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Wake Forest School of Medicine

9 ARTICLES PUBLISHED IN JoVE

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Biology

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue
Thomas Lampl 1, Jo A. Crum 1, Taylor A. Davis 1, Carol Milligan 2,3,4, Victoria Del Gaizo Moore 1
1Chemistry Department, Elon University, 2Department of Neurobiology and Anatomy, Wake Forest School of Medicine, 3Neuroscience Graduate Program, Wake Forest School of Medicine, 4ALS Center Translational Science Unit, Wake Forest School of Medicine

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

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Medicine

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy
Manish S. Bharadwaj 1, Daniel J. Tyrrell 1, Mary F. Lyles 1, Jamehl L. Demons 1, George W. Rogers 2, Anthony J. A. Molina 1
1Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine, 2Seahorse Biosciences

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

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Biochemistry

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1
Eric D. Routh *1, Steven D. Creacy *2, Peter E. Beerbower 3, Steven A. Akman 4, James P. Vaughn 1, Philip J. Smaldino 3
1Department of Cancer Biology, Wake Forest School of Medicine, 2YX Genomics, 3Department of Biology, Ball State University, 4Department of Hematology and Oncology, Roper St. Francis Hospital

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

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Immunology and Infection

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota
Shokouh Ahmadi *1,2,3, Shaohua Wang *1,2, Ravinder Nagpal *1,2, Rabina Mainali 1,2, Sabihe Soleimanian-Zad 3,4, Dalane Kitzman 5, Hariom Yadav 1,2
1Department of Internal Medicine- Molecular Medicine, Wake Forest School of Medicine, 2Department of Microbiology and Immunology, Wake Forest School of Medicine, 3Department of Food Science and Technology, Isfahan University of Technology, 4Research Institute for Biotechnology and Bioengineering, College of Agriculture, Isfahan University of Technology, 5Department of Geriatrics and Gerontology, Wake Forest School of Medicine

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

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Immunology and Infection

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages
Yong Joo Ahn 1, Luxi Wang 1, Reto Asmis 1,2
1Department of Internal Medicine, Section on Molecular Medicine, Wake Forest School of Medicine, 2Department of Biochemistry, Wake Forest School of Medicine

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

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Bioengineering

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

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

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Immunology and Infection

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery
Guy El Helou 1, Joseph F. Goodman 2, Maria Blevins 3, David L. Caudell 4, Todd A. Ponzio 3, John W. Sanders 3,5
1Department of Medicine, Division of Infectious Diseases and Global Medicine, University of Florida, 2Department of Otolaryngology, George Washington University School of Medicine, 3Department of Medicine, Section on Infectious Diseases, Wake Forest Baptist Medical Center, 4Department of Pathology, Wake Forest School of Medicine, 5Department of Medicine, Section of Infectious Diseases, Hefner Veterans Affairs Medical Center

Salivary glands have been proposed as a tissue target site for gene therapy, especially in the area of vaccination by gene transfer. We demonstrate gene delivery in a non-human primate model utilizing retrograde parotid infusion.

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Biochemistry

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density
Chelcie F. Heaney 1,2, Colin J. McArdle 2, Kimberly F. Raab-Graham 1,2
1Wake Forest Translational Alcohol Research Center, Wake Forest School of Medicine, 2Physiology and Pharmacology, Wake Forest School of Medicine

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

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Medicine

Neuromuscular Ultrasound of the Median Nerve at the Carpal Tunnel
Vanessa Baute Penry 1, Rachana K. Gandhi Mehta 1, Robert H. Alavi 2
1Department of Neurology, Atrium Health Wake Forest Baptist, 2Wake Forest School of Medicine

The present protocol describes a technique for the standardization of median nerve ultrasound in cases of suspected carpal tunnel syndrome.

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