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See detailAn ESR, Mass Spectrometry and Fluorescence microscopy approach to study the stearic acid derivatives anchoring in cells
Grammenos, Angeliki ULg; Fillet, Marianne ULg; Collodoro, Mike et al

in Applied Magnetic Resonance (2012)

Lateral phase separations in biological membranes are of great interest, making Electron Spin Resonance (ESR) spectroscopy combined with spin labelling a non destructive and sensitive technique for the ... [more ▼]

Lateral phase separations in biological membranes are of great interest, making Electron Spin Resonance (ESR) spectroscopy combined with spin labelling a non destructive and sensitive technique for the study of lipid rafts. This is currently accepted that spin probe localization is on the plasma membrane. However, no study confirms this hypothesis. Herein, we report, for the first time, an accurate multi spectral method for the quantification of lipid spin label presence in every sub-cellular fraction. Cells were incubated with 5-doxyl stearic acid derivative and then sub-fractionated. Results of our multimodal spectroscopy approach ubiquitously demonstrate that the presence of ESR spin label only sets in the plasma membranes. [less ▲]

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See detailBiological variations during a race cycling
LE GOFF, Caroline ULg; Kaux, Jean-François ULg; Goffaux, Sébastien et al

in Biomedica 2012 (2012, April)

Background: The metabolic and cardiac impact of a cycling effort on blood biology is not very well described in the literature. We aimed to measure the concentration of different biomarkers (cardiac and ... [more ▼]

Background: The metabolic and cardiac impact of a cycling effort on blood biology is not very well described in the literature. We aimed to measure the concentration of different biomarkers (cardiac and metabolic) released during an international cycling race. Materials and Methods: Venous blood samples of 15 young men (25.1 ± 6.4 y.o.) were collected just before (T1), just after (T2), 3 hours (T3) after an international cycling race of 176 kilometers in Belgium for the determination of cardiac and metabolic biomarkers: red blood cells (RBC) haemoglobin (HgB) creatinin (Cr) highly sensitive troponin T (hsTnT) myoglobin (MYO) NT-proBNP All automated assays were performed according to the manufacter’s specifications. For the statistical analysis, an Anova calculated with the Statistica Software version 9.1 was used. Results: •RBC and HgB levels varied significantly between T0 and T3 (respectively p=0.0026, and p=0.002) (Fig. 1 and 2). • Cr concentration also varied significantly between all times (T0-T1:p<0.0001, T1-T3:p=0.0326 and T0-T3 p=0.0001)(Fig.3). These changes might be related to renal flow depletion during exercise. •MYO increased significantly between T0 and T1 (p<0.0001), but quickly decreased between T1 and T3, however the T3 level stay higher than T0 (p=0.014) (Fig.4). •The stress delivered from the physical activity performed during the race induced a significant variation of hsTnT which increased significantly between T0 and T1 (p<0.0001) and stayed higher 3 hours after the end of the exercise (T0-T3: p<0.0001) (Fig.5) . •The intense exercise delivery by the race induced a significant variation of NT-proBNP, that followed the same kinetic of hsTnT but in smaller proportion. We noticed variations statistically significant between T0 and T1 and between T0 and T3 for NT-proBNP (Fig.6). •These increases of cardiac biomarkers were significant but reasonable and could not allow us to talk about cellular necrosis or irreversible injury. Conclusions: Our results show that stress generated by a cycling race could be the cause for the different metabolic variations observed. Troponin T stays without a doubt the most specific marker for stress related to myocardial tissue. Its increase can then be considered as being of interest. [less ▲]

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See detailMigration behaviour study of charged and uncharged compounds in micellar electrokinetic chromatography systems containing various proportions of SDS micelles and acetonitrile as organic modifier
Lamalle, Caroline ULg; Servais, Anne-Catherine ULg; Crommen, Jacques ULg et al

Poster (2012)

In 1984, Terabe and co-workers introduced a modified version of CZE, called micellar electrokinetic chromatography (MEKC), in which surfactant-formed micelles are included in the running buffer providing ... [more ▼]

