S'identifier

The Ohio State University

86 ARTICLES PUBLISHED IN JoVE

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Biology

Murine Renal Transplantation Procedure
Jiao-Jing Wang 1, Sara Hockenheimer 2, Alice A. Bickerstaff 1, Gregg A. Hadley 1
1Department of Surgery, The Ohio State University, 2Integrated Biomedical Sciences Graduate Program, The Ohio State University

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

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Biology

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues
Carlos M. Hernandez-Garcia 1, Joseph M. Chiera 1,2, John J. Finer 1
1Department of Horticulture and Crop Science, The Ohio State University, 2Department of Plant Pathology, North Carolina State University

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

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Medicine

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart
Surya C. Gnyawali 1,2, Sashwati Roy 1,2, Jason Driggs 1,2, Savita Khanna 1,2, Thomas Ryan 2,3, Chandan K. Sen 2
1Department of Surgery, The Ohio State University, 2Heart and Lung Research Institute, The Ohio State University, 3Department of Cardiovascular Medicine, The Ohio State University

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

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Biology

Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects
Gustavo R. Rodríguez 1, Jennifer B. Moyseenko 1, Matthew D. Robbins 1, Nancy Huarachi Morejón 1, David M. Francis 1, Esther van der Knaap 1
1Department of Horticulture and Crop Science, The Ohio State University

Tomato Analyzer (TA) quantifies attributes of two dimensional shapes and color in a reproducible and accurate manner. A step-by-step procedure for obtaining high quality digitalized images of tomato fruit, morphological and color analyses of these images and several applications using the data generated through this software are described.

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Biology

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR
Binny Bhandary 1, Swapna Priya Rajarapu 1, Loren Rivera-Vega 1, Omprakash Mittapalli 1
1Department of Entomology, The Ohio State University

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

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Medicine

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function
Ronald X. Xu 1, Kun Huang 2, Ruogu Qin 1, Jiwei Huang 1, Jeff S. Xu 1, Liya Ding 2, Urmila S. Gnyawali 3, Gayle M. Gordillo 3, Surya C. Gnyawali 3,4, Chandan K. Sen 3
1Department of Biomedical Engineering, The Ohio State University, 2Department of Biomedical Informatics, The Ohio State University, 3Comprehensive Wound Center, The Ohio State University, 4Department of Surgery, The Ohio State University

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

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Medicine

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation
Erik J. Zmuda 1, Catherine A. Powell 1,2, Tsonwin Hai 1,2,3
1Molecular and Cellular Biochemistry, Center for Molecular Neurobiology, The Ohio State University, 2Integrated Biomedical Science Graduate Program, The Ohio State University, 3Comprehensive Cancer Center, The Ohio State University

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

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Biology

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
Jeffrey Parvin 1, Natsuko Chiba 2, Derek Ransburgh 1
1Department of Biomedical Informatics, The Ohio State University, 2Departments of Molecular Immunology and Clinical Oncology, Tohoku University

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

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Biology

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems
Noah Weisleder 1, Peihui Lin 1, Xiaoli Zhao 1, Matthew Orange 1, Hua Zhu 1, Jianjie Ma 1
1Department of Physiology and Biophysics, Robert Wood Johnson Medical School

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

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Neuroscience

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging
Nicholas P. Boyer 1, Chunhe Chen 1, Yiannis Koutalos 1
1Storm Eye Institute, Medical University of South Carolina

A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.

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Biology

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps
Bhavani Gopalakrishnan 1, Kevin M. Nash 1, Murugesan Velayutham 1, Frederick A. Villamena 1,2
1The Davis Heart and Lung Research Institute, The Ohio State University, 2Department of Pharmacology, College of Medicine, The Ohio State University

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

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Immunology and Infection

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging
Jenny M. Tam 1, Carlos E. Castro 2, Robert J. W. Heath 3, Michael K. Mansour 1, Michael L. Cardenas 1, Ramnik J. Xavier 3, Matthew J. Lang 4, Jatin M. Vyas 1
1Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 2Department of Mechanical and Aerospace Engineering, The Ohio State University, 3Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, 4Dept. of Chemical and Biomolecular Engineering, Vanderbilt University

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

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JoVE Core

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers
Xiangrui Li 1, Zhong-Lin Lu 2,3,4,5
1Center for Cognitive and Behavioral Brain Imaging, The Ohio State University, 2Department of Psychology, University of Southern California, 3Biomedical Engineering, University of Southern California, 4Neuroscience Graduate Program, University of Southern California, 5Department of Psychology, The Ohio State University

Conventional computer hardware can not generate visual stimuli with sufficiently high grayscale resolution and measure response times with sufficient accuracy. We describe how to use the VideoSwitcher to produce high-resolution monochromatic displays, and the RTbox to measure response times with high accuracy on conventional computer hardware.

