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28 ARTICLES PUBLISHED IN JoVE

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Biology

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization
Phillip George 1, Maria V. Sharakhova 1, Igor V. Sharakhov 1
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.

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Immunology and Infection

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes
Vladimir A. Timoshevskiy 1, Atashi Sharma 1, Igor V. Sharakhov 1, Maria V. Sharakhova 1
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.

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Biology

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter
Oswald J. Schmitz 1, Mark A. Bradford 1, Michael S. Strickland 1,2, Dror Hawlena 3
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.

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Medicine

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis
Elizabeth S. Elvington 1, Alireza Salmanzadeh 1,2, Mark A. Stremler 1,2, Rafael V. Davalos 1,2
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.

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Immunology and Infection

2D and 3D Chromosome Painting in Malaria Mosquitoes
Phillip George 1, Atashi Sharma 1, Igor V. Sharakhov 1
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).

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Chemistry

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation
Karthik Pillai 1,2,3, Fernando Navarro Arzate 2,4, Wei Zhang 2,5, Scott Renneckar 1,2,5,6
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.

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Biology

Tissue Triage and Freezing for Models of Skeletal Muscle Disease
Hui Meng 1, Paul M.L. Janssen 2, Robert W. Grange 3, Lin Yang 4, Alan H. Beggs 5, Lindsay C. Swanson 5, Stacy A. Cossette 1,6, Alison Frase 7, Martin K. Childers 8, Henk Granzier 9, Emanuela Gussoni 5, Michael W. Lawlor 1
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, 5Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School, 6Cure Congenital Muscular Dystrophy, 7Joshua Frase Foundation, 8Department of Rehabilitation Medicine, University of Washington, 9Department of Physiology, University of Arizona

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.

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Bioengineering

Glutamine Flux Imaging Using Genetically Encoded Sensors
Julien Besnard 1, Sakiko Okumoto 1
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.

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Biology

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes
Atashi Sharma 1, Troy D. Anderson 1, Igor V. Sharakhov 1
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.

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Developmental Biology

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements
Nabil E. Boutagy 1,2, George W. Rogers 3, Emily S. Pyne 1, Mostafa M. Ali 1, Matthew W. Hulver 1,2, Madlyn I. Frisard 1,2
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.

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Biology

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements
Nabil E. Boutagy 1,2, Emily Pyne 1, George W. Rogers 3, Mostafa Ali 1, Matthew W. Hulver 1,2, Madlyn I. Frisard 1,2
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.

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Bioengineering

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor
Iulia M. Lazar 1, Jingren Deng 1, Nicole Smith 1
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.

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Behavior

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation
Courtney A. Crist 1, Susan E. Duncan 1, Daniel L. Gallagher 2
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.

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Neuroscience

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions
William Dalkin 1,2, Thomas Taetzsch 1, Gregorio Valdez 1,2,3
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.

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Biology

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function
Scott T. O'Neal 1, Troy D. Anderson 1
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.

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Environment

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars
Brittany E. Campbell 1, Dini M. Miller 2
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.

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Environment

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees
Nicholas R. Larson 1, Troy D. Anderson 2
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.

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Biology

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts
F. Parker E. Laimbeer 1, Melissa Makris 2, Richard E. Veilleux 1
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.

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Chemistry

Preparation and Characterization of C60/Graphene Hybrid Nanostructures
Chuanhui Chen 1, Adam Mills 1,2, Husong Zheng 1, Yanlong Li 1, Chenggang Tao 1
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.

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Bioengineering

Quantifying Intermembrane Distances with Serial Image Dilations
Tristan Raisch 1,2, Momina Khan 1, Steven Poelzing 1,2
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.

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Neuroscience

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster
Daniel R. Swale 1, Aaron D. Gross 2, Quentin R. R. Coquerel 3, Jeffrey R. Bloomquist 3
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.

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Biology

Bovine Mammary Gland Biopsy Techniques
Veridiana L. Daley 1,4, Charles Dye 2, Sophie H. Bogers 3, R. Michael Akers 4, Francisco C. Rodriguez 3, John P. Cant 5, John Doelman 5, Peter Yoder 4, Karan Kumar 2, Dane Webster 6, Mark D. Hanigan 4
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.

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Medicine

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response
John A. Richey 1, Holly Sullivan-Toole 2, Marlene Strege 1, Corinne Carlton 1, Dylan McDaniel 3, Matthew Komelski 4, Amy Epperley 5, Hongxiao Zhu 6, Irving C. Allen 3,7
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.

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Behavior

Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury
Oleksii Shandra 1, Stefanie Robel 1,2,3
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.

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JoVE Journal

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions
Teshani Kumarage 1,2, Julie Nguyen 1, Rana Ashkar 1,2
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.

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Behavior

Assessing Human Spatial Navigation in a Virtual Space and its Sensitivity to Exercise
Alana J. Smith 1, Noor Tasnim 2, Zach Psaras 3, Daphne Gyamfi 4, Krishna Makani 4, Wendy A. Suzuki 3, Julia C. Basso 1,4,5
1School of Neuroscience, Virginia Tech, 2Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, 3Center for Neural Science, New York University, 4Department of Human Nutrition, Foods, and Exercise, Virginia Tech, 5Center for Health Behaviors Research, Fralin Biomedical Research Institute at VTC

Here, we present a novel, brief, and active spatial navigation task that assesses both spatial navigation and episodic memory ability. Importantly, spatial navigation and episodic memory were associated with one another, and this task demonstrated sensitivity to exercise.

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Immunology and Infection

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito
Matteo Vitale *1, Jiangtao Liang *2, Igor Sharakhov 2, Federica Bernardini 1
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.

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A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow
Samantha Howerton 1,2, Yanping Liang 1, Jennifer Hammel 1,3, Benjamin Purow 4, Jennifer Munson 1,3
1Fralin Biomedical Research Institute at Virginia Tech Carilion, 2Translational Biology, Medicine, and Health Graduate Program, Virginia Tech, 3Department of Biomedical Engineering & Mechanics, Virginia Tech, 4Department of Neurology, University of Virginia School of Medicine

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

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