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Search Results: 1 - 10 of 142 matches for " Veerle Baekelandt "
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Comparison of lentiviral vector titration methods
Martine Geraerts, Sofie Willems, Veerle Baekelandt, Zeger Debyser, Rik Gijsbers
BMC Biotechnology , 2006, DOI: 10.1186/1472-6750-6-34
Abstract: We compared lentiviral vector titration methods that measure pg p24/ml, RNA equivalents/ml, transducing units (TU/ml) or mRNA equivalents. The amount of genomic RNA in vector particles proves to be reliable to assess the production quality of vectors encoding non-fluorescent proteins. However, the RNA and p24 titers of concentrated vectors are rather poor in predicting transduction efficiency, due to the high variability of vector production based on transient transfection. Moreover, we demonstrate that transgenic mRNA levels correlate well with TU and can be used for functional titration of non-fluorescent transgenes.The different titration methods have specific advantages and disadvantages. Depending on the experimental set-up one titration method should be preferred over the others.In our laboratory we routinely produce and apply vectors derived from the human immunodeficiency virus type 1 (HIV-1). Since lentiviral vectors (LV) integrate stably into the host-cell genome of non-dividing cells such as neurons and in haematopoietic stem cells [1-3], they offer great potential for gene therapeutic applications [4]. For biosafety reasons, the HIV-1 genome has been modified and cis and trans-acting viral sequences have been segregated over 3 to 4 different plasmids [5,6]. Indeed, viral structural and functional proteins can be provided in trans and are encoded by 1 or 2 packaging plasmids while the envelope plasmid encodes the glycoprotein of the vesicular stomatitis virus envelope (VSV-G) and a transfer plasmid encodes the transgene of interest flanked by all cis-acting viral sequences necessary for packaging of the RNA genome (reviewed by [7]). Production of lentiviral vectors is routinely achieved by transient transfection of human embryonic kidney (293T) cells using high concentrations of the different plasmids, implicating the presence of residual plasmid DNA in the vector preparation, even after concentration. Transduction by lentiviral vectors matches a single-r
Regulation and targeting of enzymes mediating Parkinson's disease pathogenesis: focus on Parkinson's disease kinases, GTPases, and ATPases
Jean-Marc Taymans,Veerle Baekelandt,Kirsten Harvey
Frontiers in Molecular Neuroscience , 2014, DOI: 10.3389/fnmol.2014.00071
Abstract:
Automated quantitative gait analysis in animal models of movement disorders
Caroline Vandeputte, Jean-Marc Taymans, Cindy Casteels, Frea Coun, Yicheng Ni, Koen Van Laere, Veerle Baekelandt
BMC Neuroscience , 2010, DOI: 10.1186/1471-2202-11-92
Abstract: Hemiparkinsonian rats were generated by unilateral injection of the neurotoxin 6-hydroxydopamine in the striatum or in the medial forebrain bundle. For Huntington's disease, a transgenic rat model expressing a truncated huntingtin fragment with multiple CAG repeats was used. Thirdly, a stroke model was generated by a photothrombotic induced infarct in the right sensorimotor cortex. We found that multiple gait parameters were significantly altered in all three disease models compared to their respective controls. Behavioural deficits could be efficiently measured using the cylinder test in the PD and stroke animals, and in the case of the PD model, the deficits in gait essentially confirmed results obtained by the cylinder test. However, in the HD model and the stroke model the Catwalk analysis proved more sensitive than the rotarod test and also added new and more detailed information on specific gait parameters.The automated quantitative gait analysis test may be a useful tool to study both motor impairment and recovery associated with various neurological motor disorders.Multiple neurodegenerative and vascular central nervous system (CNS) diseases are characterized by motor deficits. For many of these diseases, no satisfactory neuroprotective or neuroregenerative therapies are available thus far. Therefore, the development of appropriate animal models for CNS motor disorders and the adequate evaluation of novel therapies in these animal models is an active field of preclinical research. In addition to the emerging non-invasive molecular and anatomical imaging techniques to visualize and quantify deficits and recovery in these disorders, behavioral testing is frequently the primary experimental readout to assess therapeutic effects. For this reason, sensitive, reproducible, time-efficient and easily applicable behavioral tests for existing or newly generated animal models are warranted. The present study has focused on animal models for three different motor disord
