Texas A&M University

55 articles published in JoVE

• 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 Chavez^{1 1}Department 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. Adelman^{1 1}Department 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. Adelman^{1 1}Department 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 Liu^{1 1}Department 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. Bishop^{1 1}Department 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. Mistry^1,2, Pankaj K. Singh^1,3, Maureen G. Mancini^1,2, Fabio Stossi^1,2, Michael A. Mancini^{1,2,3,4 1}GCC 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 Song^{1,2 1}Texas 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 Grandin^{2 1}Animal 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. Hart^{2 1}Department 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. Angel^{1 1}Department 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 Wu^1,2, Clinton D Allred¹, Shaodong Guo¹, Yuxiang Sun^{1,2 1}Department 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 Muneoka^{1 1}Department 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 Panin^{1 1}Department 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. Strycker^1,2, Kai Wang¹, Alexei V. Sokolov^1,2, Marlan O. Scully^{1,2 1}Texas 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 Lee^{1 1}Regenerative 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. Deans^1,2, Gregory A. Sword¹, Paul A. Lenhart³, Eric Burkness², William D. Hutchison², Spencer T. Behmer^{1 1}Department 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. Conkle^{1 1}Department 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 Tian^{1,2 1}Hunan 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. Mukherjee^{2 1}Department 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. Maitland^{1 1}Department 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 Huang^{1 1}Centre 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. Lele^{1 1}Artie 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. Feng^{1 1}Department of Biomedical Sciences, Texas A&M University College of Dentistry We developed two sets of tracing combinations in Rosa26^tdtomato (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 Lin^2,3, 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 Richardson^{4 1}Department 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. Santschi^{4 1}Department 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. Sorg^{1 1}Department 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 Wang^2,3, Abhijeet Shinde², Zhengdong Cheng^{1,2,3,4 1}Department 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. Mukherjee^{1 1}Department 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 Zhang^2,3, Jessica L. Reinhard¹, Melissa A. Grunlan^{1,2 1}Department 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. Svoboda^{1 1}Department 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 Pellois^{1 1}Biochemistry 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 Kumar^{4 1}Department 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 Adams^{1 1}Department 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. Kelly^{2 1}Soil 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 Chen^{1,2 1}Artie 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. Bellinger^{1 1}Department 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 Zhou^{3 1}Department 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 Kolomiets^{1 1}Plant 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. Abbott^{5 1}Department 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 He^{1 1}Department 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 Dickman^{1 1}Department 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-Bageshwar^1,2, Patricia V. Pietrantonio^{1 1}Department 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. Ugaz^{3 1}Department 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. Cirillo^{1 1}Microbial & 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. Rosenthal^{2 1}Department 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 Trache^1,2, Soon-Mi Lim^{1 1}Department 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 Jayaraman^{1,2 1}McFerrin 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 Jayaraman^{1,3 1}McFerrin 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 Li^{1 1}Department 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 Han^{1 1}Department 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 Hwang^{2 1}Department 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 Finnell^{1 1}Institute 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 Finnell^{1 1}Institute 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 Finnell^{2 1}Center 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 Finnell^{2 1}Center 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.