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University of Miami Miller School of Medicine

31 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration
Yousuf O. Ali 1, Wilfredo Escala 1, Kai Ruan 1, R. Grace Zhai 1
1Department of Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

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Biology

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders
Luciana Madeira da Silva 1, Lauren Vandepas 1, Suzy D.C. Bianco 1,2
1Division of Endocrinology and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, 2Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

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Neuroscience

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging
Jenny I. Szu 1, Melissa M. Eberle 2, Carissa L. Reynolds 2, Mike S. Hsu 1, Yan Wang 2, Christian M. Oh 2, M. Shahidul Islam 2, B. Hyle Park 2, Devin K. Binder 1
1Division of Biomedical Sciences, University of California, Riverside , 2Department of Bioengineering, University of California, Riverside

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

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Medicine

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time
Midhat H. Abdulreda 1,2, Alejandro Caicedo 1,3,4, Per-Olof Berggren 1,5
1Diabetes Research Institute, University of Miami Miller School of Medicine, 2Department of Surgery, University of Miami Miller School of Medicine, 3Department of Medicine, University of Miami Miller School of Medicine, 4Department of Physiology & Biophysics, University of Miami Miller School of Medicine, 5The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

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Methods for the Delivery of Cells, Vaccines, or Infectious Agents into Neonatal Mice In Vivo
Patricia Guevara 1, James Phillips 1, Becky Adkins 2
1Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, 2Department of Microbiology and Immunology, University of Miami Miller School of Medicine

This article describes three methods for the introduction of materials into murine neonates. We demonstrate (a) intravenous injection in ≤1 day old neonates, (b) subcutaneous and intraperitoneal injection into 7 day old neonates, and (c) orogastric infusion into 7 day old neonates.

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Behavior

Development of a Virtual Reality Assessment of Everyday Living Skills
Stacy A. Ruse 1, Vicki G. Davis 1, Alexandra S. Atkins 1, K. Ranga R. Krishnan 2,3, Kolleen H. Fox 4, Philip D. Harvey 5, Richard S.E. Keefe 1,3
1NeuroCog Trials, Inc., 2Duke-NUS Graduate Medical Center, 3Duke University Medical Center, 4Fox Evaluation and Consulting, PLLC, 5University of Miami Miller School of Medicine

A challenge for proving treatment efficacy for cognitive impairments in schizophrenia is finding the optimizing measurement of skills related to everyday functioning.  The Virtual Reality Functional Capacity Assessment Tool (VRFCAT) is an interactive gaming based computerized measure aimed at skills associated with everyday functioning, including baseline impairments and treatment related changes.

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Medicine

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy
Yan Wang 1,3,4, Dale P. Brown 1, Brant D. Watson 2, Jeffrey L. Goldberg 1,3
1Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 2Departments of Neurology and Biomedical Engineering, University of Miami Miller School of Medicine, 3Shiley Eye Center, University of California, 4Department of Ophthalmology and Vision Science, Fudan University

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

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Immunology and Infection

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells
Natasa Strbo 1, Laura Romero 1, Maria Alcaide 2, Margaret Fischl 2
1Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, 2Division of Infectious Diseases, Miller School of Medicine, University of Miami

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

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Medicine

Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap
Yee-Shuan Lee 1, Siliang Wu 2, Treena Livingston Arinzeh 3, Mary Bartlett Bunge 1,4,5
1The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, 2Department of Materials Science and Engineering, New Jersey Institute of Technology, 3Department of Biomedical Engineering, New Jersey Institute of Technology, 4Department of Cell Biology, University of Miami Miller School of Medicine, 5Department of Neurological Surgery, University of Miami Miller School of Medicine

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

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Biology

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets
Madina Makhmutova 1, Tao Liang 2, Herbert Gaisano 2, Alejandro Caicedo 1, Joana Almaça 1
1Department of Medicine, University of Miami, 2Department of Medicine, University of Toronto

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

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JoVE Journal

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana
Wenhui Lyu 1, Jennifer Selinski 1, Lu Li 1, David A. Day 2, Monika W. Murcha 3, James Whelan 1, Yan Wang 1
1ARC Centre of Excellence in Plant Energy Biology, Department of Animal, Plant and Soil Science, School of Life Science, La Trobe University, 2School of Biological Sciences, Flinders University, 3ARC Centre of Excellence in Plant Energy Biology, University of Western Australia