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Immunology and Infection

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model
Bryan A. Anthony 1, Gregg A. Hadley 1
1Department of Surgery, The Ohio State University Medical Center

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

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Medicine

DNA Vector-based RNA Interference to Study Gene Function in Cancer
Daniel B. Stovall 1, Meimei Wan 1, Qiang Zhang 1, Purnima Dubey 2, Guangchao Sui 1
1Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University School of Medicine, 2Department of Pathology and Comprehensive Cancer Center, Wake Forest University School of Medicine

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

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Biology

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment
Zachery Oestreicher 1, Steven K. Lower 1,2, Wei Lin 3, Brian H. Lower 2
1School of Earth Sciences, The Ohio State University, 2School of Environment & Natural Resources, The Ohio State University, 3Institute of Geology and Geophysics, Chinese Academy of Sciences

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

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Biology

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers
Ki Ho Park 1, Noah Weisleder 2, Jingsong Zhou 3, Kristyn Gumpper 1, Xinyu Zhou 1, Pu Duann 4, Jianjie Ma 1, Pei-Hui Lin 1
1Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, 2Department of Physiology and Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, 3Department of Molecular Biophysics and Physiology, Rush University Medical Center, 4Department of Internal Medicine, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

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JoVE Core

Quantum State Engineering of Light with Continuous-wave Optical Parametric Oscillators
Olivier Morin 1, Jianli Liu 1, Kun Huang 1,2, Felippe Barbosa 3, Claude Fabre 1, Julien Laurat 1
1Laboratoire Kastler Brossel, Université Pierre et Marie Curie, Ecole Normale Supérieure, CNRS, 2State Key Laboratory of Precision Spectroscopy, East China Normal University, 3Instituto de Física, Universidade de São Paulo

We describe the reliable generation of non-Gaussian states of traveling optical fields, including single-photon states and coherent state superpositions, using a conditional preparation method operated on the non-classical light emitted by optical parametric oscillators. Type-I and type-II phase-matched oscillators are considered and common procedures, such as the required frequency filtering or the high-efficiency quantum state characterization by homodyning, are detailed.

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Medicine

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery
Zhaobin Xu 1, Jenna Alloush 1, Eric Beck 1, Noah Weisleder 1
1Davis Heart & Lung Research Institute, Department of Physiology and Cell Biology, The Ohio State University

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

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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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Medicine

Collection and Extraction of Saliva DNA for Next Generation Sequencing
Michael R. Goode 1, Soo Yeon Cheong 1, Ning Li 1, William C. Ray 1,2, Christopher W. Bartlett 1,3
1Battelle Center for Mathematical Medicine, The Research Institute at Nationwide Children's Hospital, 2The Interdisciplinary Graduate Program in Biophysics, The Ohio State University, 3Department of Pediatrics, The Ohio State University

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

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Biology

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue
Ana Clara P. Azevedo-Pouly 1, Ola A. Elgamal 1, Thomas D. Schmittgen 1
1Pharmaceutics & Pharmaceutical Chemistry, The Ohio State University

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

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Biology

Isolation of Murine Valve Endothelial Cells
Lindsey J. Miller 1,2, Joy Lincoln 2,3
1Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, 2Center for Cardiovascular and Pulmonary Research, The Heart Center, The Research Institute at Nationwide Children's Hospital, 3Department of Pediatrics, The Ohio State University

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

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Biology

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus
Monica F. Poelchau 1, Xin Huang 1, Allison Goff 1, Julie Reynolds 2, Peter Armbruster 1
1Department of Biology, Georgetown University, 2Insect Physiology Lab, EEOB, The Ohio State University

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

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Biology

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes
Shubhra Majumder 1, Harold A. Fisk 1
1Department of Molecular Genetics, The Ohio State University

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard.

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Medicine

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury
Stephana Carelli *1, Toniella Giallongo *1, Claudio Gerace 1, Anthea De Angelis 1, Michele D. Basso 2, Anna Maria Di Giulio 1, Alfredo Gorio 1
1Laboratory of Pharmacology, Department of Health Sciences, University of Milan, 2Department of Neuroscience, College of Medicine, The Ohio State University

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

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Medicine

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect
Bo Zhang 1,2, Jonathan P. Davis 1, Mark T. Ziolo 1
1Department of Physiology & Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University, 2Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

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Engineering

Electron Channeling Contrast Imaging for Rapid III-V Heteroepitaxial Characterization
Julia I. Deitz 1, Santino D. Carnevale 2, Steven A. Ringel 3, David W. McComb 3, Tyler J. Grassman 1,2
1Department of Materials Science and Engineering, The Ohio State University, 2Department of Electrical and Computer Engineering, The Ohio State University, 3Institute of Materials Research, The Ohio State University

The use of electron channeling contrast imaging in a scanning electron microscope to characterize defects in III-V/Si heteroexpitaxial thin films is described. This method yields similar results to plan-view transmission electron microscopy, but in significantly less time due to lack of required sample preparation.

