Genopole (France)

BACKGROUND AND AIM: A role for the intestinal microbial community (microbiota) in the onset and chronicity of Crohn's disease (CD) is strongly suspected. However, investigation of such a complex ecosystem is difficult, even with culture independent molecular approaches. METHODS: We used, for the first time, a comprehensive metagenomic approach to investigate the full range of intestinal microbial diversity. We used a fosmid vector to construct two libraries of genomic DNA isolated directly from faecal samples of six healthy donors and six patients with CD. Bacterial diversity was analysed by screening the two DNA libraries, each composed of 25,000 clones, for the 16S rRNA gene by DNA hybridisation. RESULTS: Among 1190 selected clones, we identified 125 non-redundant ribotypes mainly represented by the phyla Bacteroidetes and Firmicutes. Among the Firmicutes, 43 distinct ribotypes were identified in the healthy microbiota, compared with only 13 in CD (p<0.025). Fluorescent in situ hybridisation directly targeting 16S rRNA in faecal samples analysed individually (n=12) confirmed the significant reduction in the proportion of bacteria belonging to this phylum in CD patients (p<0.02). CONCLUSION: The metagenomic approach allowed us to detect a reduced complexity of the bacterial phylum Firmicutes as a signature of the faecal microbiota in patients with CD. It also indicated the presence of new bacterial species.

Abstract Background As an alternative to the frequently used "reference design" for two-channel microarrays, other designs have been proposed. These designs have been shown to be more profitable from a theoretical point of view (more replicates of the conditions of interest for the same number of arrays). However, the interpretation of the measurements is less straightforward and a reconstruction method is needed to convert the observed ratios into the genuine profile of interest (e.g. a time profile). The potential advantages of using these alternative designs thus largely depend on the success of the profile reconstruction. Therefore, we compared to what extent different linear models agree with each other in reconstructing expression ratios and corresponding time profiles from a complex design. Results On average the correlation between the estimated ratios was high, and all methods agreed with each other in predicting the same profile, especially for genes of which the expression profile showed a large variance across the different time points. Assessing the similarity in profile shape, it appears that, the more similar the underlying principles of the methods (model and input data), the more similar their results. Methods with a dye effect seemed more robust against array failure. The influence of a different normalization was not drastic and independent of the method used. Conclusion Including a dye effect such as in the methods lmbr_dye, anovaFix and anovaMix compensates for residual dye related inconsistencies in the data and renders the results more robust against array failure. Including random effects requires more parameters to be estimated and is only advised when a design is used with a sufficient number of replicates. Because of this, we believe lmbr_dye, anovaFix and anovaMix are most appropriate for practical use.

SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability-more than 1 in 50-warrant its consideration for mutation screening in clinical practice.

OBJECTIVE: The gut microbiota, which is considered a causal factor in metabolic diseases as shown best in animals, is under the dual influence of the host genome and nutritional environment. This study investigated whether the gut microbiota per se, aside from changes in genetic background and diet, could sign different metabolic phenotypes in mice. METHODS: The unique animal model of metabolic adaptation was used, whereby C57Bl/6 male mice fed a high-fat carbohydrate-free diet (HFD) became either diabetic (HFD diabetic, HFD-D) or resisted diabetes (HFD diabetes-resistant, HFD-DR). Pyrosequencing of the gut microbiota was carried out to profile the gut microbial community of different metabolic phenotypes. Inflammation, gut permeability, features of white adipose tissue, liver and skeletal muscle were studied. Furthermore, to modify the gut microbiota directly, an additional group of mice was given a gluco-oligosaccharide (GOS)-supplemented HFD (HFD+GOS). RESULTS: Despite the mice having the same genetic background and nutritional status, a gut microbial profile specific to each metabolic phenotype was identified. The HFD-D gut microbial profile was associated with increased gut permeability linked to increased endotoxaemia and to a dramatic increase in cell number in the stroma vascular fraction from visceral white adipose tissue. Most of the physiological characteristics of the HFD-fed mice were modulated when gut microbiota was intentionally modified by GOS dietary fibres. CONCLUSIONS: The gut microbiota is a signature of the metabolic phenotypes independent of differences in host genetic background and diet.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

