Enolases: Storage compounds in seeds? Evidence from a proteomic comparison of zygotic and somatic embryos of Cyclamen persicum Mill

Somatic embryogenesis is well established for the economic relevant ornamental crop Cyclamen and thus could supplement the elaborate propagation via seeds. However, the use of somatic embryogenesis for commercial large scale propagation is still limited due to physiological disorders and asynchronous development within emerged embryos. To overcome these problems, profound knowledge of the physiological processes in Cyclamen embryogenesis is essential. Thus, the proteomes of somatic and zygotic embryos were characterised in a comparative approach. Protein separation via two dimensional IEF-SDS PAGE led to a resolution of more than 1,000 protein spots/gel. Overall, 246 proteins were of differential abundance in the two tissues compared. Mass spectrometry analysis of the 300 most abundant protein spots resulted in the identification of 247 proteins, which represent 90 distinct protein species. Fifty-five percent of the 247 proteins belong to only three physiological categories: glycolysis, protein folding and stress response. The latter physiological process was especially predominant in the somatic embryos. Remarkably, the glycolytic enzyme enolase was the protein most frequently detected and thus is supposed to play an important role in Cyclamen embryogenesis. Data are presented that indicate involvement of "small enolases" as storage proteins in Cyclamen. A digital reference map was established via a novel software tool for the web-based presentation of proteome data linked to KEGG and ExPasy protein-databases and both were made publicly available online. © 2011 Springer Science+Business Media B.V

Impact of Low-Level-Viremia on HIV-1 Drug-Resistance Evolution among Antiretroviral Treated-Patients

to determine the emergence and evolution of DRAM during LLV in HIV-1-infected patients while receiving antiretroviral therapy (ART).Retrospective analysis of patients presenting a LLV episode defined as pVL between 40 and 500 c/mL on at least 3 occasions during a 6-month period or longer while on the same ART. Resistance genotypic testing was performed at the onset and at the end of LLV period. Emerging DRAM was defined during LLV if never detected on baseline genotype or before.48 patients including 4 naive and 44 pretreated (median 9 years) presented a LLV episode with a median duration of 11 months. Current ART included 2NRTI (94%), ritonavir-boosted PI (94%), NNRTI (23%), and/or raltegravir (19%). Median pVL during LLV was 134 c/mL. Successful resistance testing at both onset and end of the LLV episode were obtained for 37 patients (77%), among who 11 (30%) acquired at least 1 DRAM during the LLV period: for NRTI in 6, for NNRTI in 1, for PI in 4, and for raltegravir in 2. During the LLV period, number of drugs with genotypic resistance increased from a median of 4.5 to 6 drugs. Duration and pVL level of LLV episode, duration of previous ART, current and nadir CD4 count, number of baseline DRAM and GSS were not identified as predictive factors of resistance acquisition during LLV, probably due to limited number of patients.Persistent LLV episodes below 500 c/ml while receiving ART is associated with emerging DRAM for all drug classes and a decreasing in further therapeutic options, suggesting to earlier consider resistance monitoring and ART optimization in this setting

Characterization of leaf apoplastic peroxidases and metabolites in Vigna unguiculata in response to toxic manganese supply and silicon

Previous work suggested that the apoplastic phenol composition and its interaction with apoplastic class III peroxidases (PODs) are decisive in the development or avoidance of manganese (Mn) toxicity in cowpea (Vigna unguiculata L.). This study characterizes apoplastic PODs with particular emphasis on the activities of specific isoenzymes and their modulation by phenols in the Mn-sensitive cowpea cultivar TVu 91 as affected by Mn and silicon (Si) supply. Si reduced Mn-induced toxicity symptoms without affecting the Mn uptake. Blue Native-PAGE combined with Nano-LC-MS/MS allowed identification of a range of POD isoenzymes in the apoplastic washing fluid (AWF). In Si-treated plants Mn-mediated induction of POD activity was delayed. Four POD isoenzymes eluted from the BN gels catalysed both H2O2-consuming and H2O2-producing activity with pH optima at 6.5 and 5.5, respectively. Four phenols enhanced NADH-peroxidase activity of these isoenzymes in the presence of Mn2+ (p-coumaric=vanillic>>benzoic>ferulic acid). p-Coumaric acid-enhanced NADH-peroxidase activity was inhibited by ferulic acid (50%) and five other phenols (50–90%). An independent component analysis (ICA) of the total and apoplastic GC-MS-based metabolome profile showed that Mn, Si supply, and the AWF fraction (AWFH2O, AWFNaCl) significantly changed the metabolite composition. Extracting non-polar metabolites from the AWF allowed the identification of phenols. Predominantly NADH-peroxidase activity-inhibiting ferulic acid appeared to be down-regulated in Mn-sensitive (+Mn, –Si) and up-regulated in Mn-tolerant (+Si) leaf tissue. The results presented here support the previously hypothesized role of apoplastic NADH-peroxidase and its activity-modulating phenols in Mn toxicity and Si-enhanced Mn tolerance

