NIHRDataInsights/HRCSData huggingface dataset:
Title: Single molecule quantification of the activation, biophysics and pharmacology of GlyREM, a new structural model for pentameric ligand-gated channels
Funder: Medical Research Council

Abstract: The functional properties of the GlyR-EM channel construct that has produced the new cryoEM structures (Du et al., 2015) are not known in any detail. We will characterize this receptor by patch-clamp electrophysiology. We will use the whole-cell mode to describe the channel's pharmacology cf. agonists, antagonists, blockers and modulators, and fast agonist applications (ca 0.15 ms exchange) to outside-out patches to measure its macroscopic kinetics of activation/deactivation. We will also record in conditions approximating those in the structural experiments, to estimate desensitisation rates and the distribution of the channel across functional states during very long agonist exposures. Finally, low-noise single-channel recording will measure agonist efficacy as maximum open probability. Kinetic analysis of these data by global mechanism fitting with full missed event correction will establish a detailed quantitative mechanism for the glycine activation/deactivation of this channel. Extensive preliminary experiments data show that the work is feasible. Our pilot data show also higher agonist efficacy and much slower deactivation for this channel vs its human homologue. We will investigate these properties with electrophysiology on wild type and mutant channels. Another question arises from the structural data, namely whether the glycine/ivermectin bound channel is partially open. We will obtain precise single channel conductance measurements to clarify that.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Investigating the functional significance of lipid-binding properties of human Notch ligands in health and disease
Funder: Medical Research Council

Abstract: Understanding the molecular basis of Notch signaling is of significant biomedical importance. We will investigate the role of lipid as a novel modulator of Notch signaling using the following methods: i) Create affinity-matured forms of ligand receptor complexes using established expression systems for use in lipid-binding assays which utilize liposomes or Nanodiscs. This will provide biochemical (plate assays, lipid specificity) and biophysical information (eg SPR, NMR, mass spec) about the lipid/ligand interaction. ii) Different high-throughput screening methods including commercial lipid arrays, LiMA, native mass spectrometry and lipid pathway inhibitors will be used to identify candidate lipid ligands for each ligand family. iii) The effects of somatic mutations associated with various cancers found in the lipid binding region of C2 domains of Notch ligands on lipid/Notch binding and Notch activity will be tested, and why hepatic cells appear sensitive to the effects of lipid binding mutations giving rise to disease will be investigated. These data will give new insights into the modulatory role of lipid binding to mammalian C2 domains in Notch signaling which can be tested in animal models. It will facilitate the understanding of disease-causing mutations affecting this domain. Results will be disseminated at international conferences on Notch function which are held annually. Any commercial exploitation will be handled by Oxford University Innovation.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: The structure and function of the SLX4 nuclease complex
Funder: Medical Research Council

Abstract: Loss of genomic stability directly contributes to the initiation of human ageing and disease. During every cell division, structural aberrations in the DNA can block the progression of replication forks. The SLX4 nuclease platform plays a critical role in spatially targeting and modulating the activity of three structure-selective nucleases, XPF-ERCC1, MUS81-EME1 and SLX1 during replication fork repair and homologous recombination. Our recent work with the human SLX4 complex has demonstrated that its constituent proteins are difficult to produce in amounts needed for structural studies, and suffer from aggregation problems during in vitro complex formation. For this reason, we have turned to the Xenopus laevis SLX4/SMX complex, which is functionally homologous to the human complex, but during purification behaves very differently from the human complex. This complex is far less prone to aggregation and can be produced in a soluble single-state form with sufficient yield to permit structural studies. Here, we propose to solve the structure of the SLX4/SMX complex using cryo-electron microscopy. We will initially structurally characterise several biochemically active SLX4 subcomplexes. Our preliminary data suggest that we will subsequently be able to tackle the full-length SLX4 protein in association with XPF-ERCC1, MUS81-EME1 and SLX1 allowing us reveal the structural basis for nuclease targeting and stimulation by SLX4. Our structural studies will be complemented with functional biochemistry and cellular phenotype analysis, that will allow us to iteratively test the functional importance of the structural features identified during DNA repair and recombination. Together, this will provide an unparalleled insight into the structure and mechanism of one of the most important, yet poorly understood DNA repair complexes, and one which is critical to maintaining health, suppressing the hallmarks of ageing and represents a highly attractive target in oncology.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Definition of the naturally-processed drug-peptide adducts that can act as functional T-cell antigens
Funder: Medical Research Council

