Louisiana State University View Institution's Website 32 articles published in JoVE Biology Evaluation of Photosynthetic Efficiency in Photorespiratory Mutants by Chlorophyll Fluorescence Analysis Joseph Qian1, Nicholas Ferrari1, Richard Garcia1, Mary Beth L. Rollins2, Paul F. South1,2 1Department of Biological Sciences, Louisiana State University, 2Department of Plant Pathology and Crop Physiology, Louisiana State University AgCenter We describe an approach to measure changes in photosynthetic efficiency in plants after treatment with low CO2 using chlorophyll fluorescence. Cancer Research Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System Loren M. Brown1, Katherine L. Hebert2, Rakesh R. Gurrala3, C. Ethan Byrne4, Matthew Burow5, Elizabeth C. Martin4, Frank H. Lau1 1Department of Surgery, Louisiana State University Health Sciences Center, 2Department of Bioinnovation, Tulane University, 3Tulane University School of Medicine, 4Department of Biological and Agricultural Engineering, Louisiana State University, 5Department of Medicine, Tulane University School of Medicine This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials. Neuroscience Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus Amita Shrestha1, Philip A. Adeniyi1, Olalekan M. Ogundele1 1Department of Comparative Biomedical Sciences, Louisiana State University School of Veterinary Medicine The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo. Biochemistry Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR Alex Kirui*1, Malitha C. Dickwella Widanage*1, Frederic Mentink-Vigier2, Ping Wang3, Xue Kang1, Tuo Wang1 1Department of Chemistry, Louisiana State University, 2National High Magnetic Field Laboratory, 3Departments of Pediatrics, and Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented. Genetics Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula Guannan Wang1, Pramod Pantha1, Kieu-Nga Tran1, Dong-Ha Oh1, Maheshi Dassanayake1 1Department of Biological Sciences, Louisiana State University Agrobacterium-mediated transformation using a floral-dip method can be successfully employed to create stable transgenic lines of the extremophyte model Schrenkiella parvula. We present a protocol modified from that for Arabidopsis thaliana, considering different growth habits and physiological characteristics of the extremophyte. Neuroscience Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster Daniel R. Swale1, Aaron D. Gross2, Quentin R. R. Coquerel3, Jeffrey R. Bloomquist3 1Department of Entomology, Louisiana State University AgCenter, 2Department of Entomology, Virginia Tech, 3Department of Entomology and Nematology, Emerging Pathogens Institute, University of Florida This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways. Environment Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens Cai Wang1, Huifang Wang1, Tao Ma1,2, Qiang Xiao3, Panrong Cao4, Xuan Chen5, Hongpeng Xiong1, Wenquan Qin1, Zhaohui Sun1, Xiujun Wen1 1Guangdong Key Laboratory for Innovation Development and Utilization of Forest Plant Germplasm, College of Forestry and Landscape Architecture, South China Agricultural University, 2College of Agriculture, South China Agricultural University, 3Tea Research Institute, Chinese Academy of Agricultural Sciences, 4College of Horticulture, South China Agricultural University, 5College of Coast and Environment, Louisiana State University Here, we present a protocol to investigate the pupation preference of mature larvae of Ectropis grisescens in response to soil factors (e.g., substrate type and moisture content) using choice bioassays. We also present a protocol of no-choice bioassays to determine the factors that affect the pupation behaviors and survivorship of E. grisescens. Chemistry Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies Alexandra Noël1, Christina M. Verret1, Farhana Hasan2, Slawomir Lomnicki2, John Morse3, Annette Robichaud3, Arthur L. Penn1 1Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, 2Department of Environmental Sciences, College of the Coast & Environment, Louisiana State University, 3SCIREQ Scientific Respiratory Equipment Inc. Electronic cigarette (e-cig) users are increasing worldwide. Little, however, is known about the health effects induced by inhaled e-cig aerosols. This article describes an e-cig aerosol generation technique suitable for animal exposures and subsequent toxicological studies. Such protocols are required to establish experimentally reproducible and standardized e-cig exposure systems. Environment Sampling, Sorting, and Characterizing Microplastics in Aquatic Environments with High Suspended Sediment Loads and Large Floating Debris Katherine M. Martin1, Elizabeth A. Hasenmueller2, John R. White3, Lisa G. Chambers4, Jeremy