Virologic Failure of Protease Inhibitor-Based Second-Line Antiretroviral Therapy without Resistance in a Large HIV Treatment Program in South Africa

Background: We investigated the prevalence of wild-type virus (no major drug resistance) and drug resistance mutations at second-line antiretroviral treatment (ART) failure in a large HIV treatment program in South Africa. Methodology/ Principal Findings HIV-infected patients $\geq$15 years of age who had failed protease inhibitor (PI)-based second-line ART (2 consecutive HIV RNA tests >1000 copies/ml on lopinavir/ritonavir, didanosine, and zidovudine) were identified retrospectively. Patients with virologic failure were continued on second-line ART. Genotypic testing for drug resistance was performed on frozen plasma samples obtained closest to and after the date of laboratory confirmed second-line ART failure. Of 322 HIV-infected patients on second-line ART, 43 were adults with confirmed virologic failure, and 33 had available plasma for viral sequencing. HIV-1 RNA subtype C predominated (n = 32, 97%). Mean duration on ART (SD) prior to initiation of second-line ART was 23 (17) months, and time from second-line ART initiation to failure was 10 (9) months. Plasma samples were obtained 7(9) months from confirmed failure. At second-line failure, 22 patients (67%) had wild-type virus. There was no major resistance to PIs found. Eleven of 33 patients had a second plasma sample taken 8 (5.5) months after the first. Median HIV-1 RNA and the genotypic resistance profile were unchanged. Conclusions/ Significance: Most patients who failed second-line ART had wild-type virus. We did not observe evolution of resistance despite continuation of PI-based ART after failure. Interventions that successfully improve adherence could allow patients to continue to benefit from second-line ART therapy even after initial failure

Immunoregulatory molecule expression on extracellular microvesicles in people living with HIV

IntroductionPeople living with HIV (PLWH) now benefit from combined antiviral treatments that durably control viral replication. These antiretroviral treatments decrease mortality and improve quality of life in PLWH, but do not completely control the excessive non-specific activation of the immune system in PLWH. This chronic immune activation is a key element of HIV immunopathology that contributes to the pathophysiology of inflammatory comorbid conditions, such as cardiovascular disorders, cancer and autoimmune diseases. Circulating non-exosomal extracellular vesicles, also known as microparticles (MPs) are detected in these diseases and have been linked to immune activation. The objective of this study was to characterize the MPs present in PLWH and to assess their association with chronic immune activation.MethodsWe performed flow cytometry for the complete phenotypic characterization of MPs from fresh plasma from PLWH and from people without HIV as the control group. The absolute number, size and cellular origin of MPs were evaluated. The immunoregulatory profile was determined by cell origin, for MPs derived from platelets (PMPs), monocytes (MMPs) and T lymphocytes (LMPs).ResultsPLWH had significantly more circulating MPs than controls, for MPs of all sizes originating from T lymphocytes, red blood cells, neutrophils, dendritic cells, B lymphocytes and endothelial cells. PMPs and MMPs were not more numerous in PLWH, but the immunoregulatory phenotypes of these MPs differed between PLWH and controls. These differences in immunoregulatory molecule expression profile were also observed for LMPs. PDL1, ICOSL, CCR5, TGFβ1, MHC classes I and II, TRAIL, CXCR4, OX40, DC-SIGN, CTLA4 and PDL2 were more strongly expressed on the surface of MPs from PLWH than on those from controls.ConclusionMPs are an important element in intercellular communication, making it possible to transfer phenotypes and functions to immune cells. The significantly higher numbers of MPs expressing diverse immunomodulatory molecules in PLWH may make a major contribution to the maintenance and/or the development of immune-cell activation in these individuals

New proteomic methodologies to aid in genome annotation and protein sequence validation

