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International audience; The aim of this study was to characterize the effects of tannins on plant protein during sheep digestion using a digestomic approach combining in vivo (rumen) conditions and an in vitro digestive system (abomasum and small intestine). Ruminal fluid from wethers infused with a tannin solution or water (control) was introduced into the digester, and protein degradation was followed by liquid chromatography-tandem mass spectrometry (LC−MS/MS). Tannin infusion in the rumen led to a clear decrease in protein degradation-related fermentation end-products, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) protein was more abundant than in control wethers. In the simulated abomasum, peptidomic analysis showed more degradation products of RuBisCo in the presence of tannins. The effect of RuBisCo protection by tannins continued to impact Rubisco digestion into early-stage intestinal digestion but was no longer detectable in late-stage intestinal digestion. The peptidomics approach proved a potent tool for identifying and quantifying the type of protein hydrolyzed throughout the gastrointestinal tract.

Background and Aims The mechanism of action of anti-tumour necrosis factor [anti-TNF] agents could implicate macrophage modulation in Crohn’s disease [CD]. As CD macrophages are defective in controlling CD-associated adherent-invasive Escherichia coli [AIEC], anti-TNF agents could limit AIEC replication within macrophages. We assessed the effect of anti-TNF agents on AIEC survival within monocyte-derived macrophages [MDMs] from CD patients and attempted to identify the proteins involved. Methods Peripheral blood MDMs were obtained from 44 CD patients [22 with and 22 without anti-TNF agents]. MDMs were infected with reference strain AIEC-LF82. Proteomic analysis was performed before and 6 h after AIEC-LF82 infection. Results AIEC-LF82 survival was lower in MDMs from CD patients receiving anti-TNF agents compared to those who did not [−73%, p = 0.006]. After AIEC-LF82 infection, the levels of CD82 [p = 0.007], ILF3 [Interleukin enhancer-binding factor 3; p = 0.001], FLOT-1 [Flotillin-1; p = 0.007] and CHI3L1 [Chitinase 3-like 1; p = 0.035] proteins were different within CD-MDMs depending on anti-TNF exposure. FLOT-1 [ϱ = −0.44; p = 0.038] and CHI3L1 [ϱ = 0.57, p = 0.006] levels were inversely and positively correlated with AIEC survival within MDMs from CD patients with or without anti-TNF, respectively. We observed a dose-dependent decrease of AIEC-LF82 survival after adjunction of anti-TNF within MDMs, inducing an increase of FLOT-1 and decrease of CHI3L1 mRNA levels. Neutralization of intra-macrophagic CHI3L1 protein using anti-CHI3L1 antibodies reduced AIEC survival within macrophages 6 h after infection [p < 0.05]. Conclusion Anti-TNF agents are able to restrict replication of pathobionts, such as AIEC, within macrophages by modulating FLOT-1 and CHI3L1 expression in CD patients.

Listeria monocytogenes presents a dimorphism associated to the SecA2 activity with cells having a normal rod shape or a dysmorphic elongated filamentous form. Besides variation of the cell and colony morphotype, this cell differentiation has profound ecophysiological and physiopathological implications with collateral effects on virulence and pathogenicity, biotope colonisation, bacterial adhesion and biofilm formation. This suggests the SecA2-only protein export could influence the listerial cell surface, which was investigated first by characterising its properties in L. monocytogenes wt and secA2. The degree of hydrophilicity and Lewis acid-base properties appeared significantly affected upon SecA2 inactivation. As modification of electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteosurfaceome was further investigated by shotgun label-free proteomic analysis with a comparative relative quantitative approach. Following secretomic analysis, the protein secretion routes of the identified proteins were mapped considering the cognate transport and post-translocational maturation systems, as well as protein categories and subcellular localisation. Differential protein abundance profiles coupled to network analysis revealed the SecA2 dependence of 48 proteins, including some related to cell envelope biogenesis, translation and protein export, which could account for modifications of adhesion and cell properties of L. monocytogenes upon SecA2 inactivation. This investigation unravelled the profound influence of SecA2 activity on the cell surface properties and proteosurfaceome of L. monocytogenes, which provides advanced insights about its ecophysiopathology. Significance L. monocytogenes is a foodborne zoonotic pathogen and etiological agent of human listeriosis. This species presents a cellular dimorphism associated to the SecA2 activity that has profound physiopathological and ecophysiological implications with collateral effects on bacterial virulence and colonisation. To explore the influence of the SecA2-only protein export on the listerial cell, the surface properties of L. monocytogenes expressing or depleted of SecA2 was characterised by microelectrophoresis, microbial affinity to solvents and contact angles analyses. As modifications of hydrophilicity and Lewis acid-base electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteinaceous subset of the surfaceome, i.e. the proteosurfaceome, was investigated further by shotgun label-free proteomic analysis. This subproteome appeared quite impacted upon SecA2 inactivation with the identification of proteins accounting for modifications in the cell surface properties. The profound influence of SecA2 activity on the cell surface of L. monocytogenes was unravelled, which provides advanced insights about its ecophysiopathology., INRAE (“Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement”, previously called INRA, “Institut National de la Recherche Agronomique”), NCN (“Narodowe Centrum Nauki”) National Science Centre Poland (n°2013/09/B/NZ6/00710), ANR (“Agence National de la Recherche”) PathoFood project (n°ANR-17-CE21-0002), COST (European Cooperation in Science and Technology) Action FA1202 BacFoodNet, RMT (“Réseau Mixte Technologique”) CHLEAN (“Conception Hygiénique des Lignes et Equipements et Amélioration de la Nettoyabilité pour une alimentation saine et sure”) and France-Poland CampusFrance EGIDE PHC (“Programme Hubert Curien”) POLONIUM 2013 (n°28298ZE), info:eu-repo/semantics/publishedVersion

