References of "Servais, Anne-Catherine"
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See detailCapillary electrophoresis method to determine siRNA complexation with cationic liposomes
Furst, Tania ULg; Bettonville, Virginie ULg; Farcas, Elena ULg et al

in Electrophoresis (2016)

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally ... [more ▼]

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally nanoparticles. The nanoparticulate complex requires an optimal physiochemical characterization and the complexation efficiency has to be precisely determined. The methods usually used to measure complexation are gel electrophoresis and RiboGreen® fluorescence-based assay. However, those approaches are not automated and present some drawbacks such as the low throughput and the use of carcinogenic reagents. The aim of this work is to develop a new simple and fast method to accurately quantify the complexation efficiency. In this research, capillary electrophoresis (CE) was used to determine the siRNA complexation with cationic liposomes. The short-end injection mode applied enabled siRNA detection in less than 5 min. Moreover, the CE technique offers many advantages compared to the other classical methods. It is automated, does not require sample preparation and expensive reagents. Moreover, no mutagenic risk is associated to CE approach since no carcinogenic product is used. Finally, this methodology can also be extended to the characterization of other types of nanoparticles encapsulating siRNA, such as cationic polymeric nanoparticles. [less ▲]

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See detailApproches intégrées en Sciences Pharmaceutiques
Servais, Anne-Catherine ULg; Ziemons, Eric ULg

Conference (2016, May 13)

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See detailVolumetric Absorptive Microsampling for Hepcidin Peptide Extraction from Whole Blood
Houbart, Virginie ULg; COBRAIVILLE, Gaël ULg; Nys, Gwenaël ULg et al

in LCGC North America (2016), 34(5),

Whole blood analysis is an emerging trend in the field of bioanalysis. We developed a fast and simple protocol to extract and analyze a peptide, hepcidin, from whole blood. Sampling and extraction were ... [more ▼]

Whole blood analysis is an emerging trend in the field of bioanalysis. We developed a fast and simple protocol to extract and analyze a peptide, hepcidin, from whole blood. Sampling and extraction were carried out using volumetric absorptive microsampling (VAMS), a novel blood collection method that allows the sampling of a known blood volume independently from hematocrit. The composition of the extraction medium was optimized using an experimental design to get the most intense signal of hepcidin, considering different organic solvents and acidic additives. [less ▲]

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See detailFully automated electrophoretically mediated microanalysis for CYP1A1 activity monitoring optimized by multivariate approach
Farcas, Elena ULg; Servais, Anne-Catherine ULg; Lamalle, Caroline ULg et al

in Electrophoresis (2016), 37(2), 248-255

In this study, a fully automatized in-capillary system was developed to monitor the activity of CYP1A1 in physiological conditions. Ethoxycoumarin, the selected substrate, undergoes an in-line bioreaction ... [more ▼]

In this study, a fully automatized in-capillary system was developed to monitor the activity of CYP1A1 in physiological conditions. Ethoxycoumarin, the selected substrate, undergoes an in-line bioreaction in the presence of CYP1A1 supersomes and NADPH as co-factor, giving rise to hydroxycoumarin, the product that was assayed. The optimization of the experimental conditions was supported by the application of a design of experiment, providing a better understanding of electrophoretic mixing parameters that influence the metabolic reactions. The results obtained in optimal conditions were compared not only to those achieved after off-line metabolization but also with liver microsomes. Finally, inhibition studies were conducted showing an important decrease of hydroxycoumarin formation using apigenin as CYP1A1 potent inhibitor. This study demonstrates the usefulness of our in-line system for the fully automated in vitro metabolism studies and the screening of new CYP1A1 inhibitors. [less ▲]

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See detailStudy of intact virus-like particles of human papillomavirus by capillary electrophoresis
Bettonville, Virginie ULg; Nicol, Jérôme ULg; Thelen, Nicolas ULg et al

in Electrophoresis (2016), 37

Virus-like particles of human papillomavirus (HPV-VLP), resulting from the self-assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native ... [more ▼]

