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33 ARTICLES PUBLISHED IN JoVE

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Biology

Measuring Caenorhabditis elegans Life Span on Solid Media
George L. Sutphin 1,2, Matt Kaeberlein 1
1Department of Pathology, University of Washington, 2Molecular and Cellular Biology Program, University of Washington

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

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Medicine

Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty
Hilary C. Rees 1, Voichita Ianas 1, Patricia McCracken 1, Shannon Smith 1, Anca Georgescu 1, Tirdad Zangeneh 1, Jane Mohler 2, Stephen A. Klotz 1
1Division of Infectious Diseases, University of Arizona, 2Arizona Center on Aging, University of Arizona

Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.

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Medicine

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice
Hao Chen 1, Jessica N. Perez 1, Eleni Constantopoulos 1, Laurel McKee 1, Jessica Regan 1, Patricia B. Hoyer 1, Heddwen L. Brooks 1, John Konhilas 1
1Department of Physiology, University of Arizona

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

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Bioengineering

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis
Sang-Hyuck Park 1, Rebecca Garlock Ong 2, Chuansheng Mei 3, Mariam Sticklen 4
1The School of Plant Sciences, University of Arizona, 2Department of Chemical Engineering and Materials Science, DOE Great Lakes Bioenergy Research Center, Michigan State University, 3The Institute for Sustainable and Renewable Resources, The Institute for Advanced Learning and Research, 4Department of Plant, Soil and Microbial Sciences, Michigan State University

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

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Biology

Tissue Triage and Freezing for Models of Skeletal Muscle Disease
Hui Meng 1, Paul M.L. Janssen 2, Robert W. Grange 3, Lin Yang 4, Alan H. Beggs 5, Lindsay C. Swanson 5, Stacy A. Cossette 1,6, Alison Frase 7, Martin K. Childers 8, Henk Granzier 9, Emanuela Gussoni 5, Michael W. Lawlor 1
1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 2Department of Physiology and Cell Biology, The Ohio State University, 3Department of Human Nutrition, Foods and Exercise, Virginia Tech, 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, 5Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School, 6Cure Congenital Muscular Dystrophy, 7Joshua Frase Foundation, 8Department of Rehabilitation Medicine, University of Washington, 9Department of Physiology, University of Arizona

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

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Environment

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)
Rousel A. Orozco 1, Ming-Min Lee 1, S. Patricia Stock 1
1Department of Entomology, University of Arizona

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

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Bioengineering

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)
John G. McMullen II 1, S. Patricia Stock 2
1School of Animal and Comparative Biomedical Sciences, University of Arizona, 2Department of Entomology, University of Arizona

The goal of this presentation is to demonstrate in vivo and in vitro techniques for the rearing of entomopathogenic nematodes. In vivo methods consider the rearing of these nematodes with an insect host, whereas the in vitro methods utilize rich agar media.

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Bioengineering

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids
Bradley P. Weegman 1, Ahmad Essawy 2, Peter Nash 2, Alexandra L. Carlson 2, Kristin J. Voltzke 3, Zhaohui Geng 2, Marjan Jahani 2, Benjamin B. Becker 2, Klearchos K. Papas 4, Meri T. Firpo 2
1Radiology, University of Minnesota, 2Medicine, University of Minnesota, 3School of Public Health, University of Minnesota, 4Surgery, University of Arizona

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

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Neuroscience

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii
Carla M. Cabral 1, Anita A. Koshy 1,2,3
1Bio5 Institute, University of Arizona, 2Department of Neurology, University of Arizona, 3Department of Immunobiology, University of Arizona

Using this protocol, we were able to image 160 µm thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

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Biology

High Throughput Danio Rerio Energy Expenditure Assay
Savannah Y. Williams 1, Benjamin J. Renquist 2
1Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 2School of Animal and Comparative Biomedical Sciences, University of Arizona

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

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Medicine

Exergaming in Older People Living with HIV Improves Balance, Mobility and Ameliorates Some Aspects of Frailty
Suhitha Veeravelli 1, Bijan Najafi 3, Ivan Marin 3, Fernando Blumenkron 2, Shannon Smith 2, Stephen A. Klotz 2
1Department of Surgery, Interdisciplinary Consortium on Advanced Motion Performance (iCAMP), College of Medicine, University of Arizona, 2Department of Medicine, Division of Infectious Disease, College of Medicine, University of Arizona, 3Interdisciplinary Consortium on Advanced Motion Performance (iCAMP), Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine

Persons infected with HIV are often frail, depressed and live a sedentary lifestyle for which conventional exercise is too taxing. Here, we present an exercise protocol that ameliorates aspects of frailty in HIV-infected persons. An exergame integrating cognitive control was developed using biosensors that measured balance, weight-shifting and obstacle crossing.

