Solid phase extraction for removal of matrix effects in lipophilic marine toxin analysis by liquid chromatography-tandem mass spectrometry

The potential of solid phase extraction (SPE) clean-up has been assessed to reduce matrix effects (signal suppression or enhancement) in the liquid chromatography-tandem mass spectrometry (LC¿MS/MS) analysis of lipophilic marine toxins. A large array of ion-exchange, silica-based, and mixed-function SPE sorbents was tested. Polymeric sorbents were found to retain most of the toxins. Optimization experiments were carried out to maximize recoveries and the effectiveness of the clean-up. In LC¿MS/MS analysis, the observed matrix effects can depend on the chromatographic conditions used, therefore, two different HPLC methods were tested, using either an acidic or an alkaline mobile phase. The recovery of the optimized SPE protocol was around 90% for all toxins studied and no break-through was observed. The matrix effects were determined by comparing signal response from toxins spiked in crude and SPE-cleaned extracts with those derived from toxins prepared in methanol. In crude extracts, all toxins suffered from matrix effects, although in varying amounts. The most serious effects were observed for okadaic acid (OA) and pectenotoxin-2 (PTX2) in the positive electrospray ionization mode (ESI+). SPE clean-up on polymeric sorbents in combination with the alkaline LC method resulted in a substantial reduction of matrix effects to less than 15% (apparent recovery between 85 and 115%) for OA, yessotoxin (YTX) in ESI¿ and azaspiracid-1 (AZA1), PTX2, 13-desmethyl spirolides C (SPX1), and gymnodimine (GYM) in ESI+. In combination with the acidic LC method, the matrix effects after SPE were also reduced but nevertheless approximately 30% of the matrix effects remained for PTX2, SPX1, and GYM in ESI+. It was concluded that SPE of methanolic shellfish extracts can be very useful for reduction of matrix effects. However, the type of LC and MS methods used is also of great importance. SPE on polymeric sorbents in combination with LC under alkaline conditions was found the most effective method

The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent of hemorrhagic colitis and the hemolytic uremic syndrome (HUS), produces long bundles of type IV pili (TFP) called hemorrhagic coli pili (HCP). HCP are capable of mediating several phenomena associated with pathogenicity: i) adherence to human and bovine epithelial cells; ii) invasion of epithelial cells; iii) hemagglutination of rabbit erythrocytes; iv) biofilm formation; v) twitching motility; and vi) specific binding to laminin and fibronectin. HCP are composed of a 19 kDa pilin subunit (HcpA) encoded by the hcpA chromosomal gene (called prepilin peptidase-dependent gene [ppdD] in E. coli K-12).In this study we investigated the potential role of HCP of E. coli O157:H7 strain EDL933 in activating the release of pro- and anti-inflammatory cytokines from a variety of host epithelial cells. We found that purified HCP and a recombinant HcpA protein induced significant release of IL-8 and TNF-alpha, from cultured polarized intestinal cells (T84 and HT-29 cells) and non-intestinal HeLa cells. Levels of proinflammatory IL-8 and TNF-alpha, but not IL-2, IL6, or IL-10 cytokines, were increased in the presence of HCP and recombinant HcpA after 6 h of incubation with >or=50 ng/ml of protein, suggesting that stimulation of IL-8 and TNF-alpha are dose and time-dependent. In addition, we also demonstrated that flagella are potent inducers of cytokine production. Furthermore, MAPK activation kinetics studies showed that EHEC induces p38 phosphorylation under HCP-producing conditions, and ERK1/2 and JNK activation was detectable after 3 h of EHEC infection. HT-29 cells were stimulated with epidermal growth factor stimulation of HT-29 cells for 30 min leading to activation of three MAPKs.The HcpA pilin monomer of the HCP produced by EHEC O157:H7 is a potent inducer of IL-8 and TNF-alpha release, an event which could play a significant role in the pathogenesis of hemorrhagic colitis caused by this pathogen

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe

Amnesic shellfish poisoning toxins in bivalve molluscs in Ireland

In December 1999, domoic acid (DA) a potent neurotoxin, responsible for the syndrome Amnesic Shellfish Poisoning (ASP) was detected for the first time in shellfish harvested in Ireland. Two liquid chromatography (LC) methods were applied to quantify DA in shellfish after sample clean-up using solid-phase extraction (SPE) with strong anion exchange (SAX) cartridges. Toxin detection was achieved using photodiode array ultraviolet (LC-UV) and multiple tandem mass spectrometry (LC-MSn). DA was identified in four species of bivalve shellfish collected along the west and south coastal regions of the Republic of Ireland. The amount of DA that was present in three species was within EU guideline limits for sale of shellfish (20 μg DA/g); mussels (Mytilus edulis), <1.0 μg DA/g; oysters (Crassostrea edulis), <5.0 μg DA/g and razor clams (Ensis siliqua), <0.3 μg DA/g. However, king scallops (Pecten maximus) posed a significant human health hazard with levels up to 240 μg DA/g total tissues. Most scallop samples (55%) contained DA at levels greater than the regulatory limit. The DA levels in the digestive glands of some samples of scallops were among the highest that have ever been recorded (2820 μg DA/g). © 2005 Elsevier Ltd. All rights reserved

Stability Analysis of Discontinuous Galerkin Approximations to the Elastodynamics Problem

We consider semi-discrete discontinuous Galerkin approximations of both displacement and displacement-stress formulations of the elastodynamics problem. We prove the stability analysis in the natural energy norm and derive optimal a-priori error estimates. For the displacement-stress formulation, schemes preserving the total energy of the system are introduced and discussed. We verify our theoretical estimates on two and three dimensions test problems

« Etre aidé, aider autrui, s’aider soi-même : des ressources pour des jeunes en situation de précarité »

International audienceLe texte restitue les ressources et contraintes propres à l’expérience sociale de l’aide et ses déclinaisons en « être aidé », « aider autrui » et « s’aider soi-même » chez les jeunes des classes populaires en les envisageant comme une manière de ne pas toujours s’en remettre aux normalisations institutionnelles de l’injonction à l’autonomie. Il s’agit de mettre à l’épreuve les représentations dominantes de l’aide en s’intéressant aux ressorts et aux conséquences objectives et subjectives des relations d’interdépendance, à distance d’une vision manichéenne et moralisante des « bienfaits » ou des « méfaits » de l’aide.Cette recherche interroge également les modalités de résistance et/ou d’assignation aux discours institutionnels des jeunes quant à leur situation actuelle et leur destin social probable