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See detailNew Advances for In Situ Protein Identification by MALDI In-Source Decay FTMS Imaging
Calligaris, David ULg; Zimmerman, Tyler ULg; Debois, Delphine ULg et al

Poster (2012, April 18)

MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular analysis could be performed directly from ... [more ▼]

MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular analysis could be performed directly from tissue sections in the region of the diseased area. The use of in-source decay (ISD), allowing fast and reliable sequences assignments of proteins termini, has proven to be a crucial tool for proteins identification in solution and tissue slices. However, it is necessary to develop additional tools that allow unambiguous assignment of proteins sequences in complex tissue slices. The development of bioinformatic tools and the use of ultra-high mass resolution and high mass accuracy of Fourier transform ion-cyclotron (FTICR) mass spectrometry are ideal for this purpose. In this study, we show that FTICR mass spectrometry combined with data filtering with a software that subtracts matrix peaks aid protein identification. All measurements were carried out on a SolariX FTMS (9.4 Tesla) equipped with a Dual Source with a smartbeamTMII laser (Bruker Daltonics). Mouse brain tissue slices of 14 µm thickness were rinsed to obtain optimal sensitivity and high-quality ions. Before matrix application, a spot of myelin was deposited near mouse brain. 1,5-Diaminionaphtalene was sprayed using an ImagePrep (Bruker Daltonics). Results were interpreted using BioToolsTM 3.2 in combination with MascotTM (Matrix Science) for ISD spectra and FlexImagingTM 3.0 for MALDI-ISD imaging experiments. Matrix peaks were subtracted using an in-house written Java code that sequentially scans all peak lists from acquired spectra against the DAN mass list. Then, another Java code allows to create 2D ion images at selected m/z ratios. The studies were carried out by MALDI-ISD imaging to create interest on FTICR mass spectrometer for proteins identification in the field of biomarkers characterization. It is demonstrated that protein ISD leads to the same pattern of fragmentation observed during MALDI-TOF analyzes. Fragmentation generates cn- and zn-series ions of myelin in presence of DAN. The internal calibration of all the data provides a mass accuracy neighboring 2.5 ppm over the m/z range of interest (300-2500 Da) and a mass resolution of 70000 at m/z 400 Da. It allows the assignment of ISD fragments of proteins in the low mass range (m/z between 300 and 900) that is unambiguously validated by the “ISD signal” recorded from the spots of pure protein solution (myelin) near tissue slice. Moreover, the use of our software “cleans” MS imaging data by reducing/eliminating MALDI matrix peaks that are isobaric to an analyte peak. Novel aspect This study evidences the main input of FTICR mass spectrometer for pathologies diagnosis based on biomarkers localization and identification by MALDI-ISD imaging. [less ▲]

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See detailHemocompatibility of nanocarriers designed to transport biopharmaceutical drugs
Sevrin, Chantal ULg; Cerda, Bernardino; Lombart, François ULg et al

Poster (2012, April 18)

The optimization of nanoparticles (NP) for drug delivery, in particular to target the BBB, imposes to verify their hemocompatibility both for toxicological and efficiency of targeting perspectives. Indeed ... [more ▼]

The optimization of nanoparticles (NP) for drug delivery, in particular to target the BBB, imposes to verify their hemocompatibility both for toxicological and efficiency of targeting perspectives. Indeed the large surface they are able to expose to the biological environment promotes their interaction with various biochemicals, in particular proteins which can after adsorption elicit the activation of biological cascades either responsible from NP clearance or/and harmful body reaction (inflammatory / coagulation). In the frame of the European Integrated Project : “Nanobiopharmaceutics”, we have the opportunity to compare the hemoreactivity of about 145 different NP samples differing in core and surface chemistry and classified according to their expected difference in hydrophobicity based on the nature of their core materials. According to this classification, PLGA nanoparticles, polyglycidol-polyethyethylene oxide nanoparticles, polyglycidol thyolated or polyacrylamide nanogels, and polyelectrolyte complexes either based on polyamidoamine or poly(N,N-dimethylamino-2-ethylmethacrylate) have been evaluated within a concentration ranging from 0.3 to 1000 =g/mL. These in vitro tests have been realized for screening purpose adopting normal human bloods and according to Iso 10993. As a summary of this extensive study, our results clearly highlight that most of the polymeric nanoparticles evaluated give rise to some alterations of the blood components. In particular the platelets, intrinsic pathway of coagulation and complement activation are the most reactive biological parameters in the presence of these nanostuctures. Although not strictly related to the surface chemistry our classification has also allowed us to derive some clear correlations between nanomaterial properties and their hemoreactivity. Within the class of polyelectrolyte electrolyte complexes, the modifications brought in the surface chemistry has drastically improved their hemoreactivity. [less ▲]

