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  • Journal article
    Sparks H, Dvinskikh L, Firth J, Francis A, Harding S, Paterson C, MacLeod K, Dunsby Cet al., 2020,

    Development a flexible light-sheet fluorescence microscope for high-speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure

    , Journal of Biophotonics, Vol: 13, ISSN: 1864-063X

    We report a flexible light‐sheet fluorescence microscope (LSFM) designed for studying dynamic events in cardiac tissue at high speed in 3D and the correlation of these events to cell microstructure. The system employs two illumination‐detection modes: the first uses angle‐dithering of a Gaussian light sheet combined with remote refocusing of the detection plane for video‐rate volumetric imaging; the second combines digitally‐scanned light‐sheet illumination with an axially‐swept light‐sheet waist and stage‐scanned acquisition for improved axial resolution compared to the first mode. We present a characterisation of the spatial resolution of the system in both modes. The first illumination‐detection mode achieves dual spectral‐channel imaging at 25 volumes per second with 1024 × 200 × 50 voxel volumes and is demonstrated by time‐lapse imaging of calcium dynamics in a live cardiomyocyte. The second illumination‐detection mode is demonstrated through the acquisition of a higher spatial resolution structural map of the t‐tubule network in a fixed cardiomyocyte cell.
  • Journal article
    Dubois MAJ, Lazaridou A, Choi C, Mousseau JJ, Bull JAet al., 2019,

    Synthesis of 3-Aryl-3-Sulfanyl Azetidines by Iron-Catalyzed Thiol Alkylation with N-Cbz Azetidinols

    , Journal of Organic Chemistry, Vol: 84, Pages: 5943-5956, ISSN: 0022-3263

    © 2019 American Chemical Society. New small-ring derivatives can provide valuable motifs in new chemical space for drug design. 3-Aryl-3-sulfanyl azetidines are synthesized directly from azetidine-3-ols in excellent yield by a mild Fe-catalyzed thiol alkylation. A broad range of thiols and azetidinols bearing electron-donating aromatics are successful, proceeding via an azetidine carbocation. The N-carboxybenzyl group is a requirement for good reactivity and enables the NH-azetidine to be revealed. Further reactions of the azetidine sulfides demonstrate their potential for incorporation in drug discovery programs.
  • Journal article
    Supramaniam P, Ces O, Salehi-Reyhani A, 2019,

    Microfluidics for artificial life: techniques for bottom-up synthetic biology

    , Micromachines, Vol: 10, Pages: 1-27, ISSN: 2072-666X

    Synthetic biology is a rapidly growing multidisciplinary branch of science that exploits the advancement of molecular and cellular biology. Conventional modification of pre-existing cells is referred to as the top-down approach. Bottom-up synthetic biology is an emerging complementary branch that seeks to construct artificial cells from natural or synthetic components. One of the aims in bottom-up synthetic biology is to construct or mimic the complex pathways present in living cells. The recent, and rapidly growing, application of microfluidics in the field is driven by the central tenet of the bottom-up approach—the pursuit of controllably generating artificial cells with precisely defined parameters, in terms of molecular and geometrical composition. In this review we survey conventional methods of artificial cell synthesis and their limitations. We proceed to show how microfluidic approaches have been pivotal in overcoming these limitations and ushering in a new generation of complexity that may be imbued in artificial cells and the milieu of applications that result.
  • Journal article
    Gilburt J, Girvan P, Blagg J, Ying L, Dodson CAet al., 2019,

    Ligand discrimination between active and inactive activation loop conformations of Aurora-A kinase is unmodified by phosphorylation

    , Chemical Science, Vol: 10, Pages: 4069-4076, ISSN: 2041-6520

    Structure-based drug design is commonly used to guide the development of potent and specific enzyme inhibitors. Many enzymes – such as protein kinases – adopt multiple conformations, and conformational interconversion is expected to impact on the design of small molecule inhibitors. We measured the dynamic equilibrium between DFG-in-like active and DFG-out-like inactive conformations of the activation loop of unphosphorylated Aurora-A alone, in the presence of the activator TPX2, and in the presence of kinase inhibitors. The unphosphorylated kinase had a shorter residence time of the activation loop in the active conformation and a shift in the position of equilibrium towards the inactive conformation compared with phosphorylated kinase for all conditions measured. Ligand binding was associated with a change in the position of conformational equilibrium which was specific to each ligand and independent of the kinase phosphorylation state. As a consequence of this, the ability of a ligand to discriminate between active and inactive activation loop conformations was also independent of phosphorylation. Importantly, we discovered that the presence of multiple enzyme conformations can lead to a plateau in the overall ligand Kd, despite increasing affinity for the chosen target conformation, and modelled the conformational discrimination necessary for a conformation-promoting ligand.
  • Journal article
    Storck Saha E, Morales Sanfrutos J, Serwa R, Panyain N, Lanyon-Hogg T, Tolmachova T, Ventimiglia L, Martin-Serrano J, Seabra M, Wojciak-Stothard B, Tate Eet al., 2019,

    Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics

    , Nature Chemistry, Vol: 11, Pages: 552-561, ISSN: 1755-4330

    Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.
  • Journal article
    Hansel C, Crowder S, Cooper S, Gopal S, Pardelha da Cruz J, De Oliveira Martins L, Keller D, Rothery S, Becce M, Cass A, Bakal C, Chiappini C, Stevens Met al., 2019,

