NIHRDataInsights/HRCSData huggingface dataset:
Title: Assembly of human ATP synthase
Funder: Medical Research Council

Abstract: We will use CRISPR-Cas9 to disrupt nuclear encoded subunits of ATP synthase in human cells, and study the effects on the functioning of the ATP synthase and other components of the mitochondrial respiratory chain in a Seahorse XF analyzer. We will also study the impact on the assembly of the ATP synthase itself, and characterize the subunit compositions of the vestigial partially assembled ATP synthase complexes in mitochondria (or mitoplasts) and in purified vestigial complexes by SDS-PAGE, blue native- and clear native-gel electrophoresis, combined with mass spectrometric analyses of tryptic and chymotryptic digests of stained gel regions and protein bands. In addition, we will study the compositions of vestigial complexes by quantitative mass spectrometry of samples labelled in vivo with heavy isotopes (known as stable isotope labelling in cell culture, or SILAC). This approach allows not only the subunit compositions of vestigial complexes to be studied, but also detects other associated proteins, which can then be tested for their being assembly factors by disrupting their genes by CRISPR-Cas9. Where necessary (for example when there are no protein specific antibodies for purifying remaining vestigial complexes), we will introduce tagged subunits into subunit depleted HEK293 Flp-InTM T-RexTM cells by stably incorporating DNA encoding the tagged constructs into the Flp-In site, and induce controlled expression with doxycycline. We will also reintroduce mutated forms of IF1 into cell lines where the gene for IF1 has been disrupted, and characterize their effects by respiration measurements with a Seahorse instrument and by advanced light microscopy. With access to four confocal microscopes (Zeiss LSM880 and Apotome, Andor Dragonfly spinning disk and Nikon N-SIM instruments) we will be able to quantify changes in mitochondrial morphology e.g. branching or fragmentation, and with the N-SIM instrument visualise significant changes to the cristae ultrastructure.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: METABOLIC AND ENDOCRINE
Title: Novel non-invasive assessment of de novo lipogenesis and its mechanistic role in human insulin resistance
Funder: Medical Research Council

Abstract: Background Insulin resistance links obesity to type 2 diabetes and accounts for almost all of the associated comorbidities such as non-alcoholic fatty liver and cardiovascular disease. Insulin resistance is closely associated with an accumulation of lipid stored ectopically in liver and skeletal muscle. Emerging evidence suggests de novo lipogenesis (DNL), the generation of lipid from non-lipid sources, is involved in the pathogenesis of insulin resistance. There are no non-invasive ways to measure the storage of DNL-derived lipid. Existing methods require a biopsy sample which is a procedure associated with substantial risk, and this has significantly limited its investigation. Aim To develop a non-invasive method to measure the storage of DNL-derived lipids, and to use this to safely examine the relevance of this lipid storage in human insulin resistance. Approach Magnetic Resonance Spectroscopy (MRS) is a completely non-invasive technique that can provide biochemical information from within tissues in vivo. I plan to devise and validate a novel MRS method that will safely and completely non-invasively measure the incorporation of DNL-derived lipid into stored lipid pools. Using unique cohort insulin resistant patients and matched controls, we shall utilise this new method to investigate the role of DNL-stored lipids in the mechanisms of insulin resistance. Importance Understanding the pathogenesis of insulin resistance will provide new therapeutic targets for early intervention in a number of diseases linked to the metabolic syndrome, when mechanisms may still be reversible. The novel methodology developed also has the potential to provide a non-invasive biomarker for early detection and/or monitoring response to therapy.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: METABOLIC AND ENDOCRINE
Title: STRESS recovery
Funder: Medical Research Council