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See detailPrazeres do raio-x: estudo de um striptease radiográfico
Sturnack, Lionel ULg

Conference (2012, April 18)

O striptease que propomos analisar é na verdade o título de uma obra que pertence à produção da artista italiana Benedetta Bonichi. Trata-se de uma composição mista, a meio caminho entre os raios-x e a ... [more ▼]

O striptease que propomos analisar é na verdade o título de uma obra que pertence à produção da artista italiana Benedetta Bonichi. Trata-se de uma composição mista, a meio caminho entre os raios-x e a fotografia. “Striptease” chamou a nossa atenção por várias razões. Com certeza, por seu caráter surpreendente, sendo uma cena erótica e ao mesmo tempo uma radiografia. Mas também por sua estética particular em relação aos outros artistas que desenvolvem uma técnica similar. O propósito de nossos passos é levar em consideração o lugar da obra estudada no espaço de realizações em raio-x artístico, numa perspectiva semiótica. Pretendemos desta maneira chegar à descrição fina de “Striptease, por meio do estabelecimento de um conjunto de formantes plásticos estáveis funcionando como base sobre a qual se correlacionam e se diferenciam os raios-x artísticos estudados. Por isso, desenvolveremos o estudo segundo três etapas gerais. Com a primeira, tentaremos efetuar uma descrição de “Striptease” segundo os três eixos da leitura plástica padrão, tal como foram trabalhados por Jean-Marie Floch: cromático, eidético e topológico. Na segunda etapa, adotaremos o mesmo ponto de vista para a descrição plástica de cinco obras de quatro outros artistas. O interesse da segunda parte consiste em ver como se atualizam as unidades plásticas em cada realização particular, a fim de observar as suas possíveis convergências e divergências. Na última etapa, poderemos considerar a estética de cada obra à luz das atualizações plásticas e das integrações entre elas com outros patamares temáticos. Conseguiremos, finalmente, entender a pertinência e a particularidade de “Striptease” como obra com vocação reflexiva a respeito da prática do raio-x artístico. [less ▲]

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See detailOptimization of recombinant root-secreted IgGs production in Arabidopsis thaliana by screening cell wall mutants
Boulanger, Benoit ULg; Périlleux, Claire ULg; Tocquin, Pierre ULg

Poster (2012, April 18)

The production of complex heterologous proteins (e.g. monoclonal antibodies, mAbs) in plants has several advantages animal based systems such as low cost, scalability and limited risk of contamination by ... [more ▼]

The production of complex heterologous proteins (e.g. monoclonal antibodies, mAbs) in plants has several advantages animal based systems such as low cost, scalability and limited risk of contamination by human pathogens. mAbs are glycoproteins that require to be targeted to the plant secretory pathway in order to be properly folded and matured. They are ultimately delivered in the cell wall and are expected to be freely released in the extracellular space and the external medium, which would greatly simplify downstream processing. However, a significant part of plant produced and secreted mAbs remains bound to the cell wall, therefore hindering recovery. In this study, we evaluated the extra-cellular release of root-secreted proteins of wild-type plants and cell wall mutants of Arabidopsis thaliana. Recovered protein were either analyzed by SDS-PAGE for full proteome profiling or by gelatin zymography to reveal the activity of cell wall-bound proteases. The production, secretion and release of recombinant IgG will be eventually studied in transgenic hairy-roots generated from selected mutants. [less ▲]

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See detailNew alginate-chitosan hydrogel to repair cartilage
Henrotin, Yves ULg

Conference (2012, April 18)

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See detailMolecular biomimetics applied to medical devices
Van de Weerdt, Cécile ULg; Vreuls, Christelle ULg; Genin, Alexis ULg et al

Poster (2012, April 18)

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See detailA sustainable method inspired by nature for the production of original bi-functional nutraceuticals containing prebiotic fibers and highly bio-available forms of calcium and magnesium
Goffin, Dorothée ULg; Blecker, Christophe ULg; Paquot, Michel ULg

Poster (2012, April 18)

A sustainable method inspired by nature is proposed leading to an original bifunctional product active on gut health, metabolism regulation, immunity and mineral fortification, fighting against topical ... [more ▼]

A sustainable method inspired by nature is proposed leading to an original bifunctional product active on gut health, metabolism regulation, immunity and mineral fortification, fighting against topical chronic diseases (type2 diabetes, osteoporosis, colon cancer…). [less ▲]

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See detailReading an Arthurian Prose Romance through its Textual Tradition.
Morato, Nicola ULg

Scientific conference (2012, April 18)

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See detailOptimisation of new biodegradable microcarriers tailored for tissue engineering
Demina, T; Akopova, T; Tsoy, A et al

