George Washington University

26 articles published in JoVE

• Neuroscience

  
  Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics Ashley Frankenfield*¹, Jiawei Ni*¹, Ling Hao^{1 1}Department of Chemistry, The George Washington University A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

• Chemistry

  
  Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo Aparna B. Baxi^1,2, Leena R. Pade¹, Peter Nemes^{1,2 1}Department of Chemistry & Biochemistry, University of Maryland, ²Department of Anatomy & Cell Biology, The George Washington University Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

• Immunology and Infection

  
  Culturing and Genetically Manipulating Entomopathogenic Nematodes Christa Heryanto¹, Ramesh Ratnappan², Damien M. O'Halloran¹, John M. Hawdon², Ioannis Eleftherianos^{1 1}Department of Biological Sciences, The George Washington University, ²Department of Microbiology, Immunology, and Tropical Medicine, School of Medicine and Health Sciences, The George Washington University Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

• Immunology and Infection

  
  Drosophila melanogaster Larva Injection Protocol Ghada Tafesh-Edwards¹, Eric Kenney¹, Ioannis Eleftherianos^{1 1}Infection and Innate Immunity Laboratory, Department of Biological Sciences, Institute for Biomedical Sciences, The George Washington University Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

• Engineering

  
  Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix Kartik V. Bulusu¹, Samantha Racan¹, Michael W. Plesniak^{1 1}Department of Mechanical and Aerospace Engineering, The George Washington University This protocol presents a method to perform rheology characterization of mucus that resides on gill rakers (GRs) of the silver carp. Viscoelastic characteristics of GR-mucus, obtained by measuring viscosity, storage and loss moduli, are evaluated for the apparent yield stress to understand the filter feeding mechanism in GRs.

• Bioengineering

  
  Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices Sharon A. George*¹, Jaclyn A. Brennan*¹, Igor R. Efimov^{1 1}Department of Biomedical Engineering, The George Washington University This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

• Medicine

  
  Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts Luther M. Swift^1,2, Rafael Jaimes III^1,2, Damon McCullough^1,2, Morgan Burke^1,2, Marissa Reilly^1,2, Takuya Maeda^1,2,3, Hanyu Zhang⁴, Nobuyuki Ishibashi^1,2,3, Jack M. Rogers⁴, Nikki Gillum Posnack^{1,2,5 1}Center for Neuroscience Research, Children's National Hospital, ⁴Department of Biomedical Engineering, School of Engineering, University of Alabama at Birmingham, ⁵Department of Pediatrics, Department of Pharmacology & Physiology, School of Medicine and Health Sciences, George Washington University The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

• Cancer Research

  
  Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids Erin R. Bonner^1,2, Karim Saoud¹, Sulgi Lee^1,2, Eshini Panditharatna³, Madhuri Kambhampati¹, Sabine Mueller^4,5, Javad Nazarian^{1,2,5 1} Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

• Bioengineering

  
  Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts Armel N. Femnou^1,2, Abigail Giles¹, Robert S. Balaban^{1 1}Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, ²Department of Biomedical Engineering, The George Washington University Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

• Chemistry

  
  Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos Rosemary M. Onjiko¹, Erika P. Portero¹, Sally A. Moody², Peter Nemes^{1,3 1}Department of Chemistry, George Washington University, ²Department of Anatomy & Regenerative Biology, George Washington University, ³Department of Chemistry & Biochemistry, University of Maryland, College Park We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

• Engineering

  
  A Robotic Platform to Study the Foreflipper of the California Sea Lion Aditya A. Kulkarni¹, Rahi K. Patel¹, Chen Friedman¹, Megan C. Leftwich^{1 1}Department of Mechanical and Aerospace Engineering, The George Washington University A robotic platform is described that will be used to study the hydrodynamic performance—forces and flowfields—of the swimming California sea lion. The robot is a model of the animal's foreflipper that is actuated by motors to replicate the motion of its propulsive stroke (the 'clap').