Abstract: Background: Drug hypersensitivity reactions cannot be predicted and show no simple dose-response relationship. Recently, we have demonstrated that beta-lactam-protein conjugates can function as antigens for T-cells and that the same conjugates are formed in patients. We have also shown that is possible to characterize natural drug-modified MHC binding peptides. Despite this, the peptide repertoire displayed by MHC molecules on antigen presenting cells exposed to drugs, the structure of the drug-modified peptide T-cell receptor binding interaction and the drug-modified peptides that activate T-cells have not been defined. Objectives: We propose to conduct a kinetic analysis of the drug-modified and unmodified immunopeptidome. Moreover, we will synthesize drug-modified peptides to identify the antigenic determinants that activate T-cells from hypersensitive patients and healthy donors. Structural assessment of MHC associated drug-modified peptides in complex with T-cell receptors will be performed, while tetramers will be used to ascertain antigen-specific T-cell numbers in hypersensitive and tolerant patients and healthy donors. Methods: We will characterize drug-modified MHC binding peptides and the kinetics of MHC peptide display using mass spectrometry and directly relate the chemistry of antigen formation to immune function using PBMC from hypersensitive patients and healthy donors. Immune responses will be measured using a battery of established methods and through the production of tetramers. The GOLD programme will be used for in silico modelling, while X-ray diffraction will define the structure of drug-modified peptides bound to MHC and T-cell receptors. Impact: A better understanding of drug hypersensitivity will improve our ability to diagnose, predict and prevent such reactions. Synthesis of drug-modified peptides as reagents will allow the development of more evidence-based culture systems and thereby improve drug safety for the good of public health.


Research activity codes: 6.1 PHARMACEUTICALS
Health categories: GENERIC HEALTH RELEVANCE
Title: Unravelling c-Met signalling from autophagic endomembranes
Funder: Medical Research Council

Abstract: The receptor tyrosine kinase c-Met, implicated in cancer and chronic inflammation, signals on endosomes, enhancing cell migration, growth and malignant transformation. We have discovered that c-Met traffics and signals from autophagic related endomembranes, ARE, leading to enhanced cell viability in anchorage independent conditions. We hypothesize that autophagy, considered conduit for degradation only, also has a signalling role, which may be targeted in diseases such as cancer. Moreover, this signalling occurs on novel non-canonical autophagy endomembranes, poorly characterized. We aim to understand the signalling of c-Met on non-canonical autophagy endomembranes. Using state-of-the-art cell biology and microscopy, we will characterise the ARE and the role of non-canonical autophagy on c-Met trafficking and signalling. Using a recently described approach of proximal proteomic mapping in live cells by engineered ascorbic peroxidase, APEX, we will determine the molecular partners of c-Met trafficking and signalling on ARE. We will assess the physiological and pathological relevance of c-Met signalling on ARE. This project will be performed using panels of normal and cancer cell lines modified by CRISPR technology, with impaired canonical or impaired non-canonical autophagy. Shedding light and further the understanding of a totally novel function of autophagy (signalling) will have major impacts in cell biology. Moreover, a new physiological role of a recently discovered non-canonical autophagy pathway will be uncovered. This project could lead to the development of strategies to reduce or enhance c-Met signalling and more generally of other receptors. The relevance of our results will be applied to cancer in the view of finding new targets. Our results will have larger implications as autophagy and RTK signalling both play major roles in numerous physiological and pathological processes.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Transcriptional control of cell fate decisions by chromatin remodelling proteins
Funder: Medical Research Council

