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  • Journal article
    Elani Y, Trantidou T, Wylie D, Dekker L, Polizzi K, Law R, Ces Oet al., 2018,

    Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules

    , Scientific Reports, Vol: 8, Pages: 1-8, ISSN: 2045-2322

    There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.

  • Journal article
    Kim Y, Warren S, Favero F, Stone J, Clegg J, Neil M, Paterson C, Knight J, French P, Dunsby CWet al., 2018,

    Semi-random multicore fibre design for adaptive multiphoton endoscopy

    , Optics Express, Vol: 26, Pages: 3661-3673, ISSN: 1094-4087

    This paper reports the development, modelling and application of a semi-random multicore fibre (MCF) design for adaptive multiphoton endoscopy. The MCF was constructed from 55 sub-units, each comprising 7 single mode cores, in a hexagonally close-packed lattice where each sub-unit had a random angular orientation. The resulting fibre had 385 single mode cores and was double-clad for proximal detection of multiphoton excited fluorescence. The random orientation of each sub-unit in the fibre reduces the symmetry of the positions of the cores in the MCF, reducing the intensity of higher diffracted orders away from the central focal spot formed at the distal tip of the fibre and increasing the maximum size of object that can be imaged. The performance of the MCF was demonstrated by imaging fluorescently labelled beads with both distal and proximal fluorescence detection and pollen grains with distal fluorescence detection. We estimate that the number of independent resolution elements in the final image – measured as the half-maximum area of the two-photon point spread function divided by the area imaged – to be ~3200.

  • Conference paper
    Tahirbegi B, Magness AJ, Boillat A, Willison KR, Klug DR, Knopfel T, Ying Let al., 2018,

    Probing synaptic amyloid-beta aggregation promoted by copper release

    , 62nd Annual Meeting of the Biophysical-Society, Publisher: Biophysical Society, Pages: 430A-430A, ISSN: 0006-3495

    Whether or not the metal ions released during synaptic transmission induce amyloid-beta oligomer formation in the vicinity of synapses is a central question pertinent to the molecular mechanism of Alzheimer's disease. Recently, through a combination of experimental kinetics studies and coupled reaction-diffusion simulations, we predicted that Cu(II) rather than Zn(II) plays an important role in the very early stages (i.e., dimer formation) of Aβ aggregation in the synapse. Single molecule photobleaching analysis is a powerful tool to determine the stoichiometry of amyloid-beta oligomers which enables us to examine the time course of small amyloid-beta oligomer formation in solution, immobilised to a solid-phase substrate or artificial lipid membrane, and in live neurons in the presence of Cu(II). Preliminary results indicate that small amyloid-beta oligomers can be locked in their oligomeric state without dissociation on a poly-lysine coated surface and that Cu(II) increases the diversity and abundance of amyloid-beta oligomers.

  • Conference paper
    Marston SB, Messer AE, Eiros-Zamora J, Gould I, Papadaki M, Choudry A, Sheehan Aet al., 2018,

    The Molecular Defects in Ca<SUP>2+</SUP> Regulation due to Mutations that Cause Hypertrophic Cardiomyopathy can be Reversed by Small Molecules that Bind to Troponin

    , 62nd Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 37A-37A, ISSN: 0006-3495
  • Journal article
    Barlow NE, Bolognesi G, Haylock S, Flemming AJ, Brooks NJ, Barter LMC, Ces Oet al., 2017,

    Rheological Droplet Interface Bilayers (rheo-DIBs): Probing the Unstirred Water Layer Effect on Membrane Permeability via Spinning Disk Induced Shear Stress

    , Scientific Reports, Vol: 7, ISSN: 2045-2322

    A new rheological droplet interface bilayer (rheo-DIB) device is presented as a tool to apply shear stress on biological lipid membranes. Despite their exciting potential for affecting high-throughput membrane translocation studies, permeability assays conducted using DIBs have neglected the effect of the unstirred water layer (UWL). However as demonstrated in this study, neglecting this phenomenon can cause significant underestimates in membrane permeability measurements which in turn limits their ability to predict key processes such as drug translocation rates across lipid membranes. With the use of the rheo-DIB chip, the effective bilayer permeability can be modulated by applying shear stress to the droplet interfaces, inducing flow parallel to the DIB membranes. By analysing the relation between the effective membrane permeability and the applied stress, both the intrinsic membrane permeability and UWL thickness can be determined for the first time using this model membrane approach, thereby unlocking the potential of DIBs for undertaking diffusion assays. The results are also validated with numerical simulations.

  • Journal article
    de Bruin A, Friddin MS, Elani Y, Brooks N, Law R, Seddon J, Ces Oet al., 2017,

    A transparent 3D printed device for assembling droplet hydrogel bilayers (DHBs)

    , RSC Advances, Vol: 7, Pages: 47796-47800, ISSN: 2046-2069

    We report a new approach for assembling droplet hydrogel bilayers (DHBs) using a transparent 3D printed device. We characterise the transparency of our platform, confirm bilayer formation using electrical measurements and show that single-channel recordings can be obtained using our reusable rapid prototyped device. This method significantly reduces the cost and infrastructure required to develop devices for DHB assembly and downstream study.

