Virginia Polytechnic Institute and State University View Institution's Website 34 articles published in JoVE Chemistry A Technical Guide for Performing Spectroscopic Measurements on Metal-Organic Frameworks Daniel R. Cairnie1, Amanda J. Morris1 1Department of Chemistry, Virginia Polytechnic and State Institute Here, we use a polymer stabilizer to prepare metal-organic framework (MOF) suspensions that exhibit markedly decreased scatter in their ground-state and transient absorption spectra. With these MOF suspensions, the protocol provides various guidelines to characterize the MOFs spectroscopically to yield interpretable data. Neuroscience TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis Alisa A. Omelchenko1,2, Hua Bai1, Sibtain Hussain1, Jordan J. Tyrrell1,3, Mason Klein4, Lina Ni1 1School of Neuroscience, Virginia Polytechnic Institute and State University, 2CMU-Pitt Joint Computational Biology, School of Medicine, University of Pittsburgh, 3Eastern Virginia Medical School, 4Department of Physics, University of Miami TrackMate Analysis of Calcium Imaging (TACI) is an open-source ImageJ plugin for 3D calcium imaging analysis that examines motion on the z-axis and identifies the maximum value of each z-stack to represent a cell's intensity at the corresponding time point. It can separate neurons overlapping in the lateral (x/y) direction but on different z-planes. Immunology and Infection Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice Xavier Cabana-Puig1, Xin M. Luo1 1Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University The present protocol describes a method to track lupus progression in mice. Two additional procedures are presented to characterize lupus nephritis based on cell infiltration and protein deposition in the kidneys. Immunology and Infection Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method Xavier Cabana Puig1, Christopher M. Reilly1, Xin M. Luo1 1Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University This protocol provides a simple, cost-effective DNA isolation method for the analysis of murine gut microbiota alterations during the development of autoimmune disease. Biology Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium Meng Zhang1,2, Mohamed N. Seleem3, Ji-Xin Cheng1,2,4,5 1Department of Electrical and Computer Engineering, Boston University, 2Boston University Photonics Center, Boston University, 3Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 4Department of Biomedical Engineering, Boston University, 5Department of Chemistry, Boston University This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic. Biology A Step-by-Step Guide to Mosquito Electroantennography Chloé Lahondère1 1Department of Biochemistry; The Fralin Life Science Institute; The Global Change Center; Department of Entomology and the Center for Emerging Zoonotic and Arthropod-Borne Pathogens, Virginia Polytechnic Institute and State University The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males. Cancer Research Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model Margaret A. Nagai-Singer1, Alissa Hendricks-Wenger1,2,3, Rebecca M. Brock1,2, Holly A. Morrison1, Juselyn D. Tupik1, Sheryl Coutermarsh-Ott1, Irving C. Allen1,2,4 1Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, 2Graduate Program in Translational Biology, Medicine and Health, Virginia Polytechnic Institute and State University, 3Department of Biomedical Engineering and Mechanics, Virginia Polytechnic Institute and State University, 4Department of Basic Science Education, Virginia Tech Carilion School of Medicine, Virginia Polytechnic Institute and State University We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ. Behavior Noninvasive, In-pen Approach Test for Laboratory-housed Pigs Lindsey E. Hulbert1, Eduarda M. Bortoluzzi1, Yunzhi Luo1, Jared M. Mumm1, Morgan J. Coffin1, Gina Y. Becker2, Pamela J. Vandevord3, Elizabeth M. McNeil3, Tim Walilko4, Zin Z. Khaing5,6, Laila Zai4,5 1Animal Sciences and Industry, Kansas State University, 2DynaSim Technical Services, INC, 3Center for Injury Biomechanics, Virginia Polytechnic Institute and State University, 4Applied Research Associates, Inc., 5Lucent Research, LLC, 6Department of Neurological Surgery, University of Washington This protocol describes a new behavioral test—the human approach test in the pigs' home pen—to detect functional deficits in laboratory pigs after subconcussive traumatic brain injury. Neuroscience Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain Taylor McFadden1, Rishi K. Devulapalli2, Timothy J. Jarome1,2 1Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, 2School of Neuroscience, Virginia Polytechnic Institute and State University This protocol is designed to efficiently quantify ubiquitin-proteasome system (UPS) activity in different cellular