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The chemical composition of wine is known to be influenced by multiple factors including some viticulture practices and winemaking processes. 1H-NMR metabolomics has been successfully applied to the study of wine authenticity. In the present study, 1H-NMR metabolomics in combination with multivariate analysis was applied to investigate the effects of grape maturity and enzyme and fining treatments on Cabernet Sauvignon wines. A total of forty wine metabolites were quantified. Three different stages of maturity were studied (under-maturity, maturity and over-maturity). Enzyme treatments were carried out using two pectolytic enzymes (E1 and E2). Finally, two proteinaceous fining treatments were compared (vegetable protein, fining F1, pea protein and PVPP, fining F2). The results show a clear difference between the three stages of maturity, with an impact on different classes of metabolites including amino acids, organic acids, sugars, phenolic compounds, alcohols and esters. A clear separation between enzymes E1 and E2 was observed. Both fining agents had a significant effect on metabolite concentrations. The results demonstrate that 1H-NMR metabolomics provides a fast and robust approach to study the effect of winemaking processes on wine metabolites. These results support the interest to pursue the development of 1H-NMR metabolomics to investigate the effects of winemaking on wine quality.

International audience; Gut interleukin-17A (IL-17)-producing γδ T cells are tissue-resident cells that are involved in both host defense and regulation of intestinal inflammation. However, factors that regulate their functions are poorly understood. In this study, we find that the gut microbiota represses IL-17 production by cecal γδ T cells. Treatment with vancomycin, a Gram-positive bacterium-targeting antibiotic, leads to decreased production of short-chain fatty acids (SCFAs) by the gut microbiota. Our data reveal that these microbiota-derived metabolites, particularly propionate, reduce IL-17 and IL-22 production by intestinal γδ T cells. Propionate acts directly on γδ T cells to inhibit their production of IL-17 in a histone deacetylase-dependent manner. Moreover, the production of IL-17 by human IL-17-producing γδ T cells from patients with inflammatory bowel disease (IBD) is regulated by propionate. These data contribute to a better understanding of the mechanisms regulating gut γδ T cell functions and offer therapeutic perspectives of these cells.

International audience; Pistacia lentiscus L. (Anacardeaceae), a largely distributed plant in the Mediterranean region, has long been used in human food, pharmaceutical industry and traditional medicine. P. lentiscus leaves are well known as a rich source of antioxidant polyphenols that may play a potential role in the prevention of neurodegeneration. Aluminium is a puissant neurotoxin metal; is proposed to be involved in the development of Alzheimer's disease (AD). The present study aims to examine the neuroprotective effect of P. lentiscus L. leaves extract (PL) on behavioral, histological and biochemical impairments caused by Al in male albinos mice. First, Al (100 ppm) and PL (150 and 300 ppm) were co-administered daily for two months to the mice. The extract significantly reversed all Al effects; it decreased the anxiety in head-dipping, black/ white and elevated plus maze tests, improved memory performance in Morris water maze test and inhibited histological alterations. On the other hand, in vitro study was added to determine the active principles of PL and to clarify their mode of action in the neuroprotective effect. The extract and its phenolic compounds (Myricetin rhamnoside, quercetin rhamnoside, gallic acid, myricetin and quercetin) were tested against Al-activated iron-induced lipid peroxidation in synaptosomal membrane. Results showed that PL and its bioactive compounds had a dose dependent effect where the highest effect was obtained with myricetin rhamnoside (IC50 = 0.04 mM). Therefore, PL is a source of phenolics possessing a neuroprotective potential which is validated by their ability to reverse the toxic effects caused by Al.

International audience; Grapevine canes are vine growing byproducts studied for their antimicrobial activities. These properties are directly connected to the stilbene content; oligomeric stilbenes being the most active. In this study, we propose a chemical process, based on oxidative coupling, using metals to increase the oligostilbene rate and the biological effectivity of cane extract against grapevine pathogens. A total of ten compounds were obtained and identified by combining LCMS and NMR spectroscopies, including four newly reported compounds: trans-oxistilbenin C, trans-oxistilbenin D, and cis- and trans-oxistilbenin E. The extract and the main stilbene formed were evaluated for their preventive effects on Plasmopara viticola and Botrytis cinerea growth. The processed extract was highly effective against both pathogens.

International audience; Alzheimer disease's (AD) is a neurodegenerative disease induced by amyloid-beta (A beta) aggregation and accumulation of neurotoxic metals in the brain. Fraxinus angustifolia Vahl. (Oleaceae) is a Mediterranean plant traditionally used to treat several human problems as nervous system problems. This study aimed to evaluate the neuroprotective effects of F. angustifolia Vahl. bark extract (FAB) in vitro and in vivo against A beta-aggregation and aluminium induced-neurotoxicity in mice. FAB was characterized by colorimetric methods and its individual compounds were identified and quantified by LC-MS. First, the neuroprotective effect of FAB was evaluated against A beta(25-35)-aggregation where it was directly incubated with A beta(25-35) and the kinetic of aggregation was measured by spectrophotometer at 200 nm. Then, the extract was tested against A beta(25-35)-induced cytotoxicity on PC12 cells and the cells viability was determined by MTT test. On the other hand, FAB (0.01 - 0.5 mg/mL) was tested against aluminium-activated lipid peroxidation in mice synaptosomal membranes, and in vivo against aluminium-caused neurotoxicity in male N.M.R.I. (Naval Medical Research Institute) mice; this test consisted of daily co-administration of the extract with Al for 60 days. At the end of the treatment, behavioral and memory tests (locomotor activity, black and white and Morris water maze tests) and histological analysis were realized. The identification and quantification of FAB phenolics revealed the presence of different phenolic classes with high concentration of phenylethanoids and hydroxycoumarins. FAB showed a high A beta(25-35) anti-aggregative effect and a dose dependent protective effect on PC12 cells. The extract also demonstrated a significant inhibition of lipid peroxidation and was found to prevent the Al harmful effects where it significantly increased the locomotor activity, decreased the anxiety, improved memory and reduced histological alterations. In conclusion, FAB is rich of bioactive compounds that gave it the ability to inhibit A beta-aggregation and Al-caused neurotoxicity in mice.

