North Carolina State University View Institution's Website 35 articles published in JoVE Biology Scalable, Flexible, and Cost-Effective Seedling Grafting Victoria Yell1,2, Xu Li1,2 1Department of Plant and Microbial Biology, North Carolina State University, 2Plants for Human Health Institute, North Carolina State University This protocol describes a robust seedling grafting method that requires no prior experience or training and can be executed at a very low cost using materials easily accessible in most molecular biology labs. Bioengineering Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells Madelyn VanBlunk1, Pritha Agarwalla1,2, Sharda Pandit1,2, Yevgeny Brudno1,2,3 1Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, 2Comparative Medicine Institute, North Carolina State University, 3Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill Herein is a protocol for creating dry macroporous alginate scaffolds that mediate efficient viral gene transfer for use in genetic engineering of T cells, including T cells for CAR-T cell therapy. The scaffolds were shown to transduce activated primary T cells with >85% transduction. Medicine A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft Michelle Mendiola Pla1, Amy Evans2, Franklin H. Lee3, Yuting Chiang1, Muath Bishawi1, Andrew Vekstein1, Lillian Kang1, Diego Zapata4, Ryan Gross3, Alexis Carnes2, Lynden E. Gault5, Julie A. Balko6, Desiree Bonadonna2, Sam Ho5, Paul Lezberg7, Benjamin S. Bryner1, Jacob N. Schroder1, Carmelo A. Milano1, Dawn E. Bowles3 1Division of Cardiothoracic Surgery, Department of Surgery, Duke University, 2Perfusion Services, Duke University, 3Division of Surgical Sciences, Department of Surgery, Duke University, 4Division of Laboratory Animal Resources, Duke University, 5Gift of Hope Organ and Tissue Donor Network, 6College of Veterinary Medicine, North Carolina State University, 7TransMedics, Inc We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model. Immunology and Infection Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays Alba Frias-De-Diego1, Elisa Crisci1 1College of Veterinary Medicine, Department of Population Health and Pathobiology, North Carolina State University This protocol shows an accurate and objective approach to visualize viral titrations using crystal violet, by comparing it with optical microscopy and immunocytochemical staining. Neuroscience Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing Rachael M. Cunningham1,2, Rachel M. Park1,2, David Knazovicky3, B. Duncan X. Lascelles2,4,5,6, Margaret E. Gruen1,2,4 1Comparative Behavioral Research, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 2Translational Research in Pain (TRiP) Program, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 3Small Animal Orthopedic Surgery, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4Comparative Pain Research and Education Center, College of Veterinary Medicine, North Carolina State University, 5Thurston Arthritis Center, UNC School of Medicine, University of North Carolina - Chapel Hill, 6Center for Translational Pain Research, Department of Anesthesiology, Duke University This work describes a standard protocol for mechanical and hot thermal quantitative sensory testing to evaluate the somatosensory system in dogs. Sensory thresholds are measured using an electronic von Frey anesthesiometer, pressure algometer, and hot contact thermode. Chemistry Quantitative 31P NMR Analysis of Lignins and Tannins Dimitris S. Argyropoulos2, Nicolò Pajer1, Claudia Crestini1 1Department of Molecular Sciences and Nanosystems, Ca’ Foscari University of Venezia, 2Departments of Chemistry and Forest Biomaterials, North Carolina State University 31P NMR is a powerful tool for the structural elucidation of polyphenols. This fast, easy, precise, quantitative, and highly reproducible analytical procedure, that allows for the quantification and differentiation of the different types of hydroxy, phenolic, and carboxylic groups in lignins and tannins has now become a routine analytical tool. Bioengineering Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale Irini M. Dijkhoff1, Benedetta Petracca1,2, Roxane Prieux3, Giuseppe Valacchi3,4, Barbara Rothen-Rutishauser1, Marc Eeman2 1Adolphe Merkle Institute, University of Fribourg, 2Dow Silicones Belgium SRL, 3Department of Biomedical and Specialist Surgical Sciences, University of Ferrara, 4Department of Animal Sciences, Plants for Human Health Institute, North Carolina State University This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented. Bioengineering Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing William Klobasa*1, Fu-Chyun Chu*1, Ordom Huot1, Nathaniel Grubbs1, Dorith Rotenberg1, Anna E. Whitfield1, Marcé D. Lorenzen1 1Department of Entomology and Plant Pathology, North Carolina State University Herein are protocols for collecting and microinjecting precellular corn planthopper embryos for the purpose of modifying their genome via CRISPR/Cas9-based genome editing or for the addition of marked transposable elements through germline transformation. Chemistry Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures Gabriela C. Schröder1,2, Flora Meilleur1,2 1Department of Molecular and Structural Biochemistry, North Carolina State University, 2Neutron Scattering Division, Oak Ridge National Laboratory Neutron protein crystallography is a structural technique