University of Miami Miller School of Medicine

10 ARTICLES PUBLISHED IN JoVE

Neuroscience

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue

Karolina J. Janczura^1,2, Claude-Henry Volmar^1,2, Claes Wahlestedt^1,2

¹Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, ²Center for Therapeutic Innovation, University of Miami Miller School of Medicine

The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues.

Immunology and Infection

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

Petoria Gayle¹, Nancy E. Freitag², Natasa Strbo¹, Kurt Schesser¹

¹Department of Microbiology & Immunology, University of Miami Miller School of Medicine, ²Department of Microbiology & Immunology, University of Illinois College of Medicine

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Immunology and Infection

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

Duneia McManus^1,2,3,4, Horacio J. Novaira^1,2,3,4, Anouk A.J. Hamers^1,2,3,4, Asha B. Pillai^1,2,3,4,5

¹Department of Pediatrics, Division of Hematology / Oncology and Bone Marrow Transplantation, University of Miami Miller School of Medicine, ²Batchelor Children's Research Institute, University of Miami Miller School of Medicine, ³Department of Microbiology & Immunology, University of Miami Miller School of Medicine, ⁴Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, ⁵Holtz Children's Hospital, University of Miami Miller School of Medicine

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Medicine

A Computerized Functional Skills Assessment and Training Program Targeting Technology Based Everyday Functional Skills

Philip D. Harvey^1,2, Lize Tibiriçá^2,3, Peter Kallestrup², Sara J. Czaja^1,2,4

¹University of Miami Miller School of Medicine, ²i-Function, ³Albizu University, ⁴Weill-Cornell Medical Center

This training protocol uses computerized training to teach technology-related everyday functional skills. These skills include financial skills, travel and transit, as well as medication management.

Developmental Biology

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Amir Bagheri^1,2, Seyedeh Fatemeh Razavipour³, Claes Wahlestedt², Seyed Javad Mowla¹, Mohammad Ali Faghihi^2,4

¹Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, ²Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, ³Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, ⁴Persian BayanGene Research and Training Center

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

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 Enck³, Sean M. Muir⁵, Justine Mariam Aziz⁶, Sandrine Lablanche⁷, Emily Tubbs⁷, Alice A. Tomei^8,9, Mark Van Dyke¹⁰, Shay Soker³, Emmanuel C. Opara³, 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.

Medicine

Electroretinogram Recording for Infants and Children under Anesthesia to Achieve Optimal Dark Adaptation and International Standards

Byron L. Lam¹, Carlos Mendoza-Santiestaban¹, Alex Gonzalez^1,2, Cornelis Rowaan^1,2, Mu Liu¹, Joanne Martin¹, Steven Gayer^1,3, Osmany Gil Figueredo¹, Jean-Marie Parel^1,2

¹Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, ²Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, ³Department of Anesthesiology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine

Adhering to international standards and maintaining retinal dark adaptation are critical to acquire valid full-field electroretinogram responses in the diagnosis and management of inherited retinal diseases. A practical protocol using a portable darkroom is provided to obtain full-field electroretinogram for infants and children under sedation or general anesthesia in the operating room setting.

Medicine

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Antoine J. Ribieras¹, Yulexi Y. Ortiz^1,2, Sophie Shrestha³, C. Theodore Huerta¹, Hongwei Shao^1,2, Maria E. Boulina⁴, Roberto I. Vazquez-Padron^1,2, Zhao-Jun Liu^1,2, Omaida C. Velazquez^1,2

¹Dewitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, ²Vascular Biology Institute, University of Miami Miller School of Medicine, ³University of Miami, ⁴Analytical Imaging Core Facility, University of Miami

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Cancer Research

Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells

Jacob Jahn¹, Sana Chaudhry¹, Maurizio Affer¹, Alejandro Pardo¹, Gabriel Pardo¹, Justin Taylor¹

¹Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine

The present protocol offers an efficient and flexible method to isolate RNA from nuclear and cytoplasmic fractions using cultured cells, and then validate using qPCR. This effectively serves as a replacement for other RNA preparation kits.

Immunology and Infection

Comprehensive Approach for Microbial Isolation from Hidradenitis Suppurativa Tunnels

Tammy Gonzalez^*1, Elina V. Zhivov^*1, Raji R. Nagalla¹, Rebecca Verpile¹, Viviane Abreu Nunes¹, Marjana Tomic-Canic¹, Barry Resnik¹, Hadar Lev-Tov¹, Irena Pastar¹

¹Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine

We showcase a method to successfully isolate fastidious and anaerobic organisms from cutaneous sinus tracts (tunnels) in tissues excised from patients with Hidradenitis Suppurativa.