Background Host-microbe balance maintains intestinal homeostasis and strongly influences inflammatory conditions such as inflammatory bowel diseases (IBD). Here we focused on bacteria-fungi interactions and their implications on intestinal inflammation, a poorly understood area. Methods Dextran sodium sulfate (DSS)-induced colitis was assessed in mice treated with vancomycin (targeting gram-positive bacteria) or colistin (targeting Enterobacteriaceae) and supplemented with either Saccharomyces boulardii CNCM I-745 or Candida albicans. Inflammation severity as well as bacterial and fungal microbiota compositions was monitored. Results While S. boulardii improved DSS-induced colitis and C. albicans worsened it in untreated settings, antibiotic treatment strongly modified DSS susceptibility and effects of fungi on colitis. Vancomycin-treated mice were fully protected from colitis, while colistin-treated mice retained colitis phenotype but were not affected anymore by administration of fungi. Antibacterial treatments not only influenced bacterial populations but also had indirect effects on fungal microbiota. Correlations between bacterial and fungal relative abundance were dramatically decreased in colistin-treated mice compared to vancomycin-treated and control mice, suggesting that colistin-sensitive bacteria are involved in interactions with fungi. Restoration of the Enterobacteriaceae population by administrating colistin-resistant Escherichia coli reestablished both beneficial effects of S. boulardii and pathogenic effects of C. albicans on colitis severity. This effect was at least partly mediated by an improved gut colonization by fungi. Conclusions Fungal colonization of the gut is affected by the Enterobacteriaceae population, indirectly modifying effects of mycobiome on the host. This finding provides new insights into the role of inter-kingdom functional interactions in intestinal physiopathology and potentially in IBD. Electronic supplementary material The online version of this article (10.1186/s40168-018-0538-9) contains supplementary material, which is available to authorized users.

International audience; Aim: Stilbenes are well-known phytoalexins present in vine and wine. Stilbene glucosides have been identified in wine; however, other than piceid, these compounds have never been reported to be present in the woody parts of grapevine. The aims of this study were to investigate the presence of stilbene glucosides in the woody parts of the vine and to evaluate their cytotoxic activity, in comparison with that of resveratrol, in human hepatoma HepG2 cells. Methods and results: Stilbene glucosides were isolated from a Vitis vinifera root extract. The extract was partitioned with ethyl acetate and fractionated by polyamide gel column chromatography. Pure compounds were obtained by semipreparative high-performance liquid chromatography. These were then identified by mass spectrometry and nuclear magnetic resonance (NMR) analyses, including analysis of two-dimensional NMR spectra. In addition to resveratrol, five stilbene glucosides were found: resveratroloside, resveratrol rutinoside, trans-epsilon-viniferin diglucoside, cis-epsilon-viniferin diglucoside and piceid. Of these, the first four showed cytotoxic effects against HepG2 cells when the crystal violet assay was used to determine cell viability. Conclusion: In addition to resveratrol and piceid, this is the first report of the presence of four glucosylated derivatives of resveratrol (resveratroloside, resveratrol rutinoside, and trans- and cis-epsilon-viniferin diglucosides) in the woody parts of vine. These compounds showed significant cytotoxicity against HepG2 cells. Significance and impact of the study: Stilbenes are well-known biological compounds. Grapevine is one of the main sources of this family of polyphenols. Other than piceid, stilbene glucosides have been identified in wine but never in the woody parts of vine. This is the first study in which five glucosylated derivatives of resveratrol were isolated from woody parts of vine. They were also shown to exert antiproliferative effects in human hepatoma HepG2 cells.

International audience; Ligand-receptor interactions can be implicated in many pathological events such as chronic neurodegenerative diseases. Thus, the discovery of molecules disrupting this type of interactions could be an interesting therapeutic approach. Polyphenols are well known for their affinity for proteins and several studies have characterized these direct interactions. But studying the direct influence of multi-therapeutic drugs on a ligand-receptor complex relevant to a neurodegenerative disorder is a challenging issue. Solution NMR, molecular modeling and iterative calculations were used to obtain information about the interaction between a phenolic compound, α-glucogallin (α-2) and a ligand/fragment receptor complex neurotensin (NT) and its receptor NTS1. The α-2 was shown to bind to NT and a peptidic fragment of its NTS1 receptor, independently. Although the formation of the corresponding ligand-receptor complex did not seem to be affected, this experimental modeling protocol will enable the evaluation of other anti-amyloidogenic compounds such as blockers of NT-NTS1 binding. These types of studies help in understanding the specificity and influence in binding and can provide information to develop new molecules with a putative pharmacological interest.