In 1984, Terabe and co-workers introduced a modified version of CZE, called micellar electrokinetic chromatography (MEKC), in which surfactant-formed micelles are included in the running buffer providing a two phases chromatographic system for the separation of neutral compounds. Depending on their hydrophobicity, compounds can interact with the core of the micelles. Many MEKC methods have been described for the separation of neutral and basic compounds and for various applications. However, the migration behaviour of cationic, anionic and neutral analytes mixture is not well established regarding to the surfactant concentration and to the proportion of organic solvent in the background electrolyte (BGE). With such a mixture, it is important to remember that the separation of charged solutes in MEKC involves a combination of chromatographic and electrophoretic separation mechanisms. Moreover, it is worth noting that with charged analytes, two kinds of interactions with the micelles may occur; not only with the hydrophobic core but also with the head groups of the micelles through electrostatic interactions. In systems in which the surfactant has an opposite charge to that of the solute, ion pairing may occur. On the contrary, when a solute and the surfactant have similar charges, coulombic forces may cause the repulsion of these molecules. The goal of this study is to improve our understanding of the migration behaviour of charged and uncharged analytes in MEKC systems. With this aim in view, effective mobility (electrophoretic mobility under the influence of micelles) of neutrals, cations and anions were mesured at neutral, basic and acidic pH with BGE containing different SDS concentrations and ACN proportions. This study is also focused on the changes of migration order between CZE and MEKC systems using different BGE compositions. In the MEKC systems investigated in the study, SDS concentration and ACN proportion show a tremendous effect on the effective mobilities and migration order of the model compounds. While anions interact very weakly with SDS micelles, neutrals and cations interact with SDS through hydrophobic and ionic bonds. These interactions become stronger with the increase of SDS concentration and weaker with high ACN proportion. With 20 mM of SDS in the BGE, CZE behaviour is observed till 40% of ACN. But, when the SDS concentration is high and the ACN proportion is low, the migration order of analytes is reversed compared to CZE: EOF first, then the anions, followed by the neutrals and finally the cations. The migration order inside each group (cations, neutrals and anions) depends on the hydrophobicity of the analytes. Different organic solvents were also investigated to study the ion-pair formation. Those observations confirm the interest of using MEKC not only for the separation of neutral compounds but also variously charged analytes. [less ▲]

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See detailMicellar electrokinetic chromatography (MEKC) systems for the separation of mixtures of charged and uncharged compounds
Lamalle, Caroline ULg; Servais, Anne-Catherine ULg; Fradi, Inès et al

Poster (2012)

The migration behaviour of charged and uncharged analytes in MEKC was investigated under different conditions. Effective mobilities – electrophoretic mobilities under the influence of the negatively ... [more ▼]

The migration behaviour of charged and uncharged analytes in MEKC was investigated under different conditions. Effective mobilities – electrophoretic mobilities under the influence of the negatively charged micelles – of cations, anions and neutrals were measured at neutral, basic and acidic pH values (7.5, 11 and 2.2) using background electrolytes containing different sodium dodecyl sulfate (SDS) concentrations (0-90 mM) and acetonitrile proportions (0-75 %, v/v). The SDS concentration and acetonitrile proportion were found to have a tremendous effect on the effective mobilities and the migration order of the tested compounds. Although the SDS micelles interact more strongly with neutrals and cations, the migration of anionic compounds is also affected by the SDS concentration, indicating that hydrophobic interactions can occur between the micelles and these compounds. Since cationic, anionic and neutral solutes exhibit rather different migration behaviours, it is possible to considerably enhance the separation selectivity by properly adjusting the SDS concentration and the acetonitrile proportion in the background electrolyte. These observations confirm the interest of using MEKC not only for the separation of neutral substances but also for the analysis of mixtures of charged compounds. [less ▲]

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See detailMicellar electrokinetic chromatography systems for the separation of mixtures of charged and uncharged compounds
Lamalle, Caroline ULg; Servais, Anne-Catherine ULg; Fradi, Ines ULg et al

in Journal of Separation Science (2012), 35(15), 1933-1939

In this study, the migration behavior of charged and uncharged analytes was investigated under different conditions. Effective mobilities - electrophoretic mobilities under the influence of micelles - of ... [more ▼]

