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41 ARTICLES PUBLISHED IN JoVE

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Medicine

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma
Amanda Gatesman Ammer 1, Karen E. Hayes 1, Karen H. Martin 1, Lingqing Zhang 2, George A. Spirou 3, Scott A. Weed 1
1Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, 2Sensory Neuroscience Research Center, West Virginia University , 3Departments of Otolaryngology and Physiology, Center for Neuroscience, West Virginia University

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

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Biology

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes
Yanning Wu 1, Shuo Wang 1, Chunying Li 1,2,3
1Department of Biochemistry & Molecular Biology, Wayne State University School of Medicine, 2Cardiovascular Research Institute, Wayne State University School of Medicine, 3Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

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Biology

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts
Karen H. Martin 1, Karen E. Hayes 1, Elyse L. Walk 1, Amanda Gatesman Ammer 1, Steven M. Markwell 1, Scott A. Weed 1
1Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

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Behavior

Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking
Evan D. Morris 1,2,3,4, Su Jin Kim 1,3, Jenna M. Sullivan 1,3,4, Shuo Wang 3,4, Marc D. Normandin 5, Cristian C. Constantinescu 6, Kelly P. Cosgrove 1,2,3
1Diagnostic Radiology, Yale University, 2Psychiatry, Yale University, 3Yale PET Center, Yale University, 4Biomedical Engineering, Yale University, 5Nuclear Medicine, Massachusetts General Hospital, 6Radiological Sciences, University of California, Irvine

We present a novel PET imaging approach for capturing dopamine fluctuations induced by cigarette smoking. Subjects smoke in the PET scanner. Dynamic PET images are modeled voxel-by-voxel in time by lp-ntPET, which includes a time-varying dopamine term. The results are 'movies' of dopamine fluctuations in the striatum during smoking.

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Bioengineering

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip
Xiang Li 1, Samantha Marie Mearns 1, Manuela Martins-Green 2, Yuxin Liu 1
1Lane Department of Computer Science and Electrical Engineering, West Virginia University, 2Department of Cell Biology and Neuroscience, University of California at Riverside

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging and can be applied for microvascular research.

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Behavior

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment
Daniel S. Weitzner 1, Elizabeth B. Engler-Chiurazzi 2, Linda A. Kotilinek 3, Karen Hsiao Ashe 3,4,5, Miranda Nicole Reed 1,6
1Department of Psychology, Behavioral Neuroscience, West Virginia University, 2Department of Physiology and Pharmacology, West Virginia University, 3Department of Neurology, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, 4Department of Neuroscience, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, 5GRECC, VA Medical Center, 6Center for Neuroscience, Center for Basic and Translational Stroke Research, West Virginia University

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

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Medicine

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes
Elyse L. Walk *1,3, Sarah L. McLaughlin *2,3, Scott A. Weed 1,3
1Department of Neurobiology and Anatomy, West Virginia University, 2Animal Models and Imaging Facility, West Virginia University, 3Mary Babb Randolph Cancer Center, West Virginia University

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

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Behavior

Multifunctional Setup for Studying Human Motor Control Using Transcranial Magnetic Stimulation, Electromyography, Motion Capture, and Virtual Reality
William J. Talkington 1, Bradley S. Pollard 1, Erienne V. Olesh 1, Valeriya Gritsenko 1
1Department of Human Performance and Applied Exercise Science, Division of Physical Therapy, West Virginia University

Transcranial magnetic stimulation, electromyography, and 3D motion capture are commonly used non-invasive techniques for investigating neuromuscular function in humans. In this paper, we describe a protocol that synchronously samples data generated by all three of these tools along with the unique addition of virtual reality stimulus presentation and feedback.