International audience; Purpose : Objective markers of usual diet are of interest as alternative or validating tools in nutritional epidemiology research. The main purpose of the work was to assess whether saliva protein composition can reflect dietary habits in older adults, and how type 2 diabetes impacted on the saliva-diet correlates. Methods : 214 participants were selected from two European cohorts of community-dwelling older adults (3C-Bordeaux and Seniors-ENRICA-2), using a case-control design nested in each cohort. Cases were individuals with type 2 diabetes. Dietary information was obtained using the Mediterranean Diet Adherence Screener (MEDAS). Saliva was successfully obtained from 211 subjects, and its proteome analyzed by liquid chromatography-tandem mass spectrometry. Results : The relative abundance of 246 saliva proteins was obtained across all participants. The salivary proteome differed depending on the intake level of some food groups (especially vegetables, fruits, sweet snacks and red meat), in a diabetic status-and cohort-specific manner. Gene Set Enrichment Analysis suggested that some biological processes were consistently affected by diet across cohorts, for example enhanced platelet degranulation in high consumers of sweet snacks. Minimal models were then fitted to predict dietary variables by sociodemographic, clinical and salivary proteome variables. For the food group « sweet snacks », selected salivary proteins contributed to the predictive model and improved its performance in the Seniors-ENRICA-2 cohort and when both cohorts were combined. Conclusion : Saliva proteome composition of elderly individuals can reflect some aspects of dietary patterns.

International audience; Food processing affects the structure and chemical state of proteins. In particular, protein oxidation occurs and may impair protein properties. These chemical reactions initiated during processing can develop during digestion. Indeed, the physicochemical conditions of the stomach (oxygen pressure, low pH) favor oxidation. In that respect, digestive proteases may be affected as well. Yet, very little is known about the link between endogenous oxidation of digestive enzymes, their potential denaturation, and, therefore, food protein digestibility. Thus, the objective of this study is to understand how oxidative chemical processes will impact the pepsin secondary structure and its hydrolytic activity. The folding and unfolding kinetics of pepsin under oxidative conditions was determined using Synchrotron Radiation Circular Dichroism. SRCD gave us the possibility to monitor the rapid kinetics of protein folding and unfolding in real-time, giving highly resolved spectral data. The proteolytic activity of control and oxidized pepsin was investigated by MALDI-TOF mass spectrometry on a meat protein model, the creatine kinase. MALDI-TOF MS allowed a rapid evaluation of the proteolytic activity through peptide fingerprint. This study opens up new perspectives by shifting the digestion paradigm taking into account the gastric digestive enzyme and its substrate.

Introduction. The peripheral layers (PL) of wheat, including the aleurone layer (AL), are an important source of micronutrients. AL is excluded, and mainly used for animal feed, although these tissues have significant nutritional potential as they contain most micronutrients, phytochemicals and fiber, and could improve the nutritional quality of foods. Objective. A proteomic approach was used to reveal major protein differences in the aleurone layer (AL) of mature grain between four wheat species: T. Aestivum, and T. spelta (6X), T. durum (4X), and T. monococum (2X). Materiel and methods. In each species, one representative cultivar was used for total AL protein extraction. Two-dimensional electrophoresis of AL proteins revealed 1380, 1355, 1120, and 973 Coomassie stained spots, respectively. Results. A total of 334 spots showed quantitative or qualitative (presence/absence) differences between the four cultivars. T. Aestivum, and T. spelta had 90.9% and 92.6% of their AL proteome, identical, respectively. Only 25 spots were significantly different between the two hexaploid cultivars. AL proteins, encoded by A genome, were mainly expressed in polyploid species. Percentages of proteins, encoded by A genome, was 85%, in tetraploid species, and 70% in the hexaploid spelt. Eighty spots proteins, that are different qualitatively and quantitatively, were identified by mass spectrometry, and data mining, and were classified in 5 biological processes, and 12 functional categories. Conclusion. The major differences between species are, particularly, due to storage globulins (22, 11, and 1 of Glo-3, Glo-3B, Glo-3C, respectively), stress related proteins, including antioxidant proteins, like 1-Cys peroxiredoxin, and manganese superoxide dismutase, defence proteins like xylanase, serpin3, and heat shock proteins.

Crosstalk between adipose and muscular tissues is hypothesized to regulate the number of muscular and adipose cells during fetal growth, with post-natal consequences on lean and fat masses. Such crosstalk largely remains, however, to be described. We hypothesized that a characterization of the proteomes of adipose and muscular tissues from bovine fetuses may enhance the understanding of the crosstalk between these tissues through the prediction of their secretomes and surfaceomes. Proteomic experiments have identified 751 and 514 proteins in fetal adipose tissue and muscle. These are mainly involved in the regulation of cell proliferation or differentiation, but also in pathways such as apoptosis, Wnt signalling, or cytokine-mediated signalling. Of the identified proteins, 51 adipokines, 11 myokines, and 37 adipomyokines were predicted, together with 26 adipose and 13 muscular cell surface proteins. Analysis of protein&ndash, protein interactions suggested 13 links between secreted and cell surface proteins that may contribute to the adipose&ndash, muscular crosstalk. Of these, an interaction between the adipokine plasminogen and the muscular cell surface alpha-enolase may regulate the fetal myogenesis. The in silico secretome and surfaceome analyzed herein exemplify a powerful strategy to enhance the elucidation of the crosstalk between cell types or tissues.

