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51 ARTICLES PUBLISHED IN JoVE

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Biology

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions
Jeongyun Kim 1, Manjunath Hegde 1, Arul Jayaraman 1,2
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.

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Biology

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients
Derek L. Englert 1, Michael D. Manson 2, Arul Jayaraman 1,3
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.

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Biology

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy
Andreea Trache 1,2, Soon-Mi Lim 1
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.

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Biology

Rapid PCR Thermocycling using Microscale Thermal Convection
Radha Muddu 1, Yassin A. Hassan 2, Victor M. Ugaz 3
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.

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Biology

A β-glucuronidase (GUS) Based Cell Death Assay
Mehdi Kabbage 1, Maria Ek-Ramos 1, Martin Dickman 1
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.

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Biology

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton
Xiquan Gao 1, Robert C. Britt Jr. 1, Libo Shan 2, Ping He 1
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.

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Bioengineering

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
Yoonsuck Choe 1, David Mayerich 2, Jaerock Kwon 3, Daniel E. Miller 1, Chul Sung 1, Ji Ryang Chung 1, Todd Huffman 4, John Keyser 1, Louise C. Abbott 5
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.

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Immunology and Infection

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays
Shawn Christensen *1, Eli Borrego *1, Won-Bo Shim 1, Tom Isakeit 1, Michael Kolomiets 1
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.

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Immunology and Infection

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages
Wei Ying 1, Patali S. Cheruku 2, Fuller W. Bazer 1, Stephen H. Safe 3, Beiyan Zhou 3
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.

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Environment

Design and Construction of an Urban Runoff Research Facility
Benjamin G. Wherley 1, Richard H. White 1, Kevin J. McInnes 1, Charles H. Fontanier 1, James C. Thomas 1, Jacqueline A. Aitkenhead-Peterson 1, Steven T. Kelly 2
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.

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Bioengineering

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars
Mahtab Hassanpourfard 1, Xiaohui Sun 2, Amin Valiei 1, Partha Mukherjee 3, Thomas Thundat 1, Yang Liu 2, Aloke Kumar 4
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.

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Immunology and Infection

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages
Jing Wu 1, Roberta Pugh 1, Richard C. Laughlin 1, Helene Andrews-Polymenis 2, Michael McClelland 3, Andreas J. Bäumler 4, L. Garry Adams 1
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.

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Bioengineering

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold
Lindsay N. Nail 1, Dawei Zhang 2,3, Jessica L. Reinhard 1, Melissa A. Grunlan 1,2
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.

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Bioengineering

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT
Kristina Najjar 1, Alfredo Erazo-Oliveras 1, Jean-Philippe Pellois 1
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.

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Engineering

Non-aqueous Electrode Processing and Construction of Lithium-ion Coin Cells
Malcolm Stein IV 1, Chien-Fan Chen 1, Daniel J. Robles 1, Christopher Rhodes 2, Partha P. Mukherjee 1
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.

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Chemistry

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals
Yi-Hsien Yu 1, Xuezhen Wang 2,3, Abhijeet Shinde 2, Zhengdong Cheng 1,2,3,4
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.

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Biology

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)
Daniel E. Miller 1, Zubin H. Patel 1,2,3, Xiaoming Lu 1,3, Arthur T. Lynch 1, Matthew T. Weirauch *1,4, Leah C. Kottyan *1
1Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital, 2Medical Scientist Training Program, University of Cincinnati, 3Immunology Graduate Program, University of Cincinnati, 4Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

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Immunology and Infection

Detecting Cortex Fragments During Bacterial Spore Germination
Michael B. Francis 1, Joseph A. Sorg 1
1Department of Biology, Texas A&M University

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

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Cancer Research

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases
Mercedes Rodriguez-Teja 1, Claudia Breit 2, Mitchell Clarke 3, Kamil Talar 3, Kai Wang 3, Mohammad A. Mohammad 3, Sage Pickwell 3, Guillermina Etchandy 1, Graeme J. Stasiuk 3, Justin Sturge 3
1Departamento de Genética, Facultad de Medicina, Universidad de la República (UDELAR), 2Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 3School of Biological, Biomedical & Environmental Sciences, University of Hull