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Medicine

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots
Lu Xu 1, Qiwen Shen 1, Zhongqi Mao 2, L. James Lee 3, Ouliana Ziouzenkova 1
1Department of Human Sciences, The Ohio State University, 2Department of Minimally Invasive Surgery, The First Affiliated Hospital of Soochow University, 3NSF Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

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Behavior

A Protocol for Housing Mice in an Enriched Environment
Andrew M. Slater 1, Lei Cao 1
1Department of Molecular Virology, Immunology, and Medical Genetics, and Comprehensive Cancer Center, The Ohio State University

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

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Chemistry

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues
R. Wolfgang Rumpf 1, William C. Ray 1
1Battelle Center for Mathematical Medicine, The Research Institute at Nationwide Children's Hospital

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

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Chemistry

Chemical Vapor Deposition of an Organic Magnet, Vanadium Tetracyanoethylene
Megan Harberts 1, Yu Lu 2, Howard Yu 1, Arthur J. Epstein 1,2, Ezekiel Johnston-Halperin 1
1Department of Physics, The Ohio State University, 2Department of Chemistry, The Ohio State University

We present the synthesis of the organic-based ferrimagnet vanadium tetracyanoethylene (V[TCNE]x, x~2) via low temperature chemical vapor deposition (CVD). This optimized recipe yields an increase in Curie temperature from 400 K to over 600 K and a dramatic improvement in magnetic resonance properties.

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JoVE Core

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles
W. David Arnold 1,2,3, Kajri A. Sheth 1, Christopher G. Wier 4, John T. Kissel 1,3, Arthur H. Burghes 1,3,4, Stephen J. Kolb 1,3,4
1Department of Neurology, The Ohio State University Wexner Medical Center, 2Department of Physical Medicine and Rehabilitation, The Ohio State University, 3Department of Neuroscience, The Ohio State University Wexner Medical Center, 4Department of Biochemistry and Pharmacology, The Ohio State University Wexner Medical Center

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

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Engineering

Experiments on Ultrasonic Lubrication Using a Piezoelectrically-assisted Tribometer and Optical Profilometer
Sheng Dong 1, Marcelo Dapino 1
1Smart Vehicle Concepts Center, Department of Mechanical and Aerospace Engineering, The Ohio State University

We present a protocol for using a piezoelectrically-assisted tribometer and optical profilometer to investigate the dependence of ultrasonic wear and friction reduction on linear velocity, contact pressure, and surface properties.

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Bioengineering

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect
Daniel S. Oh 1, Alia Koch 1, Sidney Eisig 1, Sahng Gyoon Kim 2, Yoon Hyuk Kim 3, Do-Gyoon Kim 4, Jae Hyuck Shim 5
1Oral and Maxillofacial Surgery, Columbia University, 2Endodontics, Columbia University, 3Mechanical Engineering, Kyung Hee University, South Korea, 4Orthodontics, The Ohio State University, 5Pathology, Weill Cornell Medical College

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

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Medicine

Design, Fabrication, and Administration of the Hand Active Sensation Test (HASTe)
Alexandra Borstad 1, Alex Altenburger 1, Aaron Hannigan 1, Joshua LaPorte 1, Rachael Mott 1, Deborah S. Nichols-Larsen 2
1Division of Physical Therapy, The Ohio State University, 2School of Health and Rehabilitation Sciences, The Ohio State University

The Hand Active Sensation Test (HASTe) is a valid and reliable measure of haptic performance, which has been used successfully to identify impaired haptic touch in individuals with stroke. The purpose of this paper is to describe the design, fabrication and administration of the HASTe.

