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Georgia Institute of Technology

31 ARTICLES PUBLISHED IN JoVE

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Biology

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography
Matthew C. Johnson *1, Frederik Rudolph *1,2, Tina M. Dreaden *3, Gengxiang Zhao *1, Bridgette A. Barry 3, Ingeborg Schmidt-Krey 1,3
1School of Biology, Georgia Institute of Technology, 2Department of Molecular Pharmacology, RWTH Aachen University, 3School of Chemistry and Biochemistry, Georgia Institute of Technology

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 – 90kDa.

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Neuroscience

Simultaneous fMRI and Electrophysiology in the Rodent Brain
Wen-ju Pan 1,2, Garth Thompson 1,2, Matthew Magnuson 1,2, Waqas Majeed 1,2, Dieter Jaeger 3, Shella Keilholz 1,2
1Biomedical Engineering, Emory University, 2Biomedical Engineering, Georgia Institute of Technology, 3Biology, Emory University

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

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Biology

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides
Ying Shen 1, Francesca Storici 1
1School of Biology, Georgia Institute of Technology

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

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Bioengineering

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface
Veronika I. Zarnitsyna 1, Cheng Zhu 1
1Biomedical Engineering Department, Georgia Institute of Technology

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin αLβ2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

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Chemistry

Spectrophotometric Determination of an Equilibrium Constant
Michael Evans 1
1Georgia Institute of Technology

Spectrophotometric Determination of an Equilibrium Constant

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Bioengineering

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry
Rachel V. Bennett *1, Chaminda M. Gamage *1, Facundo M. Fernández 1
1School of Chemistry and Biochemistry, Georgia Institute of Technology

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

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Chemistry

Conducting Miller-Urey Experiments
Eric T. Parker 1, James H. Cleaves 2,3, Aaron S. Burton 4, Daniel P. Glavin 5, Jason P. Dworkin 5, Manshui Zhou 1, Jeffrey L. Bada 6, Facundo M. Fernández 1
1School of Chemistry and Biochemistry, Georgia Institute of Technology, 2Earth-Life Science Institute, Tokyo Institute of Technology, 3Institute for Advanced Study, 4Astromaterials Research and Exploration Science Directorate, NASA Johnson Space Center, 5Goddard Center for Astrobiology, NASA Goddard Space Flight Center, 6Geosciences Research Division, Scripps Institution of Oceanography, University of California at San Diego

The Miller-Urey experiment was a pioneering study regarding the abiotic synthesis of organic compounds with possible relevance to the origins of life. Simple gases were introduced into a glass apparatus and subjected to an electric discharge, simulating the effects of lightning in the primordial Earth’s atmosphere-ocean system. The experiment was conducted for one week, after which, the samples collected from it were analyzed for the chemical building blocks of life.

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JoVE Core

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems
Pamela Pollet 1, Amy L. Ethier 2, James C. Senter 2, Charles A. Eckert 2, Charles L. Liotta 1
1School of Chemistry and Biochemistry, Georgia Institute of Technology, 2School of Chemical and Biomolecular Engineering, Georgia Institute of Technology

The high pressure sapphire cell apparatus is a unique tool to study, without sampling, phase behavior under a wide range of pressures. Using a cathetometer, very precise volume measurements can be recorded to measure liquid expansion and phase composition. Thus, this synthetic method enables the study of (1) phase equilibria of multi-component mixtures and (2) the partition behavior of catalyst or model compounds as a function of pressure.

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Biology

Studying DNA Looping by Single-Molecule FRET
Tung T. Le 1, Harold D. Kim 1
1School of Physics, Georgia Institute of Technology

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

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JoVE Core

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States
Natalie K. Fan 1, Philip M. Keegan 1, Manu O. Platt 1,2, Rodney D. Averett 2,3
1Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University School of Medicine, 2Parker H. Petit Institute for Bioengineering & Bioscience, Georgia Institute of Technology, 3George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

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Bioengineering

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell
Yunfeng Chen *1, Baoyu Liu *2, Lining Ju *3, Jinsung Hong *1, Qinghua Ji 4,5, Wei Chen 6, Cheng Zhu 1
1Woodruff School of Mechanical Engineering, Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, 2Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 3Charles Perkins Centre, The University of Sydney, 4Institute of Biophysics, Laboratory of RNA Biology, Chinese Academy of Sciences, 5University of Chinese Academy of Sciences, 6School of Medicine and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

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Developmental Biology

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration
Mianbo Huang 1, Jin Xu 1, Chong Hyun Shin 1
1School of Biology, Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

