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28 ARTICLES PUBLISHED IN JoVE

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Biology

Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase
Matthew L. Fowler 1, Cheryl J. Ingram-Smith 1, Kerry S. Smith 1
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.

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Bioengineering

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer
Alexander B. Owczarczak 1, Stephen O. Shuford 1, Scott T. Wood 1, Sandra Deitch 1, Delphine Dean 1
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.

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Engineering

Construction and Testing of Coin Cells of Lithium Ion Batteries
Archana Kayyar 1, Jiajia Huang 1, Mojtaba Samiee 1, Jian Luo 1,2
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.

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Medicine

A Contusion Model of Severe Spinal Cord Injury in Rats
Vibhor Krishna 1, Hampton Andrews 1, Xing Jin 2, Jin Yu 1, Abhay Varma 1, Xuejun Wen 3, Mark Kindy 1
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.

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Bioengineering

Graphene Coatings for Biomedical Implants
Ramakrishna Podila 1,2, Thomas Moore 3, Frank Alexis 3, Apparao Rao 1,4
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.

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Chemistry

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis
Derek D. Lovingood 1, Jeffrey R. Owens 2, Michael Seeber 3, Konstantin G. Kornev 3, Igor Luzinov 3
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.

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Behavior

How to Study Placebo Responses in Motion Sickness with a Rotation Chair Paradigm in Healthy Participants
Katja Weimer 1, Björn Horing 2, Eric R. Muth 2, Paul Enck 1
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.

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Biology

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer
Jiajie Yan *1, Justin K. Thomson *1, Weiwei Zhao 1, Vladimir G. Fast 2, Tong Ye 3, Xun Ai 1
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.

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Immunology and Infection

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans
Tonya Taylor 1, Indrani Bose 2, Taylor Luckie 1, Kerry Smith 1
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.

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Chemistry

Synthesis of Non-uniformly Pr-doped SrTiO3 Ceramics and Their Thermoelectric Properties
Arash Mehdizadeh Dehkordi 1, Sriparna Bhattacharya 2, Taghi Darroudi 3, Xiaoyu Zeng 2, Husam N. Alshareef 4, Terry M. Tritt 1,2
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.

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Bioengineering

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer
Sarah Grace Dennis 1, Thomas Trusk 2, Dylan Richards 3, Jia Jia 3, Yu Tan 3, Ying Mei 3, Stephen Fann 1, Roger Markwald 3, Michael Yost 1
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.

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Engineering

Electrospray Deposition of Uniform Thickness Ge23Sb7S70 and As40S60 Chalcogenide Glass Films
Spencer Novak 1, Pao-Tai Lin 2,3, Cheng Li 4, Nikolay Borodinov 1, Zhaohong Han 5, Corentin Monmeyran 5, Neil Patel 5, Qingyang Du 5, Marcin Malinowski 4, Sasan Fathpour 4, Chatdanai Lumdee 4, Chi Xu 4, Pieter G. Kik 4, Weiwei Deng 6, Juejun Hu 7, Anuradha Agarwal 7, Igor Luzinov 1, Kathleen Richardson 4
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.

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Biology

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm
Vera B. S. Chan 1, Takashi Toyofuku 2, George Wetzel 3, Laxmikant Saraf 3, Vengatesen Thiyagarajan 4, Andrew S. Mount 1
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.

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Biology

A Customizable Chamber for Measuring Cell Migration
Aniqa N. Chowdhury 1, Huu Tri Vo 1, Sharon Olang 1, Elliott Mappus 1, Brian Peterson 1, Nora Hlavac 1, Tyler Harvey 1, Delphine Dean 1
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.

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Biochemistry

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination
Junyan Ma 1, Inna S. Yanez-Orozco 2, Soheila Rezaei Adariani 2, Drew Dolino 3, Vasanthi Jayaraman 3, Hugo Sanabria 2
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.

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JoVE Journal

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory
Thea M. Edwards 1,2, Howard E. Morgan 2,3, Coralia Balasca 4, Naveen K. Chalasani 5, Lauren Yam 4,6, Alison M. Roark 4
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.

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Biology

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects
Matthew S. Lehnert 1, Kristen E. Reiter 1, Andrew Bennett 1, Patrick D. Gerard 2, Qi-Huo Wei 3, Miranda Byler 1, Huan Yan 3, Wah-Keat Lee 4
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.

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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 Arvay 1,2, Sapna Sarupria 1, Rachel B. Getman 1
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.

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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 Micchelli 1,2, L. Brock Thornton 1,2, Sophie Millard 1,2, Zhicheng Dou 1,2
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.

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Behavior

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster
Tatum N. Sass *1, Rebecca A. MacPherson *1, Trudy F. C. Mackay 1, Robert R. H. Anholt 1
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.

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Immunology and Infection

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions
Savini Thrikawala 1, Emily E. Rosowski 1
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.

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Determining the Influence of Soil Microbial Biomass Size on Soil Organic Matter Priming and Plant Residue Decomposition
Lu-Jun Li 1,2, Xia Zhu-Barker 3, Rongzhong Ye 4, William R. Horwath 3
1National Field Observation and Research Station of Hailun Agroecosystems, Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3Department of Land, Air and Water Resources, University of California, Davis, 4Department of Plant & Environmental Sciences, Pee Dee Research & Education Center, Clemson University

This protocol describes a method to determine the influence of ryegrass residue addition on soil organic matter mineralization (i.e., priming effect) as well as explore the changes in soil microbial biomass size induced by soil organic matter priming, which involves artificially changing the size of microbial biomass.

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Bioengineering

Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure
Christopher Pierce 1, Cara Katterman 2, Jessica Larsen 1,3
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.

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Behavior

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila
Joshua D. Walters *1, Jeffrey S. Hatfield *1, Brandon B. Baker 1, Trudy F. C. Mackay 1, Robert R. H. Anholt 1
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.

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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. King 1, Justin M. Napolitano 1, Theodore J. Swann 3, Lesslie G. Pekarek 4, 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.

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Bioengineering

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes
Tanner Rathbone *1, Ilayda Ates *1, Callie Stuart 1, Tina Parker 2, Renee N. Cottle 1
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.

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Chemistry

High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue
Apeksha C. Rajamanthrilage 1, Erin Levon 2, Unaiza Uzair 1, Cedric Taylor 2, Tzuen-Rong Tzeng 2, Jeffrey N. Anker 1,3
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.

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Immunology and Infection

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei
Daniel Call *1, Sabrina S. Pizarro *2,3, Erica Tovey 1, Emily Knight 2,3, Carrie Baumgardner 2,4, Kenneth A. Christensen 1, James C. Morris 2
1Department Chemistry and Biochemistry, Brigham Young University, 2Eukaryotic Pathogens Innovation Center, Clemson University, 3Department of Genetics and Biochemistry, Clemson University, 4Department of Physics and Astronomy, Clemson University

We describe a method to study how pH responds to environmental cues in the glycosomes of the bloodstream form of African trypanosomes. This approach involves a pH-sensitive heritable protein sensor in combination with flow cytometry to measure pH dynamics, both as a time-course assay and in a high-throughput screen format.

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