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JoVE Journal

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations
Aditi Sengupta 1, Yadi Wang 2, Antonio A. Meira Neto 3, Katarena A. Matos 3, Katerina Dontsova 1,2, Rob Root 2, Julie W. Neilson 2, Raina M. Maier 2, Jon Chorover 1,2, Peter A. Troch 1,3
1Biosphere 2, University of Arizona, 2Department of Soil, Water and Environmental Science, University of Arizona, 3Department of Hydrology and Water Resources, University of Arizona

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

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Genetics

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms
Blake L. Joyce 1,2, Asher K. Haug-Baltzell 3, Jonathan P. Hulvey 4, Fiona McCarthy 5, Upendra Kumar Devisetty 1,6, Eric Lyons 1,2,3
1BIO5 Institute, University of Arizona, 2The School of Plant Sciences, University of Arizona, 3Genetics GIDP, University of Arizona, 4Biology Department, University of Massachusetts Amherst, 5School of Animal and Comparative Biomedical Sciences, University of Arizona, 6CyVerse, University of Arizona

This protocol outlines a comparative de novo transcriptome assembly and annotation workflow for novice bioinformaticians. The workflow is available for free entirely through CyVerse and connected by the Data Store. Command line and graphical user interfaces are used, but all code needed is available to copy and paste.

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Immunology and Infection

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds
Kevin L. Hockett 1, David A. Baltrus 1
1School of Plant Sciences, University of Arizona

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

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Behavior

Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children – Working Memory (CABC-WM)
Kathryn Cabbage 1, Shara Brinkley 2, Shelley Gray 2, Mary Alt 3, Nelson Cowan 4, Samuel Green 5, Trudy Kuo 6, Tiffany P. Hogan 1
1Communication Sciences and Disorders, MGH Institute of Health Professions, 2Speech and Hearing Science, Arizona State University, 3Speech, Language, and Hearing Sciences, University of Arizona, 4Department of Psychological Sciences, University of Missouri-Columbia, 5Sanford School of Social and Family Dynamics, Arizona State University, 6School of Social and Behavioral Sciences, New College of Interdisciplinary Arts and Sciences, Arizona State University - West

Working memory predicts a significant amount of variance for a variety of cognitive tasks, including speaking, reading, and writing. However, few tools are available to assess working memory in children. We present an innovative, computer-based battery that comprehensively assesses different components of working memory in school-age children.

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Medicine

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection
Hao Chen 1, Robert Klein 1, Stacy Arnold 1, Yiying Wang 2, Setsuko Chambers 3,4, Wenxin Zheng 5,6
1Department of Pathology, University of Arizona College of Medicine, 2Department of Obstetrics and Gynecology, Henan Provincial People's Hospital, 3Department of Obstetrics and Gynecology, University of Arizona College of Medicine, 4University of Arizona Cancer Center, 5Department of Pathology, University of Texas Southwestern Medical Center, 6Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

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Neuroscience

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ
Kimberly Young 1, Helena Morrison 1
1College of Nursing, University of Arizona

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

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Cancer Research

Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System
Hao Chen *1, Thomas M.B. Ware *1, Josephine Iaria 1, Hong-Jian Zhu 1
1Department of Surgery (RMH), University of Melbourne

Here, we present a protocol for live cell imaging of TGF-β/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

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Environment

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures
Malak M. Tfaily *1,2, Rachel M. Wilson *3, Heather M. Brewer 1, Rosalie K. Chu 1, Heino M. Heyman 4, David W. Hoyt 1, Jennifer E. Kyle 5, Samuel O. Purvine 1
1Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 2Department of Soil, Water and Environmental Science, University of Arizona, 3Department of Earth Ocean and Atmospheric Sciences, Florida State University, 4Bruker Daltonics Inc., 5Biological Sciences Division, Pacific Northwest National Laboratory

