NIHRDataInsights/HRCSData huggingface dataset:
Title: Elucidating the contributions of ubiquitin ligase complexes to endoplasmic reticulum associated degradation (ERAD)
Funder: Medical Research Council

Abstract: ERAD represents the principal remediation mechanism for non-native, maturation-incompetent proteins whose unabated accumulation can disrupt optimal ER function. ERAD employs a network of accessory factors localised to both sides the ER membrane that coordinate with the ubiquitin-proteasome system (UPS) to degrade misfolded integral membrane and secreted proteins. Membrane embedded ubiquitin ligases (E3s) such as Hrd1 form nexuses for ERAD activity by assembling macromolecular complexes in the ER along the transmembrane axis. This proposal aims to identify the individual components and domains essential for formation of Hrd1-containing complexes using velocity sedimentation to monitor size, rationally designed truncations/deletions to map the interaction domains, RNAi (and possibly TALEN-based genomic editing) to determine essential accessory factors and radiolabeling-densitometry to ascertain stoichiometry. This proposal also draws on a proven affinity purification-LC-MS/MS strategy to establish the interaction networks (and potentially substrates) of the 26 ER-resident E3s putatively involved in ERAD. Quantitative analysis using the Nanostring hybridization platform will be used to track changes gene expression of components in the ERAD network (with a focus on ER-resident E3s and their interactors) that result in response to ER stress conditions. This study will provide the first comprehensive analysis of ER-resident E3 complexes, their organisation and their expression with the goal of ultimately determining the contributions they individually make to the ERAD capacity and its ability to restore ER functionality during periods of stress-induced global misfolding.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Functional dissection of the genetic interaction network that affects growth of cells with telomere defects: implications for health and disease
Funder: Medical Research Council

Abstract: We will use high-throughput robotic genetic procedures to measure fitness of nearly 24,000 genetically different yeast strains at a range of growth temperatures. To ensure that our measurements are accurate we will create 8 independent biological replicates of each of these 24,000 different yeast strains. All the strains will contain cdc13-1 a recessive mutation that causes temperature dependent telomere cap defects, and two null mutations in genes that affect the telomere. We will examine fitness at four temperatures (23, 27, 30 and 33oC), resulting in a spectrum of telomere defects, from minor to severe. We will use appropriate photographic, computational and statistical procedures to measure the fitness of each genetically distinct yeast strain. From these data we will learn how gene mutations interact (epistasis) in this system and develop new Bayesian statistical methods to identify and quantify epistatic interactions revealed by this novel experimental design. We will also determine how interactions are affected by the severity of the telomere defect (temperature). There is already precedent that very strong genetic interactions occur, allowing in some cases yeast strains to completely bypass the requirement for the normally essential CDC13 gene. Significant genetic interactions identified in yeast will be used to determine if analogous interactions could be relevant in humans using relevant genome-wide association study (GWAS) datasets.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: GENERIC HEALTH RELEVANCE
Title: School Health Action Research Partnership and Network (SHARPEN)
Funder: Medical Research Council

Abstract: Schools are important settings for young people's health improvement, but research in UK secondary schools has become increasingly challenging with health improvement intervention trials undertaken on an ad hoc basis. A network of 'trial ready' schools with research literate staff would support more efficient health improvement research that could better inform public health policy and practice in schools. With the aim of facilitating a step change in the quantity and quality of school-based health improvement research and informed by the success of UK primary care research networks, this project will develop and evaluate a School Health Action Research Partnership and Network (SHARPEN) in Wales. Working with Welsh Government, NHS and third sector partners we will identify the infrastructure and processes needed to support SHARPEN through a staged scoping and feasibility study with 3 strands. Strand 1 will establish and scope a basic school health research network (SHRN), using qualitative methods to explore staff, pupil and other stakeholder views on such issues as incentives to participate in SHRN and expectations of membership. New and efficient approaches to key elements of the research process, e.g. informed consent and whole school recruitment, will also be developed. Strand 2 will explore the feasibility of an enhanced, action research partnerships (ARP) model to support knowledge transfer and development of research-practitioners in schools. Transdisciplinary school action groups will review student health behaviour data, then develop and implement action plans. The ARP model will be evaluated using a mixed methods process evaluation. The potential for linking student health behaviour data with other routinely collected datasets will also be explored. Strand 3 will use surveys, interviews and consultation with policy and practice stakeholders to scope the barriers and facilitators to sustaining SHARPEN in Wales and the potential for transfer to England.


