Texas A&M University View Institution's Website 57 articles published in JoVE Biology Manipulation of Rhythmic Food Intake in Mice Using a Custom-Made Feeding System Aishwarya Sahasrabudhe1,2, Chanté R. Guy1,2, Ben J. Greenwell1,2,3, Jerome S. Menet1,2,3 1Department of Biology, Texas A&M University, 2Center for Biological Clocks Research, Texas A&M University, 3Interdisciplinary Program of Genetics, Texas A&M University Restricting the timing of food intake has emerged as a promising intervention to attenuate diet-induced metabolic diseases. This manuscript details the construction and use of an efficient system built in-house for measuring and manipulating rhythmic food intake in mice. Biochemistry A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors Caixing Xiong1, Dwight Baker2, Patricia Pietrantonio1 1Department of Entomology, Texas A&M University, 2Department of Biochemistry and Biophysics, Texas A&M University In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay. Biology Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles Brenda Leal-Galvan1, Cristina Harvey1, Donald Thomas2, Perot Saelao3, Adela S. Oliva Chavez1 1Department of Entomology, Texas A&M University, 2USDA-ARS Cattle Fever Tick Research Laboratory, 3USDA-ARS Veterinary Pest Research Unit The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced. Genetics Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti Bianca B. Kojin1, Hitoshi Tsujimoto1, Emma Jakes1, Sarah O'Leary1, Zach N. Adelman1 1Department of Entomology and Agrilife Research, Texas A&M University This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis. Biology Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates) Hitoshi Tsujimoto1, Zach N. Adelman1 1Department of Entomology, Texas A&M University Described is a time and space-saving method to count eggs and determine hatch rates of individual mosquitoes using 24 well tissue culture plates, which can substantially increase the scale and speed of fecundity and fertility assays. Biochemistry Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli Chesley Marie Rowlett1, Wenshe Ray Liu1 1Department of Chemistry, Texas A&M University Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro. Biology Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform Shreedevi Arun Kumar1, Jihui Lee1, Corey J. Bishop1 1Department of Biomedical Engineering, Texas A&M University This article describes the protocols used to produce a novel vaccine delivery platform, "polybubbles," to enable delayed burst release. Polyesters including poly(lactic-co-glycolic acid) and polycaprolactone were used to form the polybubbles and small molecules and antigen were used as cargo. Biochemistry Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH Ragini M. Mistry1,2, Pankaj K. Singh1,3, Maureen G. Mancini1,2, Fabio Stossi1,2, Michael A. Mancini1,2,3,4 1GCC Center for Advanced Microscopy and Image Informatics, 2Department of Molecular and Cellular Biology, Baylor College of Medicine, 3Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, 4Department of Pharmacology and Chemical Biology, Baylor College of Medicine Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs. Environment Potato Virus X-Based microRNA Silencing (VbMS) In Potato. Jinping Zhao1, Carlos Garcia Rios1, Junqi Song1,2 1Texas A&M AgriLife Research Center at Dallas, Texas A&M University System, 2Department of Plant Pathology, Microbiology, Texas A&M University We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family. Behavior The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle Courtney Lynd Daigle1, Amanda J. Hubbard1, Temple Grandin2 1Animal Behavior & Welfare Laboratory, Department of Animal Science, Texas A&M University, 2Department of Animal Sciences, Colorado State University Here, we present a protocol to conduct a variety of behavioral tests in cattle that have been designed to evaluate emotions. A battery of behavioral tests (open field test, startle test, bovine zero maze, exit velocity, pen score, and chute score) were conducted to evaluate different components of animal temperament. Behavior Using Cholesky Decomposition to Explore Individual Differences in Longitudinal Relations between Reading Skills Florina Erbeli1, Aaron R. Campbell1, Sara A. Hart2 1Department of Educational Psychology, Texas A&M University, 2Department of Psychology and Florida Center for Reading Research, Florida State University This paper demonstrates use of the gold standard method in behavioral genetics, the Cholesky decomposition method, to estimate unique, overlapping genetic and