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University of Michigan, Ann Arbor

40 ARTICLES PUBLISHED IN JoVE

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Biology

Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy
Mark H. Shalinsky 1, Iouila Kovelman 1, Melody S. Berens 2, Laura-Ann Petitto 2
1Department of Psychology, University of Michigan, Ann Arbor, 2Department of Psychology, University of Toronto Scarborough

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

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Immunology and Infection

Plaque Assay for Murine Norovirus
Mariam B. Gonzalez-Hernandez 1, Juliana Bragazzi Cunha 1, Christiane E. Wobus 1
1Department of Microbiology and Immunology, University of Michigan, Ann Arbor

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV’s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

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Medicine

Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases
Dongshan Yang 1, Jifeng Zhang 1, Jie Xu 1, Tianqing Zhu 1, Yanbo Fan 1, Jianglin Fan 2, Y. Eugene Chen 1
1Center for Advanced Models for Translational Sciences and Therapeutics, Department of Internal Medicine, University of Michigan Medical Center, 2Department of Molecular Pathology, University of Yamanashi

Recent development in gene targeting tools makes production of knockout (KO) rabbits possible. In the present work, we generated five Apolipoprotein (Apo) C-III KO rabbits using Zinc Finger Nucleases (ZFN). This work demonstrated that ZFN is a highly efficient method to produce KO rabbits.

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Bioengineering

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting
Christopher W. Coyne 1, Karan Patel 1, Johanna Heureaux 1, Jeanne Stachowiak 3, Daniel A. Fletcher 4,5, Allen P. Liu 1,2
1Department of Mechanical Engineering, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Department of Biomedical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 4Department of Bioengineering, University of California, Berkeley, 5Physical Biosciences Division, Lawrence Berkeley National Laboratory

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

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Medicine

Direct Mouse Trauma/Burn Model of Heterotopic Ossification
Jonathan R. Peterson 1, Shailesh Agarwal 1, R. Cameron Brownley 1, Shawn J. Loder 1, Kavitha Ranganathan 1, Paul S. Cederna 1, Yuji Mishina 2, Stewart C. Wang 1, Benjamin Levi 1
1Department of Surgery, University of Michigan Medical School, 2Department of Biologic and Materials Sciences, University of Michigan School of Dentistry

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

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Medicine

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet
Pierre-Emmanuel Noly 1, Julien Guihaire 1,4, Matthieu Coblence 1, Peter Dorfmüller 2,5, Elie Fadel 1,3, Olaf Mercier 1,3
1Surgical Research Lab, Marie Lannelongue Hospital, 2Department of Pathology, Marie Lannelongue Hospital, 3Department of Thoracic and Vascular Surgery, Marie Lannelongue Hospital, 4Thoracic and Cardiovascular Surgery, University Hospital of Rennes, 5INSERM U999 Paris-Sud University

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

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JoVE Journal

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope
Zackary N. Scholl 1, Qing Li 1, Eric Josephs 1, Dimitra Apostolidou 1, Piotr E. Marszalek 1
1Mechanical Engineering and Materials Science, Duke University

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

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Cancer Research

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity
Yanlan Wang 1,2, Jiayu Liu 1,2, Haitao Pan 1,2, Jieyu Xing 1,2, Xiaoqiong Wu 1,2, Qing Li 1,2, Zhong Wang 1,2
1School of Pharmaceutical Sciences, Sun Yat-Sen University, 2Center for Cellular & Structural Biology, Sun Yat-Sen University

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

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Bioengineering

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope
Yashar Bashirzadeh *1, Siddharth Chatterji *1, Dakota Palmer *2, Sandeep Dumbali *1, Shizhi Qian 1, Venkat Maruthamuthu 1
1Department of Mechanical & Aerospace Engineering, Old Dominion University, 2Department of Biological Sciences, Old Dominion University

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

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Biochemistry

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts
Ye Guan 1,2, Shiyuan Wang 1, Minjun Jin 2, Haotian Xu 3, Qiong Yang 1,4
1Department of Biophysics, University of Michigan, Ann Arbor, 2Department of Chemistry, University of Michigan, Ann Arbor, 3Department of Computer Science, Wayne State University, 4Department of Physics, University of Michigan, Ann Arbor

