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University of Rochester Medical Center

33 ARTICLES PUBLISHED IN JoVE

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Medicine

Experimental Metastasis Assay
Sonali Mohanty 1, Lei Xu 1,2
1Department of Biomedical Genetics, University of Rochester Medical Center, 2Department of Dermatology, University of Rochester Medical Center

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

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Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA
Luis Martínez-Sobrido 1, Adolfo García-Sastre 2
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Microbiology and Medicine, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

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Immunology and Infection

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci
Marlise I. Klein 1, Jin Xiao 1,2, Arne Heydorn 3, Hyun Koo 1,4
1Center for Oral Biology, University of Rochester Medical Center, 2State Key Laboratory of Oral Diseases, Sichuan University, 3Department of General Medicine, Glostrup Hospital, Glostrup, Denmark, 4Department of Microbiology and Immunology, University of Rochester Medical Center

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

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Neuroscience

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence
Oksana Polesskaya 1, Anita Sun 2, Gheorghe Salahura 2, Jharon N. Silva 1, Stephen Dewhurst 1, Karl Kasischke 3
1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Center for Neural Development and Disease, University of Rochester Medical Center, 3Deptartment of Neurology, Center for Neural Development and Disease, University of Rochester Medical Center

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

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Medicine

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis
Michele M. Pelter 1, Teri M. Kozik 2, Denise L. Loranger 1, Mary G. Carey 3
1Orvis School of Nursing, University of Nevada, Reno, 2St. Joseph's Medical Center, 3Strong Memorial Hospital, University of Rochester Medical Center

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

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Immunology and Infection

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells
Benson Y.H. Cheng *1, Emilio Ortiz-Riaño *1, Juan Carlos de la Torre 2, Luis Martínez-Sobrido 1
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Immunology and Microbial Sciences, The Scripps Research Institute

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

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Immunology and Infection

Rescue of Recombinant Newcastle Disease Virus from cDNA
Juan Ayllon 1,2, Adolfo García-Sastre 1,2,3, Luis Martínez-Sobrido 4
1Department of Microbiology, Icahn School of Medicine at Mount Sinai, 2Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, 3Department of Medicine, Icahn School of Medicine at Mount Sinai, 4Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

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Chemistry

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Amy H. Van Hove 1, Brandon D. Wilson 2, Danielle S. W. Benoit 1,2,3
1Department of Biomedical Engineering, University of Rochester, 2Department of Chemical Engineering, University of Rochester, 3Center for Musculoskeletal Research, University of Rochester Medical Center

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

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Neuroscience

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation
Christopher Nishimura 1, Oksana Polesskaya 2, Stephen Dewhurst 1, Jharon N. Silva 1
1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Department of Human Genetics, University of Chicago

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

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Medicine

Murine Flexor Tendon Injury and Repair Surgery
Jessica E. Ackerman 1, Alayna E. Loiselle 1
1Center for Musculoskeletal Research, Department of Orthopaedics & Rehabilitation, University of Rochester Medical Center

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

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JoVE Journal

Influenza A Virus Studies in a Mouse Model of Infection
Laura Rodriguez 1, Aitor Nogales 1, Luis Martínez-Sobrido 1
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

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JoVE Core

Preparation of Aligned Steel Fiber Reinforced Cementitious Composite and Its Flexural Behavior
Ru Mu 1, Luansu Wei 1, Xiaowei Wang 1, Hui Li 1, Longbang Qing 1, Jian Zhou 1, Quanming Zhao 2
1School of Civil and Transportation Engineering, Hebei University of Technology, 2School of Information Engineering, Hebei University of Technology

This protocol describes an approach for manufacturing aligned steel fiber reinforced cementitious composite by applying a uniform electromagnetic field. Aligned steel fiber reinforced cementitious composite exhibits superior mechanical properties to ordinary fiber reinforced concrete.

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Neuroscience

Cannula Implantation into the Cisterna Magna of Rodents
Anna L.R. Xavier 1, Natalie Linea Hauglund 1, Stephanie von Holstein-Rathlou 1, Qianliang Li 1, Simon Sanggaard 1,2, Nanhong Lou 3, Iben Lundgaard 3,4, Maiken Nedergaard 1,3
1Center for Translational Neuromedicine, Division of Glial Therapeutics, University of Copenhagen, 2Department of Anesthesiology, Yale School of Medicine, 3Center for Translational Neuromedicine, Division of Glial Therapeutics, University of Rochester Medical Center, 4Department of Experimental Medical Science, Wallenberg Center for Molecular Medicine, Lund University

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

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Bioengineering

Retroductal Nanoparticle Injection to the Murine Submandibular Gland
Jomy J. Varghese 1, Isaac L. Schmale 2, Yuchen Wang 1, Mollie Eva Hansen 1, Shawn D. Newlands 2, Catherine E. Ovitt 3, Danielle S. W. Benoit 1
1Department of Biomedical Engineering, University of Rochester, 2Department of Otolaryngology Head and Neck Surgery, University of Rochester Medical Center, 3Center for Oral Biology, University of Rochester Medical Center

