University of California, San Diego View Institution's Website 94 articles published in JoVE Chemistry Experimental Approaches for the Synthesis of Low-Valent Metal-Organic Frameworks from Multitopic Phosphine Linkers Samuel E. Griffin1, Grant P. Domecus1, Seth M. Cohen1 1Department of Chemistry and Biochemistry, University of California, San Diego Here, we describe a protocol for the synthesis of low-valent metal-organic frameworks (LVMOFs) from low-valent metals and multitopic phosphine linkers under air-free conditions. The resulting materials have potential applications as heterogeneous catalyst mimics of low-valent metal-based homogeneous catalysts. Bioengineering Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids Pegah Bagheri*1, Khang Hoang*1, Chan-yu Kuo1, Hetvi Trivedi1, Hongje Jang1, Lingyan Shi1 1Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF). Immunology and Infection RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines Dalaena E. Rivera1, Vladimir Lažetić2, Emily R. Troemel2, Robert J. Luallen1 1Department of Biology, San Diego State University, 2School of Biological Sciences, University of California, San Diego Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab. Biochemistry Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon Sriram Aiyer1, Timothy S. Strutzenberg1, Marianne E. Bowman2, Joseph P. Noel2,3, Dmitry Lyumkis1,4,5 1Laboratory of Genetics, The Salk Institute for Biological Studies, 2Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, 3Department of Chemistry and Biochemistry, University of California San Diego, 4Graduate School of Biological Sciences, Section of Molecular Biology, University of California San Diego, 5Department of Integrative Structural and Computational Biology, The Scripps Research Institute The present protocol describes a generalized and easy-to-implement scheme for tilted single-particle data collection in cryo-EM experiments. Such a procedure is especially useful for obtaining a high-quality EM map for samples suffering from preferential orientation bias due to adherence to the air-water interface. Immunology and Infection Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria Sydney R. Morrill1,2,3, Kavita Agarwal2,3, Sudeshna Saha2,3, Warren G. Lewis2,3, Nicole M. Gilbert4, Amanda L. Lewis2,3 1Division of Biology and Biomedical Sciences, Washington University School of Medicine, 2Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, 3Glycobiology Research and Training Center, UCSD, 4Department of Pediatrics, Washington University School of Medicine The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis, while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria. Medicine Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level Naseeb Kaur Malhi*1, Yingjun Luo*1, Xiaofang Tang*1, Kiran Sriram1,3, Riccardo Calandrelli2, Sheng Zhong2, Zhen Bouman Chen1,3 1Department of Diabetes Complications and Metabolism, City of Hope, 2Department of Bioengineering, University of California San Diego, 3Irell and Manella Graduate School of Biological Sciences, City of Hope The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery. Medicine Self-Administration of Drugs in Mouse Models of Feeding and Obesity Rizaldy C. Zapata1, Dinghong Zhang1, Besma Chaudry1, Olivia Osborn1 1Division of Endocrinology and Metabolism, Department of Medicine, University of California San Diego The overall goal of this procedure is to describe a method for self-administration of drugs that can be used in mouse models of feeding and obesity. Bioengineering Building a Simple and Versatile Illumination System for Optogenetic Experiments Phillip Kyriakakis1, Lourdes Fernandez de Cossio2, Patrick Wade Howard1, Sivleng Kouv1, Marianne Catanho1, Vincent J. Hu3, Robert Kyriakakis1, Molly E. Allen1, Yunhan Ma4, Marcelo Aguilar-Rivera1, Todd P. Coleman1 1Department of Bioengineering, University of California, San Diego, 2University of California, San Diego, 3Department of Mathematical Computational, and Systems biology, University of California, Irvine, 4Department of Biomedical Engineering, Duke University This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer. Immunology and Infection Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice Valerie P. O'Brien1,2,6, Matthew S. Joens3,7, Amanda L. Lewis1,2,4,8, Nicole M. Gilbert2,5 1Department of Molecular Microbiology, Washington University School of Medicine in Saint Louis, 2Center for Women’s Infectious Disease Research, Washington University School of Medicine in Saint Louis, 3Center for Cellular Imaging, Washington University School of Medicine in Saint Louis, 4Department of Obstetrics and Gynecology, Washington University School of Medicine in Saint Louis, 5Department of Pediatrics, Washington University School of Medicine in Saint Louis, 6Fred Hutchinson Cancer Research Center, 7TESCAN USA, Inc., 8University of California San Diego A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy. Biology Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis Elena Dalla Benetta*1, Duverney Chaverra-Rodriguez*1, Jason L. Rasgon2, Omar S. Akbari1 1Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, 2Department of Entomology, The Center for Infectious Disease Dynamics, The Huck Institutes of the Life Sciences, The Pennsylvania State University Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing. Engineering Fabrication and Characterization of Thickness Mode Piezoelectric Devices for Atomization and Acoustofluidics Aditya Vasan1, William Connacher1, James Friend1 1Medically Advanced Devices Laboratory, Center for Medical Devices, Department