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Emory University School of Medicine

25 ARTICLES PUBLISHED IN JoVE

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Biology

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)
Chadwick M. Hales 1,2, John D. Rolston 2,3, Steve M. Potter 2
1Department of Neurology, Emory University School of Medicine, 2Coulter Department of Biomedical Engineering, Laboratory for Neuroengineering, Georgia Institute of Technology and Emory, University School of Medicine, 3Emory University School of Medicine

This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

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Bioengineering

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases
David R. Myers *1,2,3,4, Yumiko Sakurai *1,2,3,4, Reginald Tran 1,2,3,4, Byungwook Ahn 1,2,3,4, Elaissa Trybus Hardy 1,2,3,4, Robert Mannino 1,2,3,4, Ashley Kita 1,2,3,4, Michelle Tsai 1,2,3,4, Wilbur A. Lam 1,2,3,4
1Department of Pediatrics, Emory University School of Medicine, 2Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 3Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta , 4Winship Cancer Institute of Emory University

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (<30 μm) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

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Neuroscience

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium
Karolina Piotrowska-Nitsche 1,2, Tamara Caspary 1
1Department of Human Genetics, Emory University School of Medicine, 2Department of Experimental Embryology, IGAB Polish Academy of Sciences

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

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Biology

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA
Yujing Li 1, Chun-Xiao Song 2, Chuan He 2, Peng Jin 1
1Department of Human Genetics, Emory University School of Medicine, 2Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago

Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

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Immunology and Infection

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment
Adrianne N. Edwards 1, Jose M. Suárez 1, Shonna M. McBride 1
1Department of Microbiology and Immunology, Emory University School of Medicine

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

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Neuroscience

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Robert S. McNeill 1, Ralf S. Schmid 2, Ryan E. Bash 3, Mark Vitucci 4, Kristen K. White 1, Andrea M. Werneke 3, Brian H. Constance 5, Byron Huff 6, C. Ryan Miller 2,3,7
1Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, 2Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 3Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, 4Curriculum in Genetics and Molecular Biology, University of North Carolina School of Medicine, 5Biological and Biomedical Sciences Program, University of North Carolina School of Medicine, 6Department of Radiation Oncology, Emory University School of Medicine, 7Department of Neurology, Neurosciences Center, University of North Carolina School of Medicine

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

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Neuroscience

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents
Joy A. Franco 1, Heidi E. Kloefkorn 2, Shawn Hochman 3, Katherine A. Wilkinson 4
1Department of Mechanical Engineering, San José State University, 2J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, 3Department of Physiology, Emory University School of Medicine, 4Department of Biological Sciences, San José State University

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

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JoVE Journal

Community-based Adapted Tango Dancing for Individuals with Parkinson's Disease and Older Adults
Madeleine E. Hackney 1, Kathleen McKee 2
1Center for Visual and Neurocognitive Rehabilitation, Atlanta VAMC, Department of Medicine, Division of General Medicine and Geriatrics, Emory University School of Medicine, 2Harvard Neurology Residency Program, Brigham and Woman‘s Hospital and Massachusetts General Hospital

Adapted tango has demonstrated efficacy for improving mobility and balance. We describe the dissemination of adapted tango teaching methods to dance instructor trainees and the implementation of adapted tango by the trainees in the community for improving mobility and balance in older adults and individuals with Parkinson’s disease.

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Immunology and Infection

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice
Lauren E. Hudson 1, Taryn P. Stewart 1, Milo B. Fasken 2, Anita H. Corbett 2, Tracey J. Lamb 1
1Department of Pediatrics, Emory University School of Medicine, 2Department of Biochemistry, Emory University School of Medicine

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

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JoVE Journal

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals
Kateena Addae Konadu 1, Ming Bo Huang 1, William Roth 1, Wendy Armstrong 2, Michael Powell 1, Francois Villinger 3,4, Vincent Bond 1
1Department of Microbiology, Biochemistry, Immunology, Morehouse School of Medicine, 2Department of Medicine, Emory University School of Medicine, 3Department of Pathology and Laboratory Medicine, Emory University School of Medicine, 4Yerkes National Primate Research Center

