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University of Wisconsin, Madison

8 ARTICLES PUBLISHED IN JoVE

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Biology

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development
Katie E. Holmes 1, Matthew J. Wyatt 2, Yu-chi Shen 2, Deborah A. Thompson 2,3, Kate F. Barald 2,4
1Department of Veterinary Science, University of Wisconsin, Madison, 2Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 3Present address: Department of Pulmonary Medicine, University of Michigan, Ann Arbor, MI, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

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Biology

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil
Chao Liang 1,2, Harry W. Read 2, Teri C. Balser 2,3
1DOE-Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, 2Department of Soil Science, University of Wisconsin, Madison, 3Department of Soil and Water Science, University of Florida

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

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Bioengineering

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-µCP) and Sequential Nucleophilic Substitution
Gavin T. Knight *1, Tyler Klann *1, Jason D. McNulty 1,2, Randolph S. Ashton 1
1Department of Biomedical Engineering, University of Wisconsin, Madison, 2Department of Mechanical Engineering, University of Wisconsin, Madison

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

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Environment

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples
Lawrence G. Oates 1, Harry W. Read 2, Jessica L. M. Gutknecht 3, David S. Duncan 1, Teri B. Balser 4, Randall D. Jackson 1
1Department of Agronomy and Great Lakes Bioenergy Research Center, University of Wisconsin - Madison, 2Department of Soil Science, University of Wisconsin - Madison, 3Department of Soil, Water, and Climate, University of Minnesota, 4Faculty of Science and Engineering, Curtin University

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

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Biology

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor
Thomas R. Vincent 1, James Canham 1, Masatsugu Toyota 2,3,4, Marieta Avramova 1, Sam T. Mugford 5, Simon Gilroy 2, Anthony J. Miller 1, Saskia Hogenhout 5, Dale Sanders 1
1Department of Metabolic Biology, John Innes Centre, Norwich Research Park, 2Department of Botany, University of Wisconsin, Madison, 3Department of Biochemistry and Molecular Biology, Saitama University, 4Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 5Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

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JoVE Journal

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis
Takuya Uemura 1, Jiaqi Wang 1, Yuri Aratani 1, Simon Gilroy 2, Masatsugu Toyota 1,2
1Department of Biochemistry and Molecular Biology, Saitama University, 2Department of Botany, University of Wisconsin

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

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Biochemistry

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows
Bryan S. Sibert *1,2,3, Joseph Y. Kim *1,4, Jie E. Yang 1,2,3, Elizabeth R. Wright 1,2,3,5
1Department of Biochemistry, University of Wisconsin, Madison, 2Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, 3Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, 4Department of Chemistry, University of Wisconsin, Madison, 5Morgridge Institute for Research

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

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Behavior

A Common Marmoset Model of Mother-Infant Intervention for Breastfeeding Disorders in the Presence of Paternal Inhibition and Maternal Neglect
Mayuko Yoda 1,2, Yoshimasa Kamei 3, Hayato Sakurai 4, Hiroko Kakei 4, Ting Tao 1,2,4, Hideo Yamanouchi 4, Tetsuya Kunikata 4, Masanori Hariyama 5, Ricki Colman 6, Mamiko Koshiba 1,4,5
1Graduate School of Sciences and Technology for Innovation, Yamaguchi University, 2Faculty of Health Sciences and Nursing, Juntendo University, 3Obstetrician, Medical Department, Saitama Medical University, 4Department of Pediatrics, Saitama Medical University, 5Graduate School of Information Sciences, Tohoku University, 6Department of Cell and Regenerative Biology and Wisconsin National Primate Research Center, University of Wisconsin, Madison

Here, we present a nonhuman primate model of mother-infant intervention for breastfeeding disorders in the presence of paternal inhibition and maternal neglect. The mate model video complements education to support primate and human caregivers with respect to infants with breastfeeding problems such as pain.

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