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

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Behavior

Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest
Celeste Y. C. Wu 1,2, Francesca M. Lerner 4, Alexandre Couto e Silva 3, Harlee E. Possoit 1,2, Tsung-Han Hsieh 1,2, Jake T. Neumann 5, Alireza Minagar 1, Hung Wen Lin 1,2,3, Reggie H. C. Lee 1,2
1Department of Neurology, Louisiana State University Health Science Center, 2Center for Brain Health, Louisiana State University Health Science Center, 3Department of Cellular Biology and Anatomy, Louisiana State University Health Science Center, 4Department of Neurology, Cerebral Vascular Disease Research Laboratories, University of Miami Miller School of Medicine, 5Department of Biomedical Sciences, West Virginia School of Osteopathic Medicine

This protocol describes the use of a modified T-maze to evaluate functional learning/memory in asphyxia cardiac arrest-induced cerebral ischemia.

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Biology

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Xiaofeng Zheng 1,2, Calvin Qing Wei Ho 1, Xiaowei Zheng 3, Kian Leong Lee 4, Katarina Gradin 5, Teresa S. Pereira 3, Per-Olof Berggren 1,2,3, Yusuf Ali 1,2
1Lee Kong Chian School of Medicine, Nanyang Technological University, 2Singapore Eye Research Institute (SERI), Singapore General Hospital, 3The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, 4Cancer and Stem Cell Biology Program, Duke-NUS Medical School, 5Department of Cell and Molecular Biology, Karolinska Institutet

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

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Medicine

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy
Eriko Katsuta 1, Masanori Oshi 1, Omar M. Rashid 2,3,4,5, Kazuaki Takabe 1,6,7,8,9,10
1Breast Surgery, Department of Surgical Oncology, Roswell Park Comprehensive Cancer Center, 2Holy Cross Hospital Michael and Dianne Bienes Comprehensive Cancer Center, 3Department of Surgery, Massachusetts General Hospital, 4Department of Surgery, University of Miami Miller School of Medicine, 5Department of Surgery, Nova Southeastern University School of Medicine, 6Department of Surgery, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, 7Department of Breast Surgery and Oncology, Tokyo Medical University, 8Department of Surgery, Yokohama City University, 9Department of Surgery, Niigata University Graduate School of Medical and Dental Sciences, 10Department of Surgery, Fukushima Medical University

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

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Neuroscience

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ
Jennifer M. Brazill 1, Yi Zhu 1, Chong Li 1, R. Grace Zhai 1,2
1Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, 2School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

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Biology

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy
Elisa Oltra 1,2, Alejandro Caicedo 3
1School of Medicine and Dentistry, Universidad Católica de Valencia San Vicente Mártir, 2Unidad Mixta CIPF-UCV, Centro de Investigación Príncipe Felipe, 3Department of Medicine, University of Miami

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

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Neuroscience

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue
Karolina J. Janczura 1,2, Claude-Henry Volmar 1,2, Claes Wahlestedt 1,2
1Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, 2Center for Therapeutic Innovation, University of Miami Miller School of Medicine

The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues.

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Immunology and Infection

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses
Petoria Gayle 1, Nancy E. Freitag 2, Natasa Strbo 1, Kurt Schesser 1
1Department of Microbiology & Immunology, University of Miami Miller School of Medicine, 2Department of Microbiology & Immunology, University of Illinois College of Medicine