that permits the localization of hydrogen atoms, thereby providing important mechanistic details of protein function. We present here the workflow for mounting a protein crystal, neutron diffraction data collection, structure refinement and analysis of the neutron scattering length density maps. Biochemistry Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress Caitlin E Tolbert1, Lindsey Palmquist2, Hannah Lee Dixon2, Melissa C. Srougi2,3 1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, 2Department of Chemistry, High Point University, 3Biotechnology Program and the Department of Molecular Biomedical Sciences, North Carolina State University This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways. Neuroscience Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors Yu Yamanoi1,2,3,4, Hiroki Kittaka1,2,5, Makoto Tominaga1,2,3,6 1Thermal Biology Group, Exploratory Research Center on Life and Living Systems (ExCELLS), 2Division of Cell Signaling, National Institute for Physiological Sciences, 3Department of Physiological Sciences, Graduate University for Advanced Studies (SOKENDAI), 4Research Laboratory, Ikedamohando Co., Ltd., 5Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, 6Institute for Environmental and Gender-Specific Medicine, Juntendo University Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors. Environment Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline Joseph E. Weaver1, Jon C. Williams1, Joel J. Ducoste1, Francis L. de los Reyes III1 1Department of Civil, Construction, and Environmental Engineering, North Carolina State University The size and shape of particles in activated sludge are important parameters that are measured using varying methods. Inaccuracies arise from non-representative sampling, suboptimal images, and subjective analysis parameters. To minimize these errors and ease measurement, we present a protocol specifying every step, including an open source software pipeline. Biology Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face Anne M. Burrows1,2, Kailey M Omstead1, Ashley R Deutsch3,5, Justin T. Gladman4, Adam Hartstone-Rose3 1Department of Physical Therapy, Duquesne University, 2Department of Anthropology, University of Pittsburgh, 3College of Sciences, North Carolina State University, 4Shared Materials Instrumentation Facility, Duke University, 5Department of Anthropology, University of Florida Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects. Environment Construction of a Compact Low-Cost Radiation Shield for Air-Temperature Sensors in Ecological Field Studies Adam J. Terando1,2, Sara G. Prado2, Elsa Youngsteadt3 1U.S. Geological Survey, Southeast Climate Adaptation Science Center, 2Department of Applied Ecology, NC State University, 3Department of Entomology and Plant Pathology, NC State University With the advent of small, low-cost environmental sensors, it is now possible to deploy high-density networks of sensors to measure hyper localized temperature variation. Here, we provide a detailed methodology for constructing a compact version of a previously described custom-fabricated radiation shield for use with inexpensive thermochrons. Environment Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants David P. Livingston III1, Tan D. Tuong1, Mark Hoffman2, Gina Fernandez2 1United States Department of Agriculture and North Carolina State University, 2Department of Horticulture Sciences, North Carolina State University Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions. Biochemistry Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells Sayan Chakraborty1, Krittin Trihemasava1, Guozhou Xu1 1Department of Molecular and Structural Biochemistry, College of Agriculture and Life Sciences, North Carolina State University Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells. Chemistry Solvothermal Synthesis of MIL-96 and UiO-66-NH2 on Atomic Layer Deposited Metal Oxide Coatings on Fiber Mats Heather F. Barton1, Alexandra K. Davis1, Dennis T. Lee1, Gregory N. Parsons1 1Department of Chemical and Biomolecular Engineering, North Carolina State University Metal-organic frameworks are effective in gas storage and heterogeneous catalysis, but typical synthesis methods result in loose powders that are difficult to incorporate into smart materials. We demonstrate a method of first coating fabrics with ALD metal oxides, resulting in conformal films of MOF on the fabrics during solvothermal synthesis. Medicine Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia Amy Stieler Stewart1, John M Freund1, Anthony T Blikslager1, Liara M Gonzalez1 1Department of Clinical Sciences, North Carolina State University This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury. Engineering A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals Robert W Epps1, Kobi C Felton1, Connor W Coley2, Milad Abolhasani1 1Chemical and Biomolecular Engineering, North Carolina State University, 2Chemical Engineering, Massachusetts Institute of Technology Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space. Bioengineering Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing Fu-Chyun Chu1, Pei-Shan Wu1, Sofia Pinzi1, Nathaniel Grubbs1, Marcé D. Lorenzen1 1Department of Entomology and Plant Pathology, North Carolina State University Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing. Medicine Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts Amanda R. Hale1, Ann H. Ross1 1Department of Biological Sciences, North Carolina State University Bone mineral density (BMD) is an important factor in understanding nutritional intake. For human skeletal remains, it is a useful metric to assess quality of life in both juveniles and adults, particularly in fatal starvation and neglect cases. This paper provides guidelines for scanning human skeletal remains for forensic purposes. Immunology and Infection Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity Matthew D. Moore1, Brittany S. Mertens2, Lee-Ann Jaykus1 1Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, 2Department of Chemical and Biomolecular Engineering, North Carolina State University Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy. Bioengineering Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments Seema Nandi1,2, Ashley C. Brown1,2 1Joint Department of Biomedical Engineering, North Carolina State University and The University of North Carolina - Chapel Hill, 2Comparative Medicine Institute, North Carolina State University The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix. Bioengineering Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests Amanda L. Karam1, Catherine C. McMillan1, Yi-Chun Lai1, Francis L. de los Reyes III1, Heike W. Sederoff2, Amy M. Grunden2, Ranji S. Ranjithan1, James W. Levis1, Joel J. Ducoste1 1Department of Civil, Construction, and Environmental Engineering, North Carolina State University, 2Department of Plant and Microbial Biology, North Carolina State University This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software. Environment Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems Matthew D. Jeffries1, Travis W. Gannon1, Patrick J. Maxwell1 1Department of Crop and Soil Sciences, North Carolina State University Transferable pesticide residue experiments in turfgrass systems are integral components of human risk exposure assessments. Experimental approaches to measure transferable residues should be adjusted to the human interaction of interest and turfgrass system dynamics. Three transferable pesticide residue protocols are presented and the suitability across three turfgrass systems is discussed. Bioengineering Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Milad Nazari1, Mark T. Bokhart1, David C. Muddiman1 1W. M. Keck FTMS Laboratory for Human Health Research, Department of Chemistry, North Carolina State University A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse. Chemistry A Quantitative Glycomics and Proteomics Combined Purification Strategy Elizabeth S. Hecht*1, James P. McCord*1, David C. Muddiman1 1Department of Chemistry, North Carolina State University A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy. Chemistry A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction Collin B. Eaker*1, M. Rashed Khan*1, Michael D. Dickey1 1Department of Chemical and Biomolecular Engineering, North Carolina State University We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages. Developmental Biology Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes Adam C. Vandergriff1,2, Michael Taylor Hensley1,2, Ke Cheng1,2,3 1Department of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, 2Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, North Carolina State University, 3The Cyrus Tang Hematology Center, Soochow University The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time. Behavior Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring Alexander Verderber*1, Michael McKnight*1, Alper Bozkurt1 1Department of Electrical and Computer Engineering, North Carolina State University We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw. Environment Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation Audrey R. Matteson*1, Denis J. Mahoney*2, Travis W. Gannon2, Matthew L. Polizzotto1 1Department of Soil Science, North Carolina State University, 2Department of Crop Science, North Carolina State University Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments. Environment Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes Stephanie L. Mathews1,2, Ali S. Ayoub2, Joel Pawlak2, Amy M. Grunden1 1Department of Plant and Microbial Biology, North Carolina State University, 2Department of Forest Biomaterials, North Carolina State University Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste. Bioengineering Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing Philip R. Miller1,2, Shelby A. Skoog1, Thayne L. Edwards2, David R. Wheeler2, Xiaoyin Xiao2, Susan M. Brozik2, Ronen Polsky2, Roger J. Narayan1 1Joint Department of Biomedical Engineering, University of North Carolina and North Carolina State University, 2Department of Biosensors and Nanomaterials, Sandia National Laboratories This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors. Neuroscience An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain Benoit V. Jacquet1, Philip Ruckart1, H. Troy Ghashghaei1 1Department of Molecular Biomedical Sciences, Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream. Biology Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues Carlos M. Hernandez-Garcia1, Joseph M. Chiera1,2, John J. Finer1 1Department of Horticulture and Crop Science, The Ohio State University, 2Department of Plant Pathology, North Carolina State University We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.