Johns Hopkins Bloomberg School of Public Health 10 articles published in JoVE Biology Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes Mabel Taracena1,2, Catherine Hunt1, Pamela Pennington3, Deborah Andrew4,5, Marcelo Jacobs-Lorena5,6, Ellen Dotson1, Michael Wells5,7,8 1Division of Parasitic Diseases and Malaria, Entomology Branch, Centers for Disease Control and Prevention, 2Department of Entomology, Cornell University, 3Centro de Estudios en Biotecnologia, Universidad del Valle de Guatemala, 4Department of Cell Biology, Johns Hopkins School of Medicine, 5Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, 6Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health and Malaria Research Institute, 7Department of Cell Biology, Johns Hopkins School of Medicine, 8Biomedical Sciences Department, Idaho College of Osteopathic Medicine The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection. Biochemistry A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure Jenny L. Carey1, Olivia H. Cox1, Fayaz Seifuddin1, Leonard Marque1, Kellie L.K. Tamashiro1, Peter P. Zandi1,3, Gary S. Wand1,2, Richard S. Lee1 1Departments of Psychiatry and Behavioral Sciences, Johns Hopkins School of Medicine, 2Department of Medicine, Johns Hopkins School of Medicine, 3Department of Mental Health, Johns Hopkins School of Public Health Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats. Genetics Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II Grace H. Hwang*1, Jessica L. Hopkins*1, Philip W. Jordan1 1Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis. Developmental Biology A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model Stephen R Wellard*1, Jessica Hopkins*1, Philip W. Jordan1 1Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis. Immunology and Infection Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body Quigly Dragotakes1, Arturo Casadevall1 1Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects. Biology Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy Shuo Li*1,2, Sumana Raychaudhuri*1, Shigeki Watanabe1,3 1Department of Cell Biology, Johns Hopkins School of Medicine, 2Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, 3Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine We developed a novel technique in electron microscopy, "flash-and-freeze," that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail. Biology In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila Ho Lam Tang1, Ho Man Tang1, Ming Chiu Fung2, J. Marie Hardwick1 1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, 2School of Life Sciences, Chinese University of Hong Kong To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli. Biology Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis Ho Lam Tang1,2,3, Ho Man Tang1,2,3, J. Marie Hardwick1, Ming Chiu Fung2 1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, 2School of Life Sciences, Chinese University of Hong Kong, 3Center for Cell Dynamics, Department of Biological Chemistry, Johns Hopkins University School of Medicine The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis. Biology Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity Nathachit Limjunyawong1, Jonathan Fallica1, Amritha Ramakrishnan2, Kausik Datta3, Matthew Gabrielson1, Maureen Horton1, Wayne Mitzner1 1Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, 2Department of International Health, Johns Hopkins University Bloomberg School of Public Health, 3Department of Medicine, Johns Hopkins University School of Medicine We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans. Medicine Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers Nancy A. Hodgson1, Douglas A. Granger2,3,4 1Center for Innovative Care in Aging, Johns Hopkins University School of Nursing, 2Institute for Interdisciplinary Salivary Bioscience Research, Arizona State University, 3Johns Hopkins University School of Nursing, 4Johns Hopkins University Bloomberg School of Public Health We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.
