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University of Wyoming

20 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce
Bledar Bisha 1, Byron F. Brehm-Stecher 2
1Center for Meat Safety and Quality, Department of Animal Sciences, Colorado State University, 2Rapid Microbial Detection and Control Laboratory, Department of Food Science and Human Nutrition, Iowa State University

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

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Medicine

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window
Lifeng Zhang *1, Andree Lapierre *1, Brittany Roy 1, Maili Lim 1, Jennifer Zhu 1, Wei Wang 1, Stephen B. Sampson 1, Kyuson Yun 1, Bonnie Lyons 1, Yun Li 1, Da-Ting Lin 1
1The Jackson Laboratory

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

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Neuroscience

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons
Yun Li 1, Brittany D. Roy 1, Wei Wang 1, Lifeng Zhang 1, Stephen B. Sampson 1, Da-Ting Lin 1
1The Jackson Laboratory

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

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Environment

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water
Bledar Bisha 1, Jaclyn A. Adkins 2, Jana C. Jokerst 3, Jeffrey C. Chandler 1, Alma Pérez-Méndez 4, Shannon M. Coleman 4, Adrian O. Sbodio 5, Trevor V. Suslow 5, Michelle D. Danyluk 6, Charles S. Henry 2, Lawrence D. Goodridge 7
1Department of Animal Science, University of Wyoming, 2Department of Chemistry, Colorado State University, 3Department of Environmental and Radiological Health Sciences, Colorado State University, 4Department of Animal Sciences, Colorado State University, 5Department of Plant Sciences, University of California, Davis, 6Department of Food Science and Human Nutrition, Citrus Research and Education Center, University of Florida, 7Department of Food Science and Agricultural Chemistry, McGill University

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (µPAD) imbedded with bacterial-indicative colorimetric substrates.

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JoVE Journal

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis
Choong Yong Ung 1, Feng Guo 2, Xiaoling Zhang 3, Zhihui Zhu 3, Shizhen Zhu 1,3
1Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic College of Medicine, Center for Individualized Medicine, 2Tufts University School of Medicine, 3Department of Biochemistry and Molecular Biology, Mayo Clinic

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

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Environment

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability
Jodi R. Schilz 1, K. J. Reddy 2, Sreejayan Nair 3, Thomas E. Johnson 4, Ronald B. Tjalkens 5, Kem P. Krueger 3, Suzanne Clark 6
1Division of Physical Therapy, Department of Orthopedics & Rehabilitation, University of New Mexico, 2Department of Ecosystem Science and Management, University of Wyoming, 3School of Pharmacy, University of Wyoming, 4Department of Environmental and Radiological Health Sciences, Colorado State University, 5Center for Environmental Medicine, Colorado State University, 6College of Pharmacy, California Northstate University

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

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Biology

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation
Lisa J. Edens 1, Daniel L. Levy 1
1Department of Molecular Biology, University of Wyoming

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

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Biology

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus
Najie Zhu *1, Liqun Bai *1, Stefan Schütz 2, Baojun Liu 3, Zhenyu Liu 3, Xingyao Zhang 4, Hongshi Yu 1, Jiafu Hu 1,5
1Department of Forest Protection, Zhejiang Agricultural & Forestry University, 2Institute of Forest Zoology and Forest Conservation, Georg-August University Göttingen, 3College of Plant Protection, Shandong Agricultural University, 4Institute of Forest Protection, Chinese Academy of Forestry, 5Collaborative Innovation Center of Sustainable Forestry in Southern China, Nanjing Forestry University

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

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Biochemistry

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development
Robyn M. Johnston 1, Anne W. Sylvester 2, Michael J. Scanlon 1
1Plant Biology Section, School of Integrative Plant Science, Cornell University, 2Department of Developmental Genetics, University of Wyoming

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

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Biochemistry

Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads
Padmamalini Baskaran 1, Baskaran Thyagarajan 1
1School of Pharmacy, University of Wyoming

This protocol describes the method of determining basal and forskolin-stimulated lipolysis in inguinal fat pads obtained from normal chow diet (NCD) or high fat diet (HFD) ± capsaicin fed wild type mice. As an index for lipolysis, glycerol release was measured from inguinal adipose fat pads.