    Nanoneedle-mediated stimulation of cell mechanotransduction machinery

    , ACS Nano, Vol: 13, Pages: 2913-2019, ISSN: 1936-0851

    Biomaterial substrates can be engineered to present topographical signals to cells which, through interactions between the material and active components of the cell membrane, regulate key cellular processes and guide cell fate decisions. However, targeting mechanoresponsive elements that reside within the intracellular domain is a concept that has only recently emerged. Here, we show that mesoporous silicon nanoneedle arrays interact simultaneously with the cell membrane, cytoskeleton, and nucleus of primary human cells, generating distinct responses at each of these cellular compartments. Specifically, nanoneedles inhibit focal adhesion maturation at the membrane, reduce tension in the cytoskeleton, and lead to remodeling of the nuclear envelope at sites of impingement. The combined changes in actin cytoskeleton assembly, expression and segregation of the nuclear lamina, and localization of Yes-associated protein (YAP) correlate differently from what is canonically observed upon stimulation at the cell membrane, revealing that biophysical cues directed to the intracellular space can generate heretofore unobserved mechanosensory responses. These findings highlight the ability of nanoneedles to study and direct the phenotype of large cell populations simultaneously, through biophysical interactions with multiple mechanoresponsive components.
  • Journal article
    Khan H, Seddon JM, Law RV, Brooks NJ, Robles E, Cabral JT, Ces Oet al., 2019,

    Effect of glycerol with sodium chloride on the Krafft point of sodium dodecyl sulfate using surface tension

    , Journal of Colloid and Interface Science, Vol: 538, Pages: 75-82, ISSN: 0021-9797

    The effect of glycerol with sodium chloride (NaCl) on the phase behaviour of sodium dodecyl sulfate (SDS) near the Krafft point was studied by surface tension analysis using the pendant drop method. The critical micelle concentration (CMC) and Krafft Temperature (TK) of SDS in water: glycerol mixtures, across the full composition range, and in NaCl solutions within 0.005–0.1 M were obtained. The pendant drop method successfully allowed us to determine the Krafft point of SDS in high glycerol systems where other traditional methods (e.g. conductivity) have been ineffective. Overall the addition of glycerol increases the CMC and the TK, thus shifting the Krafft point of SDS to higher temperatures (increasing crystallisation temperatures) and higher SDS content in the presence of glycerol, which is interpreted as a result of the reduction in solvent polarity which opposes micellization. The addition of NaCl to the SDS – water-glycerol systems brings the CMC back down, while having no significant effect on the TK. Our results establish a robust route for tuning the Krafft point of model surfactant SDS by adjusting solvent quality and salt content.
  • Journal article
    Ces O, Elani Y, 2019,

    Community building in synthetic biology.

    , Experimental biology and medicine (Maywood, N.J.), Vol: 244, Pages: 281-282, ISSN: 0037-9727
  • Conference paper
    Girvan P, Teng X, Brooks NJ, Baldwin GS, Ying Let al., 2019,

    Redox Kinetics of the Amyloid-Beta-Copper Complex and Its Biological Implications

    , 63rd Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 28A-28A, ISSN: 0006-3495
  • Journal article
    Rains JGD, ODonnelly K, Oliver T, Woscholski R, Long NJ, Barter LMCet al., 2019,

    Bicarbonate inhibition of carbonic anhydrase mimics hinders catalytic efficiency: Elucidating the mechanism and gaining insight toward improving speed and efficiency

    , ACS Catalysis, Vol: 9, Pages: 1353-1365, ISSN: 2155-5435

    Carbonic anhydrase (CA) mimics are often studied with a focus on the hydration of CO2 for atmospheric carbon capture. Consequently, the reverse reaction (dehydration of HCO3–) has received minimal attention, so much so that the rate-limiting step of the dehydration reaction in CA mimics is currently unknown. The rate-limiting step of the hydration reaction is reported to be the bicarbonate-bound intermediate step, and thus is susceptible to product inhibition. It is not, however, clear if this inhibition is a consequence of an increase in the rate of the competing dehydration reaction or resulting from the strong affinity of bicarbonate to the mimic. To address this, insight into the dehydration reaction kinetics is needed. We therefore report the most comprehensive study of a CA mimic to date. The dehydration profile of the fastest small-molecule CA mimic, ZnL1S, was characterized, and consequently evidence for the rate-limiting step for the dehydration reaction was seen to be the bicarbonate-bound intermediate step, much like the hydration reaction. This experimental validation of the rate-limiting step was achieved through a variety of methods including NMR experiments and the effect of inhibitors, substrate concentration, and metal center on activity. With this understanding, an improvement in the favorability of the rate-limiting step was achieved, resulting in decreased bicarbonate inhibition. Thus, an increase in the mimic’s kcat for both reactions was observed, resulting in the largest rate constants of any small-molecule CA mimic reported to date (28 093 and 579 M–1 s–1 for hydration and dehydration, respectively). Enzyme-like kcat/km values were obtained for ZnL1S (5.9 × 105 M–1 s–1 for CO2 hydration), and notably there is only a difference of 2.5 orders of magnitude from the enzyme, the closest of any CA mimic reported in the literature. The results from this work can be applied to the development and improvement

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