Abstract: Chronic stress (CS) is a major risk factor for development of a wide range of human disorders including diabetes, obesity, cardiovascular function as well as neurological and behavioural deficits. Despite the impact of CS on health, its long-term effect on hypothalamic-pituitary-adrenal (HPA) axis function and how this relates to the resilience or susceptibility to the development of disorders is poorly understood. The anterior pituitary corticotroph has been proposed as a major locus for CS-induced changes in HPA axis function, however mechanisms are not defined. Remarkably, we have recently identified that cessation of an episode of CS dramatically invokes a persistent change to corticotroph physiology with unpredictable consequences for ACTH output and HPA activity. RNAseq analysis revealed a surprising paucity of changes in corticotroph gene transcription during CS but a profound downregulation of gene transcription persisting up to 4 weeks post CS. Importantly, this shows that CS leads to more dramatic alteration of the HPA axis at the level of the pituitary than previously recognised. To address whether these persistent changes in corticotroph function alter HPA axis regulation and response to further stress, we will determine the consequences of the long-term alteration of corticotroph function following CS. We will integrate functional studies in vitro and in vivo and interrogate molecular pathways defined by our RNA-Seq and modelling approaches. Importantly, our ability for the first time to measure corticotroph physiology in real-time in vivo will allow longitudinal monitoring of axis function before, during and after CS in the same animals, and define whether intervention in vivo can alter or reset the changes caused by CS. Taken together we aim to unravel the molecular mechanisms by which alterations at the level of the pituitary corticotrophs underlie susceptibility to dysfunction and resilience following CS and potential targets for therapy


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: BUD23 drives system-wide adaptations to energy metabolism
Funder: Medical Research Council

Abstract: BUD23 as an important regulator of systemic metabolism, with selective deletion of BUD23 from adipose tissue driving a reprogramming of energy expenditure and storage with a resultant lean phenotype. We will now further characterise the AdipoCREBud23FL/FL mice, and extend these studies to include BAT-specific targeting (via Ucp1CRE) to delineate white and brown adipose contributions. This will be important in defining the broader impact of Bud23-dependent alteration in BAT activity and thermogenesis on systemic energy expenditure and adiposity. We also include a tamoxifen inducible hepatocyte-specific BUD23 null mouse line (via AlbcreERTBud23FL/FL). As the mechanism of action of BUD23 is through differential translation of mRNA species it is essential to take a global view of the tissue proteome. We did this for heart, and it was highly revealing, identifying a role in mitochondrial function. Here, we will characterise in detail mitochondrial number, structure and activity in each of our tissues of interest. We will also interrogate tissue metabolic activity using the Oroboros technology, which allows for high-resolution respirometry with monitoring of O2 consumption, ROS production (AmR), and mt-membrane potential in small tissue samples or isolated mitochondria. We will also use Seahorse technology to study intact cells in culture. We will investigate de-novo lipogenesis (DNL), and fatty acid content of adipose beds, and liver will be determined post-mortem. In order to identify candidate mRNA targets, we shall apply ribosome profiling (ribo-seq) to generate quantitative, transcriptome-wide and high-resolution maps of the tissue translatomes across genotypes. Quantifying footprint vs. RNA abundances (RNA-seq data is always produced in parallel from the samples) allows calculating relative translation efficiencies transcriptome-wide.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: Kynurenine-3-monooxygenase inhibition as a novel therapeutic strategy to enhance islet cell viability and improve outcomes of Islet Transplantation
Funder: Medical Research Council

Abstract: Severe hypoglycaemic unawareness is a life-threatening and debilitating manifestation of diabetes mellitus for which pancreatic islet cell transplantation is the treatment of choice. Because islet cells are very susceptible to hypoxia, the viability of isolated islets is significantly impaired and pancreases with prolonged cold ischaemia are often discarded. There is therefore a pressing and urgent unmet need to discover novel therapeutics that improve islet cell yield, viability, and function. This project proposes to evaluate a new class of small molecule enzyme inhibitors that target kynurenine monooxygenase (KMO), a mitochondrial outer membrane protein and critical gatekeeper in the kynurenine pathway of tryptophan metabolism that regulates inflammation and immunity. KMO inhibitors have been shown to protect against systemic inflammation and organ failure in experimental pancreatic inflammation. I aim to evaluate the efficacy of KMO inhibitors to prevent organ injury during hypothermic oxygenated machine perfusion of the pancreas. Donor human pancreases that are not being transplanted (usually due to capsular damage) will be used to assess the efficacy of KMO inhibition to reduce pancreas inflammation and tissue injury during whole organ perfusion preservation, and measure the effect of KMO inhibitors on islet cell isolation yield, viability and function. This Clinical Academic Research Partnership is built on the strength of both partners. Andrew Sutherland, is Clinical Lead for Pancreas Transplant in Scotland, and has strong clinical and research background in hypoxia research and organ perfusion technologies. Professor Damian Mole is an MRC Senior Clinical Fellow, whose laboratory has focused on KMO inhibition as a therapeutic strategy in systemic inflammation and in the protection of organ injury for 15 years, and who has co-developed a portfolio of best-in-class KMO inhibitors with GSK through a Discovery Partnership with Academia.


