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International audience; Background: Soybean lecithin, a plant-based emulsifier widely used in food, is capable of modulating postprandial lipid metabolism. With arising concerns of sustainability, alternative sources of vegetal lecithin are urgently needed, and their metabolic effects must be characterized.Objectives: We evaluated the impact of increasing doses of rapeseed lecithin (RL), rich in essential alpha-linolenic acid (ALA), on postprandial lipid metabolism and ALA bioavailability in lymph-cannulated rats.Methods: Male Wistar rats (8 weeks old) undergoing a mesenteric lymph duct cannulation were intragastrically administered 1 g of an oil mixture containing 4% ALA and 0, 1, 3, 10, or 30% RL (5 groups). Lymph fractions were collected for 6 h. Lymph lipids and chylomicrons (CMs) were characterized. The expression of genes implicated in intestinal lipid metabolism was determined in the duodenum at 6 h. Data was analyzed using either sigmoidal or linear mixed-effects models, or one-way ANOVA, where appropriate.Results: RL dose-dependently increased the lymphatic recovery (AUC) of total lipids (1100 mu g/mL.h per additional RL%; P = 0.010) and ALA (50 mu g/mL.h per additional RL%; P = 0.0076). RL induced a faster appearance of ALA in lymph, as evidenced by the exponential decrease of the rate of appearance of ALA with RL (R-2 = 0.26; P= 0.0064). Although the number of CMs was unaffected by RL, CM diameter was increased in the 30%-RL group, compared to the control group (0% RL), by 86% at 3-4 h (P = 0.065) and by 81% at 4-6 h (P = 0.0002) following administration. This increase was positively correlated with the duodenal mRNA expression of microsomal triglyceride transfer protein (Mttp; rho= 0.63; P= 0.0052). The expression of Mttp and secretion-associated, ras-related GTPase 1 gene homolog B (Sarl b, CM secretion), carnitine palmitoyltransferase IA (Cpt1a) and acyl-coenzyme A oxidase 1 (Acoxl, beta-oxidation), and fatty acid desaturase 2 (Fads2, bioconversion of ALA into long-chain n-3 PUFAs) were, respectively, 49%, 29%, 74%, 48%, and 55% higher in the 30%-RL group vs. the control group (P < 0.05).Conclusions: In rats, RL enhanced lymphatic lipid output, as well as the rate of appearance of ALA, which may promote its subsequent bioavailability and metabolic fate.

