References of "De Pauw, Edwin"
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See detailMALDI mass spectrometry imaging of secreted lipopeptides in a bacterial biofilm colonizing plant roots
Debois, Delphine ULg; Jourdan, Emmanuel ULg; Ongena, Marc ULg et al

Conference (2011, June 06)

During the aggression of a plant by a pathogen, different immune reactions may occur. "Induced Systemic Resistance” (ISR) is triggered by the specific interaction between plant and non-pathogenic ... [more ▼]

During the aggression of a plant by a pathogen, different immune reactions may occur. "Induced Systemic Resistance” (ISR) is triggered by the specific interaction between plant and non-pathogenic microorganism. The first step (of three) consists in the perception by plant cells of elicitors produced by the inducing agents that initiates the phenomenon. One class of known elicitors is antibiotics including surfactin- and fengycin-type lipopeptides. Recent studies in biology, genetics or biochemistry allowed a better understanding of the interactions between plants and microorganism but few has been done at the molecular level. MALDI MS imaging has been used to study the nature of the secreted lipopeptides, their relative quantity and their distribution in the root’s environment. Disinfected tomato seeds were first incubated at 28°C in sterile conditions for germination. Germinated seeds were then treated with freshly-grown cells of Bacillus amyloliquefaciens S499 and placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution (Hoagland) containing 1,75% of agar. Petri dishes were finally incubated vertically in phytotron during 10 days (28°C, photoperiod 16h). For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution (9-aminoacridine) was applied with an ImagePrep automated sprayer (Bruker Daltonics). An UltraFlex II TOF/TOF mass spectrometer was used to record molecular cartographies. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. Only the surfactins have been detected when working with the S499 strain. The most abundant surfactins were those with longer fatty acyl chain lengths, such as C14- and C15-homologues. Such a surfactin signature is interesting since homologues with the longest acyl chains are also the more active biologically. The distribution of surfactins showed a gradient representing the diffusion of the molecules during the root growth. The more the fatty acyl chain is long, the more the surfactin is detected near the root. Other compounds detected during the analysis showed a clear anti-colocalization with the surfactins. Future work will be focused on the influence of the plant species (tobacco, salad, Arabidopsis thaliana) on the secretion of lipopeptides (type, concentration…) and the influence of the strain of Bacillus amyloliquefaciens regarding its ability to selectively produce specific lipopeptide families (overproducing or repressed mutants). This MS imaging technique thus appears to be a very powerful method to study in situ production of bioactive lipopeptides by bacteria developing on roots. This is crucial for a better understanding of the molecular dialogue governing perception of beneficial Bacillus strains by the host plant. This study provides a first analysis over a long root section of lipopeptides secreted by a bacterial biofilm colonizing plant. [less ▲]

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See detailInvestigation of Ion Mobility coupled with mass spectrometry (IMMS) for the screening of pesticide residues in food
Goscinny, Séverine ULg; Touilloux, Romain; Joly, Laure et al

Conference (2011, June)

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See detailStudy on the susceptibility of the bovine milk fat globule membrane proteins to enzymatic hydrolysis and organization of some of the proteins
Vanderghem, Caroline ULg; Francis, Frédéric ULg; Danthine, Sabine ULg et al

in International Dairy Journal (2011), 21(5), 312-318

Isolated milk fat globules were subjected to enzyme hydrolysis by a specific protease (trypsin) and a nonspecific protease (pronase E) to study the asymmetric arrangement of milk fat globule membrane ... [more ▼]

Isolated milk fat globules were subjected to enzyme hydrolysis by a specific protease (trypsin) and a nonspecific protease (pronase E) to study the asymmetric arrangement of milk fat globule membrane (MFGM) proteins. The remaining proteins on the globules after proteolysis were resolved by two-dimensional gel electrophoresis and identified by mass spectrometry. By this proteomic approach, the results confirmed different susceptibility of the MFGM proteins to proteolysis by enzymes. Butyrophilin and adipophilin were completely digested by trypsin and by pronase E, whereas lactadherin and xanthine dehydrogenase/oxidase were almost resistant to hydrolysis by trypsin and partially attacked by pronase E. Based on our results and recent bibliographic data, an up-dated model of the organization of some MFGM proteins is proposed and discussed. (c) 2011 Elsevier Ltd. All rights reserved. [less ▲]

