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University of California, Los Angeles

92 ARTICLES PUBLISHED IN JoVE

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Biology

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling
John Marshall 1, Koji Morikawa 1, Nicholas Manoukis 1, Charles Taylor 1
1Department of Ecology and Evolutionary Biology, University of California, Los Angeles

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

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Biology

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window
Ricardo Mostany 1, Carlos Portera-Cailliau 1
1Department of Neurology, University of California, Los Angeles

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

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Biology

A Craniotomy Surgery Procedure for Chronic Brain Imaging
Ricardo Mostany 1, Carlos Portera-Cailliau 1
1Department of Neurology, University of California, Los Angeles

This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

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Biology

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice
Peyman Golshani 1, Carlos Portera-Cailliau 1
1Department of Neurology, University of California, Los Angeles

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

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Biology

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs
Jin Zhang 1, Ivan Khvorostov 1, Michael Teitell 1
1David Geffen School of Medicine, University of California, Los Angeles

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

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Biology

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells
Ivan Khvorostov 1, Jin Zhang 1, Michael Teitell 1
1David Geffen School of Medicine, University of California, Los Angeles

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

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JoVE Journal

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta
Christos Gekas 1, Katrin E. Rhodes 1, Hanna K. A. Mikkola 1
1Jonsson Comprehensive Cancer Center, University of California, Los Angeles

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

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Biology

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel
Ivan Khvorostov 1, Jin Zhang 1, Michael Teitell 1
1David Geffen School of Medicine, University of California, Los Angeles

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

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Biology

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel
Jin Zhang 1, Ivan Khvorostov 1, Michael Teitell 1
1David Geffen School of Medicine, University of California, Los Angeles

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

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Biology

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila
Brian J. Duistermars 1, Mark A. Frye 1
1Department of Physiological Science, University of California, Los Angeles

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

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Biology

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides
Farid Rahimi 1, Panchanan Maiti 1, Gal Bitan 2,3
1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, 2Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, 3Department of Neurology, University of California, Los Angeles

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid β-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

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Biology

Visually Mediated Odor Tracking During Flight in Drosophila
Mark A. Frye 1, Brian J. Duistermars 1
1Department of Physiological Science, University of California, Los Angeles

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

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Biology

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos
Georgeann S. O'Brien 1, Sandra Rieger 1, Seanna M. Martin 1, Ann M. Cavanaugh 1, Carlos Portera-Cailliau 2, Alvaro Sagasti 1
1Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, 2Departments of Neurology and Neurobiology, University of California, Los Angeles

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

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Biology

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures
Yali Zhao 1, Dan O. Wang 1, Kelsey C. Martin 1,2,3
1Dept. of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, 2Dept. of Biological Chemistry, University of California, Los Angeles, 3Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

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Biology

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics
Onur Mudanyali 1, Anthony Erlinger 1, Sungkyu Seo 1, Ting-Wei Su 1, Derek Tseng 1, Aydogan Ozcan 1,2
1Electrical Engineering Department, University of California, Los Angeles, 2California NanoSystems Institute, University of California, Los Angeles

Lensfree on-chip imaging and characterization of cells is illustrated. This on-chip cell imaging approach provides a compact and cost-effective tool for medical diagnostics and high-throughput cell biology applications, making it especially suitable for resource poor settings.

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Neuroscience

Low-stress Route Learning Using the Lashley III Maze in Mice
Amanda Bressler 1, David Blizard 2, Anne Andrews 3,4,5,6
1Department of Chemistry, Pennsylvania State University, 2Center for Developmental and Health Genetics, Pennsylvania State University, 3Department of Veterinary and Biomedical Sciences, Pennsylvania State University, 4Huck Institute of the Life Sciences, Pennsylvania State University, 5California NanoSystems Institute, University of California, Los Angeles, 6Semel Institute of Neuroscience and Human Behavior, University of California, Los Angeles

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

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Neuroscience

Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease
Farid Rahimi 1, Gal Bitan 1,2,3
1Department of Neurology, David Geffen School of Medicine, 2Molecular Biology Institute, University of California, Los Angeles, 3Brain Research Institute, University of California, Los Angeles

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.