Research activity codes: 8.1 ORGANISATION AND DELIVERY OF SERVICES
Health categories: GENERIC HEALTH RELEVANCE
Title: Non-genetic heterogeneity in response to anti-mitotic chemotherapeutics
Funder: Medical Research Council

Abstract: Genetically identical daughter cells can exhibit different fates when exposed to anti-mitotic drugs. This non-genetic heterogeneity arises due to the stochasticity of biochemical reactions, generating cells with identical genomes but different proteomes. We have discovered that inhibiting the transcription factor c-Myc modulates this fate choice. Our hypothesis is that Myc regulates transcriptional subprograms that have downstream effects on protein networks that influence cell fate decisions operating in mitosis. Because Myc is itself dynamic, stochastic variation in Myc activity could contribute to heterogeneity in response to anti-mitotics. We have designed three work packages to test these hypotheses: WORK PACKAGE #1. Using tried and tested approaches, we will probe Myc as a modulator of mitotic cell fate. We will use RNAi, dominant negatives, overexpression and small molecule inhibitors to alter Myc function, then use time-lapse microscopy to monitor mitosis and apoptosis in order to generate cell fate profiles measuring the frequency of "death in mitosis" vs. "slippage". WORK PACKAGE #2. We will generate cell lines expressing Myc biosensors, e.g. using plasmids and/or BACs with reporters downstream of Myc target promoters. Stable cell lines harbouring the transgenes will be used for pedigree analyses; sister cells will be monitored using time-lapse fluorescence microscopy to measure endogenous Myc activity, challenged with anti-mitotic drug, and their fate determined. WORK PACKAGE #3. The links between Myc and mitotic cell fate are largely unexplored, but we hypothesize that inhibition of Myc does not effect mitosis per se, rather the ability to apoptose while in mitosis. To test this, and to determine which aspect of apoptosis account for the fate shift, we will take two complementary approaches: a top-down descriptive approach, namely focused gene expression profiling, and a bottom-up functional approach, namely mitochondrial BH3 profiling.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Linking Regulatory Elements Harboring Common Disease-Associated Variants to Their Target Genes
Funder: Medical Research Council

Abstract: Numerous genome-wide association studies (GWAS) have identified thousands of regions of the human genome containing structural variants or single nucleotide polymorphisms (SNPs) that are associated with various common diseases and traits. The vast majority of GWAS SNPs lie in non-coding regions and more than half are located at considerable distances from nearby genes making it very difficult to predict if the associated variant is causal, or to identify a potential causal gene. Thus a large amount of potentially useful information on genetic variability and its links with disease is currently un-exploitable. A recent study (Maurano et al., 2012) shows that many GWAS SNPs map to DNAse I hypersensitive sites (DHS), which are a characteristic feature of gene regulatory elements such as enhancers. Enhancers can be up to a megabase away from the genes they regulate, often jumping over several intervening genes, and can be located within one gene while regulating another. Non-coding structural variants may therefore contribute to disease by disrupting transcription factor binding sites in enhancers or other regulatory elements that control distal genes, leading to gene mis-regulation. Our preliminary findings strongly support this hypothesis. In this application we propose to systematically identify all long-range genomic elements that interact with all human gene promoters in a panel of primary human cell types using a new method that we have developed called promoter capture Hi-C. Our preliminary data show that the long-range elements that we identify through interaction with promoters are enriched in active histone marks and transcription factor binding sites characteristic of enhancers. These genomic regions are significantly enriched in disease associated SNPs. This work will allow us to identify hundreds to thousands of new potential disease-causing genes, creating new biomarkers and opening the door to new drug targets and avenues of clinical intervention.


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE
Title: Mechanisms underlying developmental programming of lifelong health
Funder: Medical Research Council

