NIHRDataInsights/HRCSData huggingface dataset:
Title: Dissecting the molecular basis of cell-cell variability in mouse embryonic stem cell populations using systems approaches
Funder: Biotechnology and Biological Sciences Research Council

Abstract: A number of recent studies using single cell gene expression profiling have uncovered a surprising degree of cell-to-cell variability within apparently functionally homogeneous embryonic cell (ESC) populations. In particular, a number of regulators of the pluripotent stem cell identity, including key transcription factors such as Nanog, Rex1, Stella and Klf4, have been found to exhibit significant expression fluctuations at the single cell level. Such fluctuations give rise to robust functional heterogeneity within ESC cell populations, profoundly affecting their long-term regenerative potency. Collectively, these studies indicate a remarkably dynamic view of stem cell identity: individual stem cells are apparently not closely regulated, yet well-defined and robust population-level behaviour emerges from apparently stochastic dynamics at the single cell level. However, the molecular basis for these intracellular fluctuations and their functional consequences for stem cell populations remain unclear. This project aims to take an integrated quantitative approach, employing high-throughput single cell profiling with time-lapse microscopy, image analysis and bioinformatic/mathematical analyses, to dissect further the molecular basis of cell-cell variability and its functional consequences in mouse ESC populations. This is important since a more complete understanding of these mechanisms will advance our understanding of early development. Furthermore, understanding of individual ESC identity and the relationships between single cell identity and population function will facilitate the maintenance of more defined pluripotent populations and the development of more robust differentiation protocols which will be necessary if human pluripotent stem cells are to be used safely and routinely in clinical applications.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Crowding and Complexity: Simulation Studies of Biologically Realistic Membrane Models
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Cell membranes are crowded environments, composed of up to ca. 50% protein by mass corresponding to a membrane area fraction (theta) of ca. 25% or more occupied by proteins. In addition to crowding per se, the spatial and compositional complexities of membranes may result in the formation of membrane protein nano-clusters. However, detailed molecular simulations of crowded membrane systems have been limited. Given advances in high performance computing, it is timely to extend the scale and complexity of molecular simulations in order to understand the organization and dynamics of proteins and lipids in biologically realistic models of crowded and complex membrane systems The overall aim of this proposal is to characterise dynamic properties of crowded and complex biomembranes using multiscale molecular dynamics (MD) simulations. In particular we will examine these properties in terms of their implications for our understanding of drug/receptor interactions, and of membranes for use in synthetic metabolons. We will building upon our preliminary studies to develop biologically realistic membrane models composed of complex mixtures of lipids. These complex lipid bilayers will be combined with multiple membrane proteins to yield crowded-complex membrane systems Quantification of protein-protein and protein-lipid-protein interactions in these crowded systems will yield free energy landscapes for biologically realistic membranes. These free energy landscapes will be used to parameterize ultra-coarse-grained models based on hybrid Brownian Dynamics/Monte Carlo approaches which will allow us to simulate emergent properties of very large (1000s of proteins) planar and vesicular membrane systems The resultant membrane models and simulation methodologies will be used to explore two key applications: (i) receptor/ligand interactions and drug efficacy; and (ii) crowding effects in membrane-based synthetic metabolons.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Delivering ELIXIR-UK
Funder: Biotechnology and Biological Sciences Research Council

Abstract: ELIXIR is a project that is designed to construct and operate a sustainable infrastructure for biological information in Europe. Owing to the dearth of individuals who can take advantage of large biological data sets, we have decided to focus the UK's ELIXIR node on training. This is the century of biology, and genomics, transcriptomics, proteomics and metabolomics ('omics and bioinformatics) lie at its heart. The computer is an essential tool and now drives many critical aspects of the scientific process hence scientists need to know how to gain most advantage from it. Despite the central role of computing in science today: - Most research scientists are ill-equipped to take advantage of the data 'bonanza' that is emanating from new technologies. Critical training needs across the community are, broadly, in the analysis and the interpretation of genome-scale data of many types, including next generation sequencing (NGS) data. Genomics is the area of greatest training need as it lies at the intersection of many fast-moving research disciplines. - Many infrastructure technologists (career bioinformaticians) are ill-equipped to offer scalable, sustainable solutions for managing and interpreting data arising from this flood of technological advances. Critical training needs are in techniques and practices for data management, integration, compute and software, tools, services and standards. The ELIXIR-UK Core Organisations are: the Universities of Oxford, Manchester, Liverpool, Birmingham, and Edinburgh; the NERC Environmental Omics Synthesis Centre; the Wellcome Trust Sanger Institute; the Genome Analysis Centre; and, University College London. Additional participating organisations are: the Software Sustainability Institute UK; the Hartree Centre; and the University of Dundee. Other organisations (UK and ELIXIR-wide) will be incorporated to provision training.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);8.5 RESOURCES AND INFRASTRUCTURE (HEALTH SERVICES);8.4 RESEARCH DESIGN AND METHODOLOGIES (HEALTH SERVICES);2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: GENERIC HEALTH RELEVANCE
Title: Molecular and cellular mechanisms of selective autophagy and their relevance to ageing
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Ageing is associated with the lifelong, gradual accumulation of molecular and cellular damage and this has been observed in species ranging from yeast to humans. One of the phenotypic hallmarks of ageing cells is the intracellular accumulation of damaged proteins and organelles. Autophagy is an evolutionarily conserved lysosomal, self-degradation process. It is involved in protein and organelle degradation and plays an important role in both cellular and whole-organism homeostasis. Recent evidence indicates that autophagic activity declines with age and this gradual reduction of autophagy plays a causative role in the functional impairment of biological systems during ageing. Although it was initially believed that autophagy occurs randomly inside the cell, recently there has been growing evidence that sequestration and degradation of cytoplasmic material by autophagy can be selectively mediated through receptor and adaptor proteins. To understand cellular ageing, it is therefore important to identify the proteins required for recognition and targeting of the various autophagic cargos for degradation and to elucidate the molecular links between selective autophagy of damaged proteins and organelles and the regulation of ageing at the organismal level. We will use drosophila melanogaster as a genetically modifiable model organism The first aim of this project is to understand the role of Ref(2)P (the drosophila homologue of mammalian p62) during ageing. The second aim is to identify novel selective autophagy receptors and adaptors in drosophila and examine their role during ageing. We will use a multidisciplinary approach by combining genetic, biochemical, bioinformatics, imaging and behavioral analysis. Using this approach we expect to identify novel mechanisms via which selective autophagy regulates ageing, and to elucidate the molecular details of selective autophagy in the context of cell and tissue physiology.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: DNA damage induced phosphorylation and regulation of NF-kappaB
Funder: Biotechnology and Biological Sciences Research Council

