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37 ARTICLES PUBLISHED IN JoVE

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Biology

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus
Wendy Sparks 1, Huarong Li 1, Bryony Bonning 1
1Department of Entomology, Iowa State University

In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

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Biology

Protocols for Microapplicator-assisted Infection of Lepidopteran Larvae with Baculovirus
Huarong Li 1, Wendy Sparks 1, Bryony Bonning 1
1Department of Entomology, Iowa State University

In this video, we demonstrate two microapplicator techniques used to infect of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

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Biology

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling
Weili Cai 1, Ye Jin 1, Jack Girton 1, Jorgen Johansen 1, Kristen M. Johansen 1
1Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

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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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Biology

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats
Curtis R. Youngs 1
1Animal Science Department, Iowa State University

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

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Neuroscience

Single-cell Profiling of Developing and Mature Retinal Neurons
Jillian J. Goetz 1, Jeffrey M. Trimarchi 1
1Department of Genetics, Development and Cell Biology, Neuroscience Program, Iowa State University

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

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Bioengineering

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries
Latrisha K. Petersen 1, Ana V. Chavez-Santoscoy 1, Balaji Narasimhan 1
1Department of Chemical and Biological Engineering, Iowa State University

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

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Bioengineering

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles
Ana V. Chavez-Santoscoy 1, Lucas M. Huntimer 2, Amanda E. Ramer-Tait 2, Michael Wannemuehler 2, Balaji Narasimhan 1
1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Veterinary Microbiology and Preventive Medicine, Iowa State University

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

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Bioengineering

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry
Yashdeep Phanse 1, Amanda E. Ramer-Tait 1, Sherree L. Friend 2, Brenda Carrillo-Conde 3, Paul Lueth 1, Carrie J. Oster 1, Gregory J. Phillips 1, Balaji Narasimhan 3, Michael J. Wannemuehler 1, Bryan H. Bellaire 1
1Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, 2Amnis Corporation, 3Department of Chemical and Biological Engineering, Iowa State University

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

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Bioengineering

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles
Brenda R. Carrillo-Conde *1, Rajarshi Roychoudhury *2, Ana V. Chavez-Santoscoy *1, Balaji Narasimhan 1, Nicola L.B. Pohl 1,2
1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Chemistry, Iowa State University

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

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Biology

Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm
Sergey Rabotyagov 1, Todd Campbell 2, Adriana Valcu 2, Philip Gassman 2, Manoj Jha 3, Keith Schilling 4, Calvin Wolter 4, Catherine Kling 2
1School of Environmental and Forest Sciences, University of Washington, 2Center for Agricultural and Rural Development, Department of Economics, Iowa State University, 3Department of Civil, Architectural, and Environmental Engineering, North Carolina A&T University, 4Iowa Geological and Water Survey

This work demonstrates an integration of a water quality model with an optimization component utilizing evolutionary algorithms to solve for optimal (lowest-cost) placement of agricultural conservation practices for a specified set of water quality improvement objectives. The solutions are generated using a multi-objective approach, allowing for explicit quantification of tradeoffs.

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Engineering

High-resolution, High-speed, Three-dimensional Video Imaging with Digital Fringe Projection Techniques
Laura Ekstrand 1, Nikolaus Karpinsky 1, Yajun Wang 1, Song Zhang 1
13D Machine Vision Laboratory, Department of Mechanical Engineering, Iowa State University

This video describes the fundamentals of digital fringe projection techniques, which provide dense 3D measurements of dynamically changing surfaces. It also demonstrates the design and operation of a high-speed binary defocusing system based on these techniques.

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Education

Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis
Jeremy D. Smith 1, Abbie E. Ferris 1, Gary D. Heise 1, Richard N. Hinrichs 2, Philip E. Martin 3
1School of Sport & Exercise Science, University of Northern Colorado, 2Kinesiology Program, Arizona State University, 3Department of Kinesiology, Iowa State University

Body segmental inertial properties are required for inverse dynamics modeling. Using an oscillation and reaction board technique, inertial properties of below-knee prostheses were measured. Using direct measures of prosthesis inertia in the inverse dynamics model of the prosthetic leg resulted in lower magnitudes of resultant joint forces and moments.

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JoVE Journal

Characterization of Thermal Transport in One-dimensional Solid Materials
Guoqing Liu 1, Huan Lin 1, Xiaoduan Tang 1, Kevin Bergler 1, Xinwei Wang 1
1Department of Mechanical Engineering, Iowa State University

The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials.