Ces dernières années, l’analyse protéomique par spectrométrie de masse a considérablement progressé grâce à des développements technologiques jusqu’à devenir la méthode de choix pour l’étude des protéines. Les domaines d’application de la protéomique sont aujourd’hui très vastes et parmi ceux-ci un nouveau domaine est en pleine émergence : la protéogénomique dans laquelle la protéomique sert directement d’outil d’aide à l’annotation génomique. L’objectif principal de ce travail de thèse était de développer de nouvelles approches de protéogénomique basées sur les méthodologies de la protéomique pour améliorer la qualité des banques de séquences protéiques disponibles pour la communauté scientifique. Pour atteindre cet objectif, nous avons notamment développé : 1-Une nouvelle stratégie pour améliorer la détermination des peptides N-terminaux des protéines (stratégie N-TOP) afin de faciliter l’identification des codons d’initiation des protéines qui représente sans doute un des plus grands défis de l’annotation génomique. 2-Des stratégies optimisées de recherche de données MS/MS dans les banques de séquences (génomiques ou protéiques) pour faciliter la mise en évidence d’erreurs dans les banques protéiques générées par prédiction in silico. Ces stratégies ont pu être appliquées avec succès dans le cadre de différentes études en permettant d’améliorer la qualité des banques protéiques La stratégie N-TOP a également ouvert d’autres perspectives dans deux grands champs très important de la protéomique : la caractérisation des phosphorylations des protéines et la protéomique quantitative.During last years, proteomic analysis by mass spectrometry has considerably progressed thanks to technological developments to become the reference method for protein study. Today, the areas of application of proteomics are very wide and among them a new field is emerging : the proteogenomics in which proteomics is used directly as a tool to aid in genome annotation. The main objective of this PhD thesis was to develop new proteogenomic approaches based on proteomic methodologies to improve the quality of protein sequence databases available to the scientific community. To reach this goal, we have developed : 1. A new strategy to improve the identification of N-terminal peptides (N-TOP strategy) in order to facilitate the identification of protein start codons which is probably one of the greatest challenges of genome annotation. 2. Optimized MS/MS search strategies in sequence databanks (genome or protein) in order to facilitate the detection of errors in protein databases generated by bioinformatic prediction. These strategies have been successfully applied in various studies to improve protein database reliability. The N-TOP strategy has also opened up outlook in two important proteomic fields : characterization of protein phosphorylation and quantitative proteomics

New proteomUªUªethodologies to aid in genome annotation and protein sequence validation

Ces dernières années, l'analyse protéomique par spectrométrie de masse a considérablement progressé grâce à des développements technologiques jusqu à devenir la méthode de choix pour l'étude des protéines. Les domaines d'application de la protéomique sontDuring last years, proteomic analysis by mass spectrometry has considerably progressed thanks to technological developments to become the reference method for protein study. Today, the areas of application of proteomics are very wide and among them a ne

Amyotrophie spinale infantile proximale (prise en charge et perspectives thérapeutiques)

LYON1-BU Santé (693882101) / SudocRENNES1-BU Santé (352382103) / SudocSudocFranceF

Accès au suivi virologique pour les patients vivant avec le VIH sous traitement en zone décentralisée d' Afrique sub-Saharienne (apport de la méthode DBS)

Pour le suivi du traitement anti-VIH dans les pays à faibles ressources, l'OMS a développé une approche de santé publique fondée sur la décentralisation des soins. Notre objectif est l'évaluation virologique et l'émergence de résistance grâce à la technique du papier-filtre (DBS), de patients infectés par le VIH suivi dans le district de Kolofata au Cameroun. De février 2009 à mars 2011, 126 patients infectés par le VIH suivis après l'initiation d'un traitement ARV à M12, M24 et M36 (+/- 2 mois) ont été inclus. Sang total et plasma ont été prélevés. Le sang total a été déposé sur DBS puis acheminé au laboratoire de référence afin de quantifier la charge virale (CV) et réaliser un génotypage de résistance. Un total de 19 (38,8%), 21 (40,4%) et 8 (34,8%) avaient une CV> 1000 cpmL à respectivement 12, 24 et 36 mois de traitement. Chez les patients ayant une CV>5000 cpmL, au moins une mutation de résistance a été retrouvée chez respectivement 7/13 (53,8%), 8/11 (72,7%) et 5/5 (100%) patients. Aucune souche virale (0/5) de patient ayant une CV comprise entre 1000 et 5000 cpmL n'avait de mutation majeure de résistance. De plus 17 (85%) étaient résistantes à 1 (n=10, 50%) et à 2 (n=7, 35%) molécules de la seconde ligne de traitement recommandée et 5 (25%) étaient résistantes à l'étravirine. Dans la zone de Kolofata, le contrôle virologique est modéré avec un taux de résistance élevé réduisant l'efficacité des traitements ultérieurs. Ces données suggèrent que l'absence de suivi virologique favoriserait l'émergence de la résistance et que le DBS représente un outil de prélèvement fiable dans des conditions de décentralisation et climatiques extrêmes.PARIS6-Bibl.Pitié-Salpêtrie (751132101) / SudocSudocFranceF

Étude rétrospective sur l'aspergillose invasive dans le service de maladies infectieuses et tropicales de l'hôpital Saint-Louis de 1995 à 2004

PARIS6-Bibl.Pitié-Salpêtrie (751132101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF