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Virginia Polytechnic Institute and State University

26 ARTICLES PUBLISHED IN JoVE

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Medicine

In Vivo Canine Muscle Function Assay
Martin K. Childers 1, Robert W. Grange 2, Joe N. Kornegay 3
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.

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Bioengineering

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
Elvira Romero 1, Reeder Robinson 1, Pablo Sobrado 1
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.

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Immunology and Infection

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice
Irving C. Allen 1
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.

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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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Biology

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers
Joseph S. Najem 1, Myles D. Dunlap 2, Anthony Yasmann 3, Eric C. Freeman 4, John W. Grant 5, Sergei Sukharev 3, Donald J. Leo 4
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.

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Immunology and Infection

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids
Daniel E. Rothschild 1, Tara Srinivasan 2, Linette A. Aponte-Santiago 1, Xiling Shen 2,3,4, Irving C. Allen 1
1Department of Biomedical Sciences and Pathobiology, Virginia Maryland College of Veterinary Medicine, Virginia Tech, 2Department of Biomedical Engineering, Cornell University, 3School of Electrical and Computer Engineering, Cornell University, 4Department of Biomedical Engineering, Duke University

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

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Immunology and Infection

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow
Xiaofeng Liao 1, Melissa Makris 2, Xin M. Luo 1
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.

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Environment

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes
Gregory E. Welbaum 1, Osamah S. Khan 1, Nezar H. Samarah 1,2
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.

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Bioengineering

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials
Keith C. Heyde *1,2, Felicia Y. Scott *3, Sung-Ho Paek 3, Ruihua Zhang 3, Warren C. Ruder 3,4
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.

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Chemistry

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes
Quanyou Feng 1,2, Rong Tong 2
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.

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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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Bioengineering

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications
Ming-Cheng Chen *1, John R. Lake *1, Keith C. Heyde 2, Warren C. Ruder 1,2
1Department of Bioengineering, University of Pittsburgh, 2Department of Mechanical Engineering, Carnegie Mellon University

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

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Education

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles
Saeed Mozaffari *1, Wenhui Li *1, Coogan Thompson 1, Sergei Ivanov 2, Soenke Seifert 3, Byeongdu Lee 4, Libor Kovarik 5, Ayman M. Karim 1
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.

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JoVE Journal

Determining Tribocorrosion Rate and Wear-Corrosion Synergy of Bulk and Thin Film Aluminum Alloys
Jia Chen 1,2, Hesham Mraied 3, Wenjun Cai 1,2
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.

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Behavior

Noninvasive, In-pen Approach Test for Laboratory-housed Pigs
Lindsey E. Hulbert 1, Eduarda M. Bortoluzzi 1, Yunzhi Luo 1, Jared M. Mumm 1, Morgan J. Coffin 1, Gina Y. Becker 2, Pamela J. Vandevord 3, Elizabeth M. McNeil 3, Tim Walilko 4, Zin Z. Khaing 5,6, Laila Zai 4,5
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.

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JoVE Core

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes
Joseph S. Najem 1,2, Graham J. Taylor 2,3, Nick Armendarez 4, Ryan J. Weiss 5, Md Sakib Hasan 5, Garrett S. Rose 5, Catherine D. Schuman 6, Alex Belianinov 7, Stephen A. Sarles 2, C. Patrick Collier 2,3,7
1Joint Institute for Biological Sciences, Oak Ridge National Laboratory, 2Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee, 3Bredesen Center for Interdisciplinary Research, University of Tennessee, 4Department of Biosystems and Agriculture Engineering, University of Kentucky, 5Department of Electrical Engineering and Computer Science, University of Tennessee, 6Computer Science and Mathematics Division, Oak Ridge National Laboratory, 7Center for Nanophase Materials Sciences, Oak Ridge National Laboratory

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

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JoVE Journal

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain
Taylor McFadden 1, Rishi K. Devulapalli 2, Timothy J. Jarome 1,2
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.

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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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Medicine

Technical Modification of the Terminal Ureter During Total Transperitoneal Laparoscopic Nephroureterectomy for Upper Urinary Tract Urothelial Carcinoma
Chunlong Fu *1,2, Yaqun Zhang *1, Xin Wang 1, Shengcai Zhu 1, Huimin Hou 1, Shengjie Liu 1, Cheng Pang 1, Xinda Song 1, Jia Chen 1, Jianye Wang 1, Ming Liu 1,2
1(Urology) Beijing Hospital, National Center of Gerontology, The Joint Unit of the Chinese Academy of Medical Sciences, 2Graduate School of Peking Union Medical College, Beijing Hospital

Here, we present a protocol to increase the surgical field of view and reduce the difficulty of total transperitoneal laparoscopic nephroureterectomy surgery by precutting the umbilical ligament before treating the terminal ureter.

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Bioengineering

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production
Riccardo Tamburrini 1,2,3, Deborah Chaimov 1,3, Amish Asthana 1,3, Carlo Gazia 3,4, Kevin Enck 3, Sean M. Muir 5, Justine Mariam Aziz 6, Sandrine Lablanche 7, Emily Tubbs 7, Alice A. Tomei 8,9, Mark Van Dyke 10, Shay Soker 3, Emmanuel C. Opara 3, Giuseppe Orlando 1,3
1Department of Surgery, Wake Forest Baptist Medical Center, 2Department of General Surgery, PhD Program in Experimental Medicine, University of Pavia, 3Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, 4Department of Surgery, Tor Vergata University of Rome, 5Wake Forest University College of Arts and Science, 6Wake Forest University School of Medicine, 7Laboratory of Fundamental and Applied Bioenergetics (LBFA), and Environmental and System Biology (BEeSy), Grenoble Alps University, 8Department of Biomedical Engineering, University of Miami, 9Diabetes Research Institute, University of Miami Miller School of Medicine, 10Department of Biomedical Engineering and Mechanics, School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets’ microenvironment in vitro and, potentially, in vivo settings.

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Cancer Research

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model
Margaret A. Nagai-Singer 1, Alissa Hendricks-Wenger 1,2,3, Rebecca M. Brock 1,2, Holly A. Morrison 1, Juselyn D. Tupik 1, Sheryl Coutermarsh-Ott 1, Irving C. Allen 1,2,4
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.

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Biology

A Step-by-Step Guide to Mosquito Electroantennography
Chloé Lahondère 1
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.

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Biology

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium
Meng Zhang 1,2, Mohamed N. Seleem 3, Ji-Xin Cheng 1,2,4,5
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.

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Immunology and Infection

Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice
Xavier Cabana-Puig 1, Xin M. Luo 1
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.

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Immunology and Infection

Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method
Xavier Cabana Puig 1, Christopher M. Reilly 1, Xin M. Luo 1
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.

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Neuroscience

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis
Alisa A. Omelchenko 1,2, Hua Bai 1, Sibtain Hussain 1, Jordan J. Tyrrell 1,3, Mason Klein 4, Lina Ni 1
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.

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