University of Texas Health Science Center at Houston View Institution's Website 26 articles published in JoVE Cancer Research Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform Lindsey K. Sablatura1, Kristin M. Bircsak2, Peter Shepherd3, Karla Queiroz4, Mary C. Farach-Carson1,5,6, Pamela E. Constantinou1,7, Anthony Saleh2, Nora Navone3, Daniel A. Harrington1,5,6 1Department of BioSciences, Rice University, 2Mimetas US Inc, 3Department of Genitourinary Medical Oncology Research, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, 4Mimetas B.V., 5Department of Bioengineering, Rice University, 6Department of Diagnostic and Biomedical Sciences, The University of Texas Health Science Center, 7Sheikh Ahmed Center for Pancreatic Cancer Research, The University of Texas MD Anderson Cancer Center This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform. Cancer Research Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans Dharini van der Hoeven1,2, Thuy Nhu L. Truong1, Ali Naji1, Sabita Thapa1, John F. Hancock2, Ransome van der Hoeven1 1Department of Diagnostic and Biomedical Sciences, School of Dentistry, University of Texas Health Science Center, 2Department of Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins. Immunology and Infection Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System David Chetrit1, Donghyun Park1,2, Bo Hu3, Jun Liu1,2, Craig R. Roy1 1Department of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, 2Microbial Sciences Institute, Yale University, 3Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions. Medicine Oral Health Assessment by Lay Personnel for Older Adults Natalia S. Rozas1, June M. Sadowsky2, Jordyn A. Stanek1, Cameron B. Jeter1 1Department of Diagnostic and Biomedical Sciences, The University of Texas Health Science Center at Houston School of Dentistry, 2Department of General Practice and Dental Public Health, The University of Texas Health Science Center at Houston School of Dentistry The goal of this protocol is to enable non-dental professionals to assess oral health status for research or health-screening purposes. Aspects assessed include lips, tongue, soft and hard tissues, natural and artificial teeth, oral cleanliness, plaque, swallowing, and impact of oral health on quality of life. Developmental Biology A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos Sanat Upadhyay1, Leoncio Vergara2, Pranjali Shah2, Jan-Åke Gustafsson3,4, Ioannis Kakadiaris1,4, Maria Bondesson5 1Computational Biomedicine Lab, Texas Institute of Measurement Evaluation and Statistics, University of Houston, 2Institute of Biosciences and Technology, Texas A&M Health Science Center, 3Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, 4Department of Biosciences and Nutrition, Novum, Karolinska Institutet, 5Department of Intelligent Systems Engineering, Indiana University This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations. Developmental Biology Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans Ali Naji1, Ali Al Hatem1, Ransome van der Hoeven1 1Department of Diagnostic and Biomedical Sciences, School of Dentistry, University of Texas Health Science Center The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms. Immunology and Infection Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue Xin Li1, Zhengmei Mao2, Li Yang1, Kai Sun1 1Center for Metabolic and Degenerative Diseases, the Brown Foundation of Institute of Molecular Medicine, University of Texas Health Science Center at Houston, 2Microscopy Core, the Brown Foundation of Institute of Molecular Medicine, University of Texas Health Science Center at Houston New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues. Immunology and Infection Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae Erika Flores1, Laurel Thompson1, Natalie Sirisaengtaksin1, Anh Trinh Nguyen1, Abigail Ballard1, Anne-Marie Krachler1 1McGovern Medical School, Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston Zebrafish (Danio rerio) are becoming a widely-used vertebrate animal model for microbial colonization and pathogenesis. This protocol describes the use of the protozoan Paramecium caudatum as a vehicle for food-borne infection in zebrafish larvae. P. caudatum readily internalizes bacteria and get taken up by larval zebrafish through natural preying behavior. Cancer Research Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells Ruoji Zhou1,2, An Xu1, Jian Tu1,3, Mo Liu1, Julian A Gingold4, Ruiying Zhao1, Dung-Fang Lee1,2,5,6 1Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, 2Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center UTHealth, 3Department of Musculoskeletal Oncology, The First Affiliated Hospital of Sun Yat-sen University, 4Women's Health Institute, Cleveland Clinic Foundation, 5Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, 6Center for Precision Health, School of Biomedical Informatics and School of Public Health, The University of Texas Health Science Center at Houston Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts. Neuroscience Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFRα+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis Xiaomin Dong1, Raquel Cuevas-Diaz Duran1, Yanan You1, Jia Qian Wu1 1The Vivian L. Smith Department of Neurosurgery, Center for Stem Cell and Regenerative Medicine, University of Texas Health Science Center Here we present a protocol which is designed to analyze the genome-wide binding of the oligodendrocyte transcription factor 2 (Olig2) in acutely purified brain oligodendrocyte precursor cells (OPCs) by performing low-cell chromatin immunoprecipitation (ChIP), library preparation, high-throughput sequencing and bioinformatic data analysis. Biochemistry A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation Kazunari Nohara1, Zheng Chen1, Seung-Hee Yoo1 1Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies. Neuroscience A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains Antonio J. Tito1, Shebna Cheema2, Mian Jiang2, Sheng Zhang1,3 1Center for Metabolic and Degenerative Diseases, The Brown Foundation Institute of Molecular Medicine, Programs