International audience; INTRODUCTION: Grapevine protection is an important issue in viticulture. To reduce pesticide use, sustainable disease control strategies are proposed, including a promising alternative method based on the elicitor-triggered stimulation of the grapevine natural defense responses. However, detailed investigations are necessary to characterize the impact of such defense induction on the primary metabolism. OBJECTIVES: Our aim was to use a metabolomics approach to assess the impact on grapevine of different elicitors dependent on the salicylic acid (SA) and/or jasmonic acid (JA) pathway. For this purpose, leaves of grapevine foliar cuttings were treated with methyl jasmonate, acibenzolar-S-methyl or phosphonates. METHODS: According to the elicitor, common and discriminating metabolites were elucidated using 1H NMR measurements and principal component analysis. RESULTS: A wide range of compounds including carbohydrates, amino acids, organic acids, phenolics and amines were identified. The score plots obtained by combining PC1 versus PC2 and PC1 versus PC3 allowed a clear separation of samples, so metabolite fingerprinting showed an extensive reprogramming of primary metabolic pathways after elicitation. CONCLUSION: The methods applied were found to be accurate for the rapid determination and differential characterization of plant samples based on their metabolic composition. These investigations can be very useful because the application of plant defense stimulators is gaining greater importance as an alternative strategy to pesticides in the vineyard.

International audience; The q-NMR metabolomics has already demonstrated its potential for classifying wines of different geographical origins, grape varieties, or vintages. This study focuses on the characterisation of Bordeaux red wines, seeking to discriminate them from others produced in the major French wine regions. A sampling of 224 commercial French wines was analysed by 1H NMR and forty compounds were quantified. Non-supervised and supervised statistical analyses revealed a singular imprint of Bordeaux wines in comparison with other French wines, with classification rates ranging from 71% to 100%. Within the Bordeaux vineyards, red wines from the different Bordeaux subdivisions were analysed from different vintages. Our results indicate that q-NMR metabolomics enables the differentiation of Médoc and Libournais vineyard highlighting the most discriminant constituents. In addition, the effects of wine evolution during bottle aging and vintage on Bordeaux red wines were pointed out and discussed.

International audience; Food counterfeit is a major issue for food industries. In the case of wines, there can be multiple forgeries such as geographical, vintage, or varietal mislabeling. For the last 10 years, the use of quantitative 1H NMR (q-NMR) for food authentication has experienced an enormous increase. This study was based on a comparative evaluation of the results obtained for three sets of authentic high-valued wines and suspected wines. Two methodologies have been followed: (i) the usual wine analysis, based on the utilization of multiple techniques, which is the traditional way of analysis for wine authentication and (ii) q-NMR profiling, the alternative proposed method. For wine comparison, an original approach based on similarity score (S-score) by analogy to the Z-score calculation was developed. A limit of four S-scores outside the range − 2/2 has been, arbitrary, defined to reveal that wines are different using q-NMR analysis. Data treatments allowed the extraction of discriminating parameters, some of which were common to both approaches while others could be linked to the techniques’ capabilities showing the complementarity of the two approaches. This study demonstrated the potential of q-NMR in wine authentication by comparative analysis with authentic samples. The q-NMR alternative method has also the advantages of rapidity (around 20 min including sample preparation, analysis, and data treatment) and low volume (0.5 mL), which is a prerequisite for analyzing priceless wines.

Fraxinus angustifolia leaves and bark are used in traditional medicine against various inflammatory-related pathologies incumbent to reactive oxygen species (ROS) generation by the NADH oxidase activity of enzymes such as xanthine oxidoreductase (XOR). This study was designed to investigate the in vitro and in vivo inhibitory activities of this enzyme by Fraxinus angustifolia extracts. The leaf organic phase of ethyl acetate (LFA) and its bark aqueous counterpart (BFA) showed the strongest anti-NADH oxidase activity in vitro (IC50 = 38.51 and 42.04 μg mL-1, respectively). They consequently suppressed superoxide generation both enzymatically (53% and 19%, respectively) and nonenzymatically (34% and 19%, respectively). These results were corroborated in vivo, with high anti- NADH oxidase potential of the leaves and bark extracts (75.32% and 51.32%, respectively) concomitant with moderate hypouricemic activities (36.84% and 38.59%, respectively). Bio-guided fractionation led to the identification, by LC-DAD-MS/MS, of esculin and calcelarioside in bark and kaempferol glucoside in leaves as the main compounds responsible for the anti-NADH oxidase activity of XOR. These results plead in favor of the use of F. angustifolia as a source of potentially interesting therapeutic substances.

