University of Arizona, Tucson View Institution's Website 32 articles published in JoVE Developmental Biology In Vivo Real-Time Study of Drug Effects on Carotid Blood Flow in the Ovine Fetus Alexander L. Pendleton1, Sean W. Limesand1, Ravi Goyal1 1School of Animal and Comparative Biomedical Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson The present protocol describes a method to deliver drugs and gene expression-modifying agents perivascularly in an in utero developing fetus. Importantly, the effect of drugs/agents on blood flow can be measured with the progression of pregnancy. Cancer Research Spontaneous Murine Model of Anaplastic Thyroid Cancer Huayun Yan1, Yingfang Ma1, Xinyue Zhou2, Yushuang He3, Yang Liu2, Carlos Caulin4, Leiming Wang5, Heng Xu1,6, Han Luo2,6 1State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, 2Division of Thyroid Surgery, Laboratory of Thyroid and Parathyroid Disease, Frontiers Science Center for Disease-Related Molecular Network, Department of General Surgery, West China Hospital, Sichuan University, 3Department of Ultrasound, West China Hospital of Sichuan University, Sichuan University, 4Department of Otolaryngology-Head & Neck Surgery and University of Arizona Cancer Center, University of Arizona, 5Center for Translational Medicine, Shenzhen Bay Laboratory, 6Division of Laboratory Medicine/Research Centre of Clinical Laboratory Medicine, West China Hospital, Sichuan University Here, we present a standard pipeline to obtain murine ATC tumors by spontaneous genetically engineered mouse models. Further, we present tumor dynamics and pathological information about the primary and metastasized lesions. This model will help researchers to understand tumorigenesis and facilitate drug discoveries. Biology Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices Emily A. Gardea1, Destiny DeNicola1, Samuel Freitas1, Will Peterson1, Hope Dang1, Karissa Shuck1, Christopher Fang-Yen2, George L. Sutphin1 1Department of Molecular & Cellular Biology, University of Arizona, 2Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival. Biology Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions Luis Espejo*1, Bradford Hull*1, Leah M. Chang1, Destiny DeNicola1, Samuel Freitas1, Vanessa Silbar1, Anne Haskins1, Emily A. Turner1, George L. Sutphin1 1Department of Molecular & Cellular Biology, University of Arizona, Tucson Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors. Medicine Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies Hannah E. Korah1, Kevin Cheng1, Stephanie M. Washington2, Matthew E. Flowers1, Harrison J. Stratton1, Amol Patwardhan5, Mohab M. Ibrahim*1,2,3,4, Laurent F. Martin*1,2 1Department of Pharmacology, College of Medicine, The University of Arizona, Tucson, 2Department of Anesthesiology, College of Medicine, The University of Arizona, Tucson, 3Neuroscience Graduate Interdisciplinary Program, College of Medicine, The University of Arizona, Tucson, 4Comprehensive Pain and Addiction Center, The University of Arizona, Tucson, 5Department of Anesthesiology and Pain Management, University of Texas Southwestern Medical Center Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility. Neuroscience Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy Suvithanandhini Loganathan*1, Hannah E. Ball*1, Ernesto Manzo1,2, Daniela C. Zarnescu1,3 1Department of Molecular and Cellular Biology, University of Arizona, Tucson, 2Vollum Institute, Oregon Health and Science University, 3Department of Neuroscience, University of Arizona, Tucson Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor. Environment Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation Margarita Acedo1, Juan R. Gonzalez Cena2, Kasi M. Kiehlbaugh3, Kimberly L. Ogden1,2 1Department of Chemical and Environmental Engineering, University of Arizona, 2Department of Biosystems Engineering, University of Arizona, 3Department of Biomedical Engineering, University of Arizona A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density. Neuroscience Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9 Irina Sinakevitch1,2, Zev Kurtzman*2, Hyun G. Choi*2, David Arturo Ruiz Pardo3, Romain A. Dahan2, Nathaniel Klein1, Branimir Bugarija4, Erik Wendlandt4, Brian H. Smith2 1Department of Neuroscience, University of Arizona, 2School of Life Sciences, Arizona State University, 3Department of Scientific and Technologic Investigations, University of Sonora, 4Integrated DNA Technologies, Inc. Presented here is a protocol to use the CRISPR-Cas9 system for reducing the production of a protein in the adult honeybee brain to test antibody specificity. Behavior Examining Changes in HRV and Emotion Following Artmaking with Three Different Art Materials Johanna Czamanski-Cohen1, Giora Galili2, John J. B. Allen3 1The Emili Sagol Creative Arts Therapies Research Center, The School of Creative Arts Therapies, The Faculty of Health and Welfare, University of Haifa, 2The Department of Education and Psychology, The Open University of Israel, 3Department of Psychology, University of Arizona The goal of the protocol is to guide researchers