  • Journal article
    Sherlock B, Warren SC, Alexandrov Y, Yu F, Stone J, Knight J, Neil MAA, Paterson C, French PMW, Dunsby CWet al., 2017,

    In vivo multiphoton microscopy using a handheld scanner with lateral and axial motion compensation

    , Journal of Biophotonics, Vol: 11, ISSN: 1864-063X

    This paper reports a handheld multiphoton fluorescence microscope designed for clinical imaging that incorporates axial motion compensation and lateral image stabilization. Spectral domain optical coherence tomography is employed to track the axial position of the skin surface, and lateral motion compensation is realised by imaging the speckle pattern arising from the optical coherence tomography beam illuminating the sample. Our system is able to correct lateral sample velocities of up to ~65 μm s-1. Combined with the use of negative curvature microstructured optical fibre to deliver tunable ultrafast radiation to the handheld multiphoton scanner without the need of a dispersion compensation unit, this instrument has potential for a range of clinical applications. The system is used to compensate for both lateral and axial motion of the sample when imaging human skin in vivo.

  • Conference paper
    Sim S, Sowley H, Kidley N, Barter L, Klug Det al., 2017,

    Investigation of inhibitor-protein interactions in plants & mammalians from EVV 2DIR data

    , 254th National Meeting and Exposition of the American-Chemical-Society (ACS) on Chemistry's Impact on the Global Economy, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
  • Journal article
    Cornell CE, McCarthy NLC, Levental KR, Levental I, Brooks NJ, Keller SLet al., 2017,

    Lengths of n-alcohols govern how Lo-Ld mixing temperatures shift in synthetic and cell-derived membranes

    , Biophysical Journal, Vol: 113, Pages: 1-13, ISSN: 1542-0086

    A persistent challenge in membrane biophysics has been to quantitatively predict how membrane physical properties change upon addition of new amphiphiles (e.g., lipids, alcohols, peptides, or proteins) in order to assess whether the changes are large enough to plausibly result in biological ramifications. Because of their roles as general anesthetics, n-alcohols are perhaps the best-studied amphiphiles of this class. When n-alcohols are added to model and cell membranes, changes in membrane parameters tend to be modest. One striking exception is found in the large decrease in liquid-liquid miscibility transition temperatures (Tmix) observed when short-chain n-alcohols are incorporated into giant plasma membrane vesicles (GPMVs). Coexisting liquid-ordered and liquid-disordered phases are observed at temperatures below Tmix in GPMVs as well as in giant unilamellar vesicles (GUVs) composed of ternary mixtures of a lipid with a low melting temperature, a lipid with a high melting temperature, and cholesterol. Here, we find that when GUVs of canonical ternary mixtures are formed in aqueous solutions of short-chain n-alcohols (n ≤ 10), Tmix increases relative to GUVs in water. This shift is in the opposite direction from that reported for cell-derived GPMVs. The increase in Tmix is robust across GUVs of several types of lipids, ratios of lipids, types of short-chain n-alcohols, and concentrations of n-alcohols. However, as chain lengths of n-alcohols increase, nonmonotonic shifts in Tmix are observed. Alcohols with chain lengths of 10–14 carbons decrease Tmix in ternary GUVs of dioleoyl-PC/dipalmitoyl-PC/cholesterol, whereas 16 carbons increase Tmix again. Gray et al. observed a similar influence of the length of n-alcohols on the direction of the shift in Tmix. These results are consistent with a scenario in which the relative partitioning of n-alcohols between liquid-ordered and liquid-disordered phases evolves as the chain length of the n-alcohol increases.

  • Journal article
    Trantidou T, Friddin M, Elani Y, Brooks NJ, Law RV, Seddon JM, Ces Oet al., 2017,

    Engineering compartmentalized biomimetic micro- and nanocontainers

    , ACS Nano, Vol: 11, Pages: 6549-6565, ISSN: 1936-086X

    Compartmentalization of biological content and function is a key architectural feature in biology, where membrane bound micro- and nanocompartments are used for performing a host of highly specialized and tightly regulated biological functions. The benefit of compartmentalization as a design principle is behind its ubiquity in cells and has led to it being a central engineering theme in construction of artificial cell-like systems. In this review, we discuss the attractions of designing compartmentalized membrane-bound constructs and review a range of biomimetic membrane architectures that span length scales, focusing on lipid-based structures but also addressing polymer-based and hybrid approaches. These include nested vesicles, multicompartment vesicles, large-scale vesicle networks, as well as droplet interface bilayers, and double-emulsion multiphase systems (multisomes). We outline key examples of how such structures have been functionalized with biological and synthetic machinery, for example, to manufacture and deliver drugs and metabolic compounds, to replicate intracellular signaling cascades, and to demonstrate collective behaviors as minimal tissue constructs. Particular emphasis is placed on the applications of these architectures and the state-of-the-art microfluidic engineering required to fabricate, functionalize, and precisely assemble them. Finally, we outline the future directions of these technologies and highlight how they could be applied to engineer the next generation of cell models, therapeutic agents, and microreactors, together with the diverse applications in the emerging field of bottom-up synthetic biology.

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