of Mechanical and Aerospace Engineering, Jacobs School of Engineering and Department of Surgery, School of Medicine, University of California San Diego, 2Department of Surgery, School of Medicine, University of California San Diego Fabrication of piezoelectric thickness mode transducers via direct current sputtering of plate electrodes on lithium niobate is described. Additionally, reliable operation is achieved with a transducer holder and fluid supply system and characterization is demonstrated via impedance analysis, laser doppler vibrometry, high-speed imaging, and droplet size distribution using laser scattering. Biology Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing Ziad Sabry1, Christina Rabeler1, Danielle Ireland1, Kevin Bayingana1, Eva-Maria S. Collins1,2 1Biology Department, Swarthmore College, 2Physics Department, UC San Diego Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout. Engineering Fabrication of Surface Acoustic Wave Devices on Lithium Niobate Jiyang Mei1, Naiqing Zhang1, James Friend1 1Medically Advanced Devices Laboratory, Center for Medical Devices and Instrumentation, Department of Mechanical and Aerospace Engineering, Jacobs School of Engineering and Department of Surgery, School of Medicine, University of California San Diego Two fabrication techniques, lift-off and wet etching, are described in producing interdigital electrode transducers upon a piezoelectric substrate, lithium niobate, widely used to generate surface acoustic waves now finding broad utility in micro to nanoscale fluidics. The as-produced electrodes are shown to efficiently induce megahertz order Rayleigh surface acoustic waves. Genetics Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus Michelle Bui1, Ming Li1, Robyn R Raban1, Nannan Liu2, Omar S Akbari1,3 1Section of Cell and Developmental Biology, University of California, San Diego, 2Department of Entomology and Plant Patholog, Auburn University, 3Tata Institute for Genetics and Society, University of California, San Diego This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector. Neuroscience Simulator Training for Endovascular Neurosurgery Yasmeen Elsawaf1, Robert C. Rennert1, Jeffrey A. Steinberg1, David R. Santiago-Dieppa1, Scott E. Olson1, Alexandar A. Khalessi1, J. Scott Pannell1 1Department of Neurosurgery, University of California Simulation of complex, high-risk procedures is critical to the education of medical trainees. A protocol for simulator-based endovascular neurosurgery training in a controlled academic environment is described. The protocol includes stepwise guidelines for trainees of varying levels, with a discussion of the advantages and limitations of this model. Genetics A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila Lin Zhang1, Kenny Dao1, Angela Kang1, Andre C. Loyola1, Robin Shang1, Jinghong Li1, Willis X. Li1 1Department of Medicine, University of California San Diego Drosophila is a widely used experimental model suitable for screening drugs with potential applications for cancer therapy. Here, we describe the use of Drosophila variegated eye color phenotypes as a method for screening small-molecule compounds that promote heterochromatin formation. Engineering Fabrication of Nanoheight Channels Incorporating Surface Acoustic Wave Actuation via Lithium Niobate for Acoustic Nanofluidics Naiqing Zhang1, James Friend1 1Medically Advanced Devices Laboratory, Center for Medical Devices, Department of Mechanical and Aerospace Engineering, Jacobs School of Engineering, and the Department of Surgery, School of Medicine, University of California San Diego We demonstrate fabrication of nanoheight channels with the integration of surface acoustic wave actuation devices upon lithium niobate for acoustic nanofluidics via liftoff photolithography, nano-depth reactive ion etching, and room-temperature plasma surface-activated multilayer bonding of single-crystal lithium niobate, a process similarly useful for bonding lithium niobate to oxides. Cancer Research Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts Sanghee Lee1,2, Danielle N. Burner1,2, Theresa R. Mendoza1,2, Michelle T. Muldong1,2, Catalina Arreola1,2, Christina N. Wu2,3, Nicholas A. Cacalano4, Anna A. Kulidjian2,5, Christopher J. Kane1,2, Christina A. M. Jamieson1,2 1Department of Urology, University of California, San Diego, 2Moores Cancer Center, University of California, San Diego, 3Department of Medicine, University of California, San Diego, 4Department of Radiation Oncology, University of California, Los Angeles, 5Department of Orthopedic Surgery, University of California, San Diego Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC. Cancer Research Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models Hervé Tiriac1,2, Randall French1,2, Andrew M. Lowy1,2 1Moores Cancer Center, University of California San Diego, 2Department of Surgery, Division of Surgical Oncology, University of California San Diego Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models. Developmental Biology A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development Renat N. Khaliullin1,2,3, Jeffrey M. Hendel1,2, Adina Gerson-Gurwitz1,2, Shaohe Wang1,4,5, Stacy D. Ochoa1,6, Zhiling Zhao1,7, Arshad Desai1,2, Karen Oegema1,2, Rebecca A. Green1,2 1Ludwig Institute for Cancer Research, San Diego, 2Department of Cellular and Molecular Medicine, University of California, San Diego, 3Recursion Pharmaceuticals, 4Biomedical Sciences Graduate Program, University of California, San Diego, 5Cell Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, 6Department of Biology, San Diego