environmental influences on different variables to answer longitudinally motivated research questions. Chemistry Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides Enas N. Yousef1, Ramakrishna Sesham1, Jacob W. McCabe1, Rajpal Vangala1, Laurence A. Angel1 1Department of Chemistry, Texas A&M University-Commerce Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance. Biology A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice Daniel Villarreal1, Geetali Pradhan2, Chia-Shan Wu1,2, Clinton D Allred1, Shaodong Guo1, Yuxiang Sun1,2 1Department of Nutrition and Food Science, Texas A&M University, 2Children's Nutrition Research Center, Baylor College of Medicine This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250–350 high quality and fully functional islets per mouse. Developmental Biology Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification Lindsay A. Dawson1, Regina Brunauer1, Katherine N. Zimmel1, Osama Qureshi1, Alyssa R. Falck2, Patrick Kim3, Connor P. Dolan1, Ling Yu1, Yu-Lieh Lin1, Benjamin Daniel1, Mingquan Yan1, Ken Muneoka1 1Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 2Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 3Department of Neurosurgery, University of Mississippi Medical Center Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography. Developmental Biology Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo Ishita Chandel1, Ryan Baker1, Naosuke Nakamura1, Vlad Panin1 1Department of Biochemistry and Biophysics, Texas A&M University Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner. Engineering Femtosecond Laser Filaments for Use in Sub-Diffraction-Limited Imaging and Remote Sensing Matthews M. Springer1, Benjamin D. Strycker1,2, Kai Wang1, Alexei V. Sokolov1,2, Marlan O. Scully1,2 1Texas A&M University, 2Baylor University High-intensity femtosecond pulses of laser light can undergo cycles of Kerr self-focusing and plasma defocusing, propagating an intense sub-millimeter-diameter beam over long distances. We describe a technique for generating and using these filaments to perform remote imaging and sensing beyond the classical diffraction limits of linear optics. Bioengineering Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation Doris A. Taylor1, Luiz C. Sampaio1, Rafael Cabello1, Abdelmotagaly Elgalad1, Rohan Parikh1, R Patrick Wood2, Kevin A. Myer2, Alvin T. Yeh3, Po-Feng Lee1 1Regenerative Medicine Research, Texas Heart Institute, 2Lifegift Organ Donation Center, 3Biomedical Engineering, Texas A&M University This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow. Environment Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar Carrie A. Deans1,2, Gregory A. Sword1, Paul A. Lenhart3, Eric Burkness2, William D. Hutchison2, Spencer T. Behmer1 1Department of Entomology, Texas A&M University, 2Department of Entomology, University of Minnesota, 3Department of Entomology, University of Kentucky The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology. 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. Immunology and Infection A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals Jingshu Chen*2, Lei Zhong*1, Jing Wu1, Sui Ke2, Benjamin Morpurgo3, Andrei Golovko3, Nengtai Ouyang4, Yuxiang Sun2, Shaodong Guo2, Yanan Tian1,2 1Hunan Engineering Technology Research Center of Featured Aquatic Resources Utilization, Hunan Agriculture University, 2Texas A&M University, 3Texas A&M Institute for Genomic Medicine, 4Sun Yat-Sen Memorial Hospital Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals. Engineering Three-electrode Coin Cell Preparation and Electrodeposition Analytics for Lithium-ion Batteries Robert D. Minter*1, Daniel Juarez-Robles*2, Conner Fear2, Yevgen Barsukov3, Partha P. Mukherjee2 1Department of Mechanical Engineering, Texas A&M University, 2School of Mechanical Engineering, Purdue University, 3Battery Management Systems, Texas Instruments Inc. Three-electrode cells are useful in studying the electrochemistry of lithium-ion batteries. Such an electrochemical setup allows the phenomena associated with the cathode and anode to be decoupled and examined independently. Here, we present a guide for construction and use of a three-electrode coin cell with emphasis on lithium plating analytics. Bioengineering Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure Madeleine S. Durkee1, Landon D. Nash1, Fatemeh Nooshabadi1, Jeffrey D. Cirillo2, Duncan J. Maitland1, Kristen C. Maitland1 1Department of Biomedical Engineering, Texas A&M University, 2Deparment of Molecular Pathogenesis and Immunology, Texas A&M College of