University of Bonn View Institution's Website 20 articles published in JoVE Biology Cost-Efficient Transcriptomic-Based Drug Screening Jacqueline Leidner1,2, Heidi Theis3, Michael Kraut3, Alice Ragogna1, Marc Beyer1,3,4, Joachim Schultze1,2,3, Jonas Schulte-Schrepping1,2, Caterina Carraro1,2, Lorenzo Bonaguro1,2 1Systems Medicine, German Center for Neurodegenerative Diseases (DZNE) eV., 2Genomics & Immunoregulation, LIMES Institute, University of Bonn, 3PRECISE Platform for Single Cell Genomics and Epigenomics, DZNE and University of Bonn, 4Immunogenomics & Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE) eV. This protocol describes a workflow from ex vivo or in vitro cell cultures to transcriptomic data pre-processing for cost-effective transcriptome-based drug screening. Medicine A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence Leon von der Emde*1, Merten Mallwitz*1, Frank G. Holz1, Kenneth R. Sloan2, Thomas Ach1 1Department of Ophthalmology, University of Bonn, 2Department of Ophthalmology and Visual Sciences, University of Alabama at Birmingham This research describes a workflow to determine and compare autofluorescence levels from individual regions of interest (e.g., drusen and subretinal drusenoid deposits in age-related macular degeneration [AMD]) while accounting for varying autofluorescence levels throughout the fundus. Biochemistry Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC Philipp Gaugler1, Verena Gaugler1, Marília Kamleitner1, Gabriel Schaaf1 1Department of Plant Nutrition, Institute of Crop Science and Resource Conservation, University of Bonn Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants. Chemistry Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs Stefanie Lindner*1, Christian Steinebach*2, Hannes Kehm1, Martin Mangold2, Michael Gütschow2, Jan Krönke1 1Department of Internal Medicine III, University Hospital Ulm, 2Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn This work describes the synthesis and characterization of a pomalidomide-based, bifunctional homo-PROTAC as a novel approach to induce ubiquitination and degradation of the E3 ubiquitin ligase cereblon (CRBN), the target of thalidomide analogs. Chemistry Synthesis and Structure Determination of µ-Conotoxin PIIIA Isomers with Different Disulfide Connectivities Pascal Heimer*1, Thomas Schmitz*1, Charlotte A. Bäuml*1, Diana Imhof1 1Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies. Medicine Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo Tim R. Glowka1, Pascal Paschenda2, Michael Czaplik3, Jörg C. Kalff1, René H. Tolba2 1Department of Surgery, University of Bonn, 2Institute for Laboratory Animal Science & Experimental Surgery, RWTH Aachen University, 3Department of Anesthesiology, RWTH Aachen University Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments. Medicine A Closed-chest Model to Induce Transverse Aortic Constriction in Mice Lars Eichhorn*1, Christina Katharina Weisheit*1, Christopher Gestrich2, Konrad Peukert1, Georg Daniel Duerr2, Muhammad Ajmal Ayub3, Felix Erdfelder1, Florian Stöckigt4 1Department of Anaesthesiology, University Hospital Bonn, 2Department of Cardiac Surgery, University Hospital Bonn, 3Institute of Physiology 2, University of Bonn, 4Department of Medicine - Cardiology, University Hospital Bonn Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings. Developmental Biology Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells Olivia Krefft*1, Ammar Jabali*1, Vira Iefremova1, Philipp Koch1,2,3, Julia Ladewig1 1Institute of Reconstructive Neurobiology, University of Bonn, 2Central Institute of Mental Health, University of Heidelberg/Medical Faculty Mannheim, 3Hector Institute for Translational Brain Research (HITBR gGmbH) Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps. Developmental Biology Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes Alexandra Raulf*1, Nadine Voeltz*1, Daniel Korzus1, Bernd K. Fleischmann1, Michael Hesse1 1Institute of Physiology I, University of Bonn To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations. Medicine A Model to Simulate Clinically Relevant Hypoxia in Humans Lars Eichhorn1, Florian Kessler1, Volker Böhnert2, Felix Erdfelder1, Anja Reckendorf3, Rainer Meyer4, Richard K. Ellerkmann1 1Department of Anaesthesiology and Intensive Care Medicine, University Hospital of Bonn, 2Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, 3Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, 4Institute of Physiology 2, University of Bonn Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2). Medicine A Step by Step Protocol for Subretinal Surgery in Rabbits Sami Al-Nawaiseh1, Fabian Thieltges1, Zengping Liu1,2, Claudine Strack1, Ralf Brinken1, Norbert Braun3, Marc Wolschendorf3, Arvydas Maminishkis5, Nicole Eter4, Boris V. Stanzel1,6 1Department of Ophthalmology, University of Bonn, 2Department of Ophthalmology, National University of Singapore, 3Geuder AG, 4Department of Ophthalmology, University of Münster, 5Section on Epithelial and Retinal Physiology and Disease, National Eye Institute/National Institutes of Health, 6Surgical Retina Department, Singapore National Eye Centre Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient. Developmental Biology Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells Caroline Kubaczka1, Hubert Schorle1 1Department of Developmental Pathology, Institute of Pathology, University of Bonn Medical School Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2. Neuroscience Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells Sandra Meyer1,2, Philipp Wörsdörfer1, Katharina Günther1, Marc Thier2,3, Frank Edenhofer1,2 1Institute of Anatomy and Cell Biology, University of Würzburg, 2Institute of Reconstructive Neurobiology, University of Bonn, 3German Cancer Research Center, Heidelberg Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages. Neuroscience Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration Christina Müller1,2, Stefan Remy1,2 1NRW Research Group 'Dendritic integration in the CNS', Department of Epileptology, University of Bonn, 2Neuronal Networks Group, Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE) In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision. Medicine Orthotopic Small Bowel Transplantation in Rats Koji Kitamura*1,2, Martin W. von Websky*1, Ichiro Ohsawa1, Azin Jaffari1, Thomas C. Pech1, Tim Vilz1, Sven Wehner1, Shinji Uemoto2, Joerg C. Kalff1, Nico Schaefer1 1Department of Surgery, University of Bonn, Germany, 2Department of Surgery, Kyoto University Hospital Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation. Medicine Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation Tim O. Vilz1, Marcus Overhaus1, Burkhard Stoffels1, Martin von Websky1, Joerg C. Kalff1, Sven Wehner1 1Department of Surgery, University of Bonn Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options. Medicine A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion Se-Chan Kim1, Olaf Boehm1, Rainer Meyer2, Andreas Hoeft1, Pascal Knüfermann1, Georg Baumgarten1 1Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Germany, 2Institute of Physiology, University of Bonn, Germany Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy. Neuroscience Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro Gabriela Oana Bodea1, Sandra Blaess1 1Institute of Reconstructive Neurobiology, University of Bonn A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons. Neuroscience Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells Kristian Pajtler*1, Anna Bohrer*1, Jochen Maurer2, Hubert Schorle2, Alexander Schramm1, Angelika Eggert1, Johannes Hubertus Schulte1 1Department of Pediatric Oncology, University Children's Hospital Essen, 2Department of Developmental Pathology, Bonn Medical School, Institute of Pathology To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes. Biology Engineering Cell-permeable Protein Bernhard Münst1, Christoph Patsch1, Frank Edenhofer1 1Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, University of Bonn - Life & Brain Center and Hertie Foundation Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.
