Recent Submissions

  • S. cerevisiae Srs2 helicase ensures normal recombination intermediate metabolism during meiosis and prevents accumulation of Rad51 aggregates

    Hunt, L.J.; Ahmed, E.A.; Kaur, H.; Ahuja, J.S.; Hulme, L.; Chou, T.C.; Lichten, M.; Goldman, Alastair S.H. (2019)
    We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalised with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards interhomologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division.
  • A Grounded Theory approach to understanding the role of medication safety within a hospital early discharge team

    Tomlinson, Justine; Silcock, Jonathan; Karban, Kate; Blenkinsopp, Alison; Smith, H. (2019-02)
  • Examining the utility of the connect with pharmacy (CWP) intervention in reducing elderly readmission

    Sabir, F.; Tomlinson, Justine; Strickland-Hodge, B.; Smith, H. (2019-02)
  • Kinase regulation of HOX transcription factors

    Primon, Monika; Hunter, K.D.; Pandha, H.S.; Morgan, Richard (2019-04)
    The HOX genes are a group of homeodomain-containing transcription factors that play important regulatory roles in early development, including the establishment of cell and tissue identity. HOX expression is generally reduced in adult cells but is frequently re-established as an early event in tumour formation and supports an oncogenic phenotype. HOX transcription factors are also involved in cell cycle regulation and DNA repair, along with normal adult physiological process including stem cell renewal. There have been extensive studies on the mechanism by which HOX proteins regulate transcription, with particular emphasis on their interaction with cofactors such as Pre-B-cell Leukaemia Homeobox (PBX) and Myeloid Ecotropic Viral Integration Site 1 (MEIS). However, significantly less is known of how the activity of HOX proteins is regulated. There is growing evidence that phosphorylation may play an important role in this context, and in this review, we draw together a number of important studies published over the last 20 years, and discuss the relevance of phosphorylation in the regulation and function of HOX proteins in development, evolution, cell cycle regulation, and cancer.
  • A new route to tricyclane sesquiterpenoids: total synthesis of α-ekasantalic acid

    Lomba, L.; Afarinkia, Kamyar; Vinader, Victoria (2019)
    Chemical manipulation of the cycloadduct of citraconic anhydride and cyclopentadiene enables a new synthetic route to tricyclane sesquiterpenoids. This methodology is applied to the first total synthesis of α-ekasantalic acid.
  • A community pharmacist medicines optimisation service for patients with advanced cancer pain: a proof of concept study

    Edwards, Zoe; Bennett, M.I.; Blenkinsopp, Alison (2019)
    Background Patients with advanced cancer commonly experience pain and it is least controlled in community settings. Community pharmacists in the UK already offer medicines optimisation consultations although not for this patient group. Objective To determine whether medicines consultations for patients with advanced cancer pain are feasible and acceptable. Setting Community-dwelling patients with advanced cancer pain were recruited from primary, secondary and tertiary care using purposive sampling in one UK city. Methods One face-to-face or two telephone delivered medicines optimisation consultations by pharmacists were tested. These were based on services currently delivered in UK community pharmacies. Feedback was obtained from patients and healthcare professionals involved to assess feasibility and acceptability. Main outcome measure Recruitment, acceptability and drug related problems. Results Twenty-three patients, (range 33–88 years) were recruited, 19 completed consultation(s) of whom 17 were receiving palliative care services. Five received face-to-face consultations and 14 by telephone during which 47 drug related problems were identified from 33 consultations (mean 2.5). Advice was provided for 34 drug related problems in 17 patients and referral to other healthcare professionals for 13 in 8 patients, 2 patients had none. Eleven patients returned questionnaires of which 8 (73%) would recommend the consultations to others. Conclusion The consultations were feasible as patients were recruited, retained, consultations delivered, and data collected. Patients found the 20–30 min intervention acceptable, found a self-perceived increase in medicines knowledge and most would recommend it to others. Community pharmacists were willing to carry out these services however they had confidence issues in accessing working knowledge. Most drug related problems were resolved by the pharmacists and even among patients receiving palliative care services there were still issues concerning analgesic management. Pharmacist-conducted medicines consultations demonstrate potential which now needs to be evaluated within a larger study in the future.
  • Tumor growth suppression using a combination of taxol-based therapy and GSK3 inhibition in non-small cell lung cancer