in conducting experiments that are intended to measure changes in self-reported emotional response and heart rate variability following art making with different materials. The protocol can easily be adapted for use in a variety of behavioral conditions and activities. Medicine Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics Rachel Meyer1, Wen Yu Wong2, Roberto Guzman3, Randy Burd1, Kirsten Limesand1,2 1Department of Nutritional Sciences, University of Arizona, 2Cancer Biology Graduate Interdisciplinary Program, University of Arizona, 3Department of Chemical Engineering, University of Arizona Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50–90 μm thick salivary gland sections prior to and following exposure to ionizing radiation. Genetics Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique Elizabeth C. Bondy1, Martha S. Hunter2 1Graduate Interdisciplinary Program in Entomology and Insect Science, University of Arizona, 2Department of Entomology, University of Arizona We present a simple cytogenetic technique using 4′,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci. Environment Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures Malak M. Tfaily*1,2, Rachel M. Wilson*3, Heather M. Brewer1, Rosalie K. Chu1, Heino M. Heyman4, David W. Hoyt1, Jennifer E. Kyle5, Samuel O. Purvine1 1Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 2Department of Soil, Water and Environmental Science, University of Arizona, 3Department of Earth Ocean and Atmospheric Sciences, Florida State University, 4Bruker Daltonics Inc., 5Biological Sciences Division, Pacific Northwest National Laboratory This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change. Neuroscience Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ Kimberly Young1, Helena Morrison1 1College of Nursing, University of Arizona Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape. Neuroscience A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels Hrvoje Brzica*1, Wazir Abdullahi*1, Bianca G. Reilly1, Patrick T. Ronaldson1 1Department of Pharmacology, College of Medicine, University of Arizona, Tucson Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium. Behavior Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children – Working Memory (CABC-WM) Kathryn Cabbage1, Shara Brinkley2, Shelley Gray2, Mary Alt3, Nelson Cowan4, Samuel Green5, Trudy Kuo6, Tiffany P. Hogan1 1Communication Sciences and Disorders, MGH Institute of Health Professions, 2Speech and Hearing Science, Arizona State University, 3Speech, Language, and Hearing Sciences, University of Arizona, 4Department of Psychological Sciences, University of Missouri-Columbia, 5Sanford School of Social and Family Dynamics, Arizona State University, 6School of Social and Behavioral Sciences, New College of Interdisciplinary Arts and Sciences, Arizona State University - West Working memory predicts a significant amount of variance for a variety of cognitive tasks, including speaking, reading, and writing. However, few tools are available to assess working memory in children. We present an innovative, computer-based battery that comprehensively assesses different components of working memory in school-age children. Genetics Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms Blake L. Joyce1,2, Asher K. Haug-Baltzell3, Jonathan P. Hulvey4, Fiona McCarthy5, Upendra Kumar Devisetty1,6, Eric Lyons1,2,3 1BIO5 Institute, University of Arizona, 2The School of Plant Sciences, University of Arizona, 3Genetics GIDP, University of Arizona, 4Biology Department, University of Massachusetts Amherst, 5School of Animal and Comparative Biomedical Sciences, University of Arizona, 6CyVerse, University of Arizona This protocol outlines a comparative de novo transcriptome assembly and annotation workflow for novice bioinformaticians. The workflow is available for free entirely through CyVerse and connected by the Data Store. Command line and graphical user interfaces are used, but all code needed is available to copy and paste. Developmental Biology Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization Mandi J. Corenblum1, Lalitha Madhavan1 1Department of Neurology, The University of Arizona Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry. Immunology and Infection Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds Kevin L. Hockett1, David A. Baltrus1 1School of Plant Sciences, University of Arizona We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria. Bioengineering Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids Bradley P. Weegman1, Ahmad Essawy2, Peter Nash2, Alexandra L. Carlson2, Kristin J. Voltzke3, Zhaohui Geng2, Marjan Jahani2, Benjamin B. Becker2, Klearchos K. Papas4, Meri T. Firpo2 1Radiology, University of Minnesota, 2Medicine, University of Minnesota, 3School of Public Health, University of Minnesota, 4Surgery, University of Arizona Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures. Environment Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations Aditi Sengupta1, Yadi Wang2, Antonio A. Meira Neto3, Katarena A. Matos3, Katerina Dontsova1,2, Rob