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University of Manchester

35 ARTICLES PUBLISHED IN JoVE

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Biology

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation
Enrique Amaya 1, Kristen Kroll 2
1The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, 2Department of Developmental Biology, Washington University School of Medicine

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

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Medicine

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies
Mitra Tavakoli *1, Rayaz A. Malik *1
1Division of Cardiovascular Medicine, University of Manchester

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

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Biology

Preparation of Highly Coupled Rat Heart Mitochondria
Irina Gostimskaya 1, Alexander Galkin 2
1Faculty of Life Sciences, University of Manchester, 2School of Biological Sciences, Queen's University Belfast

We describe а protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

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Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts
Urszula M. Polanska *1, Ahmet Acar *1, Akira Orimo 1,2
1CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, University of Manchester, 2Atopy Research Center, Juntendo University

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

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Biology

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae
Liam O'Ryan 1, Tracy Rimington 1, Natasha Cant 1, Robert C. Ford 1
1Faculty of Life Sciences, University of Manchester

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

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Biology

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors
Açelya Yilmazer 1, Irene de Lázaro 1, Cyrill Bussy 1, Kostas Kostarelos 2
1Nanomedicine Lab, UCL School of Pharmacy, University College London, 2Nanomedicine Lab, Faculty of Medical & Human Sciences, University of Manchester

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

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Biology

Rapid Generation of Amyloid from Native Proteins In vitro
Stephanie M Dorta-Estremera 1, Jingjing Li 1, Wei Cao 1
1Department of Immunology, The University of Texas MD Anderson Cancer Center

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

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JoVE Core

A Simple Stimulatory Device for Evoking Point-like Tactile Stimuli: A Searchlight for LFP to Spike Transitions
Antonio G. Zippo 1, Sara Nencini 1, Gian Carlo Caramenti 2, Maurizio Valente 1, Riccardo Storchi 3, Gabriele E.M. Biella 1
1Institute of Molecular Bioimaging and Physiology (IBFM), Department of Biomedicine, National Research Council, 2Institute of Biomedical Technologies (ITB), Department of Biomedicine, National Research Council, 3Faculty of Life Sciences, University of Manchester

To elucidate the complex transition from Local Field Potentials (LFPs) to spikes a suitable stimulator for light mechanical peripheral stimuli was built. As an application, the spiking activities recorded from somatosensory cortex were analyzed by a multi-objective optimization strategy. The results demonstrated that the proposed stimulator was able to deliver tactile stimuli with millisecond and millimeter precisions.

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Biology

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae
Naomi Pollock 1, Natasha Cant 1, Tracy Rimington 1, Robert C. Ford 1
1Faculty of Life Sciences, University of Manchester

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies. 

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Upper Extremity Transplantation in Non-Human Primates: An Orthotopic Model for Translational Research.
David A Leonard 1,2,3, Harrison Powell 1, Alexander Albritton 1, Kumaran Shanmugarajah 1,2, Melissa Mastroianni 1,2, Sarah Lofgren 1, James Winter 1, Josef M Kurtz 1,4, Curtis L Cetrulo, Jr 1,2
1Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, 2Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, 3Department of Plastic & Reconstructive Surgery Research, University of Manchester, 4Department of Biology, Emmanuel College

Vascularized composite allotransplantation (VCA) offers unparalleled restoration of function and form following devastating musculoskeletal and soft tissue injury. Surgical techniques have developed rapidly, but immunological and functional outcomes require further study. We described a rigorous non-human primate model of orthotopic upper extremity transplantation for pre-clinical studies immediately preceding clinical translation.

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Medicine

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology
Aamer Sandoo 1,2, George D. Kitas 2,3
1School of Sport, Health and Exercise Sciences, Bangor University, 2Department of Rheumatology, Dudley Group of Hospitals NHS Trust, Russells Hall Hospital, 3Arthritis Research UK Epidemiology Unit, University of Manchester

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

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Engineering

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles
Thomas J. A. Slater 1, Edward A. Lewis 1, Sarah J. Haigh 1
1School of Materials, University of Manchester

The use of energy dispersive X-ray tomography in the scanning transmission electron microscope to characterize elemental distributions within single nanoparticles in three dimensions is described.