STUDY QUESTION Are the large cells derived from cultured DEAD box polypeptide 4 (DDX4)-positive oogonial stem cells (OSCs), isolated from the ovarian cortex of non-menopausal and menopausal women, oocyte-like cells? SUMMARY ANSWER Under appropriate culture conditions, DDX4-positive OSCs from non-menopausal and menopausal women differentiate into large haploid oocyte-like cells expressing the major oocyte markers growth differentiation factor 9 (GDF-9) and synaptonemal complex protein 3 (SYCP3) and then enter meiosis. WHAT IS KNOWN ALREADY The recent reports of OSCs in the ovaries of non-menopausal and menopausal women suggest that neo-oogenesis is inducible during ovarian senescence. However, several questions remain regarding the isolation of these cells, their spontaneous maturation in vitro, and the final differentiation state of the resulting putative oocytes. STUDY DESIGN, SIZE, DURATION DDX4-positive OSCs were obtained from 19 menopausal and 13 non-menopausal women (who underwent hysterectomy for uterine fibroma, ovarian cyst, or other benign pathologies) and cultured for up to 3 weeks. Large and small cells were individually isolated and typed for early and late differentiation markers. PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian cortex fragments were processed by immuno-magnetic separation using a rabbit anti-human DDX4 antibody and the positive populations were measured by assessing both FRAGILIS and stage-specific embryonic antigen 4 (SSEA-4) expression. After 3 weeks in culture, large oocyte-like cells were individually isolated by DEPArray based on PKH26 red staining and cell size determination. GDF-9 and SYCP3 as final, and developmental pluripotency-associated protein 3 (DPPA3) as primordial, germline markers were measured by droplet digital PCR. The haploid versus diploid chromosomal content of chromosomes X and 5 was investigated using fluorescence in situ hybridization (FISH). MAIN RESULTS AND THE ROLE OF CHANCE SSEA-4 + and FRAGILIS + subsets of DDX4-positive populations were present at lower mean levels in menopausal (SSEA-4 +: 46.7%; FRAGILIS +: 47.5%) than in non-menopausal (SSEA-4 +: 64.9%; FRAGILIS +: 64.8) women (P < 0.05). A comparison of the women's age with the ratio of DDX4-positive cells/cm 3 of ovarian cortex revealed an inverse correlation with OSC number (P < 0.05). Once cultured, cells from both groups differentiated to form large (up to 80 μm) mature oocyte-like cells with typical oocyte morphology. Despite the higher numbers of these cells in cultures from non-menopausal women (37.4 versus 23.7/well; P < 0.001), the intra-culture percentages of large oocyte-like cells did not differ significantly between the two groups. Single large oocyte-like cells isolated from non-menopausal and menopausal women expressed equivalent levels of GDF-9 (e.g. 2.0 and 2.6 copies/μl RNA, respectively) and SYCP3 (e.g. 1.2 and 1.5 copies/μl RNA, respectively) mRNA. The remaining small cells isolated from the cultures expressed large amounts of DPPA3 mRNA (e.g. 5.0 and 5.1 copies/μl RNA, from menopausal and non-menopausal women, respectively), which was undetectable in the large oocyte-like cells. FISH analysis of the large and small cells using probes for chromosomes X and 5 revealed a single signal in the large cells, indicative of chromosome haploidy, whereas in the small cells two distinct signals for each chromosome indicated diploidy. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION Our study demonstrated the final differentiation of OSCs, collected from the ovarian cortex of adult women, to oocyte-like cells. However, because the rate of differentiation was low, a major role of the stem cell niche housing these OSCs cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS Since the ability of OSCs to generate mature oocytes in vitro is highly variable, the viability of these cells in the ovarian cortex of non-menopausal and menopausal women may well be determined by the stem cell niche and the woman's concurrent reproductive state. Our study showed that the oocyte-like cells obtained by OSC differentiation in vitro, including those from the OSCs of menopausal women, express markers of meiosis. This model of ovarian neo-oogenesis will contribute to the development of approaches to treat female infertility. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by Italian Association for Cancer Research (IG grant 17536), and from the Apulia Region ('Oncogenomic Project' and 'Jonico-Salentino Project'). All Authors declare no competing interests.

Context Increased prevalence of type 2 diabetes mellitus and prediabetes worldwide is attributed in part to an unhealthy diet. Objective To evaluate whether 12 weeks of high monounsaturated fatty acid (MUFA) or fiber-rich weight-maintenance diet lowers hepatic fat and improves glucose tolerance in people with prediabetes. Design Subjects underwent a [6, 6-2H2]-labeled 75-g oral glucose tolerance test to estimate hepatic insulin sensitivity and liver fat fraction (LFF) using magnetic resonance spectroscopy before and after intervention. Setting Mayo Clinic Clinical Research Trials Unit. Participants 43 subjects with prediabetes. Intervention Subjects were randomized into three isocaloric weight-maintaining diets containing MUFA (olive oil), extra fiber, and standard US food (control-habitual diet). Outcome Measures LFF, glucose tolerance, and indices of insulin action and secretion. Results Body weight was maintained constant in all groups during the intervention. Glucose and hormonal concentrations were similar in all groups before, and unchanged after, 12 weeks of intervention. LFF was significantly lower after intervention in the MUFA group (P < 0.0003) but remained unchanged in the fiber (P = 0.25) and control groups (P = 0.45). After 12 weeks, LFF was significantly lower in the MUFA than in the control group (P = 0.01), but fiber and control groups did not differ (P = 0.41). Indices of insulin action and secretion were not significantly different between the MUFA and control groups after intervention (P ≥ 0.11), but within-group comparison showed higher hepatic (P = 0.01) and total insulin sensitivity (P < 0.04) with MUFA. Conclusions Twelve weeks of a MUFA diet decreases hepatic fat and improves both hepatic and total insulin sensitivity.

