Emory University View Institution's Website 33 articles published in JoVE Environment Visualizing Field Data Collection Procedures of Exposure and Biomarker Assessments for the Household Air Pollution Intervention Network Trial in India Karthikeyan D. Rajamani1, Sankar Sambandam1, Krishnendu Mukhopadhyay1, Naveen Puttaswamy1, Gurusamy Thangavel1, Durairaj Natesan1, Rengaraj Ramasamy1, Saritha Sendhil1, Amudha Natarajan1, Vigneswari Aravindalochan1, Ajay Pillarisetti2, Michael Johnson3, Joshua Rosenthal*4, Kyle Steenland5, Ricardo Piedhrahita3, Jennifer Peel6, Maggie L. Clark6, Dana Boyd Barr5, Sarah Rajkumar6, Bonnie Young6, Shirin Jabbarzadeh7, Ghislaine Rosa8, Miles Kirby9, Lindsay J. Underhill10, Anaite Diaz-Artiga11, Amy Lovvorn5, William Checkley12, Thomas Clasen5, Kalpana Balakrishnan1 1Department of Environmental Health Engineering, ICMR Center for Advanced Research on Air Quality, Climate and Health, Faculty of Public Health, Sri Ramachandra Institute of Higher Education and Research (Deemed University), 2Division of Environmental Health Sciences, University of California, Berkeley, 3Berkeley Air Monitoring Group, 4Division of International Epidemiology and Population Studies, National Institutes of Health, 5Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, 6Department of Environmental and Radiological Health Sciences, Colorado State University, 7Department of Biostatistics and Informatics, Rollins School of Public Health, Emory University, 8Department of Disease Control, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, 9Department of Global Health & Population, Harvard, T.H. Chan School of Public Health, 10Cardiovascular Division, Washington University School of Medicine, Washington University, 11Centro de Estudios en Salud, Universidad del Valle de Guatemala, 12Division of Pulmonary and Critical Care, School of Medicine, Johns Hopkins University We detail the consistent, high-quality procedures used throughout air and biological sampling processes at Indian field sites during a large randomized controlled trial. Insights gathered from the oversight of applications of innovative technologies, adapted for exposure assessment in rural regions, enable better field data collection practices with more reliable outcomes. Biology Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions Megan N. Taylor1, Satya Spandana Boddu2, Nic M. Vega1,2 1Department of Biology, Emory University, 2Department of Physics, Emory University This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms. Biology Contribution of the Na+/K+ Pump to Rhythmic Bursting, Explored with Modeling and Dynamic Clamp Analyses Ricardo Javier Erazo-Toscano1,2, Parker J. Ellingson2, Ronald L. Calabrese*1, Gennady S. Cymbalyuk*2 1Department of Biology, Emory University, 2Neuroscience Institute, Georgia State University Presented here is a method for investigation of the roles of the Na+/K+ pump and persistent Na+ current in leech heart interneurons using dynamic clamp. Bioengineering DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells Rong Ma1, Anna V. Kellner2, Yuesong Hu1, Brendan R. Deal1, Aaron T. Blanchard2, Khalid Salaita1,2 1Department of Chemistry, Emory University, 2Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces >4.7pN in real-time and can integrate forces over time. Behavior Behavioral Assessment of Visual Function via Optomotor Response and Cognitive Function via Y-Maze in Diabetic Rats Kaavya Gudapati*1,2, Anayesha Singh*1,3, Danielle Clarkson-Townsend1,4, Stephen Q. Phillips1, Amber Douglass1, Andrew J. Feola1,2, Rachael S. Allen1,2 1Center for Visual and Neurocognitive Rehabilitation, Atlanta VA Medical Center, 2Department of Biomedical Engineering, Georgia Institute of Technology, 3Department of Neuroscience, Emory University, 4Gangarosa Department of Environmental Health, Emory University Neural degeneration in both eyes and brain as a result of diabetes can be observed through behavioral tests carried out on rodents. The Y-maze, a measure of spatial cognition, and the optomotor response, a measure of visual function, both provide insight into potential diagnoses and treatments. Bioengineering In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography Rachael S. Allen1,2, Katie Bales1,3, Andrew Feola1,2, Machelle T Pardue1,2,3 1Center of Excellence for Visual and Neurocognitive Rehabilitation, Atlanta Veterans Affairs Medical Center, 2Department of Biomedical Engineering, Georgia Institute of Technology, 3Department of Ophthalmology, Emory University Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Neuroscience Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles Daniela Ávila-González1, Larry J. Young2, Francisco Camacho1, Raúl G. Paredes1,3, Néstor F. Díaz4, Wendy Portillo1 1Departamento de Neurobiología Conductual y Cognitiva, Instituto de Neurobiología, Universidad Nacional Autónoma de México, 2Silvio O. Conte Center for Oxytocin and Social Cognition, Center for Translational Social Neuroscience, Department of Psychiatry and Behavioral Sciences, Yerkes National Primate Research Center, Emory University, 3Escuela Nacional de Estudios Superiores Juriquilla, Universidad Nacional