rRNA gene sequencing analysis identified Clostridiales, Bacteroidaceae, and Lactobacillaceae species as the most abundant species in the gut. For metaproteomics analysis, peptides were generated by using the Fasp method and subsequently fractionated by strong anion exchanges. Metaproteomics analysis identified 3,673 proteins. Among the most frequently identified proteins, 380 proteins belonged to Lactobacillus spp., 155 belonged to Clostridium spp., and 66 belonged to Streptococcus spp. The most frequently identified proteins were heat shock chaperones, including 349 GroEL proteins, from many bacterial species, whereas the most abundant enzymes were pyruvate kinases, as judged by the number of peptides identified per protein (spectral counting). Gene ontology and KEGG pathway analyses revealed the functions and locations of the identified proteins. The findings of both metaproteomics and 16S rRNA sequencing analyses are discussed.

Transcriptome analysis for discovering candidate genes involve in embryogenesis in coconut (Cocos nucifera L.) through 454 pyrosequencing

Coconut, Cocos nucifera L. is a major plantation crop, which ensures income for millions of people in the tropical region. Detailed molecular studies on zygotic embryo development would provide valuable clues for the identification of molecular markers to improve somatic embryogenesis. Since there is no ongoing genome project for this species, coconut expressed sequence tags (EST) would be an interesting technique to identify important coconut embryo specific genes as well as other functional genes in different biochemical pathways. The goal of this study was to analyse the ESTs by examining the transcriptome data of the different embryo tissue types together with one somatic tissue. Here, four cDNA libraries from immature embryo, mature embryo, microspore derived embryo and mature leaves were constructed. cDNA was sequenced by the Roche-454 GS-FLX system and assembled into 32621 putative unigenes and 155017 singletons. Of these unigenes, 18651 had significant sequence similarities to non-redundant protein database, from which 16153 were assigned to one or more gene ontology categories. Homologue genes, which are responsible for embryo development such as chitinase, beta-1,3-glucanase, ATP synthase CF0 subunit, thaumatin-like protein and metallothionein-like protein were identified among the embryo EST collection. Of the unigenes, 6694 were mapped into 139 KEGG pathways including carbohydrate metabolism, energy metabolism, lipid metabolism, amino acid metabolism and nucleotide metabolism. This collection of 454-derived EST data generated from different tissue types provides a significant resource for genome wide studies and gene discovery of coconut, a non-model species.

Identification and validation of candidate genes associated with domesticated and improved traits in soybean

Soybean, an important source of vegetable oils and proteins for humans, has undergone significant phenotypic changes during domestication and improvement. However, there is limited knowledge about genes related to these domesticated and improved traits, such as flowering time, seed development, alkaline-salt tolerance, and seed oil content (SOC). In this study, more than 106,000 single nucleotide polymorphisms (SNPs) were identified by restriction site associated DNA sequencing of 14 wild, 153 landrace, and 119 bred soybean accessions, and 198 candidate domestication regions (CDRs) were identified via multiple genetic diversity analyses. Of the 1489 candidate domestication genes (CDGs) within these CDRs, a total of 330 CDGs were related to the above four traits in the domestication, gene ontology (GO) enrichment, gene expression, and pathway analyses. Eighteen, 60, 66, and 10 of the 330 CDGs were significantly associated with the above four traits, respectively. Of 134 traitassociated CDGs, 29 overlapped with previous CDGs, 11 were consistent with candidate genes in previous trait association studies, and 66 were covered by the domesticated and improved quantitative trait loci or their adjacent regions, having six common CDGs, such as one functionally characterized gene Glyma15 g17480 (GmZTL3). Of the 68 seed size (SS) and SOC CDGs, 37 were further confirmed by gene expression analysis. In addition, eight genes were found to be related to artificial selection during modern breeding. Therefore, this study provides an integrated method for efficiently identifying CDGs and valuable information for domestication and genetic research.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.

The CATH Hierarchy Revisited-Structural Divergence in Domain Superfamilies and the Continuity of Fold Space

This paper explores the structural continuum in CATH and the extent to which superfamilies adopt distinct folds. Although most superfamilies are structurally conserved, in some of the most highly populated superfamilies (4% of all superfamilies) there is considerable structural divergence. While relatives share a similar fold in the evolutionary conserved core, diverse elaborations to this core can result in significant differences in the global structures. Applying similar protocols to examine the extent to which structural overlaps occur between different fold groups, it appears this effect is confined to just a few architectures and is largely due to small, recurring super-secondary motifs (e.g., alpha beta-motifs, alpha-hairpins). Although 24% of superfamilies overlap with superfamilies having different folds, only 14% of nonredundant structures in CATH are involved in overlaps. Nevertheless, the existence of these overlaps suggests that, in some regions of structure space, the fold universe should be seen as more continuous.

The Aedes aegypti larval transcriptome: a comparative perspective with emphasis on trypsins and the domain structure of peritrophins

