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33 ARTICLES PUBLISHED IN JoVE

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Medicine

Microvascular Decompression: Salient Surgical Principles and Technical Nuances
Jonathan Forbes 1, Calvin Cooper *2, Walter Jermakowicz *2, Joseph Neimat *1, Peter Konrad *1
1Department of Neurosurgery, Vanderbilt University Medical Center, 2Vanderbilt School of Medicine, Vanderbilt University Medical Center

There are many available options for management of the patient with trigeminal neuralgia. Microvascular decompression, while the most invasive of all options, is also the most effective at achieving long term remission of symptoms. Video instruction on how to maximize efficacy and minimize complications with this procedure is described.

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Behavior

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease
Sheila M. Fleming 1,2, Osunde R. Ekhator 1,2, Valentins Ghisays 1
1Department of Psychology, University of Cincinnati, 2Department of Neurology, University of Cincinnati

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

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JoVE Core

Olfactory Assays for Mouse Models of Neurodegenerative Disease
Andrew M. Lehmkuhl 1, Emily R. Dirr 2,3, Sheila M. Fleming 1,2
1Department of Psychology, University of Cincinnati, 2Department of Neurology, University of Cincinnati, 3Department of Neuroscience, Cell Biology, and Physiology, Wright State University

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

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Medicine

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS
Touraj Shokati 1, Nicholas Bodenberger 1, Holly Gadpaille 1, Björn Schniedewind 1, Alexander A. Vinks 2, Wenlei Jiang 3, Rita R. Alloway 4, Uwe Christians 1
1iC42 Clinical Research and Development, University of Colorado, Anschutz Medical Campus, 2Division of Clinical Pharmacology, Cincinnati Children's Hospital Medical Center, 3Food and Drug Administration (FDA), Center of Drug Evaluation Research - Office of Generic Drugs, 4Transplant Clinical Research, University of Cincinnati

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

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Behavior

Vision Training Methods for Sports Concussion Mitigation and Management
Joseph F. Clark 1, Angelo Colosimo 2, James K. Ellis 3, Robert Mangine 3, Benjamin Bixenmann 4, Kimberly Hasselfeld 2, Patricia Graman 5, Hagar Elgendy 2, Gregory Myer 6, Jon Divine 2
1Neurology and Rehabilitative Medicine, University of Cincinnati, 2Division of Sports Medicine, Department of Orthopaedic Surgery, University of Cincinnati, 3Department of Athletics, University of Cincinnati, 4Department of Neurosurgery, University of Cincinnati, 5College of Education, Criminal Justice, and Human Services, University of Cincinnati, 6Division of Sports Medicine, Cincinnati Children's Hospital Medical Center

This paper describes a protocol to conduct, quantitatively monitor, and assess the success of vision training initiated as part of a sports medical management program including intervention for concussion prevention and performance enhancement.

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Engineering

Phase Diagram Characterization Using Magnetic Beads as Liquid Carriers
Nicholas Blumenschein 1, Daewoo Han 1, Andrew J. Steckl 1
1Department of Electrical Engineering and Computing Systems, University of Cincinnati

Here, we present a protocol to investigate multi-component phase diagrams using externally controlled magnetic beads as liquid carriers in a lab-in-tube approach. This approach can aid in applications that seek to gather further information on phase change in complex liquid systems.

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Medicine

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail
Chun Yu Wong 1,2,3, Margreet R. de Vries 2,3, Yang Wang 4, Joost R. van der Vorst 3, Alexander L. Vahrmeijer 3, Anton-Jan van Zonneveld 1,2, Jaap F. Hamming 3, Prabir Roy-Chaudhury 4, Ton J. Rabelink 1,2, Paul H. A. Quax 2,3, Joris I. Rotmans 1,2
1Department of Nephrology, Leiden University Medical Center, 2Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 3Department of Surgery, Leiden University Medical Center, 4Division of Nephrology, University of Cincinnati

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

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Biology

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)
Daniel E. Miller 1, Zubin H. Patel 1,2,3, Xiaoming Lu 1,3, Arthur T. Lynch 1, Matthew T. Weirauch *1,4, Leah C. Kottyan *1
1Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital, 2Medical Scientist Training Program, University of Cincinnati, 3Immunology Graduate Program, University of Cincinnati, 4Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