    O'Flaherty, L.; Shnyder, Steven D.; Cooper, Patricia A.; Cross, S.J.; Wakefield, J.G.; Pardo, O.E.; Seckl, M.J.; Tavare, J.M. (2019-04-10)
    Glycogen synthase kinase-3 (GSK3) is over-expressed and hyperactivated in non-small cell lung carcinoma (NSCLC) and plays a role in ensuring the correct alignment of chromosomes on the metaphase plate during mitosis through regulation of microtubule stability. This makes the enzyme an attractive target for cancer therapy. We examined the effects of a selective cell-permeant GSK3 inhibitor (CHIR99021), used alone or in combination with paclitaxel, using an in vitro cell growth assay, a quantitative chromosome alignment assay, and a tumor xenograft model. CHIR99021 inhibits the growth of human H1975 and H1299 NSCLC cell lines in a synergistic manner with paclitaxel. CHIR99021 and paclitaxel promoted a synergistic defect in chromosomal alignment when compared to each compound administered as monotherapy. Furthermore, we corroborated our in vitro findings in a mouse tumor xenograft model. Our results demonstrate that a GSK3 inhibitor and paclitaxel act synergistically to inhibit the growth of NSCLC cells in vitro and in vivo via a mechanism that may involve converging modes of action on microtubule spindle stability and thus chromosomal alignment during metaphase. Our findings provide novel support for the use of the GSK3 inhibitor, CHIR99021, alongside taxol-based chemotherapy in the treatment of human lung cancer.
  • Spectroscopic (FT-IR, FT-Raman, and 13C SS-NMR) and quantum chemical investigations to provide structural insights into nitrofurantoin–4-hydroxybenzoic acid cocrystals

    Shukla, A.; Khan, E.; Alsirawan, MHD Bashir; Mandal, R.; Tandon, P.; Vangala, Venu R. (2019-05-14)
    Cocrystallization is an attractive approach to improving the physicochemical properties of active pharmaceutical ingredients (APIs), which have great potential in drug development. Accordingly, there is a growing need to understand the physicochemical changes that occur upon co-crystallisation. This work focuses on the combined use of spectroscopy and density functional theory (DFT) calculations to understand the molecular structure, hydrogen bond interactions and physicochemical properties of a pharmaceutical cocrystal. Solid-state NMR (ssNMR) spectroscopy can provide detailed molecular structure information on pharmaceutical cocrystals and complexes. It is non-destructive and usually provides deep structural insights that complement well with vibrational spectroscopy. In this work, a cocrystal of an antibiotic drug, nitrofurantoin (NF), with 4-hydroxybenzoic acid (4HBA) is examined to understand the capability of multiple spectroscopic techniques such as infrared (IR), Raman and solid-state NMR spectroscopies, and to confirm the molecular structure and hydrogen bonding of cocrystal systems. The results of IR and Raman spectroscopy showed that for the cocrystal formation, NF and 4HBA molecules interact through N–H⋯O–H interactions between the imide N–H of nitrofurantoin and the phenolic –OH of 4-hydroxybenzoic acid, and these interactions are also confirmed by natural bond orbital (NBO) and quantum theory of atoms in molecules (QTAIM) analyses. It is critical to understand whether a given cocrystal, upon conceiving a modified crystalline structure compared to that of its API, shows enhanced physical and chemical properties or not. Computationally, it is found that the NF–4HBA cocrystal shows softer (more reactive) behaviour in comparison to NF as its cocrystal, NF–4HBA, has a low band gap in comparison to the API, NF. These results demonstrate that the quantum chemical approach predicts accurately how to relate cocrystal with its physical and chemical properties.
  • Gaps in Propolis Research: Challenges Posed to Commercialisation and the Need for an Holistic Approach