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Biology

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny
Maureen R. Lynch 1, Judith C. Gasson 2, Helicia Paz 1
1Department of Medicine, Hematology-Oncology, University of California, Los Angeles, 2Department of Biological Chemistry, University of California, Los Angeles

mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

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Immunology and Infection

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins
Marija Pinne 1,2, David Haake 3,4
1Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, 2Research service, 151, Veterans Affairs Greater Los Angeles Healthcare System, 3Departments of Medicine, Urology at David Geffen School of Medicine and Department of Microbiology, Immunology and Molecular Gentics, University of California Los Angeles (UCLA), 4Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Health Care System

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

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Biology

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies
Nicholas Manoukis 1, Scott Geib 1, Danny Seo 1, Michael McKenney 1, Roger Vargas 1, Eric Jang 1
1Agricultural Research Service, US Dept. of Agriculture, US Pacific Basin Agricultural Research Center

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

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JoVE Journal

Use of Human Perivascular Stem Cells for Bone Regeneration
Aaron W. James *1, Janette N. Zara *2, Mirko Corselli 2, Michael Chiang 1, Wei Yuan 2, Virginia Nguyen 1, Asal Askarinam 1, Raghav Goyal 1, Ronald K. Siu 3, Victoria Scott 1, Min Lee 3, Kang Ting 1, Bruno Péault 2,4, Chia Soo 2
1Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, UCLA, 2UCLA and Orthopaedic Hospital, Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA, 3Department of Bioengineering, UCLA, 4Center for Cardiovascular Science, University of Edinburgh

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

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Neuroscience

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis
Rei Kitani 1, Federico Kalinec 1
1Division of Cell Biology and Genetics, House Ear Institute

A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

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Biology

Drosophila Pupal Abdomen Immunohistochemistry
Wei Wang 1, John H. Yoder 1
1Department of Biological Sciences, University of Alabama

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

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Bioengineering

Lensless Fluorescent Microscopy on a Chip
Ahmet F. Coskun 1, Ting-Wei Su 1, Ikbal Sencan 1, Aydogan Ozcan 1
1Department of Electrical Engineering, University of California, Los Angeles

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., >0.6-8 cm2 with <4μm resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

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Behavior

Method for Simultaneous fMRI/EEG Data Collection during a Focused Attention Suggestion for Differential Thermal Sensation
Pamela K. Douglas 1,2, Maureen Pisani 2, Rory Reid 1, Austin Head 2, Edward Lau 2, Ebrahim Mirakhor 3, Jennifer Bramen 2, Billi Gordon 2, Ariana Anderson 2, Wesley T. Kerr 2, Chajoon Cheong 4, Mark S. Cohen 1,2
1Neuropsychiatric Institute, University of California, Los Angeles, 2Laboratory of Neuroimaging Technology, University of California, Los Angeles, 3Yale School of Medicine, 4Korean Basic Science Institute

We present a protocol for concurrent collection of EEG/fMRI data, and synchronized MR clock signal recording. We demonstrate this method using a unique paradigm whereby subjects receive ‘cold glove’ instructions during scanning, and EEG/fMRI data are recorded along with hand temperature measurements both before and after hypnotic induction.

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Bioengineering

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution
Serhan O. Isikman 1, Waheb Bishara 1, Aydogan Ozcan 1,2,3
1Electrical Engineering Department, University of California, Los Angeles , 2Bioengineering Department, University of California, Los Angeles , 3California NanoSystems Institute, University of California, Los Angeles

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of <1 μm × <1 μm × <3 μm in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

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Medicine

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window
Lifeng Zhang *1, Andree Lapierre *1, Brittany Roy 1, Maili Lim 1, Jennifer Zhu 1, Wei Wang 1, Stephen B. Sampson 1, Kyuson Yun 1, Bonnie Lyons 1, Yun Li 1, Da-Ting Lin 1
1The Jackson Laboratory

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

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Immunology and Infection

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation
Hon Wai Koon 1, Samantha Ho 1, Michelle Cheng 1, Ryan Ichikawa 1, Charalabos Pothoulakis 1
1Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School of Medicine, The University of California Los Angeles, Los Angeles

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

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Neuroscience

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons
Yun Li 1, Brittany D. Roy 1, Wei Wang 1, Lifeng Zhang 1, Stephen B. Sampson 1, Da-Ting Lin 1
1The Jackson Laboratory

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

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Neuroscience

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato
Ana Marie S. Palanca 1, Alvaro Sagasti 1
1Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

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Neuroscience

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish
Yali Zhao 1, Nancy L. Wayne 1
1Department of Physiology, University of California, Los Angeles

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

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Bioengineering

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors
Jae Youn Hwang 1, Daniel L. Farkas 1, Lali K. Medina-Kauwe 2,3
1Department of Biomedical Engineering, University of Southern California, 2Department of Biomedical Sciences, Cedars-Sinai Medical Center, 3Geffen School of Medicine, University of California, Los Angeles

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

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Bioengineering

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone
Hongying Zhu 1, Aydogan Ozcan 1,2,3
1Electrical Engineering Department, University of California, Los Angeles , 2Bioengineering Department, University of California, Los Angeles , 3California NanoSystems Institute (CNSI), University of California, Los Angeles

