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Among all posttranslational modifications that occur on histone tails, phosphorylation is the one that establishes a direct link between chromatin remodeling and intracellular signaling pathways. Specific, conserved serine residues are present on the N-terminal tails of each histone. These are phosphoacceptor sites for a number of kinases, whose identification is essential to decipher the transduction routes leading to various physiological responses. In the case of histone H3, phosphorylation at the Ser10 residue may lead to either activated gene expression or chromatin condensation during mitosis. In addition, phosphorylation at specific sites may be coupled to other distinct modifications, such as acetylation and methylation, generating the so-called "histone code" which postulates that well defined combinatorial modifications at histone tails correspond to specific physiological responses. Here we describe a number of experimental methodologies that are essential for the study of histone phosphorylation. While chromatin immunoprecipitation is useful in recognizing gene targets, the in-gel kinase assay is a first, essential step in establishing the identity of the kinase(s) that operates in response to a specific signaling pathway. The subsequent use of in vitro kinase assays is helpful in validating the implication of a candidate kinase. These powerful approaches are important as identification of the signaling transduction routes leading to chromatin remodeling is critical to an understanding of all cellular processes. 相似文献
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Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry
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Romain Carrière Jordane Biarc Catherine Fonbonne Arnaud Salvador Céline Huillet Yves Berard Olivier Adam Catherine Manin Jérôme Lemoine 《Proteomics》2015,15(19):3320-3330
Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells. 相似文献
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Romain Ferru-Clément Fleur Fresquet Caroline Norez Thierry Métayé Frédéric Becq Alain Kitzis Vincent Thoreau 《PloS one》2015,10(3)
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation. 相似文献
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Véronique Pantaléon Anna Philibertine Soavelomandroso Sylvie Bouttier Romain Briandet Bryan Roxas Michele Chu Anne Collignon Claire Janoir Gayatri Vedantam Thomas Candela 《PloS one》2015,10(4)
Clostridium difficile is responsible for 15-20% of antibiotic-associated diarrheas, and nearly all cases of pseudomembranous colitis. Among the cell wall proteins involved in the colonization process, Cwp84 is a protease that cleaves the S-layer protein SlpA into two subunits. A cwp84 mutant was previously shown to be affected for in vitro growth but not in its virulence in a hamster model. In this study, the cwp84 mutant elaborated biofilms with increased biomass compared with the parental strain, allowing the mutant to grow more robustly in the biofilm state. Proteomic analyses of the 630Δerm bacteria growing within the biofilm revealed the distribution of abundant proteins either in cell surface, matrix or supernatant fractions. Of note, the toxin TcdA was found in the biofilm matrix. Although the overall proteome differences between the cwp84 mutant and the parental strains were modest, there was still a significant impact on bacterial surface properties such as altered hydrophobicity. In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo. Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins. 相似文献
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Jessalyn K. Holodinsky Marilynne A. Hebert David A. Zygun Romain Rigal Simon Berthelot Deborah J. Cook Henry T. Stelfox 《PloS one》2015,10(12)
Objective
To describe rounding practices in Canadian adult Intensive Care Units (ICU) and identify opportunities for improvement.Design
Mixed methods design. Cross sectional survey of Canadian Adult ICUs (n = 180) with purposefully sampled follow-up interviews (n = 7).Measurements and Main Results
Medical directors representing 111 ICUs (62%) participated in the survey. Rounding practices varied across ICUs with the majority reporting the use of interprofessional rounds (81%) that employed an open (94%) and collaborative (86%) approach, occurred at the patient’s bedside (82%), and started at a standard time (79%) and standard location (56%). Most participants reported that patients (83%) and family members (67%) were welcome to attend rounds. Approximately half of ICUs (48%) used tools to facilitate rounds. Interruptions during rounds were reported to be common (i.e., ≥1 interruption for ≥50% of patients) in 46% of ICUs. Four themes were identified from qualitative analysis of participant responses to open-ended survey questions and interviews: multidisciplinarity, patient and family involvement, factors influencing productivity, and teaching and learning.Conclusions
There is considerable variation in current rounding practices in Canadian medical/surgical ICUs. Opportunities exist to improve ICU rounds including ensuring the engagement of essential participants, clearly defining participant roles, establishing a standardized approach to the rounding process, minimizing interruptions, modifying the role of teaching, utilizing a structured rounding tool, and developing a metric for measuring rounding quality. 相似文献68.
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