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University of Leeds

11 ARTICLES PUBLISHED IN JoVE

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Engineering

Sputter Growth and Characterization of Metamagnetic B2-ordered FeRh Epilayers
Chantal Le Graët 1, Mark A. de Vries 1,2, Mathew McLaren 1,3, Richard M.D. Brydson 3, Melissa Loving 4, Don Heiman 5, Laura H. Lewis 4, Christopher H. Marrows 1
1School of Physics and Astronomy, University of Leeds, 2Institute of Materials Research, University of Leeds, 3School of Chemistry, University of Edinburgh, 4Department of Chemical Engineering, Northeastern University, 5Department of Physics, Northeastern University

A method to prepare epitaxial layers of ordered alloys by sputtering is described. The B2-ordered FeRh compound is used as an example, as it displays a metamagnetic transition that depends sensitively on the degree of chemical order and the exact composition of the alloy.

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Chemistry

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes
Yogita Patil-Sen 1, Amin Sadeghpour 2, Michael Rappolt 2, Chandrashekhar V. Kulkarni 1
1Centre for Materials Science, School of Physical Sciences and Computing, University of Central Lancashire, 2School of Food Science & Nutrition, University of Leeds

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

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Biochemistry

Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems
Charlotte A. Scarff 1, Martin J. G. Fuller 2, Rebecca F. Thompson 2, Matthew G. Iadanza 1
1Astbury Centre for Structural Molecular Biology, University of Leeds, 2Astbury Biostructure Laboratory, University of Leeds

Negative stain EM is a powerful technique for visualizing macromolecular structure, but different staining techniques can produce varying results in a sample dependent manner. Here several negative staining approaches are described in detail to provide an initial workflow for tackling the visualization of challenging systems.

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Engineering

Controllable Nucleation of Cavitation from Plasmonic Gold Nanoparticles for Enhancing High Intensity Focused Ultrasound Applications
James R. McLaughlan 1,2
1School of Electronic and Electrical Engineering, University of Leeds, 2Leeds Institute of Cancer and Pathology, University of Leeds

This protocol demonstrates the controllable nucleation of cavitation in gel phantoms, through simultaneous exposure to both near-infrared pulsed laser light and high intensity focused ultrasound (HIFU). The cavitation activity can then be used for enhancing imaging and/or therapeutic uses of HIFU.

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Biochemistry

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy
Ievgen Mazurenko 1, Nikos S. Hatzakis 2, Lars J.C. Jeuken 1
1School of Biomedical Sciences & the Astbury Centre for Structural Molecular Biology, University of Leeds, 2Department of Chemistry and Nano-Science Center, University of Copenhagen

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

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Biochemistry

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors
Keni Vidilaseris *1, Niklas G. Johansson *2, Ainoleena Turku 2, Alexandros Kiriazis 2, Gustav Boije af Gennäs 2, Jari Yli-Kauhaluoma 2, Henri Xhaard 2, Adrian Goldman 1,3
1Research Program in Molecular and Integrative Biosciences, University of Helsinki, 2Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, 3School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds

Here we present a screening method for membrane-bound pyrophosphatase (from Thermotoga maritima) inhibitors based on the molybdenum blue reaction in a 96 well plate format.

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Biology

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling
Jay Miles 1, Patricija van Oosten-Hawle 1
1School of Molecular and Cell Biology & Astbury Centre for Structural Molecular Biology, University of Leeds

Maintenance of organismal proteostasis requires the coordination of protein quality control responses such as chaperone expression from one tissue to another. Here, we provide tools used in C. elegans that allow monitoring of proteostasis capacity in specific tissues and determine intercellular signaling responses.

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Biochemistry

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy
David P. Klebl 1, Frank Sobott 2, Howard D. White 3, Stephen P. Muench 1
1School of Biomedical Sciences, Faculty of Biological Sciences & Astbury Centre for Structural and Molecular Biology, University of Leeds, 2School of Molecular and Cellular Biology, Faculty of Biological Sciences & Astbury Centre for Structural and Molecular Biology, University of Leeds, 3Department of Physiological Sciences, Eastern Virginia Medical School

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

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JoVE Journal

Single Particle Cryo-Electron Microscopy: From Sample to Structure
Joshua B.R. White 1, Daniel P. Maskell 1, Andrew Howe 2, Martin Harrow 2, Daniel K. Clare 2, C. Alistair Siebert 2, Emma L. Hesketh 1, Rebecca F. Thompson 1
1Astbury Centre Structural Molecular Biology, School Molecular and Cellular Biology, Faulty Biological Sciences, University of Leeds, 2Diamond Light Source, Harwell Science and Innovation Campus

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

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Medicine

Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound
Nahla Ibrahim 1, Johannes Klopf 1, Sonja Bleichert 1, Marc A. Bailey 2,3, Albert Busch 4, Alexander Stiglbauer-Tscholakoff 5, Wolf Eilenberg 1, Christoph Neumayer 1, Christine Brostjan 1
1Division of Vascular Surgery, Department of General Surgery, Medical University of Vienna and Vienna General Hospital, 2Leeds Institute for Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, 3Leeds Vascular Institute, Leeds General Infirmary, 4Department for Visceral, Thoracic and Vascular Surgery, Technical University of Dresden and University Hospital Carl-Gustav Carus, 5Division of Cardiovascular and Interventional Radiology, Division of Molecular and Gender Imaging, Department of Biomedical Imaging and Image Guided Therapy, Medical University of Vienna and Vienna General Hospital

This protocol describes the consecutive implantation of an osmotic pump to induce abdominal aortic aneurysm by angiotensin II release in apolipoprotein E (ApoE) deficient mice and of a vascular access port with a jugular vein catheter for repeated drug treatment. Monitoring of aneurysm development by 3D ultrasound is effectively conducted despite dorsal implants.

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Medicine

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue
Oliver I. Brown 1, Katherine I. Bridge 1, Sam Straw 1, Natallia Makava 1, Jason Scragg 1, Sunti Limumpornpetch 1, Cheukyau Luk 1, Jessica Smith 1, Anna Skromna 1, Alexander F. Bruns 1, Piruthivi Sukumar 1, Lee D. Roberts 1, Richard Cubbon 1, Klaus K. Witte 1, Stephen Wheatcroft 1, Mark T. Kearney 1
1Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

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