Vancomycin-resistant enterococci (VRE), due to their intrinsic resistance to various commonly used antibiotics and their malleable genome, make the treatment of infections caused by these bacteria less effective. The aims of this work were to characterize isolates of Enterococcus spp. that originated from processed meat, through phenotypic and genotypic techniques, as well as to detect putative antibiotic resistance biomarkers. The 19 VRE identified had high resistance to teicoplanin (89%), tetracycline (94%), and erythromycin (84%) and a low resistance to kanamycin (11%), gentamicin (11%), and streptomycin (5%). Based on a Next-Generation Sequencing NGS technique, most isolates were vanA-positive. The most prevalent resistance genes detected were erm(B) and aac(6')-Ii, conferring resistance to the classes of macrolides and aminoglycosides, respectively. MALDI-TOF mass spectrometry (MS) analysis detected an exclusive peak of the Enterococcus genus at m/z (mass-to-charge-ratio) 4428 &plusmn, 3, and a peak at m/z 6048 &plusmn, 1 allowed us to distinguish Enterococcus faecium from the other species. Several statistically significant protein masses associated with resistance were detected, such as peaks at m/z 6358.27 and m/z 13237.3 in ciprofloxacin resistance isolates. These results reinforce the relevance of the combined and complementary NGS and MALDI-TOF MS techniques for bacterial characterization.

Background Crohn’s disease (CD) is a disabling inflammatory bowel disease. Currently, the available treatments only alleviate the symptoms. The anti-TNFα represent the most efficient therapeutic class, but its mechanisms are poorly known. Experimental data have highlighted the role of intestinal macrophages in the pathogenesis of CD. These macrophages present a defect in the control of CD-associated adherent-invasive E. coli (AIEC) replication, which is linked to altered autophagy. The main aim of this study was to compare the proteomic profile of macrophages from CD patients with and without anti-TNFα treatment, to those from ulcerative colitis (UC) patients or healthy subjects, both at the basal state and after AIEC infection. Methods Peripheral blood monocyte-derived macrophages (MDM) were obtained from 44 CD patients including 22 with and 22 without anti-TNFα treatment, 22 UC patients and 22 healthy subjects. MDM was infected or not with AIEC LF82 reference strain. The numbers of intracellular bacteria were determined at 1h and 6h post-infection, using gentamicin protection assay. The ‘bottom-up’ proteomic analysis of macrophages after 6h of AIEC LF82 infection or not, was assessed by a mass spectrometer, using the label-free quantification. Results AIEC survival was reduced within MDM from CD patients with anti-TNFα treatment compared with those from CD without TNFα treatment (p = 0.0235). The proteomic analysis identified 1359 proteins, of which, 71 were differentially expressed between MDM at the basal state and infected by AIEC LF82 whatever the origin, 30 proteins were specific in CD patients with anti-TNFα treatment and 16 in CD patients without anti-TNFα treatment (fold change >1.3). The bioinformatics analysis shows an impact of several pathways involved in cellular activation, immune process and lysosomal activity. Interestingly, CD macrophages expression levels of β3 integrin (ITGB3), glutathione peroxidase-1 (GPx-1) and peroxiredoxin 3 (PRDX3) are significantly correlated with AIEC survival at 6h post-infection and faecal calprotectin. The stratification of MDM from CD patients according to the level of faecal calprotectin highlighted that levels of calprotectin and α-L-Fucosidase 1 (FUCA-1) increased in a similar way, both at the basal state and after AIEC LF82 infection. Conclusion Our study highlighted that the anti-TNFα treatment limits bacteria replication and acts on macrophages function. For the first time, global proteomic analysis suggests that CD macrophages-AIEC interaction could be orchestrated by ITGB3, GPx-1, PRDX3 and FUCA-1. These proteins could represent potential therapeutic targets in CD patients that need to be further investigated.

International audience; Food-derived bioactive peptides have generated an increasing interest in the field of health and well-being research. They can act either against the metabolic syndrome, participate in regulating the oxidation balance or act on the immune system. The aim of this study is to develop a workflow to generate bioactive peptides from three porcine offals namely, heart, liver, and lung and one muscle the Longissimus Dorsi, by combining in silico and in vitro approaches. Bioinformatics tools (e.i. BIOPEP and Uniprot) permitted to orientate the choice of enzymes for generating abundant bioactive peptides from the four studied porcine products. With papain and subtilisin, the main bioactivities potentially released were ACE inhibitors, DPP4 inhibitors and antioxidant peptides. An in vitro validation study using papain and subtilisin demonstrated high DPP4 inhibitors and antioxidant bioactivities for the generation of peptides. This work allowed: i) the identification of all proteins that composed porcine heart, liver, lung and LD muscle that could be useful for the scientific community, ii) the development of a workflow to select most abundant proteins in a product while considering abundance factors and iii) the potential of porcine meat and offals to generate DPP4 inhibitors and antioxidant peptides. However, there is still a need in developing new tools in order to face limitations of mass spectrometry for the identification of peptides with less than six amino acids. Such a work may contribute to the development of the circular economy and the innovative creation of value-added products from animal production.