Abstract: Mother:offspring interactions during pregnancy and lactation are defining features of mammals. Poor maternal diet, maternal obesity and gestational diabetes directly influence offspring growth and lead to prevalent health problems including obesity, type 2 diabetes and cardiovascular disease. Developmental programming of offspring health is well recognised but the underlying mechanisms are poorly understood. Progress through human studies is hampered by the long time-scales involved and the obvious ethical barriers to experimental interventions. A number of useful animal models have been developed involving manipulation of offspring growth, typically through manipulation of maternal diet, but rapid further progress will require a genetic model of developmental programming. We have produced such a model using a knockout of the imprinted Grb10 gene. Grb10 knockout mice are large at birth and as adults are lean with enhanced insulin signalling and improved ability to clear a glucose load. Cross-fostering experiments allowed us to separate effects of Grb10 acting in offspring from those of maternal Grb10. Our findings indicate that Grb10 has evolved coadapted functions in mother and offspring in order to optimize offspring growth and physiology. These properties indicate a role for Grb10 as a developmental programming gene. In support of this, in recent studies of two different maternal dietary restriction models Grb10 was found to be up-regulated in the liver of affected offspring. Here, we propose to directly test the role of Grb10 as a developmental programming gene by combining our genetic model with an established maternal dietary restriction model. This will be the first functional validation of a developmental programming gene. We will also use our combined model to identify further developmental programming genes as well as signature epigenetic changes (DNA methylation and histone modifications) associated with them.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Harnessing Our Understanding Of Water To Identify druggable Protein Targets and drive The Development Of Effective Therapeutics
Funder: Medical Research Council

Abstract: Water is the most important molecule in biology. The interaction between any two biological molecules must compete with the interactions of the individual molecules with water and thus it is involved in every single intermolecular interaction in an aqueous environment. This also makes water the most important molecule in medicine, as it controls the interactions of every therapeutic with its target. In order to account for hydration thermodynamics, this proposal is focused on inhomogeneous fluid solvation theory (IFST), a statistical mechanical method to determine the enthalpy and entropy of solvation. IFST has a number of advantages that make it superior to many of its alternatives. Explicitly including the thermodynamics of interfacial water molecules is vital, as the subtle balance of enthalpy and entropy means that immobilising a water can favour or disfavour binding. In addition, visualisation of the contributions of spatial subvolumes is a revolutionary approach that transforms the way in which water is viewed, understood and harnessed. The combination of accuracy with elucidative results, calculable on a reasonable timescale, makes IFST a powerful method for studying hydration. Novel extensions to the theory developed in this project will allow computation of binding free energies. IFST will then be used to solve two fundamental problems in drug discovery: the identification of druggable targets and the design of small molecule inhibitors. After validation, the software will be used to identify protein targets containing hot spots suitable for small molecule binding. IFST will also be combined with a molecular design algorithm that incorporates the contributions of structural waters. Collaborations with experimental groups in Cambridge and at the MRCT will allow the software to be applied directly to active drug development projects. These two advances will increase the cost effectiveness of drug development and thus support one of the UK's key industries.


Research activity codes: 5.1 PHARMACEUTICALS;5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: GENERIC HEALTH RELEVANCE
Title: Maximising the potential of wild-derived laboratory mouse strains for medical research
Funder: Medical Research Council

Abstract: The 1.5-2M years of genetic divergence between M.m domesticus and the wild derived strains (SPRET/EiJ, PWK/PhJ, CAST/EiJ, and WSB/EiJ) makes it difficult to use standard methods of mapping back to the C57B6/J reference. We have observed 4-8 times more SNPs, short indels and larger structural variants (>100bp) in the wild derived strains so it is not appropriate to use the C57B6/J reference genome as a comparator for these strains. If we consider just protein coding regions of the genome alone, we can see that in Mus spretus just under 3,000 large structural variants occur in these regions and so are likely to result in significantly altered gene models. This problem poses a significant difficulty for any outbred crossing based projects (such the CC and the Diversity Outbred Cross (DO)) where a subset of or interspecific crosses where the founder strains are wild-derived. Without near contiguous sequences assemblies (at least in protein coding regions), we currently cannot accurately predict gene modes and hence predict function of polymorphism. The task of generating accurate whole-genome draft assemblies of eukaryotic genomes from second-generation sequencing data remains challenging. The key ingredients required to generate high quality draft genome sequences of large eukaryotes are high sequencing depth from short fragment (200-600bp) paired-end sequencing libraries, lower depth from a range of large mate-pair fragment libraries (3-40Kbp), and a optical or physical map. This strategy has been used recently to produce several draft genome sequences for the goat, hamster, and gorilla.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Structural and mechanistic understanding of Unfolded Protein Response (UPR) regulation by ER Hsp70 resident chaperone BIP.
Funder: Medical Research Council

