David Geffen School of Medicine at UCLA 16 articles published in JoVE Biology Determining Basal Energy Expenditure and the Capacity of Thermogenic Adipocytes to Expend Energy in Obese Mice Michael Shum1, Zhiqiang Zhou2, Marc Liesa2,3,4 1Department of Molecular Medicine, Faculty of Medicine, Universite Laval, 2Department of Medicine, Division of Endocrinology, David Geffen School of Medicine at UCLA, 3Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, 4Molecular Biology Institute at UCLA This manuscript describes a protocol to measure the basal metabolic rate and the oxidative capacity of thermogenic adipocytes in obese mice. Biology Rodent Estrous Cycle Monitoring Utilizing Vaginal Lavage: No Such Thing As a Normal Cycle Hannah Robert1, Lindsay Ferguson1, Olivia Reins2, Tiffany Greco1, Mayumi L. Prins1, Michael Folkerts2 1Department of Neurosurgery, Brain Injury Research Center, UCLA, David Geffen School of Medicine, 2Department of Psychology, Pepperdine University, Seaver College This study details the crucial factors to consider in experimental designs involving female rats. In a larger sense, these data serve to decrease stigma and assist in the development of more inclusive diagnostic and intervention tools. Chemistry Optimization of Radiochemical Reactions using Droplet Arrays Alejandra Rios1,2, Travis S. Holloway2,3, Jia Wang2,4, R. Michael van Dam1,2,3,4 1Physics and Biology in Medicine Interdepartmental Graduate Program, University of California Los Angeles (UCLA), 2Crump Institute of Molecular Imaging, UCLA, 3Department of Molecular & Medical Pharmacology, David Geffen School of Medicine, 4Department of Bioengineering, UCLA This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents. Cancer Research Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane Moe Ishihara*1, Junhui Hu*1, Xiaoyu Zhang2, YongHyeon Choi3, Anthony Wong4, Celine Cano-Ruiz5, Rongwei Zhao6, Ping Tan7, Jonathan L. Tso1, Lily Wu1,8 1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, 3Department of Bioengineering, Hanyang University, 4Department of Ecology and Evolutionary Biology, University of California, Los Angeles, 5Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, 6School of Life Sciences, Beijing Normal University, 7Department of Urology, West China Hospital, Sichuan University, 8Department of Urology, David Geffen School of Medicine, University of California, Los Angeles Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases. Behavior A Step-by-Step Implementation of DeepBehavior, Deep Learning Toolbox for Automated Behavior Analysis Sanjay Shukla1, Ahmet Arac1 1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles The purpose of this protocol is to utilize pre-built convolutional neural nets to automate behavior tracking and perform detailed behavior analysis. Behavior tracking can be applied to any video data or sequences of images and is generalizable to track any user-defined object. Developmental Biology Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish Junjie Chen1, René R. Sevag Packard1,2,3 1Division of Cardiology, Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, 2Ronald Reagan UCLA Medical Center, 3Veterans Affairs West Los Angeles Medical Center The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions. Biology In Vivo Surface Electrocardiography for Adult Zebrafish Yali Zhao1, Morgan Yun1, Sean A. Nguyen1, Michelle Tran1, Thao P. Nguyen1 1Department of Medicine, Division of Cardiology, the Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish. Chemistry Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production Eric Schopf*1, Christopher M. Waldmann*2,3, Jeffrey Collins2,4, Christopher Drake1, Roger Slavik2,3, R. Michael van Dam2,4 1SOFIE, 2Department of Molecular & Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles (UCLA), 3Ahmanson Translational Imaging Division, University of California, Los Angeles (UCLA), 4Crump Institute for Molecular Imaging, University of California, Los Angeles (UCLA) Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [18F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use. Cancer Research Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer Larry C. Cheng*1,2, Zhen Li*3, Thomas G. Graeber4, Nicholas A. Graham5, Justin M. Drake1,2,3,6,7 1Graduate Program in Cellular and Molecular Pharmacology, School of Graduate Studies, Rutgers