University of Colorado, Boulder View Institution's Website 23 articles published in JoVE Bioengineering 3D Printing Bacteria to Study Motility and Growth in Complex 3D Porous Media R. Kōnane Bay1,2, Anna M. Hancock2, Arabella S. Dill-Macky2, Hao Nghi Luu2, Sujit S. Datta2 1Department of Chemical and Biological Engineering, University of Colorado Boulder, 2Department of Chemical and Biological Engineering, Princeton University This protocol describes a procedure for three-dimensional (3D) printing of bacterial colonies to study their motility and growth in complex 3D porous hydrogel matrices that are more akin to their natural habitats than conventional liquid cultures or Petri dishes. Bioengineering Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting Jessica J. Senior*1, Richard J. A. Moakes*2, Megan E. Cooke3, Samuel R. Moxon4, Alan M. Smith1, Liam M. Grover2 1Department of Pharmacy, University of Huddersfield, 2School of Chemical Engineering, University of Birmingham, 3Chemical and Biological Engineering, University of Colorado Boulder, 4School of Biological Sciences, University of Manchester Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described. Biology Isolation and Characterization of the Murine Uterosacral Ligaments and Pelvic Floor Organs Catalina S. Bastías1, Lea M. Savard2, Kevin N. Eckstein2, Kathleen Connell3, Callan M. Luetkemeyer2,5, Virginia L. Ferguson1,2,4, Sarah Calve1,2,4 1Biomedical Engineering Program, University of Colorado Boulder, 2Paul M. Rady Department of Mechanical Engineering, University of Colorado Boulder, 3Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, 4BioFrontiers Institute, University of Colorado Boulder, 5Department of Mechanical Science and Engineering, University of Illinois Urbana-Champaign This article presents a detailed protocol for dissecting uterosacral ligaments and other pelvic floor tissues, including the cervix, rectum, and bladder in mice, to expand the study of female reproductive tissues. Bioengineering Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications Victoria G. Muir1, Margaret E. Prendergast1, Jason A. Burdick1,2,3 1Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, 2BioFrontiers Institute, University of Colorado Boulder, 3Department of Chemical and Biological Engineering, College of Engineering and Applied Science, University of Colorado Boulder This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications. Bioengineering Voxel Printing Anatomy: Design and Fabrication of Realistic, Presurgical Planning Models through Bitmap Printing Nicholas M. Jacobson1, Lawrence Smith2, Jane Brusilovsky1, Erik Carrera1, Hayden McClain1, Robert MacCurdy2 1Inworks Innovation Initiative, University of Colorado-Anschutz, 2Department of Mechanical Engineering, University of Colorado-Boulder This method demonstrates a voxel-based 3D printing workflow, which prints directly from medical images with exact spatial fidelity and spatial/contrast resolution. This enables the precise, graduated control of material distributions through morphologically complex, graduated materials correlated to radiodensity without loss or alteration of data. Biochemistry Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling Steven E. Guard1, Christopher C. Ebmeier1, William M. Old1,2 1Department of Molecular, Cellular and Developmental Biology, University of Colorado, 2Linda Crnic Institute for Down Syndrome, University of Colorado School of Medicine Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline. Genetics Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins Ravinder Singh1 1Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins. Genetics A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates Ravinder Singh1 1Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA. Environment Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas Xu Wang1,2, Joseph Schwan1,2, Noah Hood1,2, Hsiang-Wen Hsu1,2, Eberhard Grün1,2, Mihály Horányi1,2 1Laboratory for Atmospheric and Space Physics, University of Colorado, 2 Dust charging and mobilization is demonstrated in three experiments with exposure to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. These experiments present the advanced understanding of electrostatic dust transport and its role in shaping the surfaces of airless planetary bodies. Behavior Behavioral Assessment of Hearing in 2 to 4 Year-old Children: A Two-interval, Observer-based Procedure Using Conditioned Play-based Responses Angela Yarnell Bonino1,2, Lori J. Leibold3 1Department of Allied Health Sciences, The University of North Carolina at Chapel Hill, 2Department of Speech, Language, and Hearing Sciences, University of Colorado Boulder, 3Center for Hearing Research, Boys Town National Research Hospital This paper describes a procedure for measuring hearing sensitivity in 2 to 4 year-old children. Children are trained to perform play-based responses when they hear a target signal. Thresholds are then estimated in a two-interval, two-alternative forced-choice paradigm, based on observations of the child's