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

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Medicine

Parabiosis in Mice: A Detailed Protocol
Paniz Kamran 1,2, Konstantina-Ioanna Sereti 1,2, Peng Zhao 1,2, Shah R. Ali 3, Irving L. Weissman 3, Reza Ardehali 1,2
1Department of Medicine-Division of Cardiology, University of California, Los Angeles, 2Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

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Immunology and Infection

A Protocol for Analyzing Hepatitis C Virus Replication
Songyang Ren *1, Deisy Contreras *1, Vaithilingaraja Arumugaswami 1,2
1Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery, Cedars-Sinai Medical Center, 2Department of Surgery, David Geffen School of Medicine at UCLA

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

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Neuroscience

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina
Allison Sargoy 1, Steven Barnes 1,2,3, Nicholas C. Brecha 1,2,4, Luis Pérez De Sevilla Müller 1
1Department of Neurobiology, University of California, Los Angeles, 2Veterans Administration Greater Los Angeles Healthcare System, 3Departments of Physiology & Biophysics and Ophthalmology & Visual Sciences, Dalhousie University, 4Departments of Neurobiology and Medicine, Jules Stein Eye Institute, CURE-Digestive Diseases Research Center, David Geffen School of Medicine, University of California, Los Angeles

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

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Medicine

Network Analysis of the Default Mode Network Using Functional Connectivity MRI in Temporal Lobe Epilepsy
Zulfi Haneef 1,2, Agatha Lenartowicz 3, Hsiang J. Yeh 4, Jerome Engel Jr. 4, John M. Stern 4
1Department of Neurology, Baylor College of Medicine, 2Neurology Care Line, Michael E. DeBakey VA Medical Center, 3Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, 4Department of Neurology, University of California, Los Angeles

The Default Mode Network (DMN) in Temporal Lobe Epilepsy (TLE) is analyzed in the resting state of the brain using seed-based functional connectivity MRI (fcMRI).

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JoVE Journal

A Microfluidic Technique to Probe Cell Deformability
David J. Hoelzle 1,2, Bino A. Varghese 1,3, Clara K. Chan 1, Amy C. Rowat 1
1Department of Integrative Biology and Physiology, University of California, Los Angeles, 2Department of Aerospace and Mechanical Engineering, University of Notre Dame, 3Molecular Imaging Center, University of Southern California

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

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Bioengineering

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)
Michael Tu 1, Fang Wei 1, Jieping Yang 2, David Wong 1
1School of Dentistry, University of California, Los Angeles, 2School of Medicine, Clinical Nutrition, University of California, Los Angeles

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

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Developmental Biology

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans
Daniela A. Parodi 1, Robert Damoiseaux 3, Patrick Allard 1,2
1Institute for Society and Genetics, University of California, Los Angeles, 2Environmental Health Sciences, University of California, Los Angeles, 3California Nanosystems Institute, Department of Molecular and Medical Pharmacology, University of California, Los Angeles

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

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Developmental Biology

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy
Thomas S. Lisse 1, Elizabeth A. Brochu 1, Sandra Rieger 1
1Davis Center for Regenerative Biology and Medicine, MDI Biological Laboratory

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

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Chemistry

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization
Wei Wang 1, John M. Lester 1, Anthony E. Amorosa 2, Deborah L. Chance 3, Valeri V. Mossine 1, Thomas P. Mawhinney 1,4
1Department of Biochemistry, University of Missouri, 2Department of Biological Sciences, University of Missouri, 3Department of Molecular Microbiology & Immunology, University of Missouri, 4Department of Child Health, University of Missouri

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

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Behavior

Measurement of Neurophysiological Signals of Ignoring and Attending Processes in Attention Control
Agatha Lenartowicz 1, Gregory V. Simpson 2, Samantha R. O'Connell 1, Mark S. Cohen 3
1Department of Psychiatry, University of California Los Angeles, 2Attention Research Institute, 3Departments of Psychiatry, Radiology, Neurology, Biomedical Physics, Psychology and Bioengineering, University of California Los Angeles

Attention control comprises enhancement of target signals and attenuation of distractor signals. We describe an approach to measure separately but concurrently, the neurophysiology of attending and ignoring in sustained intermodal attention, utilizing a passive control condition during which neither process is continuously engaged.