International audience; Rationale: The hallmark of Severe influenza virus infection is excessive inflammation of the lungs. Platelets are activated during influenza, but their role in influenza virus pathogenesis and inflammatory responses is unknown. Objectives: To determine the role of platelets during influenza A virus infections and propose new therapeutics against influenza. Methods: We used targeted gene deletion approaches and pharmacologic interventions to investigate the role of platelets during influenza virus infection in mice. Measurements and Main Results: Lungs of infected mice were Massively infiltrated by aggregates of activated platelets. Platelet activation promoted influenza A virus pathogenesis. Activating protease-activated receptor 4, a platelet receptor for thrombin that is crucial for platelet activation, exacerbated influenza-induced acute lung injury and death. In contrast, deficiency in the major platelet receptor glycoprotein IIIa protected mice from death caused by influenza viruses, and treating the mice with a specific glycoprotein IIb/IIIa antagonist, eptifibatide, had the same effect. Interestingly, mice treated with other antiplatelet compounds (antagonists of protease-activated receptor 4, MRS 2179, and clopidogrel) were also protected from severe lung injury and lethal infections induced by several influenza strains. Conclusions: The intricate relationship between hemostasis and inflammation has major consequences in influenza virus pathogenesis, and antiplatelet drugs might be explored to develop new antiinflammatory treatment against influenza virus infections.

International audience; It has recently been established that exosomes can mediate intercellular cross-talk under normal and pathological conditions through the transfer of specific miRNAs. As muscle cells secrete exosomes, we addressed the question of whether skeletal muscle (SkM) exosomes contained specific miRNAs, and whether they could act as "endocrine signals" during myogenesis. We compared the miRNA repertoires found in exosomes released from C2C12 myoblasts and myotubes and found that 171 and 182 miRNAs were exported into exosomes from myoblasts and myotubes, respectively. Interestingly, some miRNAs were expressed at higher levels in exosomes than in their donor cells and vice versa, indicating a selectivity in the incorporation of miRNAs into exosomes. Moreover miRNAs from C2C12 exosomes were regulated during myogenesis. The predicted target genes of regulated exosomal miRNAs are mainly involved in the control of important signaling pathways for muscle cell differentiation (e.g., Wnt signaling pathway). We demonstrated that exosomes from myotubes can transfer small RNAs (C. elegans miRNAs and siRNA) into myoblasts. Moreover, we present evidence that exosome miRNAs secreted by myotubes are functionally able to silence Sirt1 in myoblasts. As Sirt1 regulates muscle gene expression and differentiation, our results show that myotube-exosome miRNAs could contribute to the commitment of myoblasts in the process of differentiation. Until now, myokines in muscle cell secretome provided a conceptual basis for communication between muscles. Here, we show that miRNA exosomal transfer would be a powerful means by which gene expression is orchestrated to regulate SkM metabolic homeostasis.

International audience; Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.

BackgroundWe conducted a systematic review of incidence rates in England over a sixty-year period to determine the extent to which rates varied along accepted (age, sex) and less-accepted epidemiological gradients (ethnicity, migration and place of birth and upbringing, time).ObjectivesTo determine variation in incidence of several psychotic disorders as above.Data sourcesPublished and grey literature searches (MEDLINE, PSycINFO, EMBASE, CINAHL, ASSIA, HMIC), and identification of unpublished data through bibliographic searches and author communication.Study eligibility criteriaPublished 1950-2009; conducted wholly or partially in England; original data on incidence of non-organic adult-onset psychosis or one or more factor(s) pertaining to incidence.ParticipantsPeople, 16-64 years, with first -onset psychosis, including non-affective psychoses, schizophrenia, bipolar disorder, psychotic depression and substance-induced psychosis.Study appraisal and synthesis methodsTitle, abstract and full-text review by two independent raters to identify suitable citations. Data were extracted to a standardized extraction form. Descriptive appraisals of variation in rates, including tables and forest plots, and where suitable, random-effects meta-analyses and meta-regressions to test specific hypotheses; rate heterogeneity was assessed by the I²-statistic.Results83 citations met inclusion. Pooled incidence of all psychoses (N = 9) was 31.7 per 100,000 person-years (95%CI: 24.6-40.9), 23.2 (95%CI: 18.3-29.5) for non-affective psychoses (N = 8), 15.2 (95%CI: 11.9-19.5) for schizophrenia (N = 15) and 12.4 (95%CI: 9.0-17.1) for affective psychoses (N = 7). This masked rate heterogeneity (I²: 0.54-0.97), possibly explained by socio-environmental factors; our review confirmed (via meta-regression) the typical age-sex interaction in psychosis risk, including secondary peak onset in women after 45 years. Rates of most disorders were elevated in several ethnic minority groups compared with the white (British) population. For example, for schizophrenia: black Caribbean (pooled RR: 5.6; 95%CI: 3.4-9.2; N = 5), black African (pooled RR: 4.7; 95%CI: 3.3-6.8; N = 5) and South Asian groups in England (pooled RR: 2.4; 95%CI: 1.3-4.5; N = 3). We found no evidence to support an overall change in the incidence of psychotic disorder over time, though diagnostic shifts (away from schizophrenia) were reported.LimitationsIncidence studies were predominantly cross-sectional, limiting causal inference. Heterogeneity, while evidencing important variation, suggested pooled estimates require interpretation alongside our descriptive systematic results.Conclusions and implications of key findingsIncidence of psychotic disorders varied markedly by age, sex, place and migration status/ethnicity. Stable incidence over time, together with a robust socio-environmental epidemiology, provides a platform for developing prediction models for health service planning.

