Institut National de la Sante et de la Recherche Medicale 6 articles published in JoVE Biochemistry Quaternary Structure Modeling Through Chemical Cross-Linking Mass Spectrometry: Extending TX-MS Jupyter Reports Hamed Khakzad1,2, Swen Vermeul3, Lars Malmström4,5,6 1Equipe Signalisation Calcique et Infections Microbiennes, Ecole Normale Supérieure Paris-Saclay, 2Institut National de la Santé et de la Recherche Médicale, 3Scientific IT Services, ETH Zurich, 4Institute for Computational Science, University of Zurich, 5S3IT, University of Zurich, 6Division of Infection Medicine, Department of Clinical Sciences Lund, Faculty of Medicine, Lund University Targeted cross-linking mass spectrometry creates quaternary protein structure models using mass spectrometry data acquired using up to three different acquisition protocols. When executed as a simplified workflow on the Cheetah-MS web server, the results are reported in a Jupyter Notebook. Here, we demonstrate the technical aspects of how the Jupyter Notebook can be extended for a more in-depth analysis. 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. Developmental Biology Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells Karim Ben M'Barek1,2,3, Walter Habeler1,2,3, Alexandra Plancheron1,2,3, Mohamed Jarraya4, Olivier Goureau5, Christelle Monville1,2 1U861, I-Stem, Association Française contre les Myopathies (AFM), Institut National de la Santé et de la Recherche Médicale (INSERM), 2I-Stem, Association Française contre les Myopathies (AFM), Centre pour L’Etude des Cellules Souches (CECS), 4Banque de tissus humain, Hôpital Saint Louis, Assistance Publique - Hôpitaux de Paris (AP-HP), 5Sorbonne Université, INSERM, CNRS, Institut de la Vision, F-75012 We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models. 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. Neuroscience Purification of Mouse Brain Vessels Anne-Cécile Boulay1,2,3, Bruno Saubaméa4, Xavier Declèves4, Martine Cohen-Salmon1,2,3 1Collège de France, Center for Interdisciplinary Research in Biology (CIRB), Centre National de la Recherche Scientifique CNRS, Unité Mixte de Recherche 7241, Institut National de la Santé et de la Recherche Médicale, 2Centre Interdisciplinaire de Recherche en Biologie, 3MEMOLIFE Laboratory of Excellence and Paris Science, Lettre Research University, 4Université Paris Descartes, Faculté de Pharmacie, Université Paris Diderot We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes. 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.
