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Université Paris-Saclay

9 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos
Audrey Bernut 1,2, Christian Dupont 1,2, Alain Sahuquet 1, Jean-Louis Herrmann 3, Georges Lutfalla 1, Laurent Kremer 1,2
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.

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Engineering

Growth and Electrostatic/chemical Properties of Metal/LaAlO3/SrTiO3 Heterostructures
Diogo Castro Vaz 1, Edouard Lesne 1,2, Anke Sander 1, Hiroshi Naganuma 1,3, Eric Jacquet 1, Jacobo Santamaria 1,4, Agnès Barthélémy 1, Manuel Bibes 1
1Unité Mixte de Physique CNRS/Thales, Université Paris-Saclay, 2Max Planck Institut für Mikrostrukturphysik, 3Department of Applied Physics, Tohoku University, 4Instituto de Magnetismo Aplicado, Universidad Complutense de Madrid

We fabricate metal/LaAlO3/SrTiO3 heterostructures using a combination of pulsed laser deposition and in situ magnetron sputtering. Through magnetotransport and in situ X-ray photoelectron spectroscopy experiments, we investigate the interplay between electrostatic and chemical phenomena of the quasi two-dimensional electron gas formed in this system.

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Immunology and Infection

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes
Violaine Dubois *1, Laura Laencina *1, Anouchka Bories 1, Vincent Le Moigne 1, Alexandre Pawlik 2, Jean-Louis Herrmann 1, Fabienne Girard-Misguich 1
12I, UVSQ, INSERM UMR1173, Université Paris-Saclay, 2Institut Pasteur, Unité de Pathogénomique Mycobactérienne

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

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Genetics

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs
Erwin L. van Dijk 1, Evangelia Eleftheriou 1, Claude Thermes 1
1Institute for Integrative Biology of the Cell, UMR9198, CNRS CEA Univ Paris-Sud, Université Paris-Saclay

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2'-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

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Biology

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe
Aline Stedman 1,3, Antonin Levy 1,4, Philippe J. Sansonetti 1,2,5, Giulia Nigro 1,6
1Molecular Microbial Pathogenesis Unit, Institut Pasteur, 2Chaire de Microbiologie et Maladies Infectieuses, Collège de France, 3Institut de Biologie Paris Seine (IBPS) - Developmental Biology Unit, Sorbonne Université, CNRS UMR7622, INSERM U1156, 4Molecular Radiotherapy, INSERM U1030, Gustave Roussy, Université Paris-Saclay, 5The Center for Microbes, Development and Health, Institut Pasteur Shanghai and Chinese Academy of Sciences, 6Microenvironment and Immunity Unit, Institut Pasteur

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

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JoVE Core

Postural Organization of Gait Initiation for Biomechanical Analysis Using Force Platform Recordings
Arnaud Simonet 1,2,3, Arnaud Delafontaine 2,3,4, Paul Fourcade 2,3, Eric Yiou 2,3
1LADAPT Loiret, Centre de Soins de Suite et de Réadaptation, 2CIAMS, Université Paris-Saclay, 3CIAMS, Université d'Orléans, 4Université Libre de Bruxelles, Belgique

This paper describes the material and method developed to investigate the postural organization of gait initiation. The method is based on force platform recordings and on the direct principle of mechanics to compute center of gravity and center of pressure kinematics.

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Bioengineering

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates
Mahnaz Sabeti Azad *1, Angelo Cardoso Batista *1, Jean-Loup Faulon 1, Chase L. Beisel 2,3, Jerome Bonnet 4, Manish Kushwaha 1
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.

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Biology

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays
Vera Filipa Monteiro-Cardoso 1,2, Romain Le Bars 1,3, Francesca Giordano 1,2
1Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, 2Inserm U1280, 3Imagerie-Gif, Light Microscopy Facility, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at MCSs.

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Developmental Biology

Generating Retinal Injury Models in Xenopus Tadpoles
Karine Parain *1, Alicia Donval *1, Albert Chesneau *1, Jing Xian Lun 1, Caroline Borday 1, Muriel Perron 1
1Paris-Saclay Institute of Neuroscience, CNRS, Université Paris-Saclay

We have developed several protocols to induce retinal damage or retinal degeneration in Xenopus laevis tadpoles. These models offer the possibility of studying retinal regeneration mechanisms.

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