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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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Immunology and Infection

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens
Swinburne A. J. Augustine 1, Tarsha N. Eason 2, Kaneatra J. Simmons 1, Clarissa L. Curioso 3, Shannon M. Griffin 1, Malini K. D. Ramudit 1, Trevor R. Plunkett 4
1National Exposure Research Laboratory, U.S. Environmental Protection Agency, 2National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 3Oak Ridge Institute for Science and Education, 4Department of Biological Sciences, McMicken College of Arts and Sciences, University of Cincinnati

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

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Genetics

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
Huan-Huan Wei *1, Yuanlong Liu *1, Yang Wang 2, Qianyun Lu 1, Xuerong Yang 1, Jiefu Li 1, Zefeng Wang 1
1Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences (SIBS), 2Institute of Cancer Stem Cell, Second Affiliated Hospital, Cancer Center, Dalian Medical University

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

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JoVE Core

Ultrasound Images of the Tongue: A Tutorial for Assessment and Remediation of Speech Sound Errors
Jonathan L. Preston 1,2, Tara McAllister Byun 3, Suzanne E. Boyce 2,4, Sarah Hamilton 4, Mark Tiede 2, Emily Phillips 2, Ahmed Rivera-Campos 4, Douglas H. Whalen 2,5,6
1Department of Communication Sciences and Disorders, Syracuse University, 2Haskins Laboratories, 3Department of Communicative Sciences and Disorders, New York University, 4Department of Communication Sciences and Disorders, University of Cincinnati, 5Program in Speech-Language-Hearing Sciences, City University of New York Graduate Center, 6Department of Linguistics, Yale University

Ultrasound imaging can be used to display the shape and movements of the tongue in real time during speech. The images can be used to determine the nature of speech sound errors. Visual feedback of the tongue can be used to facilitate improvements in speech sound production in clinical populations.

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Medicine

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease
Victoria N. Jensen *1, Shannon H. Romer *2, Sarah M. Turner 3, Steven A. Crone 3
1Neuroscience Graduate Program, University of Cincinnati, 2Department of Biological Sciences, Wright State University, 3Division of Neurosurgery, Cincinnati Children's Hospital Medical Center

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

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Neuroscience

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult
Stephanie A. Herrlinger 1, Qiang Shao 2, Li Ma 2, Melinda Brindley 3, Jian-Fu Chen 2
1Biomedical and Health Sciences Institute, University of Georgia, 2Center for Craniofacial Molecular Biology, University of Southern California, 3Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

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Neuroscience

Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition
Michael D. Guthrie 1, Donald L. Gilbert 2, David A. Huddleston 2, Ernest V. Pedapati 2,3, Paul S. Horn 2, Stewart H. Mostofsky 4, Steve W. Wu 2
1College of Medicine, University of Cincinnati, 2Division of Neurology, Cincinnati Children's Hospital Medical Center, 3Division of Psychiatry, Cincinnati Children's Hospital Medical Center, 4Center for Neurodevelopmental and Imaging Research, Kennedy Krieger Institute

We describe an experimental procedure to quantify excitability and inhibition of primary motor cortex during a motor response inhibition task by using Transcranial Magnetic Stimulation throughout the course of a Stop Signal Task.

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Education

Collecting Hair Samples for Hair Cortisol Analysis in African Americans
Kathy D. Wright 1, Jodi L. Ford 1, Joseph Perazzo 2, Lenette M. Jones 3, Sherrilynn Mahari 4, Brent A. Sullenbarger 5,6,7, Mark L. Laudenslager 8
1College of Nursing, The Ohio State University, 2College of Nursing, University of Cincinnati, 3School of Nursing, University of Michigan, 4Licensed Cosmetologist Consultant, 5Clinical Research Manager, The Ohio State University, 6Biomedical Core Research Coordinator, The Ohio State University, 7College of Nursing Biomedical Core Research Laboratory, The Ohio State University, 8Department of Psychiatry, Behavioral Immunology & Endocrinology Laboratory, University of Colorado Denver Anschutz Medical Campus