International audience; Background and Aims: Gut microbiota is involved in many physiological functions and its imbalance is associated with several diseases, particularly with inflammatory bowel diseases. Mucosa-associated microbiota could have a key role in induction of host immunity and in inflammatory process. Although the role of fungi has been suggested in inflammatory disease pathogenesis, the fungal microbiota has not yet been deeply explored. Here we analysed the bacterial and fungal composition of the mucosa-associated microbiota of Crohn's disease patients and healthy subjects.Methods: Our prospective, observational study evaluated bacterial and fungal composition of mucosa-associated microbiota of 23 Crohn's disease patients [16 in flare, 7 in remission] and 10 healthy subjects, using 16S [MiSeq] and ITS2 [pyrosequencing] sequencing, respectively. Global fungal load was assessed by real time quantitative polymerase chain reaction. Results: Bacterial microbiota in Crohn's disease patients was characterised by a restriction in biodiversity. with an increase of Proteobacteria and Fusobacteria. Global fungus load was significantly increased in Crohn's disease flare compared with healthy subjects [p < 0.05]. In both groups, the colonic mucosa-associated fungal microbiota was dominated by Basidiomycota and Ascomycota phyla. Cystofilobasidiaceae family and Candida glabrata species were overrepresented in Crohn's disease. Saccharomyces cerevisiae and Filobasidium uniguttulatum species were associated with non-inflamed mucosa, whereas Xylariales order was associated with inflamed mucosa.Conclusions: Our study confirms the alteration of the bacterial microbiota and is the first demonstration of the existence of an altered fungal microbiota in Crohn's disease patients, suggesting that fungi may play a role in pathogenesis.

In the courses of studies on ellagitannin changes during oak wood toasting, two C-glycosidic ellagitannins were isolated from the french oak wood for the first time. These two compounds exhibited [M−H]− ion peak at m/z 1055.0631 (compound A) and at m/z 1011.0756 (compound B). A compound is named Castacrenin E and is produced by Castacrenin D oxidation. Castacrenin D is a vescalagin with an additional aromating ring to the C-1 through a C-C bond. These compounds are not only found under laboratory conditions but also in commercial oak wood representing different toasting methods and sizes. Their levels are conditioned by oak wood dimensions and toasting degree. The wood pieces with the smallest size present almost two times more compounds A and B. Moreover, the compound B is the only compound to be present in medium toasting temperatures of the smallest wood pieces. Both of them can influence either astringency sensation or bitterness taste.

International audience; Resveratrol is a well-known wine constituent. Its concentration can vary according to the cultivar choice and the winemaking process. Due to its phenolic structure, resveratrol could be transformed under high temperature or oxidative conditions, leading to the formation of various derivatives including oligomers. Hence, the goal of this study is to investigate the presence of these derivatives in wine. In the first stage, hemisynthesis of oligomeric stilbenes was achieved from resveratrol in ethanol by oxidative coupling using metals. Four de novo synthetized resveratrol derivatives were identified by MS and NMR spectroscopy including two new molecules, oxistilbenin F and oxistilbenin G. In the second stage, analysis of red wine after heat treatment by LC-MS confirmed the presence of some of these compounds in wine. Finally, the anti-inflammatory effects of the compounds were evaluated by studying their ability to prevent lipopolysaccharide (LPS)-induced upregulation of nitric oxide (NO) and reactive oxygen species (ROS) production in RAW 264.7 macrophage cell line.

Dietary lipids favor the growth of the pathobiont Bilophila wadsworthia, but the relevance of this expansion in metabolic syndrome pathogenesis is poorly understood. Here, we showed that B. wadsworthia synergizes with high fat diet (HFD) to promote higher inflammation, intestinal barrier dysfunction and bile acid dysmetabolism, leading to higher glucose dysmetabolism and hepatic steatosis. Host-microbiota transcriptomics analysis reveal pathways, particularly butanoate metabolism, which may underlie the metabolic effects mediated by B. wadsworthia. Pharmacological suppression of B. wadsworthia-associated inflammation demonstrate the bacterium’s intrinsic capacity to induce a negative impact on glycemic control and hepatic function. Administration of the probiotic Lactobacillus rhamnosus CNCM I-3690 limits B. wadsworthia-induced immune and metabolic impairment by limiting its expansion, reducing inflammation and reinforcing intestinal barrier. Our results suggest a new avenue for interventions against western diet-driven inflammatory and metabolic diseases., Lipid intake is known to promote Bilophila wadsworthia growth. Here the authors show that B. wadsworthia aggravates high fat diet induced metabolic dysfunctions and its suppression, both pharmacologically or mediated by Lactobacillus rhamnosus, limits the severity of metabolic impairment.

International audience; Scope: Amyloid-beta peptide is the main component of senile plaques in Alzheimer's disease. The inhibition of amyloid-beta peptide assembly, the destabilization of amyloid-beta peptide aggregates, and the decrease of its cytotoxicity for the prevention of neuronal death are considered neuroprotective effects. In this work, the protective effects against amyloid-beta peptide aggregation and cytotoxicity of eight indolic compounds are evaluated: tryptophan, tryptamine, serotonin, tryptophol, N-acetylserotonin, 3-indoleacetic acid, tryptophan ethyl ester, and melatonin. Methods and results: Thioflavin T spectroscopic assay, transmission electron microscopy, western blotting, circular dichroism, NMR, cell viability (thiazolyl blue tetrazolium bromide assay), quantitative PCR, and heme oxygenase activity are used. Serotonin is the most effective compound for inhibiting amyloid-beta peptide aggregation. Almost all the indolic compounds tested prevent amyloid-beta peptide-induced and increase cell viability, being between 9 and 25%. Melatonin and serotonin are the most active. Moreover, serotonin increased the expression of SIRT-1 and 2, heat shock protein 70, and heme oxygenase activity, this being a possible mechanism underlying the observed neuroprotective effect. Conclusion: Melatonin and other related indolic compounds, mainly serotonin, show an inhibitory and destabilizing effect on amyloid-beta peptide fibril formation and they possess neuroprotective properties related to the vitagenes system.