BACKGROUND: Glucocorticoids (GC) play a major role in muscle atrophy. As skeletal muscle is a secretory organ, characterization of the muscle secretome elicited by muscle atrophy should allow to better understand the cellular mechanisms and to identify circulating biomarkers of this condition. Our project aimed to identify the changes in the muscle secretome associated with GC-induced muscle atrophy and susceptible to translate into circulation. METHODS: We have identified the GC-induced changes in the secretome of C2 C12 muscle cells by proteomic analysis, and then, we have determined how these changes translate into the circulation of mice or human subjects exposed to high concentrations of GC. RESULTS: This approach led us to identify Serpina3n as one of the most markedly secreted protein in response to GC. Our original in vitro results were confirmed in vivo by an increased expression of Serpina3n in skeletal muscle (3.9-fold; P < 0.01) and in the serum (two-fold; P < 0.01) of mice treated with GC. We also observed increased levels of the human orthologue Serpina3 in the serum of Cushing's syndrome patients compared with healthy controls matched for age and sex (n = 9/group, 2.5-fold; P < 0.01). An increase of Serpina3n was also demonstrated in muscle atrophy models mediated by GC such as cancer cachexia (four-fold; P < 0.01), sepsis (12.5-fold; P < 0.001), or diabetes (two-fold; P < 0.01). In contrast, levels of Serpina3n both in skeletal muscle and in the circulation were reduced in several models of muscle hypertrophy induced by myostatin inhibition (P < 0.01). Furthermore, a cluster of data suggests that the regulation of muscle Serpina3n involves mTOR, an essential determinant of the muscle cell size. CONCLUSIONS: Taken together, these data suggest that Serpina3n may represent a circulating biomarker of muscle atrophy associated to GC and, broadly, a reflection of dynamic changes in muscle mass.

Matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a powerful tool that opens new research opportunities in the field of biology. In this work, predictive model was developed to discriminate metabolic myofiber types using the MALDI spectral data. Rat skeletal muscles are constituted of type I and type IIA fiber, which have an oxidative metabolism for glycogen degradation, and type IIX and type IIB fiber which have a glycolytic metabolism, present in different proportions according to the muscle function and physiological state. So far, myofiber type is determined by histological methods that are time consuming. Thanks to the predictive model, we were able to predict not only the metabolic fiber type but also their location, on the same muscle section that was used for MALDI imaging. Copyright © 2017 John Wiley & Sons, Ltd.

Les auteurs remercient l’institut Carnot Qualiment pour son soutien financier (Projet QualigrasPhys coordonné par E Guichard) Les auteurs remercient l’institut Carnot Qualiment pour son soutien financier (Projet QualigrasPhys coordonné par E Guichard)Les auteurs remercient l’institut Carnot Qualiment pour son soutien financier (Projet QualigrasPhys coordonné par E Guichard); Introduction et but de l’étude: La population Sénior ne cesse d’augmenter avec des besoins nutritionnels spécifiques. La viande est une source de protéines de bonne qualité en termes de composition en acides aminés essentiels et de vitesse de digestion. Peu de données existent sur la biodisponibilité des protéines chez le Sénior en fonction de son statut oral. Ce travail a pour objectif de quantifier la bioaccessibilité des nutriments protéiques pour le Sénior, pour une meilleure évaluation des conséquences d’une malnutrition.Matériel et méthodes: Le dispositif expérimental combine l’utilisation d’un masticateur in vitro (AM2), programmé pour reproduire les troubles de la mastication et de la physiologie orale du Sénior avec l’utilisation d’un digesteur instrumenté (DIDGI) mimant les conditions physiologiques de digestion du Sénior (réduction des concentrations en lipase et pepsine gastrique, en trypsine et chymotrypsine, acidification gastrique réduite, …). Le modèle d’aliment choisi est une émulsion de type knack pour s’affranchir des effets structure inhérents à la viande. Les cinétiques de relargage des peptides au niveau gastrique sont évaluées par le dosage des peptides entre 30 et 150 minutes (Direct detect). Les peptides sont identifiés et quantifiés en analyses LC/MS (Q Exactive plus Thermo) et Progenesis QI.Résultats et Analyse statistique : Les spectres peptidiques obtenus en LC/MS sont analysés par le logiciel Progenesis QI. Les intensités cumulées des peptides identifiés correspondant à une protéine (degrés d’hydrolyse de cette protéine) sont ensuite traités par classification hiérarchique ascendante permettant de définir 4 clusters.. La cinétique d’hydrolyse des protéines pour chacun des clusters est analysée par la méthode des kmeans.. Seules les protéines du cluster 1 (n=14) ne présentent pas ou peu de différences de cinétiques entre les conditions de digestion Adulte et Senior. En revanche, pour les 54 autres protéines, on observe globalement une diminution, une phase de latence, pour les conditions Sénior… Ces clusters referment des protéines de structure fibreuse, des protéines structurelles du muscle qui sont en quantité majoritaire. Pour la première fois il est établi que la nature de la protéine, ses propriétés, impacte sa cinétique de digestion en conditions digestives du Sénior.Conclusion: Le couplage des outils de mastication et de digestion in vitro ont permis de mettre en évidence pour la première fois des différences de cinétique de digestion des protéines chez le Sénior. L’impact de la physiologie orale est bien présent mais moindre comparé à la physiologie digestive. Ces approches vont permettre à termes d’évaluer le potentiel nutritionnel des aliments notamment pour des populations spécifiques comme les Séniors.