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Biology

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production
Sulei Xu *1, Xiang Li *1, Yuxin Liu 2, Pingnian He 1
1Department of Cellular and Molecular Physiology, College of Medicine, Penn State University, 2Lane Department of Computer Science and Electrical Engineering, West Virginia University

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

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Bioengineering

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo
Joshua R. Biller 1,2, Deborah G. Mitchell 1, Mark Tseytlin 3,4, Hanan Elajaili 1, George A. Rinard 5, Richard W. Quine 6, Sandra S. Eaton 1, Gareth R. Eaton 1
1Department of Chemistry and Biochemistry, University of Denver, 2Magnetic Imaging Group, Applied Physics Division, Physical Measurements Laboratory, National Institute of Standards and Technology, 3Department of Radiology, Geisel School of Medicine, Dartmouth University, 4Department of Biochemistry, West Virginia University, 5Department of Electrical and Computer Engineering, University of Denver, 6Department of Engineering, University of Denver

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

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Environment

Watershed Planning within a Quantitative Scenario Analysis Framework
Eric R. Merriam 1, J. Todd Petty 1, Michael P. Strager 2
1Division of Forestry and Natural Resources, West Virginia University, 2Division of Resource Management, West Virginia University

There is a critical need for tools and methodologies capable of managing aquatic systems in the face of uncertain future conditions. We provide methods for conducting a targeted watershed assessment that enables resource managers to produce landscape-based cumulative effects models for use within a scenario analysis management framework.

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Education

Design and Use of a Full Flow Sampling System (FFS) for the Quantification of Methane Emissions
Derek R. Johnson 1,2, April N. Covington 1,2, Nigel N. Clark 1
1Mechanical and Aerospace Engineering Department, West Virginia University, 2Center for Alternative Fuels, Engines, and Emissions, West Virginia University

We have designed, developed, and implemented a novel full flow sampling system (FFS) for quantification of methane emissions and greenhouse gases from across the natural gas supply chain.

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Cancer Research

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases
Mercedes Rodriguez-Teja 1, Claudia Breit 2, Mitchell Clarke 3, Kamil Talar 3, Kai Wang 3, Mohammad A. Mohammad 3, Sage Pickwell 3, Guillermina Etchandy 1, Graeme J. Stasiuk 3, Justin Sturge 3
1Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR), 2Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 3School of Biological, Biomedical & Environmental Sciences, University of Hull

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

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Bioengineering

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior
Kai Wang 1, Kam W. Leong 2, Yong Yang 1
1Department of Chemical and Biomedical Engineering, West Virginia University, 2Department of Biomedical Engineering, Columbia University

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

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Neuroscience

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells
Stephanie L. Rellick 1,2,4, Heng Hu 1,3,4, James W. Simpkins 1,4, Xuefang Ren 1,3,4
1Department of Physiology and Pharmacology, West Virginia University, 2Mitochondrial Evaluation Core, West Virginia University, 3Experimental Stroke Core, West Virginia University, 4Center for Basic and Translational Stroke Research, West Virginia University

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

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Bioengineering

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice
Kai Wang *1, Xi Wang *1, Cheng-sheng Han *2, Liang-yi Chen 2, Ying Luo 1
1Department of Biomedical Engineering, Peking University, 2Institute of Molecular Medicine, Peking University

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

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Neuroscience

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models
Holly C. Hunsberger 1, Sharay E. Setti 2, Ryan T. Heslin 2, Jorge E. Quintero 3, Greg A. Gerhardt 3, Miranda N. Reed 2
1Department of Psychology, West Virginia University, 2Department of Drug Discovery & Development, Auburn University, 3Department of Neuroscience, University of Kentucky Medical Center

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

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Engineering

Resonance Fluorescence of an InGaAs Quantum Dot in a Planar Cavity Using Orthogonal Excitation and Detection
Disheng Chen 1, Gary R. Lander 1, Edward B. Flagg 1
1Department of Physics and Astronomy, West Virginia University

Resonant excitation of a single self-assembled quantum dot can be achieved using an excitation mode orthogonal to the fluorescence collection mode. We demonstrate a method using the waveguide and Fabry-Perot modes of a planar microcavity surrounding the quantum dots. The method allows complete freedom in the detection polarization.

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Cancer Research

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment
Andrey A. Bobko 1,2, Timothy D. Eubank 1,3, Benoit Driesschaert 1,2, Valery V. Khramtsov 1,2
1In Vivo Multifunctional Magnetic Resonance center, Robert C. Byrd Health Sciences Center, West Virginia University, 2Department of Biochemistry, West Virginia University School of Medicine, 3Department of Microbiology, Immunology & Cell Biology, West Virginia University School of Medicine

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

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Biology

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model
Kang Qin 1, Yifan Wu 1, Yiwa Pan 1, Kai Wang 1, Deling Kong 1, Qiang Zhao 1
1State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials (Ministry of Education), College of Life Sciences, Nankai University

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

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Environment

Necropsy-based Wild Fish Health Assessment
Vicki S. Blazer 1, Heather L. Walsh 2, Ryan P. Braham 1, Cheyenne Smith 2
1National Fish Health Research Laboratory, Leetown Science Center, U.S. Geological Survey, 2School of Natural Resources, West Virginia University

The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses.