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

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Biology

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method
Clarisse van der Feltz 1, Daniel Pomeranz Krummel 1,2
1Department of Biochemistry, Brandeis University, 2Winship Cancer Institute, Emory University School of Medicine

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

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Engineering

3D Printing of Biomolecular Models for Research and Pedagogy
Eduardo Da Veiga Beltrame 1, James Tyrwhitt-Drake 2, Ian Roy 3, Raed Shalaby 4, Jakob Suckale 4, Daniel Pomeranz Krummel 5
1Department of Physics, Brandeis University, 2Bioinformatics and Computational Biosciences Branch (BCBB), NIH/NIAID/OD/OSMO/OCICB, 3Library/LTS/MakerLab, Brandeis University, 4Interfaculty Institute of Biochemistry (IFIB), University of Tübingen, 5Winship Cancer Institute, Emory University School of Medicine

Physical models of biomolecules can facilitate an understanding of their structure-function for the researcher, aid in communication between researchers, and serve as an educational tool in pedagogical endeavors. Here, we provide detailed guidance for the 3D printing of accurate models of biomolecules using fused filament fabrication desktop 3D printers.

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Biochemistry

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells
Joshua T. Maxwell 1, Chin-Hsien Tsai 1, Tahmina A. Mohiuddin 2, Jennifer Q. Kwong 1
1Department of Pediatrics, Emory University School of Medicine, 2Emory College of Arts and Sciences, Emory University

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

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Genetics

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy
Baoheng Gui *1,2,3, Yingxin Zhang *4, Bo Liang 5, Yvonne Ka Yin Kwok 4, Wai Ting Lui 4, Queenie Sum Yee Yeung 4, Lingyin Kong 6, Liming Xuan 6, Jacqueline Pui Wah Chung 4, Kwong Wai Choy 3,4
1Department of Genetics and Metabolism, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, 2Birth Defects Prevention and Control Institute of Guangxi Zhuang Autonomous Region, 3Shenzhen Research Institute, The Chinese University of Hong Kong, 4Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, 5State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 6Basecare Medical Device Co., Ltd

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

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Biology

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry
M. Edward Quach 1,2, Anum K. Syed 1,2, Renhao Li 1,2
1Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, 2Department of Pediatrics, Emory University School of Medicine

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

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Medicine

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat
Daniella Corporan 1, Takanori Kono 1, Daisuke Onohara 1, Muralidhar Padala 1,2
1Structural Heart Research and Innovation Laboratory, Carlyle Fraser Heart Center, Emory University Hospital Midtown, 2Division of Cardiothoracic Surgery, Joseph P. Whitehead Department of Surgery, Emory University School of Medicine

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance. 

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Neuroscience

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury
Rowan O. Brothers *1, Sara Bitarafan *2,3, Alyssa F. Pybus 1,3, Levi B. Wood *1,2,3, Erin M. Buckley *1,4,5
1Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 2George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, 3Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 4Department of Pediatrics, Emory University School of Medicine, 5Children's Research Scholar, Children's Healthcare of Atlanta

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

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Medicine

Segmentation and Linear Measurement for Body Composition Analysis using Slice-O-Matic and Horos
Sean Steele 1, Fangyi Lin 1, Thien-Linh Le 1, Alexandra Medline 1, Michelle Higgins 1, Alex Sandberg 1, Sean Evans 1, Gordon Hong 2, Milton A. Williams 3, Mehmet A. Bilen 4, Sarah Psutka 5, Kenneth Ogan 1, Viraj A. Master 1
1Department of Urology, Emory University School of Medicine, 2Northeast Ohio Medical University, 3Department of Urology, University of Alabama at Birmingham, 4Department of Medical Oncology, Winship Cancer Institute of Emory University, 5Department of Urology, University of Washington

Segmentation and linear measurements quantify skeletal muscle mass and adipose tissues using Computed Tomography and/or Magnetic Resonance Imaging images. Here, we outline the use of Slice-O-Matic software and Horos image viewer for rapid and accurate analysis of body composition. These methods can provide important information for prognosis and risk stratification.