compartments of the rodent brain. Users are able to examine UPS functioning in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the amount of time and number of animals needed to perform these complex analyses. Neuroscience Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster Daniel R. Swale1, Aaron D. Gross2, Quentin R. R. Coquerel3, Jeffrey R. Bloomquist3 1Department of Entomology, Louisiana State University AgCenter, 2Department of Entomology, Virginia Tech, 3Department of Entomology and Nematology, Emerging Pathogens Institute, University of Florida This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways. Engineering Determining Tribocorrosion Rate and Wear-Corrosion Synergy of Bulk and Thin Film Aluminum Alloys Jia Chen1,2, Hesham Mraied3, Wenjun Cai1,2 1Department of Mechanical Engineering, University of South Florida, 2Department of Materials Science and Engineering, Virginia Polytechnic Institute and State University, 3Department of Civil and Environmental Engineering, University of South Florida Here, we present a protocol to measure the tribocorrosion rate and wear-corrosion synergy of thin film and bulk Al alloys in simulated sea water at room temperature. Chemistry Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles Saeed Mozaffari*1, Wenhui Li*1, Coogan Thompson1, Sergei Ivanov2, Soenke Seifert3, Byeongdu Lee4, Libor Kovarik5, Ayman M. Karim1 1Department of Chemical Engineering, Virginia Polytechnic Institute and State University, 2Center for Integrated Nanotechnologies, Los Alamos National Laboratory, 3Advanced Photon Source, Argonne National Laboratory, 4X-ray Science Division, Argonne National Laboratory, 5Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling. Chemistry Preparation and Characterization of C60/Graphene Hybrid Nanostructures Chuanhui Chen1, Adam Mills1,2, Husong Zheng1, Yanlong Li1, Chenggang Tao1 1Department of Physics, Center for Soft Matter and Biological Physics, Virginia Tech, 2Department of Physics, Princeton University Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene. Biology Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts F. Parker E. Laimbeer1, Melissa Makris2, Richard E. Veilleux1 1Department of Horticulture, Virginia Tech, 2Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis. Chemistry Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes Quanyou Feng1,2, Rong Tong2 1Center for Molecular Systems and Organic Devices, Institute of Optoelectronic Materials, Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced Materials, Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing University of Posts and Telecommunications, 2Department of Chemical Engineering, Virginia Polytechnic Institute and State University A protocol for the controlled photoredox ring-opening polymerization of O-carboxyanhydrides mediated by Ni/Zn complexes is presented. Environment Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees Nicholas R. Larson1, Troy D. Anderson2 1Entomology, Virginia Tech, 2Entomology, University of Nebraska-Lincoln The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees. Environment A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars Brittany E. Campbell1, Dini M. Miller2 1Department of Entomology and Nematology, University of Florida, 2Department of Entomology, Virginia Tech Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages. Bioengineering Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials Keith C. Heyde*1,2, Felicia Y. Scott*3, Sung-Ho Paek3, Ruihua Zhang3, Warren C. Ruder3,4 1Department of Mechanical Engineering, Carnegie Mellon University, 2Engineering Science and Mechanics Program, Virginia Polytechnic Institute and State University, 3Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 4Department of Bioengineering, University of Pittsburgh This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces. Biology Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function Scott T. O'Neal1, Troy D. Anderson1 1Department of Entomology, Virginia Tech The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate. Immunology and Infection Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow Xiaofeng Liao1, Melissa Makris2, Xin M. Luo1 1Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 2Flow Cytometry Laboratory, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC. Environment A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes Gregory E. Welbaum1, Osamah S. Khan1, Nezar H. Samarah1,2 1Department of Horticulture, Virginia Polytechnic Institute and State University, 2Department of Plant Production, Faculty of Agriculture, Jordan University of Science and Technology Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications. Behavior Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation Courtney A. Crist1, Susan E. Duncan1, Daniel