Modification of peptides by disulfide bridging: a biochemical and analytical investigation

The use of chemical reagents for the modification of peptides has broad applications and, as a result, is a rapidly expanding area of research. Such methods serve to improve the pharmacokinetic properties of the peptide or add functionality for a desired application. Chemical modification of peptides has facilitated the development of a variety of bioconjugates for use as analytical probes, diagnostic agents and therapeutics. This project focuses on the modification of peptides by targeting disulfide bonds. Many peptides contain disulfide bonds which serve a crucial role in retaining their structure, function and stability. Reduction of these disulfides affords two reactive cysteine thiolates whose nucleophilicity can be exploited in peptide modification. To this end, a family of 3,4-disubstituted maleimide reagents were synthesised, designed to efficiently re-bridge a reduced, accessible disulfide bond. The bridging reagents vary in reactivity, properties and functionality but all serve to maintain the structural integrity conferred by a disulfide bond. With these reagents in hand, the scope of their utility was tested on peptides of biological and medicinal interest. One such peptide was tertiapin Q, a neurotoxin derived from the venom of the honey bee and consisting of two disulfide bonds in close proximity. Both singly and doubly bridged variants of the peptide were synthesised and isolated. Biological activity of the modified peptide was analysed by whole-cell patch clamp experiments on cells expressing the target of the peptide toxin, the G-coupled inwardly rectifying K+ (GIRK) channel, which tertiapin Q is known to inhibit. Loss of biological activity was observed upon modification, which led into a full structural characterisation study to determine the explanation for this intriguing result. A second peptide target, octreotide, a stabilised analogue of the hormone somatostatin with a single disulfide bond, was modified with relative ease. Biological activity was examined by whole-cell patch clamp on cells expressing the target of octreotide, the somatostatin receptor (SSTR) subtype 2. Pleasingly, nanomolar activity of the modified peptide was observed. A novel candidate for the next generation of diagnostics for SSTR positive tumours was subsequently developed, characterised and tested by confocal microscopy

Adaption of the ex vivo mycobacterial growth inhibition assay for use with murine lung cells.

In the absence of a correlate(s) of protection against human tuberculosis and a validated animal model of the disease, tools to facilitate vaccine development must be identified. We present an optimised ex vivo mycobacterial growth inhibition assay (MGIA) to assess the ability of host cells within the lung to inhibit mycobacterial growth, including Bacille Calmette-Guérin (BCG) and Mycobacterium tuberculosis (MTB) Erdman. Growth of BCG was reduced by 0.39, 0.96 and 0.73 log10 CFU following subcutaneous (s.c.) BCG, intranasal (i.n.) BCG, or BCG s.c. + mucosal boost, respectively, versus naïve mice. Comparatively, a 0.49 (s.c.), 0.60 (i.n.) and 0.81 (s.c. + mucosal boost) log10 reduction in MTB CFU was found. A BCG growth inhibitor, 2-thiophenecarboxylic acid hydrazide (TCH), was used to prevent quantification of residual BCG from i.n. immunisation and allow accurate MTB quantification. Using TCH, a further 0.58 log10 reduction in MTB CFU was revealed in the i.n. group. In combination with existing methods, the ex vivo lung MGIA may represent an important tool for analysis of vaccine efficacy and the immune mechanisms associated with vaccination in the organ primarily affected by MTB disease

Differences in the imaging of Crohn's disease patients between North America and Europe: are we ready to bridge the divide?

The emphasis of treatment in Crohn's disease has evolved from a reactive model to "treat-to-target" approaches. Cross-sectional imaging has rapidly evolved in parallel, with a growing evidence base supporting its abilities for diagnosis, monitoring and prognostication. Whilst there are differences in emphasis between Europe and North America, particularly around the type of imaging modalities and patterns of multidisciplinary care, there is increasing convergence. This perspective piece provides an overview of the evolving role of cross-sectional imaging in Crohn's disease, discusses practice differences between North America and Europe and provides suggestions on areas for future collaboration and research priorities

Photochemically re-bridging disulfide bonds and the discovery of a thiomaleimide mediated photodecarboxylation of C-terminal cysteines

Described in this work is a novel method for photochemically manipulating peptides and proteins via the installation of cysteine-selective photoactive tags. Thiomaleimides, generated simply by the addition of bromomaleimides to reduced disulfide bonds, undergo [2 + 2] photocycloadditions to reconnect the crosslink between the two cysteine residues. This methodology is demonstrated to enable photoactivation of a peptide by macrocyclisation, and reconnection of the heavy and light chains in an antibody fragment to form thiol stable conjugates. Finally we report on an intriguing thiomaleimide mediated photochemical decarboxylation of C-terminal cysteines, discovered during this study