L. Conkle1 1Department of Physical and Environmental Sciences, Texas A&M University-Corpus Christi, 2Department of Earth and Atmospheric Sciences, Saint Louis University, 3Department of Oceanography and Coastal Sciences, Louisiana State University, 4Department of Biology, University of Central Florida Most microplastic research to date has occurred in marine systems where suspended solid levels are relatively low. Focus is now shifting to freshwater systems, which may feature high sediment loads and floating debris. This protocol addresses collecting and analyzing microplastic samples from aquatic environments that contain high suspended solid loads. Bioengineering Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1 Merrel T. Holley*1, Neerajha Nagarajan*2, Christian Danielson1, Pinar Zorlutuna*2, Kidong Park*1 1Division of Electrical and Computer Engineering, Louisiana State University, 2Department of Aerospace and Mechanical Engineering, Bioengineering Graduate Program, University of Notre Dame In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot. Genetics QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii Bang Shen1, Robin H. Powell2, Michael S. Behnke2 1State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, 2Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene. Bioengineering Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2 Neerajha Nagarajan*1, Merrel T. Holley*2, Christian Danielson2, Kidong Park*2, Pinar Zorlutuna*1 1Department of Aerospace and Mechanical Engineering, Bioengineering Graduate Program, University of Notre Dame, 2Division of Electrical and Computer Engineering, Louisiana State University In this study, a biological actuator and a self-stabilizing, swimming biorobot with functionalized elastomeric cantilever arms are seeded with cardiomyocytes, cultured, and characterized for their biochemical and biomechanical properties over time. Bioengineering Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2 Aurel Radulescu1, Noemi Kinga Szekely1, Marie-Sousai Appavou1, Vitaliy Pipich1, Thomas Kohnke1, Vladimir Ossovyi1, Simon Staringer1, Gerald J. Schneider2, Matthias Amann3, Bo Zhang-Haagen3, Georg Brandl1, Matthias Drochner4, Ralf Engels4, Romuald Hanslik5, Günter Kemmerling1 1Jülich Centre for Neutron Science Outstation at MLZ, Forschungszentrum Jülich GmbH, 2Department of Chemistry, Louisiana State University, 3Jülich Centre for Neutron Science JCNS-1 & Institute of Complex Systems ICS-1, Forschungszentrum Jülich GmbH, 4Central Institute of Engineering, Electronics and Analytics — Electronic Systems (ZEA-2), Forschungszentrum Jülich GmbH, 5Central Institute of Engineering, Electronics and Analytics — Engineering and Technology (ZEA-1), Forschungszentrum Jülich GmbH Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to µm that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution. Environment Assessment of Labile Organic Carbon in Soil Using Sequential Fumigation Incubation Procedures Michael A. Blazier1, Hal O. Liechty2 1Hill Farm Research Station, Louisiana State University Agricultural Center, 2School of Forestry and Natural Resources, University of Arkansas at Monticello Labile organic carbon (LOC) and the potential carbon turnover rate are sensitive indicators of changes in soil nutrient cycling processes. Details are provided for a method based on fumigating and incubating soil in a series of cycles and using the CO2 accumulated during the incubation periods to estimate these parameters. Medicine Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice Shantel A. Vital1, Felicity N. E. Gavins1 1Department of Molecular & Cellular Physiology, Health Sciences Center Shreveport, Louisiana State University In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion. Behavior A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration Vinita Batra1, Glenn F. Guerin2, Nicholas E. Goeders2, Jessica A. Wilden1,2 1Department of Neurosurgery, Louisiana State University, 2Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence. Engineering Mechanical Expansion of Steel Tubing as a Solution to Leaky Wellbores Mileva Radonjic1, Darko Kupresan1 1Craft and Hawkins Department of Petroleum Engineering, Louisiana State University This article reports on a laboratory scale investigation of an existing field procedure and its adaptation for sealing of leaky wellbores. It consists of mechanical expansion of metal pipe, which results in an improved metal/cement bond, ultimate sealing of hydraulic pathways and prevention of gas leaks caused by the presence of a microannular channel. Biology Chromosome Preparation From Cultured Cells Bradley Howe1, Ayesha Umrigar1, Fern Tsien1 1Department of Genetics, Louisiana State University Health Science