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Medicine

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology
Stefanie Nunez 1, M. Mehdi Doroudchi 1, Amy J. Gleichman 1, Kwan L. Ng 1, Irene L. Llorente 1, Elif G. Sozmen 1, S. Thomas Carmichael 1, Jason D. Hinman 1
1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

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Neuroscience

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY
Dustin G. Roberts 1, Hadley B. Johnsonbaugh 1, Rory D. Spence 2, Allan MacKenzie-Graham 1
1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, 2W.M. Keck Science Department, Claremont McKenna, Pitzer & Scripps Colleges

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

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Medicine

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model
Anjie Zhen 1, Valerie Rezek 1, Cindy Youn 1, Jonathan Rick 1, Brianna Lam 1, Nelson Chang 1, Jerome Zack 1, Masakazu Kamata 1, Scott Kitchen 1
1David Geffen School of Medicine, University of California, Los Angeles

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

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Developmental Biology

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells
Andrei A. Kramerov 1, Mehrnoosh Saghizadeh 1,2, Alexander V. Ljubimov 1,2
1Eye Program, Board of Governors Regenerative Medicine Institute, Departments of Biomedical Sciences and Neurosurgery, Cedars-Sinai Medical Center, 2David Geffen School of Medicine, University of California, Los Angeles

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

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Bioengineering

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery
Raymond Wing Moon Lam 1, Sunny Akogwu Abbah 2, Wang Ming 1, Mathanapriya Naidu 1, Felly Ng 1, Hu Tao 1, James Goh Cho Hong 3,4, Kang Ting 5, Wong Hee Kit 1,4
1Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 2Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, 3Department of Bioengineering, Faculty of Engineering, National University of Singapore, 4Tissue Engineering Program, National University of Singapore, 5Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

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Biology

Working with Auditory HEI-OC1 Cells
Gilda M. Kalinec 1, Channy Park 1, Pru Thein 1, Federico Kalinec 1
1Head and Neck Surgery, David Geffen School of Medicine, University of California, Los Angeles

House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

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Medicine

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model
Aditya S. Shirali 1, Austin I. McDonald 2, Julia J. Mack 3, M. Luisa Iruela-Arispe 3
1Department of Surgery, University of California, Los Angeles, 2Molecular Biology Institute, University of California, Los Angeles, 3Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

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Immunology and Infection

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons
Deisy Contreras 1, Vaithilingaraja Arumugaswami 1,2,3
1Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 2Department of Surgery, Cedars-Sinai Medical Center, 3Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

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Neuroscience

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach
Sachin Parikh 1, Andrew Le 1, Julian Davenport 1, Michael B. Gorin 1, Steven Nusinowitz 1, Anna Matynia 1
1Jules Stein Eye Institute, University of California, Los Angeles

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

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Biology

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks
Michelle Bradley 1, Ivan Ramirez 1, Keith Cheung 1, Ankur A. Gholkar 1, Jorge Z. Torres 1,2,3
1Department of Chemistry and Biochemistry, University of California, Los Angeles, 2Molecular Biology Institute, University of California, Los Angeles, 3Jonsson Comprehensive Cancer Center, University of California, Los Angeles

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

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Neuroscience

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease
Theresa M. Harrison 1, Donald G. McLaren 2, Teena D. Moody 1, Jamie D. Feusner 1, Susan Y. Bookheimer 1
1Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, 2Biospective, Inc.

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

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Immunology and Infection

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity
Fadi Kandarian 1, Gemalene M Sunga 1, Diana Arango-Saenz 1, Maura Rossetti 1
1UCLA Immunogenetics Center, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

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Immunology and Infection

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions
Thomas A. Angelovich 1,2, Anna C. Hearps 1,3, Anna Maisa 1, Theodoros Kelesidis 4, Anthony Jaworowski 1,3
1Centre for Biomedical Research, Burnet Institute, 2School of Health and Biomedical Sciences, RMIT University, 3Department of Infectious Diseases, Monash University, 4University of California, Los Angeles

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

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Immunology and Infection

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein
Shubhendu Sen Roy 1, Huy Cong Xuan Nguyen 1, Thomas A. Angelovich 2,3, Anna C. Hearps 2, Diana Huynh 1,4, Anthony Jaworowski 2,4, Theodoros Kelesidis 1
1University of California, Los Angeles, 2Centre for Biomedical Research, Burnet Institute, 3School of Health and Biomedical Sciences, RMIT University, 4Department of Infectious Diseases, Monash University

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

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Genetics

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts
Mithun Mitra 1,2, Ha Neul Lee 1,2,3, Hilary A. Coller 1,2,3
1Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, 2Department of Biological Chemistry, David Geffen School of Medicine, 3Molecular Biology Institute Interdepartmental Program, University of California, Los Angeles

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

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JoVE Journal

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System
Marlin Touma 1,2
1Neonatal/Congenital Heart Laboratory, Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, 2Children's Discovery and Innovation Institute, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

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Behavior

Assessing Activity-based Anorexia in Mice
Amanda C. Welch 1, William R. Katzka 1,2, Stephanie C. Dulawa 1
1Department of Psychiatry, University of California, San Diego, 2University of California, Los Angeles

Mice individually housed with a running wheel while given limited access to food develop reductions in food consumption and increase activity on the running wheel. This experimental phenomenon is called activity-based anorexia. This paradigm provides an experimental tool for studying the neurobiology and behaviors underlying aspects of anorexia nervosa.