The genome sequence of Aedes aegypti was recently reported. A significant amount of Expressed Sequence Tags (ESTs) were sequenced to aid in the gene prediction process. In the present work we describe an integrated analysis of the genomic and EST data, focusing on genes with preferential expression in larvae (LG), adults (AG) and in both stages (SG). A total of 913 genes (5.4% of the transcript complement) are LG, including ion transporters and cuticle proteins that are important for ion homeostasis and defense. From a starting set of 245 genes encoding the trypsin domain, we identified 66 putative LG, AG, and SG trypsins by manual curation. Phylogenetic analyses showed that AG trypsins are divergent from their larval counterparts (LG), grouping with blood-induced trypsins from Anopheles gambiae and Simulium vittatum. These results support the hypothesis that blood-feeding arose only once, in the ancestral Culicomorpha. Peritrophins are proteins that interlock chitin fibrils to form the peritrophic membrane (PM) that compartmentalizes the food in the midgut. These proteins are recognized by having chitin-binding domains with 6 conserved Cys and may also present mucin-like domains (regions expected to be highly O-glycosylated). PM may be formed by a ring of cells (type 2, seen in Ae. aegypti larvae and Drosophila melanogaster) or by most midgut cells (type 1, found in Ae. aegypti adult and Tribolium castaneum). LG and D. melanogaster peritrophins have more complex domain structures than AG and T. castaneum peritrophins. Furthermore, mucin-like domains of peritrophins from T. castaneum (feeding on rough food) are lengthier than those of adult Ae. aegypti (blood-feeding). This suggests, for the first time, that type 1 and type 2 PM may have variable molecular architectures determined by different peritrophins and/or ancillary proteins, which may be partly modulated by diet.

Screening the Schistosoma mansoni transcriptome for genes differentially expressed in the schistosomulum stage in search for vaccine candidates

Schistosomiasis affects more than 200 million people worldwide; another 600 million are at risk of infection. The schistosomulum stage is believed to be the target of protective immunity in the attenuated cercaria vaccine model. In an attempt to identify genes up-regulated in the schistosomulum stage in relation to cercaria, we explored the Schistosoma mansoni transcriptome by looking at the relative frequency of reads in EST libraries from both stages. The 400 genes potentially up-regulated in schistosomula were analyzed as to their Gene Ontology categorization, and we have focused on those encoding-predicted proteins with no similarity to proteins of other organisms, assuming they could be parasite-specific proteins important for survival in the host. Up-regulation in schistosomulum relative to cercaria was validated with real-time reverse transcription polymerase chain reaction (RT-PCR) for five out of nine selected genes (56%). We tested their protective potential in mice through immunization with DNA vaccines followed by a parasite challenge. Worm burden reductions of 16-17% were observed for one of them, indicating its protective potential. Our results demonstrate the value and caveats of using stage-associated frequency of ESTs as an indication of differential expression coupled to DNA vaccine screening in the identification of novel proteins to be further investigated as potential vaccine candidates.

Identifying co-regulating microrna groups

Background: Current miRNA target prediction tools have the common problem that their false positive rate is high. This renders identification of co-regulating groups of miRNAs and target genes unreliable. In this study, we describe a procedure to identify highly probable co-regulating miRNAs and the corresponding co-regulated gene groups. Our procedure involves a sequence of statistical tests: (1) identify genes that are highly probable miRNA targets; (2) determine for each such gene, the minimum number of miRNAs that co-regulate it with high probability; (3) find, for each such gene, the combination of the determined minimum size of miRNAs that co-regulate it with the lowest p-value; and (4) discover for each such combination of miRNAs, the group of genes that are co-regulated by these miRNAs with the lowest p-value computed based on GO term annotations of the genes.
Results: Our method identifies 4, 3 and 2-term miRNA groups that co-regulate gene groups of size at least 3 in human. Our result suggests some interesting hypothesis on the functional role of several miRNAs through a "guilt by association" reasoning. For example, miR-130, miR-19 and miR-101 are known neurodegenerative diseases associated miRNAs. Our 3-term miRNA table shows that miR-130/19/101 form a co-regulating group of rank 22 (p-value =1.16 × 10^-2). Since miR-144 is co-regulating with miR-130, miR-19 and miR-101 of rank 4 (p-value = 1.16 × 10^-2) in our 4-term miRNA table, this suggests hsa-miR-144 may be neurodegenerative diseases related miRNA. Conclusions: This work identifies highly probable co-regulating miRNAs, which are refined from the prediction by computational tools using (1) signal-to-noise ratio to get high accurate regulating miRNAs for every gene, and (2) Gene Ontology to obtain functional related co-regulating miRNA groups. Our result has partly been supported by biological experiments. Based on prediction by TargetScanS, we found highly probable target gene groups in the Supplementary Information. This result might help biologists to find small set of miRNAs for genes of interest rather than huge amount of miRNA set.

Transcriptional insights on the regenerative mechanics of axotomized neurons in vitro

Axotomized neurons have the innate ability to undergo regenerative sprouting but this is often impeded by the inhibitory central nervous system environment. To gain mechanistic insights into the key molecular determinates that specifically underlie neuronal regeneration at a transcriptomic level, we have undertaken a DNA microarray study on mature cortical neuronal clusters maintained in vitro at 8, 15, 24 and 48 hrs following complete axonal severance. A total of 305 genes, each with a minimum fold change of ±1.5 for at least one out of the four time points and which achieved statistical significance (one-way ANOVA, P < 0.05), were identified by DAVID and classified into 14 different functional clusters according to Gene Ontology. From our data, we conclude that post-injury regenerative sprouting is an intricate process that requires two distinct pathways. Firstly, it involves restructuring of the neurite cytoskeleton, determined by compound actin and microtubule dynamics, protein trafficking and concomitant modulation of both guidance cues and neurotrophic factors. Secondly, it elicits a cell survival response whereby genes are regulated to protect against oxidative stress, inflammation and cellular ion imbalance. Our data reveal that neurons have the capability to fight insults by elevating biological antioxidants, regulating secondary messengers, suppressing apoptotic genes, controlling ion-associated processes and by expressing cell cycle proteins that, in the context of neuronal injury, could potentially have functions outside their normal role in cell division. Overall, vigilant control of cell survival responses against pernicious secondary processes is vital to avoid cell death and ensure successful neurite regeneration.