State University, 7Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80–100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis. Neuroscience Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals Ekaterina Martianova1, Sage Aronson2,3, Christophe D. Proulx1 1CERVO Brain Research Center, Department of Psychiatry and Neurosciences, Université Laval, 2Center for Neural Circuits and Behavior, Department of Neuroscience and Section of Neurobiology, Division of Biology, University of California at San Diego, 3Neurophotometrics Ltd. This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging. Genetics Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas Francesca Boscolo Sesillo1,2,3, Alessandra Sacco2 1Graduate School of Biomedical Sciences, Sanford Burnham Prebys Medical Discovery Institute, 2Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, 3Department of Obstetrics, Gynecology, and Reproductive Sciences, Division of Female Pelvic Medicine and Reconstructive Surgery, University California San Diego This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures. Neuroscience Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation Barbara Calabrese1,2, Regina M. Powers1,2, Alexandria J. Slepian1,2, Shelley Halpain1,2 1Division of Biological Sciences, University of California, San Diego, 2Sanford Consortium for Regenerative Medicine This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening. Behavior Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease Virginia Wertman1,2, Anastasia Gromova1,2,3, Albert R. La Spada1,2,4,5, Constanza J. Cortes1,2 1Department of Neurology, Duke University School of Medicine, 2Duke Center for Neurodegeneration & Neurotherapeutics, Duke University School of Medicine, 3Biomedical Sciences Graduate Program, University of California San Diego, 4Department of Neurobiology, Duke University School of Medicine, 5Department of Cell Biology, Duke University School of Medicine Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models. Medicine Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent Eric T. Geier1, Rebecca J. Theilmann2, Chantal Darquenne1, G. Kim Prisk1, Rui Carlos Sá1 1Pulmonary Imaging Laboratory, Department of Medicine, University of California, San Diego, 2Pulmonary Imaging Laboratory, Department of Radiology, University of California, San Diego Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data. Neuroscience An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons Brent Aulston1, Qing Liu1, Patrick Reilly1, Shauna H. Yuan1 1Department of Neurosciences, University of California, San Diego This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies. Engineering Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery Byungji Kim1, Michael J. Sailor1,2 1Materials Science and Engineering Program, University of California, San Diego, 2Department of Chemistry and Biochemistry, University of California, San Diego We demonstrate the synthesis of fusogenic porous silicon nanoparticles for effective in vitro and in vivo oligonucleotide delivery. Porous silicon nanoparticles are loaded with siRNA to form the core, which is coated by fusogenic lipids through extrusion to form the shell. Targeting moiety functionalization and particle characterization are included. Genetics ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes Hannah D. Rickner1, Sheng-Yong Niu1,2, Christine S. Cheng1,3 1Department of Biology, Boston University, 2Department of Computer Science and Engineering, University of California San Diego, 3Bioinformatics Program, Boston University Here, we present a protocol to perform an assay for transposase-accessible chromatin sequencing (ATAC-seq) on activated CD4+ human lymphocytes. The protocol has been modified to minimize contaminating mitochondrial DNA reads from 50% to 3% through the introduction of a new lysis buffer. Medicine Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer Shizuko Tachibana1, Chao Chen1, Oliver R. Zhang1, Sarah V. Schurr1, Cameron Hill1, Ruixia Li1, Ana M. Manso1, Jianlin Zhang1, Aleksander Andreyev2, Anne N. Murphy2, Robert S. Ross1,3, Yoshitake Cho1 1Division of Cardiology, Department of Medicine, University of California San Diego, 2Department of Pharmacology, University of California San Diego, 3Cardiology Section, Department of Medicine, Veterans Administration Healthcare, San Diego The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart. Neuroscience Disruption of Frontal Lobe Neural Synchrony During Cognitive Control by Alcohol Intoxication Ksenija Marinkovic1,2, Lauren E. Beaton1, Burke Q. Rosen1,3, Joseph P. Happer4, Laura C. Wagner1 1Department of Psychology, San Diego State University, 2Department of Radiology, University of California San Diego, 3Department of Neurosciences, University of California San Diego, 4San Diego State University/UC San Diego Joint Doctoral Program in Clinical Psychology This experiment uses an anatomically-constrained magnetoencephalography (aMEG) method to examine brain oscillatory dynamics and long-range functional synchrony during engagement of cognitive control as a function of acute alcohol intoxication. Cancer Research Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays Anagha Deshpande1, Bo Rui Chen1, Luyi Zhao1, Kelsey Saddoris1, Mayuri Kerr1, Nan Zhu2, Prashant Mali3, Aniruddha J. Deshpande1 1Tumor Initiation and Maintenance Program, Sanford Burnham Prebys Medical Discovery Institute, 2Blood Research Institute, Blood Center of Wisconsin, 3Department of Bioengineering, University of California, San Diego This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well. Biochemistry