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

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Medicine

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay
Gemma Pearson 1, Scott A. Soleimanpour 1,2
1Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, 2VA Ann Arbor Healthcare System

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

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Genetics

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation
Linyuan Ma *1, Lydia Jang *1, Jian Chen 2, Jun Song 1, Dongshan Yang 1, Jifeng Zhang 1, Y. Eugene Chen 1, Jie Xu 1
1Center for Advanced Models for Translational Sciences and Therapeutics, Department of Internal Medicine, University of Michigan Medical Center, 2Celetrix LLC

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

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Bioengineering

Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat
Shelby R. Svientek 1, Dan C. Ursu 1, Paul S. Cederna 1,2, Stephen W. P. Kemp 1,2
1Department of Surgery, Division of Plastic Surgery, University of Michigan, Ann Arbor, 2Department of Biomedical Engineering, University of Michigan, Ann Arbor

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

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Bioengineering

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels
Ariel A. Szklanny 1, Dylan B. Neale 2, Joerg Lahann 2, Shulamit Levenberg 1
1Faculty of Biomedical Engineering, Technion, 2Department of Chemical Engineering and Biointerfaces Institute, University of Michigan, Ann Arbor

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

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Genetics

R-Loop Analysis by Dot-Blot
Prisila Ramirez 1, Robert J. Crouch 2, Vivian G. Cheung 1,3,4, Christopher Grunseich 5
1Life Sciences Institute, University of Michigan, Ann Arbor, 2Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 3Department of Pediatrics, University of Michigan, Ann Arbor, 4Howard Hughes Medical Institute, 5Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health

This protocol details a simple method that quantifies R-loop, a three-stranded nucleic acid structure that comprises of an RNA-DNA hybrid and a displaced DNA strand.

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Immunology and Infection

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model
Kevin Boerner 1, Anny-Claude Luissint 1, Charles A. Parkos 1
1Department of Pathology, University of Michigan, Ann Arbor

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

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Immunology and Infection

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model
Vicky Garcia-Hernandez 1, Philipp-Alexander Neumann 2, Stefan Koch 3,4, Renae Lyons 1, Asma Nusrat 1, Charles A. Parkos 1
1Department of Pathology, University of Michigan, 2Department of Surgery, Klinikum Rechts der Isar, Technische Universität München, 3Wallenberg Centre for Molecular Medicine, Linköping University, 4Department of Biomedical and Clinical Sciences (BKV), Linköping University

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

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Biology

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer
Surinder Kumar 1, Morgan Jones 2, Qing Li 2,3,4, David B. Lombard 1,4,5
1Department of Pathology, University of Michigan, Ann Arbor, 2Department of Internal Medicine, University of Michigan, Ann Arbor, 3Department of Cellular and Developmental Biology, University of Michigan, Ann Arbor, 4Rogel Cancer Center, University of Michigan, Ann Arbor, 5Institute of Gerontology, University of Michigan, Ann Arbor

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

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Bioengineering

Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording
Julianna M. Richie 1, Paras R. Patel 1, Elissa J. Welle 1, Tianshu Dong 2, Lei Chen 3, Albert J. Shih 2, Cynthia A. Chestek 1,4,5,6
1Department of Biomedical Engineering, University of Michigan, Ann Arbor, 2Department of Mechanical Engineering, University of Michigan, Ann Arbor, 3Department of Mechanical Engineering, University of Massachusetts Lowell, 4Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, 5Neuroscience Graduate Program, University of Michigan, Ann Arbor, 6Robotics Graduate Program, University of Michigan, Ann Arbor

Here, we describe fabrication methodology for customizable carbon fiber electrode arrays for recording in vivo in nerve and brain.

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Bioengineering

The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals
Shelby R. Svientek 1, Justin P. Wisely 1, Amir Dehdashtian 1, Jarred V. Bratley 1, Paul S. Cederna 1,2, Stephen W. P. Kemp 1,2
1Department of Surgery, Section of Plastic Surgery, The University of Michigan Health System, 2Department of Biomedical Engineering, The University of Michigan, Ann Arbor

This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI). This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices.

