Universitat-Basel View Institution's Website 22 articles published in JoVE Immunology and Infection Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications Stephanie Mauti1, Monique Léchenne2, Service Naïssengar3, Abdallah Traoré4, Vessaly Kallo5,6, Casimir Kouakou7, Emmanuel Couacy-Hymann7, Morgane Gourlaouen8, Céline Mbilo9,10, Pati Patient Pyana11, Enos Madaye3, Ibrahima Dicko4, Pascal Cozette1, Paola De Benedictis8, Hervé Bourhy1, Jakob Zinsstag9,10, Laurent Dacheux1 1Unit Lyssavirus Epidemiology and Neuropathology, National Reference Center for Rabies and WHO Collaborating Center for Reference and Research on Rabies, Institut Pasteur, 2Environment and Sustainability Institute, University of Exeter, Penryn Campus, 3Institut de Recherche en Elevage pour le Développement, 4Laboratoire Central Vétérinaire, 5 We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping. Biochemistry Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells Dominik P. Buser1, Martin Spiess1 1Biozentrum, University of Basel Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells. Medicine Measurements of Motor Function and Other Clinical Outcome Parameters in Ambulant Children with Duchenne Muscular Dystrophy Sara Nagy1,2, Simone Schmidt1,2, Patricia Hafner1,3, Andrea Klein1,4,5, Daniela Rubino-Nacht1, Vanya Gocheva1, Oliver Bieri6, Carole Vuillerot7, Ulrike Bonati1,2, Dirk Fischer1,2,3 1Division of Pediatric Neurology, University Children's Hospital Basel (UKBB), University of Basel, 2Department of Neurology, University Hospital Basel, University of Basel, 3Division of Neurology, University Clinic of Medicine, Cantonal Hospital Baselland, Bruderholz, 4Division of Pediatric Neurology, CHUV, 5Division of Neuropaediatrics, Inselspital, University Children's Hospital Bern, 6Department of Radiology, Division of Radiological Physics, University Hospital Basel, University of Basel, 7Hospices Civils de Lyon, Hôpital Femme-Mère-Enfant, L'Escale, Service de Médecine Physique et de Réadaptation Pédiatrique The aim of this study is to present the most reliable clinical outcome measures and their correlations with quantitative muscle MRI in ambulant patients with Duchenne muscular dystrophy. Neuroscience Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice Charlotte Amalie Navntoft1,2, Jeremy Marozeau1, Tania Rinaldi Barkat2 1Hearing Systems Group, Department of Electrical Engineering, Technical University of Denmark, 2Brain and Sound Lab, Department of Biomedicine, Basel University Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response. Environment A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci Simon Blaser1,2,3, Hanspeter Diem4, Andreas von Felten4, Morgan Gueuning1, Michael Andreou5, Neil Boonham6,7, Jennifer Tomlinson6, Pie Müller2,3, Jürg Utzinger2,3, Beatrice Frey1, Jürg E. Frey1, Andreas Bühlmann8 1Department of Method Development and Analytics, Agroscope, 2Swiss Tropical and Public Health Institute, 3University of Basel, 4Swiss Federal Plant Protection Service, Federal Office for Agriculture, 5OptiGene Limited, 6Fera Science Limited, 7School of Natural and Environmental Sciences, Newcastle University, 8Department of Plants and Plant Products, Agroscope This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports. Biology Miniaturized Sample Preparation for Transmission Electron Microscopy Claudio Schmidli*1,2, Luca Rima*1, Stefan A. Arnold*1,2, Thomas Stohler1, Anastasia Syntychaki1, Andrej Bieri1, Stefan Albiez1, Kenneth N. Goldie1, Mohamed Chami3, Henning Stahlberg1, Thomas Braun1 1Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, 2Swiss Nanoscience Institute, University of Basel, 3BioEM lab, Biozentrum, University of Basel An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics. Immunology and Infection Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach Katarina Radulovic1, Rachel Mak’Anyengo1, Berna Kaya1, Anna Steinert1, Jan Hendrik Niess1,2 1Department of Biomedicine, University of Basel, 2Division of Gastroenterology and Hepatology, University Hospital of Basel Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon. Medicine An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers Lukas Kaufmann*1, Mohammedyaseen Syedbasha*1, Dominik Vogt1, Yvonne Hollenstein1, Julia Hartmann1, Janina E. Linnik1,2,3, Adrian Egli1,4 1Applied Microbiology Research, Department of Biomedicine, University of Basel, 2Department of Biosystems Science and Engineering, ETH Zurich, 3Swiss Institute of Bioinformatics, 4Clinical Microbiology, University Hospital Basel The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition. Biology 3' End Sequencing Library Preparation with A-seq2 Georges Martin1, Ralf Schmidt1, Andreas J. Gruber1, Souvik Ghosh1, Walter Keller1, Mihaela Zavolan1,2 1Computational and Systems Biology, Biozentrum, University of Basel, 2Swiss Institute of Bioinformatics, Biozentrum, University of Basel This protocol describes a method for mapping pre-mRNA 3' end processing sites. Biochemistry Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay Xinzhou Zhu1, Andrea Zelmer1, Sven Wellmann1,2 1University Children's Hospital Basel (UKBB), University of Basel, 2Department of Clinical Research, University of Basel Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm. Developmental Biology An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery Martin H. M. Sailer1, Durga Sarvepalli2, Catherine Brégère3, Urs Fisch3, Marin Guentchev4, Michael