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The George Washington University

29 ARTICLES PUBLISHED IN JoVE

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Biology

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry
Peter Nemes 1, Akos Vertes 1
1Department 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.

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Immunology and Infection

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites
Sittiporn Pattaradilokrat 1, Jian Li 1,2, Xin-zhuan Su 1
1National Institute of Allergy and Infectious Diseases, National Institutes of Health, 2School of Life Science, Xiamen University

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

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Bioengineering

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart
Qing Lou 1, Wenwen Li 1, Igor R. Efimov 1
1Department of Biomedical Engineering, Washington University in St. Louis

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

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Bioengineering

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart
Di Lang 1, Matthew Sulkin 1, Qing Lou 1, Igor R. Efimov 1
1Department of Biomedical Engineering, Washington University in St. Louis

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

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Bioengineering

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma
Jian Li 1, Alexey Shashurin 1, Madhusudhan Kundrapu 1, Michael Keidar 1
1Department 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.

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Medicine

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts
Huda Asfour 1, Anastasia M. Wengrowski 1, Rafael Jaimes III 1, Luther M. Swift 2, Matthew W. Kay 1
1Electrical 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.

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Biology

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars
Teresa M. Stoepler 1, Julio C. Castillo 1, John T. Lill 1, Ioannis Eleftherianos 1
1Department 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.

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Neuroscience

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans
Chao He 1, Jin I. Lee 2, Noelle L'Etoile 3, Damien O'Halloran 1
1Department 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.

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JoVE Journal

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development
Paaqua A. Grant 1, Mona B. Herold 1, Sally A. Moody 2
1Department 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.

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Engineering

Fabrication of VB2/Air Cells for Electrochemical Testing
Jessica Stuart 1, Ruben Lopez 2, Jason Lau 1, Xuguang Li 2, Mahesh Waje 2, Matthew Mullings 2, Christopher Rhodes 2, Stuart Licht 1
1Department 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.

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Biology

A Practical Guide to Phylogenetics for Nonexperts
Damien O'Halloran 1
1Department 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. 

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Bioengineering

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp
Kelley C. Stewart 1, Byron D. Erath 2, Michael W. Plesniak 1
1Department 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.

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Bioengineering

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180° Curved Artery Test Section
Kartik V. Bulusu 1, Michael W. Plesniak 1
1Department 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.

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Engineering

A Robotic Platform to Study the Foreflipper of the California Sea Lion
Aditya A. Kulkarni 1, Rahi K. Patel 1, Chen Friedman 1, Megan C. Leftwich 1
1Department 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').

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Bioengineering

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair
Jian Li 1, Holger Jahr 2,3, Wei Zheng 4, Pei-Gen Ren 1
1Center for Translational Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 2Department of Orthopedic Surgery, Maastricht UMC+, 3Department of Orthopaedic Surgery, University Hospital RWTH, 4Research Laboratory for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

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Medicine

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients
Elena K. Schneider 1, Felisa Reyes-Ortega 1, Jian Li 1,2, Tony Velkov 1
1Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 2Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

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Chemistry

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos
Rosemary M. Onjiko 1, Erika P. Portero 1, Sally A. Moody 2, Peter Nemes 1,3
1Department 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.

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JoVE Journal

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction
Dan Zhou *1,2, Lei Tan *1, Jian Li *3, Tanbin Liu 1, Yi Hu 1, Yalan Li 1, Sachiyo Kawamoto 4, Chengyu Liu 5, Shiyin Guo 3, Aibing Wang 1
1Lab of Animal Models and Functional Genomics (LAMFG), The Key Laboratory of Animal Vaccine & Protein Engineering, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), 2Department of Pathology, Georgetown University Medical School, 3College of Food Science and Technology, Hunan Agricultural University (HUNAU), 4Lab of Molecular Cardiology (LMC), National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH), 5Transgenic Core, National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH)

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

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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 Giles 1, Robert S. Balaban 1
1Laboratory 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.

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JoVE Core

Home-Based Prescribed Pulmonary Exercise in Patients with Stable Chronic Obstructive Pulmonary Disease
Xiaodan Liu 1,2, Peijun Li 3, Jian Li 3, Lu Xiao 1, Ning Li 3, Yufan Lu 3, Zhengrong Wang 3, Jianqing Su 3, Zhenwei Wang 4, Chunlei Shan 1,2, Weibing Wu 3
1School of Rehabilitation Science, Shanghai University of Traditional Chinese Medicine, 2Institute of Rehabilitation Medicine, Shanghai Academy of Traditional Chinese Medicine, 3Department of Sports Medicine, Shanghai University of Sport, 4Department of Respiratory Medicine, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine

Presented here is a protocol to investigate the effects of home-based prescribed pulmonary exercise in stable chronic obstructive pulmonary disease (COPD) patients, which is modified based on traditional Chinese exercises according to dyspnea and limited exercise capacity observed in COPD patients.

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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 Zhang 4, Nobuyuki Ishibashi 1,2,3, Jack M. Rogers 4, Nikki Gillum Posnack 1,2,5
1Sheikh Zayed Institute for Pediatric Surgical Innovation, Children's National Hospital, 2Children's National Heart Institute, Children's National Hospital, 3Center 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.

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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
1Department 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.

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Engineering

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix
Kartik V. Bulusu 1, Samantha Racan 1, Michael W. Plesniak 1
1Department 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.

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Immunology and Infection

Drosophila melanogaster Larva Injection Protocol
Ghada Tafesh-Edwards 1, Eric Kenney 1, Ioannis Eleftherianos 1
1Infection 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.

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Neuroscience

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study
Sara Oberrauch 1, Jing Lu 1, Linda Cornthwaite-Duncan 1, Maytham Hussein 1, Jian Li 2, Gauri Rao 3, Tony Velkov 1
1Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 2Department of Microbiology, Biomedicine Discovery Institute, Monash University, 3UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

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Chemistry

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo
Aparna B. Baxi 1,2, Leena R. Pade 1, Peter Nemes 1,2
1Department 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.

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Immunology and Infection

Culturing and Genetically Manipulating Entomopathogenic Nematodes
Christa Heryanto 1, Ramesh Ratnappan 2, Damien M. O'Halloran 1, John M. Hawdon 2, Ioannis Eleftherianos 1
1Department 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.

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Neuroscience

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics
Ashley Frankenfield *1, Jiawei Ni *1, Ling Hao 1
1Department 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.

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Immunology and Infection

Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection
Rajnikant Sharma 1, Shekhar Yeshwante 1, Quentin Vallé 1, Maytham Hussein 2, Varsha Thombare 2, Sean Michael McCann 1, Robert Maile 3,4,5, Jian Li 6, Tony Velkov 2, Gauri Rao 1
1UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 2Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 3Department of Microbiology & Immunology, University of North Carolina School of Medicine, 4Department of Surgery, University of North Carolina at Chapel Hill, 5Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, 6Department of Microbiology, Monash Biomedicine Discovery Institute, Monash University

A model mimicking the clinical scenario of burn injury and infection is necessary for furthering burn research. The present protocol demonstrates a simple and reproducible rat burn infection model comparable to that in humans. This facilitates the study of burn and infections following burn for developing new topical antibiotic treatments.

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