Medicine Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure. Chemistry An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling Minjung Lee1, Yubin Zhou2, Yun Huang1 1Centre for Epigenetics and Disease Prevention, Department of Molecular and Cellular Medicine, Institute of Biosciences and Technology, College of Medicine, Texas A&M University, 2Centre for Translational Cancer Research, Department of Medical Physiology, Institute of Biosciences and Technology, College of Medicine, Texas A&M University Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented. Biology Biophysical Characterization of Flagellar Motor Functions Katie M. Ford1, Ravi Chawla1, Pushkar P. Lele1 1Artie McFerrin Department of Chemical Engineering, Texas A&M University Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors. Developmental Biology Co-localization of Cell Lineage Markers and the Tomato Signal Yan Jing1, Robert J. Hinton1, Kevin S. Chan1, Jian Q. Feng1 1Department of Biomedical Sciences, Texas A&M University College of Dentistry We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells. Engineering Electrospray Deposition of Uniform Thickness Ge23Sb7S70 and As40S60 Chalcogenide Glass Films Spencer Novak1, Pao-Tai Lin2,3, Cheng Li4, Nikolay Borodinov1, Zhaohong Han5, Corentin Monmeyran5, Neil Patel5, Qingyang Du5, Marcin Malinowski4, Sasan Fathpour4, Chatdanai Lumdee4, Chi Xu4, Pieter G. Kik4, Weiwei Deng6, Juejun Hu7, Anuradha Agarwal7, Igor Luzinov1, Kathleen Richardson4 1Department of Materials Science and Engineering, Clemson University, 2Department of Materials Science and Engineering, Texas A&M University, 3Department of Electrical and Computer Engineering, Texas A&M University, 4College of Optics and Photonics, Center for Research and Education in Optics and Lasers (CREOL), University of Central Florida, 5Department of Materials Science and Engineering, Massachusetts Institute of Technology, 6Department of Mechanical Engineering, Virginia Polytechnic Institute, 7Microphotonics Center, Massachusetts Institute of Technology A method of uniform thickness solution-derived chalcogenide glass film deposition is demonstrated using computer numerical controlled motion of a single-nozzle electrospray. Environment Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies Kuo-Tung Jiann*1, Liang-Saw Wen*2,3, Peter H. Santschi4 1Department of Oceanography, National Sun Yat-sen University, 2Institute of Oceanography, National Taiwan University, 3Taiwan Ocean Research Institute, National Research Laboratories, 4Department of Oceanography and Marine Sciences, Texas A&M University at Galveston Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented. Immunology and Infection Detecting Cortex Fragments During Bacterial Spore Germination Michael B. Francis1, Joseph A. Sorg1 1Department of Biology, Texas A&M University Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination. Chemistry Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals Yi-Hsien Yu1, Xuezhen Wang2,3, Abhijeet Shinde2, Zhengdong Cheng1,2,3,4 1Department of Materials Science and Engineering, Texas A&M University, 2Artie McFerrin Department of Chemical Engineering, Texas A&M University, 3Mary Kay O'Connor Process Safety Center, Texas A&M University, 4Professional Program in Biotechnology, Texas A&M University A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers. Engineering Non-aqueous Electrode Processing and Construction of Lithium-ion Coin Cells Malcolm Stein IV1, Chien-Fan Chen1, Daniel J. Robles1, Christopher Rhodes2, Partha P. Mukherjee1 1Department of Mechanical Engineering, Texas A&M University, 2Department of Chemistry and Biochemistry, Texas State University Non-aqueous electrode processing is central to the construction of coin cells and the evaluation of new electrode chemistries for lithium-ion batteries. A step-by-step guide to the basic practices needed as an electrochemical engineer working with batteries in an academic experimental setting is furnished. Bioengineering Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold Lindsay N. Nail1, Dawei Zhang2,3, Jessica L. Reinhard1, Melissa A. Grunlan1,2 1Department of Biomedical Engineering, Texas A&M University, 2Department of Material Science and Engineering, Texas A&M University, 3Institute of Advanced Materials and Technology, University of Science & Technology Beijing Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating. Developmental Biology Method of Studying Palatal Fusion using Static Organ Culture Isra Ibrahim1, Maria J. Serrano1, Kathy K.H. Svoboda1 1Department of Biomedical