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

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Medicine

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation
Jomy J. Varghese 1, Isaac L. Schmale 2, Mollie Eva Hansen 1, Shawn D. Newlands 2, Danielle S.W. Benoit 1, Catherine E. Ovitt 3
1Department of Biomedical Engineering, University of Rochester, 2Department of Otolaryngology, University of Rochester Medical Center, 3Center for Oral Biology, University of Rochester Medical Center

We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

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Genetics

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan
Adam B. Cornwell 1, Jesse R. Llop 1, Peter Salzman 2,3, Juilee Thakar 4, Andrew V. Samuelson 1
1Department of Biomedical Genetics, University of Rochester Medical Center, 2Department of Biostatistics and Computational Biology, University of Rochester Medical Center, 3Non-Clinical Statistics, Bristol-Myers Squibb, 4Department of Microbiology and Immunology, University of Rochester Medical Center

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

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Bioengineering

Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation
Doran S. Mix 1, Michael C. Stoner 1, Steven W. Day 2, Michael S. Richards 3
1Division of Vascular Surgery, University of Rochester Medical Center, 2Department of Biomedical Engineering, Rochester Institute of Technology, 3Department of Surgery, University of Rochester Medical Center

Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments.

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Bioengineering

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA
Tatjana Michel 1, Antonia Link 1, Meike-Kristin Abraham 1,2, Christian Schlensak 1, Karlheinz Peter 2,3, Hans-Peter Wendel 1, Xiaowei Wang 2,3, Stefanie Krajewski 1
1Department of Thoracic and Cardiovascular Surgery, Clinical Research Laboratory, University Medical Center, 2Atherothrombosis and Vascular Biology, Baker Heart & Diabetes Institute, 3Department of Medicine, Monash University

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

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Immunology and Infection

Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone
Ginés Ávila-Pérez 1, Jun-Gyu Park 1, Aitor Nogales 1, Fernando Almazán 2, Luis Martínez-Sobrido 1
1Department of Microbiology and Immunology, University of Rochester Medical Center, 2Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid

The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter.

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Neuroscience

Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping
Bradford Z. Mahon 1,2, Jeffrey A. Mead 3, Benjamin L Chernoff 2, Maxwell H. Sims 5, Frank E. Garcea 4, Emily Prentiss 5, Raouf Belkhir 2, Sam J. Haber 1, Sarah B. Gannon 5, Steve Erickson 5, Kelly A. Wright 5, Michael Z. Schmidt 5, Audrey Paulzak 1, Vanessa C. Milano 1, David A. Paul 1, Kenneth Foxx 1, Madalina Tivarus 7,8, Jacob W. Nadler 6, Jacqueline M Behr 1, Susan O. Smith 1, Yan Michael Li 1, Kevin Walter 1, Webster H. Pilcher 1
1Department of Neurosurgery, University of Rochester Medical Center, 2Department of Psychology, Carnegie Mellon University, 3Public Relations and Communications, University of Rochester Medical Center, 4MOSS Rehabilitation Research Institute, Cognitive Neuroscience, 5University of Rochester Medical Center, 6Department of Anesthesiology and Perioperative Medicine, University of Rochester Medical Center, 7Department of Imaging Sciences, University of Rochester Medical Center, 8Department of Neuroscience, University of Rochester Medical Center

This article provides an overview of a multi-modal brain mapping program designed to identify regions of the brain that support critical cognitive functions in individual neurosurgery patients.

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Neuroscience

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy
Amanda M Sweeney *1, Virginia Plá *1, Ting Du 1, Guojun Liu 1, Qian Sun 1, Sisi Peng 1, Benjamin A. Plog 1, Benjamin T. Kress 1, Xiaowei Wang 1, Humberto Mestre 1, Maiken Nedergaard 1,2
1Center for Translational Neuromedicine, University of Rochester Medical Center, 2Center for Translational Neuromedicine, University of Copenhagen

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

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Immunology and Infection

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection
Kevin Chiem 1, Javier Rangel-Moreno 2, Aitor Nogales 1,3, Luis Martinez-Sobrido 1
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Division of Allergy/Immunology and Rheumatology, Department of Medicine, University of Rochester, 3Center for Animal Health Research, INIA-CISA

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

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Biology

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans
Maria I. Lazaro-Pena 1, Adam B. Cornwell 1, Andrew V. Samuelson 1
1Department of Biomedical Genetics, University of Rochester Medical Center