Virus-like particles of human papillomavirus (HPV-VLP), resulting from the self-assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native virion. Moreover, two prophylactic vaccines, Gardasil® and Cervarix®, are based on HPV-VLP L1. Analytical techniques currently used to characterize HPV-VLP, such as SDS-PAGE, Western blot, ELISA, are time-consuming and semi-quantitative. In this study, capillary electrophoresis (CE) was evaluated for the analysis of intact HPV16-VLP. The usefulness of capillary inner wall coating as well as various BGEs, pH and detergent additives were investigated. Reproducible HPV-VLP analysis in CE was achieved using poly(ethylene oxide) coated capillary and a BGE containing high salt concentration and low SDS concentration. The developed method enables HPV-VLP detection in less than 10 min (migration times RSD : 1.6 %). The identity of HPV-VLP peak was confirmed by comparison with a sample obtained from a wild-type baculovirus and with VLP-based vaccine, Gardasil®, after adjuvant dissolution. Finally, we applied the developed methodology to VLP-based vaccines, demonstrating that CE could be successfully used for vaccine quality control. [less ▲]

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See detailFULLY AUTOMATED ELECTROPHORETICALLY MEDIATED MICROANALYSIS SYSTEM FOR CYP1A1 ACTIVITY MONITORING
Farcas, Elena ULg; Servais, Anne-Catherine ULg; Lamalle, Caroline ULg et al

Poster (2015, June 23)

Introduction Since the efficacy and toxicity of drugs are closely related to their pharmacokinetics, a good understanding of metabolic pathways is important at an early stage of development. The ... [more ▼]

Introduction Since the efficacy and toxicity of drugs are closely related to their pharmacokinetics, a good understanding of metabolic pathways is important at an early stage of development. The identification of the enzymes involved in drug metabolism is thus of critical importance for the design of further clinical studies. The availability of specifically expressed human CYPs, namely supersomes, allows the investigation of the contribution of a single metabolic enzyme to the biotransformation pathway of the compound under investigation. CYP1A1, a member of the cytochrome P450 superfamily, was studied in this project. Interestingly, it has been described to be over expressed in various types of cancer. Consequently, CYP1A1 has emerged as a particularly interesting target for cancer therapy. Methods All the experiments were carried out on a HP3DCE system equipped with an on-column DAD. The EMMA procedure was performed by injecting a plug containing CYP1A1 supersomes, followed by a plug that contained the co-factor and the substrate, then another plug of CYP1A1 supersomes. The reaction was triggered by the application of a voltage switch. The voltage was then turned off to allow the metabolic reaction to occur. The separation of the components was then performed at -25 kV. Results The present study describes the development of a fully automatized in-capillary method to follow metabolization of 7-hydroxycoumarin and screen CYP1A1 inhibitors. After preliminary studies, satisfying results were obtained using CYP1A1 at a concentration of 200 pmol/mL, while the incubation time was settled to 15 min. Equal reactant plugs were injected at -50 mbar for 6 sec. The short-end injection performed gave rise to a baseline separation of the molecules (substrate, product, CYP1A1 and NADPH) in less than 2 minutes. Adequate plugs overlap was obtained using electrophoretic mixing. The DoE performed highlighted that the voltage switch has a great impact on the metabolite formation. The amount of product obtained in the optimal conditions was found to be comparable to the one detected after conventional off-line metabolization. Besides the interest of developing an automatized CE approach for metabolisation studies, we also wanted to investigate the potentiality of this approach to screen CYP1A1 inhibitors. The ability of our system to monitor CYP1A1 inhibition was undertaken with apigenin, a well-known inhibitor. It is noteworthy that the compatibility of our system with MEKC ensures its applicability to a large variety of molecules. Novel aspect Monitoring CYP1A1 activity using a rapid and fully automated EMMA method that could be used for new anticancer agents screening. [less ▲]

