Augusta University 9 articles published in JoVE Biology Isolation of Primary Mouse Retinal Pigmented Epithelium Cells Ryan Tomaszewski1,2, Pragya Rajpurohit3, Mei Cheng1,2, Amany Tawfik1,2,3 1Eye Research Institute, Oakland University, 2Eye Research Center (OUWB)/ERC, William Beaumont School of Medicine, 3Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells. Engineering Glass-Based Devices to Generate Drops and Emulsions Josefa Guerrero1, Javier Rojo2, Alexis de la Cotte2, Luis Manuel Aguilera-Sáez3, Enric Vila3, Alberto Fernandez-Nieves2,4,5 1Department of Chemistry and Physics, Augusta University, 2Department of Condensed Matter Physics, University of Barcelona, 3Agrobío S.L., 4ICREA - Institució Catalana de Recerca i Estudis Avançats, 5Institute of Complex Systems (UBICS), University of Barcelona Here, a protocol to manufacture glass-based microfluidic devices used for generating highly monodisperse emulsions with controlled drop size is presented. Medicine Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy WonMo Ahn1, Bhupesh Singla1, Brendan Marshall2, Gábor Csányi1,3 1Vascular Biology Center, Medical College of Georgia at Augusta University, 2Department of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University, 3Department of Pharmacology and Toxicology, Medical College of Georgia at Augusta University Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy. Cancer Research Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients Divya Bhagirath1, Rajvir Dahiya2, Shahana Majid2, Z. Laura Tabatabai3, Sharanjot Saini1 1Department of Biochemistry and Molecular Biology, Augusta University, 2Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, 3Department of Pathology, Veterans Affairs Medical Center and University of California, San Francisco Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer. Developmental Biology CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors Yue Jin1, Yan Shen1, Xuan Su1, Neal Weintraub1, Yaoliang Tang1 1Medical College of Georgia, Augusta University Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmdmdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system. Genetics Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice Xuan Su*1,2, Yan Shen*1, Yue Jin*1,2, Meng Jiang2, Neal Weintraub1, Yaoliang Tang1 1Vascular Biology Center, Medical College of Georgia, Augusta University, 2Renji Hospital, School of Medicine, Shanghai Jiaotong University Here, we present a protocol to transiently improve cardiac function in Duchenne muscular dystrophy mice by transplanting exosomes derived from normal myogenic progenitor cells. Developmental Biology Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes Andrew S. Riching1, Yuanbiao Zhao1, Yingqiong Cao1, Pilar Londono1, Hongyan Xu2, Kunhua Song1 1Division of Cardiology, Department of Medicine, University of Colorado Anschutz Medical Campus, 2Department of Population Health Sciences, Medical College of Georgia, Augusta University Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-β signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency. Medicine An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis Amy V. Paschall1,2,3, Kebin Liu1,2,3 1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, 2Georgia Cancer Center, Augusta University, 3Charlie Norwood VA Medical Center An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging. Medicine Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test Paula Rodriguez-Miguelez1, Nichole Seigler1, Ryan A. Harris1,2 1Division of Clinical and Translational Sciences, Georgia Prevention Institute, Georgia Regents University, 2Sport and Exercise Science Research Institute, University of Ulster The flow mediated dilation (FMD) test is the most commonly utilized, non-invasive, ultrasound assessment of endothelial function in humans. Although the FMD test has been related with the prediction of future cardiovascular disease and events, it is a physiological assessment with many inherent confounding factors that need to be considered.