Wake Forest School of Medicine 8 articles published in JoVE Biochemistry DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density Chelcie F. Heaney1,2, Colin J. McArdle2, Kimberly F. Raab-Graham1,2 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. Immunology and Infection Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery Guy El Helou1, Joseph F. Goodman2, Maria Blevins3, David L. Caudell4, Todd A. Ponzio3, John W. Sanders3,5 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. Immunology and Infection Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages Yong Joo Ahn1, Luxi Wang1, Reto Asmis1,2 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. 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 Mainali1,2, Sabihe Soleimanian-Zad3,4, Dalane Kitzman5, Hariom Yadav1,2 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. Genetics Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells Hector Guillen-Ahlers1,2, Prahlad K. Rao1, Danu S. Perumalla1, Maria J. Montoya1, Avinash Y.L. Jadhav1, Michael R. Shortreed3, Lloyd M. Smith3, Michael Olivier1,2 1Department of Genetics, Texas Biomedical Research Institute, 2Department of Internal Medicine-Molecular Medicine, Wake Forest University School of Medicine, 3Department of Chemistry, University of Wisconsin This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells. Biochemistry A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1 Eric D. Routh*1, Steven D. Creacy*2, Peter E. Beerbower3, Steven A. Akman4, James P. Vaughn1, Philip J. Smaldino3 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. Biology Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue Thomas Lampl1, Jo A. Crum1, Taylor A. Davis1, Carol Milligan2,3,4, Victoria Del Gaizo Moore1 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. Medicine Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy Manish S. Bharadwaj1, Daniel J. Tyrrell1, Mary F. Lyles1, Jamehl L. Demons1, George W. Rogers2, Anthony J. A. Molina1 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.