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Neuroscience

Enrichment of Bruch's Membrane from Human Donor Eyes
Selina McHarg 1,2, Nicole Brace 1,2, Paul N. Bishop 1,2, Simon J. Clark 1,2
1Centre for Ophthalmology & Vision Sciences, Institute of Human Development, University of Manchester, 2Centre for Advanced Discovery and Experimental Therapeutics, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre

Identifying proteins specifically associated with Bruch’s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

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Immunology and Infection

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network
Nicolas Dutzan *1, Loreto Abusleme *1, Joanne E. Konkel 2,3, Niki M. Moutsopoulos 1
1Oral Immunity and Inflammation Unit, NIDCR, NIH, 2Manchester Immunology Group, Faculty of Life Sciences, University of Manchester, 3Manchester Collaborative Centre for Inflammation Research, University of Manchester

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

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Neuroscience

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining
Siwei Zhang *1,2, Jingjing Li *1,3, Robert Lea 1, Enrique Amaya 1
1The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, 2Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, 3Department of Craniofacial Development and Stem Cell Biology, Dental Institute, King's College London

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

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Biology

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling
Zai Hao Zhao 1, Chun Li Jin 1, Ji Hyun Jang 1, Yu Na Wu 1, Sung Joon Kim 1, Hong Hua Jin 2, Lan Cui 2, Yin Hua Zhang 1,2,3
1Department of Physiology & Biomedical Sciences, Ischemic/hypoxic Disease Institute, Seoul National University College of Medicine, 2Yan Bian University Hospital, 3Institute of Cardiovascular Sciences, University of Manchester

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

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Developmental Biology

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution
Wasay M. Shaikh Qureshi 1, Lianjie Miao 1, David Shieh 1, Jingjing Li 1, Yangyang Lu 1, Saiyang Hu 1, Margarida Barroso 1, Joseph Mazurkiewicz 2, Mingfu Wu 1
1Department of Molecular and Cellular Physiology, Albany Medical College, 2Department of Neuroscience and Experimental Therapeutics, Albany Medical College

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

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Cancer Research

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures
Eamonn Morrison 1,2, Patty Wai 1,2, Andri Leonidou 1,2, Philip Bland 1,2, Saira Khalique 1,2, Gillian Farnie 3, Frances Daley 1, Barrie Peck 1,2, Rachael Natrajan 1,2
1The Breast Cancer Now Toby Robins Research Centre, Division of Breast Cancer, The Institute of Cancer Research, 2Division of Molecular Pathology, The Institute of Cancer Research, 3Institute of Cancer Sciences, University of Manchester

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

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JoVE Journal

Experimental Procedure for Warm Spinning of Cast Aluminum Components
Matthew J. Roy 1, Daan M. Maijer 2
1School of Mechanical, Aerospace, and Civil Engineering, University of Manchester, 2Materials Engineering, University of British Columbia

An experimental protocol for instrumented warm rotary forming of cast aluminum alloys employing a bespoke industrially scaled apparatus is presented. Experimental considerations including thermal and mechanical effects are discussed, as well as similitude with full-scale processing of automotive wheels.

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Bioengineering

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement
Jun Liu 1, John Wilson 2, Claire Davis 1, Anthony Peyton 2
1Advanced Steel Research Centre, Warwick Manufacturing Group, University of Warwick, 2School of Electrical and Electronic Engineering, University of Manchester

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

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Bioengineering

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies
I-Ning Lee *1,2, Joseph Hosford *1, Shuai Wang 3, John A. Hunt 4, Judith M. Curran 2, William P. Heath 3, Lu Shin Wong 1
1Manchester Institute of Biotechnology & School of Chemistry, University of Manchester, 2School of Engineering, University of Liverpool, 3School of Electrical and Electronic Engineering, University of Manchester, 4School of Science and Technology, Nottingham Trent University

