Endogenous Plasma Peptide Detection and Identification in the Rat by a Combination of Fractionation Methods and Mass Spectrometry

Mass spectrometry-based analyses are essential tools in the field of biomarker research. However, detection and characterization of plasma low abundance and/or low molecular weight peptides is challenged by the presence of highly abundant proteins, salts and lipids. Numerous strategies have already been tested to reduce the complexity of plasma samples. The aim of this study was to enrich the low molecular weight fraction of rat plasma. To this end, we developed and compared simple protocols based on membrane filtration, solid phase extraction, and a combination of both. As assessed by UV absorbance, an albumin depletion >99% was obtained. The multistep fractionation strategy (including reverse phase HPLC) allowed detection, in a reproducible manner (CV < 30%–35%), of more than 450 peaks below 3000 Da by MALDI-TOF/MS. A MALDI-TOF/MS-determined LOD as low as 1 fmol/μL was obtained, thus allowing nanoLC-Chip/MS/MS identification of spiked peptides representing ~10−6% of total proteins, by weight. Signal peptide recovery ranged between 5%–100% according to the spiked peptide considered. Tens of peptide sequence tags from endogenous plasma peptides were also obtained and high confidence identifications of low abundance fibrinopeptide A and B are reported here to show the efficiency of the protocol. It is concluded that the fractionation protocol presented would be of particular interest for future differential (high throughput) analyses of the plasma low molecular weight fraction

A qualitative study of urban hospital transitional care

This study is part of a mixed methods evaluation of a large urban medical center transitional care practice (NMG-TC). The NMG-TC provides integrated physical and behavioral health care for high need patients referred from the hospital emergency department or inpatient units and who lack a usual source of primary care. The study was designed for internal quality improvement and sought to evaluate staff perceptions of successful transitions for their medically and socially complex patients, and alternatively, the obstacles most likely to negatively impact patient outcomes. All 16 NMG-TC patient care staff were interviewed in a collaborative effort to produce empowered testimony that might go beyond expected clinical narratives. The interview schedule included questions on risk stratification, integrated mental health care, provider to provider handoffs, and how staff deal with key social determinates of patients’ health. The constant comparative method was used to deductively derive themes reflecting key domains of transitional care practice. Seven themes emerged: i) the need to quickly assess patient complexity; ii) emphasizing caring for major mental health and substance use issues; iii) obstacles to care for uninsured, often undocumented patients; iv) the intractability of homelessness; v) expertise in advancing patients’ health literacy, engagement and activation; vi) fragmented handoffs from hospital care and vii) to primary care in the community. Respondent stories emphasized methods of nurturing patients’ self-efficacy in a very challenging urban health environment. Findings will be used to conceptualize pragmatic, potentially high-impact transitional care quality improvement initiatives capable of better addressing frequent hospital use

ICDS database: interrupted CoDing sequences in prokaryotic genomes

Unrecognized frameshifts, in-frame stop codons and sequencing errors lead to Interrupted CoDing Sequence (ICDS) that can seriously affect all subsequent steps of functional characterization, from in silico analysis to high-throughput proteomic projects. Here, we describe the Interrupted CoDing Sequence database containing ICDS detected by a similarity-based approach in 80 complete prokaryotic genomes. ICDS can be retrieved by species browsing or similarity searches via a web interface (). The definition of each interrupted gene is provided as well as the ICDS genomic localization with the surrounding sequence. Furthermore, to facilitate the experimental characterization of ICDS, we propose optimized primers for re-sequencing purposes. The database will be regularly updated with additional data from ongoing sequenced genomes. Our strategy has been validated by three independent tests: (i) ICDS prediction on a benchmark of artificially created frameshifts, (ii) comparison of predicted ICDS and results obtained from the comparison of the two genomic sequences of Bacillus licheniformis strain ATCC 14580 and (iii) re-sequencing of 25 predicted ICDS of the recently sequenced genome of Mycobacterium smegmatis. This allows us to estimate the specificity and sensitivity (95 and 82%, respectively) of our program and the efficiency of primer determination

Genetic analysis of egg production traits in turkeys (Meleagris gallopavo) using a single-step genomic random regression model.