Original genome annotations need to be regularly updated if the information they contain is to remain accurate and relevant. Here the complete re-annotation of the genome sequence of Mycobacterium tuberculosis strain H37Rv is presented almost 4 years after the first submission. Eighty-two new protein-coding sequences (CDS) have been included and 22 of these have a predicted function. The majority were identified by manual or automated re-analysis of the genome and most of them were shorter than the 100 codon cut-off used in the initial genome analysis. The functional classification of 643 CDS has been changed based principally on recent sequence comparisons and new experimental data from the literature. More than 300 gene names and over 1000 targeted citations have been added and the lengths of 60 genes have been modified. Presently, it is possible to assign a function to 2058 proteins (52% of the 3995 proteins predicted) and only 376 putative proteins share no homology with known proteins and thus could be unique to M. tuberculosis.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors exerting several functions in development and metabolism. The physiological functions of PPARdelta remain elusive. By using a CRE-Lox recombination approach, we generated an animal model for muscle-specific PPARdelta overexpression to investigate the role of PPARdelta in this tissue. Muscle-specific PPARdelta overexpression results in a profound change in fiber composition due to hyperplasia and/or shift to more oxidative fiber and, as a consequence, leads to the increase of both enzymatic activities and genes implicated in oxidative metabolism. These changes in muscle are accompanied by a reduction of body fat mass, mainly due to a large reduction of adipose cell size. Furthermore, we demonstrate that endurance exercise promotes an accumulation of PPARdelta protein in muscle of wild-type animals. Collectively, these results suggest that PPARdelta plays an important role in muscle development and adaptive response to environmental changes, such as training exercise. They strongly support the idea that activation of PPARdelta could be beneficial in prevention of metabolic disorders, such as obesity or type 2 diabetes.

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While many rare variants in synaptic proteins have been identified in patients with ASD, little is known about their effects at the synapse and their interactions with other genetic variations. Here, following the discovery of two de novo SHANK2 deletions by the Autism Genome Project, we identified a novel 421 kb de novo SHANK2 deletion in a patient with autism. We then sequenced SHANK2 in 455 patients with ASD and 431 controls and integrated these results with those reported by Berkel et al. 2010 (n = 396 patients and n = 659 controls). We observed a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and in 16 of 1,090 (1.5%) controls (P = 0.004, OR = 2.37, 95% CI = 1.23-4.70). In neuronal cell cultures, the variants identified in patients were associated with a reduced synaptic density at dendrites compared to the variants only detected in controls (P = 0.0013). Interestingly, the three patients with de novo SHANK2 deletions also carried inherited CNVs at 15q11-q13 previously associated with neuropsychiatric disorders. In two cases, the nicotinic receptor CHRNA7 was duplicated and in one case the synaptic translation repressor CYFIP1 was deleted. These results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the "multiple hit model" for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.

RepeatMasker is a program that screens DNA sequences for interspersed repeats and low-complexity DNA sequences. In this chapter, we present the procedure to routinely use this program on a personal computer.

Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is rampant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed >98% nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS2404, 91 copies of IS2606, 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans-specific by comparative genomics were verified as such by PCR screening a diverse collection of 33 strains of M. ulcerans and M. marinum. This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.

The sirtuin family of histone deacetylases (HDACs) was named after their homology to the Saccharomyces cerevisiae gene silent information regulator 2 (Sir2). In the yeast, Sir2 has been shown to mediate the effects of calorie restriction on the extension of life span and high levels of Sir2 activity promote longevity. Like their yeast homologs, the mammalian sirtuins (SIRT1-7) are class III HDACs and require NAD(+) as a cofactor to deacetylate substrates ranging from histones to transcriptional regulators. Through this activity, sirtuins are shown to regulate important biological processes ranging from apoptosis, adipocyte and muscle differentiation, and energy expenditure to gluconeogenesis. We review here the current knowledge regarding the role of sirtuins in metabolism, longevity, and discuss the possible therapeutic applications that could result from the understanding of their function in different organs and pathologies.