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Bioengineering

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles
Ningquan Wang 1, Ruxiu Liu 1, A. Fatih Sarioglu 1,2,3
1School of Electrical and Computer Engineering, Georgia Institute of Technology, 2Institute of Electronics and Nanotechnology, Georgia Institute of Technology, 3Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

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Medicine

Transvaginal Mesh Insertion in the Ovine Model
Iva Urbankova 1,2, Geertje Callewaert 1,3, Nikhil Sindhwani 1, Alice Turri 1, Lucie Hympanova 1,2, Andrew Feola 1, Jan Deprest 1,3
1Centrum for Surgical Technologies, Department of Development and Regeneration, Clinical Specialties Research Groups, Faculty of Medicine, KU Leuven, 2Institute for the Care of Mother and Child and Third Faculty of Medicine, Charles University, Prague, 3Pelvic Floor Unit, University Hospitals KU Leuven

This protocol describes mesh implantation in the ovine rectovaginal septum using a single vaginal incision technique, with and without the trocar-guided insertion of anchoring arms.

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Bioengineering

Melt Electrospinning Writing of Three-dimensional Poly(ε-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications
Felix M. Wunner 1, Onur Bas 1, Navid T. Saidy 1, Paul D. Dalton 2, Elena M. De-Juan Pardo 1, Dietmar W. Hutmacher 1,3,4
1ARC ITTC in Additive Biomanufacturing, Institute for Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), 2Department for Functional Materials in Medicine and Dentistry and Bavarian Polymer Institute, University of Würzburg, 3Institute for Advanced Study, Technical University of Munich (TUM), 4George W Woodruff School of Mechanical Engineering, Georgia Institute of Technology

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

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JoVE Journal

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans
Dhaval S Patel *1, Giovanni Diana *1, Eugeni V. Entchev 1, Mei Zhan 2,3,4, Hang Lu 2,3,4, QueeLim Ch'ng 1
1Centre for Developmental Neurobiology, King's College London, 2Interdisciplinary Bioengineering Graduate Program, Georgia Institute of Technology, 3Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 4School of Chemical & Biomolecular Engineering, Georgia Institute of Technology

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

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Education

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid
Alex Aw 1, Marshall Fritz 2, Jonathan W. Napoline 1, Pamela Pollet 1, Charles L. Liotta 1
1School of Chemistry & Biochemistry, Georgia Institute of Technology, 2School of Chemical & Biomolecular Engineering, Georgia Institute of Technology

Flow chemistry carries environmental and economic advantages by leveraging superior mixing, heat transfer and cost benefits. Herein, we provide a blueprint to transfer chemical processes from batch to flow mode. The reaction of diphenyldiazomethane (DDM) with p-nitrobenzoic acid, conducted in batch and flow, was chosen for proof of concept.

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Biochemistry

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling
Ryan C. Oliver 1, Swe-Htet Naing 2, Kevin L. Weiss 1, Sai Venkatesh Pingali 1, Raquel L. Lieberman 2, Volker S. Urban 1
1Neutron Scattering Division, Oak Ridge National Laboratory, 2School of Chemistry and Biochemistry, Georgia Institute of Technology

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

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Medicine

Porcine As a Training Module for Head and Neck Microvascular Reconstruction
Mohammad Ali Alessa *1,2, Sang Hyun Kwak *2, Young Woo Lee 2, Mi-Lan Kang 3, Hak-Joon Sung 3,4, Soon Hyun Ahn 5, Eun Chang Choi 2, Won Shik Kim 5
1Department of Otolaryngology, Head and Neck Surgery, King Abdullah Medical City, 2Department of Otorhinolaryngology, Yonsei University, College of Medicine, 3Severance Biomedical Science Institute, Yonsei University, College of Medicine, 4The George W.Woodruff School of Mechanical Engineering, Georgia Institute of Technology, 5Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University College of Medicine

Here we present a protocol for the use of the pig superior epigastric artery perforator flap as a learning module for head and neck microvascular reconstruction.

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Chemistry

Stereolithographic 3D Printing with Renewable Acrylates
Vincent S.D. Voet 1, Geraldine H.M. Schnelting 1, Jin Xu 2, Katja Loos 2, Rudy Folkersma 1, Jan Jager 1
1Professorship Sustainable Polymers, NHL Stenden University of Applied Sciences, 2Macromolecular Chemistry and New Polymeric Materials, Zernike Institute for Advanced Materials, University of Groningen

A protocol for additive manufacturing with renewable photopolymer resins on a stereolithography apparatus is presented.

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Biology

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly
Chang Li *1,2, Zhu Hui Liu *1,2, Jia Wei Chen 1,2, Xin Yi Shu 1,2, Ying Shen 1, Feng Hua Ding 1, Rui Yan Zhang 1, Wei Feng Shen 1, Lin Lu 1, Xiao Qun Wang 1
1Department of Cardiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 2Institute of Cardiovascular Diseases, Shanghai Jiao Tong University

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of atherosclerosis.