Autónoma de México, 4Departamento de Fisiología y Desarrollo Celular, Instituto Nacional de Perinatología We established the conditions to culture neural progenitor cells from the subventricular zone and dentate gyrus of the adult brain of prairie voles, as a complementary in vitro study, to analyze the sex-dependent differences between neurogenic niches that could be part of functional plastic changes associated with social behaviors. Medicine An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat Daniella Corporan1, Takanori Kono1, Daisuke Onohara1, Muralidhar Padala1,2 1Structural Heart Research and Innovation Laboratory, Carlyle Fraser Heart Center, Emory University Hospital Midtown, 2Division of Cardiothoracic Surgery, Joseph P. Whitehead Department of Surgery, Emory University School of Medicine A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance. Genetics Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury Zhihong Yuan1,2, Brahmchetna Bedi1, Ruxana T. Sadikot1,2 1Department of Veterans Affairs, Atlanta VAMC, 2Division of Pulmonary and Critical Care Medicine, Department of Medicine, Emory University Mice exposed to intraperitoneal LPS secrete exosomes in Broncho-alveolar lavage (BAL) fluid that are packaged with miRNAs. Using a co-culture system, we show that exosomes released in the BAL fluid disrupt expression of tight junction proteins in bronchial epithelial cells and increase expression of pro-inflammatory cytokines that accentuate lung injury. Biochemistry Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells Joshua T. Maxwell1, Chin-Hsien Tsai1, Tahmina A. Mohiuddin2, Jennifer Q. Kwong1 1Department of Pediatrics, Emory University School of Medicine, 2Emory College of Arts and Sciences, Emory University Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM. Medicine Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung Ashley E. Van Avermaete1, Phi T. Trac1, Theresa W. Gauthier1, My N. Helms2 1Department of Pediatrics, Neonatology Division, Emory Children's Center, Emory University, 2Department of Internal Medicine, Pulmonary Division, University of Utah We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology. Biology Cryosectioning Method for Microdissection of Murine Colonic Mucosa Attila E. Farkas1, Christian Gerner-Smidt1, Loukia Lili1, Asma Nusrat1, Christopher T. Capaldo1 1Epithelial Pathobiology and Mucosal Inflammation Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells. Immunology and Infection Pseudomonas aeruginosa Induced Lung Injury Model Varsha Suresh Kumar1, Ruxana T. Sadikot2, Jeanette E. Purcell3, Asrar B. Malik1, Yuru Liu1 1Department of Pharmacology, University of Illinois College of Medicine, University of Illinois at Chicago, 2Section of Pulmonary and Critical Care Medicine, Atlanta VAMC, Emory University, 3Biologic Resources Lab, University of Illinois at Chicago We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant. Immunology and Infection A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses Daniel T. Claiborne*1, Jessica L. Prince*1, Eric Hunter1,2 1Emory Vaccine Center at Yerkes National Primate Research Center, Emory University, 2Department of Pathology and Laboratory Medicine, Emory University HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus. Immunology and Infection Culturing and Maintaining Clostridium difficile in an Anaerobic Environment Adrianne N. Edwards1, Jose M. Suárez1, Shonna M. McBride1 1Department of Microbiology and Immunology, Emory University School of Medicine Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation. Biology A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation Jon Waters1, Vishal Dhere1, Adam Benjamin1, Arvind Sekar1, Archana Kumar1, Sampath Prahalad2, David T. Okou1, Subra Kugathasan1 1Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Emory University School of Medicine and Children's Health Care of Atlanta, 2Division of Rheumatology, Department of Pediatrics, Emory University School of Medicine and Children's Health Care of Atlanta We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood. Neuroscience Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium Karolina Piotrowska-Nitsche1,2, Tamara Caspary1 1Department of Human Genetics, Emory University School of Medicine, 2Department of Experimental Embryology, IGAB Polish Academy of Sciences Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium Medicine Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs Thais Federici1, Carl V. Hurtig1, Kentrell L. Burks1, Jonathan P. Riley1, Vibhor Krishna2, Brandon A. Miller1, Eric A. Sribnick1, Joseph H. Miller3, Natalia Grin1, Jason J. Lamanna1,4,5, Nicholas M. Boulis1 1Department of Neurosurgery, Emory University, 2Department of Neuroscience, Medical University of South Carolina, 3Division of Neurosurgery, University of Alabama, Birmingham, 4Department of Biomedical Engineering, Georgia Institute of Technology, 5Department of Biomedical Engineering, Emory University Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application. Neuroscience A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds Lukas A. Hoffmann1,2, Conor W. Kelly1,3, David A. Nicholson1,2, Samuel J. Sober1 