In this study, the migration behavior of charged and uncharged analytes was investigated under different conditions. Effective mobilities - electrophoretic mobilities under the influence of micelles - of cations, anions, and neutrals were measured at neutral, basic, and acidic pH (7.5, 11, and 2.2) using background electrolytes containing different sodium dodecyl sulfate (SDS) concentrations (0-90 mM) and acetonitrile (ACN) proportions (0-75%). SDS concentration and ACN proportion were found to have a tremendous effect on the effective mobilities and migration order of the model compounds. Although the SDS micelles preferably interact with neutrals and cations, hydrophobic bonds can also occur with anions. Cations, anions, and neutrals having rather different migration behaviors, it is possible to considerably enhance the selectivity of the method by adjusting properly the SDS concentration and the ACN proportion. These observations confirm the interest of using micellar electrokinetic chromatography not only for the separation of neutral substances but also to analyze charged compounds. [less ▲]

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See detailChemo- and enantio-selective method for the analysis of amino acids by capillary electrophoresis with in-capillary derivatization.
Fradi, Ines ULg; Servais, Anne-Catherine ULg; Lamalle, Caroline ULg et al

in Journal of Chromatography. A (2012), 1267

A novel dual chiral CE method was developed for the separation of l- and d-amino acids (AAs), using in-capillary derivatization with 9-fluoroenylmethyl chloroformate (FMOC). Firstly, using pre-column ... [more ▼]

A novel dual chiral CE method was developed for the separation of l- and d-amino acids (AAs), using in-capillary derivatization with 9-fluoroenylmethyl chloroformate (FMOC). Firstly, using pre-column derivatization, the enantioseparation of FMOC-AAs was optimized according to the nature of cyclodextrins (CD). A background electrolyte (BGE) composed of 30mM beta-CD, 30mM octakis(2,3-dihydroxy-6-O-sulfo)-gamma-CD (OS-gamma-CD), 40mM tetraborate and 15% isopropanol (IPA) was selected and led to 17 baseline resolved pairs (R(s)=1.7-5.8) and two partially resolved pairs (Lys, R(s)=0.5 and Arg, R(s)=1.2). Experimental conditions for in-capillary derivatization were then optimized. Several parameters, such as mixing voltage and time, concentration of labeling solution and the length of the spacer plug were studied. The optimal conditions for in-capillary derivatization procedure were obtained using successive hydrodynamic injections (30mbar) of AAs for 2s, borate buffer for 4s and 10mM FMOC solution for 6s, followed by a mixing at 3kV for 72s and wait time of 1min. Moreover, a particular attention was paid to improve separation chemoselectivity. The effect on stereoselectivity and chemoselectivity of different factors, such as decrease of pH and tetraborate concentration and the addition of sodium dodecyl sulfate (SDS), was investigated using the in-capillary derivatization procedure. The best separation of a standard mixture of ten AA racemates was observed using a BGE containing 30mM beta-CD, 30mM OS-gamma-CD, 25mM SDS, 40mM sodium tetraborate and 17% IPA. [less ▲]

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See detailComparative enantioseparation of talinolol in aqueous and non-aqueous capillary electrophoresis and study of related selector-selectand interactions by nuclear magnetic resonance spectroscopy.
Chankvetadze, Lali; Servais, Anne-Catherine ULg; Fillet, Marianne ULg et al

in Journal of Chromatography. A (2012), 1267

The enantiomers of the chiral beta-blocker drug talinolol were separated with two single component sulfated beta-cyclodextrin (CD) derivatives, namely heptakis (2,3-di-O-methyl-6-sulfo)-beta-CD) (HDMS ... [more ▼]