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Biochemistry

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue
David Jelinek 1,2, Aimee Flores 1,3, Melanie Uebelhoer 1, Vincent Pasque 2, Kathrin Plath 2,3, M. Luisa Iruela-Arispe 1,3, Heather R. Christofk 2,3, William E. Lowry 1,3, Hilary A. Coller 1,2,3
1Department of Molecular, Cell and Developmental Biology, UCLA, 2Department of Biological Chemistry, David Geffen School of Medicine, 3Molecular Biology Institute Interdepartmental Program, UCLA

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

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Cancer Research

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice
Gilbert Gastelum 1, Eric Y. Chang 2, David Shackleford 3, Nicholas Bernthal 3, Jeffery Kraut 1,3, Kevin Francis 4, Victoria Smutko 1, Patrick Frost 1,3
1Greater Los Angeles Veteran Administration Healthcare System, 2San Diego Veterans Administration Healthcare System, 3University of California, Los Angeles, 4Perkin Elmer

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

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Bioengineering

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells
Weikun Xiao 1, Arshia Ehsanipour 1, Alireza Sohrabi 1, Stephanie K. Seidlits 2
1Department of Bioengineering, University of California, Los Angeles, 2Department of Bioengineering, Jonsson Comprehensive Cancer Center, Broad Stem Cell Research Center, Brain Research Institute, University of California, Los Angeles

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

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Behavior

Continuous Theta Burst Stimulation of the Posterior Medial Frontal Cortex to Experimentally Reduce Ideological Threat Responses
Colin Holbrook 1, Chelsea L. Gordon 1, Marco Iacoboni 2
1Department of Cognitive and Information Sciences, University of California, Merced, 2Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles

Threats reliably evoke shifts in high-level ideological investment, but little work to date has explored the neural mechanisms underlying these dynamics. This paper describes how continuous theta burst transcranial magnetic stimulation may be employed to test the contribution of the posterior medial frontal cortex (and/or other regions) to threat-related ideological shifts.

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Behavior

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder
Abha K. Rajbhandari 1,2, Sarah T. Gonzalez 1,2, Michael S. Fanselow 1,2,3
1Department of Psychology, University of California, Los Angeles, 2Staglin Center for Brain and Behavioral Health, University of California, Los Angeles, 3Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

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Medicine

A Metadata Extraction Approach for Clinical Case Reports to Enable Advanced Understanding of Biomedical Concepts
John Harry Caufield 1,2, David A. Liem 1,2,3, Anders O. Garlid 1,2, Yijiang Zhou 4, Karol Watson 1,3, Alex A. T. Bui 1,5,6,7, Wei Wang 1,7,8,9, Peipei Ping 1,2,3,7,8
1The NIH BD2K Center of Excellence in Biomedical Computing, University of California, Los Angeles, 2Department of Physiology, University of California, Los Angeles, 3Department of Medicine/Cardiology, University of California, Los Angeles, 4Department of Cardiology, First Affiliated Hospital, Zhejiang University School of Medicine, 5Department of Radiological Sciences, University of California, Los Angeles, 6Department of Bioengineering, University of California, Los Angeles, 7Scalable Analytics Institute (ScAi), University of California, Los Angeles, 8Department of Bioinformatics, University of California, Los Angeles, 9Department of Computer Science, University of California, Los Angeles

We present a protocol and associated metadata template for the extraction of text describing biomedical concepts in clinical case reports. The structured text values produced through this protocol can support deep analysis of thousands of clinical narratives.

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Bioengineering

Meso-Scale Particle Image Velocimetry Studies of Neurovascular Flows In Vitro
Ryan A. Peck *1, Edver Bahena *1, Reza Jahan 2, Guillermo Aguilar 1,3,4, Hideaki Tsutsui 1,4, Marko Princevac 1, Monica M. Wilhelmus 1, Masaru P. Rao 1,3,4
1Department of Mechanical Engineering, University of California, Riverside, 2Division of Interventional Neuroradiology, University of California, Los Angeles, 3Materials Science and Engineering Program, University of California, Riverside, 4Department of Bioengineering, University of California, Riverside

Here we present simplified methods for fabricating transparent neurovascular phantoms and characterizing the flow therein. We highlight several important parameters and demonstrate their relationship to field accuracy.

