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See detailDetermination of inhibitory potency of argatroban toward thrombin by electrophoretically mediated microanalysis
Pochet, Lionel; Servais, Anne-Catherine ULg; Farcas, Elena ULg et al

in Talanta (2013), 116

Developing an EMMA method for enzymatic assay remains a challenge, particularly using UV detection. Indeed, it is necessary to optimize the separation conditions while allowing the enzymatic reaction to ... [more ▼]

Developing an EMMA method for enzymatic assay remains a challenge, particularly using UV detection. Indeed, it is necessary to optimize the separation conditions while allowing the enzymatic reaction to occur within the capillary respecting kinetic constraints and achieving enough sensitivity. In this work, such EMMA methodology was set up to evaluate the inhibitory potency of drugs toward thrombin, a serine protease implicated in the coagulation cascade. To achieve our goal, the separation buffer, the injection sequence, the internal standard and the chromogenic substrate were investigated. The newly developed system was then assessed determining the kinetic Km constant for the selected substrate and compared with the results obtained with a continuous spectrophotometer cell assay. Secondly, the Ki inhibitory constant of the thrombin inhibitor argatroban was determined and found in agreement with the published value. [less ▲]

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See detailIdentification of protein biomarkers associated with cardiac ischemia by a proteomic approach.
Fillet, Marianne ULg; Deroyer, Céline ULg; COBRAIVILLE, G. et al

in Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals (2013), 18(7), 614-24

Angina is chest pain induced by ischemia of the heart muscle, generally due to obstruction or spasm of the coronary arteries. People that suffer from average to severe cases of angina have an increased ... [more ▼]

Angina is chest pain induced by ischemia of the heart muscle, generally due to obstruction or spasm of the coronary arteries. People that suffer from average to severe cases of angina have an increased percentage of death before the age of 55, usually around 60%. Therefore, prevention of major complications, optimizing diagnosis, prognosis and therapeutics are of primary importance. The main objective of this study was to uncover biomarkers by comparing serum protein profiles of patients suffering from stable or unstable angina and controls. We identified by non-targeted proteomic approach and confirmed by the means of independent techniques, the differential expression of several proteins indicating significantly increased vascular inflammation response, disturbance in the lipid metabolism and in atherogenic plaques stability. [less ▲]

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See detailKinetics of IL-7 and IL-15 Levels after Allogeneic Peripheral Blood Stem Cell Transplantation following Nonmyeloablative Conditioning
De Bock, Muriel; Fillet, Marianne ULg; Hannon, Muriel ULg et al

in PLoS ONE (2013), 8(2), 55876

Background: We analysed kinetics of IL-7 and IL-15 levels in 70 patients given peripheral blood stem cells after nonmyeloablative conditioning. Methods: EDTA-anticoagulated plasma and serum samples were ... [more ▼]

Background: We analysed kinetics of IL-7 and IL-15 levels in 70 patients given peripheral blood stem cells after nonmyeloablative conditioning. Methods: EDTA-anticoagulated plasma and serum samples were obtained before conditioning and about once per week after transplantation until day 100. Samples were aliquoted and stored at 280uC within 3 hours after collection until measurement of cytokines. IL-7 and IL-15 levels were measured by ELISAs. Results: Median IL-7 plasma levels remained below 6 pg/L throughout the first 100 days, although IL-7 plasma levels were significantly higher on days 7 (5.1 pg/mL, P = 0.002), 14 (5.2 pg/mL, P,0.001), and 28 (5.1 pg/mL, P = 0.03) (but not thereafter) than before transplantation (median value of 3.8 pg/mL). Median IL-15 serum levels were significantly higher on days 7 (12.5 pg/mL, P,0.001), 14 (10.5 pg/mL, P,0.001), and 28 (6.2 pg/mL, P,0.001) than before transplantation (median value of 2.4 pg/mL). Importantly, IL-7 and IL-15 levels on days 7 or 14 after transplantation did not predict grade II–IV acute GVHD. Conclusions: These data suggest that IL-7 and IL-15 levels remain relatively low after nonmyeloablative transplantation, and that IL-7 and IL-15 levels early after nonmyeloablative transplantation do not predict for acute GVHD. [less ▲]