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See detailDisulfide bonds assignment and folding characterization of peptide toxins by Ion Mobility Mass Spectrometry
Echterbille, Julien ULg; Quinton, Loïc ULg; De Pauw, Edwin ULg et al

Conference (2011, April 29)

Main component of animal venoms is peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges fill different roles as increasing the toxins efficiency by lowering their ... [more ▼]

Main component of animal venoms is peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges fill different roles as increasing the toxins efficiency by lowering their immunogenicity or providing the adequate conformation to efficiently bind to the biological receptor. The sequencing and the determination of the cysteine pairing is still challenging and therefore an important step in structural analysis. In this work, we present a new strategy to sequence structured toxins and assign S-S bridges using ion mobility resolved MS/MS. The method relies on the analysis of partially reduced multiple-disulfide peptide. The mixture of the different forms is resolved by ion mobility, followed by MS/MS acquisition on each mobility separated species. The proof of concept has been successfully conducted on α-CnI, a toxin purified from the venom of Conus consors marine snail. The toxin’s sequence contains four cysteines linked together with two disulfide bridges. α-CnI was partially reduced by a small excess of tris(carboxyethyl)phosphine (10:1). The resulting mixture was purified before analysis by infusion nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Partial reduction of α-CnI results in a mixture of oxidized (the two disulfides are formed), reduced (the two disulfides have been reduced) and partially reduced forms (one of the two disulfides has been reduced). The arrival time distribution of triply charged ions reveals the presence of 4 different species, characterized by different relative cross sections in the gas-phase. Mass matching allows identifying the species: the first mobility (the most compact structure) was identified to be the oxidized folded toxin (M). The latest peak, corresponding to the larger cross-section, was identified as the fully reduced toxin (M+4Da). The second and the third mobility peaks were attributed to the two partially reduced forms in which only one disulfide bridge was reduced (M+2Da). The change in ion mobility depends on which S-S bridge is reduced. Ion mobility separated species give characteristic fragment ions upon fragmentation in the transfer cell (i.e. after ion mobility separator). Interestingly, fragment ions coming from partially reduced species, especially the C-S or S-S bond cleavages, clearly indicates that the disulfide linkage of α-CnI is (Cys1-Cys3) and (Cys2-Cys4) as expected from literature. The method is now being applied with success to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications. [less ▲]

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See detailMass spectrometry applied to biomolecules analysis
Far, Johann ULg; Mazzucchelli, Gabriel ULg; Meuwis, Marie-Alice ULg et al

Conference (2011, March 31)

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See detailBIOMARKER FOR OSTEOARTHRITIS AND/OR OTHER AGEING-RELATED DISEASES, AND USE THEREOF
Henrotin, Yves ULg; Gharbi, Myriam; Deberg, Michelle et al

Patent (2011)

The invention relates to the identification of abiomarker whose abundance in biological sampleis changed in subjects with osteoarthritis and/or other ageing-related diseases. Thebiomarker hasapplications ... [more ▼]

The invention relates to the identification of abiomarker whose abundance in biological sampleis changed in subjects with osteoarthritis and/or other ageing-related diseases. Thebiomarker hasapplications in the diagnosis of osteoarthritis and/or other ageing-related diseases, in determining the prognosis for an individual diagnosed with osteoarthritis and/or other ageing-related diseases, and in monitoring the efficacy of treatment for osteoarthritis and/or other ageing-related diseases. [less ▲]

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See detailBIOMARKER FOR OSTEOARTHRITIS AND/OR OTHER AGEING-RELATED DISEASES, AND USE THEREOF
Henrotin, Yves ULg; Gharbi, Myriam; Deberg, Michelle et al

Patent (2011)