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Medicine

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies
Anup D. Sharma 1, Pavel A. Brodskiy 1,3, Emma M. Petersen 2,3, Melih Dagdeviren 2, Eun-Ah Ye 2, Surya K. Mallapragada 1, Donald Sakaguchi 2,3
1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Genetics, Development and Cell Biology, Iowa State University, 3Biology Program, Iowa State University

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

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Immunology and Infection

Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle
Mayara F. Maggioli 1,2, Mitchell V. Palmer 1, H. Martin Vordermeier 3, Adam O. Whelan 3, James M. Fosse 4, Brian J. Nonnecke 1, W. Ray Waters 1
1Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, 2Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, 3UK Veterinary Laboratories Agency, 4Visual Services, National Centers for Animal Health, Animal and Plant Health Inspection Service, United States Department of Agriculture

Long-term cultured interferon-γ enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

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Biology

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension
Ganesh P. Subedi 1, Roy W. Johnson 2, Heather A. Moniz 2, Kelley W. Moremen 2, Adam W. Barb 1
1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 2Complex Carbohydrate Research Center, University of Georgia

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

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Biology

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)
Johanna E. Kowalko 1, Li Ma 2, William R. Jeffery 3
1Genetics, Development and Cell Biology, Iowa State University, 2Department of Biological Sciences, University of Cincinnati, 3Department of Biology, University of Maryland

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

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Environment

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria
Markus Maisch 1,2, Wenfang Wu 1, Andreas Kappler 1, Elizabeth D. Swanner 1,2
1Department of Geosciences, University of Tuebingen, 2Department of Geological and Atmospheric Sciences, Iowa State University

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

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Immunology and Infection

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release
Unity Jeffery 1, Robert D. Gray 2, Dana N. LeVine 3
1Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, 2MRC Centre for Inflammation Research, University of Edinburgh, 3Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

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Neuroscience

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells
Lauren A. Laboissonniere 1, Takuma Sonoda 2, Seul Ki Lee 2, Jeffrey M. Trimarchi 1, Tiffany M. Schmidt 2
1Department of Genetics, Development, and Cell Biology, Iowa State University, 2Department of Neurobiology, Northwestern University

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

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Neuroscience

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility
Souvarish Sarkar *1, Emir Malovic *1, Brandon Plante 1, Gary Zenitsky 1, Huajun Jin 1, Vellareddy Anantharam 1, Arthi Kanthasamy 1, Anumantha G. Kanthasamy 1
1Biomedical Sciences & Iowa Center for Advanced Neurotoxicology, Iowa State University

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

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Developmental Biology

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents
Megan M. Johnson 1, Julianna Deardorff 1, Kimberly Parra 1, Abbey Alkon 2, Brenda Eskenazi 1, Elizabeth Shirtcliff 3
1Center for Environmental Research and Children's Health (CERCH), Berkley School of Public Health, University of California, Berkeley, 2San Francisco (UCSF) School of Nursing, University of California, San Francisco, 3Human Development and Family Studies, Iowa State University

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

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Medicine

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats
John R. Wagner 1, Takashi Taguchi 1, Jane Y. Cho 2, Chandrashekhar Charavaryamath 3, Dominique J. Griffon 1
1College of Veterinary Medicine, Western University of Health Sciences, 2Applied Medical, 3College of Veterinary Medicine, Iowa State University

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

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Education

Separation of Uranium and Thorium for 230Th-U Dating of Submarine Hydrothermal Sulfides
Lisheng Wang 1,2,3, Xuefeng Wang 1,2, Jun Ye 4, Zhibang Ma 1,2, Weifang Yang 5, Jule Xiao 1,2
1Key Laboratory of Cenozoic Geology and Environment, Institute of Geology and Geophysics, Chinese Academy of Sciences, 2Institutions of Earth Science, Chinese Academy of Sciences, 3University of Chinese Academy of Sciences, 4First Institute of Oceanography, State Oceanic Administration, 5Key Laboratory of Submarine Geosciences, Second Institute of Oceanography, State Oceanic Administration

The protocol describes a method to purify and separate the U and Th nuclide in submarine hydrothermal sulfide sample with Fe co-precipitation and extraction chromatography for 230Th-U disequilibrium dating.

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Behavior

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte)
Eric Yu Yu 1, Aaron J. Gassmann 1, Thomas W. Sappington 2
1Department of Entomology, Iowa State University, 2Corn Insects & Crop Genetics Research Unit, USDA-ARS

Flight mills are important tools for comparing how age, sex, mating status, temperature, or various other factors may influence an insects flight behavior. Here we describe protocols to tether and measure the flight propensity and performance of western corn rootworm under different treatments.

