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university of texas medical branch

24 ARTICLES PUBLISHED IN JoVE

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Medicine

Acute Myocardial Infarction in Rats
Yewen Wu 1, Xing Yin 2, Cori Wijaya 2, Ming-He Huang 1, Bradley K. McConnell 2
1Department of Internal Medicine, Division of Cardiology, University of Texas Medical Branch, 2Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston (UH), Texas Medical Center

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

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Biology

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor
Askar M. Akimzhanov 1, Darren Boehning 1
1Department of Neuroscience and Cell Biology, University of Texas Medical Branch

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

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Immunology and Infection

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy
Birte Kalveram 1, Olga Lihoradova 1, Sabarish V. Indran 1, Tetsuro Ikegami 1
1Department of Pathology, University of Texas Medical Branch

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

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Neuroscience

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury
Deborah R. Boone 1, Stacy L. Sell 1, Helen Lee Hellmich 1
1Department of Anesthesiology, University of Texas Medical Branch

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

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Immunology and Infection

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis
Birte Kalveram 1, Olga Lihoradova 1, Sabarish V. Indran 1, Jennifer A. Head 1, Tetsuro Ikegami 1
1Department of Pathology, Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

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Bioengineering

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes
Jaimy Miller *1, Daniela Dalm *1, Alexey Y. Koyfman 2,3, Kirill Grushin 1, Svetla Stoilova-McPhie 1,3
1Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 2Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 3Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

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Behavior

Fat Preference: A Novel Model of Eating Behavior in Rats
James M Kasper 1, Sarah B Johnson 1, Jonathan D. Hommel 1
1Center for Addiction Research, Department of Pharmacology and Toxicology, University of Texas Medical Branch

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats’ preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

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Immunology and Infection

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures
Krisztina Janosko 1, Michael R. Holbrook 1, Ricky Adams 1, Jason Barr 1, Laura Bollinger 1, Je T'aime Newton 2, Corrie Ntiforo 2, Linda Coe 1, Jiro Wada 1, Daniela Pusl 1, Peter B. Jahrling 1, Jens H. Kuhn 1, Matthew G. Lackemeyer 1
1Integrated Research Facility at Frederick, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), 2Environmental Health and Safety, Biological and Chemical Safety Program, University of Texas Medical Branch

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

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Medicine

A Simple Critical-sized Femoral Defect Model in Mice
Bret H. Clough 1, Matthew R. McCarley 2, Carl A. Gregory 1,3
1Institute for Regenerative Medicine at Scott & White Hospital, Texas A&M Health Science Center, 2Department of Orthopedic Surgery, University of Texas Medical Branch, 3Molecular and Cellular Medicine, Texas A&M Health Science Center

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

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Bioengineering

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles
Sandra L. Arias 1, Akshath R. Shetty 2, Angana Senpan 3, Mónica Echeverry-Rendón 4, Lisa M. Reece 5,6, Jean Paul Allain 1,2,3,7
1Department of Bioengineering, University of Illinois at Urbana-Champaign, 2Department of Nuclear, Plasma and Radiological Engineering, University of Illinois at Urbana-Champaign, 3Micro and Nanotechnology Laboratory, University of Illinois at Urbana-Champaign, 4Program of Study and Control of Tropical Diseases (PECET), University of Antioquia, 5Sealy Center for Vaccine Development, University of Texas Medical Branch, 6WHO Collaborating Center for Vaccine Research, Evaluation and Training on Emerging Infectious Diseases, University of Texas Medical Branch, 7Beckman Institute, University of Illinois at Urbana-Champaign

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

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Developmental Biology

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens
Paul Johnson 1, Ellen J. Beswick 3, Celia Chao 1, Don W. Powell 2, Mark R. Hellmich 1, Iryna V. Pinchuk 2
1Surgery, University of Texas Medical Branch, 2Internal Medicine, University of Texas Medical Branch, 3Molecular Genetics and Microbiology, University of New Mexico

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, α-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

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Developmental Biology

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions
Joni H. Ylostalo 1, Nikolay Bazhanov 2, Arezoo Mohammadipoor 3, Thomas J. Bartosh 4
1Department of Biology, University of Mary Hardin-Baylor, 2Department of Pediatrics, University of Texas Medical Branch, 3Multi-Organ Support Technology Task Area, U.S. Army Institute of Surgical Research, 4Internal Medicine, Texas A&M University Health Science Center

