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University of Reading

10 ARTICLES PUBLISHED IN JoVE

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Biology

Assaying the Kinase Activity of LRRK2 in vitro
Patrick A. Lewis 1
1Department of Molecular Neuroscience, UCL Institute of Neurology

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

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Research

Measuring Lactase Enzymatic Activity in the Teaching Lab
Cattleya S. Leksmono 1, Claudia Manzoni 1,2, James E. Tomkins 1, Walter Lucchesi 1,3, Graeme Cottrell 1, Patrick A. Lewis 1
1School of Pharmacy, University of Reading, 2Department of Molecular Neuroscience, UCL Institute of Neurology, 3School of Biological Sciences, Royal Hollway, University of London

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

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Bioengineering

Micropatterning and Assembly of 3D Microvessels
Meredith A. Roberts *1, Surya S. Kotha *1, Kiet T. Phong 2, Ying Zheng 1,2
1Department of Bioengineering, University of Washington, 2Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

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Neuroscience

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent
Sungmin Kang 1, Michael Bruyns-Haylett 2, Yurie Hayashi 1, Ying Zheng 1
1School of Biological Sciences, Whiteknights, University of Reading, 2Department of Bioengineering, Imperial College

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

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Medicine

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model
Alejandro Giraldo *1, Jesús Talavera López *2, Maria Josefa Fernandez-Del-Palacio 2, Obdulio García-Nicolás 3,4, Juan Seva 4, Gavin Brooks 1, José María Moraleda 5
1Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, 2Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad de Murcia, 3Institute of Virology and Immunology (IVI), 4Departamento de Anatomía y Anatomía Comparada, Facultad de Veterinaria, Universidad de Murcia, 5Unidad de Trasplante Hematopoyético y Terapia Celular, Departamento de Hematología, Hospital Universitario Virgen de la Arrixaca, IMIB, Universidad de Murcia

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

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Bioengineering

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel
Harrison L. Hiraki 1, Ryan J. Nagao 2, Jonathan Himmelfarb 3, Ying Zheng 2
1Department of Bioengineering, University of Washington, 2Department of Bioengineering, Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, 3Department of Medicine, Kidney Research Institute, University of Washington

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

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Neuroscience

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice
Yunan Chen 1,2, Ming Li 3, Ying Zheng 3, Li Yang 1
1School of Life Sciences, Guangzhou University, 2Institute for Brain Research and Rehabilitation, South China Normal University, 3School of Life Sciences, South China Normal University

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

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Medicine

Transthoracic Echocardiographic Examination in the Rabbit Model
Alejandro Giraldo *1, Jesús Talavera López *2, Gavin Brooks 1, María Josefa Fernández-del-Palacio 2
1Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, 2Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad de Murcia

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

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Biology

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data
Gunjan Dixit 1, Ying Zheng 1, Brian Parker 2, Jiayu Wen 1
1Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, 2Department of Biology, New York University

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

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Biochemistry

Kinetx: A Combined Flow Cytometry Assay and Analysis Software Framework to Quantitatively Measure and Categorize Platelet Activation in Real-time.
Joanne L. Dunster *1, Jo L. Mitchell *1,2, Yasmin M. M. Mohammed 1, Kirk A. Taylor 1, Jonathan M. Gibbins 1, Chris I. Jones 1
1ICMR, School of Biological Sciences, University of Reading, 2Institute of Cardiovascular Sciences, University of Birmingham

Platelets react rapidly to a range of stimuli. This paper describes a real-time flow cytometry-based platelet function assay and a newly developed bespoke open-source software (Kinetx) to enable quantitative kinetic measurements of platelet granule release, fibrinogen binding, and intracellular calcium flux.

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