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Engineering

Femtosecond Laser Filaments for Use in Sub-Diffraction-Limited Imaging and Remote Sensing
Matthews M. Springer 1, Benjamin D. Strycker 1,2, Kai Wang 1, Alexei V. Sokolov 1,2, Marlan O. Scully 1,2
1Texas A&M University, 2Baylor University

High-intensity femtosecond pulses of laser light can undergo cycles of Kerr self-focusing and plasma defocusing, propagating an intense sub-millimeter-diameter beam over long distances. We describe a technique for generating and using these filaments to perform remote imaging and sensing beyond the classical diffraction limits of linear optics.

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Behavior

Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients
Shuo Wang 1, Nand Chandravadia 2, Adam N. Mamelak 2, Ueli Rutishauser 2,3,4
1Department of Chemical and Biomedical Engineering, and Rockefeller Neuroscience Institute, West Virginia University, 2Departments of Neurosurgery and Neurology, Cedars-Sinai Medical Center, 3Center for Neural Science and Medicine, Department of Biomedical Sciences, Cedars-Sinai Medical Center, 4Division of Biology and Biological Engineering, California Institute of Technology

We describe a method to conduct single-neuron recordings with simultaneous eye tracking in humans. We demonstrate the utility of this method and illustrate how we used this approach to obtain neurons in the human medial temporal lobe that encode targets of a visual search.

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Biology

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana
Kai Wang 1, Kayin Law 1, Manhin Leung 1, Waishing Wong 1, Ning Li 1,2
1Division of Life Science, Energy Institute, Institute for the Environment, The Hong Kong University of Science and Technology, 2Shenzhen Research Institute, The Hong Kong University of Science and Technology

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

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Medicine

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)
PeiLun Hu *1, JiSheng Lin *1, JunChuan Xu 1, Hai Meng 1, Nan Su 1, Yong Yang *1, Qi Fei 1
1Department of Orthopedics, Beijing Friendship Hospital, Capital Medical University

Herein, we present a three-dimensional printing guide template for percutaneous vertebraplasty. A patient with a T11 vertebral compression fracture was selected as a case study.

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Medicine

Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction
Anna L. Lang 1,2, William T. Goldsmith 3,4, Regina D. Schnegelberger 5,6, Gavin E. Arteel 6,7, Juliane I. Beier 6,7
1Department of Pharmacology and Toxicology, University of Louisville, 2Hepatobiology and Toxicology Program, University of Louisville, 3Department of Physiology and Pharmacology, West Virginia University, 4Center for Inhalation Toxicology, West Virginia University, 5Department of Pharmacology and Chemical Biology, University of Pittsburgh, 6Pittsburgh Liver Research Center, University of Pittsburgh, 7Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh

The goal of this protocol was to develop a murine model of low-level toxicant exposure that does not cause overt liver injury but rather exacerbates pre-existing liver damage. This paradigm better recapitulates human exposure and the subtle changes that occur upon exposure to toxicant concentrations that are considered safe.

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Cancer Research

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging
Nour Ghaddar 1,2, Shuo Wang 1, Véronique Michaud 1, Urszula Kazimierczak 1,3, Nicolas Ah-son 1, Antonis E. Koromilas 1,4
1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 2Division of Experimental Medicine, Faculty of Medicine, McGill University, 3Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, 4Gerald Bronfman Department of Oncology, Faculty of Medicine, McGill University

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

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Neuroscience

Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents
Aric F. Logsdon *1,2, Brandon P. Lucke-Wold *3, Ryan C. Turner *4, Sean M. Collins 5, Evan L. Reeder 5, Jason D. Huber 4, Charles L. Rosen 6, Matthew J. Robson *5, Florian Plattner *7
1Geriatrics Research Education and Clinical Center, Veterans Affairs, 2Division of Gerontology and Geriatric Medicine, University of Washington, 3Department of Neurosurgery, University of Florida, 4Department of Neurosurgery, West Virginia University, 5Division of Pharmaceutical Sciences, University of Cincinnati, 6Central Illinois Neuro Health Sciences, Bloomington, IL, 7Neuro-research, Dallas, TX

We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury.

