François Fenaille

A highly sensitive competitive enzyme immunoassay of broad specificity quantifying microcystins and nodularins in water samples

Microcystins (MCs) form a group of cyclic heptapeptides produced by common cyanobacteria (blue green algae) and cause both acute and chronic toxicity. For immunization purposes, an amino derivative of MC-LR was prepared before coupling to BSA. Among the different monoclonal antibodies produced, mAb MC159 was selected due to its broad specificity to develop a sensitive enzyme immunoassay (EIA). This method measures MC-LR, MC-YR, MC-LA, nodularins in a similar way and exhibits an important recognition (cross reactivity up to 69%) for Adda analogues. Using MC-LR as standard, the present EIA proved to be very sensitive with a limit of detection close to 10 fmol/ml, largely below the provisional guideline level for drinking water proposed by the WHO (1 pmol/ml for MC-LR). This assay showed a high accuracy (CV% < 12) and a high recovery rate for MC-LR in spiked surface water (up to 96.5%). Moreover due to its broad spectrum of recognition, this method allows a real quantification of the sum of MCs in water bloom and cyanobacteria culture samples. Indeed, in parallel analysis of these samples using HPLC, EIA shows a good relationship between both measurements while LC-MS/MS demonstrates the presence of different variants of MCs whose heterogeneity did not impair EIA measurement.

Deacetylation of H4-K16Ac and heterochromatin assembly in senescence

BackgroundCellular senescence is a stress response of mammalian cells leading to a durable arrest of cell proliferation that has been implicated in tumor suppression, wound healing, and aging. The proliferative arrest is mediated by transcriptional repression of genes essential for cell division by the retinoblastoma protein family. This repression is accompanied by varying degrees of heterochromatin assembly, but little is known regarding the molecular mechanisms involved.ResultsWe found that both deacetylation of H4-K16Ac and expression of HMGA1/2 can contribute to DNA compaction during senescence. SIRT2, an NAD-dependent class III histone deacetylase, contributes to H4-K16Ac deacetylation and DNA compaction in human fibroblast cell lines that assemble striking senescence-associated heterochromatin foci (SAHFs). Decreased H4-K16Ac was observed in both replicative and oncogene-induced senescence of these cells. In contrast, this mechanism was inoperative in a fibroblast cell line that did not assemble extensive heterochromatin during senescence. Treatment of senescent cells with trichostatin A, a class I/II histone deacetylase inhibitor, also induced rapid and reversible decondensation of SAHFs. Inhibition of DNA compaction did not significantly affect the stability of the senescent state.ConclusionsVariable DNA compaction observed during senescence is explained in part by cell-type specific regulation of H4 deacetylation and HMGA1/2 expression. Deacetylation of H4-K16Ac during senescence may explain reported decreases in this mark during mammalian aging and in cancer cells.

Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression

The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases.

Comparative analysis of native and permethylated human milk oligosaccharides by liquid chromatography coupled to high resolution mass spectrometry

Human milk oligosaccharides (HMOs) represent the third most abundant components of milk after lactose and lipids. HMOs are indigestible by the suckling infant but can act as prebiotics and have significant biological functions regarding the organism defense against pathogens (such as bacteria or viruses) by preventing interactions with their receptors. Although constituted of only five distinct monosaccharide building blocks, HMOs are highly structurally diverse compounds with many co-existing structural isomers. Here we report the development and comparison of two distinct glycomic platforms based on liquid chromatography coupled to high resolution mass spectrometry (LC-MS) for analyzing HMOs. We have implemented and thoroughly compared the LC-MS of permethylated and native HMOs on reversed phase (RP) and porous graphitic carbon (PGC) columns for their ability to resolve the natural heterogeneity of milk oligosaccharides at the highest sensitivity. Our data essentially underlines the usefulness of analyzing HMOs as permethylated derivatives especially for getting more precise structural information at high sensitivity. For instance, permethylation annihilates gas-phase fucose migration during MS/MS experiments, thus facilitating spectra interpretation and giving access to relevant information regarding oligosaccharide branching and isomer distinction. At the opposite, LC-MS profiling of native HMOs (using PGC) in milk performed best in terms of detected species, while also being much faster in terms of sample preparation. Although less efficient than PGC chromatography, RPLC proved successful for separating pairs of permethylated isomeric HMOs. A key advantage of RP over PGC liquid chromatography is that retention times can be correlated to molecular weights, which can greatly facilitate further HMO identification using retention time prediction. Altogether these data lead us to think that LC-MS analysis of native HMOs (using PGC) can be used as first-line profiling approach while permethylation can be performed afterwards for facilitating structural characterization.

Low-Protein Diet Induces IRE1α-Dependent Anticancer Immunosurveillance

Dietary restriction (DR) was shown to impact on tumor growth with very variable effects depending on the cancer type. However, how DR limits cancer progression remains largely unknown. Here, we demonstrate that feeding mice a low-protein (Low PROT) isocaloric diet but not a low-carbohydrate (Low CHO) diet reduced tumor growth in three independent mouse cancer models. Surprisingly, this effect relies on anticancer immunosurveillance, as depleting CD8+ Txa0cells, antigen-presenting cells (APCs), or using immunodeficient mice prevented the beneficial effect of the diet. Mechanistically, we established that a Low PROT diet induces the unfolded protein response (UPR) in tumor cells through the activation of IRE1α and RIG1 signaling, thereby resulting in cytokine production and mounting an efficient anticancer immune response. Collectively, our data suggest that a Low PROT diet induces an IRE1α-dependent UPR in cancer cells, enhancing a CD8-mediated Txa0cell response against tumors.

Data acquisition workflows in liquid chromatography coupled to high resolution mass spectrometry-based metabolomics: Where do we stand?

