University of Alabama View Institution's Website 12 articles published in JoVE Biochemistry Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B Zekiye Ceren Arituluk1,2, Bishnu Adhikari1, Urmila Maitra1, Caroline Goodman1, Lukasz M. Ciesla1 1Department of Biological Sciences, The University of Alabama, 2Department of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained. Immunology and Infection Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells Leo M. Endres1, Sarah F. Hathcock2, Alexandra Schubert-Unkmeir1, Brandon J. Kim1,2 1Institute for Hygiene and Microbiology, University of Würzburg, 2Department of Biological Sciences, University of Alabama The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses. Environment Atom Probe Tomography Analysis of Exsolved Mineral Phases Kimberly Genareau1, Alberto Perez-Huerta1, Fernando Laiginhas1 1Department of Geological Sciences, University of Alabama Analysis of the morphology, composition, and spacing of exsolution lamellae can provide essential information to understand geological processes related to volcanism and metamorphism. We present a novel application of APT for the characterization of such lamellae and compare this approach to the conventional use of electron microscopy and FIB-based nanotomography. Genetics In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster Olivia Gevedon1, Harris Bolus1, Shu Hui Lye1, Keaton Schmitz1, Jesualdo Fuentes-González1, Kathryn Hatchell2, Lyndsey Bley2, Jason Pienaar1, Carin Loewen2, Stanislava Chtarbanova1 1Department of Biological Sciences, University of Alabama, 2Department of Genetics, University of Wisconsin-Madison We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection. Genetics The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster Kelsey E. Lowman1, Brélahn J. Wyatt1, Owen P. Cunneely1, Laura K. Reed1 1Department of Biological Sciences, University of Alabama The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies' innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health. Genetics Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens Saman Saeidi*1, Michael R. McKain*1,2, Elizabeth A. Kellogg1 1Donald Danforth Plant Science Center, 2Department of Biological Sciences, The University of Alabama This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA. Bioengineering Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM Paul G. Allison1, Rogie I. Rodriguez2, Robert D. Moser1, Brett A. Williams1, Aimee R. Poda3, Jennifer M. Seiter3, Brandon J. Lafferty3, Alan J. Kennedy3, Mei Q. Chandler1 1Geotechnical and Structures Laboratory, U.S. Army Engineer Research and Development Center, 2Department of Mechanical Engineering, University of Alabama, 3Environmental Laboratory, U.S. Army Engineer Research and Development Center This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials. Immunology and Infection Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response Rami R. Ajjuri1, Janis M. O'Donnell1 1Department of Biological Sciences, University of Alabama Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method. Biology Drosophila Pupal Abdomen Immunohistochemistry Wei Wang1, John H. Yoder1 1Department of Biological Sciences, University of Alabama Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen. Biology Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants Cody Locke1, Kalen Berry1, Bwarenaba Kautu1, Kyle Lee1, Kim Caldwell1, Guy Caldwell1 1Department of Biological Sciences, University of Alabama This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures. Biology Generation of Stable Transgenic C. elegans Using Microinjection Laura A. Berkowitz1, Adam L. Knight1, Guy A. Caldwell1, Kim A. Caldwell1 1Department of Biological Sciences, University of Alabama This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals. Biology Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes Laura A. Berkowitz1, Shusei Hamamichi1, Adam L. Knight1, Adam J. Harrington1, Guy A. Caldwell1, Kim A. Caldwell1 1Department of Biological Sciences, University of Alabama This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.