Abstract: A cell's identity is defined by its gene expression patterns, and gene expression potential is encoded in chromatin. Changes to cell state which occur during normal development require very precise changes in chromatin structure and gene expression. Failure to properly regulate gene expression patterns can prevent successful execution of developmental decisions, but can also lead to tumourigenesis. Chd4 and Brg1 are two mammalian chromatin remodelling proteins which use energy derived from ATP hydrolysis to alter the structure of chromatin and thereby influence gene expression patterns. Both proteins play essential roles in early mammalian development, and both proteins have been found to be mutated in a wide variety of different tumour types. A body of evidence indicates that these two proteins often act in opposition. I hypothesise that these two proteins normally act in opposition to precisely correlate gene expression patterns. This proposal will test this hypothesis in pluripotent cells undergoing cell fate decisions both in culture and during early mouse embryogenesis. We will provide precise molecular details of how control of enhancer chromatin structure by Chd4 and Brg1 impacts the binding and subsequent activities of enhancer-binding transcription factors, and how this then influences the activity of RNA polymerase at target promoters. Using super resolution microscopy, we will determine how chromatin remodellers control transcription factor binding kinetics in live cells in culture and in preimplantation stage embryos undergoing the first cell fate decision. We will further define how these chromatin remodelling systems are used by embryonic cells to drive developmental transitions. This project combines cutting edge imaging and single cell biology to reveal fundamental transcriptional mechanisms at unprecedented molecular detail, greatly increasing the resolution of our understanding of how cell fate decisions are enacted in mammalian cells.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Defining the Deleterious Effects of Environmental Pollutants at a Mechanistic Level
Funder: Medical Research Council

Abstract: Aim: Investigate whether pollutants associated with diesel emissions cause oxidative stress, DNA damage, and AhR pathway activation in vivo. We will evaluate in utero, neonatal and adult exposures and establish whether the responses observed are also observed in exposed human populations. Methodology: This is a new collaboration between the University of Dundee and Queen Mary University London. The UoD will use its novel Hmox1, p21, and Cyp1a1 reporter mouse lines to evaluate oxidative stress, DNA damage, and AhR pathway activity, respectively, in response to chemicals associated with the semi-volatile organic fraction of diesel exhaust particles. We will also test ambient polluted air sampled from areas with high diesel emissions and provided by QMUL. Reporter mice will be randomized to control or treatment groups and reporter activities measured non-invasively by bioluminescent imaging for luciferase activity and post-mortem by LacZ staining of organ sections. The relevance of these rodent studies to humans will be assessed by measuring HMOX1, p21, and CYP1A1 levels in airway nasal epithelial biopsies and cultures taken before and after human volunteers have been exposed to polluted urban environments. Finally, to track the fate of cells damaged pre- and neonatally, we will exploit novel stress-shadowing (fate-mapping) mouse lines in which Cre recombinase driven from the Hmox1 or Cyp1a1 promoters and as a consequence permanently activates a silenced reporter system. For these studies, pure chemicals or particle mixtures will be administered to pregnant dams. Benefits: Mechanistic insights into the relationship between diesel emissions and disease, including the highly susceptible in utero and neonatal phases of life. Pinpointing the organs and cell types affected will allow predictions of specific health outcomes for specific pollutants. Facilitate future studies on how the pathogenesis of human disease may be potentiated by the environment, i.e. asthma.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE
Title: Statistical methods and applications in growth and development - SITAR, LMS and IOTF
Funder: Medical Research Council

Abstract: The SITAR method of growth curve analysis (Cole et al 2010) summarises individual growth patterns of e.g height or weight in three parameters (size, timing and intensity) that are estimated as subject-specific random effects, plus a natural cubic spline estimate of the average growth curve. It explains over 95% of the age-specific variance in the outcome measure, and as such is an effective summary of individual growth patterns. The subject-specific random effects, representing individual growth, can then be related to earlier growth-affecting exposures or later health outcomes. Thus it is relevant in translational medicine and life course epidemiology. The first objective of the project is to further develop the SITAR methodology, by applying it to a series of existing growth studies, and developing the existing SITAR software library in the R statistical language for its wider dissemination. The LMS method (Cole and Green 1992) is widely used for constructing growth references and centile charts, particularly for skew measurements such as weight, BMI or waist circumference. The second objective is to port existing software to fit and use the LMS method (LMSgrowth and LMSchartmaker) from the desktop to an R-shiny web-based platform, which will improve access to the software and extend its capabilities. A programmer post is included in the costings.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: HMS: Identifying and Measuring Spillover Effects of Changes to Healthcare Organisation and Delivery
Funder: Medical Research Council

