Virginia Commonwealth University View Institution's Website 29 articles published in JoVE Cancer Research Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis April E. Boyd1, Jeremy Allegood2, Santiago Lima1,3 1Department of Biology, Virginia Commonwealth University, 2VCU Lipidomics/Metabolomics Shared Resource, Virginia Commonwealth University School of Medicine, 3Massey Cancer Center Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS. Biochemistry Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis Kyle G. Kroeck1,2, Weihua Qiu1,2, Claudio Catalano1,2, Thi Kim Hoang Trinh1,2, Youzhong Guo1,2 1Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, 2Institute for Structural Biology, Drug Discovery and Development, School of Pharmacy, Virginia Commonwealth University Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy. Environment Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems Daniel J. McGarvey1, Taylor E. Woods1,2, Andrew J. Kirk3 1Center for Environmental Studies, Virginia Commonwealth University, 2Department of Ecology and Evolutionary Biology, University of Tennessee, 3Department of Environmental Quality This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis. Environment A New Portable In Vitro Exposure Cassette for Aerosol Sampling Lynn E. Secondo1, Nathaniel J. Wygal1, Nastassja A. Lewinski1 1Department of Chemical and Life Science Engineering, Virginia Commonwealth University Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release. Engineering Fabrication of Schottky Diodes on Zn-polar BeMgZnO/ZnO Heterostructure Grown by Plasma-assisted Molecular Beam Epitaxy Kai Ding1, Vitaliy Avrutin1, Natalia Izioumskaia1, Md Barkat Ullah1, Ümit Özgür1, Hadis Morkoç1 1Department of Electrical and Computer Engineering, Virginia Commonwealth University Attainment of high-quality Schottky contacts is imperative for achieving efficient gate modulation in heterostructure field effect transistors (HFETs). We present the fabrication methodology and characteristics of Schottky diodes on Zn-polar BeMgZnO/ZnO heterostructures with high-density two dimensional electron gas (2DEG), grown by plasma-assisted molecular beam epitaxy on GaN templates. Chemistry Measurement of Ultrafast Vibrational Coherences in Polyatomic Radical Cations with Strong-Field Adiabatic Ionization Derrick Ampadu Boateng1, Katharine Moore Tibbetts1 1Department of Chemistry, Virginia Commonwealth University We present a protocol for probing ultrafast vibrational coherences in polyatomic radical cations that result in molecular dissociation. Behavior Paradigms of Lower Extremity Electrical Stimulation Training After Spinal Cord Injury Ashraf S. Gorgey1,2, Refka E. Khalil1, Robert M. Lester1, Gary A. Dudley3, David R. Gater4 1Spinal Cord Injury and Disorders Service, Hunter Holmes McGuire VAMC, 2Department of Physical Medicine and Rehabilitation, Virginia Commonwealth University, 3Deceased, Department of Kinesiology, The University of Georgia, 4Department of Physical Medicine and Rehabilitation, Penn State Milton S. Hershey Medical Center Spinal cord injury is a traumatic medical condition that may result in elevated risks of chronic secondary metabolic disorders. Here, we presented a protocol using surface neuromuscular electrical stimulation-resistance training in conjunction with functional electrical stimulation-lower extremities cycling as a strategy to ameliorate several of these medical problems. Bioengineering A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex Paul T. Arsenovic1, Kranthidhar Bathula1, Daniel E. Conway1 1Department of Biomedical Engineering, Virginia Commonwealth University A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex. Bioengineering Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture Patrick A. Link1, Robert A. Pouliot1, Nabil S. Mikhaiel1, Bethany M. Young1, Rebecca L. Heise1,2 1Department of Biomedical Engineering, Virginia Commonwealth University, 2Department of Physiology and Biophysics, Virginia Commonwealth University This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions. Biology Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein Mulugeta Seneshaw1, Faridoddin Mirshahi1, Hae-Ki Min1, Amon Asgharpour1, Shervin Mirshahi1, Kalyani Daita1, Sherry Boyett1, Prasanna K. Santhekadur2, Michael Fuchs1, Arun J. Sanyal1 1Division of Gastroenterology, Hepatology and Nutrition, Virginia Commonwealth University, 2McGuire Veterans Affairs Medical Center, Research BC-101 MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma. Chemistry Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos Sarah C. Rothschild1, Ludmila Francescatto2, Robert M. Tombes1 1Department of Biology, Virginia Commonwealth University, 2Center for Human Disease Modeling, Duke University Medical Center Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins. Biology Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines Derek M. Kendig1,2, Norm R. Hurst1, John R. Grider1 1Department of Physiology and Biophysics, Virginia Commonwealth University, 2Department of Biology, Loyola University Maryland Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system. Immunology and Infection Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells Christopher M. Wunsch1, Janina P. Lewis1,2,3 1Philips Institute for Oral Health Research, Virginia Commonwealth University, 2Department of Microbiology and Immunology, Virginia Commonwealth University, 3Department of Biochemistry, Virginia Commonwealth University This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type. Bioengineering Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial Parthasarathy Madurantakam1, Suyog Yoganarasimha2, Fadi K. Hasan3 1Department of General Practice, School of Dentistry, Virginia Commonwealth University, 2Department of Biomedical Engineering, Virginia Commonwealth University, 3Department of Periodontics, School of Dentistry, Virginia