L. Gallagher2 1Food Science and Technology, Virginia Tech, 2Civil and Environmental Engineering, Virginia Tech A protocol for capturing and statistically analyzing emotional response of a population to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis software is described. Engineering Electrospray Deposition of Uniform Thickness Ge23Sb7S70 and As40S60 Chalcogenide Glass Films Spencer Novak1, Pao-Tai Lin2,3, Cheng Li4, Nikolay Borodinov1, Zhaohong Han5, Corentin Monmeyran5, Neil Patel5, Qingyang Du5, Marcin Malinowski4, Sasan Fathpour4, Chatdanai Lumdee4, Chi Xu4, Pieter G. Kik4, Weiwei Deng6, Juejun Hu7, Anuradha Agarwal7, Igor Luzinov1, Kathleen Richardson4 1Department of Materials Science and Engineering, Clemson University, 2Department of Materials Science and Engineering, Texas A&M University, 3Department of Electrical and Computer Engineering, Texas A&M University, 4College of Optics and Photonics, Center for Research and Education in Optics and Lasers (CREOL), University of Central Florida, 5Department of Materials Science and Engineering, Massachusetts Institute of Technology, 6Department of Mechanical Engineering, Virginia Polytechnic Institute, 7Microphotonics Center, Massachusetts Institute of Technology A method of uniform thickness solution-derived chalcogenide glass film deposition is demonstrated using computer numerical controlled motion of a single-nozzle electrospray. Neuroscience The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions William Dalkin1,2, Thomas Taetzsch1, Gregorio Valdez1,2,3 1Carilion Research Institute, Virginia Tech, 2Carilion School of Medicine, Virginia Tech, 3Department of Biological Sciences, Virginia Tech We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle. Biology Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers Joseph S. Najem1, Myles D. Dunlap2, Anthony Yasmann3, Eric C. Freeman4, John W. Grant5, Sergei Sukharev3, Donald J. Leo4 1Department of Mechanical Engineering, Virginia Polytechnic Institute and State University, 2School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, 3Department of Biology, University of Maryland, 4College of Engineering, University of Georgia, 5Department of Engineering Sciences and Mechanics, Virginia Polytechnic Institute and State University Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation. Biology Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes Atashi Sharma1, Troy D. Anderson1, Igor V. Sharakhov1 1Department of Entomology, Virginia Tech A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition. Immunology and Infection The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice Irving C. Allen1 1Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis. Bioengineering In situ TEM of Biological Assemblies in Liquid Madeline J. Dukes*1, Brian L. Gilmore*2, Justin R. Tanner*2, Sarah M. McDonald2, Deborah F. Kelly2 1Applications Science, Protochips, Inc., 2Virginia Tech Carilion Research Institute Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope. Medicine Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis Elizabeth S. Elvington1, Alireza Salmanzadeh1,2, Mark A. Stremler1,2, Rafael V. Davalos1,2 1School of Biomedical Engineering and Science, Virginia Tech, 2Department of Engineering Science and Mechanics, Virginia Tech Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments. Biology Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter Oswald J. Schmitz1, Mark A. Bradford1, Michael S. Strickland1,2, Dror Hawlena3 1School of Forestry and Environmental Studies, Yale University, 2Department of Biological Sciences, Virginia Tech, 3Department of Ecology, Evolution and Behavior, The Hebrew University of Jerusalem We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes. Immunology and Infection Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes Vladimir A. Timoshevskiy1, Atashi Sharma1, Igor V. Sharakhov1, Maria V. Sharakhova1 1Department of Entomology, Virginia Tech Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes. Biology High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization Phillip George1, Maria V. Sharakhova1, Igor V. Sharakhov1 1Department of Entomology, Virginia Tech Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development. Bioengineering Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry Elvira Romero1, Reeder Robinson1, Pablo Sobrado1 1Department of Biochemistry, Virginia Polytechnic Institute and State University We describe the use of a stopped-flow instrument to investigate both the reductive and oxidative half-reactions of Aspergillus fumigatus siderophore A (SidA), a flavin-dependent monooxygenase. We then show the spectra corresponding to the species in the reaction of SidA and we calculate the rate constants for their formation. 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.
