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Duke University

13 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition
Gloria Wong 1,2, Sanda Dolcos 1,3, Ekaterina Denkova 1, Rajendra Morey 4,5,6, Lihong Wang 4,5, Gregory McCarthy 6,7, Florin Dolcos 1,2,3,8,9
1Department of Psychiatry, University of Alberta, 2Centre for Neuroscience, University of Alberta, 3Department of Psychology, University of Illinois, 4Brain Imaging and Analysis Center, Duke University , 5Department of Psychiatry and Behavioral Sciences, Duke University , 6Mid-Atlantic Mental Illness Research Education and Clinical Center, VA Medical Center, 7Department of Psychology, Yale University, 8Neuroscience Program, University of Illinois, 9Beckman Institute for Advanced Science & Technology, University of Illinois

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

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Immunology and Infection

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging
Joel R. Meyerson 1,2, Tommi A. White 1, Donald Bliss 3, Amy Moran 3, Alberto Bartesaghi 1, Mario J. Borgnia 1, M. Jason V. de la Cruz 1, David Schauder 1, Lisa M. Hartnell 1, Rachna Nandwani 1,4, Moez Dawood 5, Brianna Kim 6, Jun Hong Kim 7, John Sununu 8, Lisa Yang 9, Siddhant Bhatia 10, Carolyn Subramaniam 1, Darrell E. Hurt 11, Laurent Gaudreault 12, Sriram Subramaniam 1
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge , 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia , 7Duke University , 8Yale University, 9University of Notre Dame , 10Washington University in St. Louis , 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

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Bioengineering

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model
Alexandra E. Jantzen 1, Whitney O. Lane 2, Shawn M. Gage 3, Justin M. Haseltine 1, Lauren J. Galinat 1, Ryan M. Jamiolkowski 4, Fu-Hsiung Lin 3, George A. Truskey 1, Hardean E. Achneck 3
1Department of Biomedical Engineering, Duke University , 2School of Medicine, Duke University , 3Department of Surgery, Duke University Medical Center, 4School of Medicine, University of Pennsylvania

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

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Bioengineering

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress
Whitney O. Lane 1, Alexandra E. Jantzen 2, Tim A. Carlon 2, Ryan M. Jamiolkowski 3, Justin E. Grenet 1, Melissa M. Ley 1, Justin M. Haseltine 2, Lauren J. Galinat 2, Fu-Hsiung Lin 1, Jason D. Allen 4, George A. Truskey 2, Hardean E. Achneck 1
1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University , 3School of Medicine, University of Pennsylvania , 4Department of Medicine, Division of Cardiology, Duke University Medical Center

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

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Bioengineering

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium
Carleen M. Bowers 1, Eric J. Toone 1, Robert L. Clark 2, Alexander A. Shestopalov 3
1Department of Chemistry, Duke University , 2Hajim School of Engineering and Applied Sciences, University of Rochester , 3Department of Chemical Engineering, University of Rochester

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

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Immunology and Infection

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes
Dana C. Upton 1, Shyam Unniraman 1
1Department of Immunology, Duke University

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

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Biology

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue
Chun-Chun Chen 1, Kazuhiro Wada 2, Erich D. Jarvis 1
1Howard Hughes Medical Institute, Department of Neurobiology, Duke University , 2Department of Biological Sciences, Hokkaido University

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

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Neuroscience

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI
Gabriel P. Howles 1, Yi Qi 2, Stephen J. Rosenzweig 3, Kathryn R. Nightingale 3, G. Allan Johnson 2
1Department of Radiology, Stanford University , 2Center for In Vivo Microscopy, Duke University Medical Center, 3Department of Biomedical Engineering, Duke University

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

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Medicine

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate
David D. Lo *1,2, Jeong S. Hyun *1,3, Michael T. Chung 1, Daniel T. Montoro 1, Andrew Zimmermann 1, Monica M. Grova 1,4, Min Lee 5, Derrick C. Wan 1, Michael T. Longaker 1
1Department of Surgery, Stanford University , 2Department of Surgery, Duke University , 3Department of Surgery, Saint Joseph Mercy Hospital, 4School of Medicine, University of California, San Francisco , 5School of Dentistry, University of California, Los Angeles

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

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Medicine

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System
Teagan J. Walter 1,2, Erin E. Sparks 3, Stacey S. Huppert 2
1Department of Cell and Developmental Biology, Center for Stem Cell Biology, Vanderbilt University, 2Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital, 3Department of Biology, Duke University

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

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Medicine

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity
Bruce A. Lessey 1, H. Lee Higdon III 1, Sara E. Miller 2, Thomas A. Price 3
1Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Greenville Hospital System, 2Department of Pathology, Duke University Health System, 3Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Duke University

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

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Neuroscience

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling
Gustavo Arriaga 1, Joshua J. Macopson 1, Erich D. Jarvis 1,2
1Department of Neurobiology, Duke University Medical Center, 2Howard Hughes Medical Institute

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

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Behavior

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs
Jonathan Chabout 1,2, Joshua Jones-Macopson 1, Erich D. Jarvis 1,2,3
1Department of Neurobiology, Duke University, 2Howard Hughes Medical Institute, 3The Rockefeller University

Mice produce a complex multisyllabic repertoire of ultrasonic vocalizations (USVs). These USVs are widely used as readouts for neuropsychiatric disorders. This protocol describes some of the practices we learned and developed to consistently induce, collect, and analyze the acoustic features and syntax of mouse songs.

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