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Bioengineering

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles
Yashar Bashirzadeh *1, Nadab Wubshet *1, Thomas Litschel 2, Petra Schwille 3, Allen P. Liu 1,4,5,6
1Department of Mechanical Engineering, University of Michigan, Ann Arbor, 2John A. Paulson School of Engineering and Applied Sciences, Harvard University, 3Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, 5Department of Biophysics, University of Michigan, Ann Arbor, 6Cellular and Molecular Biology Program, University of Michigan, Ann Arbor

This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

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Biology

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase
Chao Xue 1, Guizhen Zhao 1, Yang Zhao 1, Y Eugene Chen 1, Jifeng Zhang 1
1Department of Internal Medicine, Frankel Cardiovascular Center, University of Michigan, Ann Arbor

The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.

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Medicine

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes
Emily A. Lavey 1, Margaret V. Westfall 1
1Department of Cardiac Surgery, Michigan Medicine, University of Michigan, Ann Arbor

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

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Bioengineering

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers
Andre Monteiro da Rocha 1, Andrew Allan 2, Travis Block 3, Jeffery Creech 1, Todd J. Herron 1
1Frankel Cardiovascular Regeneration Core Laboratory, Cardiovascular Medicine-Internal Medicine, University of Michigan, Ann Arbor, 2CAIRN Research, 3StemBioSys, Inc.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

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Medicine

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model
Pierre-Emmanuel Noly 1,2, Wei Huang 1, Suyash Naik 1, Paul Tang 1, Ienglam Lei 1
1University of Michigan, Ann Arbor, 2University of Montreal

Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system.

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JoVE Core

Troubleshooting FoCUS Image Acquisition: Patient Positioning, Transducer Manipulation, and Image Optimization
Adam L. Gottula 1,2,3, Suhas Devangam 1, Jessica L. Koehler 2, Matthew J. Sigakis 1
1Department of Anesthesiology, Division of Critical Care, University of Michigan, Ann Arbor, 2Department of Emergency Medicine, University of Michigan, Ann Arbor, 3Max Harry Weil Institute for Critical Care Research and Innovation, University of Michigan, Ann Arbor

Here, we present a protocol to allow providers to perform focused cardiac ultrasound (FoCUS) in the clinical environment. We describe methods of transducer manipulation, review common pitfalls of transducer movements, and suggest tips to optimize phased array transducer use.

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Biology

Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae
Anjali Mahilkar *1,2, Prachitha Nagendra *1, Supreet Saini 1
1Department of Chemical Engineering, Indian Institute of Technology Bombay, 2Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor

In this work, a robust method for the quantification of mating efficiency in the yeast Saccharomyces cerevisiae is described. This method is particularly useful for the quantification of pre-zygotic barriers in speciation studies.

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Advancing the Assessment of Cardiac Injury and Function
Ienglam Lei 1, Pierre-Emmanuel Noly 2, Paul C. Tang 1
1Department of Cardiac Surgery, University of Michigan, Ann Arbor, 2Department of Cardiac Surgery, Montreal Heart Institute

Advancing the Assessment of Cardiac Injury and Function

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Neuroscience

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

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

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Biology

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures
Qitao Zhang 1, Gillian Autterson 1, Jason M. L. Miller 1,2
1Kellogg Eye Center, University of Michigan, Ann Arbor, 2Cellular and Molecular Biology Program, University of Michigan, Ann Arbor

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

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Cancer Research

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor
Brittany Turner-Ivey 1, Jody Fromm Longo 2, Dorea P. Jenkins 2, Stephen T. Guest 3, Steven L. Carroll 1,2
1Hollings Cancer Center, Medical University of South Carolina, 2Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 3Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

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Biology

CorrelationCalculator and Filigree: Tools for Data-Driven Network Analysis of Metabolomics Data
Gayatri Iyer 1, Marci Brandenburg 1,2, Christopher Patsalis 1, George Michailidis 3, Alla Karnovsky 1
1Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, 2Taubman Health Sciences Library, University of Michigan, Ann Arbor, 3Department of Statistics, University of Florida

We present CorrelationCalculator and Filigree, two tools for data-driven network construction and analysis of metabolomics data. CorrelationCalculator supports building a single interaction network of metabolites based on expression data, while Filigree allows building a differential network, followed by network clustering and enrichment analysis.

