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Imperial College London

15 ARTICLES PUBLISHED IN JoVE

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Biology

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis
Mark F Brady 1, Jorge Coronel 2, Robert H Gilman 3, David AJ Moore 4
1The Warren Alpert Medical School of Brown University, 2Laboratorio de Investigacion de Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, 3Department of International Health, Johns Hopkins Bloomberg School of Public Health, 4Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College London

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

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Bioengineering

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
Klaus Suhling 1, James A. Levitt 1, Pei- Hua Chung 1, Marina. K. Kuimova 2, Gokhan Yahioglu 3
1Department of Physics, King's College London, 2Department of Chemistry, Imperial College London , 3PhotoBiotics Ltd

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

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Medicine

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice
Michael T. Kuhlmann 1, Simon Cuhlmann 2,3, Irmgard Hoppe 1, Rob Krams 3, Paul C. Evans 2, Gustav J. Strijkers 4, Klaas Nicolay 4, Sven Hermann 1, Michael Schäfers 1
1European Institute for Molecular Imaging, Westfälische Wilhelms-University Münster, 2British Heart Foundation Cardiovascular Sciences Unit, Imperial College London , 3Department of Bioengineering, Imperial College London , 4Biomedical Engineering, Eindhoven University of Technology

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

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Immunology and Infection

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)
Sandra Newton 1, Adrian Martineau 2, Beate Kampmann 1
1Department of Paediatrics, Imperial College London , 2Centre for Health Sciences, Barts & The London School of Medicine and Dentistry

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). “Survival” of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

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Bioengineering

Fluorescence detection methods for microfluidic droplet platforms
Xavier Casadevall i Solvas 1, Xize Niu 1, Katherine Leeper 1, Soongwon Cho 1, Soo-Ik Chang 2, Joshua B. Edel 1, Andrew J. deMello 3
1Department of Chemistry, Imperial College London , 2Department of Biochemistry, Protein Chip Research Center, Chungbuk National University, 3Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering, ETH Zurich

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

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Biology

Identification of Protein Interacting Partners Using Tandem Affinity Purification
Dalan Bailey *1, Luis Urena *1, Lucy Thorne 1, Ian Goodfellow 1
1Section of Virology, Department of Medicine, Imperial College London

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

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Bioengineering

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis
Teresa Mortera-Blanco 1, Maria Rende 1, Hugo Macedo 1, Serene Farah 1, Alexander Bismarck 1, Athanasios Mantalaris 1, Nicki Panoskaltsis 2
1Department of Chemical Engineering and Chemical Technology, South Kensington campus, Imperial College London , 2Department of Hematology, Northwick Park & St. Mark's campus, Imperial College London

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

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Medicine

Roux-en-Y Gastric Bypass Operation in Rats
Marco Bueter 1,2, Kathrin Abegg 2,3, Florian Seyfried 4, Thomas A. Lutz 2,3, Carel W. le Roux 4
1Department of Surgery, University Hospital Zürich, 2Zürich Centre for Integrative Human Physiology, University of Zürich, 3Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zürich, 4Imperial Weight Centre, Department of Investigative Medicine, Imperial College London

Numerous studies using gastric bypass rat models have been recently conducted to uncover the underlying physiological mechanisms of Roux-en-Y gastric bypass operations. This article aims to demonstrate and discuss the technical and experimental details of our published gastric bypass rat model to understand advantages and limitations of this experimental tool.

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Medicine

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System
Anne Klomp 1, Jordi L. Tremoleda 2, Anouk Schrantee 1, Willy Gsell 2, Liesbeth Reneman 1
1Department of Radiology, Brain Imaging Center, Academic Medical Center Amsterdam, 2Biological Imaging Centre, MRC Clinical Sciences Centre, Imperial College London

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

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Biology

High-throughput Purification of Affinity-tagged Recombinant Proteins
Simone C. Wiesler 1, Robert O.J. Weinzierl 1
1Department of Life Sciences, Imperial College London

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

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Immunology and Infection

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus
Armando Arias *1, Luis Ureña *1, Lucy Thorne 1, Muhammad A. Yunus 1, Ian Goodfellow 1
1Section of Virology, Imperial College London

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

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Immunology and Infection

A Visual Assay to Monitor T6SS-mediated Bacterial Competition
Abderrahman Hachani 1, Nadine S. Lossi 1, Alain Filloux 1
1MRC Centre for Molecular Bacteriology and Infection, Division of Cell and Molecular Biology, Imperial College London

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

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Bioengineering

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder
Koon-Yang Lee 1,2, Siti Rosminah Shamsuddin 3, Marta Fortea-Verdejo 1, Alexander Bismarck 1,3
1Polymer and Composite Engineering (PaCE) Group, Institute of Materials Chemistry and Research, University of Vienna, 2Department of Chemical Engineering, University College London, 3Polymer and Composite Engineering (PaCE) Group, Department of Chemical Engineering, Imperial College London

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

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Biology

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics
Edward Emmott 1, Ian Goodfellow 1
1Division of Virology, Department of Pathology, University of Cambridge

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

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Engineering

Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging
Colin R. Crick *1, Sacha Noimark *2,3, William J. Peveler *3, Joseph C. Bear 3, Aleksandar P. Ivanov 1, Joshua B. Edel 1, Ivan P. Parkin 3
1Department of Chemistry, Imperial College London, 2Department of Medical Physics and Biomedical Engineering, University College London, 3Department of Chemistry, University College London

Here we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging.

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