The enantiomers of the chiral beta-blocker drug talinolol were separated with two single component sulfated beta-cyclodextrin (CD) derivatives, namely heptakis (2,3-di-O-methyl-6-sulfo)-beta-CD) (HDMS-beta-CD) and heptakis (2,3-di-O-acetyl-6-sulfo)-beta-CD) (HDAS-beta-CD), in aqueous and non-aqueous capillary electrophoresis (CE). The enantiomer affinity pattern of talinolol toward these two CDs was opposite in both aqueous and non-aqueous CE. However, the enantiomer affinity pattern for a given CD derivative did not change when aqueous buffer was replaced with non-aqueous background electrolyte. The structures of the analyte-selector complexes in both, aqueous and non-aqueous electrolytes were studied using rotating frame nuclear Overhauser effect (ROESY) NMR spectroscopy. Inclusion complex formation between the enantiomers of talinolol and HDAS-beta-CD was confirmed in aqueous buffer, while the complex between the enantiomers of talinolol and HDMS-beta-CD was of the external type. The complex of the talinolol enantiomers with HDAS-beta-CD in non-aqueous electrolyte was also of the external type. In spite of external complex formation excellent separation of the enantiomers was observed in non-aqueous CE. [less ▲]

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See detailThe c-jun N-terminal Kinase (JNK)-binding Protein (JNKBP1) Acts as a Negative Regulator of NOD2 Protein Signaling by Inhibiting Its Oligomerization Process
Lecat, Aurore ULg; Di Valentin, Emmanuel ULg; Somja, Joan ULg et al

in Journal of Biological Chemistry (2012), 287(35), 29213-26

NOD2 is one of the best characterized member of the cytosolic NOD-like receptors (NLR) family. NOD2 is able to sense muramyl dipeptide (MDP), a specific bacterial cell wall component, and to subsequently ... [more ▼]

NOD2 is one of the best characterized member of the cytosolic NOD-like receptors (NLR) family. NOD2 is able to sense muramyl dipeptide (MDP), a specific bacterial cell wall component, and to subsequently induce various signalling pathways leading to NF- kappaB activation and autophagy, both events contributing to an efficient innate and adaptative immune response. Interestingly, loss-of-function nod2 variants were associated with a higher susceptibility for Crohn ' s disease (CD), which highlights the physiological importance of proper regulation of NOD2 activity. We performed a biochemical screen to search for new NOD2 regulators. We identified a new NOD2 partner, c-jun N-terminal kinase binding protein 1 (JNKBP1), a scaffold protein characterized by a N-terminal WD-40 domain. JNKBP1, through its WD-40 domain, binds to NOD2 following MDP activation. This interaction attenuates NOD2-mediated NF-kappaB activation and IL-8 secretion as well as NOD2 antibacterial activity. JNKBP1 exerts its repressor effect by disturbing NOD2 oligomerization and RIP2 tyrosine phosphorylation, both steps required for downstream NOD2 signalling. We furthermore showed that JNKBP1 and NOD2 are co-expressed in the human intestinal epithelium and immune cells recruited in the lamina propria, which suggests that JNKBP1 contributes to maintain NOD2-mediated intestinal immune homeostasis. [less ▲]

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See detailPrincipes toxiques, toxicité et technologie de détoxification de la graine de Jatropha curcas L. (synthèse bibliographique)
Nesseim, Thierry Daniel Tamsir; Fillet, Marianne ULg; Mergeai, Guy ULg et al

in Biotechnologie, Agronomie, Société et Environnement = Biotechnology, Agronomy, Society and Environment [=BASE] (2012), 16(4), 531-540

The use of meal from the crushing of Jatropha curcas seed for livestock feed is limited owing to the variable amounts of seed available. This availability depends on the level and variety of toxic and ... [more ▼]

The use of meal from the crushing of Jatropha curcas seed for livestock feed is limited owing to the variable amounts of seed available. This availability depends on the level and variety of toxic and antinutritional compounds contained in the seed at a given time; the most important of these compounds are phorbol esters and curcin. The phorbol esters present in J. curcas seed are euphorbiaceae diterpenes, known for their inflammatory action resulting in irritation and toxicity to insects, fish and mammals. These compounds are sometimes completely degraded in soil and they may be reduced by physical, chemical or biological processes, with a reduction ratio of between 50 and 95%. Curcin is an irritating toxalbumin with lectin activity; it is inactivated by heat treatment at 121 °C for 30 min. Other antinutritional compounds are also present in J. curcas seed, such as saponins and an inhibitor of trypsin activity. This trypsin-inhibiting compound interferes with the digestion process and its reduction is achieved through thermal, chemical or biological treatments. The elimination of, or at least a reduction in the levels of, these molecules represents a rerequisite for using J. curcas meal in the livestock feed sector. [less ▲]