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Immunology and Infection

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine
Jacqueline M. Nolting 1, Christine M. Szablewski 1, Jody L. Edwards 1, Sarah W. Nelson 1, Andrew S. Bowman 1
1Department of Veterinary Preventive Medicine, The Ohio State University

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

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Biology

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
Dorrelyn P. Martin 1, Jharna Miya 1, Julie W. Reeser 1, Sameek Roychowdhury 1,2
1Department of Internal Medicine, Division of Medical Oncology, Comprehensive Cancer Center, The Ohio State University, 2Department of Pharmacology, The Ohio State University

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

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Medicine

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System
Kelly Regan 1, Soheil Moosavinasab 2, Philip Payne 1, Simon Lin 2
1Department of Biomedical Informatics, The Ohio State University, 2Research Information Solutions and Innovation, The Research Institute at Nationwide Children’s Hospital

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

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Medicine

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle
Vidhya Kumar 1, Henry Chang 1, David A. Reiter 2, David P. Bradley 3, Martha Belury 4, Shana E. McCormack 5, Subha V. Raman 1
1Davis Heart and Lung Research Institute, The Ohio State University, 2Laboratory of Clinical Investigation, National Institute on Aging, 3Division of Endocrinology, Diabetes and Metabolism, The Ohio State University, 4Department of Human Sciences, Human Nutrition, The Ohio State University, 5Division of Endocrinology and Diabetes, Department of Pediatrics, University of Pennsylvania

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

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Chemistry

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements
Kathryn Williamson 1, Emmanuel Hatzakis 1,2
1Department of Food Science and Technology, The Ohio State University, 2Foods for Health Discovery Theme, The Ohio State University

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

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Biology

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds
Benson E. Lindsey III *1, Luz Rivero *1, Chistopher S. Calhoun 1, Erich Grotewold 1,2, Jelena Brkljacic 1
1Arabidopsis Biological Resource Center, Center for Applied Plant Sciences, The Ohio State University, 2Department of Molecular Genetics, Center for Applied Plant Sciences, The Ohio State University

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

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Immunology and Infection

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase
Miguel A. Lopez Jr. 1, Randi M. Mackler 1, Matthew P. Altman 1, Kristine E. Yoder 1
1Department of Cancer Biology and Genetics, The Ohio State University College of Medicine

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

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Neuroscience

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons
Dustin Servello 1, Yuanzheng Gu 2,3, Chen Gu 1,2
1Molecular, Cellular, and Developmental Biology Program, The Ohio State University, 2Department of Biological Chemistry and Pharmacology, The Ohio State University, 3Biogen

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

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Education

Collecting Hair Samples for Hair Cortisol Analysis in African Americans
Kathy D. Wright 1, Jodi L. Ford 1, Joseph Perazzo 2, Lenette M. Jones 3, Sherrilynn Mahari 4, Brent A. Sullenbarger 5,6,7, Mark L. Laudenslager 8
1College of Nursing, The Ohio State University, 2College of Nursing, University of Cincinnati, 3School of Nursing, University of Michigan, 4Licensed Cosmetologist Consultant, 5Clinical Research Manager, The Ohio State University, 6Biomedical Core Research Coordinator, The Ohio State University, 7College of Nursing Biomedical Core Research Laboratory, The Ohio State University, 8Department of Psychiatry, Behavioral Immunology & Endocrinology Laboratory, University of Colorado Denver Anschutz Medical Campus

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

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Medicine

Mechanical Ventilation Boot Camp Curriculum
Jennifer Yee 1,2, Alma Benner 2, Jared Hammond 2, Bethany Malone 2, Charles Fuenning 3, Richard George 2,4, Rami A. Ahmed 2,3,4
1Department of Emergency Medicine, The Ohio State University, 2Summa Health System, Akron City Hospital, 3Western Reserve Hospital, 4Northeast Ohio Medical University

This curriculum outlines how to execute a simulation-based boot camp to teach providers how to manage mechanically ventilated patients.

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Immunology and Infection

Assembly and Purification of Prototype Foamy Virus Intasomes
Randi M. Mackler 1, Miguel A. Lopez Jr. 1, Kristine E. Yoder 1
1Department of Cancer Biology and Genetics, The Ohio State University College of Medicine

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

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Medicine

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion
Eliza W. Beal 1,2, Curtis Dumond 1, Jung-Lye Kim 1,2, Clifford Akateh 1,2, Emre Eren 1, Katelyn Maynard 1, Chandan K. Sen 3, Jay L. Zweier 4, Kenneth Washburn 2, Bryan A. Whitson 1,3, Sylvester M. Black 1,2
1Collaboration for Organ Perfusion, Protection, Engineering and Regeneration (COPPER) Lab, Division of Transplant, Department of Surgery, Comprehensive Transplant Center, Ohio State University Wexner Medical Center, 2Department of Surgery, Division of Transplant, Ohio State University Wexner Medical Center, 3Department of Surgery, Division of CardioThoracic Surgery, Ohio State University Wexner Medical Center, 4Department of Medicine, Ohio State University Wexner Medical Center