A Guide to Production, Crystallization, and Structure Determination of Human IKK1/α Smarajit Polley1,2, De-Bin Huang1, Tapan Biswas1, Gourisankar Ghosh1 1Department of Chemistry & Biochemistry, University of California, San Diego, 2Department of Biophysics, Bose Institute IκB Kinase 1/α (IKK1/α CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-κB transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein. Behavior An Electrophysiology Protocol to Measure Reward Anticipation and Processing in Children Katherine K.M. Stavropoulos1, Leslie J. Carver2 1Graduate School of Education, University of California, Riverside, 2Psychology Department, University of California, San Diego This protocol is designed to measure reward anticipation and processing in young children with and without autism. Specifically, the protocol is designed to study the neural correlates of reward during social and nonsocial conditions while controlling for reward between conditions. Environment Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior Satomi Tsuboko-Ishii1, Ronald S. Burton1 1Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior. Immunology and Infection A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells Effie E Bastounis1, Fabian E Ortega1, Ricardo Serrano2, Julie A Theriot3 1Department of Biochemistry, Stanford University School of Medicine, 2Department of Mechanical and Aerospace Engineering, University of California San Diego, 3Departments of Biochemistry, Microbiology and Immunology and Howard Hughes Medical Institute, Stanford University School of Medicine We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. Developmental Biology Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells Michael S. Yu*1,2, Sean Spiering*1, Alexandre R. Colas1 1Sanford Burnham Prebys Medical Discovery Institute, 2Department of Bioengineering, University of California at San Diego Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells. Cancer Research A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation Jayanth S. Shankara Narayanan1, Partha Ray1, Ibtehaj Naqvi2, Rebekah White1 1Moores Cancer Center, University California San Diego, 2Duke University School of Medicine Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer. Behavior Assessing Activity-based Anorexia in Mice Amanda C. Welch1, William R. Katzka1,2, Stephanie C. Dulawa1 1Department of Psychiatry, University of California, San Diego, 2University of California, Los Angeles Mice individually housed with a running wheel while given limited access to food develop reductions in food consumption and increase activity on the running wheel. This experimental phenomenon is called activity-based anorexia. This paradigm provides an experimental tool for studying the neurobiology and behaviors underlying aspects of anorexia nervosa. Neuroscience Neurovascular Network Explorer 2.0: A Simple Tool for Exploring and Sharing a Database of Optogenetically-evoked Vasomotion in Mouse Cortex In Vivo Hana Uhlirova1,2, Peifang Tian3,4, Kıvılcım Kılıç3,5, Martin Thunemann1, Vishnu B. Sridhar6, Radim Chmelik2,7, Hauke Bartsch1, Anders M. Dale1,3, Anna Devor1,3,8, Payam A. Saisan3 1Department of Radiology, University of California, San Diego, 2Central European Institute of Technology, Brno University of Technology, 3Department of Neurosciences, University of California, San Diego, 4Department of Physics, John Carroll University, 5Department of Biomedical Engineering, Boston University, 6Bioengineering Undergraduate Program, University of California, San Diego, 7Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology, 8Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School A graphical user interface for exploring and sharing a database of optogenetically-induced vascular responses in mouse somatosensory cortex in vivo measured by 2-photon microscopy is presented. It allows browsing the data, criteria-based selection, averaging, localization of measurements within a 3D volume of vasculature and exporting the data. Cancer Research Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts Jorge A. Benitez*1, Ciro Zanca*1, Jianhui Ma1, Webster K. Cavenee1,2,3, Frank B. Furnari1,2,4 1Ludwig Institute for Cancer Research, 2Moores Cancer Center, School of Medicine, University of California, San Diego, 3Department of Pathology, School of Medicine, University of California, San Diego, 4Department of Medicine, School of Medicine, University of California, San Diego Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models. Engineering Focused Ion Beam Fabrication of LiPON-based Solid-state Lithium-ion Nanobatteries for In Situ Testing Jungwoo Z. Lee*1, Thomas A. Wynn*2, Ying Shirley Meng1,2, Dhamodaran Santhanagopalan3 1Department of NanoEngineering, University of California San Diego, 2Materials Science and Engineering Program, University of California San Diego, 3Amrita Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham University A protocol for the fabrication of electrochemically active LiPON-based solid-state lithium-ion nanobatteries using a focused ion beam is presented. Developmental Biology A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes Federica Cavalera1, Mario Zanoni1, Valeria Merico1, Thi Thu Hien Bui1,2, Martina Belli1,3, Lorenzo Fassina4, Silvia Garagna1, Maurizio Zuccotti1 1Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani", University of Pavia, 2Institute of Biotechnology, University of Helsinki, 3Department of Reproductive Medicine, University of California San Diego, 4Dipartimento di Ingegneria Industriale e dell'Informazione, University of