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Medicine

Cloud-Based Phrase Mining and Analysis of User-Defined Phrase-Category Association in Biomedical Publications
Dibakar Sigdel *1,2, Vincent Kyi *1,2, Aiden Zhang *1, Shaun P. Setty 3, David A. Liem 1,2,4, Yu Shi 5, Xuan Wang 5, Jiaming Shen 5, Wei Wang 1,6,7, JiaWei Han 5, Peipei Ping 1,2,4,6
1The NIH BD2K Center of Excellence in Biomedical Computing, University of California, Los Angeles, 2Department of Physiology, University of California, Los Angeles, 3Department of Pediatric and Adult Congenital Heart Surgery, Miller Children's and Women's Hospital and Long Beach Memorial Hospital, 4Department of Medicine/Cardiology, University of California, Los Angeles, 5NIH BD2K Program Centers of Excellence for Big Data Computing -- KnowEng Center, Department of Computer Science, University of Illinois at Urbana-Champaign (UIUC), 6Scalable Analytics Institute (ScAi), University of California, Los Angeles, 7Department of Computer Science, University of California, Los Angeles

We present a protocol and associated programming code as well as metadata samples to support a cloud-based automated identification of phrases-category association representing unique concepts in user selected knowledge domain in biomedical literature. The phrase-category association quantified by this protocol can facilitate in depth analysis in the selected knowledge domain.

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Genetics

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR
Carl Robert Rankin 1, Janet Treger 1, Emmanuelle Faure-Kumar 2, Jihane Benhammou 1, Deborah Anisman-Posner 3, Alex Edward Bollinger 3, Charalabos Pothoulakis 1, David Miguel Padua 1
1Vatche and Tamar Manoukian Division of Digestive Diseases, Department of Medicine, University of California, Los Angeles, 2Vatche and Tamar Manoukian Division of Digestive Diseases, Integrated Molecular Technologies (IMT) Core, Department of Medicine, University of California, Los Angeles, 3Division of Hematology and Oncology, Department of Medicine, University of California, Los Angeles

Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.

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Medicine

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach
Ali R. Ahmadi 1, Le Qi 1, Kenichi Iwasaki 1, Wei Wang 1, Russell N. Wesson 1, Andrew M. Cameron 1, Zhaoli Sun 1
1Department of Surgery, Johns Hopkins University School of Medicine

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

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Environment

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System
Weitao Hou 1, Hanpeng Wang 1, Wei Wang 1, Zhongzhong Liu 1, Qingchuan Li 1
1Research Center of Geotechnical and Structural Engineering, Shandong University

This protocol demonstrates how to prepare a briquette sample and conduct a uniaxial compression experiment with a briquette in different CO2 pressures using a visualized and constant-volume gas-solid coupling test system. It also aims to investigate changes in terms of coal’s physical and mechanical properties induced by CO2 adsorption.

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Biology

In Vivo Surface Electrocardiography for Adult Zebrafish
Yali Zhao 1, Morgan Yun 1, Sean A. Nguyen 1, Michelle Tran 1, Thao P. Nguyen 1
1Department of Medicine, Division of Cardiology, the Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

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Medicine

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography
Di Liu *1, Xizhu Xu *2, Yuejin Li 2, Jie Zhang 1, Xiaoyu Zhang 1, Qihuan Li 1, Haifeng Hou 2, Dong Li 2, Wei Wang 1,2,3, Youxin Wang 1
1Beijing Key Laboratory of Clinical Epidemiology, School of Public Health, Capital Medical University, 2School of Public Health, Shandong First Medical University & Shandong Academy of Medical Sciences, 3School of Medical and Health Sciences, Edith Cowan University

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

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Cancer Research

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture
Preston D. Crowell *1, Jenna M. Giafaglione *1, Takao Hashimoto 2, Johnny A. Diaz 2, Andrew S. Goldstein 2,3,4,5,6
1Molecular Biology Interdepartmental Program, University of California, Los Angeles, 2Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, 3Department of Urology, David Geffen School of Medicine, University of California, Los Angeles, 4Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, 5Jonsson Comprehensive Cancer Center, University of California, Los Angeles, 6Molecular Biology Institute, University of California, Los Angeles

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

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Cancer Research

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane
Moe Ishihara *1, Junhui Hu *1, Xiaoyu Zhang 2, YongHyeon Choi 3, Anthony Wong 4, Celine Cano-Ruiz 5, Rongwei Zhao 6, Ping Tan 7, Jonathan L. Tso 1, Lily Wu 1,8
1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, 3Department of Bioengineering, Hanyang University, 4Department of Ecology and Evolutionary Biology, University of California, Los Angeles, 5Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, 6School of Life Sciences, Beijing Normal University, 7Department of Urology, West China Hospital, Sichuan University, 8Department of Urology, David Geffen School of Medicine, University of California, Los Angeles