International audience; The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity.

International audience; UNLABELLED: HepaRG human liver progenitor cells exhibit morphology and functionality of adult hepatocytes. We investigated the susceptibility of HepaRG hepatocytes to in vitro infection with serum-derived hepatitis C virus (HCV) particles (HCVsp) and the potential neutralizing activity of the E1E2-specific monoclonal antibody (mAb) D32.10. The infection was performed using HCVsp when the cells actively divided at day 3 postplating. HCV RNA, E1E2, and core antigens were quantified in HCV particles recovered from culture supernatants of differentiated cells for up to 66 days. The density distributions of particles were analyzed on iodixanol or sucrose gradients. Electron microscopy (EM) and immune-EM studies were performed for ultrastructural analysis of cells and localization of HCV E1E2 proteins in thin sections. HCV infection of HepaRG cells was documented by increasing production of E1E2-core-RNA(+) HCV particles from day 21 to day 63. Infectious particles sedimented between 1.06 and 1.12 g/mL in iodixanol gradients. E1E2 and core antigens were expressed in 50% of HCV-infected cells at day 31. The D32.10 mAb strongly inhibited HCV RNA production in HepaRG culture supernatants. Infected HepaRG cells frozen at day 56 were reseeded at low density. After only 1-3 subcultures and induction of a cell differentiation process the HepaRG cells produced high titer HCV RNA and thus showed to be sustainably infected. Apolipoprotein B-associated empty E1E2 and complete HCV particles were secreted. Characteristic virus-induced intracellular membrane changes and E1E2 protein-association to vesicles were observed. CONCLUSION: HepaRG progenitor cells permit HCVsp infection. Differentiated HepaRG cells support long-term production of infectious lipoprotein-associated enveloped HCV particles. The E1E2-specific D32.10 mAb neutralizes the infection and this cellular model could be used as a surrogate infection system for the screening of entry inhibitors.

3-4 Pigeot-Remy, S. Simonet, F. Errazuriz-Cerda, E. Lazzaroni, J. C. Atlan, D. Guillard, C.; In order to identify some of the potential bacterial targets, the effects of TiO2 nanoparticles on bacteria in aqueous suspension were assessed in the dark and under UV-A (lambda > 340 nm) radiation exposure, using the microorganism model Escherichia coli K-12. Illumination was produced with a HPK 125W lamp and suspended TiO2 Degussa P-25 was used as the photocatalyst, absorbing all the incident UV-A radiations. The impact of the photocatalyst on the bacteria was investigated by monitoring cell cultivability, cell wall integrity and nucleic acid stability. The contact of TiO2 particles with bacteria in the dark increased the bacterial sensitivity to membrane-perturbing agents, suggesting an increase in outer membrane permeability. In contrast, the contact between SiO2 particles, with an average particle size similar to that of TiO2 P-25, and bacteria did not induce any alteration of the cell permeability. The TiO2 deleterious action on the envelope integrity continued during the UV-A radiations exposure. Impacts on bacterial permeability precede the total loss of cultivability. After 2.5 h of photocatalytic treatment at 3.45 mW/cm(2), bacteria were no longer cultivable on their standard growth medium. However, some of them could become cultivable again under specific environmental conditions appropriate to their survival. These resilient bacteria exposed again to UV-A photocatalysis were more resistant to the treatment. Some chemical by-products released during photocatalytic inactivation of the bacteria were also monitored. The appearance of oxamic and oxalic acids as well as ammonium cations, sulfate and nitrate anions were observed. Transmission electron microscopy (TEM) was used to study the morphological damages to E. coli structure during the photocatalytic inactivation of the microorganisms. After 1.5 h of treatment, bacteria showed disorganized membrane structures, while bacteria were still visible although they were no longer cultivable after a longer exposure time. These results were correlated with damages of nucleic acids at in vivo level. An analysis by electrophoresis revealed that bacterial DNA and RNA molecules completely disappeared after 7 h of photocatalytic treatment. (C) 2011 Published by Elsevier B.V.