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

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Education

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
Kang-Yi Su *1,2, Steven D. Goodman *3, Hung-Ming Lai 1, Rong-Syuan Yen 1, Wei-Yao Hu 1, Wern-Cherng Cheng 2, Liang-In Lin 1,2, Ya-Chien Yang 1,2, Woei-Horng Fang 1,2
1Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, 2Department of Laboratory Medicine, National Taiwan University Hospital, 3Center for Microbial Pathogenesis, Nationwide Children's Hospital and the Department of Pediatrics, The Ohio State University

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

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Immunology and Infection

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells
Jonathan G.T. Lam 1,2,3, Chi Song 4, Stephanie Seveau 1,2,3
1Department of Microbial Infection and Immunity, The Ohio State University, 2Department of Microbiology, The Ohio State University, 3Infectious Diseases Institute, The Ohio State University, 4Division of Biostatistics, College of Public Health, The Ohio State University

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

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Behavior

Gaze in Action: Head-mounted Eye Tracking of Children's Dynamic Visual Attention During Naturalistic Behavior
Lauren K. Slone 1, Drew H. Abney 1, Jeremy I. Borjon 1, Chi-hsin Chen 2, John M. Franchak 3, Daniel Pearcy 1, Catalina Suarez-Rivera 1, Tian Linger Xu 1, Yayun Zhang 1, Linda B. Smith 1, Chen Yu 1
1Department of Psychological and Brain Sciences, Indiana University, 2Department of Otolaryngology-Head and Neck Surgery, The Ohio State University, 3Department of Psychology, University of California, Riverside

Young children do not passively observe the world, but rather actively explore and engage with their environment. This protocol provides guiding principles and practical recommendations for using head-mounted eye trackers to record infants' and toddlers' dynamic visual environments and visual attention in the context of natural behavior.

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Immunology and Infection

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis
Matthew J. Dunn 1, Andrew L. Woodruff 1, Matthew Z. Anderson 1,2
1Department of Microbiology, The Ohio State University, 2Department of Microbial Infection and Immunity, The Ohio State University

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model's effectiveness.

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Immunology and Infection

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice
Kyle Caution 1, Kacy Yount 1, Rajendar Deora 1,2, Purnima Dubey 1
1Department of Microbial Infection & Immunity, The Ohio State University, 2Department of Microbiology, The Ohio State University

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

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Cancer Research

Detection of Protease Activity by Fluorescent Peptide Zymography
Ameya A. Deshmukh 1,2, Jessica L. Weist 1, Jennifer L. Leight 1,2
1Comprehensive Cancer Center, James Cancer Hospital and Solove Research Center, Ohio State University, 2Department of Biomedical Engineering, College of Engineering, Ohio State University

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

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Bioengineering

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels
Abdulaziz S. Fakhouri 1,2,3, Jennifer L. Leight 1,2
1Department of Biomedical Engineering, The Ohio State University, 2The Ohio State University Comprehensive Cancer Center, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, 3Biomedical Technology Department, King Saud University

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

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Cancer Research

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures
Brandon M. Murphy 1, Tirzah J. Weiss 1, Christin E. Burd 1,2
1Department of Cancer Biology and Genetics, The Ohio State University, 2Department of Molecular Genetics, The Ohio State University

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

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Biochemistry

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase
Ryan K. Messer 1, Miguel A. Lopez Jr. 1, Gayan Senavirathne 1, Kristine E. Yoder 1
1Cancer Biology and Genetics, College of Medicine, The Ohio State University

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

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Medicine

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity
Jiao-Jing Wang *1, Longhui Qiu *1, Ramiro Fernandez 2, Xin Yi Yeap 1, Charlie Xiaoying Lin 3, Zheng Jenny Zhang 1,2
1Comprehensive Transplant Center, Northwestern University, 2Department of surgery, Feinberg School of Medicine, Northwestern University, 3Weinberg art and science college, Northwestern University

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

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Genetics

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)
Lauren Woodward 1, Pooja Gangras 1, Guramrit Singh 1
1Department of Molecular Genetics, Center for RNA Biology, The Ohio State University

Here, we present a protocol to enrich endogenous RNA binding sites or "footprints" of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

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Bioengineering

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro
Nicole Walters 1, Eduardo Reátegui 1,2
1William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 2Comprehensive Cancer Center, The Ohio State University

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

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Genetics

Use of the Pyrimidine Analog, 5-Iodo-2′-Deoxyuridine (IdU) with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform
Raymond D Devine 1, Gregory K Behbehani 1
1Ohio State Comprehensive Cancer Center, Division of Hematology, The Ohio State University

This protocol adapts cell cycle measurements for use in a mass cytometry platform. With the multi-parameter capabilities of mass cytometry, direct measurement of iodine incorporation allows identification of cells in S-phase while intracellular cycling markers enable characterization of each cell cycle state in a range of experimental conditions.