Pavia Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses. Genetics Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation Ming Li1,2, Michelle Bui1,2, Omar S. Akbari1,2 1Department of Entomology and Riverside Center of Disease Vector Research, Institute for Integrative Genome Biology, University of California, Riverside, 2Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism. Neuroscience Comprehensive Endovascular and Open Surgical Management of Cerebral Arteriovenous Malformations Robert C. Rennert1, Jeffrey A. Steinberg1, Vincent J. Cheung1, David R. Santiago-Dieppa1, Jeffrey Scott Pannell1, Alexander A. Khalessi1 1Department of Neurosurgery, University of California, San Diego Surgery is the gold standard for accessible arteriovenious malformations (AVMs), and pre-operative embolization can simplify this procedure. We describe our approach for staged endovascular embolization and open resection of AVMs, and provide a representative clinical example highlighting the advantages of a comprehensively trained neurovascular surgeon leading a multi-disciplinary clinical team. Bioengineering Pattern-based Search of Epigenomic Data Using GeNemo Alvin Zheng*1, Xiaoyi Cao*1, Sheng Zhong1 1Department of Bioengineering, University of California San Diego Unlike DNA sequence data, epigenomic data are not readily subjected to text-based searches. Presented here are the procedures to use an upgraded version of GeNemo, a web-based bioinformatics tool, to conduct pattern-based searches for similarities in epigenomic data comparing available online databases including Encyclopedia of DNA Elements with user's data. Neuroscience Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility Renny Ng1, Hui-Hao Lin1, Jing W. Wang1, Chih-Ying Su1 1Division of Biological Sciences, University of California San Diego The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones. Biology Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin Fengwu Li1, Christopher A. Adase1, Ling-juan Zhang1 1Department of Dermatology, School of Medicine, UC San Diego Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes. Neuroscience Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice Takahiro Tadokoro1, Atsushi Miyanohara1, Michael Navarro1, Kota Kamizato1, Stefan Juhas2, Jana Juhasova2, Silvia Marsala1, Oleksandr Platoshyn1, Erik Curtis3, Brandon Gabel3, Joseph Ciacci3, Nada Lukacova4, Katarina Bimbova4, Martin Marsala1,4 1Neuroregeneration Laboratory, Department of Anesthesiology, University of California, San Diego, 2Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 3Department of Neurosurgery, University of California, San Diego, 4Institute of Neurobiology, Slovak Academy of Sciences The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice. Neuroscience Generation and Long-term Maintenance of Nerve-free Hydra Cassidy M. Tran1, Sharon Fu1, Trevor Rowe2, Eva-Maria S. Collins1,2 1Division of Biological Sciences, UC San Diego, 2Physics Department, UC San Diego Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory. Developmental Biology Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments Liu Yang1, Sayantanee Paul1, Sarah DuBois-Coyne1, Phillip Kyriakakis2, Alexey Veraksa1 1Biology Department, University of Massachusetts Boston, 2Department of Bioengineering, University of California, San Diego The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS). Immunology and Infection The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections William J. Comstock1, Edwin Huh2, Reiley Weekes2, Connor Watson2, Tianyang Xu2, Pieter C. Dorrestein1, Robert A. Quinn1 1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 2Department of Mechanical and Aerospace Engineering, University of California, San Diego The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics. Biochemistry A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing Irena Roci1,2, Hector Gallart-Ayala3, Jeramie Watrous4, Mohit Jain4, Craig E. Wheelock3, Roland Nilsson1,2 1Department of Medicine, Unit of Computational Medicine, Karolinska Institutet, 2Center for Molecular Medicine, Karolinska Institutet, 3Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry 2, Karolinska Institutet, 4Department of Medicine, University of California San Diego This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry. Neuroscience Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice Simon H. Bayerl1, Melina Nieminen-Kelhä1, Thomas Broggini2, Peter Vajkoczy1, Vincent Prinz1 1Department of Neurosurgery and Center for Stroke-research Berlin (CSB), Charité-Universitätsmedizin, 2Department of Physics, University of California San Diego Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice. Genetics Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans Kentaro Noma1, Yishi Jin1,2 1Division of Biological Sciences, Section of Neurobiology, University of California in San Diego and Howard Hughes Medical Institute, 2Department of Cellular and Molecular Medicine, University of California in San Diego School of Medicine and Howard Hughes Medical Institute Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration. Behavior Treating Clinical Depression with Repetitive Deep Transcranial Magnetic Stimulation Using the Brainsway H1-coil David Feifel1, Katherine Pappas1 1Department of Psychiatry, University of California, San Diego Here we present a protocol outlining the methodology for treatment of Major Depressive Disorder using the deep Transcranial Magnetic Stimulation (dTMS) system. Cancer Research Fluorescent Orthotopic Mouse Model of Pancreatic Cancer Jonathan A. Moreno1, Antonio Sanchez2, Robert M. Hoffman3,4, Saima