University of Georgia

38 articles published in JoVE

• Immunology and Infection

  
  Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs Fernando Sanchez-Valdez¹, Ángel M. Padilla¹, Juan M. Bustamante¹, Caleb W. D. Hawkins¹, Rick L. Tarleton^{1,2 1}Center for Tropical and Emerging Global Diseases, University of Georgia, ²Department of Cellular Biology, University of Georgia The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.

• Biology

  
  A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates James C. Fulton*¹, Matthew A. Cullen*¹, Kristin Beckham¹, Tatiana Sanchez², Zhuxuan Xu¹, Preston Stern¹, Gary Vallad³, Geoffrey Meru^4,5, Cecilia McGregor⁶, Nicholas S. Dufault^{1 1}Department of Plant Pathology, University of Florida, ²University of Florida Institute of Food and Agricultural Sciences, ³Gulf Coast Research and Education Center, University of Florida, ⁴Horticultural Sciences Department, University of Florida, ⁵Tropical Research and Education Center, University of Florida, ⁶Department of Horticulture, University of Georgia Managing Fusarium wilt of watermelon requires knowledge of the pathogen races present. Here, we describe the root-dip, infested kernel seeding, and modified tray-dip inoculation methods to demonstrate their efficacy in race-typing of the pathogenic fungus Fusarium oxysporum f. sp niveum (Fon).

• Developmental Biology

  
  In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth Hanna M. Alcocer¹, Xiaoxing Xu¹, Morgan E. Gravely¹, John M. Gonzalez^{1 1}Department of Animal and Dairy Sciences, University of Georgia A robust methodology has been developed for conducting in ovo feeding research trials utilizing unincubated commercial broiler eggs to test the ability of natural and synthetic compounds, in this case, nicotinamide riboside, to influence muscle development and growth.

• Biology

  
  In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig Benjamin T. Corona¹, Jarrod A. Call^2,3, Matthew Borkowski⁴, Sarah M. Greising^{5 1}School of Medicine, Wake Forest University, ²Department of Kinesiology, University of Georgia, ³Regenerative Bioscience Center, University of Georgia, ⁴Aurora Scientific Inc., ⁵School of Kinesiology, University of Minnesota The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

• Biology

  
  Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana) Helen E. Dukes¹, Josey E. Dyer¹, Elizabeth A. Ottesen^{1 1}Department of Microbiology, University of Georgia This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

• Genetics

  
  Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations Amanda O. Shaver¹, Goncalo J. Gouveia², Pamela S. Kirby³, Erik C. Andersen⁴, Arthur S. Edison^{1,2,3 1}Department of Genetics, University of Georgia, ²Department of Biochemistry and Molecular Biology, University of Georgia, ³Complex Carbohydrate Research Center, University of Georgia, ⁴Department of Molecular Biosciences, Northwestern University To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

• Behavior

  
  Measuring the Behavioral Effects of Intraocular Scatter Jeffrey Nightingale¹, Billy R. Hammond^{1 1}Vision Sciences Laboratory, UGA Psychology Department, University of Georgia In this protocol, we outline the conceptual design elements and structural development of a glare acuity apparatus. Additionally, the design of a device for measuring positive dysphotopsia (halos, spokes) and two-point light thresholds is described.

• Developmental Biology

  
  Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice Wenxin Yu¹, Mohamed Ishan¹, Zhonghou Wang¹, Hong-Xiang Liu^{1 1}Regenerative Bioscience Center; Department of Animal and Dairy Science, College of Agricultural and Environmental Sciences, University of Georgia We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

• Environment

  
  Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification Owen Hudson¹, Sumyya Waliullah¹, Justin Hand², Romina Gazis-Seregina³, Fulya Baysal-Gurel⁴, Md Emran Ali^{1 1}Department of Plant Pathology, Coastal Plain Experiment Station, University of Georgia, ²University of Georgia Cooperative Extension, ³Department of Plant Pathology, Tropical Research & Education Center, University of Florida, ⁴Department of Agricultural and Environmental Sciences, Otis L. Floyd Nursery Research Center, Tennessee State University We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

• Developmental Biology

  
  Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions Hsueh Fu Wu^1,2, Nadja Zeltner^{1,2,3 1}Center for Molecular Medicine, University of Georgia, ²Department of Biochemistry and Molecular Biology, Franklin College of Arts and Sciences, University of Georgia, ³Department of Cellular Biology, Franklin College of Arts and Sciences, University of Georgia In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

• Developmental Biology

  
  Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes Melissa Ana Inal¹, Kota Banzai¹, Daichi Kamiyama^{1 1}Department of Cellular Biology, University of Georgia We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

• Environment

  
  Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies Tina L. Walters¹, Deidre M. Gibson², Marc E. Frischer^{1 1}Skidaway Institute of Oceanography, University of Georgia, ²Hampton University Doliolids, including the species Dolioletta gegenbauri, are small gelatinous marine zooplankton of ecological significance found on productive subcontinental shelf systems worldwide. The difficulty of culturing these delicate organisms limits their investigation. In this study, we describe cultivation approaches for collecting, rearing, and maintaining the doliolid Dolioletta gegenbauri.

