Clemson University View Institution's Website 26 articles published in JoVE Chemistry High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue Apeksha C. Rajamanthrilage1, Erin Levon2, Unaiza Uzair1, Cedric Taylor2, Tzuen-Rong Tzeng2, Jeffrey N. Anker1,3 1Department of Chemistry, Clemson University, 2Department of Biological Sciences, Clemson University, 3Department of Bioengineering, Clemson University Here, we present a protocol for high-resolution optical detection of chemical information around implanted medical devices with X-ray excited luminescence chemical imaging (XELCI). This novel imaging technique is developed in our lab which enables studying implant-associated infection biochemistry. Bioengineering Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes Tanner Rathbone*1, Ilayda Ates*1, Callie Stuart1, Tina Parker2, Renee N. Cottle1 1Department of Bioengineering, Clemson University, 2Godley Snell Research Center, Clemson University This protocol describes techniques for isolating primary mouse hepatocytes from the liver and electroporating CRISPR-Cas9 as ribonucleoproteins and mRNA to disrupt a therapeutic target gene associated with an inherited metabolic disease of the liver. The methods described result in high viability and high levels of gene modification after electroporation. Immunology and Infection Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots Rachel E. Ham*1, Austin R. Smothers*1,2, Kylie L. King1, Justin M. Napolitano1, Theodore J. Swann3, Lesslie G. Pekarek4, Mark A. Blenner*1,5,6, Delphine Dean*1,2 1Center for Innovative Medical Devices and Sensors (REDDI Lab), Clemson University, 2Department of Bioengineering, Clemson University, 3Swann Medicine, 4Student Health Services, Clemson University, 5Department of Chemical & Biomolecular Engineering, Clemson University, 6Department of Chemical & Biomolecular Engineering, University of Delaware The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories. Behavior A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila Joshua D. Walters*1, Jeffrey S. Hatfield*1, Brandon B. Baker1, Trudy F. C. Mackay1, Robert R. H. Anholt1 1Department of Genetics and Biochemistry and Center for Human Genetics, Clemson University The microplate feeder assay offers an economical, high throughput method for quantifying liquid food consumption in Drosophila. A 3D-printed device connects a 96-well microplate in which flies are housed to a 1536-well microplate from which flies consume a feeding solution with a tracer dye. The solution volume decline is measured spectrophotometrically. Bioengineering Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure Christopher Pierce1, Cara Katterman2, Jessica Larsen1,3 1Department of Chemical and Biomolecular Engineering, Clemson University, 2Department of Biological Sciences, Clemson University, 3Department of Bioengineering, Clemson University Polymersomes are self-assembled polymeric vesicles that are formed in spherical shapes to minimize Gibb's Free Energy. In the case of drug delivery, more elongated structures are beneficial. This protocol establishes methods to create more rod-like polymersomes, with elongated aspect ratios, using salt to induce osmotic pressure and reduce internal vesicle volumes. Immunology and Infection Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions Savini Thrikawala1, Emily E. Rosowski1 1Department of Biological Sciences, Clemson University This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating. Immunology and Infection Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay Melanie Key*1,2, Amy Bergmann*1,2, Chiara Micchelli1,2, L. Brock Thornton1,2, Sophie Millard1,2, Zhicheng Dou1,2 1Department of Biological Sciences, Clemson University, 2Eukaryotic Pathogens Innovation Center, Clemson University Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example. Behavior High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster Tatum N. Sass*1, Rebecca A. MacPherson*1, Trudy F. C. Mackay1, Robert R. H. Anholt1 1Department of Genetics and Biochemistry and Center for Human Genetics, Clemson University Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials. Chemistry Multiscale Sampling of a Heterogeneous Water/Metal Catalyst Interface using Density Functional Theory and Force-Field Molecular Dynamics Cameron J. Bodenschatz*1, Xiaohong Zhang*1, Tianjun