Virginia Tech 13 articles published in JoVE Immunology and Infection Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito Matteo Vitale*1, Jiangtao Liang*2, Igor Sharakhov2, Federica Bernardini1 1Department of Life Sciences, Sir Alexander Fleming Building, Imperial College London, 2Department of Entomology, Virginia Tech Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production. Biology Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions Teshani Kumarage1,2, Julie Nguyen1, Rana Ashkar1,2 1Department of Physics, Virginia Tech, 2Center for Soft Matter and Biological Physics, Virginia Tech This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur. Behavior Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury Oleksii Shandra1, Stefanie Robel1,2,3 1Fralin Biomedical Research Institute at Virginia Tech Carilion, 2School of Neuroscience, Virginia Tech, 3Department of Biological Sciences, Virginia Tech This systematic protocol describes a new animal model of post-traumatic epilepsy after repetitive mild traumatic brain injury. The first part details steps for traumatic brain injury induction using a modified weight drop model. The second part provides instructions on the surgical approach for single- and multi-channel electroencephalographic data acquisition systems. Medicine Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response John A. Richey1, Holly Sullivan-Toole2, Marlene Strege1, Corinne Carlton1, Dylan McDaniel3, Matthew Komelski4, Amy Epperley5, Hongxiao Zhu6, Irving C. Allen3,7 1Department of Psychology, Virginia Tech, 2Graduate Program in Translational Biology, Medicine and Health, Virginia Tech, 3Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 4Department of Human Development and Family Science, Virginia Tech, 5Wellness Center, Virginia Tech, 6Department of Statistics, Virginia Tech, 7Department of Basic Science Education, Virginia Tech Carilion School of Medicine Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response. Biology Bovine Mammary Gland Biopsy Techniques Veridiana L. Daley1,4, Charles Dye2, Sophie H. Bogers3, R. Michael Akers4, Francisco C. Rodriguez3, John P. Cant5, John Doelman5, Peter Yoder4, Karan Kumar2, Dane Webster6, Mark D. Hanigan4 1National Animal Nutrition Program, a National Research Support Project (NRSP-9), Department of Animal and Food Sciences, University of Kentucky, 2School of Performing Arts, Virginia Tech, 3Department of Large Animal Clinical Sciences, The Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 4Department of Dairy Science, Virginia Tech, 5Department of Animal Biosciences, University of Guelph, 6School of Visual Arts, Virginia Tech This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations. Bioengineering Quantifying Intermembrane Distances with Serial Image Dilations Tristan Raisch1,2, Momina Khan1, Steven Poelzing1,2 1Virginia Tech Carilion Research Institute, Virginia Tech, 2Translational Biology, Medicine and Health, Virginia Tech The purpose of this algorithm is to continuously measure the distance between two 2-dimensional edges using serial image dilations and pathfinding. This algorithm can be applied to a variety of fields such as cardiac structural biology, vascular biology, and civil engineering. Bioengineering Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor Iulia M. Lazar1, Jingren Deng1, Nicole Smith1 1Biological Sciences, Virginia Tech A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described. Biology Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements Nabil E. Boutagy1,2, Emily Pyne1, George W. Rogers3, Mostafa Ali1, Matthew W. Hulver1,2, Madlyn I. Frisard1,2 1The Department of Human Nutrition, Foods, and Exercise, Virginia Tech, 2The Metabolic Phenotyping Core, Virginia Tech, 3Seahorse Bioscience Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays. Developmental Biology Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements Nabil E. Boutagy1,2, George W. Rogers3, Emily S. Pyne1, Mostafa M. Ali1, Matthew W. Hulver1,2, Madlyn I. Frisard1,2 1The Department of Human Nutrition, Foods, and Exercise, Virginia Tech, 2The Metabolic Phenotyping Core, Virginia Tech, 3Seahorse Bioscience The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model. Bioengineering Glutamine Flux Imaging Using Genetically Encoded Sensors Julien Besnard1, Sakiko Okumoto1 1Virginia Tech This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors. Biology Tissue Triage and Freezing for Models of Skeletal Muscle Disease Hui Meng1, Paul M.L. Janssen2, Robert W. Grange3, Lin Yang4, Alan H. Beggs5, Lindsay C. Swanson5, Stacy A. Cossette1,6, Alison Frase7, Martin K. Childers8, Henk Granzier9, Emanuela Gussoni5, Michael W. Lawlor1 1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 2Department of Physiology and Cell Biology, The Ohio State University, 3Department of Human Nutrition, Foods and Exercise, Virginia Tech, 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, 5 The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies. Chemistry Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation Karthik Pillai1,2,3, Fernando Navarro Arzate2,4, Wei Zhang2,5, Scott Renneckar1,2,5,6 1Institute for Critical Technology and Applied Science, Virginia Tech, 2Macromolecules and Interfaces Institute, Virginia Tech, 3Institute for Food Safety and Health, Illinois Institute of Technology- Moffett Campus, 4Wood, Cellulose, and Paper Research Department, University of Guadalajara, 5Department of Sustainable Biomaterials, Virginia Tech, 6Sustainable Nanotechnology Interdisciplinary Graduate Education Program, Virginia Tech The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions. Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material. Immunology and Infection 2D and 3D Chromosome Painting in Malaria Mosquitoes Phillip George1, Atashi Sharma1, Igor V. Sharakhov1 1Department of Entomology, Virginia Tech Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).