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

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JoVE Core

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries
Sabrina M. Ribeiro *1, Érick D. O. Fratucelli *1, Júlia M. Fernandes *2, Paula C. P. Bueno 2,3, Alberto José Cavalheiro 2, Marlise I. Klein 1
1Department of Dental Materials and Prosthodontics, School of Dentistry, São Paulo State University (UNESP), 2Department of Organic Chemistry, Institute of Chemistry, São Paulo State University (UNESP), 3Faculty of Pharmaceutical Sciences of Ribeiraão Preto (FCFRP-USP), Department of Physics and Chemistry, University of São Paulo

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

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Neuroscience

The Impact of Motor Task Conditions on Goal-Directed Arm Reaching Kinematics and Trunk Compensation in Chronic Stroke Survivors
Jaimie Girnis 1,2, Tarek Agag 1, Tobias Nobiling 1,3, Vanessa Sweet 1,4, Bokkyu Kim 1
1Department of Physical Therapy Education, SUNY Upstate Medical University, 2College of Health and Rehabilitation Sciences: Sargent College Center for Neurorehabilitation, Boston University, 3Department of Physical Medicine and Rehabilitation, University of Rochester Medical Center, 4Rehabilitation Today

This protocol is intended to investigate the impact of task conditions on movement strategies in chronic stroke survivors. Further, this protocol can be used to examine if a restriction in elbow extension induced by neuromuscular electrical stimulation causes trunk compensation during goal-directed arm reaches in non-disabled adults.

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Medicine

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits
Cameron Bosinski 1, Kyle Wagner 1, Xiangping Zhou 2, Lynn Liu 3, David S. Auerbach 1
1Department of Pharmacology, Upstate Medical University, 2Department of Neurology, Upstate Medical University, 3Strong Epilepsy Center, Departments of Neurology, Anesthesiology / Perioperative Medicine, & Pediatrics, University of Rochester Medical Center

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

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Immunology and Infection

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System
Stephen Angeloni 1, Andrew Cameron 2, Nicole D. Pecora 2,3, Sherry Dunbar 1
1Luminex Corporation, 2Departments of Pathology and Laboratory Medicine, Clinical Microbiology, University of Rochester Medical Center, 3Department of Microbiology and Immunology, University of Rochester Medical Center

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

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Neuroscience

Direct Cannula Implantation in the Cisterna Magna of Pigs
Nicholas B. Bèchet 1,2, Nagesh C. Shanbhag 1,2, Iben Lundgaard 1,2
1Department of Experimental Medical Science, Lund University, 2Wallenberg Centre for Molecular Medicine, Lund University

This article presents a step-by-step protocol for the direct cannula implantation in the cisterna magna of pigs.

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Immunology and Infection

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2
Desarey Morales Vasquez 1, Kevin Chiem 1, Jesus Silvas 1, Jun-Gyu Park 1, Chengjin Ye 1, Luis Martínez-Sobrido 1
1Texas Biomedical Research Institute

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

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Bioengineering

Cantilever Bending of Murine Femoral Necks
Emma Knapp 1, Hani A. Awad 1,2
1Department of Orthopedics, Center for Musculoskeletal Research, University of Rochester Medical Center, 2Department of Biomedical Engineering, University of Rochester

The present protocol describes the development of a reproducible testing platform for murine femoral necks in a cantilever bending set-up. Custom 3D printed guides were used to consistently and rigidly fix the femurs in optimal alignment.

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Neuroscience

Semi-Automated Analysis of Peak Amplitude and Latency for Auditory Brainstem Response Waveforms Using R
Daxiang Na 1, Patricia M. White 2
1Department of Biomedical Genetics, University of Rochester Medical Center, 2Department of Neuroscience, Ernst J. Del Monte Institute for Neuroscience, University of Rochester Medical Center

This article describes the semi-automated measurement of the amplitudes and latencies of the first five peaks and troughs in the auditory brainstem response waveform. An additional routine compiles and annotates the data into a spreadsheet for experimenter analysis. These free computer routines are executed using the open-source statistical package R.

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Bioengineering

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement
Brian C. Wise 1,2, Keshia E. Mora 1,2, Whasil Lee 1,2,3, Mark R. Buckley 1,2
1Department of Biomedical Engineering, University of Rochester, 2Department of Orthopaedics, Center for Musculoskeletal Research, University of Rochester Medical Center, 3Department of Pharmacology and Physiology, University of Rochester Medical Center

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

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Cancer Research

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia
Christina M. Kaszuba 1,2, Benjamin J. Rodems 1,3, Sonali Sharma 1,3, Edgardo I. Franco 1,2, John M. Ashton 1,3,4, Laura M. Calvi 1,5, Jeevisha Bajaj 1,3
1Wilmot Cancer Institute, University of Rochester Medical Center, 2Department of Biomedical Engineering, University of Rochester, 3Department of Biomedical Genetics, University of Rochester Medical Center, 4Genomics Research Center, University of Rochester Medical Center, 5Division of Endocrinology and Metabolism, Department of Medicine, University of Rochester Medical Center

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

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