Research activity codes: 5.4 SURGERY
Health categories: METABOLIC AND ENDOCRINE
Title: HDHL MICA: Microbiota-Inflammation-Brain axis in heart failure ... (AMBROSIA)
Funder: Medical Research Council

Abstract: This transnational project aims to tackle undernutrition in a well-defined "older" population (i.e. heart failure and atrial fibrillation patients). Undernutrition in this patient group is a key factor leading to chronic inflammation, loss of functionality, disability and reduced life expectancy. Undernourished heart failure and atrial fibrillation patients enter a vicious cycle of poor health that may exacerbate reduced dietary intake, resulting in loss of lean body mass and cognitive function. A multipronged approach to increase nutrient status would be expected to positively impact on disease progression and other health parameters. The current project will assess the impact of dietary counselling and provision of a novel fortified food on disease status in this patient group by running a monocentric, parallel, randomised controlled trial. The first stages of this project will be to design a novel fortified food product (the AMBROSIA bar) aimed at benefiting energy, protein and targeted nutrient intake in this patient group. The primary outcome of interventions (either diet and lifestyle counselling alone or with the addition of the AMBROSIA bar) will be change in skeletal muscle mass. Improvements in dietary habit and markers of disease status, cognitive, emotional and functional status will also be measured. Finally, microbiomic, lipidomic and metabolomic analysis will be carried out on saliva, blood, urine and faecal samples collected during the randomised, controlled trial. Outcomes of these analyses will be screened via a novel, cutting-edge machine-learning platform to aid discovery of novel markers or correlates of disease, frailty, nutrient uptake and nutritional status. The UK arm of this EU-wide collaboration (which includes partners organisations across 4 countries) will focus on dietary data (using novel diet quality scoring methods) and metabolomic analyses (using high resolution mass spectrometry).


Research activity codes: 6.8 COMPLEMENTARY
Health categories: METABOLIC AND ENDOCRINE
Title: The role of DNA methylation in stratifying and understanding paediatric nephrotic syndrome - a combined bioinformatics and CRISPR-cas9 approach
Funder: Medical Research Council

Abstract: Aims: I plan to create a DNA methylation (DNAm) based tool for classifying nephrotic syndrome (NS) patients into 4 clinically useful subgroups. I aim to unveil DNAm based NS disease pathways and test these in functional laboratory work. Objectives: 1. Use peripheral blood cell DNAm profiles to establish segregation of 4 postulated NS subgroups. 2. For each NS subgroup, examine whether differences in DNAm are likely to be disease causing. 3. Determine whether modifying DNAm at candidate sites alters gene expression in lymphocyte cell lines. Methods: 1. I will use DNA extracted from peripheral blood cells of 280 NS patients from the 4 subgroups recruited NURTuRE-NephroS. DNAm data at 850,000 CpG sites will be generated using microarrays. I will use elastic net to create DNAm based subgroup classification models. I will use cross-validation when testing model performance. 2. I will compare DNAm profiles between the 4 NS subgroups using linear regression (adjusted for potential confounders). CpG sites of differing DNAm will be annotated to genes. DNAm and genetic data for each patient will be used in causal inference analyses (Mendelian Randomisation and mediation analyses). Using these results, 5 CpGs of interest which exhibit altered DNAm and are more likely to be on the causal pathway will be selected. CpGs that annotate to genes with a biologically plausible link to NS and that are expressed in lymphocytes will be prioritised. 3. I will utilise CRISPR to perform targeted DNAm editing at each of the 5 candidate CpGs in T and B lymphocyte cell lines. I will compare DNAm, gene expression and protein levels between the edited and unedited lymphocytes. Opportunities: A novel tool for accurate NS subgroup classification would allow children to avoid useless and potentially harmful treatments, whilst also enabling stratified clinical trial design. Uncovering novel disease pathways in each subgroup could lead to innovative treatments which target DNAm.


Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: METABOLIC AND ENDOCRINE
Title: Determining how glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP1) synergistically regulate beta cell function
Funder: Medical Research Council

Abstract: Background: Developing novel therapeutics to treat diabetes is crucial in the setting of epidemic prevalence and devastating, costly complications. The incretin axis has proven to be a key therapeutic target due to remarkable effects upon weight and glycaemic control. GLP1 receptor agonists (GLP1RA) are in widespread clinical use but GIP receptor agonism (GIPRA) has only recently gained traction as a therapy, and only in conjunction with GLP1RA as a dual agonist. The mechanisms behind the profound synergism of GIPRA with GLP1RA therapy is unclear but critical to further manipulation of the incretin pathway to help patients with Type 2 diabetes or obesity. Objective: Utilise advanced imaging and chemical biology to confirm mechanisms by which GIP and GLP1 synergistically influence beta cell signalling (Ca2+, ATP/ADP and cAMP) and function (insulin secretion), receptor expression levels and trafficking. Aim 1: Determine the impact of GIPR + GLP1R activation upon human beta cell stimulus-secretion coupling through a) measurement of insulin release using a perifusion system combined with highly sensitive HTRF assays; b) multicellular Ca2+ imaging and c) imaging of ATP and cAMP signalling. Aim 2: Assess the effects of GIP + GLP1 co-administration on GIPR and GLP1R expression/localisation and beta cell differentiation through QPCR and immuno-staining cryostat sections of fixed-embedded human islets. This will also require the development of a fluorescent GIPR chemical probe, since specific antibodies do not exist and I will contribute to probe development and validation. Aim 3: Compare GLPR and GIPR trafficking in response to activation by their cognate agonists and GIP + GLP1 through confocal and super-resolution imaging to understand receptor dynamics at the cell surface. Further staining with early/late endosomal or lysosomal markers will allow me to understand the propensity for recycling and degradation.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: Improving beta cell function by mesenchymal stromal cells: novel mechanisms and cell-free translational potential
Funder: Medical Research Council

Abstract: Allogeneic islet transplantation is currently used to treat a subgroup of patients with type 1 Diabetes (T1D), but current clinical protocols are inefficient with considerable loss of graft function soon after transplantation. Mesenchymal stromal cells (MSCs) improve beta-cell function and the outcomes of experimental islet transplantation but current clinical transplantation protocols do not enable co-transplantation of islets with MSCs, so we are focusing on harnessing the beneficial effects of MSCs in "cell-free" protocols. We have demonstrated that some of the effects of MSCs on islet function are mediated by soluble mediators acting via G-protein coupled receptors and that pre-treatment of islets with cocktails of MSC-derived molecules is alone sufficient to improving islet graft functional survival. We will now extend these studies to investigate the beneficial effects of MSC-derived extracellular vesicles (EVs) on islet function in vitro and on the ability of islet grafts to maintain glucose homeostasis in a mouse model of T1D. Beta-cell secretory function is dependent on mitochondrial metabolism and we have recently reported the transfer of mitochondria from MSCs to adjacent, compromised islet beta-cells. We will use combined light and electron microscopy to investigate the mechanism(s) of mitochondrial transfer (tunnelling nanotubes, EVs) and determine the functional consequences of mitochondrial transfer to beta-cells. The intended outcome of this project is to inform defined "cell-free" modifications to human islet transplantation protocols. The use of a "MSC-free" approach will avoid many of the technical hurdles associated with cellular therapy, such as upscaling MSC production whilst maintaining a stable phenotype, quality control between different MSC populations, the establishment of reliable safety and efficacy profiles, and the challenges of in vitro co-culture of MSCs and human islets on the scale required for clinical transplantation.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: METABOLIC AND ENDOCRINE
Title: Genomic Imprinting and the regulation of postnatal nutritional resources via breastmilk
Funder: Medical Research Council