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Biochemistry

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle
Sujin Kim 1, Minjeong Kang 1, Yoosik Kim 1
1Department of Chemical and Biomolecular Engineering and KI for Health Science and Technology (KIHST), Korea Advanced Institute of Science and Technology (KAIST)

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

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Biochemistry

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors
Fatima Enam 1, Thomas J. Mansell 1
1Department of Chemical and Biological Engineering, Iowa State University

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

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JoVE Journal

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor
Yuanchang Zhao 1, Nathaniel M. Wetter 1, Xuefeng Wang 2
1Department of Physics and Astronomy, Iowa State University, 2Department of Physics and Astronomy, Molecular, Cellular, and Developmental Biology Interdepartmental Program, Iowa State University

Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution.

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Biology

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes
Alicia Masters 1,2, Minjeong Kang 1,3,4, Morgan McCaw 1,3, Jacob D. Zobrist 1,3,5, William Gordon-Kamm 2, Todd Jones 2, Kan Wang 1,3
1Department of Agronomy, Iowa State University, 2Department of Applied Science and Technology, Corteva Agriscience, 3Crop Bioengineering Center, Iowa State University, 4Interdepartmental Plant Biology Major, Iowa State University, 5Interdepartmental Genetics and Genomics Major, Iowa State University

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

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Biology

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones
Bliss M. Beernink *1, Katerina L. Holan *1, Ryan R. Lappe 1, Steven A. Whitham 1
1Department of Plant Pathology and Microbiology, Iowa State University

An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP.

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Biochemistry

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the µs-ms Timescale
Aayushi Singh 1, Jeffrey A. Purslow 1, Vincenzo Venditti 1,2
1Department of Chemistry, Iowa State University, 2Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University

Here, a detailed description of the protocol implemented in the laboratory for acquisition and analysis of 15N relaxation dispersion profiles by solution NMR spectroscopy is provided.

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Cancer Research

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1α Nanoparticles
M. M. Hasibuzzaman 1,2, Kathleen A. Ross 3,4, Aliasger K. Salem 1,4,5,6, Balaji Narasimhan 3,4, Andrean L. Simons 1,2,3,5,6,7
1Interdisciplinary Graduate Program in Human Toxicology, University of Iowa, 2Department of Pathology, University of Iowa, 3Department of Chemical and Biological Engineering, College of Engineering, Iowa State University, 4Nanovaccine Institute, Iowa State University, 5Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa, 6Holden Comprehensive Cancer Center, University of Iowa, 7Department of Oral Pathology, Radiology and Medicine, College of Dentistry, University of Iowa

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1α in a syngeneic mouse model of HNSCC.

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Biochemistry

High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States
Trang T. Nguyen 1, Steven Siang 2, Julien Roche 2
1Department of Chemistry, Iowa State University, 2Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University

We provide a detailed description of the steps required to assemble a high-pressure cell, set up and record high-pressure NMR experiments, and finally analyze both peak intensity and chemical shift changes under pressure. These experiments can provide valuable insights into the folding pathways and structural stability of proteins.

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Bioengineering

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems
Jared L. Dopp 1, Nigel F. Reuel 1
1Chemical and Biological Engineering, Iowa State University

The study describes a protocol for creating large (µg-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

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Medicine

Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research
Vojtech Gabriel 1, Christopher Zdyrski 1, Dipak K. Sahoo 2, Kimberly Dao 3, Agnes Bourgois-Mochel 2, Jamie Kopper 2, Xi-Lei Zeng 4, Mary K. Estes 4, Jonathan P. Mochel 1,3, Karin Allenspach 2,3
1Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, 2Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, 33D Health Solutions Inc., 4Department of Molecular Virology and Microbiology, Baylor College of Medicine

Experimental methods to harvest adult stem cells from canine intestinal and hepatic tissues to establish 3D organoid cultures are described. Furthermore, the laboratory techniques to ensure consistent growth and provide standard operating procedures to harvest, biobank, and revive canine intestinal and hepatic organoid cultures are discussed.

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Immunology and Infection

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System
Vojtech Gabriel 1, Christopher Zdyrski 1, Dipak K. Sahoo 2, Kimberly Dao 3, Agnes Bourgois-Mochel 2, Todd Atherly 3, Marilyn N. Martinez 4, Donna A. Volpe 5, Jamie Kopper 2, Karin Allenspach 2,3, Jonathan P. Mochel 1,3
1Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, 2Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, 33D Health Solutions Inc., 4Office of New Animal Drug Evaluation, Center for Veterinary Medicine, Food and Drug Administration, 5Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, Food and Drug Administration

Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.

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