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

Des ressources pour comprendre, éduquer et sécuriser : histoire, cultures et réalités autochtones

Webographie

Translational strategies in drug development for knee osteoarthritis.

Osteoarthritis (OA) is a common disease worldwide with large unmet medical needs. To bring innovative treatments to OA patients, we at Merck have implemented a comprehensive strategy for drug candidate evaluation. We have a clear framework for decision-making in our preclinical pipeline, to design our clinical proof-of-concept trials for OA patients. We have qualified our strategy to define and refine dose and dosing regimen, for treatments administered either systemically or intra-articularly (IA). We do this through preclinical in vitro and in vivo studies, and by back-translating results from clinical studies in OA patients

GLI2-Mediated Melanoma Invasion and Metastasis

Background The transforming growth factor-β (TGF-β) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-β signaling. Methods We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. Results Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm2, difference = 1.88 mm2, 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-β-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-β vs shCtrl + TGF-β, 31.0 vs 161.9, difference = −130.9, 95% CI = −96.2 to −165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors. Conclusion GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical stud

AdaptMap: exploring goat diversity and adaptation

Not presen

Visual hallucinations in the psychosis spectrum and comparative information from neurodegenerative disorders and eye disease

Much of the research on visual hallucinations (VHs) has been conducted in the context of eye disease and neurodegenerative conditions, but little is known about these phenomena in psychiatric and nonclinical populations. The purpose of this article is to bring together current knowledge regarding VHs in the psychosis phenotype and contrast this data with the literature drawn from neurodegenerative disorders and eye disease. The evidence challenges the traditional views that VHs are atypical or uncommon in psychosis. The weighted mean for VHs is 27% in schizophrenia, 15% in affective psychosis, and 7.3% in the general community. VHs are linked to a more severe psychopathological profile and less favorable outcome in psychosis and neurodegenerative conditions. VHs typically co-occur with auditory hallucinations, suggesting a common etiological cause. VHs in psychosis are also remarkably complex, negative in content, and are interpreted to have personal relevance. The cognitive mechanisms of VHs in psychosis have rarely been investigated, but existing studies point to source-monitoring deficits and distortions in top-down mechanisms, although evidence for visual processing deficits, which feature strongly in the organic literature, is lacking. Brain imaging studies point to the activation of visual cortex during hallucinations on a background of structural and connectivity changes within wider brain networks. The relationship between VHs in psychosis, eye disease, and neurodegeneration remains unclear, although the pattern of similarities and differences described in this review suggests that comparative studies may have potentially important clinical and theoretical implications. © 2014 The Author

TRRAP a platform protein,Fonction of an histone acetylation co-factor in DNA double-strand break repair

L'initiation de la transcription est une étape clé de la régulation de l'expression des gènes codant pour les protéines. Le complexe TFIID, formé de TBP et des TAFs (TBP associated factors), est au cœur de ce processus car il reconnaît le promoteur du gènTranscription initiation is a key event in the regulated expression of protein-coding genes. The general transcription factor TFIID, containing TBP and TAFs (TBP associated factors), plays a central role in transcription, because it recognizes the promot

TRRAP a platform protein,Fonction of an histone acetylation co-factor in DNA double-strand break repair