Accounting for refrigeration heat exchange in energy performance simulations of large food retail buildings

Heat exchange between chilled food storage and conditioned spaces in large food retail stores is not currently required as part of design stage regulatory compliance energy performance models. Existing work has identified that this exchange has a significant impact on store energy demand and subsequently leads to unrealistic assessment of building performance. Research presented in this article uses whole building dynamic thermal simulation models that are calibrated against real store performance data, quantifying the impact of the refrigeration driven heat exchange. Proxy refrigerated units are used to simulate the impact of these units for the sales floor areas. A methodology is presented that allows these models to be simplified with the aim of calculating a realistic process heat exchange for refrigeration and including this in thermal simulation models; a protocol for the measurement of chilled sales areas and their inclusion in the building models is also proposed. It is intended that this modelling approach and the calculated process heat exchange inputs can be used to improve the dynamic thermal simulation of large food retail stores, reduce gaps between predicted and actual performance and provide more representative inputs for design stage and regulatory compliance energy calculations

Fano 3-folds in P2xP2 format, Tom and Jerry

We study Q-factorial terminal Fano 3-folds whose equations are modelled on those of the Segre embedding of P^2xP^2. These lie in codimension 4 in their total anticanonical embedding and have Picard rank 2. They fit into the current state of classification in three different ways. Some families arise as unprojections of degenerations of complete intersections, where the generic unprojection is a known prime Fano 3-fold in codimension 3; these are new, and an analysis of their Gorenstein projections reveals yet other new families. Others represent the "second Tom" unprojection families already known in codimension 4, and we show that every such family contains one of our models. Yet others have no easy Gorenstein projection analysis at all, so prove the existence of Fano components on their Hilbert scheme

Quantification of crypt and stem cell evolution in the normal and neoplastic human colon.

Human intestinal stem cell and crypt dynamics remain poorly characterized because transgenic lineage-tracing methods are impractical in humans. Here, we have circumvented this problem by quantitatively using somatic mtDNA mutations to trace clonal lineages. By analyzing clonal imprints on the walls of colonic crypts, we show that human intestinal stem cells conform to one-dimensional neutral drift dynamics with a "functional" stem cell number of five to six in both normal patients and individuals with familial adenomatous polyposis (germline APC(-/+)). Furthermore, we show that, in adenomatous crypts (APC(-/-)), there is a proportionate increase in both functional stem cell number and the loss/replacement rate. Finally, by analyzing fields of mtDNA mutant crypts, we show that a normal colon crypt divides around once every 30-40 years, and the division rate is increased in adenomas by at least an order of magnitude. These data provide in vivo quantification of human intestinal stem cell and crypt dynamics.This study was supported by Cancer Research UK (to A.-M.B. and N.A.W.), the Medical Research Council (to B.C. and S.A.C.M.), the Engineering and Physical Sciences Research Council (to A.G.F.), Microsoft Research (to A.G.F.), the National Institute for Health Research University College London Hospitals Biomedical Research Centre (to M.R.J.), the Dutch Cancer Research Foundation (to M.J.), the Wellcome Trust (to B.D.S.), and Higher Education Funding Council for England (to T.A.G.)

Historical BCG vaccination combined with drug treatment enhances inhibition of mycobacterial growth ex vivo in human peripheral blood cells

© 2019, The Author(s). Tuberculosis (TB) is a leading infectious cause of death globally. Drug treatment and vaccination, in particular with Bacillus Calmette-Guérin (BCG), remain the main strategies to control TB. With the emergence of drug resistance, it has been proposed that a combination of TB vaccination with pharmacological treatment may provide a greater therapeutic value. We implemented an ex vivo mycobacterial growth inhibition assay (MGIA) to discriminate vaccine responses in historically BCG-vaccinated human volunteers and to assess the contribution of vaccine-mediated immune response towards the killing effect of mycobacteria in the presence of the antibiotics isoniazid (INH) and rifampicin (RIF), in an attempt to develop the assay as a screening tool for therapeutic TB vaccines. BCG vaccination significantly enhanced the ability of INH to control mycobacterial growth ex vivo. The BCG-vaccinated group displayed a higher production of IFN-γ and IP-10 when peripheral blood mononuclear cells (PBMC) were co-cultured with INH, with a similar trend during co-culture with RIF. A higher frequency of IFN-γ + and TNF-α + CD3 − CD4 − CD8 − cells was observed, suggesting the contribution of Natural Killer (NK) cells in the combined effect between BCG vaccination and INH. Taken together, our data indicate the efficacy of INH can be augmented following historical BCG vaccination, which support findings from previous observational and animal studies.EC HORIZON2020 TBVAC2020; Indonesian Endowment Fund for Education (LPDP); UK Medical Research Council (MRC); UK Department for International Development (DFID