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Developmental Biology

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo
Yunyun Jiang 1, Jiahao Chen 1, Jin Xu 2
1Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, 2Division of Cell, Developmental and Integrative Biology, School of Medicine, South China University of Technology

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

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Bioengineering

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion
Sebastian Eggert 1,2, Melanie Kahl 1,3, Ross Kent 1, Lukas Gaats 1, Nathalie Bock 1,3,4,5, Christoph Meinert 1,2, Dietmar W. Hutmacher 1,2,3,4,6
1Centre in Regenerative Medicine, Institute of Health and Biomedical Innovation, Queensland University of Technology, 2School of Mechanical, Medical and Process Engineering, Science and Engineering Faculty, Queensland University of Technology, 3School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, 4Australian Prostate Cancer Research Centre, Institute of Health and Biomedical Innovation, Queensland University of Technology, 5Translational Research Institute, Queensland University of Technology, 6ARC ITTC in Additive Biomanufacturing, Institute of Health and Biomedical Innovation, Queensland University of Technology

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

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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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Bioengineering

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography
Rachael S. Allen 1,2, Katie Bales 1,3, Andrew Feola 1,2, Machelle T Pardue 1,2,3
1Center of Excellence for Visual and Neurocognitive Rehabilitation, Atlanta Veterans Affairs Medical Center, 2Department of Biomedical Engineering, Georgia Institute of Technology, 3Department of Ophthalmology, Emory University

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

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Behavior

Behavioral Assessment of Visual Function via Optomotor Response and Cognitive Function via Y-Maze in Diabetic Rats
Kaavya Gudapati *1,2, Anayesha Singh *1,3, Danielle Clarkson-Townsend 1,4, Stephen Q. Phillips 1, Amber Douglass 1, Andrew J. Feola 1,2, Rachael S. Allen 1,2
1Center for Visual and Neurocognitive Rehabilitation, Atlanta VA Medical Center, 2Department of Biomedical Engineering, Georgia Institute of Technology, 3Department of Neuroscience, Emory University, 4Gangarosa Department of Environmental Health, Emory University

Neural degeneration in both eyes and brain as a result of diabetes can be observed through behavioral tests carried out on rodents. The Y-maze, a measure of spatial cognition, and the optomotor response, a measure of visual function, both provide insight into potential diagnoses and treatments.

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Environment

Methane Hydrate Crystallization on Sessile Water Droplets
Abigail M. Johnson 1, Yumeng Zhao 2, Jongchan Kim 2, Sheng Dai 2, Jennifer B. Glass 1
1Earth and Atmospheric Sciences, Georgia Institute of Technology, 2Civil and Environmental Engineering, Georgia Institute of Technology

We describe a method to form gas hydrate on sessile water droplets to study the effects of various inhibitors, promoters, and substrates on the hydrate crystal morphology.

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Bioengineering

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets
Andrew Zhao 1, Jeungyoon Lee 2, Stanislav Emelianov 1,3
1Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 2School of Electrical Engineering, Georgia Institute of Technology, 3Department of Biomedical Engineering and School of Electrical Engineering, Georgia Institute of Technology and Emory University

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

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Biochemistry

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples
Matthew C. Johnson *1,2, Arshay J. Grant *1, Ingeborg Schmidt-Krey 1,3
1School of Biological Sciences, Georgia Institute of Technology, 2Department of Structural Biology, Genentech, 3School of Chemistry and Biochemistry, Georgia Institute of Technology

The peel-blot technique is a cryo-EM grid preparation method that allows for the separation of multilayered and concentrated biological samples into single layers to reduce thickness, increase sample concentration, and facilitate image processing.

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Chemistry

Determining Surface Areas and Pore Volumes of Metal-Organic Frameworks
Tania G. Evans 1, Jamie L. Salinger 1, Lukas W. Bingel 1, Krista S. Walton 1
1School of Chemical & Biomolecular Engineering, Georgia Institute of Technology

This article describes the use of nitrogen porosimetry to characterize metal-organic frameworks, using UiO-66 as a representative material.

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Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme

Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme
Yusef Haikal 1, Maria Rain Jennings 2, Johnathan Nguyen 1, John J. Blazeck 1
1School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 2Johns Hopkins University School of Medicine

In this article, we detail methods to characterize an enzyme's ability to retain function when incubated at 37 °C in human serum, a pharmacological property referred to as its serum stability. This ability may be a key factor in predicting an enzyme's pharmacokinetic profile and its suitability for therapeutic use.

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