Individual variation in fetal hemoglobin (HbF, alpha(2)gamma(2)) response underlies the remarkable diversity in phenotypic severity of sickle cell disease and beta thalassemia. HbF levels and HbF-associated quantitative traits (e.g., F cell levels) are highly heritable. We have previously mapped a major quantitative trait locus (QTL) controlling F cell levels in an extended Asian-Indian kindred with beta thalassemia to a 1.5-Mb interval on chromosome 6q23, but the causative gene(s) are not known. The QTL encompasses several genes including HBS1L, a member of the GTP-binding protein family that is expressed in erythroid progenitor cells. In this high-resolution association study, we have identified multiple genetic variants within and 5' to HBS1L at 6q23 that are strongly associated with F cell levels in families of Northern European ancestry (P = 10(-75)). The region accounts for 17.6% of the F cell variance in northern Europeans. Although mRNA levels of HBS1L and MYB in erythroid precursors grown in vitro are positively correlated, only HBS1L expression correlates with high F cell alleles. The results support a key role for the HBS1L-related genetic variants in HbF control and illustrate the biological complexity of the mechanism of 6q QTL as a modifier of fetal hemoglobin levels in the beta hemoglobinopathies.

The interferon-induced transmembrane protein BST-2/CD317 (tetherin) restricts the release of diverse enveloped viruses from infected cells. The HIV-1 accessory protein Vpu antagonizes this restriction by an unknown mechanism that likely involves the down-regulation of BST-2 from the cell surface. Here, we show that the optimal removal of BST-2 from the plasma membrane by Vpu requires the cellular protein beta-TrCP, a substrate adaptor for a multi-subunit SCF E3 ubiquitin ligase complex and a known Vpu-interacting protein. beta-TrCP is also required for the optimal enhancement of virion-release by Vpu. Mutations in the DSGxxS beta-TrCP binding-motif of Vpu impair both the down-regulation of BST-2 and the enhancement of virion-release. Such mutations also confer dominant-negative activity, consistent with a model in which Vpu links BST-2 to beta-TrCP. Optimal down-regulation of BST-2 from the cell surface by Vpu also requires the endocytic clathrin adaptor AP-2, although the rate of endocytosis is not increased; these data suggest that Vpu induces post-endocytic membrane trafficking events whose net effect is the removal of BST-2 from the cell surface. In addition to its marked effect on cell-surface levels, Vpu modestly decreases the total cellular levels of BST-2. The decreases in cell-surface and intracellular BST-2 are inhibited by bafilomycin A1, an inhibitor of endosomal acidification; these data suggest that Vpu induces late endosomal targeting and partial degradation of BST-2 in lysosomes. The Vpu-mediated decrease in surface expression is associated with reduced co-localization of BST-2 and the virion protein Gag along the plasma membrane. Together, the data support a model in which Vpu co-opts the beta-TrCP/SCF E3 ubiquitin ligase complex to induce endosomal trafficking events that remove BST-2 from its site of action as a virion-tethering factor.