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

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Medicine

Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus
Jing Liu 1,2, Roberto Copetti 3, Erich Sorantin 4, Jovan Lovrenski 5, Javier Rodriguez-Fanjul 6, Dalibor Kurepa 7, Xing Feng 8, Luigi Cattaross 9, Huayan Zhang 10,11, Misun Hwang 12, Tsu F. Yeh 13,14, Yisrael Lipener 7, Abhay Lodha 15, Jia-Qin Wang 16, Hai-Ying Cao 2,17, Cai-Bao Hu 2,18, Guo-Rong Lyu 19, Xin-Ru Qiu 1,2, Li-Qun Jia 20, Xiao-Man Wang 20, Xiao-Ling Ren 1,2, Jiu-Ye Guo 1,2, Yue-Qiao Gao 1,2, Jian-Jun Li 1,2, Ying Liu 1,2, Wei Fu 1,2, Yan Wang 21, Zu-Lin Lu 1,2, Hua-Wei Wang 8, Li-Li Shang 22
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The Neonatal Lung Ultrasound Training Base, Chinese College of Critical Ultrasound, 3Emergency Department, Cattinara University Hospital, 4Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 5Faculty of Medicine, University of Novi Sad, Radiology Department, Institute for Children and Adolescents Health Care of Vojvodina, 6Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 7Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 8Department of Neonatology, Children's Hospital of Soochow University, 9Department of Neonatology, Udine University Hospital, 10Center for Newborn Care, Guangzhou Women and Children's Medical Center, 11Division of Neonatology, Children's Hospital of Philadelphia, 12Department of Radiology, Children's Hospital of Philadelphia, 13Division of Neonatology and NICU, Cook County Children's Hospital, University of Illinois, 14Division of Neonatology, Department of Pediatrics, Taipei Medical University, 15Department of Pediatrics and Community Health Sciences, University of Calgary, 16Department of Pediatrics, The Third Affiliated Hospital of Xinxiang Medical University, 17Department of Ultrasound, GE Healthcare, 18Intensive Care Unit, Zhejiang Hospital, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Beijing Children's Hospital Affiliated with Capital Medical University, 21Department of Neonatology and NICU, Tai'an City Central Hospital of Shandong Province, 22Department of Intensive Care Unit, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine

Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making.

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Immunology and Infection

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E
Duneia McManus 1,2,3,4, Horacio J. Novaira 1,2,3,4, Anouk A.J. Hamers 1,2,3,4, Asha B. Pillai 1,2,3,4,5
1Department of Pediatrics, Division of Hematology / Oncology and Bone Marrow Transplantation, University of Miami Miller School of Medicine, 2Batchelor Children's Research Institute, University of Miami Miller School of Medicine, 3Department of Microbiology & Immunology, University of Miami Miller School of Medicine, 4Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, 5Holtz Children's Hospital, University of Miami Miller School of Medicine

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

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Medicine

A Computerized Functional Skills Assessment and Training Program Targeting Technology Based Everyday Functional Skills
Philip D. Harvey 1,2, Lize Tibiriçá 2,3, Peter Kallestrup 2, Sara J. Czaja 1,2,4
1University of Miami Miller School of Medicine, 2i-Function, 3Albizu University, 4Weill-Cornell Medical Center

This training protocol uses computerized training to teach technology-related everyday functional skills. These skills include financial skills, travel and transit, as well as medication management.

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Cancer Research

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery
Hongwei Shao 1, Mecker Moller 1, Dazhi Wang 1, Albert Ting 1, Marcia Boulina 2, Zhao-Jun Liu 1
1Department of Surgery, University of Miami School of Medicine, 2Analytical Imaging Core Facility, University of Miami School of Medicine

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

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Developmental Biology

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons
Amir Bagheri 1,2, Seyedeh Fatemeh Razavipour 3, Claes Wahlestedt 2, Seyed Javad Mowla 1, Mohammad Ali Faghihi 2,4
1Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, 2Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, 3Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, 4Persian BayanGene Research and Training Center

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

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Biology

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants
Lim Chee Liew 1,2, Yan Wang 1,2, Marta Peirats-Llobet 1, Oliver Berkowitz 1,2,3, James Whelan 1,2,3, Mathew G. Lewsey 1,3
1Department of Animal, Plant and Soil Science, AgriBio Building, La Trobe University, 2Australian Research Council Centre of Excellence in Plant Energy Biology, La Trobe University, 3Australian Research Council Research Hub for Medicinal Agriculture, AgriBio Building, La Trobe University

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

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Bioengineering

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production
Riccardo Tamburrini 1,2,3, Deborah Chaimov 1,3, Amish Asthana 1,3, Carlo Gazia 3,4, Kevin Enck 3, Sean M. Muir 5, Justine Mariam Aziz 6, Sandrine Lablanche 7, Emily Tubbs 7, Alice A. Tomei 8,9, Mark Van Dyke 10, Shay Soker 3, Emmanuel C. Opara 3, Giuseppe Orlando 1,3
1Department of Surgery, Wake Forest Baptist Medical Center, 2Department of General Surgery, PhD Program in Experimental Medicine, University of Pavia, 3Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, 4Department of Surgery, Tor Vergata University of Rome, 5Wake Forest University College of Arts and Science, 6Wake Forest University School of Medicine, 7Laboratory of Fundamental and Applied Bioenergetics (LBFA), and Environmental and System Biology (BEeSy), Grenoble Alps University, 8Department of Biomedical Engineering, University of Miami, 9Diabetes Research Institute, University of Miami Miller School of Medicine, 10Department of Biomedical Engineering and Mechanics, School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets’ microenvironment in vitro and, potentially, in vivo settings.