The invention relates to the identification of abiomarker whose abundance in biological sampleis changed in subjects with osteoarthritis and/or other ageing-related diseases. Thebiomarker hasapplications ... [more ▼]

The invention relates to the identification of abiomarker whose abundance in biological sampleis changed in subjects with osteoarthritis and/or other ageing-related diseases. Thebiomarker hasapplications in the diagnosis of osteoarthritis and/or other ageing-related diseases, in determining the prognosis for an individual diagnosed with osteoarthritis and/or other ageing-related diseases, and in monitoring the efficacy of treatment for osteoarthritis and/or other ageing-related diseases. [less ▲]

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See detailIncreased risk of non-Hodgkin lymphoma and serum organochlorine concentrations among neighbors of a municipal solid waste incinerator
Viel, Jean-François; Floret, Nathalie; Deconinck, Eric et al

in Environment International (2011), 37(2), 449-453

Organochlorine chemicals may contribute to an increased risk of non-Hodgkin lymphoma (NHL) within nonoccupationally exposed populations. Among these chemicals, dioxins and furans were mainly released by ... [more ▼]

Organochlorine chemicals may contribute to an increased risk of non-Hodgkin lymphoma (NHL) within nonoccupationally exposed populations. Among these chemicals, dioxins and furans were mainly released by municipal solid waste incinerators (MSWIs) until a recent past in France, a source of exposure that is of public concern. We investigated organochlorines and the risk of NHL among neighbors of a French MSWI with high levels of dioxin emissions (Besançon, France), using serum concentrations to assess exposure. The study area consisted of three electoral wards, containing or surrounding the MSWI. Pesticides, dioxins, furans, and polychlorinated biphenyls (PCBs) were measured in the serum of 34 newly diagnosed NHL cases (2003– 2005) and 34 controls. Risks of NHL associated with each lipid-corrected serum concentration were estimated using exact logistic regression. The pesticides β-hexachlorocyclohexane (odds ratio [OR]=1.05, 95% confidence interval [CI]=1.00–1.12, per 10 ng/g lipid) and p,p' dichloro-diphenyl-trichloroethane (DDT) (OR=1.20, 95% CI=1.01-1.45, per 10 ng/g lipid) were associated with NHL risk. Evidence indicated an increased NHL risk associated with cumulative WHO1998-toxic equivalency factor (TEQ) concentrations (dioxins, OR=1.12, 95% CI=1.03–1.26; furans, OR=1.16, 95% CI=1.03–1.35; dioxin-like PCBs, OR=1.04, 95% CI=1.00–1.07; and total TEQ, OR=1.04, 95% CI=1.01–1.05), as well as with non dioxin-like PCBs (OR=1.02, 95% CI=1.01–1.05, per 10 ng/g lipid). Most congener-specific associations were statistically significant. This study provides strong and consistent support for an association between serum cumulative WHO1998-TEQ concentrations, at levels experienced by people residing in the vicinity of a polluting MSWI, and risk of NHL. [less ▲]

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See detailInnovative Proteomics for the Discovery of Systemically Accessible Cancer Biomarkers Suitable for Imaging and Targeted Therapies
Turtoi, Andrei ULg; De Pauw, Edwin ULg; Castronovo, Vincenzo ULg

in American Journal of Pathology (2011), 178(1), 12-18

The discovery of biomarkers that are readily accessible through the circulating blood and are selectively overexpressed in pathological tissues has become a major research objective, particularly in the ... [more ▼]

The discovery of biomarkers that are readily accessible through the circulating blood and are selectively overexpressed in pathological tissues has become a major research objective, particularly in the field of oncology. Indisputably, this group of molecules has a high potential to serve as an innovative tool for effective imaging and targeted cancer therapy approaches. In this attractive therapeutic concept, specific cancer proteins are reached by intravenously administered ligands that are coupled to cytotoxic drugs. Such compounds are able to induce cancer destruction while sparing normal tissues. Owing to the performance of mass spectrometry technology, current high-throughput proteomic analysis allows for the identification of a high number of proteins that are differentially expressed in the cancerous tissues. However, such approaches provide no information regarding the effective accessibility of the biomarkers and, therefore, the possibility for these discovered proteins to be targeted. To bypass this major limitation, which clearly slows the discovery of such biomarkers, innovative methodological strategies have been developed to enrich the clinical specimens before the mass spectrometry analysis. The focus is laid on the group of proteins that are necessarily located either at the exterior face of the plasma membrane or in the extracellular matrix. The present review addresses the current technologies meant for the discovery and analysis of accessible antigens from clinically relevant samples. [less ▲]