Poster (2012, April 18)

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See detailInfluence of protein context on the propensity of polyglutamine tracts to induce protein aggregation into amyloid fibrils
Huynen, Céline ULg

Poster (2012, April 18)

Nine neurodegenerative diseases, referred to as polyglutamine diseases, are associated with nine proteins containing an expanded polyglutamine (polyQ) tract. PolyQ tracts are encoded by a repetition of ... [more ▼]

Nine neurodegenerative diseases, referred to as polyglutamine diseases, are associated with nine proteins containing an expanded polyglutamine (polyQ) tract. PolyQ tracts are encoded by a repetition of the CAG codon in the corresponding genes, and are present in proteins of healthy people. They are however pathogenic when their length, due to mutations, becomes higher than a threshold generally comprised between 35 and 45Q. Such pathogenic tracts trigger the aggregation of the proteins into amyloid-like aggregates that could play an important role in the disease. It is therefore necessary to investigate at a molecular level the aggregation process of polyQ proteins. Since proteins associated with polyQ diseases are generally big and relatively insoluble, they are difficult to produce and manipulate. We have therefore decided to study the aggregating properties of polyQ proteins by designing and characterizing model proteins made of a well-characterized host protein, the β-lactamase BlaP, and polyQ tracts of different lengths (23 to 79Q) inserted at position 197 of BlaP. The aggregating properties of these BlaP chimeras recapitulate those of proteins associated with polyQ diseases. We indeed observed that there is a minimal number of glutamines (threshold) required for chimeras aggregation into amyloid-like fibrils and that the kinetics of aggregation are faster with longer glutamine repeats. Most importantly, the value of the threshold for amyloid-like fibril formation seems to critically depend on the structural integrity of BlaP and thus on the constraints applied to the polyQ tract. In the present work, we investigate more deeply the role of the protein context and the role of oligomers in the process of aggregation of BlaP chimeras. This study is mainly based on the use of two techniques: the quartz crystal microbalance with dissipation (QCM-D) in combination with atomic force microscopy (AFM), and the dynamic light scattering (DLS). Preliminary QCM-D results indicate that in native condition there is a minimal number of glutamines required for the elongation of BlaP197(Gln)79 fibrils by BlaP chimeras. This threshold is different to that observed for the aggregation monitored in solution in absence of seed. Most interestingly it corresponds to the threshold observed in denaturing conditions. These observations suggest that the conformation of BlaP is the limiting step for amyloid fibril formation by interfering with the nucleation step. Preliminary DLS experiments indicate that the oligomers formed by BlaP197(Gln)79 in native condition are on the pathway of amyloid fibril formation and could act as aggregation nuclei. [less ▲]

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See detailNeuropeptides and endogenous opioids expression during silent chronic inflammation
Chavarria Bolanos, D; de Jesus Pozos Guillén, A; Grandfils, Christian ULg et al

Poster (2012, April 18)

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Peer Reviewed
See detailSynthesis and evaluation of boronic acids as inhibitors of Penicillin Binding Proteins of classes A, B and C
Zervosen, Astrid ULg; Sauvage, Eric ULg; Bouillez, André ULg et al

Poster (2012, April 18)

The widespread use of beta-lactam antibiotics has lead to the worldwide appearance of drug-resistant strains. Bacteria have developed resistance to beta-lactams by three main mechanisms: the production of ... [more ▼]

The widespread use of beta-lactam antibiotics has lead to the worldwide appearance of drug-resistant strains. Bacteria have developed resistance to beta-lactams by three main mechanisms: the production of beta-lactamases that catalyze hydrolysis of beta-lactams, the production of low-affinity, drug resistant Penicillin Binding Proteins (PBPs) and the over expression of resistant PBPs. PBPs are interesting targets because they catalyse the last steps of the biosynthesis of peptidoglycan, which is unique in bacteria and has no mammalian analogs, outside the cytoplasmic membrane. Various non-ß-lactam inhibitors of PBPs have been developed with the objective of attempting to stall the development of ß-lactam resistance. Boronic acids are potent beta-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2, 6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC50: 1.3 µM). Here, we will discuss the synthesis of a number of acylaminomethylboronic acids, analogs of (2, 6-dimethoxybenzamido)methylboronic acid, and their potential as inhibitors of PBPs. Several boronic acids of this library were able to inhibit PBPs of classes A, B and C from penicillin sensitive strains. Thus (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of class A PBP (PBP1b from S. pneumoniae, IC50 = 26 µM), class B PBP (PBP2xR6 from S. pneumoniae, IC50 = 138 µM) and class C PBP (R39 from Actinomadura sp., IC50 = 0.6 µM). Crystal structures of complexes of R39 and PBP1b with boronic acid analogs of our library have already been solved and allowed an interpretation of results. We believe that this work opens new avenues towards the development of molecules that will inhibit PBPs, and eventually display bactericidal effect, on distinct bacterial species. [less ▲]

