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The University of Texas at Austin

26 ARTICLES PUBLISHED IN JoVE

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Neuroscience

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain
Sucharit Katyal 1, Clint A. Greene 1, David Ress 1
1Psychology & Neurobiology, Imaging Research Center & Center for Perceptual Systems, The University of Texas at Austin

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

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Bioengineering

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish
Sudip Mondal 1, Shikha Ahlawat 1, Sandhya P. Koushika 1,2
1Neurobiology, NCBS-TIFR, 2Department of Biological Sciences, TIFR

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

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Bioengineering

Engineering a Bilayered Hydrogel to Control ASC Differentiation
Shanmugasundaram Natesan 1, David O. Zamora 1, Laura J. Suggs 2, Robert J. Christy 1
1Department of Extremity Trauma Research and Regenerative Medicine, United States Army Institute of Surgical Research, 2Department of Biomedical Engineering, The University of Texas at Austin

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

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Neuroscience

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution
Gayathri Nattar Ranganathan 1, Helmut J. Koester 1
1Section of Neurobiology, Center for Learning and Memory, The University of Texas at Austin

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

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Neuroscience

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography
Christina J. Schier 1, Regina A. Mangieri 1, Geoffrey A. Dilly 1, Rueben A. Gonzales 1
1College of Pharmacy, Division of Pharmacology and Toxicology, The University of Texas at Austin

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

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JoVE Journal

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Jeremy C. Henderson 1, John P. O'Brien 2, Jennifer S. Brodbelt 2, M. Stephen Trent 1,3
1Section of Molecular Genetics and Microbiology, The University of Texas at Austin, 2Department of Chemistry and Biochemistry, The University of Texas at Austin, 3The Institute of Cellular and Molecular Biology, The University of Texas at Austin

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

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Biology

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland
Silva Krause 1, Amy Brock 2, Donald E. Ingber 1,2,3
1Vascular Biology Program, Department of Surgery, Boston Children's Hospital and Harvard Medical School, 2Wyss Institute for Biologically Inspired Engineering, Harvard University, 3Harvard School of Engineering and Applied Sciences

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

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JoVE Journal

Experimental Measurement of Settling Velocity of Spherical Particles in Unconfined and Confined Surfactant-based Shear Thinning Viscoelastic Fluids
Sahil Malhotra 1, Mukul M. Sharma 1
1Petroleum & Geosystems Engineering, The University of Texas at Austin

This paper demonstrates the experimental procedure to measure terminal settling velocities of spherical particles in surfactant-based shear thinning viscoelastic fluids. Fluids over a wide range of rheological properties are prepared and settling velocities are measured for a range of particle sizes in unbounded fluids and fluids between parallel walls.

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Behavior

Assessing Forelimb Function after Unilateral Cervical SCI using Novel Tasks: Limb Step-alternation, Postural Instability and Pasta Handling
Zin Z. Khaing *1, Sydney A. Geissler *1, Timothy Schallert 2, Christine E. Schmidt 1,3
1Department of Biomedical Engineering, The University of Texas at Austin, 2Department of Psychology, The University of Texas at Austin, 3The J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida

Three new behavioral tests (forelimb step-alternation, postural instability test, pasta handling test) for evaluating forelimb function after cervical spinal cord injury in rodents are described.

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Biology

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy
Patrick D. McGurk 1, C. Ben Lovely 1, Johann K. Eberhart 1
1Institute for Cell and Molecular Biology, The University of Texas at Austin

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

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Bioengineering

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting
Christopher W. Coyne 1, Karan Patel 1, Johanna Heureaux 1, Jeanne Stachowiak 3, Daniel A. Fletcher 4,5, Allen P. Liu 1,2
1Department of Mechanical Engineering, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Department of Biomedical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 4Department of Bioengineering, University of California, Berkeley, 5Physical Biosciences Division, Lawrence Berkeley National Laboratory

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

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Engineering

Electroactive Polymer Nanoparticles Exhibiting Photothermal Properties
Travis Cantu 1, Bradley Rodier 2, Zachary Iszard 2, Alissa Kilian 2, Varun Pattani 3, Kyle Walsh 2, Katharina Weber 2, James Tunnell 3, Tania Betancourt 1,2, Jennifer Irvin 1,2
1Materials Science, Engineering, and Commercialization Program, Texas State University, 2Department of Chemistry and Biochemistry, Texas State University, 3Department of Biomedical Engineering, The University of Texas at Austin

A protocol is presented for the synthesis and preparation of nanoparticles consisting of electroactive polymers.

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Chemistry

Epitaxial Growth of Perovskite Strontium Titanate on Germanium via Atomic Layer Deposition
Edward L. Lin 1, Bryce I. Edmondson 1, Shen Hu 1, John G. Ekerdt 1
1McKetta Department of Chemical Engineering, The University of Texas at Austin

This work details the procedures for the growth and characterization of crystalline SrTiO3 directly on germanium substrates by atomic layer deposition. The procedure illustrates the ability of an all-chemical growth method to integrate oxides monolithically onto semiconductors for metal-oxide semiconductor devices.