Nur1, Maria P. Lambros1 1College of Pharmacy, Western University of Health Sciences, 2UVP, LLC, 3Department of Surgery, School of Medicine, University of California San Diego, 4Anticancer Inc. A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here. Neuroscience Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo Emre Lacin1, Arnaud Muller2, Marian Fernando1, David Kleinfeld2,3, Paul A. Slesinger1 1Department of Neuroscience, Icahn School of Medicine at Mount Sinai, 2Department of Physics, University of California, San Diego, 3Section of Neurobiology, University of California, San Diego We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release. Bioengineering A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws Michael B. Frank1, Steven E. Naleway1, Taylor S. Wirth2, Jae-Young Jung1, Charlene L. Cheung2, Faviola B. Loera2, Sandra Medina2, Kirk N. Sato3, Jennifer R. A. Taylor4, Joanna McKittrick1,2 1Materials Science and Engineering Program, University of California, San Diego, 2Department of Mechanical and Aerospace Engineering, University of California, San Diego, 3Integrative Oceanography Division, Center for Marine Biodiversity and Conservation, Scripps Institution of Oceanography, 4Marine Biology Research Division, Scripps Institution of Oceanography A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure. Developmental Biology Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres Miguel A. Lopez-Ramirez*1,2, Charles-Félix Calvo*3, Emma Ristori1, Jean-Léon Thomas1,3, Stefania Nicoli1 1Yale Cardiovascular Research Center, Internal Medicine, Yale University, 2Department of Medicine, University of California, San Diego, 3APHP Groupe Hospitalier Pitié-Salpètrière, Université Pierre and Marie Curie Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres. Developmental Biology Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis Jingting Li1,2, George L. Sen1,2 1Department of Dermatology, UCSD Stem Cell Program, University of California, San Diego, 2Department of Cellular and Molecular Medicine, UCSD Stem Cell Program, University of California, San Diego The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis. Neuroscience Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats Jeffrey D. Moore1, Martin Deschênes2, David Kleinfeld1 1Department of Physics 0374, University of California, San Diego, 2Department of Psychiatry and Neuroscience, Centre de Recherche de l’Université Laval Robert-Giffard This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations. Biology Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies Matthias Garten1, Sophie Aimon2, Patricia Bassereau1, Gilman E. S. Toombes3 1Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie, 2Kavli Institute for Brain and Mind, University of California, San Diego, 3Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institute of Health The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology. Biology Methods for Skin Wounding and Assays for Wound Responses in C. elegans Suhong Xu1, Andrew D. Chisholm1 1Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity. Immunology and Infection Generation of Human Alloantigen-specific T Cells from Peripheral Blood Burhan P Jama1, Gerald P Morris1 1Department of Pathology, University of California, San Diego This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands. Environment Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology Andreas Florian Haas1, Ben Knowles1, Yan Wei Lim1, Tracey McDole Somera2, Linda Wegley Kelly1, Mark Hatay1, Forest Rohwer1 1Department of Biology, San Diego State University, 2Scripps Institution of Oceanography, University of California San Diego Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments. Neuroscience Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis Sylvia Neumann1, George E. Campbell*1, Lukasz Szpankowski*2,3, Lawrence S.B. Goldstein2,4, Sandra E. Encalada1 1Department of Molecular and Experimental Medicine, Dorris Neuroscience Center, The Scripps Research Institute, 2Department of Cellular and Molecular Medicine, University of California San Diego, 3Department of Bioengineering, University of California San Diego, 4Department of Neurosciences, University of California San Diego School of Medicine Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins. Medicine A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts Shaoping Hou1, Armin Blesch2, Paul Lu3,4 1Spinal Cord Research Center, Department of Neurobiology & Anatomy, Drexel University College of Medicine, 2Spinal Cord Injury Center, Heidelberg University Hospital, 3Veterans Administration Medical Center, San Diego, CA, 4Department of Neurosciences, University of California, San Diego We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats. Neuroscience Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons Xiaobei Zhao1, Yue Zhou2, April M. Weissmiller1, Matthew L. Pearn3,4, William C. Mobley1, Chengbiao Wu1 1Department of Neurosciences, University of California, San Diego, 2School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, 3Department of Anesthesiology, University of California, San Diego, 4VA San Diego Healthcare System Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons. Neuroscience Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury Paul Lu1,2, Lori Graham2, Yaozhi Wang2, Di Wu2, Mark Tuszynski1,2 1Veterans Administration Medical Center, San Diego, 2Department of Neurosciences, University of California, San Diego Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances. Medicine Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters Ana D. Lopez1, Ayse