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

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Cancer Research

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor
Yu Okazawa 1,2, Kosuke Mizukoshi 1,2, Yu Koyama 2,4, Shoki Okubo 5, Hiromitsu Komiyama 1, Yutaka Kojima 1, Michitoshi Goto 1, Sonoko Habu 3, Okio Hino 2, Kazuhiro Sakamoto 1, Akira Orimo 2
1Department of Coloproctological Surgery, Juntendo University Faculty of Medicine, 2Department of Molecular Pathogenesis, Juntendo University Faculty of Medicine, 3Atopy Research Center, Juntendo University Faculty of Medicine, 4Department of Oral Pathobiological Science and Surgery, Tokyo Dental College, 5Department of Gastroenterology, Juntendo University Faculty of Medicine

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

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Immunology and Infection

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α
Alba Llibre *1,2, Vincent Bondet *1,2, Mathieu P. Rodero 3, David Hunt 4, Yanick J. Crow 3,5, Darragh Duffy 1,2
1Immunobiology of Dendritic Cells, Institut Pasteur, 2INSERM U1223, 3Laboratory of Neurogenetics and Neuroinflammation, INSERM UMR1163, Institut Imagine, 4MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, 5Manchester Centre for Genomic Medicine, University of Manchester

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-α subtypes in human samples.

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Bioengineering

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects
Prasad Sawadkar 1,3, Jason Wong 2, Vivek Mudera 1
1Division of Surgery and Interventional Science, University College London, 2Blond McIndoe Laboratories, Division of Cell Matrix and Regenerative Medicine, MAHSC, University of Manchester, 3Department of Surgical Research, Northwick Park Institute for Medical Research, Northwick Park Hospital

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

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Cancer Research

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells
Gray Kueberuwa 1, Weiming Zheng 1, Milena Kalaitsidou 1, David E. Gilham 1,2, Robert E. Hawkins 1
1Manchester Cancer Research Centre Building, Department Cancer Sciences, University of Manchester, 2Celyad

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

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Chemistry

Synthesis and Characterization of Amphiphilic Gold Nanoparticles
Zekiye P. Guven *1, Paulo H. Jacob Silva *1, Zhi Luo 1, Urszula B. Cendrowska 1, Matteo Gasbarri 1, Samuel T. Jones 1,2, Francesco Stellacci 1,3
1Institute of Materials, École Polytechnique Fédérale de Lausanne, 2School of Materials, University of Manchester, 3Interfaculty Institute of Bioengineering, École Polytechnique Fédérale de Lausanne

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

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Biology

Fabrication of Amyloid-β-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease
Bushra Almari 1, David Brough 2, Michael Harte 1, Annalisa Tirella 1
1Division of Pharmacy and Optometry, School of Health Studies, Faculty of Biology, Medicine and Health, University of Manchester, 2Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester

This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-β over time and can be used to study the effects of secreted amyloid-β in in vitro and in vivo models.

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Neuroscience

Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke
Siobhan Crilly 1, Alexandra Njegic 2, Adrian R. Parry-Jones 2,3, Stuart M. Allan 1,3, Paul R. Kasher 1,3
1Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Manchester Academic Health Science Centre, University of Manchester, 2Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, 3Lydia Becker Institute of Immunology and Inflammation, University of Manchester

Here we present a protocol to quantify brain injury, locomotor deficits and neuroinflammation following bleeding in the brain in zebrafish larvae, in the context of human intracerebral hemorrhage (ICH).

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Engineering

Design, Instrumentation and Usage Protocols for Distributed In Situ Thermal Hot Spots Monitoring in Electric Coils using FBG Sensor Multiplexing
Anees Mohammed 1, Siniša Durović 1
1School of Electrical and Electronic Engineering, University of Manchester

This paper presents a protocol that enables instrumentation of random wound electric coils with fiber Bragg grating (FBG) thermal sensors for the purpose of distributed condition monitoring of internal thermal hot spots.