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Bioengineering

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device
Hyun Jung Kim 1, Jaewon Lee 1, Jin-Ha Choi 1, Anthony Bahinski 2, Donald E. Ingber 2,3,4
1Department of Biomedical Engineering, The University of Texas at Austin, 2Wyss Institute for Biologically Inspired Engineering at Harvard University, 3Vascular Biology Program, Boston Children's Hospital, Harvard Medical School, 4John A. Paulson School of Engineering and Applied Sciences, Harvard University

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

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JoVE Journal

Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand
Charles A. Seipp 1,2, Neil J. Williams 1,3, Radu Custelcean 1
1Chemical Sciences Division, Oak Ridge National Laboratory, 2Department of Chemistry, The University of Texas at Austin, 3Department of Chemistry, The University of Tennessee

A protocol for in situ aqueous synthesis of a bis(iminoguanidinium) ligand and its utilization in selective separation of sulfate is presented.

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Genetics

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks
Anirban Mukherjee 1, Karen M. Vasquez 1
1Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

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Bioengineering

Intraductal Delivery to the Rabbit Mammary Gland
Amelia Clark 1, Nora K. Bird 2, Amy Brock 1
1Department of Biomedical Engineering, The University of Texas at Austin, 2Department of Anesthesiology, UTMB Health at Galveston

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

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Behavior

Noninvasive, In-pen Approach Test for Laboratory-housed Pigs
Lindsey E. Hulbert 1, Eduarda M. Bortoluzzi 1, Yunzhi Luo 1, Jared M. Mumm 1, Morgan J. Coffin 1, Gina Y. Becker 2, Pamela J. Vandevord 3, Elizabeth M. McNeil 3, Tim Walilko 4, Zin Z. Khaing 5,6, Laila Zai 4,5
1Animal Sciences and Industry, Kansas State University, 2DynaSim Technical Services, INC, 3Center for Injury Biomechanics, Virginia Polytechnic Institute and State University, 4Applied Research Associates, Inc., 5Lucent Research, LLC, 6Department of Neurological Surgery, University of Washington

This protocol describes a new behavioral test—the human approach test in the pigs' home pen—to detect functional deficits in laboratory pigs after subconcussive traumatic brain injury.

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Neuroscience

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns
Julia Campbell 1,2, Mashhood Nielsen 1,2, Alison LaBrec 1,2, Connor Bean 1,2
1Department of Communication Sciences and Disorders, The University of Texas at Austin, 2Central Sensory Processes Laboratory, The University of Texas at Austin

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.  

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Developmental Biology

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography
C. Ben Lovely 1
1Department of Biochemistry and Molecular Genetics, Alcohol Research Center, University of Louisville

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

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Biology

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series
Jonathan D. Partridge 1, Rasika M. Harshey 1
1Department of Molecular Biosciences, The University of Texas at Austin

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

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Bioengineering

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans
Jyoti Dubey 1,2,3,4, Sudip Mondal 1,5, Sandhya P. Koushika 2
1National Centre for Biological Sciences, TIFR, 2Department of Biological Sciences, TIFR, 3InSTEM, 4Manipal Academy of Higher Education, 5Department of Mechanical Engineering, The University of Texas at Austin

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

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Biochemistry

Modeling an Enzyme Active Site using Molecular Visualization Freeware
Kristen Procko 1, Sandy Bakheet 1, Josh T. Beckham 1, Margaret A. Franzen 2, Henry Jakubowski 3, Walter R. P. Novak 4
1The University of Texas at Austin, 2Mount Mary University, 3College of St. Benedict/St. John’s University, 4Wabash College

A key skill in biomolecular modeling is displaying and annotating active sites in proteins. This technique is demonstrated using four popular free programs for macromolecular visualization: iCn3D, Jmol, PyMOL, and UCSF ChimeraX.

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Developmental Biology

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach
John R. Klem 1, Raèden Gray 1, C. Ben Lovely 1
1Alcohol Research Center, Department of Biochemistry and Molecular Genetics, University of Louisville

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

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Genetics

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish
Brittney Voigt 1, Ryoko Minowa 2, Ryan S. Gray 1,2
1Department of Molecular Biosciences, The University of Texas at Austin, 2Department of Nutritional Sciences, The University of Texas at Austin

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

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Genetics

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli
Jeffrey E. Barrick 1,2, Zachary D. Blount 2,3, Devin M. Lake 2,4, Jack H. Dwenger 1, Jesus E. Chavarria-Palma 1, Minako Izutsu 2,3, Michael J. Wiser 2,5
1Department of Molecular Biosciences, The University of Texas at Austin, 2BEACON Center for the Study of Evolution in Action, Michigan State University, 3Department of Microbiology and Molecular Genetics, Michigan State University, 4Department of Integrative Biology, Michigan State University, 5Biological Sciences Program, Michigan State University

This protocol describes how to maintain the Escherichia coli Long-Term Evolution Experiment (LTEE) by performing its daily transfers and periodic freeze-downs and how to conduct competition assays to measure fitness improvements in evolved bacteria. These procedures can serve as a template for researchers starting their own microbial evolution experiments.

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