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

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Engineering

Electrospray Deposition of Uniform Thickness Ge23Sb7S70 and As40S60 Chalcogenide Glass Films
Spencer Novak 1, Pao-Tai Lin 2,3, Cheng Li 4, Nikolay Borodinov 1, Zhaohong Han 5, Corentin Monmeyran 5, Neil Patel 5, Qingyang Du 5, Marcin Malinowski 4, Sasan Fathpour 4, Chatdanai Lumdee 4, Chi Xu 4, Pieter G. Kik 4, Weiwei Deng 6, Juejun Hu 7, Anuradha Agarwal 7, Igor Luzinov 1, Kathleen Richardson 4
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.

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Bioengineering

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior
Kai Wang 1, Kam W. Leong 2, Yong Yang 1
1Department of Chemical and Biomedical Engineering, West Virginia University, 2Department of Biomedical Engineering, Columbia University

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

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Bioengineering

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice
Kai Wang *1, Xi Wang *1, Cheng-sheng Han *2, Liang-yi Chen 2, Ying Luo 1
1Department of Biomedical Engineering, Peking University, 2Institute of Molecular Medicine, Peking University

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

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Biology

Biophysical Characterization of Flagellar Motor Functions
Katie M. Ford 1, Ravi Chawla 1, Pushkar P. Lele 1
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.

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Biochemistry

Real-time Tracking of DNA Fragment Separation by Smartphone
Chunxian Tao 1, Bo Yang 1, Zhenqing Li 1, Dawei Zhang 1, Yoshinori Yamaguchi 1,2,3
1Shanghai Key Lab of Modern Optical System, University of Shanghai for Science and Technology, 2Department of Applied Physics, Graduate School of Engineering, Osaka University, 3East China University of Science and Technology, Department of Physics Faculty of Science

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

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Chemistry

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control
Xinyi Ding 1,2,3, Yuxiang Sun 1,2,3, Yanming Chen 1,2,3, Wanchuan Ding 1,2,3, Steven Emory 1,2,3,4, Tianhao Li 1,2,3, Zixing Xu 1,2,3, Ning Han 1,2,3, Jun Wang 1,2,3, Gang Ruan 1,2,3
1Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, 2Institute of Materials Engineering, College of Engineering and Applied Sciences, Nanjing University, 3Collaborative Innovation Center of Chemistry for Life Sciences, Nanjing University, 4Department of Chemistry, Western Washington University

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

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Chemistry

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling
Minjung Lee 1, Yubin Zhou 2, Yun Huang 1
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.

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Bioengineering

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure
Madeleine S. Durkee 1, Landon D. Nash 1, Fatemeh Nooshabadi 1, Jeffrey D. Cirillo 2, Duncan J. Maitland 1, Kristen C. Maitland 1
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.

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Biology

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice
Daniel Villarreal 1, Geetali Pradhan 2, Chia-Shan Wu 1,2, Clinton D Allred 1, Shaodong Guo 1, Yuxiang Sun 1,2
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.

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Immunology and Infection

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals
Jingshu Chen *2, Lei Zhong *1, Jing Wu 1, Sui Ke 2, Benjamin Morpurgo 3, Andrei Golovko 3, Nengtai Ouyang 4, Yuxiang Sun 2, Shaodong Guo 2, Yanan Tian 1,2
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.

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Biology

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model
Kang Qin 1, Yifan Wu 1, Yiwa Pan 1, Kai Wang 1, Deling Kong 1, Qiang Zhao 1
1State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials (Ministry of Education), College of Life Sciences, Nankai University

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

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Engineering

Three-electrode Coin Cell Preparation and Electrodeposition Analytics for Lithium-ion Batteries
Robert D. Minter *1, Daniel Juarez-Robles *2, Conner Fear 2, Yevgen Barsukov 3, Partha P. Mukherjee 2
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.

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Bioengineering

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation
Doris A. Taylor 1, Luiz C. Sampaio 1, Rafael Cabello 1, Abdelmotagaly Elgalad 1, Rohan Parikh 1, R Patrick Wood 2, Kevin A. Myer 2, Alvin T. Yeh 3, Po-Feng Lee 1
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.