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See detailPossible intracellular effect of the new water-soluble form of curcumin (NDS27) on the oxidant response of stimulated neutrophils
Derochette, Sandrine ULg; Mouithys-Mickalad, Ange ULg; Deby-Dupont, Ginette et al

Poster (2012, April 18)

Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) to kill pathologic agents. But, an excessive ROS production, called “oxidative ... [more ▼]

Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) to kill pathologic agents. But, an excessive ROS production, called “oxidative stress” is associated with tissue damages and development of chronic or acute inflammatory diseases. PMNs are prime therapeutic targets to control inflammatory events associated to ROS production. Nowadays, there is a growing interest for the use of polyphenolic molecules to modulate the inflammatory response. The aim of this work was to study the antioxidant effect of NDS27 (1), a new highly water-soluble form of the polyphenolic molecule curcumin, on in vitro stimulated equine PMNs. NDS27 (10-6 to 10-4 M) was pre-incubated with cells and eliminated before their activation. The ability of NDS27 to enter into the cells was checked by HPLC from the cellular extracts. The intracellular ROS production by phorbol myristate acetate (PMA) stimulated PMNs was measured by fluorescence using 2’,7’-dichlorofluorescin diacetate. Lucigenin dependent chemiluminescence was used to measure extracellular ROS production. Additionally, the effect of NDS27 was tested on the activity of myeloperoxidase (MPO), a hemic enzyme contributing to the oxidant response of neutrophils. The activity of the released MPO by cytochalazine B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated PMNs was measured by SIEFED (“Specific Immunologic Extraction Followed by Enzymatic Detection”) (2). The HPLC results showed that NDS27 enters into PMNs and interacts with their membrane. NDS27 significantly and dose-dependently inhibited the ROS production in neutrophils without affecting their viability. Likewise, the activity of MPO released by PMNs was lowered by NDS27. Overall, our findings demonstrate that the membrane of neutrophils is permeable to NDS27 or interacts with the drug, suggesting that its inhibitory effect on ROS production is mainly associated to an intracellular effect probably by acting on the enzymes implied in respiratory burst like NADPH oxidase and MPO. The modulatory effect of NDS27 towards the oxidant activity of cells involved in immune and inflammatory response open therapeutic perspectives to control equine or human pathologies with excessive inflammatory reactions. 1. Neven et al. 2011, Patent Application Publication: US2011/0257126 A1 2. Serteyn et al. 2005, European Patent Specification : EP1711817 B1 [less ▲]

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See detailInnovative multilayered nanofibers for wound dressing application
Croisier, Florence ULg; Detrembleur, Christophe ULg; Jérôme, Christine ULg

Poster (2012, April 18)

Chitosan is a natural polymer derived from the chitin of crustacean or mushroom shells, that intrinsically presents haemostatic, mucoadhesive, antimicrobial and immunostimulant properties. This ... [more ▼]

Chitosan is a natural polymer derived from the chitin of crustacean or mushroom shells, that intrinsically presents haemostatic, mucoadhesive, antimicrobial and immunostimulant properties. This polysaccharide has shown a great potential for biomedical and pharmaceutical applications, on account of its remarkable compatibility with physiological medium. Besides, it is degraded in a physiological environment into non-toxic products, which make chitosan an outstanding candidate for short- to medium-term applications. In this respect, nanometric fibers are highly interesting as their assembly mimics the skin extracellular matrix structure. Such nanofibrous materials can be prepared by electrospinning (ESP). This technique uses a high voltage to create an electrically charged jet of polymer solution or melt which leads to fibers formation. Depending on the polymer characteristics (a.o. molecular weight, solution viscosity and conductivity) and processing conditions (electric potential, distance between syringe-capillary and collection plate, concentration, flow rate), polymer fibers ranging from nanometers to a few microns in diameter can be obtained and subsequently used as potential scaffolds, a.o. to form a temporary, artificial extracellular matrix. In the present study, electrospinning technique was combined with layer-by-layer deposition method (LBL) - a well-known method for surface coating, based on electrostatic interactions - in order to prepare multilayered chitosan-based nanofibers. The antibacterial properties of the obtained material were then assessed, and the presence of a multilayered deposit was confirmed by several techniques. (Future) possibilities for valorization: These multilayered chitosan-based nanofibers present great prospects for the preparation of new biomedical scaffolds - such as wound dressings that could improve skin regeneration. [less ▲]

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