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Wayne State University

22 ARTICLES PUBLISHED IN JoVE

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Biology

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture
Jamie L. Ifkovits 1, Harini G. Sundararaghavan 1, Jason A. Burdick 1
1Department of Bioengineering, University of Pennsylvania

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

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Medicine

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery
Richard M. Lovering 1,2, Joseph A. Roche 1, Mariah H. Goodall 2, Brett B. Clark 2, Alan McMillan 3
1Department of Physiology, University of Maryland School of Medicine, 2Department of Orthopaedics, University of Maryland School of Medicine, 3Department of Diagnostic Radiology, University of Maryland School of Medicine

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

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Neuroscience

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry
Francis K. Maina *1, Madiha Khalid *1, Aaron K. Apawu 1, Tiffany A. Mathews 1
1Department of Chemistry, Wayne State University

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

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Biology

Imaging Plasma Membrane Deformations With pTIRFM
Daniel R. Passmore 1, Tejeshwar C. Rao 1, Andrew R. Peleman 1, Arun Anantharam 1
1Department of Biological Sciences, Wayne State University

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

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Bioengineering

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds
Tonya J. Whitehead 1, Harini G. Sundararaghavan 1
1Biomedical Engineering, Wayne State University

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

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Medicine

Determining The Electromyographic Fatigue Threshold Following a Single Visit Exercise Test
Sujay S. Galen 1, Darren R. Guffey 2, Jared W. Coburn 3, Moh H. Malek 1
1Physical Therapy Program and Integrative Physiology of Exercise Laboratory, Department of Health Care Sciences, Wayne State University, 2Sports Medicine and Physical Therapy, MEDSPORT, University of Michigan Health System, 3Department of Kinesiology, California State University, Fullerton

This protocol describes the electromyographic fatigue threshold which demarcates between nonfatiguing and fatiguing exercise workloads. This information could be used to develop a more individualized training program.

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Immunology and Infection

Isolation of Leukocytes from the Human Maternal-fetal Interface
Yi Xu 1, Olesya Plazyo 1, Roberto Romero 1,2,3,4, Sonia S. Hassan 1,5, Nardhy Gomez-Lopez 1,5,6
1Perinatology Research Branch, NICHD/NIH/DHHS, 2Department of Obstetrics and Gynecology, University of Michigan, 3Department of Epidemiology and Biostatistics, Michigan State University, 4Department of Molecular Obstetrics and Genetics, Wayne State University, 5Department of Obstetrics and Gynecology, Wayne State University School of Medicine, 6Department of Immunology and Microbiology, Wayne State University School of Medicine

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

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Biology

Isolation and Primary Culture of Mouse Aortic Endothelial Cells
Jie-Mei Wang 1, Alex F. Chen 2, Kezhong Zhang 1
1Center for Molecular Medicine and Genetics, Wayne State University, 2Third Xiangya Hospital and the Institute of Vascular Disease and Translational Medicine, Central South University

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

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Medicine

Video Movement Analysis Using Smartphones (ViMAS): A Pilot Study
Monica J. Finkbiner 1, Kira M. Gaina 1, Marie C. McRandall 1, Megan M. Wolf 1, Vicky M. Pardo 1, Kristina Reid 1, Brian Adams 2, Sujay S. Galen 1
1Physical Therapy Program, Department of Healthcare Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 2Adams Sports Medicine and Physical Therapy

This manuscript describes the method to test the concurrent validity of kinematic measures recorded by the smartphone application in comparison to a 3D motion capture system in the sagittal plane. This protocol will enable clinicians to set up smartphones for video capture of human movement.

