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21 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates
Matthew L. Skiles 1, Suchit Sahai 1, James O. Blanchette 2
1Biomedical Engineering Program, University of South Carolina, 2Chemical Engineering Department, University of South Carolina

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

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Biology

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts
Jeremy Willis *1, Darla DeStephanis *1, Yogin Patel 1, Vrushab Gowda 1, Shan Yan 1
1Department of Biology, University of North Carolina at Charlotte

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

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Biology

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors
Padmavathy Nandha Premnath 1, Sandra Craig 1, Campbell McInnes 1
1Department of Drug Discovery and Biomedical Sciences, University of South Carolina

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

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Medicine

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice
Hailong Li *1, Gourav Roy Choudhury *1, Nannan Zhang 1, Shinghua Ding 1
1Department of Bioengineering, Dalton Cardiovascular Research Center, University of Missouri

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

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Bioengineering

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels
Veronica Rodriguez-Rivera 1, John W. Weidner 2, Michael J. Yost 1
1Department of Surgery - Division of General Surgery, Medical University of South Carolina, 2Department of Chemical Engineering, University of South Carolina

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

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Biology

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens
Boran Zhou 1, Suraj Ravindran 2, Jahid Ferdous 2, Addis Kidane 2, Michael A. Sutton 2, Tarek Shazly 1,2
1Biomedical Engineering Program, University of South Carolina, 2Department of Mechanical Engineering, University of South Carolina

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

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Neuroscience

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons
Hailong Li *1, Marina Aksenova *1, Sarah J. Bertrand 1, Charles F. Mactutus 1, Rosemarie Booze 1
1Laboratory Program in Behavioral, Neuroscience, Department of Psychology, University of South Carolina

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

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Chemistry

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)
Daniel B. Wiedemeier 1, Susan Q. Lang 2, Merle Gierga 3, Samuel Abiven 1, Stefano M. Bernasconi 3, Gretchen L. Früh-Green 3, Irka Hajdas 4, Ulrich M. Hanke 1, Michael D. Hilf 1, Cameron P. McIntyre 4, Maximilian P. W. Scheider 1, Rienk H. Smittenberg 5, Lukas Wacker 4, Guido L. B. Wiesenberg 1, Michael W. I. Schmidt 1
1Department of Geography, University of Zurich, 2Department of Earth and Ocean Sciences, University of South Carolina, 3Department of Earth Sciences, ETH Zurich, 4Laboratory of Ion Beam Physics, ETH Zurich, 5Department of Geological Sciences, Stockholm University

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

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Medicine

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization
Yogin Patel 1, Ji S. Lee 2, Hexin Chen 1
1Department of Biological Science, Center for Colon Cancer Research, University of South Carolina, 2Department of Pathology, Chonnam National University Medical School

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

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Environment

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples
Huan Chen *1,2, Ian Arthur Palmer *1, Jian Chen 1,2, Ming Chang 1,2, Stephen L. Thompson 3, Fengquan Liu 2, Zheng Qing Fu 1
1Department of Biological Sciences, University of South Carolina, 2Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, 3Department of Instructions and Teacher Education, University of South Carolina

Citrus Greening is a particularly destructive disease affecting citrus crops globally. Presented here is a simple method using PCR and genomic DNA extraction of citrus leaf tissue for the accurate and precise identification of the citrus greening pathogen, Candidatus liberibacter spp.

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Neuroscience

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology
Hailong Li 1, Jessica M. Illenberger 1, Kristen A. McLaurin 1, Charles F. Mactutus 1, Rosemarie M. Booze 1
1Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

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Neuroscience

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents
Kristen A McLaurin 1, Landhing M Moran 1, Hailong Li 1, Rosemarie M Booze 1, Charles F Mactutus 1
1Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

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Cancer Research

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment
Lei Wang *1, Huan Chen *2,3, Fei Fei *1, Xianfeng He 2,3, Shaoyang Sun 1, Kunpeng Lv 1, Bo Yu 2,3, Jiang Long 2,4,5,6, Xu Wang 1
1Department of Biochemistry and Molecular Biology, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, School of Basic Medical Sciences, Fudan University, 2National Human Genetic Resources Sharing Service Platform (2005DKA21300), 3Fudan University Shanghai Cancer Center, 4Department of Pancreatic Surgery, Fudan University Shanghai Cancer Center, 5Department of Oncology, Shanghai Medical College, Fudan University, 6Pancreatic Cancer Institute, Fudan University

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

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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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Medicine

A Training and Testing System for Performing Vascular Reconstruction In Vitro
Yue Wang 1,2, Lijun Mu 3, Wei Zhang 1,2, Huan Chen 1,2, Qingshan Li 1,2, Aihua Shi 1, Bo Tang 2, Xiaogang Zhang 1,2, Dinghui Dong 1,2, Yi Lv 1,2
1National Local Joint Engineering Research Center for Precision Surgery and Regenerative Medicine, First Affiliated Hospital of Xi'an Jiaotong University, 2Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi'an Jiaotong University, 3Department of General Surgery, First Affiliated Hospital of Xi'an Jiaotong University

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

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Bioengineering

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye
Marwa Belhaj 1, Balasubramaniam Annamalai 2, Nathaniel Parsons 2, Andrew Shuler 1, Jay Potts 1, Bärbel Rohrer 2,3,4
1Department of Cell Biology, University of South Carolina, 2Department of Ophthalmology, Division of Research, Medical University of South Carolina, 3Department of Neuroscience, Division of Research, Medical University of South Carolina, 4Division of Research, Ralph H. Johnson VA Medical Center

Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

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Medicine

In Vivo Mouse Model of Spinal Implant Infection
Benjamin V. Kelley 1, Christopher Hamad 1, Stephen D. Zoller 1, Danielle Greig 1, Zeinab Mamouei 1, Rene Chun 1, Kellyn Hori 1, Nicolas Cevallos 1, Chad Ishmael 1, Peter Hsiue 1, Rishi Trikha 1, Troy Sekimura 2, Brandon Gettleman 3, Autreen Golzar 2, Adrian Lin 2, Thomas Olson 2, Ameen Chaudry 2, Michael M. Le 2, Anthony A. Scaduto 1, Kevin P. Francis 1, Nicholas M. Bernthal 1
1Department of Orthopaedic Surgery, University of California Los Angeles, 2David Geffen School of Medicine, University of California Los Angeles, 3University of South Carolina School of Medicine, University of South Carolina

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

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Neuroscience

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture
Hailong Li 1, Kristen A. McLaurin 1, Charles F. Mactutus 1, Rosemarie M. Booze 1
1Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

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Neuroscience

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue
Kristin N. Kirchner 1, Hailong Li 1, Adam R. Denton 1, Steven B. Harrod 1, Charles F. Mactutus 1, Rosemarie M. Booze 1
1Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

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Neuroscience

A Rat Model of EcoHIV Brain Infection
Hailong Li 1, Kristen A. McLaurin 1, Charles F. Mactutus 1, Rosemarie M. Booze 1
1Program in Behavioral Neuroscience, Department of Psychology, University of South Carolina

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

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Developmental Biology

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity
Yvon Woappi 1,2, Geraldine Ezeka 3, Justin Vercellino 4, Sean M. Bloos 5, Kim E. Creek 6, Lucia Pirisi 1
1Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, 2Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, 3Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine Baltimore, 4Department of Pathology, Albert Einstein College of Medicine, 5Department of Pediatrics, Vanderbilt University Medical Center, 6Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

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