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See detailFully automated electrophoretically mediated microanalysis system for CYP1A1 activity monitoring
Farcas, Elena ULg; Servais, Anne-Catherine ULg; Lamalle, Caroline ULg et al

Conference (2015, May 28)

In order to evaluate the potentiality of capillary electrophoresis for CYP1A1 activity monitoring, an in-line method was developed with the well-known 7-ethoxycoumarin substrate. The electrophoretically ... [more ▼]

In order to evaluate the potentiality of capillary electrophoresis for CYP1A1 activity monitoring, an in-line method was developed with the well-known 7-ethoxycoumarin substrate. The electrophoretically mediated microanalysis approach was used with CYP1A1 supersomes to provide a rapid and fully automated method. The in-line homogenous enzyme assay was performed in physiological conditions (pH 7.4), whereas a MEKC buffer was used as background electrolyte. In order to reduce the analysis time, the short end injection was performed. Firstly a plug containing CYP1A1 supersomes was hydrodynamically injected into a fused silica capillary, followed by a plug of co-factor (NADPH) and substrate (7-ethoxycoumarin) and finally another plug of CYP1A1 (sandwich mode). The experimental conditions were finely investigated and tuned by design of experiment methodology. The metabolization rate measured in the optimized conditions was comparable with the one obtained after off-line metabolization. Finally, inhibition studies were conducted and a significant decrease of 7-hydroxycoumarin formation was observed using apigenin as CYP1A1 potent inhibitor. [less ▲]

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See detailSeparation of human, bovine, and porcine insulins, three very closely related proteins, by micellar electrokinetic chromatography.
Lamalle, Caroline ULg; Roland, Diane ULg; Crommen, Jacques ULg et al

in Electrophoresis (2015), 36(19), 2504-6

Human, bovine, and porcine insulins are small proteins with very closely related amino acid sequences, which makes their separation challenging. In this study, we took advantage of the high-resolution ... [more ▼]

Human, bovine, and porcine insulins are small proteins with very closely related amino acid sequences, which makes their separation challenging. In this study, we took advantage of the high-resolution power of CE, and more particularly of micellar electrokinetic chromatography, to separate those biomolecules. Among several surfactants, perfluorooctanoic acid ammonium salt was selected. Then, using a design of experiments approach, the optimal BGE composition was found to consist of 50 mM ammonium acetate pH 9.0, 65 mM perfluorooctanoic acid ammonium salt, and 4% MeOH. The three insulins could be separated within 12 min with a satisfactory resolution. This method could be useful to detect possible counterfeit pharmaceutical formulations. Indeed, it would be easy to determine if human insulin was replaced by bovine or porcine insulin. [less ▲]

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See detailHepcidin determination in dried blood by microfluidic LC-MS/MS: comparison of DBS and volumetric absorptive microsampling for matrix effect and recovery.
Houbart, Virginie ULg; COBRAIVILLE, Gaël ULg; Servais, Anne-Catherine ULg et al

in Bioanalysis (2015), 7(21), 2789-99

BACKGROUND: Dried blood analysis experiences a growing interest due to practical, ethical and financial advantages compared with classical wet plasma or serum analysis. Besides classical DBS, new ... [more ▼]

BACKGROUND: Dried blood analysis experiences a growing interest due to practical, ethical and financial advantages compared with classical wet plasma or serum analysis. Besides classical DBS, new alternatives are commercialized as volumetric absorptive microsampling (VAMS) that are expected to overcome hematocrit influence. RESULTS: The feasibility of hepcidin (a peptide hormone) extraction and determination from DBS and VAMS blood sampling was investigated. Experimental design was used to determine the optimal extraction conditions. Matrix effect and extraction recovery were studied and a special attention was paid to phospholipid removal. CONCLUSION: The data suggest that the combination of VAMS and phospholipid removal plates provides low matrix effect and high sensitivity, and constitutes an easy and promising protocol for hepcidin analysis. [less ▲]

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