Background: Fluoroquinolone resistance in nontyphoidal Salmonella is a situation of serious and international concern, particularly in S. Typhimurium DT104B multiresistant strains. Although known to be multifactorial, fluoroquinolone resistance is still far from a complete understanding. Methods: Subproteome changes between an experimentally selected fluoroquinolone-resistant strain (Se6-M) and its parent strain (Se6), and also in Se6-M under ciprofloxacin (CIP) stress, were evaluated in order to give new insights into the mechanisms involved. Proteomes were compared at the intracellular and membrane levels by a 2-DE~LC-MS/MS and a shotgun LC-MS/MS approach, respectively. Results: In total, 35 differentially abundant proteins were identified when comparing Se6 with Se6-M (25 more abundant in Se6 and 10 more abundant in Se6-M) and 82 were identified between Se6-M and Se6-M+CIP (51 more abundant in Se6-M and 31 more abundant under ciprofloxacin stress). Conclusion: Several proteins with known and possible roles in quinolone resistance were identified which provide important information about mechanism-related differential protein expression, supporting the current knowledge and also leading to new testable hypotheses on the mechanism of action of fluoroquinolone drugs. info:eu-repo/semantics/publishedVersion

International audience; Mass spectrometry imaging (MSI) is a powerful tool to visualize the spatial distribution of molecules on a tissue section. The main limitation of MALDI-MSI of proteins is the lack of direct identification. Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section. At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section. Then, we did a back-correlation of the m/z of the proteins detected by MALDI-MSI to those identified by label-free quantitation. This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI. We managed to link identification to nine peaks observed by MALDI-MSI. The results showed that the MSI~LC-MS/MS-LF workflow (i) allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii) was sparsely impacted by matrix deposition and laser ablation. This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation.

Saccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae. info:eu-repo/semantics/publishedVersion

The finding of new diagnostic and prognostic markers of local radiation injury, and particularly of the cutaneous radiation syndrome, is crucial for its medical management, in the case of both accidental exposure and radiotherapy side effects. Especially, a fast high-throughput method is still needed for triage of people accidentally exposed to ionizing radiation. In this study, we investigated the impact of localized irradiation of the skin on the early alteration of the serum proteome of mice in an effort to discover markers associated with the exposure and severity of impending damage. Using two different large-scale quantitative proteomic approaches, 2D-DIGE-MS and SELDI-TOF-MS, we performed global analyses of serum proteins collected in the clinical latency phase (days 3 and 7) from non-irradiated and locally irradiated mice exposed to high doses of 20, 40 and 80 Gy which will develop respectively erythema, moist desquamation and necrosis. Unsupervised and supervised multivariate statistical analyses (principal component analysis, partial-least square discriminant analysis and Random Forest analysis) using 2D-DIGE quantitative protein data allowed us to discriminate early between non-irradiated and irradiated animals, and between uninjured/slightly injured animals and animals that will develop severe lesions. On the other hand, despite a high number of animal replicates, PLS-DA and Random Forest analyses of SELDI-TOF-MS data failed to reveal sets of MS peaks able to discriminate between the different groups of animals. Our results show that, unlike SELDI-TOF-MS, the 2D-DIGE approach remains a powerful and promising method for the discovery of sets of proteins that could be used for the development of clinical tests for triage and the prognosis of the severity of radiation-induced skin lesions. We propose a list of 15 proteins which constitutes a set of candidate proteins for triage and prognosis of skin lesion outcomes.

The objective is to study the effects of nutrient restrictions, which induce a metabolic imbalance on the inflammatory response of the mammary gland in early lactation cows. The aim is to decipher the molecular mechanisms involved, by comparing a control, with a restriction group, a transcriptome and proteome, after an intra-mammary lipopolysaccharide challenge. Multi-parous cows were either allowed ad libitum intake of a lactation diet (n = 8), or a ration containing low nutrient density (n = 8; 48% barley straw and dry matter basis) for four days starting at 24 ± 3 days in milk. Three days after the initiation of their treatments, one healthy rear mammary quarter of 12 lactating cows was challenged with 50 µg of lipopolysaccharide (LPS). Transcriptomic and proteomic analyses were performed on mammary biopsies obtained 24 h after the LPS challenge, using bovine 44K microarrays, and nano-LC-MS/MS, respectively. Restriction-induced deficits in energy, led to a marked negative energy balance (41 versus 97 ± 15% of Net Energy for Lactation (NEL) requirements) and metabolic imbalance. A microarray analyses identified 25 differentially expressed genes in response to restriction, suggesting that restriction had modified mammary metabolism, specifically β-oxidation process. Proteomic analyses identified 53 differentially expressed proteins, which suggests that the modification of protein synthesis from mRNA splicing to folding. Under-nutrition influenced mammary gland expression of the genes involved in metabolism, thereby increasing β-oxidation and altering protein synthesis, which may affect the response to inflammation.

International audience; Hsp27—encoded by HspB1—is a member of the small heat shock proteins (sHsp, 12–43 kDa (kilodalton)) family. This protein is constitutively present in a wide variety of tissues and in many cell lines. The abundance of Hsp27 is highest in skeletal muscle, indicating a crucial role for muscle physiology. The protein identified as a beef tenderness biomarker was found at a crucial hub in a functional network involved in beef tenderness. The aim of this study was to analyze the proteins impacted by the targeted invalidation of HspB1 in the Tibialis anterior muscle of the mouse. Comparative proteomics using two-dimensional gel electrophoresis revealed 22 spots that were differentially abundant between HspB1-null mice and their controls that could be identified by mass spectrometry. Eighteen spots were more abundant in the muscle of the mutant mice, and four were less abundant. The proteins impacted by the absence of Hsp27 belonged mainly to calcium homeostasis (Srl and Calsq1), contraction (TnnT3), energy metabolism (Tpi1, Mdh1, PdhB, Ckm, Pygm, ApoA1) and the Hsp proteins family (HspA9). These data suggest a crucial role for these proteins in meat tenderization. The information gained by this study could also be helpful to predict the side effects of Hsp27 depletion in muscle development and pathologies linked to small Hsps.