Abstract: To investigate the mechanism of activation/regulation of LD domains by BIP and to resolve the different models that have been proposed we aim to map the interaction between the LD domains and BIP by initially using a biophysical approach to discern the specific regions involved in this weak and transient interaction. We also aim to structurally characterize the molecular nature of the interaction and to propose a model of regulation by BIP. Visualizing the molecular details of interaction will allow us to introduce mutations into the site which we can characterize by measuring its effects on the interaction affinity. These mutants would also be valuable reagents to probe further the mechanism of action within the lab and also for the UPR research field as a whole. Objectives 1) Mapping the interaction site of BIP and LD domains. 2) Crystal structure of BIP-LD complex 3) Structure validation using interaction site mutants Technical summary A major effort will be directed towards cloning and over-expressing various protein constructs and purifying these constructs using standard chromatographic techniques in order to obtain homogeneous protein for biochemical and structural purposes. For Protein - protein interaction studies between the LD and BIP constructs we will use pull-down assays, Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST). For structural studies, purified protein will be set up using commercially available screens on the crystallisation robot and optimised using hanging drop crystallisation method again using specialised plates for crystallisation. Crystal diffraction optimisation can be done using the in house X-ray source and data collection will be conducted at the diamond light source. For mutation analysis again requires cloning and purifying protein constructs and then using techniques mentioned in protein-protein interaction studies.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Integrating 3D biological data on scales from molecules to cells
Funder: Medical Research Council

Abstract: In this project we will develop two tools and a new archive that will add considerable value to EMDB data and enable its integration with other structural and bioinformatics resources: a) Volume browser: We will develop a web-accessible tool to provide an integrated view of 3D biological data at the cellular and molecular scales derived from the EMDB and PDB archives, the means to navigate seamlessly between them and to navigate to related bioinformatics resources. b) Segmentation-annotation tool: PDBe has developed a draft EMDB segmentation-file format (EMDB-SFF) that accommodates a range of segmentation representations used in the field as well as the requisite annotation. We will develop a web-based tool that will allow users to fully exploit the capabilities of this format by uploading segmentations in any common format, overlaying segmentations on the 3D volume, adding biological annotation, and then writing out the segmentations in the EMDB-SFF so that it can be included in an EMDB deposition. c) Pilot image archive: Raw image data cannot be deposited in EMDB for a number of practical reasons - the size of single-particle datasets can exceed that of the deposited reconstructions by orders of magnitude, and there is no consensus as to what constitutes "raw" data. We will develop a pilot image archive for 2D image data related to EMDB entries, which will allow us to comprehensively address these issues in consultation with the EM community, provide public access to this data, and consider options for the future integration of the archive with other resources.


Research activity codes: 1.4 METHODOLOGIES AND MEASUREMENTS
Health categories: GENERIC HEALTH RELEVANCE
Title: Software tools and online resources for the self-controlled case series method and its extensions
Funder: Medical Research Council

Abstract: The main objective of the project is to program extensions of the self-controlled case series (SCCS) method in standard statistical software, namely R, STATA and SAS. These extensions broaden the applicability of the basic SCCS method to situations in which key assumptions may be violated. The extensions we plan to program include (a) event-dependent exposures, where the conditional Poisson likelihood framework of the basic method is replaced by one based on estimating equations, with confidence intervals obtained by bootstrapping; (b) events carrying high mortality, resulting in event-dependent observation periods, which requires a 2-stage model incorporating an explicit model for post-event survival; (c) more flexible temporal adjustments including seasonal as well as age effects, and smooth age effects and post-exposure risk functions using splines or kernel smoothing; (d) dependent recurrent events, where the SCCS model can be retained when the dependence is expressed additively rather than multiplicatively on the baseline hazard. We shall also provide software to aid the design and interpretation of SCCS studies, incuding a sample size calculator and graphical tools to plot output. In addition we shall undertake a comprehensive review of how the SCCS method has been used, and of where any problems in its application might lie, so as better to address these in the new online documentation we shall produce. This will include practical data-based examples, instances of what can go wrong if assumptions are violated, and suggestions for sensitivity analyses and diagnostic procedures. These materials will be freely available on a new SCCS website, aimed at epidemiologists and applied statisticians wishing to use and learn about the SCCS method. Past experience suggests that such resources are well used. Our ultimate goal is to increase the quality and quantity of studies using the SCCS method, and hence contribute to better evidence-based medical decisions.


Research activity codes: 8.4 RESEARCH DESIGN AND METHODOLOGIES (HEALTH SERVICES);2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: GENERIC HEALTH RELEVANCE