Centre National de la Recherche Scientifique (CNRS) View Institution's Website 19 articles published in JoVE Chemistry Analyzing Melts and Fluids from Ab Initio Molecular Dynamics Simulations with the UMD Package Razvan Caracas1,2, Anais Kobsch1, Natalia V. Solomatova1, Zhi Li1, Francois Soubiran1,3, Jean-Alexis Hernandez1,2 1Ecole Normale Supérieure de Lyon, Laboratory of Geology of Lyon UMR5276, CNRS, 2Centre for Earth Evolution and Dynamics (CEED), University of Oslo, 3 Melts and fluids are ubiquitous vectors of mass transport in natural systems. We have developed an open-source package to analyze ab initio molecular-dynamics simulations of such systems. We compute structural (bonding, clusterization, chemical speciation), transport (diffusion, viscosity) and thermodynamic properties (vibrational spectrum). Biochemistry Analysis of SEC-SAXS data via EFA deconvolution and Scatter Mark D Tully*1, Nicolas Tarbouriech2, Robert P Rambo3, Stephanie Hutin*4 1European Synchrotron Radiation Facility Structural Biology Group, Structural Biology Group, 2Institut de Biologie Structurale, University Grenoble Alpes, CEA, CNRS, 3Diamond Light Source, 4Laboratoire de Physiologie Cellulaire and Végétale, Université Grenoble Alpes/CNRS/CEA/INRA/BIG SEC-BioSAXS measurements of biological macromolecules are a standard approach for determining solution structure of macromolecules and their complexes. Here, we analyze SEC-BioSAXS data from two types of commonly encountered SEC traces—chromatograms with fully resolved and partially resolved peaks. We demonstrate the analysis and deconvolution using scatter and BioXTAS RAW. Biology Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells Giulia Bertolin1, Gilles Le Marchand1, Marc Tramier1 1Univ Rennes, CNRS, IGDR (Genetics and Development Institute of Rennes), UMR 6290, F-35000 Rennes, France The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis. Neuroscience Electrophoretic Delivery of γ-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice Andrea Slezia*1,5, Christopher M. Proctor*2,3, Attila Kaszas1,4, George G. Malliaras2,3, Adam Williamson1,5 1Aix Marseille Université, Institut de Neurosciences des Systèmes (INS), 2Electrical Engineering Division, University of Cambridge, 3Department of Bioelectronics, Centre Microélectronique de Provence - Ecole Nationale Supérieure des Mines de Saint-Étienne (CMP-EMSE), 4Institut de Neurosciences de la Timone, Centre National de la Recherche Scientifique (CNRS) UMR 7289 & Aix- Marseille Université, 5Neuroengineering Research Group, Interdisciplinary Excellence Center, Department of Medical Microbiology and Immunobiology, University of Szeged The challenge of epilepsy research is to develop novel treatments for patients where classical therapy is inadequate. Using a new protocol—with the help of an implantable drug delivery system—we are able to control seizures in anesthetized mice by the electrophoretic delivery of GABA into the epileptic focus. Immunology and Infection Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns) Pierrette Courtois1,2, Patricia Nabos1,2, Romaric Nzoumbou-Boko1,2, Christine E. Reix3, Frédéric-Antoine Dauchy1,2,4, Sylvie Daulouede1,2, Frédéric Bringaud3, Derrick R. Robinson3, Philippe Vincendeau1,2,4 1Univ. Bordeaux UMR INTERTRYP 177 IRD CIRAD, 2IRD UMR 177 INTERTRYP CIRAD, 3CNRS, UMR 5234, Microbiologie Fondamentale et Pathogénicité, Univ. Bordeaux, 4Centre Hospitalier Université Bordeaux This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations. Neuroscience Long-term Sensory Conflict in Freely Behaving Mice Filipa França de Barros1,2, Julie Carcaud2, Mathieu Beraneck1,2 1Integrative Neuroscience and Cognition Center, UMR8002, CNRS, 2Sorbonne Paris Cité, Université Paris Descartes The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Biochemistry Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay Javad Alizadeh1,2, Shahla Shojaei1,2,3, Simone da Silva Rosa1, Adel Rezaei Moghadam1,2, Amir A. Zeki4, Mohammad Hashemi5, Marek J. Los6,7,8, Joseph W. Gordon1,9, Saeid Ghavami1,2,10 1Department of Human Anatomy and Cell Science, Rady Faculty of Health Sciences, Max Rady College of Medicine, University of Manitoba, 2Biology of Breathing Theme, Children Hospital Research Institute of Manitoba, University of Manitoba, 3Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, 4Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Center for Comparative Respiratory Biology and Medicine, 5Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, 6Department of Molecular Biology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, 7Centre de biophysique moléculaire - UPR 4301, Centre national de la recherche scientifique (CNRS) CS80054, 8LinkoCare Life Sciences AB, 9College of Nursing and Children's Hospital Research Institute of Manitoba, Rady Faculty of Health Sciences, University of Manitoba, 10Health Policy Research Center, Institute of Health, Shiraz University of Medical Sciences Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases. Genetics Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity Tiago Baptista1,2,3,4, Didier Devys1,2,3,4 1Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France, 2Centre National de la Recherche Scientifique, Illkirch, France, 3Institut National de la Santé et de la Recherche Médicale, Illkirch, France, 4Université de Strasbourg The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription. Immunology and Infection Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells Yasmine Smail1,2,3,4, Chunhui Sun1,2,3,4, Laurent Combettes5, Guy Tran Van Nhieu1,2,3,4 1Equipe Communication Intercellulaire et Infections