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Kansas State University

17 ARTICLES PUBLISHED IN JoVE

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Biology

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae
Xin Zhang *1, Keshava Mysore *2,3, Ellen Flannery 3,4, Kristin Michel 1, David W. Severson 3,4, Kun Yan Zhu 5, Molly Duman-Scheel 2,3,4
1Division of Biology, Kansas State University, 2Department of Medical and Molecular Genetics, Indiana University School of Medicine, 3Eck Institute for Global Health, University of Notre Dame, 4Department of Biological Sciences, University of Notre Dame, 5Department of Entomology, Kansas State University

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

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Biology

RNAi Trigger Delivery into Anopheles gambiae Pupae
Kimberly Regna 1, Rachel M. Harrison 1, Shannon A. Heyse 1, Thomas C. Chiles 1, Kristin Michel 2, Marc A. T. Muskavitch 1,3
1Biology Department, Boston College, 2Division of Biology, Kansas State University, 3Discovery Research, Biogen

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

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Chemistry

An Experimental Protocol for Femtosecond NIR/UV - XUV Pump-Probe Experiments with Free-Electron Lasers
Daniel Rolles 1, Rebecca Boll 2,3, Benjamin Erk 2, Dimitrios Rompotis 2, Bastian Manschwetus 2
1J.R. Macdonald Laboratory, Department of Physics, Kansas State University, 2Deutsches Elektronen-Synchrotron DESY, 3European XFEL GmbH

This protocol describes the key steps for performing and analyzing pump-probe experiments combining a femtosecond optical laser with a free-electron laser in order to study ultrafast photochemical reactions in gas-phase molecules.

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JoVE Core

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction
Cecilia S.N. Cheung 1,2, Donald J. Weidner 1, Li Li 1, Philip G. Meredith 3, Haiyan Chen 1, Matthew Whitaker 1, Xianyin Chen 4
1Mineral Physics Institute, Department of Geoscience, Stony Brook University, 2Geological Engineering, Department of Civil and Environmental Engineering, University of Wisconsin-Madison, 3Rock and Ice Physics Laboratory, Department of Earth Sciences, University College London, 4Department of Chemistry, Stony Brook University

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

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Engineering

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers
Mohammadali Masigol 1, Niloy Barua 1, Bradley S. Lokitz 2, Ryan R. Hansen 1
1Chemical Engineering Department, Kansas State University, 2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory

Surface fabrication methods for patterned deposition of nanometer thick brushes or micron thick, crosslinked films of an azlactone block co-polymer are reported. Critical experimental steps, representative results, and limitations of each method are discussed. These methods are useful for creating functional interfaces with tailored physical features and tunable surface reactivity.

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Cancer Research

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy
Vaibhav Murthy *1, Dalton Dacus *1, Monica Gamez 2, Changkun Hu 1, Sebastian O. Wendel 1, Jazmine Snow 1, Andrew Kahn 1, Stephen H. Walterhouse 1, Nicholas A. Wallace 1
1Division of Biology, Kansas State University, 2Bristol Medical School, Translational Health Sciences, University of Bristol

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

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Biology

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits
Consuelo Almazán 1, Sarah Bonnet 1, Martine Cote 1, Mirko Slovák 2, Yoonseong Park 3, Ladislav Šimo 1
1Unité Mixte de Recherche (UMR) Biologie moléculaire et immunologie parasitaires (BIPAR), Institut national de la recherche agronomique (INRA), Ecole Nationale Vétérinaire d'Alfort, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Université Paris-Est, 2Institute of Zoology, Slovak Academy of Sciences, 3Department of Entomology, Kansas State University

We have developed a simple and versatile system to feed hard ticks on laboratory rabbits. Our non-laborious protocol uses easily accessible materials and can be adjusted depending on the requirements of the various experimental settings. The method allows comfortable monitoring and/or sampling of ticks during the entire feeding period.

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Behavior

Noninvasive, In-pen Approach Test for Laboratory-housed Pigs
Lindsey E. Hulbert 1, Eduarda M. Bortoluzzi 1, Yunzhi Luo 1, Jared M. Mumm 1, Morgan J. Coffin 1, Gina Y. Becker 2, Pamela J. Vandevord 3, Elizabeth M. McNeil 3, Tim Walilko 4, Zin Z. Khaing 5,6, Laila Zai 4,5
1Animal Sciences and Industry, Kansas State University, 2DynaSim Technical Services, INC, 3Center for Injury Biomechanics, Virginia Polytechnic Institute and State University, 4Applied Research Associates, Inc., 5Lucent Research, LLC, 6Department of Neurological Surgery, University of Washington

This protocol describes a new behavioral test—the human approach test in the pigs' home pen—to detect functional deficits in laboratory pigs after subconcussive traumatic brain injury.