G. Kayali1, Alberto Hayek1, Charles C. King1 1Pediatric Diabetes Research Center, Department of Pediatrics, University of California, San Diego A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described. The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions. Biology Cell Labeling and Injection in Developing Embryonic Mouse Hearts Emilye Hiriart*1, Patrick van Vliet*2, Ralf J. Dirschinger2, Sylvia M. Evans2, Michel Puceat1 1INSERM UMR-910, Aix-Marseille University, 2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development. Medicine Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope Joanna L. Thomas1,2, Justin Dumouchel2, Jinghong Li2, Jenna Magat2, Dana Balitzer3, Timothy D. Bigby1,2 1Medical and Research Services, VA San Diego Healthcare System, 2Department of Medicine, University of California, San Diego, 3School of Medicine, University of California, San Diego We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques. Neuroscience Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography Sarah H. Shahmoradian1, Mauricio R. Galiano2, Chengbiao Wu3, Shurui Chen4, Matthew N. Rasband2, William C. Mobley3, Wah Chiu4 1Department of Molecular Physiology and Biophysics, Baylor College of Medicine, 2Department of Neuroscience, Baylor College of Medicine, 3Department of Neuroscience, University of California at San Diego, 4National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale. Chemistry Conducting Miller-Urey Experiments Eric T. Parker1, James H. Cleaves2,3, Aaron S. Burton4, Daniel P. Glavin5, Jason P. Dworkin5, Manshui Zhou1, Jeffrey L. Bada6, Facundo M. Fernández1 1School of Chemistry and Biochemistry, Georgia Institute of Technology, 2Earth-Life Science Institute, Tokyo Institute of Technology, 3Institute for Advanced Study, 4Astromaterials Research and Exploration Science Directorate, NASA Johnson Space Center, 5Goddard Center for Astrobiology, NASA Goddard Space Flight Center, 6Geosciences Research Division, Scripps Institution of Oceanography, University of California at San Diego The Miller-Urey experiment was a pioneering study regarding the abiotic synthesis of organic compounds with possible relevance to the origins of life. Simple gases were introduced into a glass apparatus and subjected to an electric discharge, simulating the effects of lightning in the primordial Earth’s atmosphere-ocean system. The experiment was conducted for one week, after which, the samples collected from it were analyzed for the chemical building blocks of life. Behavior Handwriting Analysis Indicates Spontaneous Dyskinesias in Neuroleptic Naïve Adolescents at High Risk for Psychosis Derek J. Dean1, Hans-Leo Teulings2, Michael Caligiuri3, Vijay A. Mittal1 1Department of Psychology and Neuroscience, Center for Neuroscience, University of Colorado Boulder, 2NeuroScript LLC, 3Department of Psychiatry, University of California, San Diego Handwriting analysis software significantly improves upon existing instruments measuring movement disorders. Individuals at risk for psychosis and healthy controls completed handwriting tasks to test for dyskinesia. Results suggest that youth at risk for psychosis exhibit dyskinesia and that handwriting analysis could significantly contribute to wider dissemination of early identification efforts Neuroscience A Single-fly Assay for Foraging Behavior in Drosophila Orel A. Zaninovich1, Susy M. Kim1, Cory R. Root1,2, David S. Green1,3, Kang I. Ko1,4, Jing W. Wang1 1Neurobiology Section, Division of Biological Sciences, University of California-San Diego, 2Department of Neuroscience, Columbia University, 3Dart NeuroScience, 4School of Dental Medicine, University of Pennsylvania In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. Bioengineering Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos. Michelle T. Juarez1, Rachel A. Patterson2, Wilson Li2, William McGinnis2 1Sophie Davis School of Biomedical Education, The City College of New York, 2Cell & Developmental Biology, University of California, San Diego The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models. Biology Isolation and Culture of Neonatal Mouse Cardiomyocytes Elisabeth Ehler1, Thomas Moore-Morris2, Stephan Lange2 1 Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays. Immunology and Infection Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria Tal Danino*1, Arthur Prindle*2, Jeff Hasty2,3,4, Sangeeta Bhatia1,5,6,7,8 1Health Sciences and Technology, Massachusetts Institute of Technology, 2Department of Bioengineering, University of California, San Diego, 3Biocircuits Institute, University of California, San Diego, 4Molecular Biology Section, Division of Biological Science, University of California, San Diego, 5Broad Institute of Harvard and MIT, 6Department of Medicine, Brigham and Women's Hospital, 7Electrical Engineering and Computer Science and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 8Howard Hughes Medical Institute The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting. Medicine Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells Charles Zhang1, Arthur T. Suckow2, Steven D. Chessler1,3 1Pediatric Diabetes Research Center, University of California, San Diego, 2Janssen Research & Development, 3Department of Medicine, University of California, San Diego Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method—adapted from a coculture model of synaptogenesis—for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed. Biology A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice Timo Rieg1,2 1Division of Nephrology-Hypertension, Department of Medicine, University