Root2, Julie W. Neilson2, Raina M. Maier2, Jon Chorover1,2, Peter A. Troch1,3 1Biosphere 2, University of Arizona, 2Department of Soil, Water and Environmental Science, University of Arizona, 3Department of Hydrology and Water Resources, University of Arizona This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period. Biology Isolation of Mouse Coronary Endothelial Cells Shizhen Luo1, Aaron H. Truong1, Ayako Makino1 1Department of Physiology, University of Arizona, Tucson This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments. Biology High Throughput Danio Rerio Energy Expenditure Assay Savannah Y. Williams1, Benjamin J. Renquist2 1Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 2School of Animal and Comparative Biomedical Sciences, University of Arizona Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery. Neuroscience 3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii Carla M. Cabral1, Anita A. Koshy1,2,3 1Bio5 Institute, University of Arizona, 2Department of Neurology, University of Arizona, 3Department of Immunobiology, University of Arizona Using this protocol, we were able to image 160 µm thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron. Bioengineering In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae) John G. McMullen II1, S. Patricia Stock2 1School of Animal and Comparative Biomedical Sciences, University of Arizona, 2Department of Entomology, University of Arizona The goal of this presentation is to demonstrate in vivo and in vitro techniques for the rearing of entomopathogenic nematodes. In vivo methods consider the rearing of these nematodes with an insect host, whereas the in vitro methods utilize rich agar media. Bioengineering Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis Sang-Hyuck Park1, Rebecca Garlock Ong2, Chuansheng Mei3, Mariam Sticklen4 1The School of Plant Sciences, University of Arizona, 2Department of Chemical Engineering and Materials Science, DOE Great Lakes Bioenergy Research Center, Michigan State University, 3The Institute for Sustainable and Renewable Resources, The Institute for Advanced Learning and Research, 4Department of Plant, Soil and Microbial Sciences, Michigan State University A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed. Biology Tissue Triage and Freezing for Models of Skeletal Muscle Disease Hui Meng1, Paul M.L. Janssen2, Robert W. Grange3, Lin Yang4, Alan H. Beggs5, Lindsay C. Swanson5, Stacy A. Cossette1,6, Alison Frase7, Martin K. Childers8, Henk Granzier9, Emanuela Gussoni5, Michael W. Lawlor1 1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 2Department of Physiology and Cell Biology, The Ohio State University, 3Department of Human Nutrition, Foods and Exercise, Virginia Tech, 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, 5 The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies. Environment Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae) Rousel A. Orozco1, Ming-Min Lee1, S. Patricia Stock1 1Department of Entomology, University of Arizona Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively. Medicine A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice Hao Chen1, Jessica N. Perez1, Eleni Constantopoulos1, Laurel McKee1, Jessica Regan1, Patricia B. Hoyer1, Heddwen L. Brooks1, John Konhilas1 1Department of Physiology, University of Arizona Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise. Medicine Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty Hilary C. Rees1, Voichita Ianas1, Patricia McCracken1, Shannon Smith1, Anca Georgescu1, Tirdad Zangeneh1, Jane Mohler2, Stephen A. Klotz1 1Division of Infectious Diseases, University of Arizona, 2Arizona Center on Aging, University of Arizona Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state. Bioengineering Plasma Lithography Surface Patterning for Creation of Cell Networks Michael Junkin1, Siu Ling Leung1, Yongliang Yang1, Yi Lu1, Justin Volmering1, Pak Kin Wong1,2 1Aerospace and Mechanical Engineering, University of Arizona, 2Biomedical Engineering IDP and BIO5 Institute, University of Arizona A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types. Biology Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer Huy Nguyen1, Cristy Loustaunau1, Alexander Facista1, Lois Ramsey1, Nadia Hassounah1, Hilary Taylor1, Robert Krouse2,3, Claire M. Payne1,4, V. Liana Tsikitis3, Steve Goldschmid5, Bhaskar Banerjee5, Rafael F. Perini5, Carol Bernstein1 1Department of Cell Biology and Anatomy, College of Medicine, University of Arizona, Tucson, 2Southern Arizona Veterans Affairs Health Care System, Tucson, AZ, 3Department of Surgery, College of Medicine, University of Arizona, Tucson, 4Biomedical Diagnostics and Research, Tucson, AZ, 5Department of Medicine, College of Medicine, University of Arizona, Tucson Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects. Biology Preparation of Drosophila S2 cells for Light Microscopy Daniel W. Buster1, Jonathan Nye1, Joseph E. Klebba1, Gregory C. Rogers1 1Department of Cell Biology and Anatomy, University of Arizona (UOA) Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.
