Examples of Research
Examples of Research.
See examples of work conducted in our facilities using our state of the art equipment and specialist techniques.
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Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment
Jennifer Ross, Zak McIver, Thomas Lambert, Cecilia Piergentili, Jasmine Emma Bird, Kelly J. Gallagher, Faye L. Cruickshank, Patrick James, Efrain Zarazúa-Arvizu4 Louise E. Horsfall, Kevin J. Waldron, Marcus D. Wilson, C. Logan Mackay, Arnaud Baslé, David J. Clarke, Jon Marles-Wright.
Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryo–electron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/ deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.
https://www.science.org/doi/full/10.1126/sciadv.abj4461?af=R#
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Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2
The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNβ or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.Catherine F. Hatton, Rachel A. Botting, Maria Emilia Dueñas, Iram J. Haq, Bernard Verdon, Benjamin J. Thompson, Jarmila Stremenova Spegarova, Florian Gothe, Emily Stephenson, Aaron I. Gardner, Sandra Murphy,Jonathan Scott,James P. Garnett,Sean Carrie, Jason Powell, C. M. Anjam Khan, Lei Huang, Rafiqul Hussain, Jonathan Coxhead, Tracey Davey, A. John Simpson, Muzlifah Haniffa, Sophie Hambleton, Malcolm Brodlie, Chris Ward,Matthias Trost, Gary Reynolds & Christopher J. A. Duncan -
3D neuronal mitochondrial morphology in axons, dendrites, and somata of the aging mouse hippocampus
The brain's ability to process complex information relies on the constant supply of energy through aerobic respiration by mitochondria. Neurons contain three anatomically distinct compartments-the soma, dendrites, and projecting axons-which have different energetic and biochemical requirements, as well as different mitochondrial morphologies in cultured systems. In this study, we apply quantitative three-dimensional electron microscopy to map mitochondrial network morphology and complexity in the mouse brain. We examine somatic, dendritic, and axonal mitochondria in the dentate gyrus and cornu ammonis 1 (CA1) of the mouse hippocampus, two subregions with distinct principal cell types and functions. We also establish compartment-specific differences in mitochondrial morphology across these cell types between young and old mice, highlighting differences in age-related morphological recalibrations. Overall, these data define the nature of the neuronal mitochondrial network in the mouse hippocampus, providing a foundation to examine the role of mitochondrial morpho-function in the aging brain.
3D neuronal mitochondrial morphology in axons, dendrites, and somata of the aging mouse hippocampus
Julie Faitg, DOI: 10.1016/j.celrep.2021.109509
Service used: Std TEM, SBF-SEM, Amira, Microscopy Image Browser ( MIB)
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Quantitative 3D Mapping of the Human Skeletal Muscle Mitochondrial Network.
Genetic and biochemical defects of mitochondrial function are a major cause of human disease, but their link to mitochondrial morphology in situ has not been defined. Here, we develop a quantitative three-dimensional approach to map mitochondrial network organization in human muscle at electron microscopyresolution. We establish morphological differences between human and mouse and among patients with mitochondrial DNA (mtDNA) diseases compared to healthy controls.
- •3D mitochondrial morphology is quantified by the mitochondrial complexity index (MCI)
- •Mouse mitochondria are larger and exhibit greater connectivity than human mitochondria
- •Non-adjacent mitochondria are connected by narrow mitochondrial nanotunnels
- •Multivariate morphology signatures distinguish mitochondrial disease patients from healthy controls
Quantitative 3D Mapping of the Human Skeletal Muscle Mitochondrial Network
Amy E. Vincent, Kathryn White, Tracey Davey, Jonathan Philips, R. Todd Ogden, Conor Lawless, Charlotte Warren, Matt G. Hall, Yi Shiau Ng, Gavin Falkous, Thomas Holden, David Deehan, Robert W. Taylor, Doug M. Turnbull, Martin Picard
2019
Service Used: Gatan 3View SBF-SEM -
A guide to analysis and reconstruction of serial block face scanning electron microscopy data
Serial block face scanning electron microscopy (SBF‐SEM) is a relatively new technique that allows the acquisition of serially sectioned, imaged and digitally aligned ultrastructural data. There is a wealth of information that can be obtained from the resulting image stacks but this presents a new challenge for researchers – how to computationally analyse and make best use of the large datasets produced. One approach is to reconstruct structures and features of interest in 3D.
A guide to analysis and reconstruction of serial block face scanning electron microscopy data
Service used: Gatan 3View. SBF Scanning Electron Microscopy. Amira. Microscopy Image Browser (MIB) Fiji. Blender
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Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy
Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases.
The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy.
Sci Rep. 2016. Vincent AE, Ng YS, White K, Davey T, Mannella C, Falkous G, Feeney C, Schaefer AM, McFarland R, Gorman GS, Taylor RW, Turnbull DM, Picard M.
Service Used: Gatan 3View TEM
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Trauma to Muller cells as the ILM is peeled from their attached end plates
A dissociated optic nerve fibre layer (DONFL) is a characteristic change noted in inner retinal morphology after internal limiting membrane (ILM) peeling. It is thought to be due to trauma to Muller cells as the ILM is peeled from their attached end plates.
Acta Ophthalmol. 2016 Steel DH, Dinah C, White K, Avery PJ.
Service used TEM , Stereology
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Isolated proteins
There is increasing interest in the role of brain cholesterol in Alzheimer's disease and the contribution of cholesterol to the formation of amyloid plaques. Transmission EM can demonstrate the binding of soluble ~10 nm diameter cholesterol-PEG 600 micelles to amyloid-β1-42 fibrils.
Cholesterol binding to amyloid-beta fibrils: a TEM study
Micron 39:1192-6, 2008 - Harris JR.Service used: TEM, Negative staining
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Ultrastructural localization of Nova in the somata and dendrites of ventral horn spinal cord neurons
The Neuronal Splicing Factor Nova Co-Localizes with Target RNAs in the Dendrite
Front Neural Circuits 4:5, 2010 - Racca C, Gardiol A, Eom T, Ule J, Triller A, Darnell RB.Service used: TEM, Pre-embedding immunogold
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Inducible overexpression of sFlt-1 in podocytes ameliorates glomerulopathy in diabetic mice
Diabetic glomerulopathy is characterised by mesangial expansion and thickening of the glomerular basement membrane. By applying stereological probes to digital images it is possible to quantify these changes in humans and experimental models.
Inducible overexpression of sFlt-1 in podocytes ameliorates glomerulopathy in diabetic mice
Diabetes 57:2824-33, 2008 - Ku CH, White KE, Dei Cas A, Hayward A, Webster Z, Bilous R, Marshall S, Viberti GC, Gnudi L.Service used: TEM, Stereology
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Effect of neurotoxins on the neuromuscular junction
Examination of neuromuscular junctions using transmission EM can demonstrate how the injection of a neurotoxin into muscle results in a loss of synaptic vesicles from terminal boutons.
The neurotoxicological effects of mastoparan Polybia-MPII at the murine neuromuscular junction: an ultrastructural and immunocytochemical study
Histochem Cell Biol 132:395-404, 2009 - Rocha T, de Souza BM, Palma MS, da Cruz- Höfling MA, Harris JB.Service used: TEM, Standard epoxy resin embedding