Immunohistochemical detection of transgene expression in the brain using small epitope tags
Evy Lobbestael, Veerle Reumers, Abdelilah Ibrahimi, Kirsten Paesen, Irina Thiry, Rik Gijsbers, Chris Van den Haute, Zeger Debyser, Veerle Baekelandt, Jean-Marc Taymans
BMC Biotechnology , 2010, DOI: 10.1186/1472-6750-10-16
Abstract: In the present study, we evaluated several small epitope tags together with corresponding anti-tag antibodies for the detection of overexpressed proteins in rat brain, using eGFP as a reference. We generated several lentiviral vectors encoding eGFP with different N-terminally fused small epitope tags (AU1, flag, 3flag, HA, myc and V5). After confirmation of their functionality in cell culture, we injected these lentiviral vectors stereotactically into the striatum of rats and prepared paraformaldehyde fixed floating sections for immunohistochemical analysis. Using multiple antibodies and antibody dilutions per epitope tag, we extensively assessed the efficiency of several anti-tag antibodies for chromogenic immunohistochemical detection of the epitope tagged eGFPs by determining the proportion of immunoreactivity detected by anti-tag antibodies compared to anti-GFP antibody. Using fluorescence immunohistochemistry and confocal microscopy, we also quantified the proportion of eGFP-positive cells detected by anti-tag antibodies. Our results show that all the examined small epitope tags could be detected by anti-tag antibodies both in cell extracts as well as in vivo, although to varying degrees depending on the tag and antibody used. Using the presented protocol, V5/anti-V5 and HA/HA11 tag/antibody combinations provided the most sensitive detection in brain tissue. We confirmed the applicability of these optimized in vivo tag detection conditions for a difficult to detect protein, firefly luciferase (fLuc), using lentiviral vector constructs expressing V5 tagged and 3flag tagged fLuc protein.We show here that several small epitope tags are useful for immunohistochemical detection of exogenous proteins in vivo. Our study also provides a generic methodology which is broadly applicable for the detection of overexpressed transgenes in mammalian brain tissue.Since the advent of recombinant DNA technology, transgenic model organisms have become powerful tools for the study
LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding
Jean-Marc Taymans, Renée Vancraenenbroeck, Petri Ollikainen, Alexandra Beilina, Evy Lobbestael, Marc De Maeyer, Veerle Baekelandt, Mark R. Cookson
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0023207
Abstract: Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound “on” state and a GDP bound “off” state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.
Impaired neurogenesis, learning and memory and low seizure threshold associated with loss of neural precursor cell survivin
Vanessa Coremans, Tariq Ahmed, Detlef Balschun, Rudi D'Hooge, Astrid DeVriese, Jonathan Cremer, Flavia Antonucci, Micha?l Moons, Veerle Baekelandt, Veerle Reumers, Harold Cremer, Amelia Eisch, Diane Lagace, Tom Janssens, Yuri Bozzi, Matteo Caleo, Edward M Conway
BMC Neuroscience , 2010, DOI: 10.1186/1471-2202-11-2
Abstract: To examine the role of neural precursor cell survivin, we first showed that survivin is normally expressed in periventricular neurogenic regions in the embryo, becoming restricted postnatally to proliferating and migrating NPCs in the key neurogenic sites, the subventricular zone (SVZ) and the subgranular zone (SGZ). We then used a conditional gene inactivation strategy to delete the survivin gene prenatally in those neurogenic regions. Lack of embryonic NPC survivin results in viable, fertile mice (SurvivinCamcre) with reduced numbers of SVZ NPCs, absent rostral migratory stream, and olfactory bulb hypoplasia. The phenotype can be partially rescued, as intracerebroventricular gene delivery of survivin during embryonic development increases olfactory bulb neurogenesis, detected postnatally. SurvivinCamcre brains have fewer cortical inhibitory interneurons, contributing to enhanced sensitivity to seizures, and profound deficits in memory and learning.The findings highlight the critical role that survivin plays during neural development, deficiencies of which dramatically impact on postnatal neural function.In the adult, two major, well-defined neurogenic regions persist [1]. In the subventricular zone (SVZ), neural precursor cells (NPCs) that arise mostly from the embryonic lateral ganglionic eminence (LGE) [2], continuously proliferate, and then migrate tangentially along the rostral migratory stream (RMS) towards the olfactory bulb (OB) where they differentiate into granular and periglomerular inhibitory interneurons [3]. In the subgranular zone (SGZ) of the hippocampus, newborn NPCs also migrate, but for shorter distances, into the granule cell layer, where they become excitatory granule cells [4]. From these neurogenic sites, adult-generated neurons can migrate to regions of brain injury [5], and establish synaptic contacts and functional connections [6,7]. Decreased neurogenesis induced by prenatal or postnatal stresses is implicated in the development of seizur