    Katekhaye, S.; Fearnley, H.; Fearnley, J.; Paradkar, Anant R. (2019)
    Both the season and region in which propolis is collected influence its chemical composition, resulting in variations in biological activity. Significant differences in composition and concentration of certain chemical compounds in propolis make standardisation and quality control challenging. In addition, the lack of uniformity in evaluation methodology and analytical techniques, make it extremely difficult to correlate data across the climatic zones. In this report, we focus on the gaps in propolis research and the challenges they pose for commercialisation, with suggestions as to how we might address them. We hope to stimulate further research which explores the holistic nature of propolis in order to derive a propolis bioactivity standard.
  • Polymorphs of Curcumin and Its Cocrystals With Cinnamic Acid

    Rathi, N.; Paradkar, Anant R.; Gaikar, V.G. (2019)
    We report formation of polymorphs and new eutectics and cocrystals of curcumin, a sparingly water-soluble active component in turmeric, structurally similar to cinnamic acid. The curcumin polymorphs were formed using liquid antisolvent precipitation, where acetone acted as a solvent and water was used as the antisolvent. The metastable form 2 of curcumin was successfully prepared in varied morphology over a wide range of solvent-to-antisolvent ratio and under acidic pH conditions. We also report formation of new eutectics and cocrystals of curcumin with cinnamic acid acting as a coformer. The binary phase diagrams were studied using differential scanning calorimetry and predicted formation of the eutectics at the curcumin mole fraction of 0.15 and 0.33, whereas a cocrystal was formed at 0.3 mole fraction of curcumin in the curcumin–cinnamic acid mixture. The formation of the cocrystal was supported with X-ray powder diffraction, the enthalpy of fusion values, Fourier-transform infrared spectroscopy, and scanning electron microscopy. The hydrogen bond interaction between curcumin and cinnamic acid was predicted from Fourier-transform infrared spectra, individually optimized curcumin and cinnamic acid structures by quantum mechanical calculations using Gaussian-09 and their respective unit cell packing structures.
  • Use of Methylmalonyl-CoA Epimerase in Enhancing Crotonase Stereoselectivity

    Hamed, Refaat B.; Gomez-Castellanos, J.R.; Sean Froese, D.; Krysztofinska, E.; Yue, W.W.; Schofield, C.J. (2016-03-15)
    The use of methylmalonyl-CoA epimerase (MCEE) to improve stereoselectivity in crotonase-mediated biocatalysis is exemplified by the coupling of MCEE, crotonyl-CoA carboxylase reductase and carboxymethylproline synthase in a three-enzyme one-pot sequential synthesis of functionalised C-5 carboxyalkylprolines starting from crotonyl-CoA and carbon dioxide.
  • Stereoselective production of dimethyl-substituted carbapenams via engineered carbapenem biosynthesis enzymes

    Hamed, Refaat B.; Henry, L.; Claridge, T.D.W.; Schofield, C. (2016-12-28)
    Stereoselective biocatalysis by crotonase superfamily enzymes is exemplified by use of engineered 5-carboxymethylproline synthases (CMPSs) for preparation of functionalized 5-carboxymethylproline (5-CMP) derivatives methylated at two positions (i.e. C2/C6, C3/C6 and C5/C6), including products with a quaternary centre, from appropriately-substituted-amino acid aldehydes and C-2 epimeric methylmalonyl-CoA. The enzymatically-produced disubstituted 5-CMPs were converted by carbapenam synthetase into methylated bicyclic Β-lactams, which manifest improved hydrolytic stability compared to the unsubstituted carbapenams. The results highlight the use of modi-fied carbapenem biosynthesis enzymes for production of new carbapenams with improved properties.
  • Crotonases: Nature’s exceedingly convertible catalysts