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Environment

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar
Carrie A. Deans 1,2, Gregory A. Sword 1, Paul A. Lenhart 3, Eric Burkness 2, William D. Hutchison 2, Spencer T. Behmer 1
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.

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Engineering

Femtosecond Laser Filaments for Use in Sub-Diffraction-Limited Imaging and Remote Sensing
Matthews M. Springer 1, Benjamin D. Strycker 1,2, Kai Wang 1, Alexei V. Sokolov 1,2, Marlan O. Scully 1,2
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.

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Neuroscience

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons
Mengye Zhu 1, Daying Zhang 1, Sicong Peng 2, Nana Liu 2, Jing Wu 2, Haixia Kuang 2, Tao Liu 2,3
1Department of Pain, First Affiliated Hospital of Nanchang University, 2Department of Pediatrics, First Affiliated Hospital of Nanchang University, 3Center for Laboratory Medicine, First Affiliated Hospital of Nanchang University

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

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Biology

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana
Kai Wang 1, Kayin Law 1, Manhin Leung 1, Waishing Wong 1, Ning Li 1,2
1Division of Life Science, Energy Institute, Institute for the Environment, The Hong Kong University of Science and Technology, 2Shenzhen Research Institute, The Hong Kong University of Science and Technology

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

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Developmental Biology

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo
Ishita Chandel 1, Ryan Baker 1, Naosuke Nakamura 1, Vlad Panin 1
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.

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Developmental Biology

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification
Lindsay A. Dawson 1, Regina Brunauer 1, Katherine N. Zimmel 1, Osama Qureshi 1, Alyssa R. Falck 2, Patrick Kim 3, Connor P. Dolan 1, Ling Yu 1, Yu-Lieh Lin 1, Benjamin Daniel 1, Mingquan Yan 1, Ken Muneoka 1
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.

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Behavior

Using Cholesky Decomposition to Explore Individual Differences in Longitudinal Relations between Reading Skills
Florina Erbeli 1, Aaron R. Campbell 1, Sara A. Hart 2
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.

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Biology

Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform
Shreedevi Arun Kumar 1, Jihui Lee 1, Corey J. Bishop 1
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.

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Behavior

The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle
Courtney Lynd Daigle 1, Amanda J. Hubbard 1, Temple Grandin 2
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.

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Environment

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.
Jinping Zhao 1, Carlos Garcia Rios 1, Junqi Song 1,2
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.

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Biology

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)
Hitoshi Tsujimoto 1, Zach N. Adelman 1
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.

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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
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.

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Immunology and Infection

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages
Sahar Eshghjoo 1, Da Mi Kim 2, Arul Jayaraman 1,3, Yuxiang Sun 2, Robert C. Alaniz 1
1Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M University Health Science Center, 2Department of Nutrition and Food Science, Texas A&M University, 3Artie McFerrin Department of Chemical Engineering, Texas A&M University

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

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JoVE Journal

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli
Chesley Marie Rowlett 1, Wenshe Ray Liu 1
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.

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Immunology and Infection

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells
Jing Yang 1, Qing-Ming Qin 1, Erin Van Schaik 1, James E. Samuel 1, Paul de Figueiredo 1,2
1Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, 2College of Veterinary Medicine, Texas A&M University

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

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Genetics

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti
Bianca B. Kojin 1, Hitoshi Tsujimoto 1, Emma Jakes 1, Sarah O'Leary 1, Zach N. Adelman 1
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.

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Biology

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles
Brenda Leal-Galvan 1, Cristina Harvey 1, Donald Thomas 2, Perot Saelao 3, Adela S. Oliva Chavez 1
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.

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Biochemistry

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors
Caixing Xiong 1, Dwight Baker 2, Patricia Pietrantonio 1
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.

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Biology

Manipulation of Rhythmic Food Intake in Mice Using a Custom-Made Feeding System
Aishwarya Sahasrabudhe 1,2, Chanté R. Guy 1,2, Ben J. Greenwell 1,2,3, Jerome S. Menet 1,2,3
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.

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