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Engineering

Multimodal 3D Printing of Phantoms to Simulate Biological Tissue
Canzhen Ma *1,2, Shuwei Shen *1,2, Guangli Liu 1, Siyue Guo 1,2, Buyun Guo 1,2, Jialuo Li 1,2, Kuiming Huang 1,2, Yidan Zheng 1, Pengfei Shao 1,2, Erbao Dong 1,2, Jiaru Chu 1, Ronald X. Xu 1,2,3
1Department of Precision Machinery and Precision Instrumentation, University of Science and Technology of China, 2Key Laboratory of Precision Scientific Instrumentation of Anhui Higher Education Institutes, University of Science and Technology of China, 3Department of Biomedical Engineering, The Ohio State University

Spin coating, polyjet printing, and fused deposition modeling are integrated to produce multilayered heterogeneous phantoms that simulate structural and functional properties of biological tissue.

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Medicine

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat
Feibo Zheng 1,3, Andy Tully 1,2, Kyle M Koss 1, Xiaomin Zhang 1, Longhui Qiu 1, Jiao-Jing Wang 1, Bilal A Naved 1,4, David Z Ivancic 1, James M Mathew 1, Jason A Wertheim 1, Zheng Jenny Zhang 1
1Comprehensive Transplant Center and Department of Surgery, Feinberg School of Medicine, Northwestern University, 2Department of Surgery, University of Illinois at Chicago, 3Department of Surgery, Tianjin Occupational Diseases Precaution and Therapeutic Hospital, 4Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

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Medicine

Repeated Orotracheal Intubation in Mice
Andrew M. Nelson 1, Katherine E. Nolan 1, Ian C. Davis 1
1Department of Veterinary Biosciences, The Ohio State University

The goal of this article is to describe a refined method of intubation of the laboratory mouse. The method is noninvasive and, therefore, ideal for studies that require serial monitoring of respiratory function and/or instillation of treatments into the lung.

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Neuroscience

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve
Nicholas P. Boyer 1, Maite Azcorra 1,2, Peter Jung 3,4, Anthony Brown 1
1Department of Neuroscience, The Ohio State University, 2Present address: Interdepartmental Neuroscience Graduate Program and Department of Neurobiology, Northwestern University, 3Quantitative Biology Institute, Ohio University, 4Department of Physics and Astronomy, Ohio University

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

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Cancer Research

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells
Katie A Thies 1, Sarah Steck 1, Sue E Knoblaugh 2, Steven T Sizemore 1
1Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University Medical Center, 2Department of Veterinary Biosciences, Comparative Pathology and Digital Imaging Shared Resource, The Ohio State University

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

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Cancer Research

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging
Jennifer A. Geisler 1,2,3, Jonathan M. Spehar 1,2, Sarah A. Steck 1,2, Anna Bratasz 1,4, Reena Shakya 1, Kimerly Powell 1,4, Gina M. Sizemore 1,2
1The Comprehensive Cancer Center, The Ohio State University, 2Department of Radiation Oncology, The Ohio State University, 3Department of Veterinary Biosciences, The Ohio State University, 4Davis Heart & Lung Research Institute, The Ohio State University

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

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JoVE Core

A Standardized Method for Measurement of Elbow Kinesthesia
Molly Watkins 1, Emily Duncanson 1, Emma Gartner 1, Samantha Paripovich 1, Christine Taylor 1, Alexandra Borstad 1
1Department of Physical Therapy, The College of St. Scholastica

Here, we present a standardized method for measurement of elbow passive kinesthesia using the threshold to detection of passive movement (TDPM) that is appropriate for a research setting.

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Biology

A 3D Printed Pollen Trap for Bumble Bee (Bombus) Hive Entrances
Houston J. Judd 1,2, Craig Huntzinger 2, Ricardo Ramirez 1, James P. Strange 2,3
1Department of Biology, Utah State University, 2USDA-ARS Pollinating Insect Research Unit, 3Department of Entomology, The Ohio State University

We present a non-lethal and automated mechanism to collect pollen from bumble bee (Bombus) workers returning to a hive. Instructions for producing, preparing, installing and using the devices are included. By using 3D-printed objects, modification to the design was timely, efficient and allowed for quick turnaround for testing.