in Human and Molecular Genetics and Neuroscience, The Graduate School of Biomedical Sciences at Houston, The University of Texas Health Science Center at Houston, 2Department of Natural Sciences, University of Houston - Downtown, 3Department of Neurobiology and Anatomy, McGovern Medical School, The University of Texas Health Science Center at Houston The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology. Developmental Biology Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue James E. Baily1, William C.W. Chen2,3, Nusrat Khan4, Iain R. Murray4, Zaniah N. González Galofre4, Johnny Huard5,6, Bruno Péault4,7 1Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 2Department of Bioengineering and Orthopaedic Surgery, University of Pittsburgh, 3Research Laboratory of Electronics and Department of Biological Engineering, Massachusetts Institute of Technology, 4MRC Centre for Regenerative Medicine, University of Edinburgh, 5Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, 6Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, 7Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital, David Geffen School of Medicine, University of California at Los Angeles Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium. Bioengineering Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography Dustin R. Morado1, Bo Hu1, Jun Liu1 1Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions. Biology Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium Tengis S. Pavlov1, Daria V. Ilatovskaya1, Oleg Palygin1, Vladislav Levchenko1, Oleh Pochynyuk2, Alexander Staruschenko1 1Department of Physiology, Medical College of Wisconsin, 2Department of Integrative Biology & Pharmacology, University of Texas Health Science Center at Houston Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys. Developmental Biology Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System Shenglan Li*1,2, Haipeng Xue*1,2, Bo Long1,2,5, Li Sun1,2,6, Tai Truong1,2,4,7, Ying Liu1,2,3 1Department of Neurosurgery, The University of Texas Health Science Center at Houston, 2Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 3The Senator Lloyd & B. A. Bentsen Center for Stroke Research, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 4Summer Research Program, Office of Educational Programs, The University of Texas Health Science Center at Houston, 5Department of Anesthesiology, Shengjing Hospital, China Medical University, 6Department of Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine, 7Biology Department, University of West Georgia Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination. Biology Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis Shan Zong*1, Shuyun Deng*1, Kenian Chen1, Jia Qian Wu1 1The Vivian L. Smith Department of Neurosurgery, Center for Stem Cell and Regenerative Medicine, University of Texas Health Science Center, The University of Texas Graduate School of Biomedical Sciences at Houston RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells. Bioengineering Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells Drew M. Dolino1, Swarna S. Ramaswamy1, Vasanthi Jayaraman1 1Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification. Biology Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae Jennifer L. Abrams1, Kevin A. Morano1 1Department of Microbiology and Molecular Genetics, Graduate School of Biomedical Sciences, University of Texas Medical School This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control. Immunology and Infection Mass Spectrometric Analysis of Glycosphingolipid Antigens Alexandra Bili Yin1, David Hawke2, Dapeng Zhou3,4 1Undergraduate Program, Rice University, 2Proteomics Facility, Department of Pathology, University of Texas MD Anderson Cancer Center, 3Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, 4University of Texas Graduate School of Biological Sciences at Houston A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards. Immunology and Infection Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse Holly A. Robinson1, SunKuk Kwon1, Mary A. Hall1, John C. Rasmussen1, Melissa B. Aldrich1, Eva M. Sevick-Muraca1 1Center for Molecular Imaging (CMI), University of Texas Health Science Center-Houston Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases. Medicine Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity Sheng Li1,2,3 1Department of Physical Medicine and Rehabilitation, University of Texas Health Science Center at Houston, 2UTHealth Motor Recovery Laboratory, TIRR Memorial Hermann Hospital, 3The Institute of Rehabilitation and Research (TIRR), TIRR Memorial Hermann Hospital The purpose is to present a new method, breathing-control electrical stimulation (BreEStim) for management of neuropathic pain and spasticity. Medicine Analytical Techniques for Assaying Nitric Oxide Bioactivity Hong Jiang1, Deepa Parthasarathy1, Ashley C. Torregrossa1, Asad Mian2, Nathan S. Bryan1 1Texas Therapeutics Institute, University of Texas Health Science Center at Houston, 2Deptartment of Pediatrics, Baylor College of Medicine The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease. Neuroscience Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae Abanti Chattopadhyay*1, A'Tondra V. Gilstrap*1,2, Michael J. Galko1,3,4 1Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, 2Scholars Academy/MARC Scholar, University of Houston-Downtown, 3Genes and Development Graduate Program, University of Texas Graduate School of Biomedical Sciences, 4Neuroscience Graduate Program, University of Texas Graduate School of Biomedical Sciences In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons. Neuroscience Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons Hee Joo Choi1, Christophe P. Ribelayga2, Stuart C. Mangel1 1Department of Neuroscience, Ohio State University College of Medicine, 2Department of Ophthalmology and Visual Science, University of Texas Medical School An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night. Medicine Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology Madonna M. McManus1, Raymond J. Grill1 1Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.