Commonwealth University Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing. Bioengineering Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management Woosik Lee1, Ohjin Kwon1, Dong Sup Lee1, Woon-Hong Yeo1,2,3 1Department of Mechanical and Nuclear Engineering, Virginia Commonwealth University, 2Center for Rehabilitation Science and Engineering, Virginia Commonwealth University, 3Massey Cancer Center, Virginia Commonwealth University This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management. Neuroscience An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila Simran Sandhu1, Arnavaz P. Kollah1, Lara Lewellyn1, Robin F. Chan1, Mike Grotewiel1,2 1Department of Human and Molecular Genetics, Virginia Commonwealth University, 2VCU Alcohol Research Center, Virginia Commonwealth University Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described. Neuroscience An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans Andrew G. Davies1,2, GinaMari G. Blackwell1,2, Richard C. Raabe1, Jill C. Bettinger1,2 1Department of Pharmacology and Toxicology, Virginia Commonwealth University, 2VCU Alcohol Research Center, Virginia Commonwealth University C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay. Biology Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction Vanessa Cabra1, Montserrat Samsó1 1Department of Physiology and Biophysics, Virginia Commonwealth University This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule. Biology Quantification of Orofacial Phenotypes in Xenopus Allyson E. Kennedy1, Amanda J. Dickinson1 1Biology Department, Virginia Commonwealth University A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects. Medicine Implantation of the Syncardia Total Artificial Heart Daniel G. Tang1, Keyur B. Shah2, Micheal L. Hess2, Vigneshwar Kasirajan1 1Division of Cardiothoracic Surgery, Virginia Commonwealth University, 2Division of Cardiology, Virginia Commonwealth University The purpose of this manuscript is to briefly review indications, management, and outcomes for the total artificial heart. Video operative techniques for device implantation are presented. Biology Facial Transplants in Xenopus laevis Embryos Laura A. Jacox*1,3, Amanda J. Dickinson*4, Hazel Sive2,3 1Biological Sciences in Dental Medicine, Harvard University, 2Biology Department, Massachusetts Institute of Technology, 3Whitehead Institute, Massachusetts Institute of Technology, 4Biology Department, Virginia Commonwealth University A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions. Neuroscience An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse Tricia H. Smith1, Joy Ngwainmbi1, John R. Grider2, William L. Dewey1, Hamid I. Akbarali1 1Pharmacology and Toxicology, Virginia Commonwealth University, 2Physiology and Biophyics, Virginia Commonwealth University We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc. Medicine Automated Midline Shift and Intracranial Pressure Estimation based on Brain CT Images Wenan Chen*1,2, Ashwin Belle*3, Charles Cockrell2,4, Kevin R. Ward2,5, Kayvan Najarian2,3 1Department of Biostatistics, Virginia Commonwealth University, 2Virginia Commonwealth University Reanimation Engineering Science (VCURES) Center, 3Department of Computer Science, Virginia Commonwealth University, 4Department of Radiology, Virginia Commonwealth University, 5Department of Emergency Medicine, Virginia Commonwealth University An automated midline shift estimation and intracranial pressure (ICP) pre-screening system based on computed tomography (CT) images for patients with traumatic brain injury (TBI) is proposed using image processing and machine learning techniques. Biology Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR Xiuchun Ge1, Ping Xu1 1The Philips Institute of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations. Biology Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins Jason M. Beckta1,2, Scott C. Henderson3, Kristoffer Valerie1,2,4 1Department of Radiation Oncology, Virginia Commonwealth University, 2Department of Biochemistry & Molecular Biology, Virginia Commonwealth University, 3Department of Anatomy & Neurobiology, Virginia Commonwealth University, 4Massey Cancer Center, Virginia Commonwealth University This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis. Immunology and Infection Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation Maciej Kmieciak1, Amir Toor2, Laura Graham3, Harry D. Bear3, Masoud H. Manjili1 1Department of Microbiology & Immunology, Virginia Commonwealth University- Massey Cancer Center, 2Department of Internal Medicine, Virginia Commonwealth University- Massey Cancer Center, 3Department of Surgery, Virginia Commonwealth University- Massey Cancer Center An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer. Bioengineering Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study Michael J. McClure1, Scott A. Sell1, David G. Simpson2, Beat H. Walpoth3, Gary L. Bowlin1 1Department of Biomedical Engineering, Virginia Commonwealth University, 2Department of Anatomy and Neurobiology, Virginia Commonwealth University, 3Department of Cardiovascular Surgery, University Hospital of Geneva The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen. Biology Imaging Protein-protein Interactions in vivo Tom Seegar1, William Barton1 1Biochemistry and Molecular Biology, Virginia Commonwealth University This protocol describes how to image protein-protein interactions using a FRET-based proximity assay. Biology Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus Enrique Y. Pascual1, Marion J. Riggs1, Raj R. Rao1 1Department of Chemical and Life Sciences Engineering, Virginia Commonwealth University Suspension immunocytochemical staining of human pluripotent stem cells (hPSCs) for cell-surface markers (SSEA-3/SSEA-4) was achieved based on use of a self-made cytospin apparatus to create a monolayer of cells for observation and quantification.