Center Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY). Neuroscience Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits Charles C. Lee1, Ying-Wan Lam2, Kazuo Imaizumi1, S. Murray Sherman2 1Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, 2Department of Neurobiology, University of Chicago We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations in vitro. Chemistry Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins Kevin J. Roberson1, Pamlea N. Brady1, Michelle M. Sweeney1, Megan A. Macnaughtan1 1Department of Chemistry, Louisiana State University Two methods for assigning the α- and ε-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine. Bioengineering Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies Katla Sai Krishna1,2, Sanchita Biswas1,2, Chelliah V. Navin1,2,3, Dawit G. Yamane1, Jeffrey T. Miller4, Challa S.S.R. Kumar1,2 1Center for Advanced Microstructures and Devices (CAMD), Louisiana State University, 2Center for Atomic-Level Catalyst Design, Cain Department of Chemical Engineering, Louisiana State University, 3Department of Biological and Agricultural Engineering, Louisiana State University, 4Argonne National Laboratory Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis. Immunology and Infection A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay Maciej T. Nogalski1,2, Gary C.T. Chan3, Emily V. Stevenson1,2, Donna K. Collins-McMillen1,2, Andrew D. Yurochko1,2,4 1Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, 2Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, 3Department of Microbiology and Immunology, SUNY Upstate Medical University, 4Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips. Neuroscience Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area Michael F. Salvatore1, Brandon S. Pruett1, Charles Dempsey1, Victoria Fields1 1Department of Pharmacology, Toxicology, & Neuroscience, Louisiana State University Health Sciences Center Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents. Neuroscience Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol Marco Pacifici1,2, Francesca Peruzzi1,2 1LSU Health Sciences Center - New Orleans, 2Medical School and Stanley S. Scott Cancer Center We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required. Biology Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy Prachi Jain1, Rebecca A. Worthylake2, Suresh K. Alahari1,3 1Department of Biochemistry and Molecular Biology, LSU School of Medicine, 2Department of Oral Biology, LSU School of Dentistry, 3Stanley S. Scott Cancer Center, LSU School of Medicine This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously. Neuroscience Methods to Assay Drosophila Behavior Charles D. Nichols1, Jaime Becnel1, Udai B. Pandey2 1Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 2Department of Genetics, Louisiana State University Health Sciences Center Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior. Immunology and Infection Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics Flavia M. Souza-Smith1, Kristine M. Kurtz1, Jerome W. Breslin1 1Department of Physiology, School of Medicine, Louisiana State University Health Sciences Center We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Immunology and Infection Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis Junko Yano1, Paul L. Fidel, Jr.1 1Louisiana State University Health Sciences Center Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract. Immunology and Infection Isolation of Mouse Lung Dendritic Cells Wallissa Lancelin1, Antonieta Guerrero-Plata1 1Pathobiological Sciences, Louisiana State University A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset. Biology Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin Travis M. Doggett1, Jerome W. Breslin1 1Department of Physiology, Louisiana State University Health Sciences Center Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli. Biology Bioassays for Monitoring Insecticide Resistance Audra L.E. Miller1, Kelly Tindall1, B. Rogers Leonard2 1Division of Plant Sciences, University of Missouri, Delta Research Center, 2Department of Entomology, Louisiana State University Agricultural Center This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests. Biology Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut) Claudia Husseneder*1, Amit Sethi*1, Lane Foil1, Jennifer Delatte1 1Department of Entomology, Louisiana State University Agricultural Center We present procedures for demonstrating that ligands bind to the surface membrane of the cellulose-digesting protozoa in the gut of Formosan subterranean termites using fluorescent microscopy and that ligands coupled with lytic peptides kill these protozoa in vitro (anaerobic protozoa culture) and in vivo (injection into the termite hindgut).