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

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Cancer Research

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts
Sanghee Lee 1,2, Danielle N. Burner 1,2, Theresa R. Mendoza 1,2, Michelle T. Muldong 1,2, Catalina Arreola 1,2, Christina N. Wu 2,3, Nicholas A. Cacalano 4, Anna A. Kulidjian 2,5, Christopher J. Kane 1,2, Christina A. M. Jamieson 1,2
1Department of Urology, University of California, San Diego, 2Moores Cancer Center, University of California, San Diego, 3Department of Medicine, University of California, San Diego, 4Department of Radiation Oncology, University of California, Los Angeles, 5Department of Orthopedic Surgery, University of California, San Diego

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

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JoVE Journal

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin
Panchanan Maiti 1,2,3,4,5, Alexandra Plemmons 1, Zackary Bowers 2,3, Charles Weaver 5, Gary Dunbar 1,2,3,4
1Field Neurosciences Institute, Ascension St. Mary's Hospital, 2Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, 3Program in Neuroscience, Central Michigan University, 4Department of Psychology, Central Michigan University, 5Department of Health Sciences, Saginaw Valley State University

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

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Biology

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke
Katrina L. Wilson 1, S. Thomas Carmichael 2, Tatiana Segura 1,3,4
1Department of Biomedical Engineering, Duke University, 2Department of Neurology, University of California Los Angeles, 3Department of Neurology, Duke University, 4Department of Dermatology, Duke University

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

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Behavior

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice
Yi Xie 1, Sai-Yue Deng 1, Si-Miao Chen 1, Xue-Jiao Chen 1, Wen-Wen Lai 1, Li-Fang Huang 1, Li Ba 1, Wei Wang 1,2, Feng-Fei Ding 1,3
1Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, China, 2Key Laboratory of Neurological Diseases of Chinese Ministry of Education, The School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000,China, 3Department of Pharmacology, School of Basic Medical Science; State Key Laboratory of Medical Neurobiology, Institutes of Brain Science and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

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Cancer Research

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth
David Jelinek 1,2, Ellen Ran Zhang 1,2,3, Aaron Ambrus 1,2, Erin Haley 3, Emily Guinn 1,2, Austin Vo 1,2, Peter Le 1,2, Ayse Elif Kesaf 1,2, Jennifer Nguyen 1,2, Lily Guo 1,2, Destiny Frederick 1,2, Zhengyang Sun 1,2, Natalie Guo 3, Parker Sevier 1,2, Eric Bilotta 1,2, Kaiser Atai 1,2,4, Laurent Voisin 1,2, Hilary A. Coller 1,2,4
1Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, 2Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Molecular Biology, Princeton University, 4Molecular Biology Institute, University of California, Los Angeles

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

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Biology

Specimen Collection and Analysis of the Duodenal Microbiome
Benjamin W. Dreskin 1, Kayti Luu 1, Tien S. Dong 1,2,3, Jihane Benhammou 1,2,4, Venu Lagishetty 2,3, John Vu 1, Daniel Sanford 1, Francisco Durazo 2,4, Vatche G. Agopian 4, Jonathan P. Jacobs 1,2,3, Joseph R. Pisegna 1,5, Shehnaz K. Hussain 6
1Division of Gastroenterology, Hepatology and Parenteral Nutrition, Veterans Administration Greater Los Angeles Healthcare System, 2The Vatche and Tamar Manoukian Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine at University of California, 3UCLA Microbiome Center, David Geffen School of Medicine at University of California, 4Department of Surgery, University of California, Los Angeles, 5Department of Medicine and Human Genetics, David Geffen School of Medicine at University of California, 6Cedars-Sinai Cancer and Department of Medicine, Cedars-Sinai Medical Center

In this manuscript, we discuss a novel method to sample and analyze the duodenal microbiome. This method provides an accurate depiction of microbial diversity and composition in the duodenum and could be useful for further investigation of the duodenal microbiome.