Microbiennes, Centre de Recherche Interdisciplinaire en Biologie (CIRB), Collège de France, 2Institut National de la Santé et de la Recherche Médicale (Inserm), U1050, 3Centre Nationale de la Recherche Scientifique (CNRS), UMR7241, 4MEMOLIFE Laboratory of Excellence and Paris Sciences et Lettres, 5Inserm, UMRS1174, Université Paris Sud Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized. Biology Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture Cedric Allier1, Romaric Vincent1, Fabrice Navarro2, Mathilde Menneteau2, Lamya Ghenim3,4, Xavier Gidrol3, Thomas Bordy1, Lionel Hervé1, Olivier Cioni1, Sabine Bardin5, Michel Bornens5, Yves Usson4,6, Sophie Morales1 1CEA, LETI, DTBS, LISA, Université Grenoble Alpes, 2CEA, LETI, DTBS, LBAM, Université Grenoble Alpes, 3CEA, INSERM, BIG, Université Grenoble Alpes, 4CNRS, FR CNRS 3425, 5CNRS, UMR 144, Molecular Mechanisms of Intracellular Transport, PSL Research University, Institut Curie, 6TIMC-IMAG Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks. Developmental Biology Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo Renee Wei-Yan Chow*1,2,3,4, Paola Lamperti*1,2,3,4, Emily Steed1,2,3,4, Francesco Boselli1,2,3,4, Julien Vermot1,2,3,4 1Institut de Génétique et de Biologie Moléculaire et Cellulaire, 2UMR7104, Centre National de la Recherche Scientifique, 3U964, Institut National de la Santé et de la Recherche Médicale, 4Université de Strasbourg This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development. Chemistry Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine Aurélie Fossey-Jouenne1, Carine Vergne-Vaxelaire1, Anne Zaparucha1 1Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Univ Paris-Saclay Chiral amino alcohols are versatile molecules for use as scaffolds in organic synthesis. Starting from L-lysine, we synthesize amino alcohols by an enzymatic cascade reaction combining diastereoselective C-H oxidation catalyzed by dioxygenase followed by cleavage of the carboxylic acid moiety of the corresponding hydroxyl amino acid by a decarboxylase. Neuroscience SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy Joerg Nikolaus1,2, Erdem Karatekin1,2,3,4 1Department of Cellular and Molecular Physiology, Yale University School of Medicine, 2Nanobiology Institute, Yale University, 3Department of Molecular Biophysics and Biochemistry, Yale University, 4Laboratoire de Neurophotonique, Université Paris Descartes, Faculté des Sciences Fondamentales et Biomédicales, Centre National de la Recherche Scientifique (CNRS) Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured. Biology Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry Ekaterina Boyarchuk1, Philippe Robin1, Lauriane Fritsch1, Véronique Joliot1, Slimane Ait-Si-Ali1 1Epigenetics and Cell Fate, UMR 7216 CNRS, Centre National de la Recherche Scientifique CNRS - Université Paris Diderot, Sorbonne Paris Cité MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners. Neuroscience Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches Dominique Martinez1, Lotfi Arhidi1, Elodie Demondion2, Jean-Baptiste Masson3, Philippe Lucas2 1UMR 7503, Laboratoire Lorrain de Recherche en Informatique et ses Applications (LORIA), Centre National de la Recherche Scientifique (CNRS), 2UMR 1392 iEES-Paris, Institut d'Ecologie et des Sciences de l'Environnement de Paris, 3Physics of Biological Systems, Institut Pasteur We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source. Medicine The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice Ipek Yalcin1, Salim Megat1,2, Florent Barthas1,2, Elisabeth Waltisperger1, Mélanie Kremer1,2, Eric Salvat1,2,3, Michel Barrot1 1Institut des Neurosciences Cellulaires et Intégratives UPR3212, Centre National de la Recherche Scientifique, 2Université de Strasbourg, 3Hôpitaux Universitaires de Strasbourg Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The “cuff model” of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments. Medicine Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl Marie-Noëlle Delyfer1,2,3,4, Najate Aït-Ali1,2,3, Hawa Camara1,2,3, Emmanuelle Clérin1,2,3, Jean-François Korobelnik4, José-Alain Sahel1,2,3, Thierry Léveillard1,2,3 1U968, Institut National de la Santé et de la Recherche Médicale, 2UMR S 968, Université Pierre et Marie Curie, 3UMR 7210, Centre National de la Recherche Scientifique, 4Départment d'Ophtalmologie, Centre Hospitalier Universitaire de Bordeaux We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis. Neuroscience Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord Perrine Inquimbert1, Martin Moll2, Tatsuro Kohno3, Joachim Scholz2 1Département Nociception et Douleur, Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique (CNRS), 2Departments of Anesthesiology and Pharmacology, Columbia University, 3Department of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system. Neuroscience A Novel Approach for Documenting Phosphenes Induced by Transcranial Magnetic Stimulation Seth Elkin-Frankston1, Peter J. Fried1, Alvaro Pascual-Leone2, R. J. Rushmore III1, Antoni Valero-Cabré1,3 1Department of Anatomy and Neurobiology, Boston University School of Medicine, 2Department of Neurology, Beth Israel Deaconess Med Center, 3Centre de Recherche de l'institut du Cerveau et la Moelle Epinière (CRICM), Centre National de la Recherche Scientifique (CNRS) Phosphenes are transient percepts of light that can be induced by applying Transcranial Magnetic Stimulation (TMS) to visually sensitive regions of cortex. We demonstrate a standard protocol for determining the phosphene threshold value and introduce a novel method for quantifying and analyzing perceived phosphenes.