Abstract: Genomic imprinting is an epigenetic process causing genes to be expressed monoallelically according to their parental origin. It is well established that imprinted genes have major prenatal effects on growth, development and placental physiology. Recent evidence suggests that imprinted genes play important roles postnatally as well. In human, defective imprinting causes a series of congenital diseases with overlapping clinical features including abnormal growth and metabolism and is associated with obesity, diabetes and cancer. Epidemiological studies indicate that some of these diseases are also associated with early postnatal nutrition; specifically, that breastfeeding is protective against early postnatal morbidity and is also beneficial against obesity, diabetes and cancer. Yet, the mechanisms underlying the links between early nutrition and the origins of health and disease remain unknown. Based on our preliminary data, we hypothesise that imprinting, also extends to postnatal nutritional control via the milk and that imprinting products secreted into the milk are transferred to the offspring and modulate growth, development and metabolism. We propose a series of experiments addressing key biomedically relevant topics concerning the presence, origin and function of imprinted proteins in milk. These studies are designed not only to better understand mechanisms of early nutrition, but also to provide major insights into the physiological significance, function and evolution of genomic imprinting. We aim to: 1 - To quantify and characterise imprinted proteins and their temporal dynamic in mammalian milk over lactation. 2 - To associate the presence of imprinting proteins with normal mammary cell transcriptomes and explore the relationship between them and milk RNA. 3 - To study the physiological significance of imprinted proteins in milk using the mouse as a model, how do they affect the offspring's growth, development and long-term health.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: METABOLIC AND ENDOCRINE
Title: Developing a decision support tool to enable precision treatment of type 2 diabetes
Funder: Medical Research Council

Abstract: In Type 2 diabetes (T2D) the many different classes of glucose-lowering therapies have an approximately similar efficacy overall and there is huge variation in prescribing patterns between doctors. We have shown in our previous MRC funded collaboration that the clinical characteristics of people with T2D are associated with altered glucose response, which allowed us to build a rational "precision" approach to match an individual to the most effective drug, by integrating routine features in prediction models. Drug decisions are complex and involve tolerability, safety and the impact of co-morbidities as well as efficacy in lowering glucose. This project will use routine clinical and individual trial data from over 1 million people with T2D to create validated integrated models that inform all these aspects of prescribing. WP1: Best drug for glycaemia. We will extend and test our validated model for glucose lowering response so that it predicts glycaemic response to all non-insulin therapies used after Metformin. The model will then be optimised to work in all major UK ethnic groups. WP2: Beyond glycaemia. We will create a model that predicts likely tolerability for a person with T2D for all therapies. We will integrate limitations to the therapeutic choice based on safety (e.g. contra-indications) and co-morbidities (e.g. if cardiovascular disease, suggest drugs with non-glycaemic cardiovascular benefit). WP3: Looking ahead. Utilising genetics and novel biomarkers. We will examine the improvement in prediction by incorporating cutting-edge technologies such as genetics, novel biomarkers and data-driven sub-classification models. WP4: Integrated tool ready for clinical use. All the inputs and outputs of the model will be integrated and put into an appropriate format for intended users. Working with people with T2D, nurses, and GPs, we will work up visualisations of outputs and create an online calculator/app version of the treatment support tool.


Research activity codes: 5.9 RESOURCES AND INFRASTRUCTURE (TREATMENT DEVELOPMENT)
Health categories: METABOLIC AND ENDOCRINE