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

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Neuroscience

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder
Andrea L. Dimet *1,2,3, Irma E. Cisneros *1,4,5, Robert G. Fox 1,6, Sonja J. Stutz 1,6, Noelle C. Anastasio 1,6, Kathryn A. Cunningham 1,6, Kelly T. Dineley 1,3,4
1Center for Addiction Research, University of Texas Medical Branch, 2Institute for Translational Sciences, University of Texas Medical Branch, 3Mitchell Center for Neurodegenerative Diseases, University of Texas Medical Branch, 4Department of Neurology, University of Texas Medical Branch, 5Department of Pathology, University of Texas Medical Branch, 6Department of Pharmacology and Toxicology, University of Texas Medical Branch

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

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Medicine

Smartphone Fundus Photography
Hossein Nazari Khanamiri 1, Austin Nakatsuka 1, Jaafar El-Annan 1
1Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch

Fundus photography normally requires specialized fundus cameras that are not always available in all clinical settings. Here, a simple method to record ocular fundus images using a smartphone camera and a conventional high-plus handheld indirect ophthalmoscopy lens is described.

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Behavior

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury
Nicole Hausser *1, Kathia Johnson *1, Margaret A. Parsley 1, Jutatip Guptarak 1, Heidi Spratt 1, Stacy L. Sell 1
1Department of Anesthesiology, University of Texas Medical Branch

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

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Neuroscience

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury
Harris A. Weisz *1, Deborah R. Boone *1, Stacy L. Sell 1, Helen L. Hellmich 1
1Department of Anesthesiology, University of Texas Medical Branch

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

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Developmental Biology

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis
Erica L. McGrath 1,2, Junling Gao 1, Ping Wu 1,3
1Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 2Department of Neurology, Johns Hopkins University, 3Beijing Institute for Brain Disorders, Capital Medical University

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

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Neuroscience

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries
Uylissa A. Rodriguez 1,2, Yaping Zeng 1,2, Margaret A. Parsley 1,2, Bridget E. Hawkins 1,2, Donald S. Prough 1,2, Douglas S. DeWitt 1,2
1Charles R. Allen Research Laboratories, Department of Anesthesiology, University of Texas Medical Branch, 2The Moody Project for Translational Traumatic Brain Injury Research, University of Texas Medical Branch

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

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Immunology and Infection

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection
Elizabeth Mateer 1, Slobodan Paessler 1, Cheng Huang 1
1Department of Pathology and Institute for Human Infections and Immunity, University of Texas Medical Branch

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

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Immunology and Infection

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus
Sasha R. Azar 1,2, Scott C. Weaver 1,3,4
1Institute for Human Infections and Immunity, University of Texas Medical Branch, 2Department of Pathology, University of Texas Medical Branch, 3Department of Microbiology and Immunology, University of Texas Medical Branch, 4World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

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Neuroscience

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging
Hossein Nazari 1,2, Maxim Ivannikov 2, Lorenzo Ochoa 2,3, Gracie Vargas 2,3, Massoud Motamedi 3,4
1Department of Ophthalmology and Visual Neuroscience, University of Minnesota, 2Department of Neuroscience, Cell Biology, and Anatomy, University of Texas Medical Branch, 3Biomedical Engineering and Imaging Sciences Group, University of Texas Medical Branch, 4Department of Ophthalmology, University of Texas Medical Branch

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

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Medicine

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice
Wenjing Yang 1, Tianming Yu 1, Yingzi Cong 1
1Department of Microbiology and Immunology, University of Texas Medical Branch

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

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Neuroscience

Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies
Brice Miller *1, Ashli Powell *1, Berenice A. Gutierrez 1, Agenor Limon 1
1The Mitchell Center for Neurodegenerative Diseases, Department of Neurology, University of Texas Medical Branch

The protocol demonstrates that by performing microtransplantation of synaptic membranes into Xenopus laevis oocytes, it is possible to record consistent and reliable responses of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and γ-aminobutyric acid receptors.

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Biology

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion
Moirangthem Kiran Singh 1, Linda J. Kenney 1
1Biochemistry & Molecular Biology, University of Texas Medical Branch

This article provides a straightforward and clear protocol to label Salmonella secreted effectors using genetic code expansion (GCE) site-specifically and image the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)

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