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See detailFuran Formation from Lipids in Starch-Based Model Systems, As Influenced by Interactions with Antioxidants and Proteins.
Owczarek-Fendor, A.; De Meulenaer, B.; Scholl, Georges ULg et al

in Journal of Agricultural and Food Chemistry (2011), 59(6), 2368-2376

The formation of furan upon sterilization of a lipid-containing starch gel was investigated in the presence of various antioxidants, namely, alpha-tocopherol, beta-carotene, and ascorbic acid, with and ... [more ▼]

The formation of furan upon sterilization of a lipid-containing starch gel was investigated in the presence of various antioxidants, namely, alpha-tocopherol, beta-carotene, and ascorbic acid, with and without proteins. Results indicated that alpha-tocopherol did not significantly influence furan formation from oxidized lipids. beta-Carotene, suggested previously to be a furan precursor itself, did influence the generation of furan in a concentration-dependent manner, although to a limited extent. Surprisingly, the presence of lipids seemed to limit the furan generation from beta-carotene. Interestingly, the addition of ascorbic acid to the emulsions containing soybean or sunflower oils considerably enhanced the formation of furan from these oils. This was also the case when fresh oils were applied, shown previously to be nearly unable to generate furan. This observation can be explained by an intensified ascorbic acid degradation stimulated by the presence of lipids. [less ▲]

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See detailNew Methodology to detect toxin-GPCR binding by MALDI-TOF Mass Spectrometry
Echterbille, Julien ULg; De Pauw, Edwin ULg; Gilles, Nicolas et al

Poster (2011)

Introduction More than 50 thousands of venomous species are currently indexed in the world. Each of their venoms is composed of 200 to 1000 different toxins which potentially exhibit a high selectivity ... [more ▼]

Introduction More than 50 thousands of venomous species are currently indexed in the world. Each of their venoms is composed of 200 to 1000 different toxins which potentially exhibit a high selectivity for membrane receptors such as ionic channels or G-protein coupled receptors (GPCRs). GPCRs constitute the larger family of receptors since around 800 different kinds of them are knows. GPCRs are the target of around 30% of the current pharmacopeia drugs. Notable examples include Novartis’s Zelnorm, Eli Lilly’s Zyprexa and Schering-Plough’s Clarinex used to treat constipation, schizophrenia and allergies, respectively. Finding new GPCRs ligands appears of prime interest to design new pharmacological tools and potentially discover the drugs of our future. Interestingly, several toxins from venoms have already been described to bind to this particular family of receptor, opening the way to the discovery of new peptide drugs from animal venoms1-2. This work presents a pioneering MALDI-TOF/TOF based strategy to fish new GPCRs ligands from complex mixtures such as venom fractions. Methods The proof of concept of this methodology was built by studying the binding of [Arg8]-vasopressin (AVP) on type 2-vasopressin receptor (V2). Experimentally, fragments of cellular membranes over-expressing V2 receptors were incubated with cone snail’s venom fraction (~30 peptide toxins) doped by a small amount of AVP. After 2 hours incubation, free and bound fractions were carefully purified with a combination of centrifugation and micro column purifications. Samples were finally analyzed with a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer and the resulting spectra were interpreted with FlexAnalysis (v3.0), BioTools (v3.2) and SequenceEditor (v3.2) bioinformatics’ softwares from Bruker Daltonics. Preliminary data After the incubation of cellular membranes overexpressing V2 GPCR with a complex mixture of peptides doped by AVP, we clearly detect that the only V2 ligand present in the fraction was the AVP. Our result demonstrates the possibility to identify a ligand of GPCRs from a complex peptide mixture, such as venom fractions. Contrary to radiobinding, this approach allows detecting the direct binding of the toxin and does not imply to know a ligand of the studied GPCR before starting the experiments. This opens the way to the deorphanization of receptors (180 orphans GPCRs over 800). Moreover, since the new ligand is detected by mass spectrometry, it is directly identified from the mixture, without additional purification. Its structural characterization can be directly performed by de novo sequencing experiments. The drawback of our approach is the very long (but crucial!) sample preparation as each sample requires 2 purification steps (for both free and bound fraction). The next step of our work will be the automation of the procedure to allow a high-throughput screening of venom fractions on different GPCRs and the discovery of new ligands. Novel aspect GPCR’s ligands discovery by MALDI-TOF/TOF based techniques: a new pharmacological tool. 1 Quinton, L. et al. Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the a1A-adrenoceptor. British Journal of Pharmacology 159, 316-325 (2010). 2 Rouget, C. et al. Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the α2-adrenoceptors. British Journal of Pharmacology 161, 1361-1374, doi:10.1111/j.1476-5381.2010.00966.x (2010). [less ▲]