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Medicine

Electroretinogram Recording for Infants and Children under Anesthesia to Achieve Optimal Dark Adaptation and International Standards
Byron L. Lam 1, Carlos Mendoza-Santiestaban 1, Alex Gonzalez 1,2, Cornelis Rowaan 1,2, Mu Liu 1, Joanne Martin 1, Steven Gayer 1,3, Osmany Gil Figueredo 1, Jean-Marie Parel 1,2
1Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 2Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 3Department of Anesthesiology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine

Adhering to international standards and maintaining retinal dark adaptation are critical to acquire valid full-field electroretinogram responses in the diagnosis and management of inherited retinal diseases. A practical protocol using a portable darkroom is provided to obtain full-field electroretinogram for infants and children under sedation or general anesthesia in the operating room setting.

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Medicine

Assessing Whole-Body Lipid-Handling Capacity in Mice
Mingyang Huang 1, Noah Mathew 1, Yi Zhu 1
1Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

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Neuroscience

Inter-Brain Synchrony in Open-Ended Collaborative Learning: An fNIRS-Hyperscanning Study
Nan Zhao 1,2, Yi Zhu 1,2, Yi Hu 1,2
1School of Psychology and Cognitive Science, East China Normal University, 2Shanghai Key Laboratory of Mental Health and Crisis Intervention, East China Normal University

The protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is outlined. Further, a pipeline to analyze the Inter-Brain Synchrony (IBS) of oxygenated hemoglobin (Oxy-Hb) signals is presented.

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Neuroscience

How to Calculate and Validate Inter-brain Synchronization in a fNIRS Hyperscanning Study
Yinying Hu 1, Zixuan Wang 1, Bei Song 2, Yafeng Pan 3, Xiaojun Cheng 4, Yi Zhu 1, Yi Hu 1
1Institute of Brain and Education Innovation, School of Psychology and Cognitive Science, East China Normal University, 2Department of Musicology, Harbin Conservatory of Music, 3Department of Clinical Neuroscience, Karolinska Institutet, 4School of Psychology, Shenzhen University

The dynamics between coupled brains of individuals have been increasingly represented by inter-brain synchronization (IBS) when they coordinate with each other, mostly using simultaneous-recording signals of brains (namely hyperscanning) with fNIRS. In fNIRS hyperscanning studies, IBS has been commonly assessed through the wavelet transform coherence (WTC) method because of its advantage on expanding time series into time-frequency space where oscillations can be seen in a highly intuitive way. The observed IBS can be further validated via the permutation-based random pairing of the trial, partner, and condition. Here, a protocol is presented to describe how to obtain brain signals via fNIRS technology, calculate IBS through the WTC method, and validate IBS by permutation in a hyperscanning study. Further, we discuss the critical issues when using the above methods, including the choice of fNIRS signals, methods of data preprocessing, and optional parameters of computations. In summary, using the WTC method and permutation is a potentially standard pipeline for analyzing IBS in fNIRS hyperscanning studies, contributing to both the reproducibility and reliability of IBS.

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Medicine

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model
Antoine J. Ribieras 1, Yulexi Y. Ortiz 1,2, Sophie Shrestha 3, C. Theodore Huerta 1, Hongwei Shao 1,2, Maria E. Boulina 4, Roberto I. Vazquez-Padron 1,2, Zhao-Jun Liu 1,2, Omaida C. Velazquez 1,2
1Dewitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, 2Vascular Biology Institute, University of Miami Miller School of Medicine, 3University of Miami, 4Analytical Imaging Core Facility, University of Miami

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

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Cancer Research

Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells
Jacob Jahn 1, Sana Chaudhry 1, Maurizio Affer 1, Alejandro Pardo 1, Gabriel Pardo 1, Justin Taylor 1
1Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine

The present protocol offers an efficient and flexible method to isolate RNA from nuclear and cytoplasmic fractions using cultured cells, and then validate using qPCR. This effectively serves as a replacement for other RNA preparation kits.

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