International audience; A NMR-based metabolomics method was developed to semiautomatically quantify the main components of wine. The method was applied to discriminate wines from two regions of China, Shanxi and Ningxia, which were produced by 6 wineries and for 6 vintages. Two different cultivars, Cabernet Sauvignon and Beihong, were used for winemaking. The method allowed the quantification of 33 metabolites including sugars, amino acids, organic acids, alcohols, and phenolic compounds. Depending on the compounds, the LOD values were in the range of 0.6 to 116 mg/L. The results showed that NMR-based metabolomics combined with multivariate statistical analysis allowed wine separation as a function of terroir and cultivar. Nevertheless, wine differentiation as a function of wineries and ageing would need to be examined more carefully.

International audience; Gut microbiota dysbiosis has been associated with inflammatory bowel diseases (IBD). In colorectal cancer, the gut microbiota has also been recognized as potentially involved in aggravating or favoring the tumor development. However, very little is known on the structure and role of the microbiota in colitis associated cancer (CAC), an important complication of IBD in human. Here we analyzed the bacterial and fungal composition of the mucosa associated microbiota of patients suffering CAC, sporadic cancer (SC) and of healthy subjects (HS) by barcode sequences analysis on the following cohort: 7 CAC patients, 10 SC patients and 10 HS using 16S (MiSeq) and ITS2 (pyrosequencing) sequencing, for bacteria and fungi respectively. Mucosa-associated bacterial microbiota in CAC was significantly different from the ones in SC or in HS, while the fungal showed no differences. Comparison between mucosa-associated microbiota on the tumor site or in normal mucosa near the tumor showed very similar patterns. The global mucosa-associated bacterial microbiota in cancer patients was characterized by a restriction in biodiversity but no change for the fungal community. Compared to SC, CAC was characterized by an increase of Enterobacteriacae family and Sphingomonas genus and a decrease of Fusobacterium and Ruminococcus genus. Our study confirms the alteration of the mucosa-associated bacterial microbiota in IBD and SC. Although the cohort is limited in number, this is the first evidence of the existence of an altered bacterial microbiota in CAC clearly different from the one in SC patients.

Grapevine stem extracts are viticulture byproducts rich in stilbenes that are increasingly studied for their potential biological activities. This study aimed to investigate some biological activities of a grape byproduct with high stilbenoid content and to point out the molecules responsible of these beneficial activities. As a consequence, the extract was subjected to a bioguided fractionation and separation by centrifugal partition chromatography. The obtained fractions were characterized by liquid chromatography coupled to mass spectrometry and nuclear magnetic resonance. Fractions were purified further by column chromatography and resulted in the purification of the main constituents. Thirteen stilbenes have been quantified. The most abundant compounds were ε-viniferin, resveratrol, and, in lesser amounts, isohopeaphenol and ampelopsin A. The extract, fractions, and major stilbenes were tested for their antioxidant activity by oxygen radical absorbance capacity and their cyprotective effects against β-amyloid on rat pheochromocytoma cells. Among them, fraction 5 showed significant antioxidant activity and fraction 2 had a significant cytoprotective effect against β-amyloid-induced toxicity. Two putative inhibitors of β-amyloid toxicity have been identified: ampelopsin A and piceatannol.

ObjectiveIn association with innate and adaptive immunity, the microbiota controls the colonisation resistance against intestinal pathogens. Caspase recruitment domain 9 (CARD9), a key innate immunity gene, is required to shape a normal gut microbiota. Card9–/– mice are more susceptible to the enteric mouse pathogen Citrobacter rodentium that mimics human infections with enteropathogenic and enterohaemorrhagic Escherichia coli. Here, we examined how CARD9 controls C. rodentium infection susceptibility through microbiota-dependent and microbiota-independent mechanisms.DesignC. rodentium infection was assessed in conventional and germ-free (GF) wild-type (WT) and Card9–/– mice. To explore the impact of Card9–/–microbiota in infection susceptibility, GF WT mice were colonised with WT (WT→GF) or Card9–/– (Card9–/–→GF) microbiota before C. rodentium infection. Microbiota composition was determined by 16S rDNA gene sequencing. Inflammation severity was determined by histology score and lipocalin level. Microbiota–host immune system interactions were assessed by quantitative PCR analysis.ResultsCARD9 controls pathogen virulence in a microbiota-independent manner by supporting a specific humoral response. Higher susceptibility to C. rodentium-induced colitis was observed in Card9–/–→GF mice. The microbiota of Card9–/– mice failed to outcompete the monosaccharide-consuming C. rodentium, worsening the infection severity. A polysaccharide-enriched diet counteracted the ecological advantage of C. rodentium and the defective pathogen-specific antibody response in Card9–/– mice.ConclusionsCARD9 modulates the susceptibility to intestinal infection by controlling the pathogen virulence in a microbiota-dependent and microbiota-independent manner. Genetic susceptibility to intestinal pathogens can be overridden by diet intervention that restores humoural immunity and a competing microbiota.

