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University of Tennessee

19 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Estimating Virus Production Rates in Aquatic Systems
Audrey R. Matteson 1, Charles R. Budinoff 1, Claire E. Campbell 1, Alison Buchan 1, Steven W. Wilhelm 1
1Department of Microbiology, University of Tennessee

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

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Bioengineering

Bacterial Immobilization for Imaging by Atomic Force Microscopy
David P. Allison 1,2, Claretta J. Sullivan 3, Ninell Pollas Mortensen 1,2, Scott T. Retterer 1,4, Mitchel Doktycz 1,4
1Biological and Nanoscale Systems Group, Biosciences Division, Oak Ridge National Laboratory, 2Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee , 3Department of Surgery, Eastern Virginia Medical School, 4Center for Nanophase Materials Sciences Division, Oak Ridge National Laboratory

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

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Bioengineering

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas
Jaana Mannik *1, Alex Meyers *2, Paul Dalhaimer 1,2
1Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, 2Department of Chemical and Biomolecular Engineering, University of Tennessee

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

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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. Gannon 2, Matthew L. Polizzotto 1
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.

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Bioengineering

Sealable Femtoliter Chamber Arrays for Cell-free Biology
Sarah Elizabeth Norred 1,2, Patrick M. Caveney 1,2, Scott T. Retterer 1,2, Jonathan B. Boreyko 1,2, Jason D. Fowlkes 2,3, Charles Patrick Collier 2, Michael L. Simpson 1,2,3
1Bredesen Center, University of Tennessee, Knoxville, 2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 3Department of Materials Science and Engineering, University of Tennessee, Knoxville

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

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Biology

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers
Joseph S. Najem 1, Myles D. Dunlap 2, Anthony Yasmann 3, Eric C. Freeman 4, John W. Grant 5, Sergei Sukharev 3, Donald J. Leo 4
1Department 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.

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Engineering

Production of Synthetic Nuclear Melt Glass
Joshua J. Molgaard 1, John D. Auxier II 2,3, Andrew V. Giminaro 2,3, Colton J. Oldham 2, Jonathan Gill 2, Howard L. Hall 2,3,4
1Department of Physics and Nuclear Engineering, United States Military Academy, 2Department of Nuclear Engineering, University of Tennessee, 3Radiochemistry Center of Excellence (RCoE), University of Tennessee, 4Institute for Nuclear Security, University of Tennessee

A protocol for the production of synthetic nuclear melt glass, similar to trinitite, is presented.

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Biology

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast
Bin Wei 1, Brian S. Hercyk 1, Julius Habiyaremye 1, Maitreyi Das 1
1Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee

The fission yeast, Schizosaccharomyces pombe is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

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JoVE Journal

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform
Andrea C. Timm 1, Michelle C. Halsted 2, Jared L. Wilmoth 1, Scott T. Retterer 1,3
1Biosciences Division, Oak Ridge National Laboratory, 2Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, 3Center for Nanophase Materials Sciences, Oak Ridge National Laboratory

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

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Bioengineering

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform
Jayde A. Aufrecht 1,2, Jennifer M. Ryan 3, Sahar Hasim 4, David P. Allison 2,3, Andreas Nebenführ 3, Mitchel J. Doktycz 1,2, Scott T. Retterer 1,2
1Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, 2Bioscience Division and Center for Nanophase Materials Sciences, Oak Ridge national Laboratory, 3Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, 4Department of Microbiology, University of Tennessee

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

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JoVE Core

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes
Joseph S. Najem 1,2, Graham J. Taylor 2,3, Nick Armendarez 4, Ryan J. Weiss 5, Md Sakib Hasan 5, Garrett S. Rose 5, Catherine D. Schuman 6, Alex Belianinov 7, Stephen A. Sarles 2, C. Patrick Collier 2,3,7
1Joint Institute for Biological Sciences, Oak Ridge National Laboratory, 2Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee, 3Bredesen Center for Interdisciplinary Research, University of Tennessee, 4Department of Biosystems and Agriculture Engineering, University of Kentucky, 5Department of Electrical Engineering and Computer Science, University of Tennessee, 6Computer Science and Mathematics Division, Oak Ridge National Laboratory, 7Center for Nanophase Materials Sciences, Oak Ridge National Laboratory