Xie*1, Jeremy Arvay1,2, Sapna Sarupria1, Rachel B. Getman1 1Department of Chemical and Biomolecular Engineering, Clemson University, 2Davidson School of Chemical Engineering, Purdue University The goal of the protocol presented here is to generate and sample trajectories of configurations of liquid water molecules around catalytic species on a flat transition metal surface. The sampled configurations can be used as starting structures in quantum mechanics-based methods. Biology Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory Thea M. Edwards1,2, Howard E. Morgan2,3, Coralia Balasca4, Naveen K. Chalasani5, Lauren Yam4,6, Alison M. Roark4 1Department of Biology, University of the South, 2School of Biological Sciences, Louisiana Tech University, 3School of Medicine, Louisiana State University Health Sciences Center, 4Department of Biology, Furman University, 5Department of Computer Science, Louisiana Tech University, 6Clemson University This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ERα) and/or beta (ERβ). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis. Biology The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects Matthew S. Lehnert1, Kristen E. Reiter1, Andrew Bennett1, Patrick D. Gerard2, Qi-Huo Wei3, Miranda Byler1, Huan Yan3, Wah-Keat Lee4 1Department of Biological Sciences, Kent State University at Stark, 2Department of Mathematical Sciences, Clemson University, 3Liquid Crystal Institute, Kent State University, 4Brookhaven National Laboratory Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles. Biochemistry High Precision FRET at Single-molecule Level for Biomolecule Structure Determination Junyan Ma1, Inna S. Yanez-Orozco2, Soheila Rezaei Adariani2, Drew Dolino3, Vasanthi Jayaraman3, Hugo Sanabria2 1Department of Chemistry, Clemson University, 2Department of Physics and Astronomy, Clemson University, 3Department of Biochemistry and Molecular Biology, Center for Membrane Biology, Graduate School for Biomedical Sciences, University of Texas Health Science Center A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments. Biology A Customizable Chamber for Measuring Cell Migration Aniqa N. Chowdhury1, Huu Tri Vo1, Sharon Olang1, Elliott Mappus1, Brian Peterson1, Nora Hlavac1, Tyler Harvey1, Delphine Dean1 1Department of Bioengineering, Clemson University This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix. Biology Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm Vera B. S. Chan1, Takashi Toyofuku2, George Wetzel3, Laxmikant Saraf3, Vengatesen Thiyagarajan4, Andrew S. Mount1 1Department of Biological Sciences, Clemson University, 2Department of Marine Biodiversity Research (BioDive), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 3Advanced Material Research Laboratory (AMRL), Clemson University, 4Swire Institute of Marine Sciences and School of Biological Sciences, The University of Hong Kong We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization. Engineering Electrospray Deposition of Uniform Thickness Ge23Sb7S70 and As40S60 Chalcogenide Glass Films Spencer Novak1, Pao-Tai Lin2,3, Cheng Li4, Nikolay Borodinov1, Zhaohong Han5, Corentin Monmeyran5, Neil Patel5, Qingyang Du5, Marcin Malinowski4, Sasan Fathpour4, Chatdanai Lumdee4, Chi Xu4, Pieter G. Kik4, Weiwei Deng6, Juejun Hu7, Anuradha Agarwal7, Igor Luzinov1, Kathleen Richardson4 1Department of Materials Science and Engineering, Clemson University, 2Department of Materials Science and Engineering, Texas A&M University, 3Department of Electrical and Computer Engineering, Texas A&M University, 4College of Optics and Photonics, Center for Research and Education in Optics and Lasers (CREOL), University of Central Florida, 5Department of Materials Science and Engineering, Massachusetts Institute of Technology, 6Department of Mechanical Engineering, Virginia Polytechnic Institute, 7Microphotonics Center, Massachusetts Institute of Technology A method of uniform thickness solution-derived chalcogenide glass film deposition is demonstrated using computer numerical controlled motion of a single-nozzle electrospray. Bioengineering Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer Sarah Grace Dennis1, Thomas Trusk2, Dylan Richards3, Jia Jia3, Yu Tan3, Ying Mei3, Stephen Fann1, Roger Markwald3, Michael Yost1 1Department of Surgery, Medical University of South Carolina, 2Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, 