Small unilamellar vesicles (SUVs) of phospholipids are often used as a membrane model system for studying the interaction of molecules. When using NMR under the standard liquid-state conditions, SUV phospholipid proton spectra can be recorded, exhibiting sharp signals. This is not only because of the fast vesicular tumbling but also because of the combination of this tumbling with the individual motion of the lipids inside the bilayer. This appears evident because addition of cholesterol is responsible of broader resonances because of the slowing down of the lipid motion. On the other hand, no (1)H signal is detected for cholesterol in the bilayer. This lack of detection of the inserted molecules explains why generally SUVs are not considered as a good model for NMR studies under the standard liquid-state conditions. Here, we use two other sterols in order to demonstrate that an increase of the molecular mobility inside the bilayer could allow the detection of their proton resonances. For desmosterol and lanosterol, which show higher mobility inside the bilayer, with increasing lateral diffusion rates, (1)H sterol signals are detected in contrast to cholesterol. For the fast diffusing lanosterol, no significant improvement in detection is observed using deuterated lipids, demonstrating that homonuclear dipolar coupling is fully averaged out. Furthermore, in the case of low mobility such as for cholesterol, the use of a fast magic angle spinning probe is shown to be efficient to recover the full proton spectrum.

International audience; Neurotensin (NT) is a tridecapeptide, hormone in the periphery and neurotransmitter in the brain. We used high-resolution nuclear magnetic resonance (NMR) to resolve the three-dimensional structure of NT in a small unilamellar vesicle (SUV) environment. We demonstrate that if the dynamic of the association-dissociation processes of peptide to SUV binding is rapid enough, structural determination can be obtained by solution NMR experiments. Thus, according to the global dynamic of the system, SUVs seem to be an effective model to mimic biological membranes, especially since the lipid composition can be modified or sterols may be added to closely mimic the biological membranes studied. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:2.

Several cultivars of peach fruit (Prunus persica L.) were investigated. Their phenolic composition and concentration were assessed by LC-MS. Concentrations were calculated in mg per g of dry weight extract. Their antioxidant capacity (Folin-Ciocalteu, ORAC, DPPH, ABTS, PFRAP and ICA), inhibitory property against β-amyloid and α-synuclein fibril formation and protective capacity against Aβ-induced toxicity on PC12 cell lines (viability assessed by MTT assay and intracellular ROS production by DCFH-DA assay) were evaluated. Fifteen different phenolic compounds were identified and quantified. In particular, new isorhamnetin derivatives were identified. Phenolic contents were ranged between 19 and 82mg/g. Spring Belle extract had the highest content and Romea the lowest. Except for the ICA assay, a good correlation between phenolic content and the antioxidant capacities of peach fruit extracts was found, indicating that phenolic compounds are major contributors to their antioxidant capacity. Results indicate that the phenolic extract of peach cultivars inhibits Aβ and αS fibril formation and protects PC12 cell lines against Aβ-induced toxicity.

International audience; Complex interactions between the host and the gut microbiota govern intestinal homeostasis but remain poorly understood. Here we reveal a relationship between gut microbiota and caspase recruitment domain family member 9 (CARD9), a susceptibility gene for inflammatory bowel disease (IBD) that functions in the immune response against microorganisms. CARD9 promotes recovery from colitis by promoting interleukin (IL)-22 production, and Card9-/- mice are more susceptible to colitis. The microbiota is altered in Card9-/- mice, and transfer of the microbiota from Card9-/- to wild-type, germ-free recipients increases their susceptibility to colitis. The microbiota from Card9-/- mice fails to metabolize tryptophan into metabolites that act as aryl hydrocarbon receptor (AHR) ligands. Intestinal inflammation is attenuated after inoculation of mice with three Lactobacillus strains capable of metabolizing tryptophan or by treatment with an AHR agonist. Reduced production of AHR ligands is also observed in the microbiota from individuals with IBD, particularly in those with CARD9 risk alleles associated with IBD. Our findings reveal that host genes affect the composition and function of the gut microbiota, altering the production of microbial metabolites and intestinal inflammation.

International audience; Centrifugal partition chromatography (CPC) and all countercurrent separation apparatus provide chemists with efficient ways to work with complex matrixes, especially in the domain of natural products. However, despite the great advances provided by these techniques, more efficient ways of analyzing the output flow would bring further enhancement. This study describe a hyphenated approach made by coupling NMR with CPC through a hybrid-indirect coupling made possible by using a solid phase extraction (SPE) apparatus intended for high-pressure liquid chromatography (HPLC)-NMR hyphenation. Some hardware changes were needed to adapt the incompatible flow-rates and a reverse-engineering approach that led to the specific software required to control the apparatus. 1D 1HNMR and 1H–1H correlation spectroscopy (COSY) spectra were acquired in reasonable time without the need for any solvent-suppression method thanks to the SPE nitrogen drying step. The reduced usage of expensive deuterated solvents from several hundreds of milliliters to the milliliter order is the major improvement of this approach compared to the previously published ones.