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

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Environment

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems
Daniel J. McGarvey 1, Taylor E. Woods 1,2, Andrew J. Kirk 3
1Center for Environmental Studies, Virginia Commonwealth University, 2Department of Ecology and Evolutionary Biology, University of Tennessee, 3Department of Environmental Quality

This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

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Neuroscience

Computer-based Multitaper Spectrogram Program for Electroencephalographic Data
Christopher B. O'Brien 1, Helen A. Baghdoyan 1,2,3, Ralph Lydic 1,2,3
1Department of Psychology, University of Tennessee, 2Department of Anesthesiology, University of Tennessee, 3Oak Ridge National Laboratory

This protocol provides an open source, compiled MATLAB program that generates multitaper spectrograms for electroencephalographic data.

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Environment

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures
Jeremy R. Chen See 1,2, Olivia Wright 1, Lavinia V. Unverdorben 1,2, Nathan Heibeck 1, Stephen M. Techtmann 3, Terry C. Hazen 4,5, Regina Lamendella 1,2
1Department of Biology, Juniata College, 2Wright Labs, LLC, 3Department of Biological Sciences, Michigan Technological University, 4Biosciences Division, Oak Ridge National Laboratory, 5Department of Civil and Environmental Engineering, University of Tennessee

Here, we present a protocol to investigate the impacts of hydraulic fracturing on nearby streams by analyzing their water and sediment microbial communities.

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Bioengineering

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer
Jessie D. Ringley 1, Stephen Andrew Sarles 1
1Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

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Bioengineering

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems
Jaime Lorenzo N. Dinglasan 1,3, David T. Reeves 2,3, Robert L. Hettich 3, Mitchel J. Doktycz 3
1Graduate School of Genome Science & Technology, University of Tennessee Knoxville, 2Bredesen Center for Interdisciplinary Research, University of Tennessee Knoxville, 3Biosciences Division, Oak Ridge National Laboratory

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

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Developmental Biology

Isolation of Preadipocytes from Broiler Chick Embryos
Minjeong Kim 1, Usuk Jung 1, Elizabeth Shepherd 1, Robert Mihelic 1, Brynn H. Voy 1
1Department of Animal Science, University of Tennessee

The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.

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Neuroscience

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy
Felix A. Kyere *1,2, Ian Curtin *1,2, Oleh Krupa 1,2, Carolyn M. McCormick 1,2, Mustafa Dere 3, Sarah Khan 3,7, Minjeong Kim 7, Tzu-Wen Winnie Wang 4,5,6, Qiuhong He 4,5,6, Guorong Wu 3, Yen-Yu Ian Shih 4,5,6, Jason L. Stein 1,2
1UNC Neuroscience Center, University of North Carolina, Chapel Hill, 2Department of Genetics, University of North Carolina, Chapel Hill, 3Department of Psychiatry, University of North Carolina, Chapel Hill, 4Center for Animal Magnetic Resonance Imaging, The University of North Carolina at Chapel Hill, 5Biomedical Research Imaging Center, The University of North Carolina at Chapel Hill, 6Department of Neurology, The University of North Carolina at Chapel Hill, 7Department of Computer Science, The University of North Carolina at Greensboro

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

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Engineering

Forming Micro-and Nano-Plastics from Agricultural Plastic Films for Employment in Fundamental Research Studies
Anton F. Astner 1, Douglas G. Hayes 1, Hugh M. O'Neill 2, Barbara R. Evans 3, Sai Venkatesh Pingali 2, Volker S. Urban 2, Timothy M. Young 4
1Department of Biosystems Engineering and Soil Science, University of Tennessee, 2Neutron Scattering, Oak Ridge National Laboratory, 3Chemical Sciences Divisions, Oak Ridge National Laboratory, 4Center for Renewable Carbon, The University of Tennessee

We show the formation and dimensional characterization of micro- and nanoplastics (MPs and NPs, respectively) using a stepwise process of mechanical milling, grinding, and imaging analysis.

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