Wake Forest University View Institution's Website 12 articles published in JoVE Biochemistry Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice Liza-Anastasia DiCecco*1,2, Samantha Berry*1, G. M. Jonaid*1,3, Maria J. Solares1,4, Liam Kaylor1,4, Jennifer L. Gray5, Carol Bator6, William J. Dearnaley1, Michael Spilman7, Madeline J. Dressel-Dukes8, Kathryn Grandfield2, Sarah M. McDonald Esstman9, Deborah F. Kelly1,5,6 1Department of Biomedical Engineering, Pennsylvania State University, 2Department of Materials Science and Engineering, McMaster University, 3Bioinformatics and Genomics Graduate Program, Huck Institutes of the Life Sciences, Pennsylvania State University, 4Molecular, Cellular, and Integrative Biosciences Graduate Program, Huck Institutes of the Life Sciences, Pennsylvania State University, 5Materials Research Institute, Pennsylvania State University, 6Huck Institutes of the Life Sciences, Pennsylvania State University, 7Applications team, Direct Electron, 8Application Scientist, Protochips, Inc., 9Department of Biology, Wake Forest University Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy. Biology In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig Benjamin T. Corona1, Jarrod A. Call2,3, Matthew Borkowski4, Sarah M. Greising5 1School of Medicine, Wake Forest University, 2Department of Kinesiology, University of Georgia, 3Regenerative Bioscience Center, University of Georgia, 4Aurora Scientific Inc., 5School of Kinesiology, University of Minnesota The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs. Genetics A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe Bahjat F. Marayati1,2, James B. Pease1, Ke Zhang1,2 1Department of Biology, Wake Forest University, 2Center for Molecular Communication and Signaling, Wake Forest University We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and selective for mutations that often occur at a single genomic locus. The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug. Medicine Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model Travis K. Mann-Gow*1, Troy R. Larson*1, Chrissie T. Wøien2, Thomas M. Andersen2, Karl-Erik Andersson3,4, Peter Zvara1,2 1Department of Surgery, University of Vermont, 2Department of Urology and Biomedical Laboratory, University of Southern Denmark, 3Wake Forest Institute for Regenerative Medicine, Wake Forest University, 4Institute for Clinical Medicine, Aarhus University This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization. Engineering High Temperature Fabrication of Nanostructured Yttria-Stabilized-Zirconia (YSZ) Scaffolds by In Situ Carbon Templating Xerogels Sixbert P. Muhoza1, Matthew A. Cottam1, Michael D. Gross1,2 1Department of Chemistry, Wake Forest University, 2Center for Energy, Environment, and Sustainability, Wake Forest University A protocol for fabricating porous, nanostructured yttria-stabilized-zirconia (YSZ) scaffolds at temperatures between 1,000 °C and 1,400 °C is presented. Immunology and Infection Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations Subhashini Arimilli1, Brad E. Damratoski1, Prasad G.L.2 1Department of Microbiology and Immunology, Wake Forest University Health Sciences, 2R&D Department, R.J. Reynolds Tobacco Company Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure. Medicine Generation of Alginate Microspheres for Biomedical Applications Omaditya Khanna1, Jeffery C. Larson2, Monica L. Moya3, Emmanuel C. Opara4, Eric M. Brey2,5 1Department of Chemical and Biological Engineering, Illinois Institute of Technology, 2Department of Biomedical Engineering, Illinois Institute of Technology, 3Department of Biomedical Engineering, University of California at Irvine, 4Wake Forest Institute for Regenerative Medicine and Department of Biomedical Engineering, Wake Forest University Health Sciences, 5Research Service, Hines Veterans Administration Hospital In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes. Biology A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy Alenka Lovy1, Anthony J.A. Molina2, Fernanda M. Cerqueira3, Kyle Trudeau3, Orian S. Shirihai3 1Department of Neuroscience, Center for Neuroscience Research, Tufts School of Medicine, 2Department of Internal Medicine, Geriatrics & Gerontology, Wake Forest Baptist Medical Center, 3Department of Medicine, Boston University Medical Center Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process. Medicine DNA Vector-based RNA Interference to Study Gene Function in Cancer Daniel B. Stovall1, Meimei Wan1, Qiang Zhang1, Purnima Dubey2, Guangchao Sui1 1Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University School of Medicine, 2Department of Pathology and Comprehensive Cancer Center, Wake Forest University School of Medicine RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo. Medicine Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography Lakeesha E. Bridges1, Cicely L. Williams1, Mildred A. Pointer1,2,3, Emmanuel M. Awumey1,2,3 1Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, 2Department of Biology, North Carolina Central University, Durham, 3Department of Physiology & Pharmacology and Hypertension & Vascular Research Center, Wake Forest University School of Medicine An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 μm, independently. Medicine In Vivo Canine Muscle Function Assay Martin K. Childers1, Robert W. Grange2, Joe N. Kornegay3 1Department of Neurology and Wake Forest Institute for Regenerative Medicine, Wake Forest University, 2Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute and State University, 3Departments of Pathology and Laboratory Medicine and Neurology and the Gene Therapy Center , University of North Carolina-Chapel Hill We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions. Neuroscience DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices Gail K. Seabold1, James B. Daunais2, Andrew Rau3, Kathleen A. Grant3, Veronica A. Alvarez1 1Section on Neuronal Structure, Laboratory for Integrative Neuroscience, NIAAA, NIH, 2Department Physiology and Pharmacology, Wake Forest University Health Sciences, 3Oregon National Primate Research Center, Division of Neuroscience, Oregon Health and Science University We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.