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See detailHigh inorganic triphosphatase activities in bacteria and mammalian cells: Identification of the enzymes involved.
Kohn, Grégory ULg; Delvaux, David ULg; Lakaye, Bernard ULg et al

in PLoS ONE (2012), 7(9), 43879

Background: We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this ... [more ▼]

Background: We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. Methodology/Principal Findings: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. Conclusions and General Significance: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling. [less ▲]

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See detailDevelopment of a generic micellar electrokinetic chromatography method for the separation of 15 antimalarial drugs as a tool to detect medicine counterfeiting
Lamalle, Caroline ULg; Marini Djang'Eing'A, Roland ULg; Debrus, Benjamin ULg et al

in Electrophoresis (2012), 33

Since antimalarial drugs counterfeiting is dramatically present on the African market, the development of simple analytical methods for their quality control is of great importance. This work consists in ... [more ▼]

Since antimalarial drugs counterfeiting is dramatically present on the African market, the development of simple analytical methods for their quality control is of great importance. This work consists in the CE analysis of 15 antimalarials (artesunate, artemether, amodiaquine, chloroquine, piperaquine, primaquine, quinine, cinchonine, mefloquine, halofantrine, sulfadoxine, sulfalen, atovaquone, proguanil, and pyrimethamine). Since all these molecules cannot be ionized at the same pH, MEKC was preferred because it also allows separation of neutral compounds. Preliminary experiments were first carried out to select the most crucial factors affecting the antimalarials separation. Several conditions were tested and four parameters as well as their investigation domain were chosen: pH (5–10), SDS concentration (20–90 mM), ACN proportion (10–40%), and temperature (20–35°C). Then, the experimental design methodology was used and a central composite design was selected. Mathematical modeling of the migration times allowed the prediction of optimal conditions (29°C, pH 6.6, 29 mM SDS, 36% ACN) regarding analyte separation. The prediction at this optimum was verified experimentally and led to the separation of 13 compounds within 8 min. Finally, the method was successfully applied to the quality control of African antimalarial medicines for their qualitative and quantitative content. [less ▲]

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See detailDERP6 (ELP5) and C3ORF75 (ELP6) regulate tumorigenicity and migration of melanoma cells as subunits of Elongator
Close, Pierre ULg; Gillard, Magali; Ladang, Aurélie ULg et al

in Journal of Biological Chemistry (2012)

The Elongator complex is composed of 6 subunits (Elp1-Elp6) and promotes RNAPII transcript elongation through histone acetylation in the nucleus as well as tRNA modification in the cytoplasm. This ... [more ▼]

The Elongator complex is composed of 6 subunits (Elp1-Elp6) and promotes RNAPII transcript elongation through histone acetylation in the nucleus as well as tRNA modification in the cytoplasm. This acetyltransferase complex directly or indirectly regulates numerous biological processes ranging from exocytosis and resistance to heat shock in yeast to cell migration and neuronal differentiation in higher eukaryotes. The identity of human ELP1 through ELP4 has been reported but human ELP5 and ELP6 have remained uncharacterized. Here, we report that DERP6 (ELP5) and C3ORF75 (ELP6) encode these subunits of human Elongator. We further investigated the importance and function of these two subunits by a combination of biochemical analysis and cellular assays. Our results show that DERP6/ELP5 is required for the integrity of Elongator and directly connects ELP3 to ELP4. Importantly, the migration and tumorigenicity of melanomaderived cells are significantly decreased upon Elongator depletion through ELP1 or ELP3. Strikingly, DERP6/ELP5 and C3ORF75/ELP6-depleted melanoma cells have similar defects, further supporting the idea that DERP6/ELP5 and C3ORF75/ELP6 are essential for Elongator function. Together, our data identify DERP6/ELP5 and C3ORF75/ELP6 as key players for migration, invasion and tumorigenicity of melanoma cells, as integral subunits of Elongator. [less ▲]