University, The State University of New Jersey, 2Graduate Program in Quantitative Biomedicine, School of Graduate Studies, Rutgers University, The State University of New Jersey, 3Department of Medicine, Division of Medical Oncology, Rutgers Robert Wood Johnson Medical School, 4Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, Jonsson Comprehensive Cancer Center, UCLA Metabolomics Center, and California NanoSystems Institute, David Geffen School of Medicine, University of California, Los Angeles, 5Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, 6Pharmacology, Rutgers Robert Wood Johnson Medical School, 7Cancer Metabolism and Growth Program, Rutgers Cancer Institute of New Jersey This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics. Biochemistry Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue David Jelinek1,2, Aimee Flores1,3, Melanie Uebelhoer1, Vincent Pasque2, Kathrin Plath2,3, M. Luisa Iruela-Arispe1,3, Heather R. Christofk2,3, William E. Lowry1,3, Hilary A. Coller1,2,3 1Department of Molecular, Cell and Developmental Biology, UCLA, 2Department of Biological Chemistry, David Geffen School of Medicine, 3Molecular Biology Institute Interdepartmental Program, UCLA We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples. Behavior A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy Elizabeth M. Gibbs1,2, Rachelle H. Crosbie-Watson1,2,3,4 1Department of Integrative Biology and Physiology, University of California, 2Center for Duchenne Muscular Dystrophy, University of California, 3Department of Neurology, David Geffen School of Medicine, University of California, 4Molecular Biology Institute, University of California This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals. Immunology and Infection High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection Kristel Lourdault1,2, James Matsunaga1,2, Karen V. Evangelista1,2, David A. Haake1,2,3,4 1Veterans Affairs Greater Los Angeles Healthcare System, 2Departments of Medicine, David Geffen School of Medicine at University of California Los Angeles, 3Departments of Urology, David Geffen School of Medicine at University of California Los Angeles, 4Departments of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies. Medicine Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice Minju Song1, Sol Kim1, Terresa Kim1, Sil Park1, Ki-Hyuk Shin1,2, Mo Kang1,2, No-Hee Park1,2,3, Reuben Kim1,2 1The Shapiro Family Laboratory of Viral Oncology and Aging Research, The UCLA School of Dentistry, 2UCLA Jonsson Comprehensive Cancer Center, 3David Geffen School of Medicine at UCLA We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo. Immunology and Infection Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes Kate L. Erickson*1, Michelle J. Hickey*1, Yuki Kato2, Colin C. Malone1, Geoffrey C. Owens1, Robert M. Prins1,2, Linda M. Liau1,4,5, Noriyuki Kasahara3,5, Carol A. Kruse1,4,5 1Department of Neurosurgery, UCLA David Geffen School of Medicine, 2Department of Molecular and Medical Pharmacology, UCLA David Geffen School of Medicine, 3Department of Medicine, UCLA David Geffen School of Medicine, 4Brain Research Institute, UCLA David Geffen School of Medicine, 5Jonsson Comprehensive Cancer Center, UCLA David Geffen School of Medicine We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality. Medicine Combined In vivo Optical and µCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice Nicholas M. Bernthal1, Brad N. Taylor2, Jeffrey A. Meganck2, Yu Wang3, Jonathan H. Shahbazian3, Jared A. Niska1, Kevin P. Francis2, Lloyd S. Miller3,4 1Orthopaedic Hospital Research Center, Orthopaedic Hospital Department of Orthopaedic Surgery, David Geffen School of Medicine at University of California, Los Angeles (UCLA), 2PerkinElmer, 3Department of Dermatology, Johns Hopkins University School of Medicine, 4Department of Medicine, Division of Infectious Diseases, Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine Combined optical and μCT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone. Immunology and Infection A Protocol for Analyzing Hepatitis C Virus Replication Songyang Ren*1, Deisy Contreras*1, Vaithilingaraja Arumugaswami1,2 1Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery, Cedars-Sinai Medical Center, 2Department of Surgery, David Geffen School of Medicine at UCLA Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.