Background Plant cell wall (PCW) polysaccharides and especially xylans constitute an important part of human diet. Xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. Xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. In order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont Bacteroides xylanisolvens XB1AT. Results Transcriptomic analyses of B. xylanisolvens XB1AT grown on insoluble oat-spelt xylan (OSX) at mid- and late-log phases highlighted genes in a polysaccharide utilization locus (PUL), hereafter called PUL 43, and genes in a fragmentary remnant of another PUL, hereafter referred to as rPUL 70, which were highly overexpressed on OSX relative to glucose. Proteomic analyses supported the up-regulation of several genes belonging to PUL 43 and showed the important over-production of a CBM4-containing GH10 endo-xylanase. We also show that PUL 43 is organized in two operons and that the knockout of the PUL 43 sensor/regulator HTCS gene blocked the growth of the mutant on insoluble OSX and soluble wheat arabinoxylan (WAX). The mutation not only repressed gene expression in the PUL 43 operons but also repressed gene expression in rPUL 70. Conclusion This study shows that xylan degradation by B. xylanisolvens XB1AT is orchestrated by one PUL and one PUL remnant that are linked at the transcriptional level. Coupled to studies on other xylanolytic Bacteroides species, our data emphasize the importance of one peculiar CBM4-containing GH10 endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. Our results also suggest that B. xylanisolvens XB1AT has specialized in the degradation of xylans of low complexity. This functional feature may provide a niche to all xylanolytic bacteria harboring similar PULs. Further functional and ecological studies on fibrolytic Bacteroides species are needed to better understand their role in dietary fiber degradation and their impact on intestinal health. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2680-8) contains supplementary material, which is available to authorized users.

et al., Escherichia coli is a commensal microorganism of the gastrointestinal tract of animals and humans and it is an excellent model organism for the study of antibiotic resistance mechanisms. The resistance transmission and other characteristics of bacteria are based on different types of gene transfer occurring throughout the bacterial evolution. One of which is horizontal gene transfer that allows us to understand the ability of bacteria to acquire new genes. One dimensional and two dimensional electrophoresis (2-DE) techniques were performed in order to identify and characterize the proteome of two E. coli strains: Electromax DH10B, a transformation-ready strain; and TF-Se20, the Electromax DH10B that contains the aac(6′)-Ib-cr4-harboring pMdT1 plasmid. After 2-DE and subsequent analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), it was possible to identify 76 distinct proteins on the TF-Se20 strain, whereas 71 had a known function. From Electromax DH10B strain, 72 different proteins were identified of which 71 were associated with a biological process. The protein of interest, aminoglycoside N-(6′)-acetyltransferase type 1, was identified by MALDI-TOF MS. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique was performed to determine its sequence. Seventy six percent of the acetylase sequence was reconstructed only in the TF-Se20 strain, representing the single protein associated to antibiotic resistance. MALDI-TOF MS and LC-MS/MS approaches allowed us to determine the total proteome of both strains, as well as the acetylase sequence. Both of them enhance the ability to obtain more accurate information about the mechanisms of antimicrobial resistance. The pMdT1 plasmid brings a new perspective in understanding the metabolic processes that lead to antibiotic resistance. Biological significance This study highlights the importance of proteomics and bioinformatics in understanding mechanisms of gene transfer and antibiotic resistance. These two approaches allow to compare the protein expression in different samples, as well as different biological processes related to each protein., The authors are grateful to the Research Unit on Applied Molecular Biosciences (UCIBIO) which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728).

In the present study, we used global approaches (proteomics, transcriptomics, and metabolomics) to assess the molecular basis of the muscle response to stress in chickens. A restraint test, combined with transport for 2 h (RT test) was chosen as the potentially stressful situation. Chickens (6 wk old) were either nontreated (control chickens) or submitted to the RT test (treated chickens). The RT test induced a 6-fold increase in corticosterone concentrations, suggesting hypothalamic-pituitary-adrenal axis activation. The RT test decreased the relative abundance of several hexose phosphates [glucose-1-P (G1P), glucose-6-P (G6P), fructose-6-P (F6P), and mannose-6-P (M6P)] in thigh muscle. In addition, 55 transcripts, among which 39 corresponded to unique annotated genes, were significantly up- (12 genes) or downregulated (27 genes) by treatment. Similarly, 45 proteic spots, among which 29 corresponded to unique annotated proteins, were overexpressed (11 proteins), underexpressed (14 proteins), or only expressed in treated chickens. Integrative analysis of differentially expressed genes and proteins showed that most transcripts and proteins belong to 2 networks whose genes were mainly related with cytoskeleton structure or carbohydrate metabolism. Whereas the decrease in energetic metabolites suggested an activation of glycogenolysis and glycolysis in response to the RT test, the reduced expression of genes and proteins involved in these pathways suggested the opposite. We hypothesized that the prolonged RT test resulted in a repression of glycogenolysis and glycolysis in thigh muscle of chickens. The down-expression of genes and proteins involved in the formation of fiber stress after the RT test suggests a reinforcement of myofibrils in response to stress.

