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Cornell University

51 ARTICLES PUBLISHED IN JoVE

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Biology

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device
Kevin Wong 1, Angel Ayuso-Sacido 2,3, Patrick Ahyow 1, Andrew Darling 4, John A. Boockvar 2, Mingming Wu 4
1Biomedical Engineering Department, Cornell University, 2Neurosurgical Laboratory for Translational Stem Cell Research, Weill Cornell Brain Tumor Center, Weill Cornell Medical College of Cornell University, 3Cell Morphology Department, Instituto de Investigacion Principe Felipe, 4Department of Chemical and Biomolecular Engineering, Cornell University

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

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Biology

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects
Martin De Vos 1, Georg Jander 1
1Boyce Thompson Institute for Plant Research, Cornell University

Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.

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Biology

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection
Cecilia Tamborindeguy 1, Stewart Gray 1, Georg Jander 2
1Plant Pathology, Cornell University, 2Boyce Thompson Institute for Plant Research, Cornell University

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

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Biology

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment
John Sawyer Ramsey 1, Georg Jander 1
1Boyce Thompson Institute for Plant Research, Cornell University

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

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Biology

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana
Zhiyang Zhai 1, Ha-il Jung 1, Olena K. Vatamaniuk 1
1Crop and Soil Sciences, Cornell University

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

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Biology

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis
Xingshan Jiang 1, David Stern 1
1Boyce Thompson Institute for Plant Research, Cornell University

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

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Biology

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato
Andrá C. Velásquez 1,2, Suma Chakravarthy 2, Gregory B. Martin 1,2
1Plant Pathology and Plant-Microbe Biology, Cornell University, 2Boyce Thompson Institute for Plant Research

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

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Immunology and Infection

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants
Suma Chakravarthy 1, André C. Velásquez 1,2, Gregory B. Martin 1,2
1Boyce Thompson Institute for Plant Research, 2Plant Pathology and Plant-Microbe Biology, Cornell University

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

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Biology

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications
Huseyin C. Yalcin 1,2, Akshay Shekhar 1, Ajinkya A. Rane 1, Jonathan T. Butcher 1
1Department of Biomedical Engineering, Cornell University, 2Current Address: Mechanical Engineering Department, Dogus University

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

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Biology

Isolation of Valvular Endothelial Cells
Russell A. Gould 1, Jonathan T. Butcher 1
1Department of Biomedical Engineering, Cornell University

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

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Biology

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells
Gretchen Mahler 1, Russell Gould 1, Johnathan Butcher 1
1Department of Biomedical Engineering, Cornell University

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

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Neuroscience

Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking
Keith J. Yoder 1,2, Matthew K. Belmonte 1,3
1Department of Human Development, Cornell University, 2Social Sciences Division, University of Chicago, 3National Brain Research Centre, Manesar, India

Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.

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Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice
Rasika Jinadasa 1, Gabriel Balmus 1, Lee Gerwitz 1, Jamie Roden 1, Robert Weiss 1, Gerald Duhamel 1
1Department of Biomedical Sciences, Cornell University

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

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Biology

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation
Maria L. Lombardi 1, Monika Zwerger 1, Jan Lammerding 1,2
1Brigham and Women's Hospital / Harvard Medical School, Department of Medicine, Cardiovascular Division, 2Weill Institute for Cell and Molecular Biology & Department of Biomedical Engineering, Cornell University

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

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Bioengineering

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke
Long-Xi Yu 1, Boris G. Dzikovski 2, Jack H. Freed 2
1CDCF-AOX Lab, 2National Biomedical Center for Advanced ESR Technology (ACERT), Department of Chemistry and Chemical Biology, Cornell University

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

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Bioengineering

Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation
Joseph G. Usack 1, Catherine M. Spirito 1, Largus T. Angenent 1
1Department of Biological and Environmental Engineering, Cornell University

Laboratory-scale anaerobic digesters allow scientists to research new ways of optimizing existing applications of anaerobic biotechnology and to evaluate the methane producing potential of various organic wastes. This article introduces a generalized model for the construction, inoculation, operation, and monitoring of a laboratory-scale continuously stirred anaerobic digester.

