Histone phosphorylation: how to proceed

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.     

Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry

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.     

Predictors of Study Attrition in a Randomized Controlled Trial Evaluating a Perinatal Home-Visiting Program with Mothers with Psychosocial Vulnerabilities

Objective

Randomised controlled trials evaluating perinatal home-visiting programs are frequently confronted with the problem of high attrition rates. The aim of the present study is to identify predictors of study attrition in a trial evaluating a perinatal home-visiting program in France.

Materials and Methods

CAPEDP is a French randomized trial comparing a perinatal home-visiting program using psychologists versus usual care (N = 440). The first assessment was at inclusion into the trial at the 27^th week of pregnancy and the final assessment when the child reached the age of two. Attrition rates were calculated at 3 and 24 months postpartum. Stepwise logistic regression was used to identify predictors of early (between inclusion and 3 months postpartum) and later (between 3 and 24 months postpartum) attrition among social, psychological and parenting factors.

Results

Attrition rates were 17% and 63% at 3 and 24 months respectively. At 24 months, there was significantly more attrition in the control arm (70.6%) compared to the intervention arm (55.2%). Five independent predictors of early attrition were identified: having already had an abortion; having greater attachment insecurity as measured with the Vulnerable Attachment Style Questionnaire (VASQ); having lower global severity of psychiatric symptoms as assessed with the Symptom Check-List (SCL-90) at inclusion, being neither currently employed nor studying; and declaring no tobacco consumption during pregnancy. Being randomized into the control arm, having undergone early parental loss before age 11 and having lower global severity of psychiatric symptoms (SCL-90) at 3 months postpartum were the only variables associated with later attrition.

Conclusion

This study provides key information for identifying mothers who may require specific support to avoid study attrition in trials evaluating a home-visiting program.     

Involvement of the Cdc42 Pathway in CFTR Post-Translational Turnover and in Its Plasma Membrane Stability in Airway Epithelial Cells

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.     

The Clostridium difficile Protease Cwp84 Modulates both Biofilm Formation and Cell-Surface Properties

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.     

A Survey of Rounding Practices in Canadian Adult Intensive Care Units

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.