University of Nebraska Medical Center View Institution's Website 24 articles published in JoVE Biochemistry Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies Piero R. Bianco1 1Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions. Medicine Creation of a High-Fidelity, Low-Cost, Intraosseous Line Placement Task Trainer via 3D Printing Nicholas W. Markin1, Nathan S. Goergen1, Priscila Rodrigues Armijo2, Alicia M. Schiller1 1Department of Anesthesiology, University of Nebraska Medical Center, 2Department of Surgery, University of Nebraska Medical Center We describe a procedure to process computed tomography (CT) scans into high-fidelity, reclaimable, and low-cost procedural task trainers. The CT scan identification processes, export, segmentation, modeling, and 3D printing are all described, along with the issues and lessons learned in the process. Medicine Use of a Percutaneous Ventricular Assist Device/Left Atrium to Femoral Artery Bypass System for Cardiogenic Shock Swethika Sundaravel1, Poonam Velagapudi1, Mamas Mamas2, Sandeep Nathan3, Alexander Truesdell4 1University of Nebraska Medical Center, 2Keele Cardiovascular Research Group, Keele University, 3University of Chicago Medicine, 4Virginia Heart/Inova Heart and Vascular Institute The following article describes the stepwise procedure for placement of a device (e.g., Tandemheart) in cardiogenic shock (CS) that is a percutaneous left ventricular assist device (pLVAD) and a left atrial to femoral artery bypass (LAFAB) system that bypasses and supports the left ventricle (LV) in CS. Neuroscience Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice Ragunathan Padmashri1, Kevin Tyner1, Anna Dunaevsky1 1Department of Neurological Sciences, University of Nebraska Medical Center Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice. Medicine The Intra-Aortic Balloon Pump Ganesh Gajanan1, Emmanouil S. Brilakis2, Jolanta M. Siller-Matula3,4,5, Ronald L. Zolty1, Poonam Velagapudi1 1Division of Cardiovascular Medicine, University of Nebraska Medical Center, 2Minneapolis Heart Institute, 3Department of Internal Medicine II, Division of Cardiology, Medical University of Vienna, 4Department of Experimental and Clinical Pharmacology, Medical University of Warsaw, Center for Preclinical Research and Technology CEPT, 5Department of Internal Medicine II, Division of Cardiology, Medical University of Vienna We describe the steps for the percutaneous implantation of the intra-aortic balloon pump (IABP), a mechanical circulatory support device. It acts by counterpulsation, inflating at the onset of diastole, augmenting diastolic aortic pressure and improving coronary blood flow and systemic perfusion, and deflating before systole, reducing left ventricular afterload. Neuroscience Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease Kelly L. Stauch1, Steven Totusek1, Trey Farmer1, Benjamin G. Lamberty1, Kelsey N. Dyball1, Mohannad A. Almikhlafi1, Howard S. Fox1 1Department of Neurological Sciences, University of Nebraska Medical Center Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters. Medicine A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice Elliott G. Richards*1, Jenna M. Rehmer*2, Melissa A. Mathes3, Emily L. Esakov1, Chad Braley1, Amy Joehlin-Price4, Andres Chiesa-Vottero4, Ofer Reizes1,5 1Cardiovascular and Metabolic Sciences, Lerner Research Institute; Women’s Health Institute, Cleveland Clinic, 2Women’s Health Institute, Cleveland Clinic, 3Department of Obstetrics and Gynecology, 983255 Nebraska Medical Center, University of Nebraska Medical Center, 4Anatomic Pathology, Cleveland Clinic, 5Gynecologic Oncology, Women’s Health Institute and Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Cleveland Clinic Many rodent models of endometriosis are limited by technical complexity, reproducibility, and/or need for immunocompromised animals or special reporter mice. We present a simplified system of lesion induction using any experimental mouse with an independently verifiable, objective scoring system and with no requirement for ovariectomy or survival surgery. Immunology and Infection Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System Kristi J. Warren1,3, Todd A. Wyatt2,3,4 1Department of Internal Medicine, University of Utah, 2Research Service, VA Nebraska Iowa Health Care System, 3Department of Internal Medicine, University of Nebraska Medical Center, 4Department of Environmental, Agricultural & Occupational Health, University of Nebraska Medical Center In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration. Bioengineering A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression Donghee Lee1, Alek Erickson2, Andrew T. Dudley1, Sangjin Ryu3,4 1Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, 2Department of Physiology and Pharmacology, Karolinska Institutet, 3Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, 4Nebraska Center for Materials and Nanoscience, University of Nebraska-Lincoln This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression. Immunology and Infection Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis Raghubendra Singh Dagur1, Weimin Wang1, Edward Makarov1, Yimin Sun2, Larisa Y. Poluektova1 1Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, 2Department of Pathology and Microbiology, University of Nebraska Medical Center This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients. Developmental Biology Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans Edward M. Germany1, Nataliya Zahayko1, Oleh Khalimonchuk1,2,3,4 1Department of Biochemistry, University of Nebraska, 2Nebraska Redox Biology Center, University of Nebraska, 3Nebraska Center for Integrated Biomolecular Communication, University of Nebraska, 4Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing. Biochemistry Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging Micah P. Stumme-Diers1, Tommy Stormberg1, Zhiqiang Sun1, Yuri L. Lyubchenko1 1Department of Pharmaceutical Sciences, University of Nebraska Medical Center Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale. Bioengineering Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System Arin L. Sutlief*1, Helena Valquier-Flynn1, Christina Wilson1, Marco Perez1, Hunter Kleinschmidt2, Brett J. Schofield2, Elizabeth Delmain3, Andrea E. Holmes1, Christopher D. Wentworth*4 1Department of Chemistry, Doane University, 2Department of Biology, Doane University, 3Department of Pathology and Microbiology, University of Nebraska Medical Center, 4Department of Physics and Engineering, Doane University Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties. Biology Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart Santosh K. Yadav1, Paras K. Mishra1,2 1Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 2Department of Anesthesiology, University of Nebraska Medical Center The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes. Genetics Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds Emily Keit1, Shixuan Chen1, Hongjun Wang1, Jingwei Xie1 1Department of Surgery-Transplant and Mary & Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers. Bioengineering Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires Madhur Upadhyay1, Raja Shah2, Sachin Agarwal3, Meenakshi Vishwanath4, Po-Jung Chen5, Takafumi Asaki6, Donald Peterson7 1Division of Orthodontics, University of Connecticut Health, 2Private Practice, 3Department of Orthodontics, University of Melbourne, 4Department of Orthodontics, University of Nebraska Medical Center, 5Department of Craniofacial Sciences, University of Connecticut Health, 6Biomedical Engineering, University of Hartford, 7Department of Mechanical Engineering, College of Engineering and Engineering Technology, Northern Illinois University The method presented here is designed to construct and validate an in vitro 3D model capable of measuring the force system generated by different archwires with V-bends placed between two brackets. Additional objectives are to compare this force system with different types of archwires and to previous models. Chemistry Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials Mehdi Bin Samad1, Krishnaiah Maddeboina1, Nathalia Rodrigues de Almeida1, Martin Conda-Sheridan1 1Department of Pharmaceutical Sciences, University of Nebraska Medical Center The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules. Biology Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology Christopher Thompson1, Katherine Keck2, Abigail Hielscher2 1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 2Department of Biomedical Sciences, GA-PCOM Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins. Genetics Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet β Cell Death Sarah A Tersey1, Jennifer B Nelson1, Marisa M Fisher2, Raghavendra G Mirmira1,3,4 1Department of Pediatrics, IU Center for Diabetes and Metabolic Disease, Indiana University School of Medicine, 2Department of Pediartics, Omaha Children's Hospital and Medical Center, University of Nebraska Medical Center, 3Departments of Biochemistry and Molecular Biology, Medicine, and Cellular and Integrative Physiology, IU Center for Diabetes and Metabolic Disease, Indiana University School of Medicine, 4Indiana Biosciences Research Institute Islet β cell death precedes development of type 1 diabetes, and detecting this process may allow for early therapeutic intervention. Here, we provide a detailed description of how to measure differentially methylated INS DNA species in human serum as a biomarker of β cell death. Bioengineering Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization Karl Khandalavala1, Jiang Jiang1, Franklin D. Shuler2, Jingwei Xie1 1Department of Pharmaceutical Sciences, Mary & Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center, 2Department of Orthopedic Surgery, Joan C. Edwards School of Medicine, Marshall University Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field. Neuroscience Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation Matthew J. Van Hook1, Wallace B. Thoreson1,2 1Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, 2Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells. Medicine A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS) Yana Yunusova1,2, Jordan R. Green3, Jun Wang3, Gary Pattee4, Lorne Zinman2,5 1Department of Speech-Language Pathology, University of Toronto, 2ALS/ MN Clinic, Sunnybrook Health Science Centre, 3Department of Special Education and Communication Disorders, University of Nebraska-Lincoln, 4Department of Neurology, Munroe-Meyer Institute, University of Nebraska Medical Center, 5Department of Neurology, University of Toronto Objective assessments of the physiological mechanisms that support speech are needed to monitor disease onset and progression in persons with ALS and to quantify treatment effects in clinical trials. In this video, we present a comprehensive, instrumentation-based protocol for quantifying speech motor performance in clinical populations. Bioengineering Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring Michael Boska1, Yutong Liu1, Mariano Uberti1, Balarininvasa R. Sajja1, Shantanu Balkundi2, JoEllyn McMillan2, Howard E. Gendelman2 1Department of Radiology, University of Nebraska Medical Center, 2Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications. Bioengineering Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs Shantanu Balkundi*1, Ari S. Nowacek*1, Upal Roy1, Andrea Martinez-Skinner1, JoEllyn McMillan1, Howard E. Gendelman1 1Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.