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Immunology and Infection

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment
Kelly L. Monaghan *1, Breanne Y. Farris *1, Wen Zheng 1, Edwin C. K. Wan 1,2,3
1Department of Microbiology, Immunology, and Cell Biology, West Virginia University, 2Department of Neuroscience, West Virginia University, 3Rockefeller Neuroscience Institute, West Virginia University

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

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Bioengineering

Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of Manganese(II) Acetylacetonate
Celia Martinez de la Torre 1, Margaret F. Bennewitz 1
1Department of Chemical and Biomedical Engineering, West Virginia University

This protocol details a facile, one-pot synthesis of manganese oxide (MnO) nanoparticles by thermal decomposition of manganese(II) acetylacetonate in the presence of oleylamine and dibenzyl ether. MnO nanoparticles have been utilized in diverse applications including magnetic resonance imaging, biosensing, catalysis, batteries, and waste water treatment.

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Biology

Irradiator Commissioning and Dosimetry for Assessment of LQ α and β Parameters, Radiation Dosing Schema, and in vivo Dose Deposition
Samuel A. Sprowls *1, Vincenzo J. Pizzuti *1,3, William Pentz 1, Divine C. Nwafor 3,4, R. Alfredo C. Siochi 2,3, Paul R. Lockman 1
1Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, 2Radiation Oncology, West Virginia University, 3School of Medicine, West Virginia University, 4Department of Neuroscience, West Virginia University

Radiation dosimetry provides a technique for enhancing the accuracy of preclinical experiments and ensuring that the radiation doses delivered are closely related to clinical parameters. This protocol describes steps to be taken at each phase during preclinical radiation experiments to ensure proper experimental design.

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Cancer Research

Robot Assisted Distal Pancreatectomy with Celiac Axis Resection (DP-CAR) for Pancreatic Cancer: Surgical Planning and Technique
Pavan Rao 1, Carl R. Schmidt 1, Brian A. Boone 1,2
1Division of Surgical Oncology, Department of Surgery, West Virginia University, 2Department of Microbiology, Immunology and Cell Biology, West Virginia University

We present our operative approach to robot assisted distal pancreatectomy, splenectomy, and celiac axis resection (DP-CAR), demonstrating that the procedure is safe and feasible with proper planning, patient selection, and surgeon experience.

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JoVE Journal

Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes
Schuyler D. Vickers *1, Dominique C. Saporito *1, Roberta Leonardi 1
1Department of Biochemistry, School of Medicine, West Virginia University

Fatty acid β-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid β-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

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Biology

Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence
Alyson Nguyen *1, Keith Battle *2,3, Sunita S. Paudel *2,3, Ningyong Xu 2, Jessica Bell 3, Linn Ayers 3, Cassandra Chapman 4, Ajay P. Singh 5, Srinivas Palanki 6, Thomas Rich 3,7, Diego F. Alvarez 8, Troy Stevens 2,3, Dhananjay T. Tambe 3,4,7
1Biomedical Sciences, Pat Capps Covey College of Allied Health Professions, University of South Alabama, 2Department of Physiology and Cell Biology, College of Medicine, University of South Alabama, 3Center for Lung Biology, College of Medicine, University of South Alabama, 4William B. Burnsed Jr. Mechanical, Aerospace, and Biomedical Engineering Department, College of Engineering, University of South Alabama, 5Mitchell Cancer Institute, College of Medicine, University of South Alabama, 6Department of Chemical and Biomedical Engineering, West Virginia University, 7Department of Pharmacology, College of Medicine, University of South Alabama, 8Department of Physiology & Pharmacology, College of Osteopathic Medicine, Sam Houston State University

The Integrative Toolkit to Analyze Cellular Signals (iTACS) platform automates the process of simultaneously measuring a wide variety of chemical and mechanical signals in adherent cells. iTACS is designed to facilitate community-driven development and enable researchers to use all platform features regardless of their educational background.