• Immunology and Infection

  
  Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens Amy V. Paschall¹, Dustin R. Middleton¹, Fikri Y. Avci^{1 1}Department of Biochemistry and Molecular Biology, Center for Molecular Medicine and Complex Carbohydrate Research Center, University of Georgia This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

• Biology

  
  Sperm Collection of Differential Quality Using Density Gradient Centrifugation Muslah Uddin Ahammad*¹, Zachery Ryan Jarrell*¹, Andrew Parks Benson^{1 1}Department of Poultry Science, University of Georgia In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

• Genetics

  
  CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum Heather M. Kudyba*^1,2, David W. Cobb*¹, Anat Florentin^1,2, Michelle Krakowiak¹, Vasant Muralidharan^{1,2 1}Department of Cellular Biology, University of Georgia, ²Center for Tropical and Emerging Global Diseases, University of Georgia We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

• Neuroscience

  
  Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult Stephanie A. Herrlinger¹, Qiang Shao², Li Ma², Melinda Brindley³, Jian-Fu Chen^{2 1}Biomedical and Health Sciences Institute, University of Georgia, ²Center for Craniofacial Molecular Biology, University of Southern California, ³Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

• Behavior

  
  Paradigms of Lower Extremity Electrical Stimulation Training After Spinal Cord Injury Ashraf S. Gorgey^1,2, Refka E. Khalil¹, Robert M. Lester¹, Gary A. Dudley³, David R. Gater^{4 1}Spinal Cord Injury and Disorders Service, Hunter Holmes McGuire VAMC, ²Department of Physical Medicine and Rehabilitation, Virginia Commonwealth University, ³Deceased, Department of Kinesiology, The University of Georgia, ⁴Department of Physical Medicine and Rehabilitation, Penn State Milton S. Hershey Medical Center Spinal cord injury is a traumatic medical condition that may result in elevated risks of chronic secondary metabolic disorders. Here, we presented a protocol using surface neuromuscular electrical stimulation-resistance training in conjunction with functional electrical stimulation-lower extremities cycling as a strategy to ameliorate several of these medical problems.

• Biology

  
  Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses Aaron M. Beedle^{1 1}Department of Pharmaceutical and Biomedical Sciences, University of Georgia Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

• Immunology and Infection

  
  High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro Payel Sil*¹, Dae-goon Yoo*¹, Madison Floyd*¹, Aaron Gingerich¹, Balazs Rada^{1 1}Department of Infectious Diseases, University of Georgia High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

• Biology

  
  Expression and Purification of Virus-like Particles for Vaccination Maria T. Arevalo¹, Terianne M. Wong¹, Ted M. Ross^{1 1}Center for Vaccines and Immunology, Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

• Biology

  
  High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension Ganesh P. Subedi¹, Roy W. Johnson², Heather A. Moniz², Kelley W. Moremen², Adam Barb^{1 1}The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, ²Complex Carbohydrate Research Center, University of Georgia Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

• Neuroscience

  
  A Simple Flight Mill for the Study of Tethered Flight in Insects Alfredo Attisano¹, James T. Murphy², Andrew Vickers³, Patricia J. Moore^{2 1}Museum and Institute of Zoology, Polish Academy of Sciences, ²Department of Entomology, University of Georgia, ³Riventa Pool Innovation Centre Flight in insects is influenced by a number of factors and the propensity to disperse is an important variable in understanding insect ecology and biological control strategies. We describe the construction and use of a simple, relatively inexpensive, and flexible flight mill for measuring parameters of tethered flight in insects.

• Biology

  
  Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers Joseph S. Najem¹, Myles D. Dunlap², Anthony Yasmann³, Eric C. Freeman⁴, John W. Grant⁵, Sergei Sukharev³, Donald J. Leo^{4 1}Department of Mechanical Engineering, Virginia Polytechnic Institute and State University, ²School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, ³Department of Biology, University of Maryland, ⁴College of Engineering, University of Georgia, ⁵Department of Engineering Sciences and Mechanics, Virginia Polytechnic Institute and State University Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

• Biology

  
  Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing Claudia Baumann¹, Maria M. Viveiros^{1 1}Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

• Biology

  
  A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples Michael J. Rothrock Jr.¹, Kelli L. Hiett², John Gamble³, Andrew C. Caudill⁴, Kellie M. Cicconi-Hogan¹, J. Gregory Caporaso^{5 1}Egg Safety and Quality Research Unit, USDA-Agricultural Research Service, ²Poultry Microbiological Safety and Processing Research Unit, USDA-Agricultural Research Service, ³Department of Biochemistry and Biophysics, Oregon State University, ⁴College of Public Health, University of Georgia, ⁵Department of Biological Sciences, Center for Microbial Genetics and Genomics, Northern Arizona University A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