Glycosylphosphatidylinositol (GPI)-anchored proteins are an important class of cell wall proteins in Candida albicans because of their localization and their function, even if more than half of them have no characterized homolog in the databases. In this study, we focused on the IFF protein family, investigating their exposure on the cell surface and the sequences that determine their subcellular localization. Protein localization and surface exposure were monitored by the addition of a V5 tag on all members of the family. The data obtained using the complete proteins showed for Iff3 (or -9), Iff5, Iff6, and Iff8 a covalent linkage to the β-1,6-glucan network but, remarkably, showed that Iff2/Hyr3 was linked through disulfide bridges or NaOH-labile bonds. However, since some proteins of the Iff family were undetectable, we designed chimeric constructions using the last 60 amino acids of these proteins to test the localization signal. These constructions showed a β-1,6-glucan linkage for Iff1/Rbr3, Iff2/Hyr3, Iff4 and Iff7/Hyr4 C-terminal–Iff5 fusion proteins, and a membrane localization for the Iff10/Flo9 C terminus-Iff5 fusion protein. Immunofluorescence analyses coupled to these cell fraction data confirmed the importance of the length of the central serine/threonine-rich region for cell surface exposure. Further analysis of the Iff2/Hyr3 linkage to the cell surface showed for the first time that a serine/threonine central region of a GPI-anchored protein may be responsible for the disulfide and the NaOH bonds to the glucan and glycoproteins network and may also override the signal of the proximal ω site region.

International audience; Several paramagnetic electron-rich Fe(III) mononuclear arylacetylide complexes of formula [(2-dppe)(5-C5Me5)Fe(CC-Ar)]+ in which Ar represents a functional aryl group were studied by means of multinuclear NMR. All signals detected for the various nuclei were assigned. Hyperfine coupling constants for selected nuclei of the arylacetylide ligand were derived from 1H or 19F NMR contact shifts. These NMR data are diagnostic of a metal-centered unpaired electron partly residing in a molecular orbital on the arylacetylide ligand, in line with DFT computations. We show here that the 1H NMR paramagnetic shifts of the ortho (H1) and meta (H2) arylacetylide protons convey decisive information on the charge distribution in the aryl ring. Estimates of the relaxation rates of the unpaired electron were also derived from half-widths of the 1H NMR signals. Finally, line-broadening studies of Fe(II)/Fe(III) mixtures allowed extracting the self-exchange rates for several redox couples among these complexes. The self-exchange rates appear slightly substituent dependent and are apparently larger for compounds with electron-withdrawing substituents on the aryl ring. Reorganization energies of ca. 4000 cm-1 could be derived for these outer-sphere electron-transfer processes.

International audience; Volatile and non-volatile molecules are directly responsible for the thrill and excitement provided by wine-tasting. Their elucidation requires powerful analytical techniques and innovative methodologies. In a recent work, two novel sweet compounds called quercotriterpenosides (QTT) were identified in oak wood used for wine-ageing. The aim of the present study is to discover structural analogs of such natural sweeteners in oak wood. For this purpose, an analytical approach was developed as an alternative to chemical synthesis. Orbitrap mass spectrometry proved to be a crucial technique both to demonstrate the presence of QTT analogs in oak wood by targeted screening and to guide the purification pathway of these molecules using complementary chromatographic tools. Four compounds were isolated and identified for the first time: two isomers, one glucosyl derivative and one galloyl derivative of QTT. Their tasting showed that only the two new isomers were sweet, thus demonstrating both the pertinence of the strategy and the influence of functional groups on gustatory properties. Finally, this paper presents some developments involving multistage Fourier transform mass spectrometry (FTMS) to provide solid structural information on these functional groups prior to any purification of compounds. Such analytical developments could be particularly useful for research on taste-active or bio-active products.

The screening of natural products in the search for new lead compounds against Alzheimer’s disease has unveiled several plant polyphenols that are capable of inhibiting the formation of toxic β-amyloid fibrils. Gallic acid based gallotannins are among these polyphenols, but their antifibrillogenic activity has thus far been examined using “tannic acid”, a commercial mixture of gallotannins and other galloylated glucopyranoses. The first total syntheses of two true gallotannins, a hexagalloylglucopyranose and a decagalloylated compound whose structure is commonly used to depict “tannic acid”, are now described. These depsidic gallotannins and simpler galloylated glucose derivatives all inhibit amyloid β-peptide (Aβ) aggregation in vitro, and monogalloylated α-glucogallin and a natural β-hexagalloylglucose are shown to be the strongest inhibitors.

Ambient pH signaling involves a cascade of conserved Rim or Pal products in ascomycetous yeasts or filamentous fungi, respectively. Recent evidences in the fungi Aspergillus nidulans , Saccharomyces cerevisiae , Yarrowia lipolytica , and Candida albicans suggested that components of endosomal sorting complexes required for transport (ESCRT) involved in endocytic trafficking were needed for signal transduction along the Rim pathway. In this study, we confirm these findings with C. albicans and show that Vps28p (ESCRT-I) and Vps32p/Snf7p (ESCRT-III) are required for the transcriptional regulation of known targets of the Rim pathway, such as the PHR1 and PHR2 genes encoding cell surface proteins, which are expressed at alkaline and acidic pH, respectively. We additionally show that deletion of these two VPS genes, particularly VPS32 , has a more drastic effect than a RIM101 deletion on growth at alkaline pH and that this effect is only partially suppressed by expression of a constitutively active form of Rim101p. Finally, in an in vivo mouse model, both vps null mutants were significantly less virulent than a rim101 mutant, suggesting that VPS28 and VPS32 gene products affect virulence both through Rim-dependent and Rim-independent pathways.