3Department of Bioengineering, Clemson University A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced. Chemistry Synthesis of Non-uniformly Pr-doped SrTiO3 Ceramics and Their Thermoelectric Properties Arash Mehdizadeh Dehkordi1, Sriparna Bhattacharya2, Taghi Darroudi3, Xiaoyu Zeng2, Husam N. Alshareef4, Terry M. Tritt1,2 1Department of Materials Science and Engineering, Clemson University, 2Department of Physics and Astronomy, Clemson University, 3Electron Microscope Facility, Clemson University, 4Materials Science and Engineering, King Abdullah University of Science and Technology A protocol for the synthesis and processing of polycrystalline SrTiO3 ceramics doped non-uniformly with Pr is presented along with the investigation of their thermoelectric properties. Biology Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer Jiajie Yan*1, Justin K. Thomson*1, Weiwei Zhao1, Vladimir G. Fast2, Tong Ye3, Xun Ai1 1Department of Cell and Molecular Physiology, Loyola University Chicago, 2Department of Biomedical Engineering, University of Alabama at Birmingham, 3Department of Bioengineering, Clemson University This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers. Immunology and Infection Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans Tonya Taylor1, Indrani Bose2, Taylor Luckie1, Kerry Smith1 1Department of Genetics and Biochemistry, Eukaryotic Pathogens Innovation Center (EPIC), Clemson University, 2Department of Biology, Western Carolina University Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into C. neoformans. Behavior How to Study Placebo Responses in Motion Sickness with a Rotation Chair Paradigm in Healthy Participants Katja Weimer1, Björn Horing2, Eric R. Muth2, Paul Enck1 1Psychosomatic Medicine and Psychotherapy, University Hospital Tübingen, 2Department of Psychology, Clemson University An experimental rotation chair paradigm is used to investigate whether a placebo intervention on motion sickness affects subjective outcome measures only or also affects behavioral and objective measures in a balanced placebo design. Chemistry Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis Derek D. Lovingood1, Jeffrey R. Owens2, Michael Seeber3, Konstantin G. Kornev3, Igor Luzinov3 1Oak Ridge Institute for Science and Education, 2Air Force Research Laboratory, Airbase Technology Division, 3School of Materials Science and Engineering, Clemson University Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction. Medicine A Contusion Model of Severe Spinal Cord Injury in Rats Vibhor Krishna1, Hampton Andrews1, Xing Jin2, Jin Yu1, Abhay Varma1, Xuejun Wen3, Mark Kindy1 1Department of Neuroscience, Division of Neurosurgery, Medical University of South Carolina, 2Bioengineering, Clemson University, 3Clemson-MUSC Bioengineering Joint Program A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model. Bioengineering Graphene Coatings for Biomedical Implants Ramakrishna Podila1,2, Thomas Moore3, Frank Alexis3, Apparao Rao1,4 1Department of Physics, Clemson University, 2Department of Pharmacology and Toxicology, East Carolina University, 3Department of Bioengineering, Clemson University, 4Center for Optical Materials Science and Engineering Technologies, Clemson University Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response. Engineering Construction and Testing of Coin Cells of Lithium Ion Batteries Archana Kayyar1, Jiajia Huang1, Mojtaba Samiee1, Jian Luo1,2 1School of Materials Science and Engineering, Clemson University, 2Center for Optical Materials Science and Engineering Technologies, Clemson University A protocol to construct and test coin cells of lithium ion batteries is described. The specific procedures of making a working electrode, preparing a counter electrode, assembling a cell inside a glovebox and testing the cell are presented. Bioengineering Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer Alexander B. Owczarczak1, Stephen O. Shuford1, Scott T. Wood1, Sandra Deitch1, Delphine Dean1 1Department of Bioengineering, Clemson University A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells. Biology Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase Matthew L. Fowler1, Cheryl J. Ingram-Smith1, Kerry S. Smith1 1Department of Genetics and Biochemistry, Clemson University A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.