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Biology

Procedure for Fabricating Biofunctional Nanofibers
Jereme Doss 1, Omotunde Olubi 1, Biswajit Sannigrahi 1, M. D. Williams 2, Deepti Gadi 3, Barbara Baird 3, Ishrat Khan 1
1Department of Chemistry, Clark Atlanta University, 2Department of Physics, Clark Atlanta University, 3Department of Chemistry and Chemical Biology, Cornell University

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

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Bioengineering

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples
Andrew D. Hughes 1, Jeff Mattison 1, John D. Powderly 2,3, Bryan T. Greene 2,3, Michael R. King 1
1Department of Biomedical Engineering, Cornell University, 2BioCytics, Inc., 3Carolina BioOncology Institute, PLLC

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

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Education

Measuring Spatially- and Directionally-varying Light Scattering from Biological Material
Todd Alan Harvey 1, Kimberly S. Bostwick 2,3, Steve Marschner 4
1Department of Biomedical Science, Cornell University, 2Department of Ecology and Evolutionary Biology, Cornell University, 3Cornell University Museum of Vertebrates, 4Department of Computer Science, Cornell University

We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.

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Biology

A Strategy for Sensitive, Large Scale Quantitative Metabolomics
Xiaojing Liu 1, Zheng Ser 1, Ahmad A. Cluntun 1,2, Samantha J. Mentch 1,2, Jason W. Locasale 1,2
1Division of Nutritional Sciences, Cornell University, 2Field of Biochemistry, and Molecular Cell Biology, Cornell University

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

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Neuroscience

In vivo Optogenetic Stimulation of the Rodent Central Nervous System
Michelle M. Sidor 1, Thomas J. Davidson 2, Kay M. Tye 3, Melissa R. Warden 4, Karl Diesseroth 2,5, Colleen A. McClung 1
1Department of Psychiatry, University of Pittsburgh Medical Center, 2Department of Bioengineering, Stanford University, 3Department of Brain and Cognitive Sciences, Picower Institute for Learning and Memory, Massachusetts Institute of Technology, 4Department of Neurobiology and Behavior, Cornell University, 5Department of Psychiatry and Behavioral Sciences, Stanford University

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

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Neuroscience

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord
Matthew J. Farrar 1,2, Chris B. Schaffer 2
1Department of Neurobiology and Behavior, Cornell University, 2Department of Biomedical Engineering, Cornell University

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

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Immunology and Infection

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster
Sarah Khalil 1, Eliana Jacobson 1, Moria C. Chambers 1, Brian P. Lazzaro 1
1Department of Entomology, Cornell University

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

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Medicine

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells
Sachi Horibata 1, Tommy V. Vo 2, Venkataraman Subramanian 3, Paul R. Thompson 3, Scott A. Coonrod 1
1Department of Baker Institute for Animal Health, Cornell University, 2Department of Molecular Biology and Genetics, Cornell University, 3Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

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Neuroscience

Conditions Affecting Social Space in Drosophila melanogaster
Alison R. McNeil 1, Sam N. Jolley 1, Adesanya A. Akinleye 2, Marat Nurilov 2, Zulekha Rouzyi 2, Austin J. Milunovich 3, Moria C. Chambers 3, Anne F. Simon 1
1Department of Biology, University of Western Ontario, 2Department of Biology, York College/CUNY, 3Department of Entomology, Cornell University

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

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Biology

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots
Thomas B. Jacobs 1, Gregory B. Martin 1,2
1Boyce Thompson Institute for Plant Research, 2Section of Plant Pathology & Plant-Microbe Biology, School of Integrative Plant Science, Cornell University

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

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Immunology and Infection

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids
Daniel E. Rothschild 1, Tara Srinivasan 2, Linette A. Aponte-Santiago 1, Xiling Shen 2,3,4, Irving C. Allen 1
1Department of Biomedical Sciences and Pathobiology, Virginia Maryland College of Veterinary Medicine, Virginia Tech, 2Department of Biomedical Engineering, Cornell University, 3School of Electrical and Computer Engineering, Cornell University, 4Department of Biomedical Engineering, Duke University

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

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Developmental Biology

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions
Melinda L. Koyle 1, Madeline Veloz 1, Alec M. Judd 1, Adam C.-N. Wong 2,4, Peter D. Newell 2,5, Angela E. Douglas 2,3, John M. Chaston 1,2
1Department of Plant and Wildlife Sciences, Brigham Young University, 2Department of Entomology, Cornell University, 3Department of Molecular Biology and Genetics, Cornell University, 4Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, 5Biological Sciences, SUNY Oswego

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

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Medicine

Transplantation Into the Mouse Ovarian Fat Pad
Andrea Flesken-Nikitin 1, Blaine A. Harlan 1, Alexander Yu. Nikitin 1
1Department of Biomedical Sciences, Cornell University

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of Müllerian origin.

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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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Immunology and Infection

Identification of Plasmodesmal Localization Sequences in Proteins In Planta
Cheng Yuan 1, Sondra G. Lazarowitz 2, Vitaly Citovsky 1
1Department of Biochemistry and Cell Biology, State University of New York, 2Department of Plant Pathology and Plant-Microbe Biology, Cornell University

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

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Engineering

Measuring the Densities of Aqueous Glasses at Cryogenic Temperatures
Chen Shen *1, Ethan F. Julius *1, Timothy J. Tyree *1, Ritwik Dan 1, David W. Moreau 2, Robert Thorne 2
1Cornell University, 2Physics Department, Cornell University

A protocol for determining the vitreous phase densities of micro- to pico-liter size drops of aqueous mixtures at cryogenic temperatures is described.