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Neuroscience

Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method
Weiyan Jiang *1, Congcong Jin *2, Weiming Xu *3, Yingxian Li 4, Yinghong Lin 5, Shengxiang Liang 6,7, Wei Wang 8
1Department of Clinical Laboratory, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 2Rehabilitation Department, Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine, 3Department of Neurosurgery, the Hospital of Changle, 4Pediatrics Ward 2, Shouguang Hospital of Traditional Chinese Medicine, 5Fujian University of Traditional Chinese Medicine, 6National-Local Joint Engineering Research Center of Rehabilitation Medicine Technology, Fujian University of Traditional Chinese Medicine, 7Rehabilitation Industry Institute, Fujian University of Traditional Chinese Medicine, 8Department of Neurosurgery, the First Affiliated Hospital of Wenzhou Medical University

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

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Immunology and Infection

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection
Michelle L. Castelletto 1, Elissa A. Hallem 1,2
1Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, 2Molecular Biology Institute, University of California, Los Angeles

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S. stercoralis and S. ratti.

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Biochemistry

Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot
Yu Han 1,3, Cody T. Thomas 1,3, Sara A. Wennersten 1,3, Edward Lau 1,3, Maggie P. Y. Lam 1,2,3
1Department of Medicine-Cardiology, University of Colorado Anschutz Medical Campus, 2Department of Biochemistry & Molecular Genetics, University of Colorado Anschutz Medical Campus, 3Consortium for Fibrosis Research & Translation, School of Medicine, University of Colorado Anschutz Medical Campus

Detailed protocol and three Python scripts are provided for operating an open-source robotic liquid handling system to perform semi-automated protein sample preparation for mass spectrometry experiments, covering detergent removal, protein digestion, and peptide desalting steps.

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Bioengineering

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers
Lara Hairapetian 1, Enrico Cortes 1, Junyi Zhao 1, Yao Wang 1, Ricky Huang 1, Robert Damoiseaux 1,2, Ivan Pushkarsky 1
1Forcyte Biotechnologies, 2University of California, Los Angeles

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

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Biology

Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes
Veena Sangkhae 1, Elizabeta Nemeth 1
1Center for Iron Disorders, David Geffen School of Medicine, University of California, Los Angeles

This article demonstrates how to prepare and administer transferrin-bound nonradioactive isotopic iron for studies of iron transport in mouse pregnancy. The approach for quantifying isotopic iron in fetoplacental compartments is also described.

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Bioengineering

Engineering Antiviral Agents via Surface Plasmon Resonance
Irene Maier 1,2
1Department of Environmental Health Sciences, University of California, Los Angeles, 2Department of General Surgery, Medical University of Vienna

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

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Immunology and Infection

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice
Wenli Mu 1, Anjie Zhen 1, Mayra A. Carrillo 1, Valerie Rezek 1, Heather Martin 1, Miguel Lizarraga 1, Scott Kitchen 1
1Division of Hematology and Oncology, David Geffen School of Medicine, University of California, Los Angeles

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

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Bioengineering

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models
Jesse Liang 1, Alireza Sohrabi 1, Mary Epperson 1, Laila M. Rad 1, Kelly Tamura 1, Mayilone Sathialingam 1, Thamira Skandakumar 1, Philip Lue 1, Jeremy Huang 1, James Popoli 1, Aidan Yackly 1, Michael Bick 1, Ze Zhong Wang 1, Chia-Chun Chen 1, Grigor Varuzhanyan 1, Robert Damoiseaux 1, Stephanie K. Seidlits 1
1University of California Los Angeles

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

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Cancer Research

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model
Junli Chang *1,2, Xingyuan Sun *1,2, Xiaoping Ma 1,2, Peng Zhao 1,2, Binhao Shi 1,2, Yongjun Wang 1,2, Xianghui Han 1,3, Yanping Yang 1,2
1Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 2Key Laboratory of Theory and Therapy of Muscles and Bones, Ministry of Education, 3Institute of Chinese Traditional Surgery, Longhua Hospital, Shanghai University of Traditional Chinese Medicine

Here, we present a protocol for the intra-cardiac injection of human prostate cancer cells to generate a mouse model with bone metastasis lesions.

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Cancer Research

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures
Katie B. Grausam *1, Joshua J. Breunig *1,2,3,4,5
1Board of Governor’s Regenerative Medicine Institute, Cedars-Sinai Medical Center, 2Center for Neural Sciences in Medicine, Cedars-Sinai Medical Center, 3Department of Biomedical Sciences, Cedars-Sinai Medical Center, 4Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, 5Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

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Biology

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy
Irene Santiago Tierno 1,2,3, Mahesh Agarwal 1,2, Nikolaos Matisioudis 2,4, Sathishkumar Chandrakumar 1,2, Kaustabh Ghosh 1,2,3
1Department of Ophthalmology, University of California, Los Angeles, 2Doheny Eye Institute, 3Molecular, Cellular, and Integrative Physiology Interdepartmental PhD Program, University of California, Los Angeles, 4Department of Bioengineering, University of Pennsylvania

We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.

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