Weller6, Raphael Guzman3, Bernhard Bettler1, Arkasubhra Ghosh*2, Gregor Hutter*5 1Dept. of Biomedicine, Pharmacenter, University of Basel, 2Molecular Signalling and Gene Therapy, Narayana Nethralaya Foundation, Narayana Health City, 3Brain Ischemia and Regeneration, Department of Biomedicine, University Hospital Basel, 4Department of Neurosurgery, Klinikum Idar-Oberstein, 5Department of Neurosurgery and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 6Department of Neurology, Laboratory of Molecular Neuro Oncology, University Hospital of Zurich Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery. Immunology and Infection Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells Alain Casanova*1, Shyan H. Low*1, Mario Emmenlauer*1,4, Raquel Conde-Alvarez*1,3, Suzana P. Salcedo2, Jean-Pierre Gorvel2, Christoph Dehio1 1Focal Area Infection Biology, Biozentrum, University of Basel, 2Centre d’Immunologie de Marseille-Luminy, Université de la Méditérannée UM2, INSERM U1104 CNRS UM7280, 3Departmento de Microbiologìa and Instituto de Salud Tropical, Universidad de Navarra, 4BioDataAnalysis GmbH Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols. Biology Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays Stefan K. Drexler1, Francesco Brogna1, Adrien Vinet1, Jean Pieters1 1Biozentrum, University of Basel The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development. Chemistry An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions Mohameedyaseen Syedbasha1, Janina Linnik1,2,3, Deanna Santer4, Daire O'Shea5, Khaled Barakat4,6, Michael Joyce4, Nina Khanna7, D. Lorne Tyrrell4, Michael Houghton4, Adrian Egli1,8 1Applied Microbiology Research, Department of Biomedicine, University of Basel, 2Department of Biosystems Science and Engineering, ETH Zurich, and Swiss Institute of Bioinformatics, 3Swiss Institute of Bioinformatics, 4Li Ka Shing Institute for Virology, University of Alberta, 5Regional Infectious Diseases Unit, University of Edinburgh, 6Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 7Infection Biology, Department of Biomedicine, University of Basel, 8Clinical Microbiology, University Hospital Basel The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions. Immunology and Infection Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization Etienne Meunier1, Petr Broz1 1Focal Area Infection Biology, Biozentrum, University of Basel We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization. Biology Comet Assay as an Indirect Measure of Systemic Oxidative Stress Lei Fang1,2, Albert Neutzner1,2, Stephanie Turtschi1, Josef Flammer1, Maneli Mozaffarieh1 1Department of Ophthalmology, University of Basel, 2Department of Biomedicine, University of Basel Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress. Neuroscience The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures Sophorn Chip1, Xinzhou Zhu2, Josef P. Kapfhammer1 1Anatomical Institute, Department of Biomedicine Basel, University of Basel, 2Department of Neonatology, University Children's Hospital (UKBB), University of Basel A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury. Immunology and Infection Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes Andreas Kühbacher1,2,3, Edith Gouin1,2,3, Jason Mercer4, Mario Emmenlauer5, Christoph Dehio5, Pascale Cossart1,2,3, Javier Pizarro-Cerdá1,2,3 1Unité des Interactions Bactéries Cellules, Pasteur Institute, 2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020, 4Institute of Biochemistry, ETH Zürich, 5Focal Area Infection Biology, Biozentrum, University of Basel Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells. Immunology and Infection Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children Peiling Yap1,2, Thomas Fürst1,2, Ivan Müller1,2, Susi Kriemler1,2, Jürg Utzinger1,2, Peter Steinmann1,2 1Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute, Basel, Switzerland, 2University of Basel, Basel, Switzerland Chronic infection with soil-transmitted helminths (STHs) causes malabsorption, stunting, and wasting in the growing child. Hence, it is plausible that these infections also reduce the physical fitness of children. Here, we visualize two techniques for the diagnosis of STHs and the 20-meter shuttle run test for assessing children's physical fitness. Neuroscience A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells Anne-Sophie Arnold1, Martine Christe1, Christoph Handschin1 1Biozentrum, University of Basel Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established. Neuroscience The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures Josef P. Kapfhammer1, Olivia S. Gugger1 1Anatomical Institute, Department of Biomedicine, University of Basel We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development. Biology PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins Markus Hafner1, Markus Landthaler2, Lukas Burger3, Mohsen Khorshid3, Jean Hausser4, Philipp Berninger4, Andrea Rothballer1, Manuel Ascano1, Anna-Carina Jungkamp2, Mathias Munschauer2, Alexander Ulrich1, Greg S. Wardle1, Scott Dewell5, Mihaela Zavolan3, Thomas Tuschl1 1Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University, 2Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, 3Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 4Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 5Genomics Resource Center, Rockefeller University RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.