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Chemistry

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor
Sarah R. Nathan 1, Justin J. Wilson 1
1Department of Chemistry and Chemical Biology, Cornell University

A protocol for the synthesis, purification, and characterization of a ruthenium-based inhibitor of mitochondrial calcium uptake is presented. A procedure to evaluate its efficacy in permeabilized mammalian cells is demonstrated.

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Biology

A Murine Model of Vertical Sleeve Gastrectomy
Darline Garibay 1, Bethany P. Cummings 1
1Department of Biomedical Sciences, Cornell University

The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

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Genetics

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System
Joseph C. Siefert 1,2, Emily A. Clowdus 1,2, Duane Goins 1, Amnon Koren 3, Christopher L. Sansam 1,2
1Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, 2Department of Cell Biology, University of Oklahoma Health Sciences Center, 3Department of Molecular Biology and Genetics, Cornell University

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

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JoVE Journal

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea
Divya A. Shiroor 1, Tisha E. Bohr 1, Carolyn E. Adler 1
1Department of Molecular Medicine, College of Veterinary Medicine, Cornell University

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

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Developmental Biology

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries
Vera D. Rinaldi 1, Jordana C. Bloom 2, John C. Schimenti 1,2
1Department of Biomedical Sciences, Cornell University, 2Department of Molecular Biology and Genetics, Cornell University

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

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Biochemistry

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Javier A. Jaimes *1,2, Jean K. Millet *1,3, Monty E. Goldstein 1,4, Gary R. Whittaker 1, Marco R. Straus 1
1Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 2Department of Microbiology, College of Agricultural and Life Sciences, Cornell University, 3Virologie et Immunologie Moléculaires, Domaine de Vilvert, INRA, 4Department of Cell Biology and Molecular Genetics, University of Maryland

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

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Immunology and Infection

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting
Jean K. Millet 1,2, Tiffany Tang 3, Lakshmi Nathan 3, Javier A. Jaimes 4, Hung-Lun Hsu 3,5, Susan Daniel 3, Gary R. Whittaker 1
1Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 2INRA, Virologie et Immunologie Moléculaires, 3Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, 4Department of Microbiology, College of Agricultural and Life Sciences, Cornell University, 5Horae Gene Therapy Center, University of Massachusetts Medical School

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

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Developmental Biology

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure
Brandon J. Gheller *1, Jamie Blum *1, Sharon Soueid-Baumgarten 2, Erica Bender 1, Benjamin D. Cosgrove 2, Anna Thalacker-Mercer 1
1Division of Nutritional Sciences, Cornell University, 2Meinig School of Biomedical Engineering, Cornell University

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

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Cancer Research

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells
Hyeongsun Moon *1, Leanne R. Donahue *1, Dahihm Kim 1, Luye An 1, Andrew C. White 1
1Department of Biomedical Sciences, Cornell University

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

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Biochemistry

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry
Michael Vilkhovoy 1, David Dai 1, Sandra Vadhin *1, Abhinav Adhikari *1, Jeffrey D. Varner 1
1Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

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Biology

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging
Hui Ji 1, Chun Han 1
1Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics, Cornell University

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

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Environment

Simulating Impacts of Ice Storms on Forest Ecosystems
John L. Campbell 1, Lindsey E. Rustad 1, Charles T. Driscoll 2, Ian Halm 3, Timothy J. Fahey 4, Habibollah Fakhraei 5, Peter M. Groffman 6,7, Gary J. Hawley 8, Wendy Leuenberger 9, Paul G. Schaberg 10
1Northern Research Station, U.S. Forest Service, Durham, NH, 2Department of Civil and Environmental Engineering, Syracuse University, 3Northern Research Station, U.S. Forest Service, North Woodstock, NH, 4Department of Natural Resources, Cornell University, 5Department of Civil and Environmental Engineering, Southern Illinois University, 6Advanced Science Research Center at the Graduate Center, City University of New York, 7Cary Institute of Ecosystem Studies, 8Rubenstein School of Environment and Natural Resources, University of Vermont, 9Department of Forestry and Natural Resources, University of Kentucky, 10Northern Research Station, U.S. Forest Service, Burlington, VT

Ice storms are important weather events that are challenging to study because of difficulties in predicting their occurrence. Here, we describe a novel method for simulating ice storms that involves spraying water over a forest canopy during sub-freezing conditions.

