Universite Libre de Bruxelles 8 articles published in JoVE Biology Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli Thierry Oms*1, Tatjana Schlechtweg*1, Julien Cayron1, Laurence Van Melderen1 1Bacterial Genetics and Physiology, Département de Biologie Moléculaire, Faculté des Sciences, Université Libre de Bruxelles (ULB) Antibiotic persistence describes the ability of small subpopulations within a sensitive isogenic population to transiently tolerate high doses of bactericidal antibiotics. The present protocol combines approaches to characterize the antibiotic persistence phenotype at the molecular and cellular levels after exposing Escherichia coli to lethal doses of ofloxacin. Developmental Biology Gene Expression Analyses in Human Follicles Melody Devos*1, Joana Dias Nunes*1, Isabelle Demeestere1,2 1Research Laboratory on Human Reproduction, Université libre de Bruxelles (ULB), 2Fertility Clinic, Erasme Hospital, Université libre de Bruxelles (ULB) Here, we describe a protocol outlining how to isolate human ovarian follicles from frozen-thawed cortical tissue to perform gene expression analyses. Neuroscience Postural Organization of Gait Initiation for Biomechanical Analysis Using Force Platform Recordings Arnaud Simonet1,2,3, Arnaud Delafontaine2,3,4, Paul Fourcade2,3, Eric Yiou2,3 1LADAPT Loiret, Centre de Soins de Suite et de Réadaptation, 2CIAMS, Université Paris-Saclay, 3 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. Biology Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method Sema Elif Eski1, Christine Dubois2, Sumeet Pal Singh1 1IRIBHM, Université Libre de Bruxelles (ULB), 2Medecine Faculty, Université Libre de Bruxelles (ULB) Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq. Biochemistry X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050 Raphael Dutoit1,2, Nathalie Brandt2, Dany Van Elder1, Louis Droogmans1 1Laboratory of Microbiology, Department of Molecular Biology, Université Libre de Bruxelles, 2Labiris Institut de Recherche This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant. Chemistry [(DPEPhos)(bcp)Cu]PF6: A General and Broadly Applicable Copper-Based Photoredox Catalyst Hajar Baguia1, Christopher Deldaele1, Bastien Michelet1, Jérôme Beaudelot1,2, Cédric Theunissen1, Cécile Moucheron2, Gwilherm Evano1 1Laboratoire de Chimie Organique, Service de Chimie et PhysicoChimie Organiques, Université libre de Bruxelles (ULB), 2Laboratoire de Chimie Organique et Photochimie, Service de Chimie et PhysicoChimie Organiques, Université libre de Bruxelles (ULB) Detailed and general protocols are presented for the synthesis of [(DPEPhos)(bcp)Cu]PF6, a general copper-based photoredox catalyst, and for its use in synthetic chemistry for the direct arylation of C-H bonds in (hetero)arenes and radical cyclization of organic halides. Biology Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry Abdulkadir Abakir1, Lee Wheldon2, Andrew D. Johnson3, Patrick Laurent1, Alexey Ruzov4 1Laboratoire de Neurophysiologie (CP601), ULB Neuroscience Institute (UNI), Université Libre de Bruxelles, 2Medical Molecular Sciences, Centre for Biomolecular Sciences, University of Nottingham, 3School of Life Sciences, University of Nottingham, 4Division of Cancer and Stem Cells, Centre for Biomolecular Sciences, School of Medicine, University of Nottingham Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification. Immunology and Infection A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes Soizic Garaud*1,2, Chunyan Gu-Trantien*1,2, Jean-Nicolas Lodewyckx1,2, Anaïs Boisson1,2, Pushpamali De Silva1,2, Laurence Buisseret1,2, Edoardo Migliori1,2, Myriam Libin3, Céline Naveaux1,2, Hugues Duvillier1,4, Karen Willard-Gallo1,2 1Molecular Immunology Unit, Université Libre de Bruxelles, 2Institut Jules Bordet, Université Libre de Bruxelles, 3Institut d'Immunologie Médicale, Université Libre de Bruxelles, 4Flow Cytometry Core Facility, Université Libre de Bruxelles This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.
