University of Montpellier 17 articles published in JoVE Biology Following the Dynamics of Structural Variants in Experimentally Evolved Populations Michael Finnegan1, Jeanne Hamet1, Erick Desmarais2, Stéphanie Bedhomme1 1CEFE, University of Montpellier, CNRS, EPHE, IRD, Montpellier, France, 2ISEM, University of Montpellier, CNRS, EPHE, IRD, Montpellier, France We developed a cost-effective method to follow non-single nucleotide polymorphism allele dynamics that can easily be adapted to experimental evolution frozen archives. A triplet PCR technique was coupled with automated parallel capillary electrophoresis to quantify the relative frequency of an insertion allele over the course of experimental evolution. Bioengineering Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates Mahnaz Sabeti Azad*1, Angelo Cardoso Batista*1, Jean-Loup Faulon1, Chase L. Beisel2,3, Jerome Bonnet4, Manish Kushwaha1 1INRAe, AgroParisTech, Micalis Institute, Université Paris-Saclay, 2Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI), 3Medical Faculty, University of Würzburg, 4Centre de Biochimie Structurale, INSERM U1054, CNRS UMR 5048, University of Montpellier Presented here is a protocol for the preparation and buffer calibration of cell extracts from exonuclease V knockout strains of Escherichia coli BL21 Rosetta2 (ΔrecBCD and ΔrecB). This is a fast, easy, and direct approach for expression in cell-free protein synthesis systems using linear DNA templates. Biochemistry Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide Karidia Konate1, Quentin Seisel1, Eric Vivès1, Prisca Boisguérin1, Sébastien Deshayes1 1PHYMEDEXP, INSERM U1046 - CNRS UMR 9214 - University of Montpellier The fluorescence leakage assay is a simple method that enables the investigation of peptide/membrane interactions in order to understand their involvement in several biological processes and especially the ability of cell-penetrating peptides to disturb phospholipids bilayers during a direct cellular translocation process. Engineering Epitaxial Nanostructured α-Quartz Films on Silicon: From the Material to New Devices Claire Jolly1, David Sanchez-Fuentes1, Ricardo Garcia-Bermejo1, Dilek Cakiroglu1, Adrian Carretero-Genevrier1 1Institut d'Electronique et des Systemes (IES), CNRS, Université de Montpellier This work presents a detailed protocol for the microfabrication of nanostructured α-quartz cantilever on a Silicon-On-Insulator(SOI) technology substrate starting from the epitaxial growth of quartz film with the dip coating method and then nanostructuration of the thin film via nanoimprint lithography. Immunology and Infection In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells Michel Jourdan1, Hugues de Boussac1, Elena Viziteu1, Alboukadel Kassambara1, Jérôme Moreaux1,2,3 1IGH, CNRS, Univ Montpellier, 2Laboratory for Monitoring Innovative Therapies, Department of Biological Hematology, CHU Montpellier, 3UFR de Médecine, Univ Montpellier Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model. Chemistry Photogeneration of N-Heterocyclic Carbenes: Application in Photoinduced Ring-Opening Metathesis Polymerization Julien Pinaud1, Emeline Placet1, Patrick Lacroix-Desmazes1, Thi Kim Hoang Trinh2,3, Jean Pierre Malval2,3, Abraham Chemtob2,3, Loïc Pichavant4, Valérie Héroguez4 1ICGM, Université de Montpellier, CNRS, ENSCM, 2Institut de Science des Matériaux de Mulhouse (IS2M UMR 7361 CNRS), Université de Haute-Alsace, 3Université de Strasbourg, 4Laboratoire de Chimie des Polymères Organiques (LCPO UMR 5629 ENSCBP), Université de Bordeaux We describe a protocol to photogenerate N-heterocyclic carbenes (NHCs) by UV irradiation of a 2-isopropylthioxanthone/imidazolium tetraphenylborate salt system. Methods to characterize the photoreleased NHC and elucidate the photochemical mechanism are proposed. The protocols for ring-opening metathesis photopolymerization in solution and miniemulsion illustrate the potential of this 2-component NHC photogenerating system. Bioengineering Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources Audrey Bloem1, Stephanie Rollero2, Pauline Seguinot1, Lucie Crépin3, Marc Perez1, Christian Picou1, Carole Camarasa1 1UMR SPO, INRA, SupAgroM, Université de Montpellier, 2IWBT Stellenbosch