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Cancer Research

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma
Noah A. Brown 1, Rajesh C. Rao 1,2,3,4,5,6,7,8, Bryan L. Betz 1
1Department of Pathology, University of Michigan, Ann Arbor, 2Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, 3Department of Human Genetics, University of Michigan, Ann Arbor, 4Rogel Cancer Center, University of Michigan, Ann Arbor, 5Center of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, 6Center for RNA Biomedicine, University of Michigan, Ann Arbor, 7A. Alfred Taubman Medical Research Institute, University of Michigan, Ann Arbor, 8Section of Ophthalmology, Surgery Service, Veterans Administration Ann Arbor Healthcare System

A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing.

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Biology

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research
Safa Beydoun *1, Elizabeth S. Kitto *1, Emily Wang 1, Shijiao Huang 1, Scott F. Leiser 1,2
1Molecular and Integrative Physiology Department, University of Michigan, Ann Arbor, 2Department of Internal Medicine, University of Michigan, Ann Arbor

The food source for Caenorhabditis elegans in the lab is live Escherichia coli. Since bacteria are metabolically active, they present a confounding variable in metabolic and drug studies in C. elegans. A detailed protocol to metabolically inactivate bacteria using paraformaldehyde is described here.

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Biology

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells
Elena Levi-D’Ancona 1,2, Vaibhav Sidarala 1, Scott A. Soleimanpour 1,3,4
1Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, 2Graduate Program in Immunology, University of Michigan Medical School, 3Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, 4VA Ann Arbor Healthcare System

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic β-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

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Neuroscience

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice
Cristina Sáenz de Miera 1, Jiane Feng 1,2, Carol F. Elias 1,2, Nathan Qi 1,2
1Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, 2Mouse Metabolic Phenotyping Center-Live, University of Michigan, Ann Arbor

Luteinizing hormone (LH) pulsatility is a hallmark of reproductive function. We describe a protocol for remote activation of specific neuronal populations linked to serial automated blood collection. This technique allows timed hormonal modulation, multiplexing, and minimizing manipulation effects on LH levels in conscious freely moving, and undisturbed animals.

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Bioengineering

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System
Kyle J. Loi *1,2, Hossein Moghimianavval *3, Allen P. Liu 2,3,4,5
1Neuroscience Program, University of Michigan, Ann Arbor, 2Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, 3Department of Mechanical Engineering, University of Michigan, Ann Arbor, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, 5Department of Biophysics, University of Michigan, Ann Arbor

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.

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Neuroscience

Development of a Low-cost Epimysial Electromyography Electrode: A Simplified Workflow for Fabrication and Testing
Luke Stoneback *1, Genaro D. Fullano *2, McKenzie S. White 1, Sairub Naaz 1, Lindsey K. Lepley 1
1School of Kinesiology, University of Michigan, Ann Arbor, 2University of Michigan Medical School

Our purpose was to provide an updated, easy-to-follow guide on the fabrication and testing of epimysial electromyography electrodes. To that end, we provide instructions for material sourcing and a detailed walkthrough of the fabrication and testing process.

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Biology

Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures
Qitao Zhang *1, Daisy Y. Shu *2, Richard A. Bryan 3, John Y. S. Han 1, Gillian A. Gulette 1, Kin Lo 3, Leo A. Kim 4, Jason M. L. Miller 1,5
1Kellogg Eye Center, University of Michigan, Ann Arbor, 2School of Optometry and Vision Science, University of New South Wales, 3Lucid Scientific, 4Schepens Eye Research Institute of Mass. Eye and Ear, Department of Ophthalmology, Harvard Medical School, 5Cellular and Molecular Biology Program, University of Michigan, Ann Arbor

We introduce a novel device for measuring oxygen consumption rates (OCR) in retinal pigment epithelial (RPE) cultures. The device can measure OCR for weeks at a time on RPE grown on standard cell culture plates with standard media while the plates are in a standard cell culture incubator.

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Neuroscience

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids
Tong Pan 1,2, Daniel C. Jaklic 1,2, Shivanshi Vaid 1,2, Grace Lin 1,2, Debora VanHeyningen 1,2, Louis T. Dang 1,2
1Division of Pediatric Neurology, Department of Pediatrics, Michigan Medicine, Ann Arbor, 2Michigan Neuroscience Institute, University of Michigan, Ann Arbor

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

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