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Biology

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion
Jing Zhang *1, Jing Huang *2, Ishani Majumdar 1, Ryan C. James 3, Julia Gichimu 1, Manikandan Paramasivam 4, Durga Pokharel 5, Himabindu Gali 6, Marina A. Bellani 1, Michael M Seidman 1
1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 2Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, 3Department of Molecular Biology and Genetics, Cornell University, 4Department of Cellular and Molecular Medicine, University of Copenhagen, 5Horizon Discovery, 6Boston University School of Medicine

While replication fork collisions with DNA adducts can induce double strand breaks, less is known about the interaction between replisomes and blocking lesions. We have employed the proximity ligation assay to visualize these encounters and to characterize the consequences for replisome composition.

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Engineering

Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy
Taylor Moon 1, Michael Colletta 1, Lena F. Kourkoutis 1,2
1School of Applied and Engineering Physics, Cornell University, 2Kavli Institute at Cornell for Nanoscale Science

Cryogenic Focused Ion Beam (FIB) and Scanning Electron Microscopy (SEM) techniques can provide key insights into the chemistry and morphology of intact solid-liquid interfaces. Methods for preparing high quality Energy Dispersive X-ray (EDX) spectroscopic maps of such interfaces are detailed, with a focus on energy storage devices.

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Neuroscience

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain
Yusaku Hontani *1, Najva Akbari *1, Kristine E. Kolkman 2, Chunyan Wu 1,3, Fei Xia 1,4, Kibaek Choe 1, Grace Zhang Wang 1, Mariya Sokolova 1, Joseph R. Fetcho 2, Chris Xu 1
1School of Applied and Engineering Physics, Cornell University, 2Department of Neurobiology and Behavior, Cornell University, 3College of Veterinary Medicine, Cornell University, 4Meinig School of Biomedical Engineering, Cornell University

Three-photon microscopy enables high-contrast fluorescence imaging deep in living biological tissues, such as mouse and zebrafish brains, with high spatiotemporal resolution.

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Biology

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes
Mabel Taracena 1,2, Catherine Hunt 1, Pamela Pennington 3, Deborah Andrew 4,5, Marcelo Jacobs-Lorena 5,6, Ellen Dotson 1, Michael Wells 5,7,8
1Division of Parasitic Diseases and Malaria, Entomology Branch, Centers for Disease Control and Prevention, 2Department of Entomology, Cornell University, 3Centro de Estudios en Biotecnologia, Universidad del Valle de Guatemala, 4Department of Cell Biology, Johns Hopkins School of Medicine, 5Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, 6Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health and Malaria Research Institute, 7Department of Cell Biology, Johns Hopkins School of Medicine, 8Biomedical Sciences Department, Idaho College of Osteopathic Medicine

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

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Bioengineering

A Fluorescent Intravital Imaging Approach to Study Load-Induced Calcium Signaling Dynamics in Mouse Osteocytes
Karl J. Lewis 1, James F. Boorman-Padgett 3, Macy Castaneda 2, David C. Spray 4, Mia M. Thi 5,6, Mitchell B. Schaffler 3
1Meinig School of Biomedical Engineering, Cornell University, 2Sibley School of Mechanical and Aerospace Engineering, Cornell University, 3Department of Biomedical Engineering, The City College of New York, 4Department of Neuroscience, Albert Einstein College of Medicine, 5Department of Orthopaedic Surgery, Albert Einstein College of Medicine, 6Department of Molecular Pharmacology, Albert Einstein College of Medicine

The current article describes performing an intravital imaging approach to observe mechanically induced calcium signaling of embedded osteocytes in vivo in real-time in response to tissue-level mechanical loading of the mouse third metatarsal.

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Biology

DeepOmicsAE: Representing Signaling Modules in Alzheimer's Disease with Deep Learning Analysis of Proteomics, Metabolomics, and Clinical Data
Elena Panizza 1
1Department of Molecular Medicine, Cornell University

DeepOmicsAE is a workflow centered on the application of a deep learning method (i.e., an autoencoder) to reduce the dimensionality of multi-omics data, providing a foundation for predictive models and signaling modules representing multiple layers of omics data.

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Developmental Biology

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm
Roy Cohen 1,2, Danielle M. Sosnicki 1, Melissa A. White 1, Jacquelyn L. Nelson 1, Chinatsu Mukai 1, Alexander J. Travis 1,2
1Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, 2Department of Public and Ecosystem Health, College of Veterinary Medicine, Cornell University

The AcroSensE mouse model and live cell imaging methods described here provide a new approach to studying calcium dynamics in the subcellular compartment of the sperm acrosome and how they regulate intermediate steps leading to membrane fusion and acrosome exocytosis.

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