• Chemistry

  
  Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry David B. Nix^1,2, Tadahiro Kumagai¹, Toshihiko Katoh^1,3, Michael Tiemeyer^1,2, Kazuhiro Aoki^{1 1}Complex Carbohydrate Research Center, University of Georgia, ²Department of Biochemistry and Molecular Biology, University of Georgia, ³Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive β-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

• Medicine

  
  Developing Neuroimaging Phenotypes of the Default Mode Network in PTSD: Integrating the Resting State, Working Memory, and Structural Connectivity Noah S. Philip¹, S. Louisa Carpenter¹, Lawrence H. Sweet^{2 1}Providence VA Medical Center, Department of Psychiatry & Human Behavior, Alpert Medical School, Brown University, ²Department of Psychology, University of Georgia This protocol describes the complementary neuroimaging techniques of resting state structural connectivity, task-induced deactivation, and structural connectivity analyses to examine the default network in post-traumatic stress disorder. The use of synergistic methods could potentially lead to improved diagnostics and assessments of severity, outcome, and other relevant clinical factors.

• Chemistry

  
  Exfoliation of Egyptian Blue and Han Blue, Two Alkali Earth Copper Silicate-based Pigments Darrah Johnson-McDaniel¹, Tina T. Salguero^{1 1}Department of Chemistry, The University of Georgia The preparation and exfoliation of CaCuSi4O10 and BaCuSi4O10 are described. Upon stirring in hot water, CaCuSi4O10 spontaneously exfoliates into monolayers, whereas BaCuSi4O10 requires ultrasonication in organic solvents. Near infrared (NIR) imaging illustrates the NIR emission properties of these materials, and aqueous dispersions of these nanomaterials are useful for solution processing.

• Biology

  
  Mouse Embryonic Development in a Serum-free Whole Embryo Culture System Vijay K. Kalaskar¹, James D. Lauderdale^{1,2 1}Neuroscience Division of the Biomedical & Health Sciences Institute, University of Georgia, ²Department of Cellular Biology, University of Georgia Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr exhibit morphological development comparable to embryos developing in utero.

• Environment

  
  A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines Suchitra Chavan¹, Shavannor M. Smith^{1 1}Department of Plant Pathology, University of Georgia The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

• Neuroscience

  
  Electrophysiological Recording in the Brain of Intact Adult Zebrafish Lindsey Johnston¹, Rebecca E. Ball², Seth Acuff³, John Gaudet⁴, Andrew Sornborger⁵, James D. Lauderdale^{2 1}Department of Genetics, University of Georgia, ²Department of Cellular Biology, University of Georgia, ³College of Engineering, Architecture and Technology, Oklahoma State University, ⁴University of Georgia, ⁵Department of Mathematics, University of California, Davis This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

• Biology

  
  Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos Qiaozhi Wei¹, Nancy R. Manley¹, Brian G. Condie^{1 1}Department of Genetics, University of Georgia This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

• Immunology and Infection

  
  A Method for Labeling Vasculature in Embryonic Mice Jerrod L. Bryson¹, Mark C. Coles², Nancy R. Manley^{3 1}Department of Cellular Biology, University of Georgia, ²Centre for Immunology and Infection, Department of Biology and HYMS, University of York, ³Department of Genetics, University of Georgia This article describes a method for labeling embryonic skin and thymus blood vessels.

• Biology

  
  Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton Steven Robbins*¹, Jisha Jacob*¹, Xinxin Lu¹, Mary Ann Moran², Xiaozhen Mou^{1 1}Biological Sciences, Kent State University, ²Marine Sciences, University of Georgia (UGA) Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

• Immunology and Infection

  
  An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium John J. Maurer¹, Margie D. Lee¹, Ying Cheng¹, Adriana Pedroso^{1 1}Department of Population Health, University of Georgia We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

• Biology

  
  An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species W. Walter Lorenz¹, Yuan-Sheng Yu¹, Jeffrey F. D. Dean^{1 1}Warnell School of Forestry and Natural Resources, University of Georgia (UGA) Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

• Biology

  
  Processing the Loblolly Pine PtGen2 cDNA Microarray W. Walter Lorenz¹, Yuan-Sheng Yu¹, Marta Simões², Jeffrey F. D. Dean^{1 1}Warnell School of Forestry and Natural Resources, University of Georgia (UGA), ²Instituto de Biologia Experimental e Tecnológica, Instituto Tecnologia Química e Biológica UNL, Av. da República The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

• Biology

  
  Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics Rachel S. Poretsky¹, Scott Gifford¹, Johanna Rinta-Kanto¹, Maria Vila-Costa¹, Mary Ann Moran^{1 1}Department of Marine Sciences, University of Georgia (UGA) We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.