Sciences and Center for Craniofacial Research and Diagnosis, Texas A&M University Baylor College of Dentistry Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion. Bioengineering Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT Kristina Najjar1, Alfredo Erazo-Oliveras1, Jean-Philippe Pellois1 1Biochemistry and Biophysics, Texas A&M University We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky. Bioengineering Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars Mahtab Hassanpourfard1, Xiaohui Sun2, Amin Valiei1, Partha Mukherjee3, Thomas Thundat1, Yang Liu2, Aloke Kumar4 1Department of Chemical and Material Engineering, University of Alberta, 2Department of Civil and Environmental Engineering, University of Alberta, 3Department of Mechanical Engineering, Texas A&M University, 4Department of Mechanical Engineering, University of Alberta Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated. Immunology and Infection High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages Jing Wu1, Roberta Pugh1, Richard C. Laughlin1, Helene Andrews-Polymenis2, Michael McClelland3, Andreas J. Bäumler4, L. Garry Adams1 1Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 2Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M University System Health Science Center, 3University of California, Irvine, 4Department of Medical Microbiology & Immunology, School of Medicine, University of California, Davis A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions. Environment Design and Construction of an Urban Runoff Research Facility Benjamin G. Wherley1, Richard H. White1, Kevin J. McInnes1, Charles H. Fontanier1, James C. Thomas1, Jacqueline A. Aitkenhead-Peterson1, Steven T. Kelly2 1Soil and Crop Sciences Department, Texas A&M University, 2The Scotts Miracle-Gro Company This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns. Bioengineering Synthesis of an Intein-mediated Artificial Protein Hydrogel Miguel A. Ramirez1, Zhilei Chen1,2 1Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, 2Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, Texas A&M University, College Station We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins. Behavior Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents Phillip R. Kramer1, Larry L. Bellinger1 1Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry A lengthening in meal duration represents orofacial nociceptive behavior in rodents similar to the guarding behavior of humans with orofacial pain. Eating is a behavior that requires no training or animal manipulation, requires cortical participation, and is not competing with other experimentally induced behaviors, distinguishing this assay from alternative reflex or operant measurements. Immunology and Infection Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages Wei Ying1, Patali S. Cheruku2, Fuller W. Bazer1, Stephen H. Safe3, Beiyan Zhou3 1Department of Animal Science, Texas A&M University, 2Department of Biology, Texas A&M University, 3Department of Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways. Immunology and Infection Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays Shawn Christensen*1, Eli Borrego*1, Won-Bo Shim1, Tom Isakeit1, Michael Kolomiets1 1Plant Pathology and Microbiology, Texas A&M University The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays. Bioengineering Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy Yoonsuck Choe1, David Mayerich2, Jaerock Kwon3, Daniel E. Miller1, Chul Sung1, Ji Ryang Chung1, Todd Huffman4, John Keyser1, Louise C. Abbott5 1Department of Computer Science and Engineering, Texas A&M University, 2Beckman Institute for Advanced Science and Technology, University of Illinois, 3Department of Electrical and Computer Engineering, Kettering University, 43Scan, 5Department of Veterinary Integrative Biosciences, Texas A&M University The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species. Biology Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton Xiquan Gao1, Robert C. Britt Jr.1, Libo Shan2, Ping He1 1Department of Biochemistry and Biophysics, Institute of Plant Genomics and Biotechnology, Texas A&M University, 2Department of Plant Pathology and Microbiology, Institute of Plant Genomics and Biotechnology, Texas A&M University We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation. Biology A β-glucuronidase (GUS) Based Cell Death Assay Mehdi Kabbage1, Maria Ek-Ramos1, Martin Dickman1 1Department of Plant Pathology and Microbiology, Institute for Plant Genomics and Biotechnology, Texas A&M University Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes. Immunology and Infection A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors Hsiao-Ling Lu1, Cymon N. Kersch1, Suparna Taneja-Bageshwar1,2, Patricia V. Pietrantonio1 1Department of Entomology, Texas A&M University (TAMU), 2Department of Molecular and Cellular Medicine, Texas A&M University (TAMU) This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery. Biology Rapid PCR Thermocycling using Microscale Thermal Convection Radha Muddu1, Yassin A. Hassan2, Victor M. Ugaz3 1Department of Mechanical Engineering, Texas A&M University, 2Department of Mechanical Engineering and Department of Nuclear Engineering, Texas A&M University, 3Department of Chemical Engineering, Texas A&M University We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible. Immunology and Infection Using Luciferase to Image Bacterial Infections in Mice Mi Hee Chang1, Suat L.G. Cirillo1, Jeffrey D. Cirillo1 1Microbial & Molecular Pathogenesis, Texas A&M Health Science Center Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging. Neuroscience Automated Interactive Video Playback for Studies of Animal Communication Trisha Butkowski1, Wei Yan1, Aaron M. Gray2, Rongfeng Cui2, Machteld N. Verzijden2, Gil G. Rosenthal2 1Department of Visualization, Texas A&M University (TAMU), 2Department of Biology, Texas A&M University (TAMU) Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior. Biology Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy Andreea Trache1,2, Soon-Mi Lim1 1Department of Systems Biology and Translational Medicine, College of Medicine, Cardiovascular Research Institute, Texas A&M Health Science Center, 2Department of Biomedical Engineering, Texas A&M University This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented. Biology Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions Jeongyun Kim1, Manjunath Hegde1, Arul Jayaraman1,2 1McFerrin Department of Chemical Engineering, Texas A&M University, 2Department of Biomedical Engineering, Texas A&M University This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains. Biology A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients Derek L. Englert1, Michael D. Manson2, Arul Jayaraman1,3 1McFerrin Department of Chemical Engineering, Texas A&M University, 2Department of Biology, Texas A&M University, 3Department of Biomedical Engineering, Texas A&M University This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors. Biology Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies Hisami Koito1, Jianrong Li1 1Department of Veterinary Integrative Biosciences, Texas A&M University (TAMU) A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes. Biology A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform Jaewon Park1, Hisami Koito2, Jianrong Li2, Arum Han1 1Department of Electrical and Computer Engineering, Texas A&M University (TAMU), 2Department of Veterinary Integrative Biosciences, Texas A&M University (TAMU) We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method. Biology Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules John Noel1, Winfried Teizer1, Wonmuk Hwang2 1Department of Physics, Texas A&M University (TAMU), 2Department of Biomedical Engineering, Texas A&M University (TAMU) We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the nanoscale. Biology Assay for Neural Induction in the Chick Embryo Delphine Psychoyos1, Richard Finnell1 1Institute of Biosciences and Technology, Center for Environmental and Genetic Medicine, Texas A&M University (TAMU) Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo. Biology Method for Whole Mount Antibody Staining in Chick Delphine Psychoyos1, Richard Finnell1 1Institute of Biosciences and Technology, Center for Environmental and Genetic Medicine, Texas A&M University (TAMU) This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell. Biology Double Whole Mount in situ Hybridization of Early Chick Embryos Delphine Psychoyos1, Richard Finnell2 1Center for Environmental and Genetic Medicine, Institute of Biosciences and Technology - Texas A&M Health Science Center, 2Center for Environmental and Genetic Medicine, Texas A&M University (TAMU) This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz. Biology Method for Culture of Early Chick Embryos ex vivo (New Culture) Delphine Psychoyos1, Richard Finnell2 1Center for Environmental and Genetic Medicine, Institute of Biosciences and Technology - Texas A&M Health Science Center, 2Center for Environmental and Genetic Medicine, Texas A&M University (TAMU) This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.