International audience; Dimeric stilbene glucosides 1-3 [two diastereomers of (-)-gnemonoside A (1a and 1b), (-)-gnemonoside C (2), and (-)-gnemonoside D (3)] as well as a mixture of the two enantiomers of gnetin C (4) were isolated from the rhizomes of Gnetum africanum. The two enantiomers of gnetin C, (+)-4 and (-)-4, were obtained from the aglycones of 1a and 1b, respectively. The configurations of these stilbenoids were investigated by NMR and vibrational circular dichroism (VCD) experiments. The absolute configurations of (-)-1a, (-)-2, (-)-3, and (-)-4 were established as 7aS,8aS by VCD spectroscopy in combination with density functional theory calculations. The antiamyloidogenic activity of the isolated stilbenes was also evaluated versus beta-amyloid fibrils. The four glucosides of gnetin C (1a, 1b, 2, and 3) were found to be the most active compounds, with inhibition percentages of 56, 56, 58, and 54 at 10 μM, respectively.

International audience; The molecular tumbling of small unilamellar vesicles is not fast enough to enable the detection of (1)H NMR signals of molecules associated with phospholipids. We show that relatively fast kinetic exchange of the interacting molecules is able to induce a strong decrease of the residual homonuclear dipolar coupling, allowing the acquisition of sharp signals. At low molecule/lipids molecular ratio, this can be lead to signal broadening due to exchange at intermediate rates on the NMR chemical timescale. However, proton resonances can be easily detected when sufficient lipids are added to prevent the occurrence of any free compounds in solution. This is demonstrated, using lipid signal suppression, in the case of paramagnetic porphyrin derivatives as well as diamagnetic hematoporphyrin. Since several peptides and proteins are expected to be associated with lipids having relatively fast dynamics, this study addresses, as a first example, the interaction of cytochrome c

Cell wall proteins are central to the virulence of Candida albicans. Hwp1, Hwp2 and Rbt1 form a family of hypha-associated cell surface proteins. Hwp1 and Hwp2 have been involved in adhesion and other virulence traits but Rbt1 is still poorly characterized. To assess the role of Rbt1 in the interaction of C. albicans with biotic and abiotic surfaces independently of its morphological state, heterologous expression and promoter swap strategies were applied. The N-terminal domain with features typical of the Flo11 superfamily was found to be essential for adhesiveness to polystyrene through an increase in cell surface hydrophobicity. A 42 amino acid-long domain localized in the central part of the protein was shown to enhance the aggregation function. We demonstrated that a VTTGVVVVT motif within the 42 amino acid domain displayed a high beta-aggregation potential and was responsible for cell-to-cell interactions by promoting the aggregation of hyphae. Finally, we showed through constitutive expression that while Rbt1 was directly accessible to antibodies in hyphae, it was not so in yeast. Similar results were obtained for another cell wall protein, namely Iff8, and suggested that modification of the cell wall structure between yeast and hyphae can regulate the extracellular accessibility of cell wall proteins independently of gene regulation.

International audience; Neurotensin (NT) is a tridecapeptide hormone in the periphery and neurotransmitter in the brain that principally activates three receptor subtypes, named NTS1, NTS2, and NTS3. Since little is known about its structure in the presence of its principal receptor NTS1, we determined it using the key domain of the receptor, i.e. the third extracellular loop. We conclude the following: (i) for the receptor fragment, NT binding modifies its central part, underlying the great flexibility and adaptability of this region; (ii) for bound NT, the extended conformation of its C-terminus is confirmed for the first time in experimental conditions and in the presence of a part of the receptor; and (iii) despite some substitutions, the human receptor residues that are involved in the interaction with NT could be similar to those of the rat receptor which play an important role in NT binding.

The complex etiology of Alzheimer's disease prompts scientists to develop multi-target strategies to combat causes and symptoms. In line with this modern paradigm and as a follow-up to our previous studies, we designed and synthesized a focused collection of new 2-arylbenzofurans and evaluated their biological properties towards specific targets involved in AD, namely human AChE and human BuChE, and Aβ fibril formation. Selected compounds were also tested for their ability to inhibit Aβ neurotoxicity in terms of neuronal viability loss, and to prevent Aβ peptide-binding to cell membrane and intracellular reactive oxygen species (ROS) formation. The different modifications introduced in the structure of our lead compound led to an increase in activity towards one or more of the selected targets: the anticholinesterase activity of some compounds was found to be significantly higher than previously obtained related molecules, and the compounds also proved to possess Aβ anti-aggregating properties and neuroprotective effects. The most interesting multi-target compounds were 18, and 1. Interestingly, 1 also showed good selectivity and moderate affinity for CB1 receptor, opening new perspectives in the field of research on AD, since cannabinoid ligands have been widely reported to have neuroprotective properties.