1Department of Biology, Emory University, 2Neuroscience Graduate Program, Emory University, 3Program in Neuroscience and Behavioral Biology, Emory University We describe the design and assembly of miniaturized headphones suitable for replacing a songbird’s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning. Engineering Compact Quantum Dots for Single-molecule Imaging Andrew M. Smith1, Shuming Nie1,2 1Department of Biomedical Engineering, Emory University, 2Department of Chemistry, Georgia Institute of Technology We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells. Biology Selective Capture of 5-hydroxymethylcytosine from Genomic DNA Yujing Li1, Chun-Xiao Song2, Chuan He2, Peng Jin1 1Department of Human Genetics, Emory University School of Medicine, 2Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing. Bioengineering Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases David R. Myers*1,2,3,4, Yumiko Sakurai*1,2,3,4, Reginald Tran1,2,3,4, Byungwook Ahn1,2,3,4, Elaissa Trybus Hardy1,2,3,4, Robert Mannino1,2,3,4, Ashley Kita1,2,3,4, Michelle Tsai1,2,3,4, Wilbur A. Lam1,2,3,4 1Department of Pediatrics, Emory University School of Medicine, 2Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 3Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, 4Winship Cancer Institute of Emory University A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (<30 μm) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases. Immunology and Infection Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine Duke Geem1, Oscar Medina-Contreras1, Wooki Kim2, Clifton S. Huang1, Timothy L. Denning1,2 1Department of Pediatrics, Emory University, 2Department of Pathology & Laboratory Medicine, Emory University Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies. Immunology and Infection Murine Model of Allergen Induced Asthma Aravind T. Reddy1, Sowmya P. Lakshmi1, Raju C. Reddy1 1Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, Emory University and Atlanta VA Medical Center Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology. Biology Direct Tracheal Instillation of Solutes into Mouse Lung My N. Helms1,2, Edilson Torres-Gonzalez2,3, Preston Goodson1, Mauricio Rojas2,3 1Department of Physiology, Emory University, 2Center for Respiratory Health, Emory University, 3Department of Medicine, Emory University Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Neuroscience Simultaneous fMRI and Electrophysiology in the Rodent Brain Wen-ju Pan1,2, Garth Thompson1,2, Matthew Magnuson1,2, Waqas Majeed1,2, Dieter Jaeger3, Shella Keilholz1,2 1Biomedical Engineering, Emory University, 2Biomedical Engineering, Georgia Institute of Technology, 3Biology, Emory University We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal. Medicine A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium Douglas Nam1, Chih-Wen Ni2, Amir Rezvan1, Jin Suo2, Klaudia Budzyn1, Alexander Llanos1, David G. Harrison1, Don P. Giddens2, Hanjoong Jo1,2,3 1Department of Medicine, Division of Cardiology, Emory University, 2Coulter Department of Biomedical Engineering, Georgia Tech and Emory University, 3Department of Bioinspired Science, Ewha Womans University This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA. Biology How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs) Chadwick M. Hales1,2, John D. Rolston2,3, Steve M. Potter2 1Department of Neurology, Emory University School of Medicine, 2Coulter Department of Biomedical Engineering, Laboratory for Neuroengineering, Georgia Institute of Technology and Emory, University School of Medicine, 3Emory University School of Medicine This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction. Neuroscience SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval Rebecca F. Rosen1, Yasushi Tomidokoro2, Jorge A. Ghiso3, Lary C. Walker1,4 1Yerkes National Primate Research Center, Emory University, 2Department of Neurology, Institute of Clinical Medicine, Tsukuba University, 3Department of Pathology, New York University School of Medicine, 4Department of Neurology, Emory University We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the Aβ peptide. Using this protocol, we consistently detect monomeric and multimeric Aβ in cortical tissue from humans with Alzheimer's pathology. Biology Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography Stephanie A. Zlatic1, Pearl V. Ryder1, Gloria Salazar2, Victor Faundez1 1Department of Cell Biology, Emory University, 2Department of Medicine, Division of Cardiology, Emory University The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation. Biology In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts Marie Cross1, Maureen Powers1 1Department of Cell Biology, Emory University Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes. Biology Preparation and Fractionation of Xenopus laevis Egg Extracts Marie K. Cross1, Maureen Powers1 1Department of Cell Biology, Emory University Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations. Biology Obtaining Eggs from Xenopus laevis Females Marie K. Cross1, Maureen Powers1 1Department of Cell Biology, Emory University The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.