    Lohans, C.T.; Wang, D.Y.; Wang, J.; Hamed, Refaat B.; Schofield, C.J. (2017-08-14)
    The crotonases comprise a widely distributed enzyme superfamily that has multiple roles in both primary and secondary metabolism. Many crotonases employ oxyanion hole-mediated stabilization of intermediates to catalyze the reaction of coenzyme A (CoA) thioester substrates (e.g., malonyl-CoA, α,β-unsaturated CoA esters) both with nucleophiles and, in the case of enolate intermediates, with varied electrophiles. Reactions of crotonases that proceed via a stabilized oxyanion intermediate include the hydrolysis of substrates including proteins, as well as hydration, isomerization, nucleophilic aromatic substitution, Claisen-type, and cofactor-independent oxidation reactions. The crotonases have a conserved fold formed from a central β-sheet core surrounded by α-helices, which typically oligomerizes to form a trimer or dimer of trimers. The presence of a common structural platform and mechanisms involving intermediates with diverse reactivity implies that crotonases have considerable potential for biocatalysis and synthetic biology, as supported by pioneering protein engineering studies on them. In this Perspective, we give an overview of crotonase diversity and structural biology and then illustrate the scope of crotonase catalysis and potential for biocatalysis.
  • Living GenoChemetics by hyphenating synthetic biology and synthetic chemistry in vivo

    Sharma, S.V.; Tong, X.; Pubill-Ulldemolins, C.; Cartmell, C.; Bogosyan, E.J.A.; Rackham, E.J.; Marelli, E.; Hamed, Refaat B.; Goss, R.J.M. (2017-08-09)
    Marrying synthetic biology with synthetic chemistry provides a powerful approach toward natural product diversification, combining the best of both worlds: expediency and synthetic capability of biogenic pathways and chemical diversity enabled by organic synthesis. Biosynthetic pathway engineering can be employed to insert a chemically orthogonal tag into a complex natural scaffold affording the possibility of site-selective modification without employing protecting group strategies. Here we show that, by installing a sufficiently reactive handle (e.g., a C–Br bond) and developing compatible mild aqueous chemistries, synchronous biosynthesis of the tagged metabolite and its subsequent chemical modification in living culture can be achieved. This approach can potentially enable many new applications: for example, assay of directed evolution of enzymes catalyzing halo-metabolite biosynthesis in living cells or generating and following the fate of tagged metabolites and biomolecules in living systems. We report synthetic biological access to new-to-nature bromo-metabolites and the concomitant biorthogonal cross-coupling of halo-metabolites in living cultures.
  • Biocatalytic production of bicyclic β-lactams with three contiguous chiral centres using engineered crotonases

    Hamed, Refaat B.; Gomez-Castellanos, J.R.; Warhaut, H.L.; Claridge, T.D.W.; Schofield, C.J. (2019-01-24)
    There is a need to develop asymmetric routes to functionalised β-lactams, which remain the most important group of antibacterials. Here we describe biocatalytic and protein engineering studies concerning carbapenem biosynthesis enzymes, aiming to enable stereoselective production of functionalised carbapenams with three contiguous chiral centres. Structurallyguided substitutions of wildtype carboxymethylproline synthases enable tuning of their C-N and C-C bond forming capacity to produce 5-carboxymethylproline derivatives substituted at C-4 and C-6, from amino acid aldehyde and malonyl-CoA derivatives. Use of tandem enzyme incubations comprising an engineered carboxymethylproline synthase and an alkylmalonylCoA forming enzyme (i.e. malonyl-CoA synthetase or crotonyl-CoA carboxylase reductase) can improve stereocontrol and expand the product range. Some of the prepared 4,6-disubstituted-5-carboxymethylproline derivatives are converted to bicyclic β-lactams by carbapenam synthetase catalysis. The results illustrate the utility of tandem enzyme systems involving engineered crotonases for asymmetric bicyclic β-lactam synthesis.
  • Vikings, peat formation and settlement abandonment: multi-method chronological approach from Shetland