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Biology

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection
Raven Shah *1, Shuiyun Lan *1, Maritza N. Puray-Chavez 2, Dandan Liu 2, Philip R. Tedbury 1, Stefan G. Sarafianos 1
1Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, 2Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

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Biochemistry

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus
Yunrong Gao 1, Claire Ogilvie 1, Anirudh Raghavan 1, Chloe Von Hoffmann 1, Bo Liang 1
1Department of Biochemistry, Emory University School of Medicine

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

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Behavior

Walk with Me Hybrid Virtual/In-Person Walking for Older Adults with Neurodegenerative Disease
Meredith D. Wells 1, Amy Morse 2, Jolie Barter 1, Kevin Mammino 3, Allison A. Bay 1, Todd Prusin 1, Madeleine E. Hackney 1,2,3,4,5
1Division of Geriatrics and Gerontology, Department of Medicine, Emory University School of Medicine, 2Department of Rehabilitation Medicine, Emory University School of Medicine, 3Atlanta VA Center for Visual and Neurocognitive Rehabilitation, 4Emory University School of Nursing, 5Birmingham/Atlanta VA Geriatric Research Education and Clinical Center

The present protocol aims to inform rehabilitation specialists and fitness instructors in safe, feasible, and evidence-based methods of delivering virtual and in-person walking classes to older adults with neurodegenerative diseases.

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Neuroscience

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging
Rashmi Thapa 1, Bo Liang 2, Rongsong Liu 3, Yun Li 1
1Department of Zoology and Physiology, University of Wyoming, 2School of Electrical Engineering & Computer Science, University of North Dakota, 3Department of Mathematics and Statistics, University of Wyoming

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

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Biology

Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope
Michael Hamersky IV 1, Khushi Tekale 1, L. Matthew Winfree 2, Matthew J. M. Rowan 1,3, Lindsey Seldin 1,4,5,6
1Department of Cell Biology, Emory University School of Medicine, 2Independent scholar, 3Center for Neurodegenerative Disease, Emory University School of Medicine, 4Department of Dermatology, Emory University School of Medicine, 5Winship Cancer Institute, Emory University School of Medicine, 6Atlanta Veterans Affairs Medical Center

The protocol presents a new tool to simplify intravital imaging using inverted confocal microscopy.

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Medicine

A Randomized, Sham-Controlled Trial of Cranial Electrical Stimulation for Fibromyalgia Pain and Physical Function, Using Brain Imaging Biomarkers
Anna Ree 1, Benjamin Rapsas 2, Chanse Denmon 1, Mark Vernon 1, Sheila AM Rauch 1,2, Ying Guo 3, Xiangqin Cui 1,3, Jennifer S Stevens 1,2, Venkatagiri Krishnamurthy 1,2, Vitaly Napadow 4, Jessica A Turner 5, Anna Woodbury 1,2
1Atlanta Veterans Affairs Healthcare System, 2Emory University School of Medicine, 3Rollins School of Public Health, 4Spaulding Rehabilitation Hospital, Harvard Medical School, 5The Ohio State University Wexner Medical Center

The current study is a randomized, placebo-controlled trial to determine the efficacy of cranial electrical stimulation (CES) for improving pain and function in fibromyalgia and further develop resting functional connectivity magnetic resonance imaging (rs-fcMRI) as a clinical tool to assess the neural correlates and mechanisms of chronic pain and analgesic response.

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Neuroscience

Surgical Lumbar Sympathectomy in Mice
Tina Tian 1,2,3, Patricia J. Ward 2,3
1Medical Scientist Training Program, Emory University School of Medicine, 2Neuroscience Graduate Program, Laney Graduate School, Emory University, 3Department of Cell Biology, Emory University School of Medicine

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

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