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Cancer Research

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis
Ravan Moret 1, Linh Hellmers 1, Xin Zhang 1, Jessie Gills 2, Nathan Hite 3, Aaron Klinger 3, Grace A. Maresh 1, Daniel Canter 2,4, Stephen Bardot 2,4, David A Margolin 3,4, Li Li 1,4
1Laboratory of Translational Cancer Research, Institute for Translational Research, Ochsner Clinic Foundation, 2Department of Urology, Ochsner Clinic Foundation, 3Department of Colon and Rectal Surgery, Ochsner Clinic Foundation, 4Ochsner Clinical School, University of Queensland, School of Medicine

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

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JoVE Journal

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells
Pragyesh Dhungel 1, Fernando Cantu 1, Candy Hernandez 1, Zhilong Yang 1
1Division of Biology, Kansas State University

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5’-untranslated region (UTR) and 3’-UTR. Different translation initiation modes can also be examined using this method.

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Immunology and Infection

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method
Raja Veerapandian 1, Anuja Paudyal 1, Adeline Chang 1, Govindsamy Vediyappan 1
1Division of Biology, Kansas State University

The objective is to fractionate and isolate bioactive small molecules, peptides from a complex plant extract, and proteins from pathogenic microbes by employing liquid-phase isoelectric focusing (IEF) method. Further, the separated molecules were identified and their bioactivity confirmed.

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Developmental Biology

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans
Li Li 1, Anna Y. Zinovyeva 1
1Division of Biology, Kansas State University

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

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Genetics

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
Changkun Hu *1, Tyler Doerksen *2, Taylor Bugbee 1, Nicholas A. Wallace 1, Rachel Palinski 2,3
1Division of Biology, Kansas State University, 2Kansas State Veterinary Diagnostic Laboratory, Kansas State University, 3Department of Diagnostic Medicine/Pathobiology, Kansas State University

Presented here is sgRNA/CAS9 endonuclease and next-generation sequencing protocol that can be used to identify the mutations associated with double strand break repair near the CD4 promoter.

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Bioengineering

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation
Niloufar Fattahi 1, Niloy Barua 1, André J. van der Vlies 2, Ryan R. Hansen 1
1Tim Taylor Department of Chemical Engineering, Kansas State University, 2Department of Materials Science and Engineering, The Pennsylvania State University

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

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Behavior

Physical Activity Measurement in Children Accepting Table Tennis Training
Xin Zhang *1, Cuiqiao Xia *2, Xiuli Zhao 3, Yu Liu 3, Hóngyi Zhào 2,3, Yonghua Huang 2
1Department of Sport, Inner Mongolia Medical University, 2Department of Neurology, The Seventh Medical Center of PLA General Hospital, 3Department of Neurology, NO 984 Hospital of PLA

This study proposes an accelerometer-based method to objectively measure physical activity (PA) and leisure time physical activity (LTPA) in Chinese children accepting table tennis training in clubs.

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Neuroscience

P300-Based Brain-Computer Interface Speller Performance Estimation with Classifier-Based Latency Estimation
Nazmun N. Khan 1, Taylor Sweet 1, Chase A. Harvey 1, Seth Warschausky 2, Jane E. Huggins 3,4, David E. Thompson 1
1Brain and Body Sensing Lab, Mike Wiegers Department of Electrical & Computer Engineering, Kansas State University, 2Adaptive Cognitive Assessment Laboratory, Department of Physical Medicine and Rehabilitation, University of Michigan, Ann Arbor, 3Direct Brain Interface Laboratory, Department of Physical Medicine and Rehabilitation, University of Michigan, Ann Arbor, 4Direct Brain Interface Laboratory, Department of Biomedical Engineering, University of Michigan, Ann Arbor

This article presents a method for estimating same-day P300 speller Brain-Computer Interface (BCI) accuracy using a small testing dataset.

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Bioengineering

A Computational Modeling Approach to Investigate the Influence of Hyperthermia on the Tumor Microenvironment
Anna Bottiglieri 1, Rahul A. Sheth 2, Punit Prakash 1
1Department of Electrical and Computer Engineering, Kansas State University, 2Department of Interventional Radiology, The University of Texas MD Anderson Cancer Center

The article describes a protocol to simulate the transient temperature profiles and the coupled spatiotemporal variation of the interstitial fluid pressure following the heating delivered by a dipolar radiofrequency hyperthermia system. The protocol can be used to assess the response of biophysical parameters characterizing the tumor microenvironment to interventional hyperthermia techniques.

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