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JoVE Core

Development and Functionalization of Electrolyte-Gated Graphene Field-Effect Transistor for Biomarker Detection
Sakib Ishraq 1, Jianbo Sun 2, Yuxin Liu 1
1Lane Department of Computer Science and Electrical Engineering, West Virginia University, 2Beijing Graphene Institute

The present protocol demonstrates the development of electrolyte-gated graphene field-effect transistor (EGGFET) biosensor and its application in biomarker immunoglobulin G (IgG) detection.

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Medicine

A Surgical Model of Heart Failure with Preserved Ejection Fraction in Tibetan Minipigs
Xiaohui Li *1,2, Weijiang Tan *1,3, Xiang Li *1, Shuang Zheng *1, Xiaoshen Zhang 2, Honghua Chen 1, Zhanhong Pan 1, Caiyi Zhu 1, Feng Hua Yang 1
1Guangdong Laboratory Animals Monitoring Institute, 2Department of Cardiovascular Surgery, the First Affiliated Hospital, Jinan University, 3College of Veterinary Medicine, South China Agricultural University

The present protocol describes a step-by-step procedure to establish a minipig model of heart failure with preserved ejection fraction using descending aortic constriction. The methods for evaluating cardiac morphology, histology, and function of this disease model are also presented.

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Genetics

Investigating the Pathogenesis of MYH7 Mutation Gly823Glu in Familial Hypertrophic Cardiomyopathy using a Mouse Model
Yu Xia *1,2, Jinlin Hu *3, Xiang Li 4, Shuang Zheng 4, Ge Wang 1,2, Songtao Tan 1,2, Zengxiao Zou 1,2, Qiong Ling 2,5, Fenghua Yang 4, Xiaoping Fan 1,2
1Department of Cardiovascular Surgery, Guangdong Provincial Hospital of Traditional Chinese Medicine, the Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 2The Second Clinical College of Guangzhou University of Chinese Medicine, 3Department of Cardiovascular Surgery, The First Affiliated Hospital, Jinan University, 4Guangdong Provincial Key Laboratory of Laboratory Animals, Guangdong Laboratory Animals Monitoring Institute, 5Department of Anesthesiology, Guangdong Provincial Hospital of Chinese Medicine, the Second Affiliated Hospital of Guangzhou University of Chinese Medicine

Based on the familial hereditary cardiomyopathy family found in our clinical work, we created a C57BL/6N mouse model with a point mutation (G823E) at the mouse MYH7 locus through CRISPR/Cas9-mediated genome engineering to verify this mutation.

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Chemistry

Synthesis of a Borylated Ibuprofen Derivative Through Suzuki Cross-Coupling and Alkene Boracarboxylation Reactions
Steven W. Knowlden *1, Randika T. Abeysinghe *1, Amanda D. Swistok 1, Alexis C. Ravenscroft 1, Brian V. Popp 1
1C. Eugene Bennett Department of Chemistry, West Virginia University

The present protocol describes a detailed benchtop catalytic method that yields a unique borylated derivative of ibuprofen. 

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Bioengineering

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles
Olivia L. Rose 1, Myra Arnold 2,3, Cerasela-Zoica Dinu 1
1Department of Chemical and Biomedical Engineering, West Virginia University, 2Department of Anthropology for Energy, West Virginia University, 3Department of Hospitality and Tourism, West Virginia University

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

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Engineering

Setup for the Quantitative Assessment of Motion and Muscle Activity During a Virtual Modified Box and Block Test
Rachel I. Taitano *1, Matthew G. Yough *2, Kacie Hanna 3, Anna S. Korol 1, Valeriya Gritsenko 1,2
1Department of Neuroscience, West Virginia University, 2Department of Human Performance, West Virginia University, 3Department of Biomedical Engineering, West Virginia University

The protocol described here aims to enhance the quantitative evaluation of upper limb deficits, with the goal of developing additional technology for remote assessment both in the clinic and at home. Virtual reality and biosensor technologies are combined with standard clinical techniques to provide insights into the functioning of the neuromuscular system.

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Biochemistry

MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a
Marvin Wright *1, Shayna Redford *1, Jacob Vehar 1, Kevin C. Courtney 1, Neil Billington 1,2, Rong Liu 1
1Department of Biochemistry & Molecular Medicine, School of Medicine, West Virginia University, 2Microscope Imaging Facility, West Virginia University

This protocol details the procedures for recombinantly producing the human myosin-7a holoenzyme using the MultiBac Baculovirus system and for studying its motility using a tailored in vitro filament gliding assay.

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