Shigella invades the human intestinal mucosa, thus causing bacillary dysentery, an acute recto-colitis responsible for lethal complications, mostly in infants and toddlers. Conversely, commensal bacteria live in a mutualistic relationship with the intestinal mucosa that is characterized by homeostatic control of innate responses, thereby contributing to tolerance to the flora. Cross-talk established between commensals and the intestinal epithelium mediate this active process, the mechanisms of which remain largely uncharacterized. Probiotics such as Lactobacillus casei belong to a subclass of these commensals that modulate mucosal innate responses and possibly display anti-inflammatory properties. We analyzed whether L. casei could attenuate the pro-inflammatory signaling induced by Shigella flexneri after invasion of the epithelial lining. Cultured epithelial cells were infected with L. casei, followed by a challenge with S. flexneri. Using macroarray DNA chips, we observed that L. casei down-regulated the transcription of a number of genes encoding pro-inflammatory effectors such as cytokines and chemokines and adherence molecules induced by invasive S. flexneri. This resulted in an anti-inflammatory effect that appeared mediated by the inhibition of the NF-kappaB pathway, particularly through stabilization of I-kappaBalpha. In a time-course experiment using GeneChip hybridization analysis, the expression of many genes involved in ubiquitination and proteasome processes were modulated during L. casei treatment. Thus, L. casei has developed a sophisticated means to maintain intestinal homeostasis through a process that involves manipulation of the ubiquitin/proteasome pathway upstream of I-kappaBalpha.

The retinoic acid (RA) signal, produced locally from vitamin A by retinaldehyde dehydrogenase (Raldh) and transduced by the nuclear receptors for retinoids (RA receptor and 9-cis-RA receptor), is indispensable for ontogenesis and homeostasis of numerous tissues. We demonstrate that Raldh3 knockout in mouse suppresses RA synthesis and causes malformations restricted to ocular and nasal regions, which are similar to those observed in vitamin A-deficient fetuses and/or in retinoid receptor mutants. Raldh3 knockout notably causes choanal atresia (CA), which is responsible for respiratory distress and death of Raldh3-null mutants at birth. CA is due to persistence of nasal fins, whose rupture normally allows the communication between nasal and oral cavities. This malformation, which is similar to isolated congenital CA in humans and may result from impaired RA-controlled down-regulation of Fgf8 expression in nasal fins, can be prevented by a simple maternal treatment with RA.

BACKGROUND: Because of the central functions of the mitochondria in providing metabolic energy and initiating apoptosis on one hand and the role that microRNA (miRNA) play in gene expression, we hypothesized that some miRNA could be present in the mitochondria for post-transcriptomic regulation by RNA interference. We intend to identify miRNA localized in the mitochondria isolated from human skeletal primary muscular cells. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the potential origin of mitochondrial miRNA, we in-silico searched for microRNA candidates in the mtDNA. Twenty five human pre-miRNA and 33 miRNA aligments (E-value<0.1) were found in the reference mitochondrial sequence and some of the best candidates were chosen for a co-localization test. In situ hybridization of pre-mir-302a, pre-let-7b and mir-365, using specific labelled locked nucleic acids and confocal microscopy, demonstrated that these miRNA were localized in mitochondria of human myoblasts. Total RNA was extracted from enriched mitochondria isolated by an immunomagnetic method from a culture of human myotubes. The detection of 742 human miRNA (miRBase) were monitored by RT-qPCR at three increasing mtRNA inputs. Forty six miRNA were significantly expressed (2(nd) derivative method Cp>35) for the smallest RNA input concentration and 204 miRNA for the maximum RNA input concentration. In silico analysis predicted 80 putative miRNA target sites in the mitochondrial genome (E-value<0.05). CONCLUSIONS/SIGNIFICANCE: The present study experimentally demonstrated for the first time the presence of pre-miRNA and miRNA in the human mitochondria isolated from skeletal muscular cells. A set of miRNA were significantly detected in mitochondria fraction. The origin of these pre-miRNA and miRNA should be further investigate to determine if they are imported from the cytosol and/or if they are partially processed in the mitochondria.

Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells using two rat brain endothelial cell lines (RBE4 and GP8), we report in this paper that ICAM-1 cross-linking induces a sustained tyrosine phosphorylation of the phosphatidylinositol-phospholipase C (PLC)gamma1, with a concomitant increase in both inositol phosphate production and intracellular calcium concentration. Our results suggest that PLC are responsible, via a calcium- and protein kinase C (PKC)-dependent pathway, for p60Src activation and tyrosine phosphorylation of the p60Src substrate, cortactin. PKCs are also required for tyrosine phosphorylation of the cytoskeleton-associated proteins, focal adhesion kinase and paxillin, but not for ICAM-1-coupled p130Cas phosphorylation. PKC's activation is also necessary for stress fiber formation induced by ICAM-1 cross-linking. Finally, cell pretreatment with intracellular calcium chelator or PKC inhibitors significantly diminishes transmonolayer migration of activated T lymphocytes, without affecting their adhesion to brain endothelial cells. In summary, our data demonstrate that ICAM-1 cross-linking induces calcium signaling which, via PKCs, mediates phosphorylation of actin-associated proteins and cytoskeletal rearrangement in brain endothelial cell lines. Our results also indicate that these calcium-mediated intracellular events are essential for lymphocyte migration through the blood-brain barrier.

Homoeologous exchanges (HEs) have been shown to generate novel gene combinations and phenotypes in a range of polyploid species. Gene presence/absence variation (PAV) is also a major contributor to genetic diversity. In this study, we show that there is an association between these two events, particularly in recent Brassica napus synthetic accessions, and that these represent a novel source of genetic diversity, which can be captured for the improvement of this important crop species. By assembling the pangenome of B. napus, we show that 38% of the genes display PAV behaviour, with some of these variable genes predicted to be involved in important agronomic traits including flowering time, disease resistance, acyl lipid metabolism and glucosinolate metabolism. This study is a first and provides a detailed characterization of the association between HEs and PAVs in B. napus at the pangenome level.

The satellite cell of skeletal muscle provides a paradigm for quiescent and activated tissue stem cell states. We have carried out transcriptome analyses on satellite cells purified by flow cytometry from Pax3(GFP/+) mice. We compared samples from adult skeletal muscles where satellite cells are mainly quiescent, with samples from growing muscles or regenerating (mdx) muscles, where they are activated. Analysis of regulation that is shared by both activated states avoids other effects due to immature or pathological conditions. This in vivo profile differs from that of previously analyzed satellite cells activated after cell culture. It reveals how the satellite cell protects itself from damage and maintains quiescence, while being primed for activation on receipt of the appropriate signal. This is illustrated by manipulation of the corepressor Dach1, and by the demonstration that quiescent satellite cells are better protected from oxidative stress than those from mdx or 1-week-old muscles. The quiescent versus in vivo activated comparison also gives new insights into how the satellite cell controls its niche on the muscle fiber through cell adhesion and matrix remodeling. The latter also potentiates growth factor activity through proteoglycan modification. Dismantling the extracellular matrix is important for satellite cell activation when the expression of proteinases is up-regulated, whereas transcripts for their inhibitors are high in quiescent cells. In keeping with this, we demonstrate that metalloproteinase function is required for efficient regeneration in vivo.

Systematic behavioral phenotyping of genetically modified mice is a powerful method with which to identify the molecular factors implicated in control of animal behavior, with potential relevance for research into neuropsychiatric disorders. A number of such disorders display sex differences, yet the use of female mice in phenotyping strategies has been a rare practice because of the potential variability related to the estrous cycle. We have now investigated the behavioral effects of the estrous cycle in a battery of behavioral tests in C57BL/6J and BALB/cByJ inbred strains of mice. Whereas the performance of BALB/cByJ female mice varied significantly depending on the phase of the estrous cycle in the open field, tail flick and tail suspension tests, the behavior of C57BL/6J females, with the exception of the tail suspension performance, remained stable across all four phases of the estrous cycle in all of the tests including open field, rotarod, startle reflex and pre-pulse inhibition, tail flick and hot plate. We also found that irrespective of the estrous cycle, the behavior of C57BL/6J females was different from that of BALB/cByJ groups in all of the behavioral paradigms. Such strain differences were previously reported in male comparisons, suggesting that the same inter-group differences can be revealed by studying female or male mice. In addition, strain differences were evident even for behaviors that were susceptible to estrous cycle modulations, although their detection might necessitate the constitution of large experimental groups.