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See detailDisulfide bond assignment and folding characterization of peptide toxins by Ion Mobility Mass Spectrometry
Echterbille, Julien ULg; Quinton, Loïc ULg; Rosu, Frédéric ULg et al

Poster (2011)

Introduction Animal venoms are mainly composed of peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges are a key feature as (i) they increase the toxins efficiency ... [more ▼]

Introduction Animal venoms are mainly composed of peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges are a key feature as (i) they increase the toxins efficiency by lowering their immunogenicity; (ii) they provide the adequate conformation for high affinity binding to the biological receptor. The sequencing and the determination of the cysteine pairing is still challenging and therefore an important step in their structure analysis and the understanding of their interactions with receptors. In this work, we present a new strategy to sequence structured toxins and assign S-S bridges using ion mobility resolved MS/MS. Methods The method relies on the analysis of partially reduced multiple-disulfide peptide. The mixture of the different forms is resolved by ion mobility, followed by MS/MS acquisition on each mobility separated species. The proof of concept has been successfully conducted on α-CnI, a toxin purified from the venom of Conus consors marine snail. The toxin sequence is GRCCHPACGKYYSC-NH2. It contains four cysteines linked together with two disulfide bridges. α-CnI was partially reduced by a small excess of tris(carboxyethyl)phosphine (10:1) at 56°C during 30min. The resulting mixture was purified by ZipTip C18 micro columns before analysis by infusion nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Mobilograms and mass spectra were analyzed using MassLynx (v4.1) and Driftscope (v2.1) from Waters. Preliminary data Partial reduction of a-CnI was performed in order to obtain a mixture of oxidized (the two disulfides are formed), reduced (the two disulfides have been reduced) and partially reduced forms (only one of the two disulfides has been reduced). The arrival time distribution of triply charged ions reveals the presence of 4 different species, characterized by a different relative cross sections in the gas-phase. The charge state of the ions influences the ion mobility separation. Mass matching allows identifying the species: the first mobility (the most compact structure) was identified to be the oxidized folded toxin (M=1541.58 Da). The latest peak, corresponding to the larger cross-section, was identified as the fully reduced toxin (M=1545.6 Da). The second and the third mobility peaks were attributed to the two partially reduced forms in which only one disulfide bridge was reduced (M=1543.59 Da). The change in ion mobility depends on which S-S bridge is reduced. Ion mobility separated species give characteristic fragment ions upon fragmentation in the transfer cell (i.e. after ion mobility separator). Interestingly, fragment ions coming from partially reduced species, especially the C-S or S-S bond cleavages, clearly indicates that the disulfide linkage of α-CnI is (Cys1-Cys3) and (Cys2-Cys4) as expected from literature. The method is now being applied with success to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications. Novel aspect Sequencing and disulfide bridges assignment of peptide toxins using ion mobility resolved MS/MS [less ▲]

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