University, 3PILI, Toulouse White Biotechnology This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined. Neuroscience Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo Jolanta Jagodzinska*1, Emmanuelle Sarzi*1, Mélanie Cavalier1, Marie Seveno1, Volker Baecker2, Christian Hamel1,3,4, Marie Péquignot1, Cecile Delettre1 1INSERM U1051, Institut of Neurosciences of Montpellier, 2CNRS UMS3426, BioCampus Montepellier, 3University of Montpellier, 4CHRU Montpellier, Centre of Reference for Genetic Sensory Diseases, CHU Gui de Chauliac Hospital This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described. Biology MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells Brice Nzigou Mombo1, Sabine Gerbal-Chaloin1, Aleksandra Bokus1, Martine Daujat-Chavanieu1, Christian Jorgensen1, Jean-Philippe Hugnot2, Marie-Luce Vignais1 1Institute for Regenerative Medicine and Biotherapy (IRMB), INSERM U1183, Montpellier University, 2Institute of Neurosciences of Montpellier (INM), INSERM U1051, Montpellier University Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types. Medicine Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases Margarita Arango-Lievano1,2,3, Patrizia Giannoni1,2,3, Sylvie Claeysen1,2,3, Nicola Marchi1,2,3, Freddy Jeanneteau1,2,3 1Inserm, U1191, Institute of Functional Genomics, 2CNRS, UMR-5203, 3Université de Montpellier This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer's disease. Biochemistry Identification of Fatty Acids in Bacillus cereus Christian Ginies1, Julien Brillard1,2, Christophe Nguyen-The1 1UMR408 SQPOV, Sécurité et Qualité des Produits d'Origine Végétale, INRA, Université d'Avignon, 2UMR1333 DGIMI, INRA, Université de Montpellier We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl). Biology Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope Vasyl Mytskaniuk*1, Fabrice Bardin*2,3, Hassan Boukhaddaoui1,5, Herve Rigneault4, Nicolas Tricaud1 1INSERM U1051, Institut des Neurosciences de Montpellier (INM), Université de Montpellier, 2Université de Nîmes, 3CNRS, IES, UMR 5214, 4Aix-Marseille Université, CNRS, École Centrale Marseille, Institut Fresnel, UMR 7249, 5Montpellier RIO Imaging (MRI) Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser. Immunology and Infection Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos Audrey Bernut1,2, Christian Dupont1,2, Alain Sahuquet1, Jean-Louis Herrmann3, Georges Lutfalla1, Laurent Kremer1,2 1Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS, UMR 535, Université Montpellier, 2Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé, CNRS, FRE 3689, Université Montpellier, 3Unité de Formation et de Recherche des Sciences de la Santé, EA3647-EPIM, Université Versailles St Quentin Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos. Immunology and Infection Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants Eric Martinez1, Franck Cantet1, Matteo Bonazzi1 1Cell Biology of Bacterial Infections, CNRS, FRE3689, CPBS, Université Montpellier Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes. Behavior Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks Simone Dalla Bella1,2,3,4, Jakub Sowiński3 1Movement to Health Laboratory (EuroMov), University of Montpellier, 2Institut Universitaire de France, 3Department of Cognitive Psychology, University of Finance and Management in Warsaw, 4International Laboratory for Brain, Music, and Sound Research (BRAMS) Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders. Neuroscience Primary Culture of Mouse Dopaminergic Neurons Florence Gaven1,2,3, Philippe Marin1,2,3, Sylvie Claeysen1,2,3 1CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, 2Inserm, U661, Montpellier, 3Universités de Montpellier Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses. Biology An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis Wenjing She1, Daniel Grimanelli2, Célia Baroux1 1Institute of Plant Biology and Zürich-Basel Plant Science Center, University of Zürich, 2Institut de Recherche pour le Développement (UMR 232), Centre National de la Recherche Scientifique (ERL 5300), Université de Montpellier II We provide here an efficient and reliable protocol for immunostaining, Fluorescence in situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.