Abstract: The Highlight Notice on spillover effects notes that the methods for modelling care delivery changes are under-developed. Because inputs, processes and outcomes are inter-related, changes to one part of a system will have multiple consequences for the whole system. We will develop a methodology to enable structured identification and measurement of spillover effects. We will use econometric evaluation techniques. The research will examine three case studies in depth to understand the longer-term consequences of the equilibrating and offsetting mechanisms affecting health service delivery. The study comprises three work packages: WP1 is a systematic review of the spillover effects identified in previous literature and the methodological approaches used to measure them. WP2 will re-examine three case studies of policy changes to (a) improve quality in primary care through financial incentives, (b) increase the proportion of surgical hospital interventions delivered as daycases through higher reimbursements and (c) extend the availability of general practice services out of hours. Administrative datasets will be re-analysed at patient and organisation level to estimate between and within, and wider system, spillovers of these changes. WP3 will develop and publish structured methodological guidance on how to identify and measure spillover effects from changes to healthcare organisation and delivery. This guidance will be developed with a project advisory group of evaluation experts, research funders, policy advisors and decision-makers. The full guidance will be published electronically and a summary paper will be submitted to an academic journal.


Research activity codes: 8.1 ORGANISATION AND DELIVERY OF SERVICES;8.2 HEALTH AND WELFARE ECONOMICS;8.4 RESEARCH DESIGN AND METHODOLOGIES (HEALTH SERVICES)
Health categories: GENERIC HEALTH RELEVANCE
Title: The case time series design: a new tool for big data analysis
Funder: Medical Research Council

Abstract: Big data technologies are transforming the landscape of biomedical research. Epidemiological investigations can now rely on repeated measurements collected at individual level in large populations, linked with a wealth of data on personal characteristics and various risk factors. These resources offer the possibility to answer research questions that could not be feasibly addressed using traditional studies. However, this more complex big data setting presents important methodological challenges, and it requires innovative analytical approaches to model complex longitudinal associations between repeated measures of health outcomes and time-varying exposures using observational data. This project contributes to address this issue through the development of a novel analytical tool called case time series design. This design offers an adaptable framework that combines the individual-level setting and ability to control for confounders of case-only methods such as the self-controlled case series, with the flexibility and temporal structure of time series models. It represents a general tool, applicable in different research areas for investigating associations with environmental exposures, clinical conditions, or drug use. The case time series design is suitable for the analysis of highly-informative big data resources, particularly those providing individual profiles with longitudinal measures of health outcomes and time-varying predictors. The research proposal is structured in a detailed work plan. This includes the definition of the design setting and assumptions of the case time series, the description of the statistical framework, and simulation studies to validate the methodology. The novel design is illustrated in four case studies that demonstrate its application in various research areas within biomedical research, such as environmental, clinical, and pharmaco-epidemiology.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY);3.4 VACCINES
Health categories: GENERIC HEALTH RELEVANCE
Title: Adaptation of evidence-informed complex population health interventions for implementation and/or re-evaluation in new contexts: New guidance
Funder: Medical Research Council

Abstract: Complex population health interventions that demonstrate effectiveness in one context may be ineffective or harmful elsewhere. Intervention adaptation and re-evaluation may be required. To date, there is no established guidance for the adaptation, implementation and/or re-evaluation of complex interventions in new contexts. This study will develop guidance for researchers, research funders, journal editors, policy-makers and practitioners. The study will comprise four work packages. WP1 will include systematic review of current guidance. It will proceed to a mapping review of studies reporting intervention adaptation, implementation and/or revaluation in new contexts. This will explore how researchers undertake and explain adaptations, and how they utilise guidance. WP2 will include qualitative interviews with researchers, representatives of international-level funding bodies, peer reviewers, policy makers and practitioners. Interviews will build on WP1 and examine decision-making around intervention adaptation, the procedures undertaken, and the uptake of guidance. Interviews with research funders and peer reviewers will focus on the perceived appropriateness of proposed or reported adaptations and evaluation. WP3 will comprise a web-based Delphi exercise to identify expert consensus on adaptation. Round 1 of the survey will generate a comprehensive list of items to be considered; round 2 will validate these items with a wider group of expertise; and round 3 will present recommendations for guidance inclusion. Following the survey, guidance will then be drafted and we will host a consensus meeting to gauge agreement and identify areas that may require further development. WP4 will oversee study coordination and dissemination. Study outputs will include guidance on the adaptation, implementation and/or re-evaluation of population health interventions in new contexts, and a research agenda for intervention adaptation in population health.


Research activity codes: 3.1 PRIMARY PREVENTION INTERVENTIONS TO MODIFY BEHAVIOURS OR PROMOTE WELL-BEING;3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS
Health categories: GENERIC HEALTH RELEVANCE