behaviors. Medicine Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics Russell T. Burke1, James D. Orth1 1Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics. Chemistry Atom Transfer Radical Polymerization of Functionalized Vinyl Monomers Using Perylene as a Visible Light Photocatalyst Jordan C. Theriot1, Matthew D. Ryan1, Tracy A. French1, Ryan M. Pearson1, Garret M. Miyake1 1Department of Chemistry and Biochemistry, University of Colorado Boulder A method for the atom transfer radical polymerization of functionalized vinyl monomers using perylene as a visible-light photocatalyst is described. Bioengineering In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling Winston Elliott1, Devon Scott-Drechsel2, Wei Tan1 1Department of Mechanical Engineering, University of Colorado at Boulder, 2Product Development, Covidien, Ltd. This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture. Biology Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing Jerome E. Lee1, Rui Yi1,2 1Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, 2Linda Crnic Institute for Down Syndrome, University of Colorado, Denver MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs. Medicine Assessment of Vascular Function in Patients With Chronic Kidney Disease Kristen L. Jablonski1, Emily Decker1, Loni Perrenoud1, Jessica Kendrick1, Michel Chonchol1, Douglas R. Seals2, Diana Jalal1 1Division of Renal Diseases and Hypertension, University of Colorado, Denver, 2Department of Integrative Physiology, University of Colorado, Boulder The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression. Medicine Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion Marshall T. Bell1, T. Brett Reece1, Phillip D. Smith1, Joshua Mares1, Michael J. Weyant1, Joseph C. Cleveland Jr.1, Kirsten A. Freeman1, David A. Fullerton1, Ferenc Puskas2 1Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado, 2Department of Anesthesiology, University of Colorado The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model. Behavior Handwriting Analysis Indicates Spontaneous Dyskinesias in Neuroleptic Naïve Adolescents at High Risk for Psychosis Derek J. Dean1, Hans-Leo Teulings2, Michael Caligiuri3, Vijay A. Mittal1 1Department of Psychology and Neuroscience, Center for Neuroscience, University of Colorado Boulder, 2NeuroScript LLC, 3Department of Psychiatry, University of California, San Diego Handwriting analysis software significantly improves upon existing instruments measuring movement disorders. Individuals at risk for psychosis and healthy controls completed handwriting tasks to test for dyskinesia. Results suggest that youth at risk for psychosis exhibit dyskinesia and that handwriting analysis could significantly contribute to wider dissemination of early identification efforts Neuroscience A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo Shinya Nakamura*1,2, Michael V. Baratta*1,2, Donald C. Cooper1,2 1Institute for Behavioral Genetics, University of Colorado Boulder, 2Department of Psychology and Neuroscience, University of Colorado Boulder The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons. Bioengineering Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles Leslie A. Morton*1,2, Jonel P. Saludes*1,2, Hang Yin1,2 1Department of Chemistry & Biochemistry, University of Colorado Boulder, 2Biofrontiers Institute, University of Colorado Boulder This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein. Medicine Use of a Hanging-weight System for Isolated Renal Artery Occlusion Almut Grenz1, Julee H. Hong1, Alexander Badulak1, Douglas Ridyard1, Timothy Luebbert2, Jae-Hwan Kim3, Holger K. Eltzschig1 1Department of Anesthesiology, University of Colorado, 2School of Medicine, University of Colorado, 3Department of Anesthesiology, Korea University College of Medicine A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion. Biology Transmembrane Domain Oligomerization Propensity determined by ToxR Assay Catherine Joce1, Alyssa Wiener1, Hang Yin1 1Department of Chemistry and Biochemistry, University of Colorado at Boulder An efficient procedure to assess the oligomerization propensity of single-pass transmembrane domains (TMDs) is described. Chimeric proteins consisting of the TMD fused to ToxR are expressed in an E. coli reporter strain. TMD-induced oligomerization causes dimerization of ToxR, activation of transcription and production of the reporter protein, -galactosidase. Medicine Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice Michael Koeppen1, Tobias Eckle1, Holger K. Eltzschig1 1Department of Anesthesiology, University of Colorado A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung. Neuroscience Assaying β-amyloid Toxicity using a Transgenic C. elegans Model Vishantie Dostal1, Christopher D. Link1,2 1Institute for Behavioral Genetics, University of Colorado, 2Integrative Physiology, University of Colorado The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human β-amyloid peptide (Aβ). Induced expression of Aβ in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate Aβ toxicity.