    Swindles, G.T.; Outram, Z.; Batt, Catherine M.; Hamilton, W.D.; Church, M.J.; Bond, J.M.; Watson, E.J.; Cook, G.T.; Sim, T.G.; Newton, A.J.; Dugmore, A.J. (2019-04-15)
    Understanding the chronology of Norse settlement is crucial for deciphering the archaeology of many sites across the North Atlantic region and developing a timeline of human-environment interactions. There is ambiguity in the chronology of settlements in areas such as the Northern Isles of Scotland, arising from the lack of published sites that have been scientifically dated, the presence of plateaus in the radiocarbon calibration curve, and the use of inappropriate samples for dating. This novel study uses four absolute dating techniques (AMS radiocarbon, tephrochronology, spheroidal carbonaceous particles and archaeomagnetism) to date a Norse house (the “Upper House”), Underhoull, Unst, Shetland Isles and to interpret the chronology of settlement and peat which envelops the site. Dates were produced from hearths, activity surfaces within the structure, and peat accumulations adjacent to and above the structure. Stratigraphic evidence was used to assess sequences of dates within a Bayesian framework, constraining the chronology for the site as well as providing modelled estimates for key events in its life, namely the use, modification and abandonment of the settlement. The majority of the absolute dating methods produced consistent and coherent datasets. The overall results show that occupation at the site was not a short, single phase, as suggested initially from the excavated remains, but instead a settlement that continued throughout the Norse period. The occupants of the site built the longhouse in a location adjacent to an active peatland, and continued to live there despite the encroachment of peat onto its margins. We estimate that the Underhoull longhouse was constructed in the period cal. AD 805–1050 (95% probability), and probably in cal. AD 880–1000 (68% probability). Activity within the house ceased in the period cal. AD 1230–1495 (95% probability), and most probably in cal. AD 1260–1380 (68% probability). The Upper House at Underhoull provides important context to the expansion and abandonment of Norse settlement across the wider North Atlantic region.
  • Investigation of Pulse electric field effect on HeLa cells alignment properties on extracellular matrix protein patterned surface

    Jamil, M. Mahadi Abdul; Zaltum, M.A.M.; Rahman, N.A.A.; Ambar, R.; Denyer, Morgan C.T.; Javed, F.; Sefat, Farshid; Mozafari, M.; Youseffi, Mansour (2018)
    Cell behavior in terms of adhesion, orientation and guidance, on extracellular matrix (ECM) molecules including collagen, fibronectin and laminin can be examined using micro contact printing (MCP). These cell adhesion proteins can direct cellular adhesion, migration, differentiation and network formation in-vitro. This study investigates the effect of microcontact printed ECM protein, namely fibronectin, on alignment and morphology of HeLa cells cultured in-vitro. Fibronectin was stamped on plain glass cover slips to create patterns of 25μm, 50μm and 100μm width. However, HeLa cells seeded on 50μm induced the best alignment on fibronectin pattern (7.66° ±1.55SD). As a consequence of this, 50μm wide fibronectin pattern was used to see how fibronectin induced cell guidance of HeLa cells was influenced by 100μs and single pulse electric fields (PEF) of 1kV/cm. The results indicates that cells aligned more under pulse electric field exposure (2.33° ±1.52SD) on fibronectin pattern substrate. Thus, PEF usage on biological cells would appear to enhance cell surface attachment and cell guidance. Understanding this further may have applications in enhancing tissue graft generation and potentially wound repair.
  • Membrane insertion and secretion of the Engrailed-2 (EN2) transcription factor by prostate cancer cells may induce antiviral activity in the stroma