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Cancer Research

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis
Iftekhar A. Showpnil 1,2, Kyle R. Miller 1, Cenny Taslim 1, Kathleen I. Pishas 1, Stephen L. Lessnick 1,3, Emily R. Theisen 1,4
1Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Children's Hospital, 2Molecular, Cellular, and Developmental Biology Program, The Ohio State University, 3Division of Pediatric Hematology/Oncology/Blood & Marrow Transplant, The Ohio State University, 4Department of Pediatrics, The Ohio State University

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

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Immunology and Infection

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes
Anthony Grigsby *1, Brendan J. Kelly *1, Neil D. Sanscrainte 2, James J. Becnel 2, Sarah M. Short 1
1Department of Entomology, The Ohio State University, 2USDA/ARS Center for Medical, Agricultural, and Veterinary Entomology

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

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Biology

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9
Megan E. Meuti 1, Robert Harrell 2
1Department of Entomology, The Ohio State University, 2Insect Transformation Facility, Institute for Bioscience and Biotechnology Research, University of Maryland

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

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Genetics

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders
Camila F. Almeida 1, Emma C. Frair 1, Nianyuan Huang 1, Reid Neinast 2, Kim L. McBride 2,3,4,6, Robert B. Weiss 5, Kevin M. Flanigan 1,6, Nicolas Wein 1,6
1Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, 2Center for Cardiovascular Research, The Research Institute at Nationwide Children’s Hospital, 3The Heart Center, Nationwide Children’s Hospital, 4Division of Genetic and Genomic Medicine, Nationwide Children’s Hospital, 5Department of Human Genetics, The University of Utah School of Medicine, 6Department of Pediatrics, The Ohio State University

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

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Developmental Biology

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells
Shiqiao Ye 1,2, Xiaoping Wan 3, Juan Su 1,2, Akshar Patel 1,2,4, Blake Justis 1,2,5, Isabelle Deschênes 3, Ming-Tao Zhao 1,2,5,6
1Center for Cardiovascular Research, The Abigail Wexner Research Institute, Nationwide Children’s Hospital, 2The Heart Center, Nationwide Children’s Hospital, 3Department of Physiology and Cell Biology, The Ohio State University College of Medicine, 4Department of Anatomy, The Ohio State University College of Medicine, 5MCDB Graduate Program, The Ohio State University, 6Department of Pediatrics, The Ohio State University College of Medicine

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

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Medicine

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes
Yong Gyu Lee 1,2, Jung-Lye Kim 1,2, Andre F. Palmer 3, Brenda F. Reader 4, Jianjie Ma 1, Sylvester M. Black 2,4, Bryan A. Whitson 1,2
1Department of Surgery Division of Cardiac Surgery, The Ohio State University Wexner Medical Center, 2The Collaboration for Organ Perfusion, Protection, Engineering and Regeneration (COPPER) Laboratory, The Ohio State University College of Medicine, 3William G. Lowrie Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, 4Comprehensive Transplant Center, The Ohio State University Wexner Medical Center

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

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Engineering

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve
Yifei Liu 1, Yong-Ung Lee 2, Tai Yi 2, Ken Wu 3, Cedric Bouchet-Marquis 3, Han Chan 3, Christopher K. Breuer 2, David W. McComb 1
1Center for Electron Microscopy and Analysis, The Department of Materials Science and Engineering, Ohio State University, 2Center for Regenerative Medicine, Nationwide Children’s Hospital, 3Thermo Fisher Scientific

Here, we describe a correlative workflow for the excision, pressurization, fixation, and imaging of the murine pulmonary valve to determine the gross conformation and local extracellular matrix structures.

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Neuroscience

A Mouse Model of Direct Anastomosis via the Prespinal Route for Crossing Nerve Transfer Surgery
Zhengrun Gao *1,2, Gaowei Lei *1, Zhen Pang *1, Yiming Chen 1, Shuai Zhu 1, Kun Huang 1, Weishan Lin 1, Yundong Shen 1,2,3,4,5, Wendong Xu 1,2,3,4,5
1Department of Hand Surgery, Huashan Hospital, Fudan University, 2Department of Hand and Upper Extremity Surgery, Jing‘an District Central Hospital, Fudan University, 3The National Clinical Research Center for Aging and Medicine, Fudan University, 4Institutes of Brain Science, Fudan University, 5State Key Laboratory of Medical Neurobiology, Collaborative Innovation Center of Brain Science, Fudan University

We simulated clinical surgery to establish a protocol of direct anastomosis of bilateral brachial plexus nerves via the prespinal route in mice, contributing to the study of the neural mechanisms underlying rehabilitation upon crossing nerve transfer after central and peripheral nervous system injuries.