Typical mass spectrometry (MS) based untargeted metabolomics protocols are tedious as well as time- and sample-consuming. In particular, they often rely on full-scan-only analyses using liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS) from which metabolites of interest are first highlighted, and then tentatively identified by using targeted MS/MS experiments. However, this situation is evolving with the emergence of integrated HRMS based-data acquisition protocols able to perform multi-event acquisitions. Most of these protocols, referring to as data dependent and data independent acquisition (DDA and DIA, respectively), have been initially developed for proteomic applications and have recently demonstrated their applicability to biomedical studies. In this context, the aim of this article is to take stock of the progress made in the field of DDA- and DIA-based protocols, and evaluate their ability to change conventional metabolomic and lipidomic data acquisition workflows, through a review of HRMS instrumentation, DDA and DIA workflows, and also associated informatics tools.

Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile

ABSTRACT Clostridium difficile is the major etiologic agent of antibiotic-associated intestinal disease. Pathogenesis of C. difficile is mainly attributed to the production and secretion of toxins A and B. Unlike most clostridial toxins, toxins A and B have no signal peptide, and they are therefore secreted by unusual mechanisms involving the holin-like TcdE protein and/or autolysis. In this study, we characterized the cell surface protein Cwp19, a newly identified peptidoglycan-degrading enzyme containing a novel catalytic domain. We purified a recombinant His6-tagged Cwp19 protein and showed that it has lytic transglycosylase activity. Moreover, we observed that Cwp19 is involved in cell autolysis and that a C. difficile cwp19 mutant exhibited delayed autolysis in stationary phase compared to the wild type when bacteria were grown in brain heart infusion (BHI) medium. Wild-type cell autolysis is correlated to strong alterations of cell wall thickness and integrity and to release of cytoplasmic material. Furthermore, we demonstrated that toxins were released into the extracellular medium as a result of Cwp19-induced autolysis when cells were grown in BHI medium. In contrast, Cwp19 did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium. These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributions in vitro depending on growth conditions. Thus, Cwp19 is an important surface protein involved in autolysis of vegetative cells of C. difficile that mediates the release of the toxins from the cell cytosol in response to specific environment conditions. IMPORTANCE Clostridium difficile-associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains of C. difficile generally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase in C. difficile, Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes to C. difficile cell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies. IMPORTANCE Clostridium difficile-associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains of C. difficile generally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase in C. difficile, Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes to C. difficile cell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies.

Bimodal Fluorescence/ 129 Xe NMR Probe for Molecular Imaging and Biological Inhibition of EGFR in Non-Small Cell Lung Cancer

Although Non-Small Cell Lung Cancer (NSCLC) is one of the main causes of cancer death, very little improvement has been made in the last decades regarding diagnosis and outcomes. In this study, a bimodal fluorescence/129Xe NMR probe containing a xenon host, a fluorescent moiety and a therapeutic antibody has been designed to target the Epidermal Growth Factor Receptors (EGFR) overexpressed in cancer cells. This biosensor shows high selectivity for the EGFR, and a biological activity similar to that of the antibody. It is detected with high specificity and high sensitivity (sub-nanomolar range) through hyperpolarized 129Xe NMR. This promising system should find important applications for theranostic use.

A capillary zone electrophoresis method for detection of Apolipoprotein C-III glycoforms and other related artifactually modified species ☆

ApolipoproteinC-III (ApoC-III) is a human plasma glycoprotein whose O-glycosylation can be altered as a result of congenital disorders of glycosylation (CDG). ApoC-III exhibits three major glycoforms whose relative quantification is of utmost importance for the diagnosis of CDG patients. Considering the very close structures of these glycoforms and their tendency to adsorb on the capillary, a thorough optimization of capillary electrophoresis (CE) parameters including preconditioning and in-between rinsing procedures was required to efficiently separate all the ApoC-III glycoforms. Permanent coatings did not contribute to high resolution separations. A fast and reliable method based on a bare-silica capillary combining the effect of urea and diamine additives allowed to separate up to six different ApoC-III forms. We demonstrated by a combination of MALDI-TOF mass spectrometry (MS) analyses and CE of intact and neuraminidase-treated samples that this method well resolved glycoforms differing not only by their sialylation degree but also by carbamylation state, an undesired chemical modification of primary amines. This method allowed to demonstrate the carbamylation of ApoC-III glycoforms for the first time. Our CZE method proved robust and accurate with excellent intermediate precision regarding migration times (RSDsu2009<u20090.7%) while RSDs for peak areas were less than 5%. Finally, the quality of three distinct batches of commercial ApoC-III obtained from different suppliers was assessed and compared. Quite similar but highly structurally heterogeneous ApoC-III profiles were observed for these samples.

Complementarity of electrophoretic, mass spectrometric, and gene sequencing techniques for the diagnosis and characterization of congenital disorders of glycosylation

Congenital disorders of glycosylation (CDG) are rare autosomal genetic diseases affecting the glycosylation of proteins and lipids. Since CDG‐related clinical symptoms are classically extremely variable and nonspecific, a combination of electrophoretic, mass spectrometric, and gene sequencing techniques is often mandatory for obtaining a definitive CDG diagnosis, as well as identifying causative gene mutations and deciphering the underlying biochemical mechanisms. Here, we illustrate the potential of integrating data from capillary electrophoresis of transferrin, two‐dimensional electrophoresis of N‐ and O‐glycoproteins, mass spectrometry analyses of total serum N‐linked glycans and mucin core1 O‐glycosylated apolipoprotein C‐III for the determination of various culprit CDG gene mutations. “Step‐by‐step” diagnosis pathways of four particular and new CDG cases, including MGAT2‐CDG, ATP6V0A2‐CDG, SLC35A2‐CDG, and SLC35A3‐CDG, are described as illustrative examples.