    Punia, N.; Primon, Monika; Simpson, G.R.; Pandha, H.S.; Morgan, Richard (2019-03)
    Engrailed-2 (EN2) is a homeodomain-containing transcription factor that has roles in boundary formation and neural guidance in early development, but which is also expressed in a range of cancers. In addition to transcriptional regulation, it is secreted by cells and taken up by others through a mechanism that is yet to be fully elucidated. In this study, the distribution of EN2 protein in cells was evaluated using immunofluorescence with a set of antibodies raised against overlapping epitopes across the protein, and through the use of an EN2-GFP construct. MX2 expression in primary prostate tumors was evaluated using immunohistochemistry. We showed that EN2 protein is present in the cell membrane and within microvesicles that can be secreted from the cell and taken up by others. When taken up by normal cells from the stroma EN2 induces the expression of MX2 (MxB), a protein that has a key role in the innate immune response to viruses. Our findings indicate that EN2 secretion by tumors may be a means of preventing viral-mediated immune invasion of tissue immediately adjacent to the tumor.
  • Influence of positive and negative dimensions of dementia caregiving on caregiver well-being and satisfaction with life: Findings from the IDEAL study

    Quinn, Catherine; Nelis, S.M.; Martyr, A.; Victor, C.; Morris, R.G. (2019)
    The aim of this study was to identify the potential impact of positive and negative dimensions of caregiving on caregiver well-being and satisfaction with life (SwL). This study used time-point one data from the Improving the experience of Dementia and Enhancing Active Life (also known as IDEAL)cohort study that involved 1,283 informal caregivers of people in the mild-to-moderate stages of dementia recruited from 29 sites within Great Britain. Multivariate linear regression modeling was used to investigate the associations between positive dimensions of caregiving (measured by caregiving competence and perceptions of positive aspects of caregiving), negative dimensions of caregiving (measured by caregiving stress and role captivity), and caregiver well-being and SwL. Lower well-being was associated with low caregiving competence (–13.77; 95% confidence interval [CI]:–16.67, –10.87), perceiving fewer positive aspects of caregiving (–7.67; 95% CI:–10.26, –5.07), high caregiving stress (–24.45; 95% CI:–26.94, –21.96), and high role captivity (–15.61; 95% CI:–18.33, –12.89). Lower SwL was associated with low caregiving competence (–4.61; 95% CI:–5.57, –3.66), perceiving fewer positive aspects of caregiving (–3.09; 95% CI:–3.94, –2.25), high caregiving stress (–7.88; 95% CI:–8.71, –7.06), and high role captivity (–6.41; 95% CI:–7.27, –5.54). When these four measures were combined within the same model, only positive aspects of caregiving and caregiving stress retained independent associations with well-being and SwL. Both positive and negative dimensions of caregiving were associated with caregiver well-being and SwL. Psychological therapies and interventions need to consider not only the negative aspects of caregiving but also positive caregiving experiences and their implications for caregiver well-being and SwL.
  • Efficient simulations of the aqueous bio-interface of graphitic nanostructures with a polarisable model

    Hughes, Zak E.; Tomasio, S.M.; Walsh, T.R. (2014-05)
    To fully harness the enormous potential offered by interfaces between graphitic nanostructures and biomolecules, detailed connections between adsorbed conformations and adsorption behaviour are needed. To elucidate these links, a key approach, in partnership with experimental techniques, is molecular simulation. For this, a force-field (FF) that can appropriately capture the relevant physics and chemistry of these complex bio-interfaces, while allowing extensive conformational sampling, and also supporting inter-operability with known biological FFs, is a pivotal requirement. Here, we present and apply such a force-field, GRAPPA, designed to work with the CHARMM FF. GRAPPA is an efficiently implemented polarisable force-field, informed by extensive plane-wave DFT calculations using the revPBE-vdW-DF functional. GRAPPA adequately recovers the spatial and orientational structuring of the aqueous interface of graphene and carbon nanotubes, compared with more sophisticated approaches. We apply GRAPPA to determine the free energy of adsorption for a range of amino acids, identifying Trp, Tyr and Arg to have the strongest binding affinity and Asp to be a weak binder. The GRAPPA FF can be readily incorporated into mainstream simulation packages, and will enable large-scale polarisable biointerfacial simulations at graphitic interfaces, that will aid the development of biomolecule-mediated, solution-based graphene processing and self-assembly strategies.

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