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Medicine

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes
Sarah L. Sturgill 1, Lorien G. Salyer 1, Brandon J. Biesiadecki 1, Mark T. Ziolo 1
1Department of Physiology and Cell Biology, The Ohio State University

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

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Biochemistry

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis
Hui-Lan Chang *1, Kang-Yi Su *1,2, Steven D. Goodman 3, Wern-Cherng Cheng 2, Liang-In Lin 1,2, Ya-Chien Yang 1,2, Sui-Yuan Chang 1,2, Woei-horng Fang 1,2
1Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, 2Department of Laboratory Medicine, National Taiwan University Hospital, 3Center for Microbial Pathogenesis, Nationwide Children’s Hospital and the Department of Pediatrics, The Ohio State University

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

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Medicine

Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers
Juan Rodriguez 1,2, Matthew Reilly 3, Robert P. Mecham 4, Steven Bassnett 2,4
1Department of Basic Sciences, University of Health Sciences and Pharmacy, 2Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, 3Department of Biomedical Engineering, The Ohio State University, 4Department of Cell Biology & Physiology, Washington University School of Medicine

The protocol describes a method for the study of extracellular matrix viscoelasticity and its dependence on protein composition or environmental factors. The matrix system targeted is the mouse zonule. The performance of the method is demonstrated by comparing the viscoelastic behavior of wild-type zonular fibers with those lacking microfibril-associated glycoprotein-1.

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Biochemistry

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo
Shashi Bhushan Kumar 1, Shanvanth Arnipalli 1, Adham Abushukur 1, Silvia Carrau 1, Priyanka Mehta 1, Ouliana Ziouzenkova 1
1Department of Human Sciences, The Ohio State University

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

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Immunology and Infection

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms
Pranav S. J. B. Rana 1, Erin S. Gloag 2, Daniel J. Wozniak 1,2
1Department of Microbiology, The Ohio State University, 2Department of Microbial Infection and Immunity, The Ohio State University

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

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Medicine

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography
Qiang Zeng 1, Doug A. Gouchoe 2,3, Mahboubeh Nabavinia 4, Yong Gyu Lee 2, Xi Wang 2, Terri A. Shaffer 5, Mitchel R. Stacy 4,5,6, Blake R. Peterson 7,8, Bryan A. Whitson 2,9, Christopher Breuer 1,4, Sylvester M. Black 1,10
1Department of Surgery, Nationwide Children’s Hospital, 2COPPER Laboratory, The Ohio State University, 388th Surgical Operation Operations Squadron, WPAFB, 4Center for Regenerative Medicine, The Research Institute at Nationwide Children’s Hospital, 5Division of Vascular Diseases and Surgery, The Ohio State University College of Medicine, 6Interdisciplinary Biophysics Graduate Program, The Ohio State University, 7Division of Medicinal Chemistry and Pharmacognosy, The Ohio State University College of Pharmacy, 8The Ohio State University Comprehensive Cancer Center, 9Division of Cardiac Surgery, Department of Surgery, The Ohio State Wexner Medical Center, 10Division of Transplantation, Department of Surgery, The Ohio State Wexner Medical Center

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

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Bioengineering

Automated Compression Testing of the Ocular Lens
Debae Alzoubi 1, Wade Rich 1, Matthew A. Reilly 1,2
1Department of Biomedical Engineering, The Ohio State University, 2Department of Ophthalmology & Visual Sciences, The Ohio State University

We present an automated method for characterizing the effective elastic modulus of an ocular lens using a compression test.

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Environment

Measuring Dissolved Methane in Aquatic Ecosystems Using An Optical Spectroscopy Gas Analyzer
Jorge A. Villa 1, Robert Bordelon 1, Yang Ju 2, Madeline Moore 1, Timothy Morin 3, Gil Bohrer 2
1School of Geosciences, University of Louisiana at Lafayette, 2Department of Civil, Environmental and Geodetic Engineering, The Ohio State University, 3Department of Environmental Resources Engineering, College of Environmental Sciences and Forestry, State University of New York

This study demonstrates an approach to measure methane gas concentrations in aqueous samples using portable optical analyzers coupled to an injection chamber in a closed loop. The results are similar to conventional gas chromatography, presenting a practical and low-cost alternative particularly suitable for remote field studies.

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Medicine

Exploring Alternative Perfusion Solutions Using Next-Generation Polymerized Hemoglobin-Based Oxygen Carriers in a Model of Rat Ex Vivo Lung Perfusion
Doug A. Gouchoe *1,2, Yong Gyu Lee *1,2, Alisyn Greenfield 3, Clayton Cuddington 3, Jung-Lye Kim 1,2, Sylvester M. Black 1, Andre F. Palmer 3, Bryan A. Whitson 1,2
1COPPER Laboratory, Department of Surgery, The Ohio State University Wexner Medical Center, 2Division of Cardiac Surgery, Department of Surgery, The Ohio State University Wexner Medical Center, 3William G. Lowrie Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University

Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat ex vivo lung perfusion.

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