International audience; Contractile and metabolic properties of bovine muscles play an important role in meat sensorial quality, particularly tenderness. Earlier studies based on Myosin heavy chain isoforms analyses and measurements of glycolytic and oxidative enzyme activities have demonstrated that the third trimester of foetal life in bovine is characterized by contractile and metabolic differentiation. In order to complete this data and to obtain a precise view of this phase and its regulation, we performed a proteomic analysis of Semitendinosus muscle from Charolais foetuses analysed at three stages of the third trimester of gestation (180, 210 and 260 days). The results complete the knowledge of important changes in the profiles of proteins from metabolic and contractile pathways. They provide new insights about proteins such as Aldehyde dehydrogenase family, Enolase, Dihydrolipoyl dehydrogenase, Troponin T or Myosin light chains isoforms. These data have agronomical applications not only for the management of beef sensorial quality but also in medical context, as bovine myogenesis appears very similar to human one.

Une période de mise à jeûne des animaux permet de diminuer les risques de contamination des carcasses lors de l’éviscération. Les objectifs de cet essai sont d’étudier en détails les effets de la durée de jeûne et de la digestibilité du maïs sur les performances zootechniques de canards mulards gavés et sur la qualité de leurs foies. Les relations entre ces performances et le métabolisme des foies ont été étudiées. Un lot de 60 canards mulards a été élevé jusqu’à 12 semaines d’âge dans des conditions standards. Puis les canards ont été gavés soit avec du maïs waxy (riche en amylopectine et rapidement digestible) soit avec du maïs témoin. Pour étudier le métabolisme du foie en fonction du type de maïs et de la durée du jeûne, des analyses biochimiques, transcriptomiques et protéomiques ont été réalisées sur les foies obtenus après 9h et 15h de jeûne. Les foies de tous les canards ont un poids équivalent même si le taux de lipides est supérieur après 15h de jeûne (p=0,019). Mais le rendement technologique est supérieur de 7,0 points pour les canards gavés au maïs waxy à 9h de jeûne (p=0,039). Ces différences de rendement technologique semblent s’expliquer par le taux de lipides qui est inférieur de 3,9 points lorsque le maïs waxy est utilisé (p=0,019) et par un niveau d’expression plus élevé des protéines et transcrits impliqués dans la glycolyse et la lipogenèse.

For many years Staphylococcus aureus has been recognized as an important human pathogen. In this study, the surfacome and exoproteome of a clinical sample of MRSA was analyzed. The C2355 strain, previously typed as ST398 and spa-t011 and showing a phenotype of multiresistance to antibiotics, has several resistance genes. Using shotgun proteomics and bioinformatics tools, 236 proteins were identified in the surfaceome and 99 proteins in the exoproteome. Although many of these proteins are related to basic cell functions, some are related to virulence and pathogenicity like catalase and isdA, main actors in S. aureus infection, and others are related to antibiotic action or eventually resistance like penicillin binding protein, a cell-wall protein. Studying the proteomes of different subcellular compartments should improve our understanding of this pathogen, a microorganism with several mechanisms of resistance and pathogenicity, and provide valuable data for bioinformatics databases., Graphical abstract, Highlights • We examine the surface proteome and exoproteome of multiresistant strains. • We identify bacterial infection proteins in the extracellular proteome. • Confirmation that moonlighting proteins will extend the localization data.

Wheat grain end-use value is determined by complex molecular interactions that occur during grain development, including those in the cell nucleus. However, our knowledge of how the nuclear proteome changes during grain development is limited. Here, we analyzed nuclear proteins of developing wheat grains collected during the cellularization, effective grain-filling, and maturation phases of development, respectively. Nuclear proteins were extracted and separated by two-dimensional gel electrophoresis. Image analysis revealed 371 and 299 reproducible spots in gels with first dimension separation along pH 4-7 and pH 6-11 isoelectric gradients, respectively. The relative abundance of 464 (67%) protein spots changed during grain development. Abundance profiles of these proteins clustered in six groups associated with the major phases and phase transitions of grain development. Using nano liquid chromatography tandem mass spectrometry to analyse 387 variant and non-variant protein spots, 114 different proteins were identified that were classified into 16 functional classes. We noted that some proteins involved in the regulation of transcription, like HMG1/2-like protein and histone deacetylase HDAC2, were most abundant before the phase transition from cellularization to grain-filling, suggesting that major transcriptional changes occur during this key developmental phase. The maturation period was characterized by high relative abundance of proteins involved in ribosome biogenesis. Data are available via ProteomeXchange with identifier PXD002999.

International audience; Background: Muscle ageing contributes to both loss of functional autonomy and increased morbidity. Muscle atrophy accelerates after 50 years of age, but the mechanisms involved are complex and likely result from the alteration of a variety of interrelated functions. In order to better understand the molecular mechanisms underlying muscle chronological ageing in human, we have undertaken a top-down differential proteomic approach to identify novel biomarkers after the fifth decade of age. Results: Muscle samples were compared between adult (56 years) and old (78 years) post-menopausal women. In addition to total muscle extracts, low-ionic strength extracts were investigated to remove high abundance myofibrillar proteins and improve the detection of low abundance proteins. Two-dimensional gel electrophoreses with overlapping IPGs were used to improve the separation of muscle proteins. Overall, 1919 protein spots were matched between all individuals, 95 were differentially expressed and identified by mass spectrometry, and they corresponded to 67 different proteins. Our results suggested important modifications in cytosolic, mitochondrial and lipid energy metabolism, which may relate to dysfunctions in old muscle force generation. A fraction of the differentially expressed proteins were linked to the sarcomere and cytoskeleton (myosin light-chains, troponin T, ankyrin repeat domain-containing protein-2, vinculin, four and a half LIM domain protein-3), which may account for alterations in contractile properties. In line with muscle contraction, we also identified proteins related to calcium signal transduction (calsequestrin-1, sarcalumenin, myozenin-1, annexins). Muscle ageing was further characterized by the differential regulation of several proteins implicated in cytoprotection (catalase, peroxiredoxins), ion homeostasis (carbonic anhydrases, selenium-binding protein 1) and detoxification (aldo-keto reductases, aldehyde dehydrogenases). Notably, many of the differentially expressed proteins were central for proteostasis, including heat shock proteins and proteins involved in proteolysis (valosin-containing protein, proteasome subunit beta type-4, mitochondrial elongation factor-Tu). Conclusions: This study describes the most extensive proteomic analysis of muscle ageing in humans, and identified 34 new potential biomarkers. None of them were previously recognized as differentially expressed in old muscles, and each may represent a novel starting point to elucidate the mechanisms of muscle chronological ageing in humans.