Biochemical Characterization of Highly Purified Leucine-Rich Repeat Kinases 1 and 2 Demonstrates Formation of Homodimers
Laura Civiero, Renée Vancraenenbroeck, Elisa Belluzzi, Alexandra Beilina, Evy Lobbestael, Lauran Reyniers, Fangye Gao, Ivan Micetic, Marc De Maeyer, Luigi Bubacco, Veerle Baekelandt, Mark R. Cookson, Elisa Greggio, Jean-Marc Taymans
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043472
Abstract: Leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2) are large multidomain proteins containing kinase, GTPase and multiple protein-protein interaction domains, but only mutations in LRRK2 are linked to familial Parkinson's disease (PD). Independent studies suggest that LRRK2 exists in the cell as a complex compatible with the size of a dimer. However, whether this complex is truly a homodimer or a heterologous complex formed by monomeric LRRK2 with other proteins has not been definitively proven due to the limitations in obtaining highly pure proteins suitable for structural characterization. Here, we used stable expression of LRRK1 and LRRK2 in HEK293T cell lines to produce recombinant LRRK1 and LRRK2 proteins of greater than 90% purity. Both purified LRRKs are folded, with a predominantly alpha-helical secondary structure and are capable of binding GTP with similar affinity. Furthermore, recombinant LRRK2 exhibits robust autophosphorylation activity, phosphorylation of model peptides in vitro and ATP binding. In contrast, LRRK1 does not display significant autophosphorylation activity and fails to phosphorylate LRRK2 model substrates, although it does bind ATP. Using these biochemically validated proteins, we show that LRRK1 and LRRK2 are capable of forming homodimers as shown by single-particle transmission electron microscopy and immunogold labeling. These LRRK dimers display an elongated conformation with a mean particle size of 145 ? and 175 ? respectively, which is disrupted by addition of 6M guanidinium chloride. Immunogold staining revealed double-labeled particles also in the pathological LRRK2 mutant G2019S and artificial mutants disrupting GTPase and kinase activities, suggesting that point mutations do not hinder the dimeric conformation. Overall, our findings indicate for the first time that purified and active LRRK1 and LRRK2 can form dimers in their full-length conformation.
Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice
Irene Bergwerf, Nathalie De Vocht, Bart Tambuyzer, Jacob Verschueren, Kristien Reekmans, Jasmijn Daans, Abdelilah Ibrahimi, Viggo Van Tendeloo, Shyama Chatterjee, Herman Goossens, Philippe G Jorens, Veerle Baekelandt, Dirk Ysebaert, Eric Van Marck, Zwi N Berneman, Annemie Linden, Peter Ponsaerts
BMC Biotechnology , 2009, DOI: 10.1186/1472-6750-9-1
Abstract: In this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-γ-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation.We here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). While injuries to
Unexpected Dramatic Response of Pretreated Invasive Thymic Malignancies on Pemetrexed-Case Report and Review of Current Treatment Modalities  [PDF]
Priscilla Raman, Veerle Surmont
Open Journal of Respiratory Diseases (OJRD) , 2012, DOI: 10.4236/ojrd.2012.24016
Abstract: Thymomas are rare and usually slowly growing tumors, originating from the epithelial layer of the thymus. Prognosis depends on the extent of invasion of adjacent tissues whereby multimodality treatment including surgery with or without adjuvant chemoradiotherapy is the preferred approach for locally advanced thymomas. For metastatic thymomas, only few chemotherapeutic options are available. We report 2 cases of patients with metastatic thymic malignancies with a dramatic response on pemetrexed treatment. The choice for this antifolate therapy is based upon a small series. Because metastatic thymic neoplasm is a rare disease, large randomised trials are not feasible. Case reports on the treatment of these malignancies are very important and can provide readers with the opportunity to deal with rare dis- eases.
S.I. Oppenhuis de Jong, De Middelnederlandse Perceval-traditie. Inleiding en editie van de bewaarde fragmenten van een Middelnederlandse vertaling van de Perceval of Conte du Graal van Chrétien de Troyes, en de Perchevael in de Lancelotcompilatie
Veerle Uyttersprot
BMGN : Low Countries Historical Review , 2005,
Abstract:
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