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See detailRace cycling: biological evolution
LE GOFF, Caroline ULg; Kaux, Jean-François ULg; Goffaux, Sébastien et al

in Meeusen, R; Duchateau, J; Roelands, B (Eds.) et al Book of Abstracts of the 17th annual Congress of the ECSS (2012, July)

Introduction: 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 ▼]

Introduction: 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 179.6 kilometers in Belgium for the determination of cardiac and metabolic biomarkers: red blood cell (RBC), haemoglobin (HgB), creatinin (Cr), highly sensitive troponin T (hsTnT), myoglobin (MYO) and 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 and discussions: RBC and HgB levels varied significantly between T0 and T3 (respectively p=0.0026, and p=0.002). Cr concentration also varied significantly between all times (T0-T1:p<0.0001, T1-T3:p=0.0326 and T0-T3 p=0.0001). These changes might be related to renal flow depletion during exercice. 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). 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). 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. These increases of cardiac biomarkers were significant but reasonable and could not allow us to talk about cellular necrosis or irreversible injury. Conclusion: 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 detailImpact of strenous exercise on the release of cardiac biomarkers
LE GOFF, Caroline ULg; MELON, Pierre ULg; Kaux, Jean-François ULg et al

in Meeusen, R; Duchateau, J; Roelands, B (Eds.) et al Book of Abstracts of the 17th annual Congress of the ECSS (2012, July)

Background: Cardiac troponins (cTn) are considered as the best biomarkers for detection of myocardial cell injury and NT-proBNP as the best for the cardiac insufficiency. In this study, cTnT was measured ... [more ▼]

Background: Cardiac troponins (cTn) are considered as the best biomarkers for detection of myocardial cell injury and NT-proBNP as the best for the cardiac insufficiency. In this study, cTnT was measured by new commercially available high-sensitive methods in subjects undergoing the Maasmarathon. Our aim was to compare cTnT and NT-proBNP levels in sportive subjects before and after a strenuous exercise. Materials and Methods: Twenty eight subjects (26 ♂, 42.5±11yo) underwent a race of 42.195 kilometers between Visé (Belgium) and Maastricht (The Netherlands). We drowned blood samples before (T0), just after (T1) and three hours after the race (T3). For all patients, cTnT concentrations were measured by high sensitive methods (hsTnT, Roche Diagnostics) on heparin plasma. The NT-proBNP was also determined with the kit Roche on heparin plasma. The protocol was approved by the ethics committee of the University of Liège (Belgium). All subjects gave their informed consent. All statistical analyses were performed using Medcalc version 8.1 for Windows. p-value <0.01 was regarded as statistically significant. Results and discussion: A significant difference between hsTnT concentrations at T0 and T1 (p<0.0001) was measured as well as between T0 and T3 (p<0.001) for NT-proBNP, but not between T1 and T3. This observation appeared only after a strenuous exercise but today this type of exercise is not reproduce easier in a laboratory of sport. Moreover, at this moment, nobody knows if these observations would have cardiac consequences at long terms. Conclusions: Measurement of cardiac troponins by high sensitive methods allows detecting significant release of biomarkers from the heart during exercise. The levels of NT-proBNP were found significantly increased but in less extent than TnThs. We think that the TnThs could be an interesting marker in the future to help sport medicine to detect risk of developing a cardiac problem. [less ▲]

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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 detailIdentification of new emerin partners in ceramide signalling pathway
DEROYER, Céline ULg; Renert, Anne-Françoise; Merville, Marie-Paule ULg et al

Conference (2012, May 25)

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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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