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

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Immunology and Infection

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration
Emma Teal 1, Nina G. Steele 2, Jayati Chakrabarti 1, Loryn Holokai 3, Yana Zavros 1
1Department of Pharmacology and Systems Physiology, University of Cincinnati, 2Department of Cell & Developmental Biology, University of Michigan, 3Department of Molecular Genetics, Biochemistry & Microbiology, University of Cincinnati

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

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Medicine

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method
Esam S.B. Salem *1,2, Kazutoshi Murakami *2, Toshimasa Takahashi 2, Elise Bernhard 2, Vishnupriya Borra 2, Mridula Bethi 2, Takahisa Nakamura 2,3,4
1Department of Pharmacology and Systems Physiology, College of Medicine, University of Cincinnati, 2Division of Endocrinology, Cincinnati Children's Hospital Medical Center, 3Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 4Department of Pediatrics, College of Medicine, University of Cincinnati

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

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Bioengineering

Novel Process for 3D Printing Decellularized Matrices
Stacey M. S. Gruber 1, Paulomi Ghosh 2, Karl Wilhelm Mueller 2, Patrick W. Whitlock 1,2,3, Chia-Ying Lin 1,3
1Department of Biomedical Engineering, University of Cincinnati, 2Division of Orthopaedic Surgery, Cincinnati Children's Hospital Medical Center, 3Department of Orthopaedic Surgery, University of Cincinnati

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

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Neuroscience

A Bedside, Single Burr Hole Approach to Multimodality Monitoring in Severe Brain Injury
Brandon Foreman 1, Daryn Cass 2, Jonathan Forbes 2, Laura B. Ngwenya 2,3
1Department of Neurology and Rehabilitation Medicine, University of Cincinnati, 2Department of Neurosurgery, University of Cincinnati, 3Neurotrauma Center, University of Cincinnati Gardner Neuroscience Institute

A method of recording multimodality monitoring signals in patients with severe brain injuries using a bedside, single burr hole technique is described.

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JoVE Journal

Wholemount In Situ Hybridization for Astyanax Embryos
Heidi Luc 1, Connor Sears 1, Andrew Raczka 1, Joshua B. Gross 1
1Department of Biological Sciences, University of Cincinnati

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

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Immunology and Infection

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages
Yuxuan Zhen 1, Wen-Hai Shao 1
1Division of Immunology, Allergy, and Rheumatology, Department of Internal Medicine, College of Medicine, University of Cincinnati

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

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JoVE Journal

Rapid Repetition Rate Fluctuation Measurement of Soliton Crystals in a Microresonator
Peng Xie *1,2, Xinyu Wang *1,2, Weiqiang Wang 1,2, Wenfu Zhang 1,2, Zhizhou Lu 1,2, Yang Wang 1,2, Wei Zhao 1,2
1State Key Laboratory of Transient Optics and Photonics, Xi'an Institute of Optics and Precision Mechanics (XIOPM), Chinese Academy of Sciences (CAS), 2University of Chinese Academy of Sciences

Here, we present a protocol to generate soliton crystals in a butterfly-packaged micro-ring resonator using a thermal tuned method. Further, the repetition rate fluctuations of a soliton crystal with a single vacancy are measured using a delayed self-heterodyne method.

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Developmental Biology

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem
Yang Wang 1, Arun Sampathkumar 1
1Max Planck Institute of Molecular Plant Physiology

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

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Biology

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes
Perwez Alam 1, Bryan D. Maliken 1, Malina J. Ivey 1, Shannon M. Jones 1, Onur Kanisicak 1
1Department of Pathology and Laboratory Medicine, College of Medicine, University of Cincinnati

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

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Neuroscience

Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents
Aric F. Logsdon *1,2, Brandon P. Lucke-Wold *3, Ryan C. Turner *4, Sean M. Collins 5, Evan L. Reeder 5, Jason D. Huber 4, Charles L. Rosen 6, Matthew J. Robson *5, Florian Plattner *7
1Geriatrics Research Education and Clinical Center, Veterans Affairs, 2Division of Gerontology and Geriatric Medicine, University of Washington, 3Department of Neurosurgery, University of Florida, 4Department of Neurosurgery, West Virginia University, 5Division of Pharmaceutical Sciences, University of Cincinnati, 6Central Illinois Neuro Health Sciences, Bloomington, IL, 7Neuro-research, Dallas, TX

We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury.