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

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Genetics

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique
Elizabeth C. Bondy 1, Martha S. Hunter 2
1Graduate Interdisciplinary Program in Entomology and Insect Science, University of Arizona, 2Department of Entomology, University of Arizona

We present a simple cytogenetic technique using 4′,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

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Medicine

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics
Rachel Meyer 1, Wen Yu Wong 2, Roberto Guzman 3, Randy Burd 1, Kirsten Limesand 1,2
1Department of Nutritional Sciences, University of Arizona, 2Cancer Biology Graduate Interdisciplinary Program, University of Arizona, 3Department of Chemical Engineering, University of Arizona

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50–90 μm thick salivary gland sections prior to and following exposure to ionizing radiation.

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Developmental Biology

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells
Aditi Bapat 1, Nakia Keita 1, Shalini Sharma 1
1Department of Basic Medical Sciences, College of Medicine-Phoenix, University of Arizona

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

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Neuroscience

Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9
Irina Sinakevitch 1,2, Zev Kurtzman *2, Hyun G. Choi *2, David Arturo Ruiz Pardo 3, Romain A. Dahan 2, Nathaniel Klein 1, Branimir Bugarija 4, Erik Wendlandt 4, Brian H. Smith 2
1Department of Neuroscience, University of Arizona, 2School of Life Sciences, Arizona State University, 3Department of Scientific and Technologic Investigations, University of Sonora, 4Integrated DNA Technologies, Inc.

Presented here is a protocol to use the CRISPR-Cas9 system for reducing the production of a protein in the adult honeybee brain to test antibody specificity.

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Behavior

Examining Changes in HRV and Emotion Following Artmaking with Three Different Art Materials
Johanna Czamanski-Cohen 1, Giora Galili 2, John J. B. Allen 3
1The Emili Sagol Creative Arts Therapies Research Center, The School of Creative Arts Therapies, The Faculty of Health and Welfare, University of Haifa, 2The Department of Education and Psychology, The Open University of Israel, 3Department of Psychology, University of Arizona

The goal of the protocol is to guide researchers in conducting experiments that are intended to measure changes in self-reported emotional response and heart rate variability following art making with different materials. The protocol can easily be adapted for use in a variety of behavioral conditions and activities.

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Environment

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation
Margarita Acedo 1, Juan R. Gonzalez Cena 2, Kasi M. Kiehlbaugh 3, Kimberly L. Ogden 1,2
1Department of Chemical and Environmental Engineering, University of Arizona, 2Department of Biosystems Engineering, University of Arizona, 3Department of Biomedical Engineering, University of Arizona

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

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JoVE Journal

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
Jason Wong 1, William Martelly 1, Shalini Sharma 1
1Department of Basic Medical Sciences, College of Medicine-Phoenix, University of Arizona

This protocol describes a minigene reporter assay to monitor the impact of 5´-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

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Biochemistry

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass
Kenneth J. Olejar *1, Min Hong *2, Sun-Yeop Lee 2, Tae-Hyung Kwon 2, Soo-Ung Lee 2, Chad A. Kinney 1,3, Joon-Hee Han 2, Sang-Hyuck Park 3
1Department of Chemistry, Colorado State University-Pueblo, 2Chuncheon Bioindustry Foundation, 3Institute for Cannabis Research, Colorado State University-Pueblo

Ultrasonic-assisted extraction (UAE) increases extraction efficiency of solvents and when applied to Cannabis spp. biomass it reduces the time required for extraction. This decreases the cost and potential cannabinoid loss due to degradation. Additionally, UAE is considered a green method due to low solvent use.

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Bioengineering

Employing Aeroponic Systems for the Clonal Propagation of Cannabis
Trevor Regas 1, Joon-Hee Han 2, Christopher S. Pauli 3, Sang-Hyuck Park 1,3
1Biology Department, Colorado State University-Pueblo, 2Chuncheon Bioindustry Foundation, 3Institute of Cannabis Research, Colorado State University-Pueblo

This protocol is designed to provide instructional information for the clonal propagation of Cannabis sativa L. by implementing aeroponic systems. The method described here includes all necessary supplies and protocols to successfully reproduce desirable morphological and chemical properties in the genus Cannabis.