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See detailOptimization of the liquid chromatography enantioseparation of chiral acidic compounds using cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector and polar organic mobile phases.
Dossou, K. S. S.; Farcas, Elena ULg; Servais, Anne-Catherine ULg et al

in Journal of Chromatography. A (2012), 1234

The LC enantioseparation of chiral acidic and zwitterionic drugs selected as model compounds was optimized using chlorine containing cellulose based chiral stationary phases and polar organic mobile ... [more ▼]

The LC enantioseparation of chiral acidic and zwitterionic drugs selected as model compounds was optimized using chlorine containing cellulose based chiral stationary phases and polar organic mobile phases. The main solvent of the mobile phase was acetonitrile, the temperature was settled at 25 degrees C and a stationary phase with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector (3-Cl-4-Me-PC) was selected. In the screening step, the nature and concentration of both acidic and basic additives were found to have a significant effect on retention, selectivity and resolution. Acetic acid (AcA) was selected as acidic additive for the optimization step since it could lead to the enantioseparation of more acidic compounds than trifluoroacetic acid (TFA) and formic acid (FA), while among the three basic additives tested, diethylamine (DEA) most often gave better results with respect to enantioresolution and selectivity than butylamine (BuA) and triethylamine (TEA). The optimization was performed using a central composite face-centered design with two factors, namely the concentration of acetic acid (0.1-0.3%) and the concentration of DEA (0.01-0.1%) in the mobile phase. On the basis of the results obtained in the screening and optimization steps, a strategy for the rapid development of methods for the enantioseparation of acidic or neutral compounds was proposed. [less ▲]

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See detailCombination of capillary electrophoresis, molecular modelling and nuclear magnetic resonance to study the interaction mechanisms between single-isomer anionic cyclodextrin derivatives and basic drug enantiomers in a methanolic background electrolyte.
Servais, Anne-Catherine ULg; Rousseau, Anne; Dive, Georges ULg et al

in Journal of Chromatography. A (2012), 1232

In order to improve our knowledge of the mechanisms of enantiomer recognition pattern in nonaqueous systems, an approach combining nonaqueous CE (NACE), molecular modelling and NMR was undertaken ... [more ▼]

In order to improve our knowledge of the mechanisms of enantiomer recognition pattern in nonaqueous systems, an approach combining nonaqueous CE (NACE), molecular modelling and NMR was undertaken. Bupivacaine and propranolol were selected as model compounds and their interactions with two single-isomer highly charged beta-CD derivatives, namely heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-CD (HDMS-beta-CD) and heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-CD (HDAS-beta-CD), were studied. The CD-bupivacaine complexes were evaluated by 2-D Rotating-frame Overhauser Effect SpectroscopY (ROESY) experiments. From these experiments, it can be assumed that inclusion complexes are not formed, whatever the CD derivative used. Molecular modelling was performed at the RHF/MINI-1 or B3LYP/6-31G(d) level. External as well as inclusion type complexes with the alkyl chain of propranolol into both CD cavities were located. Interaction energies calculated for bupivacaine and propranolol correlated with the enantiomer migration order observed in the NACE experiments using both anionic CD derivatives. The interaction of propranolol with HDMS-beta-CD or HDAS-beta-CD gives rise to a family of external and inclusion complexes in which some are more probably obtained. [less ▲]

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See detailOptimization of micro-HPLC peak focusing for the detection and quantification of low hepcidin concentrations.
Mansion, François ULg; Chiap, Patrice ULg; Houbart, Virginie ULg et al

in Journal of Separation Science (2011), 34(15), 1820-1827

Micro-high-performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ... [more ▼]

Micro-high-performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 muL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile-phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated. [less ▲]

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