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Bioengineering

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method
Cameron B. Pinnock 1, Zhengfan Xu 1, Mai T. Lam 1,2
1Department of Biomedical Engineering, Wayne State University, 2Cardiovascular Research Institute, Wayne State University

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

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Biochemistry

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples
Tyler Rhinesmith 1, Bryan A. Killinger 2, Akhil Sharma 3, Anna Moszczynska 3
1Physiology, Michigan State University, 2Center for Neurodegenerative Science Van Andel Institute, 3Pharmaceutical Sciences, Wayne State University

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

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Genetics

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach
Zuzer Dhoondia 1, Ricci Tarockoff 1, Nadra Alhusini 1, Scott Medler 1, Neha Agarwal 1, Athar Ansari 1
1Department of Biological Sciences, Wayne State University

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

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Medicine

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis
Joseph A Roche 1, Morium Begam 1, Sujay S Galen 1
1Department of Health Care Sciences, Physical Therapy Program, College of Pharmacy and Health Sciences, Wayne State University

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

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Neuroscience

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction
Alexander Vasin 1, Maria Bykhovskaia 1,2
1Department of Neurology, School of Medicine, Wayne State University, 2Department of Anatomy and Cell Biology, School of Medicine, Wayne State University

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

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Biochemistry

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts
Ye Guan 1,2, Shiyuan Wang 1, Minjun Jin 2, Haotian Xu 3, Qiong Yang 1,4
1Department of Biophysics, University of Michigan, Ann Arbor, 2Department of Chemistry, University of Michigan, Ann Arbor, 3Department of Computer Science, Wayne State University, 4Department of Physics, University of Michigan, Ann Arbor

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

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Immunology and Infection

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy
Subodh Kumar Samrat 1, Haidong Gu 1
1Department of Biological Sciences, Wayne State University

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

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Biology

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint
Thomas Wilson 1, Navdeep Kaur 1, Jason Davis 2, Shabana Amanda Ali 1,3
1Bone and Joint Center, Henry Ford Health System, 2Department of Orthopedic Surgery, Henry Ford Health System, 3Center for Molecular Medicine and Genetics, Wayne State University

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

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Biology

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion
Tejeshwar C. Rao 1, Alexa L. Mattheyses 1
1Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham

TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell spreading.

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Neuroscience

Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage
Morium Begam 1, Neha Narayan 1, Drew Mankowski 1, Robert Camaj 1, Nicholas Murphy 1, Kevin Roseni 1, Marie E. Pepin 1, Jacob M. Blackmer 1, Takako I. Jones 2, Joseph A. Roche 1
1Physical Therapy Program, Department of Health Care Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 2Department of Pharmacology, University of Nevada, Reno School of Medicine

The present protocol describes a unique technique called dosage-adjusted resistance training (DART), which can be incorporated into precision rehabilitation studies performed in small animals, such as mice.

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Medicine

Mechanical Control of Relaxation Using Intact Cardiac Trabeculae
Melissa J. Bukowski 1, Benjamin Cavanaugh 2, Anita Abbo 1, Charles S. Chung 1
1Department of Physiology, Wayne State University, 2School of Medicine, Michigan State University-Macomb and Department of Physiology, Wayne State University

Rapid myocardial and cardiac relaxation is essential for normal physiology. Mechanical relaxation mechanisms are now known to be dependent on strain rate. This protocol provides an overview of the acquisition and analysis of experiments to further study the mechanical control of relaxation.

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Biochemistry

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation
Shreenidhi Rajkumar 1, Danielle Dixon 1, Andrew M. Lipchik 2, Joshua J. Gruber 1
1Departments of Internal Medicine and the Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 2Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

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Developmental Biology

Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo
Chenjun Shi 1, Chenchen Handler 2, Haden Florn 1, Jitao Zhang 1
1Department of Biomedical Engineering, College of Engineering, Wayne State University, 2Fischell Department of Bioengineering, University of Maryland

This protocol was developed to longitudinally monitor the mechanical properties of neural plate tissue during chick embryo neurulation. It is based on the integration of a Brillouin microscope and an on-stage incubation system, enabling live mechanical imaging of neural plate tissue in ex ovo cultured chick embryos.

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