• Bioengineering

  
  Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180° Curved Artery Test Section Kartik V. Bulusu¹, Michael W. Plesniak^{1 1}Department of Mechanical and Aerospace Engineering, The George Washington University Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

• Bioengineering

  
  Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications Jennifer M. Vojtech^1,2, Juliana Cano-Mejia^1,2, Matthieu F. Dumont¹, Raymond W. Sze^1,3, Rohan Fernandes^{1,3,4 1} This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

• Behavior

  
  Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases Kathleen S. Tatem¹, James L. Quinn¹, Aditi Phadke¹, Qing Yu¹, Heather Gordish-Dressman^1,2, Kanneboyina Nagaraju^{1,2 1} Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

• Biology

  
  Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair Aurelia Defour*¹, S. C. Sreetama*¹, Jyoti K. Jaiswal^{1,2 1}Center for Genetic Medicine Research, Children's National Medical Center, ²Department of Integrative Systems Biology, George Washington University The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

• Biology

  
  A Practical Guide to Phylogenetics for Nonexperts Damien O'Halloran^{1 1}Department of Biological Sciences and Institute for Neuroscience, The George Washington University Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis. 

• Bioengineering

  
  Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp Kelley C. Stewart¹, Byron D. Erath², Michael W. Plesniak^{1 1}Department of Mechanical and Aerospace Engineering, The George Washington University, ²Department of Mechanical and Aeronautical Engineering, Clarkson University Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient's ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

• Engineering

  
  Fabrication of VB₂/Air Cells for Electrochemical Testing Jessica Stuart¹, Ruben Lopez², Jason Lau¹, Xuguang Li², Mahesh Waje², Matthew Mullings², Christopher Rhodes², Stuart Licht^{1 1}Department of Chemistry, The George Washington University, ²Lynntech A protocol is presented to study multi-electron metal/air battery systems by using previous technology developed for the zinc/air cell. Electrochemical testing is then performed on fabricated batteries to evaluate performance.

• Neuroscience

  
  Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants Mauro W. Zappaterra¹, Anthony S. LaMantia², Christopher A. Walsh^3,4, Maria K. Lehtinen^{5 1}Department of Physical Medicine and Rehabilitation, VA Greater Los Angeles Healthcare System, ²Department of Pharmacology and Physiology, Institute for Neuroscience, The George Washington University School of Medicine and Health Sciences, ³Division of Genetics, Department of Medicine, Boston Children's Hospital, ⁴Howard Hughes Medical Institute, Boston Children's Hospital, ⁵Department of Pathology, Boston Children's Hospital, Harvard Medical School The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

• Biology

  
  Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development Paaqua A. Grant¹, Mona B. Herold¹, Sally A. Moody^{2 1}Department of Biological Sciences, The George Washington University, ²Department of Anatomy and Regenerative Biology, The George Washington University The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

• Neuroscience

  
  A Molecular Readout of Long-term Olfactory Adaptation in C. elegans Chao He¹, Jin I. Lee², Noelle L'Etoile³, Damien O'Halloran^{1 1}Department of Biological Sciences and Institute for Neuroscience, George Washington University, ²Fred Hutchinson Cancer Research Center, ³Department of Cell and Tissue Biology, University of California San Francisco Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

• Biology

  
  A Simple Protocol for Extracting Hemocytes from Wild Caterpillars Teresa M. Stoepler¹, Julio C. Castillo¹, John T. Lill¹, Ioannis Eleftherianos^{1 1}Department of Biological Sciences, The George Washington University Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

• Medicine

  
  NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts Huda Asfour¹, Anastasia M. Wengrowski¹, Rafael Jaimes III¹, Luther M. Swift², Matthew W. Kay^{1 1}Electrical and Computer Engineering Department, The George Washington University, ²Pharmacology and Physiology Department, The George Washington University The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

• Bioengineering

  
  Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma Jian Li¹, Alexey Shashurin¹, Madhusudhan Kundrapu¹, Michael Keidar^{1 1}Department of Mechanical and Aerospace Engineering, The George Washington University Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

• Biology

  
  Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure Bindesh Shrestha¹, Akos Vertes^{1 1}Department of Chemistry, George Washington University Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

• Biology

  
  Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry Peter Nemes¹, Akos Vertes^{1 1}Department of Chemistry, George Washington University Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.