International audience; Epidemiologic and experimental studies showed that folate deficiency is associated with increased risk of degenerative diseases by enhancing abnormal one-carbon metabolism. We studied the changes in the proteome of liver, the main tissue of folate storage and metabolism, in a rat model of dietary folate depletion. Four-month-old rats were fed for 4 wk an amino acid-defined diet without folate and compared with pair-fed rats given the same diet adequately supplemented with folic acid. Folate deprivation decreased plasma and hepatic folate concentrations dramatically, while increasing homocysteinemia significantly. Using 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight MS, we identified 9 spots corresponding to differentially expressed proteins in the liver of folate-deficient rats compared with controls. Among those spots, 4 had a significantly increased volume, whereas the volume of the 5 other spots was decreased. Upregulated proteins included glutathione peroxidase (GPx) 1 and peroxiredoxin 6, 2 enzymes involved in the response to oxidative stress, and MAWD binding protein (MAWDBP), which has been associated with cancer. MAWDBP was simultaneously identified as a second spot with a lower isoelectric point (pI) that vanished almost completely after folate deficiency. Decreased abundance was also observed for cofilin 1, a protein linked to tumorigenesis, and for the GRP 75 precursor and preproalbumin, both of which are responsive to oxidative stress and/or inflammation. Moreover, an enzyme activity assay and/or Western blot analysis of GPx-1 and MAWDBP confirmed the proteomic findings. Our results show that folate deficiency modifies the abundance of several liver proteins consistently with adaptive tissue responses to oxidative and degenerative processes.

Organisateurs : Laboratoire de recherche en Cardiovasculaire, Mét abolisme, Diabétologie et Nutrition ( CarMeN) ; Unité de Nutrition Humaine ( UNH ) ; Laboratoire de Bioénergétique Fondamentale et Appliquée ( LBFA); National audience; Introduction et Méthodes : Une approche protéomique a été développée afin identifier de nouveaux biomarqueurs du vieillissement musculaire (sarcopénie). Des extraits totaux et sarcoplasmiques ont été préparés à partir de femmes ménopausées matures (54 ans) et âgées (78 ans). Sur un total de 1919 spots, 133 sont exprimés de façon différentielle chez les femmes âgées par rapport aux femmes matures, et la spectrométrie de masse (nanoLC-MS/MS) a permis d’identifier 74 protéines différentes. Résultats : On observe d'importantes modifications du métabolisme énergétique cytosolique (TPIS, PYGM, GPDA, créatine-kinase) et mitochondrial (pyruvate-déshydrogénase, aconitase, COX, NADH-déshydrogénase). Certaines protéines différentiellement exprimées correspondent à des protéines myofibrillaires (myosine light-chaînes, troponines T, myozenin). Tout ceci peut expliquer les modifications des propriétés contractiles chez la personne âgée. Les autres protéines montrent des perturbations dans les processus de cytoprotection et de détoxification, comme une régulation différentielle de plusieurs chaperons moléculaires (HSPA9, HSPA1A), l'homéostasie ionique (sélénium-binding protein) et le stress du réticulum endoplasmique (sarcalumenin, calséquestrine). De plus, l’expression de protéines impliquées dans la protéolyse est augmentée (VCP, UBA1, Calpain Small subunit-1). Nous avons aussi remarqué une régulation négative des protéines impliquées dans l’édition de l’ARN (apobec2) et la traduction mitochondriale (TuFM). Conclusion : Plusieurs biomarqueurs identifiés dans cette analyse étaient auparavant non reconnus comme différentiellement exprimés dans le muscle âgé, et peuvent représenter de nouveaux points de départ pour élucider certains des mécanismes de la sarcopénie.

International audience; We investigated a protein profile evolution during steatosis in ducks using 2-dimensional electrophoresis gels to better understand the mechanisms underlying liver steatosis at the level of hepatic proteins in waterfowl. Two-dimensional electrophoresis gels were performed in the liver at different stages of steatosis in the duck. Mule ducks were slaughtered after 0, 14, or 23 meals of overfeeding, according to commercial conditions. Thirty-one proteic spots were differentially expressed between 3 or 2 durations of overfeeding: 3 spots were differentially expressed between the 3 times and 28 spots were differentially expressed between 2 times. The identified proteins (14) could be regrouped into 5 categories: enzymes, translation factors, proteins involved in cell structure, proteins with antioxidant properties, and proteins that can link calcium. This study opens new research areas in the understanding of steatosis in waterfowl, such as cell structure and oxidative stress.