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Immunology and Infection

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material
Yo Ching Cheng *1, Samia Hannaoui *1, Theodore Ralph John 2, Sandor Dudas 3, Stefanie Czub 3, Sabine Gilch 1
1Dept. of Ecosystem and Public Health, Calgary Prion Research Units, Faculty of Veterinary Medicine, University of Calgary, 2Dept. of Molecular Biology, University of Wyoming, 3Canadian Food Inspection Agency, Lethbridge Laboratories

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

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Neuroscience

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons
Zhenyu Liu 1, Katelynne B. Donnelly 1, Kara G. Pratt 1
1Department of Zoology and Physiology and Program in Neuroscience, University of Wyoming

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

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Neuroscience

Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology
Adel B. Ghnenis 1, Richard E. Czaikowski 1, Zhaojie J. Zhang 2, Jared S. Bushman 1
1School of Pharmacy, University of Wyoming, 2Department of Zoology and Physiology, University of Wyoming

Here we present a protocol to visualize fine structures of peripheral nerves by obtaining and staining 1-2 µm sections with toluidine blue

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Environment

Detection of Viruses from Bioaerosols Using Anion Exchange Resin
Joshua W. Schaeffer 1, Jeffrey C. Chandler 2, Margaret Davidson 1,3, Sheryl L. Magzamen 1, Alma Pérez-Méndez 4, Stephen J. Reynolds 1, Lawrence D. Goodridge 5, John Volckens 6, Alan B. Franklin 2, Susan A. Shriner 2, Bledar Bisha 7
1High Plains Intermountain Center for Agricultural Health and Safety, Department of Environmental and Radiological Health Sciences, Colorado State University, 2National Wildlife Research Center, Wildlife Services, Animal and Plant Health Inspection Service, United States Department of Agriculture, 3Western Sydney University, 4Leprino Foods, Inc, 5Department of Food Science and Agricultural Chemistry, McGill University, 6Department of Mechanical Engineering, Colorado State University, 7Department of Animal Science, University of Wyoming

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

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Environment

Ground-level Unmanned Aerial System Imagery Coupled with Spatially Balanced Sampling and Route Optimization to Monitor Rangeland Vegetation
Michael F. Curran 1,2,3, Paddington Hodza 4, Samuel E. Cox 5, Shawn G. Lanning 4, Blair L. Robertson 6, Timothy J. Robinson 7, Peter D. Stahl 1,2,3
1Wyoming Reclamation and Restoration Center, University of Wyoming, 2Department of Ecosystem Science and Management, University of Wyoming, 3Program in Ecology, University of Wyoming, 4Wyoming Geographic Information Science Center, University of Wyoming, 5Wyoming State Office, Bureau of Land Management, 6School of Mathematics and Statistics, University of Canterbury, 7Department of Statistics, University of Wyoming

The protocol presented in this paper utilizes route optimization, balanced acceptance sampling, and ground-level and unmanned aircraft system (UAS) imagery to efficiently monitor vegetation in rangeland ecosystems. Results from images obtained from ground-level and UAS methods are compared.

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Engineering

Microfluidic Fabrication Techniques for High-Pressure Testing of Microscale Supercritical CO2 Foam Transport in Fractured Unconventional Reservoirs
Hooman Hosseini 1, Feng Guo 2, Reza Barati Ghahfarokhi 1, Saman A. Aryana 2
1Department of Chemical and Petroleum Engineering, University of Kansas, 2Department of Chemical Engineering, University of Wyoming

This paper describes a protocol along with a comparative study of two microfluidic fabrication techniques, namely photolithography/wet-etching/thermal-bonding and Selective Laser-induced Etching (SLE), that are suitable for high-pressure conditions. These techniques constitute enabling platforms for direct observation of fluid flow in surrogate permeable media and fractured systems under reservoir conditions.

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Biology

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination
Gordon F. Custer *1,2, Reilly R. Dibner *3,4
1Department of Ecosystem Science and Management, University of Wyoming, 2Program in Ecology, University of Wyoming, 3EPSCoR-IDEA, University of Wyoming, 4Department of Botany, University of Wyoming

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

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Developmental Biology

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography
Todd A. Schoborg 1
1Department of Molecular Biology, University of Wyoming

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

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Neuroscience

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging
Rashmi Thapa 1, Bo Liang 2, Rongsong Liu 3, Yun Li 1
1Department of Zoology and Physiology, University of Wyoming, 2School of Electrical Engineering & Computer Science, University of North Dakota, 3Department of Mathematics and Statistics, University of Wyoming

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

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Reconstructing Terrestrial Paleoclimate and Paleoecology with Fossil Leaves Using Digital Leaf Physiognomy and Leaf Mass Per Area

Reconstructing Terrestrial Paleoclimate and Paleoecology with Fossil Leaves Using Digital Leaf Physiognomy and Leaf Mass Per Area
Alexander J. Lowe 1, Andrew G. Flynn 2, Matthew J. Butrim 3, Aly Baumgartner 4, Dana L. Royer 5, Daniel J. Peppe 6
1Department of Biology, University of Washington, 2Department of Paleobiology, National Museum of Natural History, Smithsonian Institution, 3Department of Geology and Geophysics, Program in Ecology, University of Wyoming, 4Department of Ecology and Evolutionary Biology, University of Michigan Herbarium, 5Department of Earth and Environmental Sciences, Wesleyan University, 6Department of Geosciences, Baylor University

The protocol presented shows digital measurement and analysis of continuous leaf physiognomic traits on fossil leaves to reconstruct paleoclimate and paleoecology using the digital leaf physiognomy and leaf mass per area reconstruction methods.

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