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Biology

Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies
Sang-Hyuck Park 1, Samuel Koch 2, Katherine Richardson 3, Christopher Pauli 1, Joon-Hee Han 4, Tae-Hyung Kwon 4
1Institute of Cannabis Research, Colorado State University-Pueblo, 2Department of Biology, Colorado State University-Pueblo, 3Department of Chemistry, Colorado State University-Pueblo, 4Chuncheon Bioindustry Foundation

The present protocol provides instructional information for using tobacco hornworm Manduca sexta in cannabinoid research. The method described here includes all necessary supplies and protocols to monitor physiological and behavioral changes of the insect model in response to cannabidiol (CBD) treatment.

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Cancer Research

Spontaneous Murine Model of Anaplastic Thyroid Cancer
Huayun Yan 1, Yingfang Ma 1, Xinyue Zhou 2, Yushuang He 3, Yang Liu 2, Carlos Caulin 4, Leiming Wang 5, Heng Xu 1,6, Han Luo 2,6
1State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, 2Division of Thyroid Surgery, Laboratory of Thyroid and Parathyroid Disease, Frontiers Science Center for Disease-Related Molecular Network, Department of General Surgery, West China Hospital, Sichuan University, 3Department of Ultrasound, West China Hospital of Sichuan University, Sichuan University, 4Department of Otolaryngology-Head & Neck Surgery and University of Arizona Cancer Center, University of Arizona, 5Center for Translational Medicine, Shenzhen Bay Laboratory, 6Division of Laboratory Medicine/Research Centre of Clinical Laboratory Medicine, West China Hospital, Sichuan University

Here, we present a standard pipeline to obtain murine ATC tumors by spontaneous genetically engineered mouse models. Further, we present tumor dynamics and pathological information about the primary and metastasized lesions. This model will help researchers to understand tumorigenesis and facilitate drug discoveries.

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Biology

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices
Emily A. Gardea 1, Destiny DeNicola 1, Samuel Freitas 1, Will Peterson 1, Hope Dang 1, Karissa Shuck 1, Christopher Fang-Yen 2, George L. Sutphin 1
1Department of Molecular & Cellular Biology, University of Arizona, 2Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

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JoVE Journal

Modifying Levels of Maternal Dietary Folic Acid or Choline to Study the Impact of Deficiencies on Offspring Health Outcomes
Mitra Esfandiarei 1,2,3, Teodoro Bottiglieri 4, Nafisa M. Jadavji 5,6,7
1Department of Biomedical Sciences, Midwestern University, 2Department of Basic Medical Sciences, College of Medicine Phoenix, University of Arizona, 3Department of Anesthesiology, Pharmacology, & Therapeutics, Faculty of Medicine, University of British Columbia, 4Center of Metabolomics, Institute of Metabolic Disease, Baylor Scott & White Research Institute, 5Department of Physiology, Southern Illinois University, 6Department of Child Health, College of Medicine Phoenix, University of Arizona, 7Department of Neuroscience, Carleton University

This protocol describes the method for reducing dietary intake of folic acid or choline in female mice prior to pregnancy with the objective of investigating the impact of maternal diet on offspring health outcomes.

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Bioengineering

Motor Imagery Performance Through Embodied Digital Twins in a Virtual Reality-Enabled Brain-Computer Interface Environment
Kishor Lakshminarayanan 1, Rakshit Shah 2, Vadivelan Ramu 1, Deepa Madathil 3, Yifei Yao 4, Inga Wang 5, Brahim Brahmi 6, Mohammad Habibur Rahman 7
1Department of Sensors and Biomedical Tech, School of Electronics Engineering, Vellore Institute of Technology, 2Department of Orthopaedic Surgery, University of Arizona, 3Jindal Institute of Behavioural Sciences, O. P. Jindal Global University, 4Soft Tissue Biomechanics Laboratory, Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, 5Department of Occupational Science & Technology, University of Wisconsin-Milwaukee, 6Electrical Engineering, Collège Ahuntsic, 7Department of Mechanical Engineering, BioRobotics Lab, University of Wisconsin-Milwaukee

Motor imagery in a virtual reality environment has wide applications in brain-computer interface systems. This manuscript outlines the use of personalized digital avatars that resemble the participants performing movements imagined by the participant in a virtual reality environment to enhance immersion and a sense of body ownership.

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