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JoVE Journal

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points
Shubham Rathore 1, Jenni Hassert 1, Courtney M. Clark-Hachtel 2,3, Aaron Stahl 1,4, Yoshinori Tomoyasu 2, Elke K. Bushbeck 1
1Department of Biological Sciences, University of Cincinnati, 2Department of Biology, Miami University, 3Department of Biology, University of North Carolina at Chapel Hill, 4Department of Neuroscience, The Scripps Research Institute

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

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Bioengineering

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification
Zhenyuan Xu 1, Jacob A. Orkwis 1, Greg M. Harris 1,2,3
1Department of Chemical and Environmental Engineering, University of Cincinnati, 2Department of Biomedical Engineering, University of Cincinnati, 3Neuroscience Graduate Program, University of Cincinnati College of Medicine

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

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Developmental Biology

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus
Li Ma 1, Ruby Dessiatoun 1, Janet Shi 1, William R. Jeffery 1
1Department of Biology, University of Maryland

This article outlines the basic laboratory conditions and protocols for an incremental temperature regime to stimulate maximal spawning in the Mexican tetra Astyanax mexicanus, which is an emerging model for developmental and evolutionary studies.

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Bioengineering

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip
Varone Antonio 1, Adya Panchal 2, Magdalena Kasendra 3, Barrile Riccardo 2,3
1Merk Sharp & Dohme LLC, 2Department of Biomedical Engineering, University of Cincinnati, 3Center for Stem Cell and Organoid Medicine, Cincinnati Children's Hospital Medical Center

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

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Chemistry

A Scalable Balz-Schiemann Reaction Protocol in a Continuous Flow Reactor
Zhangtao Zhou 1,2, Yang Wang 1, Shuqiang Liu 1, Andrew Phillis 1,2, Qijun Zhang 1, Weiping Ye 1
1Shenzhen Hwagen Pharmaceutical Co., Ltd, 2Guangdong Raffles Pharmaceutical Technology Co., Ltd

A detailed scalable continuous flow protocol is presented to synthesize an aryl fluoride from an aryl amine through the Balz-Schiemann reaction.

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Biology

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens
Renata Belisário 1, Maria F. Torres 1,2, Ester A. S. Buiate 1,3, Katia V. Xavier 1,4, Etta M. Nuckles 1, Lisa J. Vaillancourt 1
1Department of Plant Pathology, University of Kentucky, 2Department of Biological Sciences, University of Cincinnati, 3Bayer Crop Science, 4Everglades Research and Education Center, University of Florida

This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues.

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Bioengineering

Hyperpolarized 129Xe Lung MRI and Spectroscopy in Mechanically Ventilated Mice
Mariah L. Costa 1,2,3, Joseph W. Plummer 1,2, Abdullah S. Bdaiwi 1, Brice J. Albert 4, Elizabeth M. Fugate 3, Peter J. Niedbalski 5,6,7, Diana M. Lindquist 3,8, Zackary I. Cleveland 1,2,3,8,9
1Center for Pulmonary Imaging Research, Division of Pulmonary Medicine, Cincinnati Children's Hospital Medical Center, 2Department of Biomedical Engineering, University of Cincinnati, 3Imaging Research Center, Department of Radiology, Cincinnati Children's Hospital Medical Center, 4Analytical Technology Group, Aurorium, 5Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, University of Kansas Medical Center, 6Department of Bioengineering, University of Kansas, 7Hoglund Biomedical Imaging Center, University of Kansas Medical Center, 8Department of Pediatrics, University of Cincinnati, 9Division of Pulmonary Medicine, Cincinnati Children's Hospital Medical Center

Hyperpolarized xenon MRI can quantify regional lung microstructure (air-space dimensions) and physiology (ventilation and gas exchange) in translational research and clinical care. Although challenging, it can provide comparable pulmonary insights in preclinical studies. This protocol describes the infrastructure and procedures needed to perform routine xenon lung MRI in mice.

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