of California, San Diego, 2San Diego VA Healthcare System Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model. Bioengineering Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering Shivanjali Joshi-Barr1, Jerome V. Karpiak2, Yogesh Ner1, Jessica H. Wen3, Adam J. Engler3, Adah Almutairi1,2 1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 2Biomedical Sciences Program, University of California, San Diego, 3Department of Bioengineering, University of California, San Diego Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues Biology Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution Miriam Cohen1, Nissi M. Varki1, Mark D. Jankowski2, Pascal Gagneux1 1Department of Cellular and Molecular Medicine, University of California, San Diego, 2Biosecurity and Public Health, Los Alamos National Laboratory Unfixed frozen tissue samples embedded in Optimal Cutting Temperature medium (OCT) can be used to study natural distribution and glycosylation of secreted mucus. In this approach tissue processing is minimal and the natural presentation of glycolipids, mucins and glycan-epitopes is preserved. Tissue sections can be analyzed by immunohistochemistry using fluorescence or chromogenic detection. Medicine Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry Wenhong Zhu1, Richard L. Gallo2,3, Chun-Ming Huang2,3,4 1Sanford-Burnham Medical Research Institute, 2Division of Dermatology, University of California, San Diego, 3VA San Diego Healthcare Center, 4Moores Cancer Center, University of California, San Diego Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable. Medicine Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR Sonia Apaza1, Susan Espetia1, Robert H. Gilman1,2, Sonia Montenegro3, Susana Pineda3, Fanny Herhold1, Romeo Pomari1, Margaret Kosek2, Nancy Vu1, Mayuko Saito1,2,4 1Laboratorio de Investigación y Desarrollo (LID), Universidad Peruana Cayetano Heredia, 2Bloomberg School of Public Health, Johns Hopkins University, 3Laboratorio de Diagnostico Molecular, Facultad de Medicina, University of Concepcion,Chile, 4University of California San Diego School of Medicine A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children’s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed. Neuroscience A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain Andy Y. Shih1, Celine Mateo1, Patrick J. Drew2,3, Philbert S. Tsai1, David Kleinfeld1,4 1Department of Physics, University of California, San Diego, 2Department of Engineering Science and Mechanics, Pennsylvania State University, 3Department of Neurosurgery, Pennsylvania State University, 4Section of Neurobiology, University of California, San Diego We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy. Neuroscience Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines Kenric J. Hoegler1, Martin Distel2, Reinhard W. Köster3, John H. Horne1 1Department of Biology, Pace University, 2Cellular and Molecular Medicine, University of California, San Diego, 3Division of Cell Biology and Cell Physiology, Zoological Institute, Braunschweig University of Technology The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb. Medicine Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling Tatsuya J. Arai1,2, G. Kim Prisk1,3, Sebastiaan Holverda1, Rui Carlos Sá1, Rebecca J. Theilmann3, A. Cortney Henderson1, Matthew V. Cronin3, Richard B. Buxton3, Susan R. Hopkins1,3 1Medicine, University of California San Diego - UCSD, 2Bioengineering, University of California San Diego - UCSD, 3Radiology, University of California San Diego - UCSD A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment. Bioengineering Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy Diana E. Schlamadinger1, Judy E. Kim1 1Chemistry and Biochemistry, University of California San Diego - UCSD This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence. Bioengineering Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering Sonya Seif-Naraghi1, Jennifer Singelyn1, Jessica DeQuach1, Pamela Schup-Magoffin1, Karen Christman1 1University of California, San Diego Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described. Neuroscience Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window Pedro Cabrales1, Leonardo J. M. Carvalho2 1Bioengineering, University of California, San Diego, 2La Jolla Bioengineering Institute Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation. Neuroscience A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia Stephan J. Guyenet1, Stephanie A. Furrer2, Vincent M. Damian1, Travis D. Baughan2, Albert R. La Spada*3, Gwenn A. Garden*2 1Department of Biochemistry, University of Washington, 2Department of Neurology, University of Washington, 3Division of Genetics, Departments of Pediatrics and Cellular and Molecular Medicine, and the Institute for Genomic Medicine, University of California, San Diego - Rady Children’s Hospital We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time. Biology Spinal Cord Electrophysiology Allyn Meyer1, Benjamin W. Gallarda1,2, Samuel Pfaff1, William Alaynick1 1The Salk Institute for Biological Studies, Howard Hughes Medical Institute and Gene Expression Laboratory, 2Biology Graduate Program, University of California San Diego - UCSD A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies. Biology Localized RNAi and Ectopic Gene Expression in the Medicinal Leech Orit Shefi1, Claire Simonnet2, Alex Groisman2, Eduardo R Macagno1 1Division of Biological Sciences, University of California San Diego - UCSD, 2Department of Physics, University of California San Diego - UCSD In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.