L'initiation de la transcription est une étape clé de la régulation de l'expression des gènes codant pour les protéines. Le complexe TFIID, formé de TBP et des TAFs (TBP associated factors), est au cœur de ce processus car il reconnaît le promoteur du gène, et déclenche la formation du complexe de pré-initiation (PIC).Le complexe TFTC (TBP fre TAF containing complex) peut lui aussi initier la transcription, malgré l'absence de TBP. Conservé de la levure à l'homme, il possède en sus une activité d'acétylation des queues d'histones, et participe à l'ouverture de la chromatine. TRRAP, sa plus grande sous unité, est le sujet d'étude de cette thèse.Le gène trrap est essentiel au développement embryonnaire, et influence le cycle cellulaire. Au niveau moléculaire, la protéine est la cible de nombreux activateurs de la transcription. Cependant, sa fonction propre était mal connue au début de ce travail, d'autant plus que TRRAP appartient par sa structure à une famille de kinase (PI3K), mais n'en possède pas l'activité enzymatique.Un protocole d'immuno-purification et d'analyse par spectrométrie de masse, complété par des contrôles biochimiques, nous a permis de mettre en évidence une interaction stable entre TRRAP et le complexe Mre11-Rad50-Nbs1 (MRN), indépendamment de TFTC. MRN est un acteur central de la réponse cellulaire aux cassures double brin de l'ADN. Notre étude de la fonctionnalité de cette interaction a montré que MRN associé à TRRAP est dépourvu d'activité d'acétylation des histones. En revanche, elle apporte les preuves in vitro et in vivo que TRRAP a un rôle spécifique dans la réparation et la signalisation des cassures double brin de l'ADN, comme ATM et ATR qui sont d'autres protéines PI3K.La participation de TRRAP à de nombreuses structures multiprotéiques, suggère que cette grande protéine se comporte en plateforme de communication et d'échange entre les machineries cellulaires de transcription, de réparation et de modification de la chromatine.Ce manuscrit présente par ailleurs des résultats indépendants, portant sur la caractérisation par spectrométrie de masse d'une modification post-traductionnelle de l'histone H3 spécifique de la mitose.Transcription initiation is a key event in the regulated expression of protein-coding genes. The general transcription factor TFIID, containing TBP and TAFs (TBP associated factors), plays a central role in transcription, because it recognizes the promoter, and triggers pre-initiation complex formation.TFTC (TBP fre TAF containing complex) is another complex able to initiate transcription. TFTC possesses Histone Acetyltransferase (HAT) activity and thus participates in chromatin opening. These studies focus on TRRAP, TFTC's largest subunit.Trrap gene is essential to embryonic development, and indirectly influences the cell cycle. Moreover, TRRAP protein is targeted by DNA binding activators of transcription. Despite the fact that TRRAP structurally belongs to the family of PI3K kinase, which regulates cellular response to genotoxic stress, its in vivo function is not well understood.Immunoprecipitation and mass spectrometry analysis, associated to biochemical controls, reveal a stable interaction between TRRAP and Mre11-Rad50-Nbs1 complex (MRN) independent of TFTC. MRN is a critical component of DNA double strand break (DSB) cellular response. Functional studies of the TRRAP-MRN complex have shown that it does not possess HAT activity. Nevertheless, in vitro and in vivo evidences demonstrate that TRRAP, like other members of the PIKK family, plays a specific role in DNA DSB repair and signalling.Taken together, our studies give an insight into TRRAP function as a transcription co-factor. We propose to discuss in which TRRAP acts as a molecular platform, allowing communication between the cellular processes of DNA transcription, DNA repair, and chromatin remodeling.Independently, this manuscript summarizes the results of another study, addressing the mass spectrometry characterisation of a mitosis-specific post-translational modification of histone H3

Pour favoriser l'inclusion des jeunes autochtones dans les collèges: une ethnographie de l'expérience collégiale des étudiants autochtones

Comprend des références bibliographiques.De nombreux collèges affichent aujourd'hui un intérêt plus marqué pour les questions autochtones dans les contenus de cours et pour la réussite des étudiants des Premières Nations, Inuit et Métis. Cela fait suite aux recommandations de la Commission de vérité et réconciliation du Canada (CVR), créée en 2008 dans un processus de réparation et de réconciliation entre les anciens pensionnaires autochtones, leur famille, leur communauté et l'ensemble des Canadiens. Rappelons que «pendant plus d'un siècle, les objectifs centraux de la politique indienne du Canada étaient les suivants : éliminer les gouvernements autochtones, ignorer les droits des Autochtones, mettre fin aux traités conclus et, au moyen d'un processus d'assimilation, faire en sorte que les peuples autochtones cessent d'exister en tant qu'entités légales, sociales, culturelles, religieuses et raciales au Canada. L'établissement et le fonctionnement des pensionnats ont été un élément central de cette politique, que l'on pourrait qualifier de génocide culturel » (CVR, 2015, p. 1). À l'issue d'un processus de consultation lors duquel des centaines de témoignages ont été recueillis, la CVR, dans un document intitulé Honorer la vérité, réconcilier pour l'avenir, énonce 94 recommandations adressées aux Canadiens, aux gouvernements fédéral, provinciaux et territoriaux et aussi aux établissements d'enseignement postsecondaire. Celles-ci concernent notamment l'éducation des jeunes Autochtones et la valorisation des cultures, des langues et de l'histoire des peuples autochtones au Canada