BACKGROUND Egg production traits are economically important in poultry breeding programs. Previous studies have shown that incorporating genomic data can increase the accuracy of genetic prediction of egg production. Our objective was to estimate the genetic and phenotypic parameters of such traits and compare the prediction accuracy of pedigree-based random regression best linear unbiased prediction (RR-PBLUP) and genomic single-step random regression BLUP (RR-ssGBLUP). Egg production was recorded on 7422 birds during 24 consecutive weeks from first egg laid. Hatch-week of birth by week of lay and week of lay by age at first egg were fitted as fixed effects and body weight as a covariate, while additive genetic and permanent environment effects were fitted as random effects, along with heterogeneous residual variances over 24 weeks of egg production. Predictions accuracies were compared based on two statistics: (1) the correlation between estimated breeding values and phenotypes divided by the square root of the trait heritability, and (2) the ratio of the variance of BLUP predictions of individual Mendelian sampling effects divided by one half of the estimate of the additive genetic variance. RESULTS Heritability estimates along the production trajectory obtained with RR-PBLUP ranged from 0.09 to 0.22, with higher estimates for intermediate weeks. Estimates of phenotypic correlations between weekly egg production were lower than the corresponding genetic correlation estimates. Our results indicate that genetic correlations decreased over the laying period, with the highest estimate being between traits in later weeks and the lowest between early weeks and later ages. Prediction accuracies based on the correlation-based statistic ranged from 0.11 to 0.44 for RR-PBLUP and from 0.22 to 0.57 for RR-ssGBLUP using the correlation-based statistic. The ratios of the variances of BLUP predictions of Mendelian sampling effects and one half of the additive genetic variance ranged from 0.17 to 0.26 for RR-PBLUP and from 0.17 to 0.34 for RR-ssGBLUP. Although the improvement in accuracies from RR-ssGBLUP over those from RR-PBLUP was not uniform over time for either statistic, accuracies obtained with RR-ssGBLUP were generally equal to or higher than those with RR-PBLUP. CONCLUSIONS Our findings show the potential advantage of incorporating genomic data in genetic evaluation of egg production traits using random regression models, which can contribute to the genetic improvement of egg production in turkey populations

Análisis del proteoma del exudado estigmático

Comunicaciones a congreso

A Novel Toxoplasma gondii Nuclear Factor TgNF3 Is a Dynamic Chromatin-Associated Component, Modulator of Nucleolar Architecture and Parasite Virulence

International audienceIn Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome activities during the intracellular proliferation of the virulent tachyzoites of T. gondii

Use of an innovative system and nanotechnology-based strategy for therapeutic applications of Gla-rich protein (GRP)

Introduction: Gla-rich protein (GRP) is a vitamin K-dependent protein (VKDP) acting as a calcification inhibitor and anti-inflammatory agent in cardiovascular and articular systems, and THP1 monocyte/macrophage cells [1,2]. Calcification and inflammation processes are known to be involved in the etiology of several calcification-related chronic inflammatory diseases such as atherosclerosis, CKD and osteoarthritis, in a complex bi-directional interplay that drives disease progression. Here, we developed an innovative system to produce human c-carboxylated GRP (cGRP), and a nanotechnology strategy based on GRP loading into extracellular vesicles (EVs) as a gold standard delivery system for GRP in therapeutic applications. Materials and methods: Human GRP protein was co-expressed with c-carboxylase enzyme (GGCX), vitamin K oxidoreductase (GGCX) and furin, in the insect cell baculovirus system in the presence of vitamin K. GRP released in the cell culture media was characterized by mass spectrometry based techniques and Western blot analysis. EVs released by the insect cells overexpressing GRP were isolated by ultracentrifugation, and characterized for GRP content through TEM-immunogold staining, Western blot, ELISA, qPCR. Functional assays using isolated EVs containing GRP were performed in primary vascular smooth muscle cells (VSMCs) and THP1 monocyte/macrophage cells, for anti-mineralizing and anti-inflammatory screening.Results: GRP released in the cell culture media when co-expressed with GGCX, VKOR and furin in the presence of vitamin K, is processed at the pro-peptide and contain Gla residues. EVs released by the insect cells in this system were shown to be loaded with GRP protein and mRNA, and capable of reducing ECM calcium deposition of calcifying VSMCs and the production of TNFa in THP1 monocyte/macrophage cells stimulated with LPS. Discussion and conclusions: While the successful production of human cGRP constitutes a major achievement, this innovative methodology will open new opportunities for the production of other biological active VKDPs. Furthermore, EVs loaded with GRP were shown to have anti-mineralizing and anti-inflammatory properties, with promising therapeutic potentialities for calcification-related chronic inflammatory diseases.Portuguese Foundation for Science and Technology (EU/PID1003201)info:eu-repo/semantics/publishedVersio

Les signes en société

Stéphane Breton, maître de conférences 1. Normativité et « forme de vie » On s’est interrogé sur le rapport entre la normativité intrinsèque des formes sociales et la normativité positive et juridique qui semble, dans les sociétés modernes, avoir complètement évincé le social. On s’est demandé si cette apparence n’est pas plutôt l’effet d’une configuration sociologique bien particulière, celle de l’ »individualisme », dont une des caractéristiques est précisément la méconnaissance de ses cond..

Les signes en société

Stéphane Breton, maître de conférences 1. Normativité et « forme de vie » On s’est interrogé sur le rapport entre la normativité intrinsèque des formes sociales et la normativité positive et juridique qui semble, dans les sociétés modernes, avoir complètement évincé le social. On s’est demandé si cette apparence n’est pas plutôt l’effet d’une configuration sociologique bien particulière, celle de l’ »individualisme », dont une des caractéristiques est précisément la méconnaissance de ses cond..