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Medicine

Slicing and Culturing Pig Hearts under Physiological Conditions
Qinghui Ou *1, Riham R.E. Abouleisa *1, Xian-Liang Tang 1, Hamzah R. Juhardeen 1, Moustafa H. Meki 2, Jessica M. Miller 2, Guruprasad Giridharan 2, Ayman El-Baz 2, Roberto Bolli 1, Tamer M.A. Mohamed 1,3,4,5,6
1Institute of Molecular Cardiology, Department of Medicine, University of Louisville, 2Department of Bioengineering, University of Louisville, 3Diabetes and Obesity Center, Department of Medicine, University of Louisville, 4Department of Pharmacology and Toxicology, University of Louisville, 5Institute of Cardiovascular Sciences, University of Manchester, 6Faculty of Pharmacy, Zagazig University

This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

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Biology

Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease
Jennifer C. Ashworth 1, Rebecca L. Morgan 1, Kataryna Lis-Slimak 1, Kate A. Meade 1, Sal Jones 1, Katherine Spence 2, Charlotte E. Slater 1, Jamie L. Thompson 1, Anna M. Grabowska 1, Robert B. Clarke 2, Gilian Farnie 3, Cathy L. R. Merry 1
1Division of Cancer & Stem Cells, School of Medicine, Nottingham Biodiscovery Institute, University of Nottingham, 2Breast Biology Group, Manchester Breast Centre, Division of Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, 3SGC, Botnar Research Centre, NDORMS, University of Oxford

We present a method for creating a 3D cell culture environment, which can be used to investigate the importance of cell/matrix interactions in cancer progression. Using a simple self-assembling octapeptide, the matrix surrounding encapsulated cells can be controlled, with independent regulation of mechanical and biochemical cues.

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Cancer Research

Characterization and Functional Prediction of Bacteria in Ovarian Tissues
Lanbo Zhao *1, Weichu Zhao *2, Qi Wang *1, Dongxin Liang 1, Yu Liu 3, Guoxing Fu 4, Lu Han 1, Yiran Wang 1, Chao Sun 1, Qing Wang 1, Qing Song 1, Qiling Li 1, Qinrui Lu 1
1Department of Obstetrics and Gynecology, First Affiliated Hospital of Xi’an Jiaotong University, 2School of Social Sciences, University of Manchester, 3Department of Pathology, First Affiliated Hospital of Xi’an Jiaotong University, 4Omega Bioservices Inc

Immunohistochemistry staining and 16S ribosomal RNA gene (16S rRNA gene) sequencing were performed in order to discover and distinguish bacteria in cancerous and noncancerous ovarian tissues in situ. The compositional and functional differences of the bacteria were predicted by using BugBase and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt).

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JoVE Journal

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
Jason Wong 1, William Martelly 1, Shalini Sharma 1
1Department of Basic Medical Sciences, College of Medicine-Phoenix, University of Arizona

This protocol describes a minigene reporter assay to monitor the impact of 5´-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

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Medicine

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening
Yu-Fen Chang 1, Wan-Chi Su 1, Chih-Chuan Su 1, Min-Wen Chung 1, Jin Chang 2, You-Yi Li 3, Yi-Ju Kao 3, Wen-Pin Chen 3, Matthew J. Daniels 4,5,6
1LumiSTAR Biotechnology, Inc., 2NEXEL Co., Ltd., 3Institute of Pharmacology, College of Medicine, National Taiwan University, 4Manchester Heart Centre, Manchester Royal Infirmary, Manchester University NHS Foundation Trust, 5Division of Cardiovascular Sciences, Manchester Academic Health Sciences Centre, University of Manchester, 6Division of Cell Matrix Biology and Regenerative Medicine, University of Manchester

The present protocol describes all-optical control and observation of triggered cellular activity in iPSC-derived cardiomyocytes (iPSC-CMs) for high throughput drug screening and toxicity testing. Multi-parametric quantification of phenotypic patterns in time, and space, are shown. Long-term effects of drugs over hours, or sequential measurements over days, are demonstrated.

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Bioengineering

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting
Jessica J. Senior *1, Richard J. A. Moakes *2, Megan E. Cooke 3, Samuel R. Moxon 4, Alan M. Smith 1, Liam M. Grover 2
1Department of Pharmacy, University of Huddersfield, 2School of Chemical Engineering, University of Birmingham, 3Chemical and Biological Engineering, University of Colorado Boulder, 4School of Biological Sciences, University of Manchester

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

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