Abstract: The NF-kB transcription factors play essential roles in the physiology and stress responses of multicellular organisms Although NF-kB activation is frequently associated with inflammation and the immune response, it also plays a critical role in the cellular response to DNA damage. This is most often studied in the context of cancer, where its activation can either inhibit or promote the effect of many chemotherapeutic drugs. However, this is also a physiologically important process, relevant to the functions of NF-kB associated with inflammation and aging, where, for example, the production of reactive oxygen species can result in DNA damage. While much has been done to start to unravel the 'nuclear-to cytoplasmic' signalling of NF-kB in response to DNA damage, it remains to be defined how the transcriptional functions of the different NF-kB dimers is specifically regulated by these types of stimuli. Numerous papers from the Perkins lab and others have demonstrated that DNA damage-induced NF-kB is functionally distinct to that found following exposure to inflammatory cytokines or engagement of Toll-like receptors. These different effects appear to be mediated, to a significant extent, by differential post-translational modifications (PTMs) of NF-kB subunits. This proposal aims to exploit state-of-the art mass spectrometric techniques to investigate DNA-damage-induced changes in post-translational modification status of the NF-kB transcription factors. Furthermore, we will elucidate how these PTMs are regulated over time, discern mechanism of regulation and evaluate how changes in these modification fingerprints correlate with NF-kB activity and transcriptional function. The experiments performed here will provide a template for future analysis to investigate other aspects of NF-kB signalling (different stimuli, context and activation pathways) while also serving more generally as a relevant example of a stimulus responsive signalling and transcription network.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: New insights into the function of the protein kinase DYRK1B, an ERK1/2 target gene
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Protein kinase signalling pathways coordinate key cell fate decisions such as cell survival, proliferation, differentiation and senescence. DYRK1B is a member of the dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs), a highly conserved family of protein kinases found within the CMGC (CDK, MAPK, GSK and CLK) group of the eukaryote kinome. However, in contrast to the CDKs and MAPKs, the normal biological functions of DYRK1B (and other DYRKs) are poorly understood. DYRK1B is inducibly expressed during myogenesis and adipogenesis and upon inhibition of the ERK1/2 signalling pathway (which causes a G1 cell cycle arrest). However, few substrates of DYRK1B have been defined that might help to place it in a biological context. We have recently collaborated with AstraZeneca in characterising a new DYRK1B-selective inhibitor, AZ191, and have used this to validate the results of a SILAC experiment to identify new DYRK1B-inducible phosphoproteins. This analysis has identified several proteins involved in mRNA degradation (e.g., Dcp1a & 1b, Edc3 & Pat1b) and translational repression (e.g. 4E-T). For two of these (Dcp1a and 4E-T) we have confirmed that they are indeed DYRK1B substrates. These proteins are all found at discrete foci within the cell called Processing Bodies (PBs); these are sites of mRNA degradation and their abundance increases upon cell stress. We find that DYRK1B co-localises with Dcp1a at processing bodies and inducible DYRK1B expression is sufficient to increase PB abundance in the absence of stress. We suggest that DYRK1B is a novel regulator of mRNA processing. In addition, we suggest that DYRK1B is part of a signalling pathway controlling mRNA processing during ERK1/2 inhibition (G1 cell cycle arrest) and myogenesis (cell cycle arrest & differentiation). In this proposal we will test these hypotheses, anticipating that we will define a new signalling pathway controlling mRNA processing.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Developing RNA aptamers as single cell biosensors for mammalian cells
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Somatic mammalian cells have limited proliferative potential, entering a state of replicative senescence after ~100 cell divisions. As cells age, increasing metabolic stress correlates with a functional decline and gradual accumulation of macromolecular damage. This process is typified by changes in the concentration of reactive oxygen species as a result of changes in mitochondrial efficacy and this type of endogenous metabolic stress results in, for example, protein misfolding and damage to DNA. As a consequence of the unpredictable and stochastic nature of metabolic decline individual cells age at different rates and accumulate unpredictable patterns of age-related defects. In order to explore the process of metabolic decline during ageing at a single cell level, and so develop detailed insights into the cellular heterogeneity associated with metabolic decline, we propose to develop single cell biosensors that will allow quantification of dNTP pool concentrations in single cells. dNTP metabolism is regulated during different cell cycle phase and different states of growth arrest to ensure that sufficient concentrations of precursor are available to support S phase that occupies a strictly controlled 9-10h of the cell cycle. If pool size is severely perturbed replication is blocked. Drug-induced reduction of the pools leads to alteration in the replication fork rate, increased origin density and often correlates with defects in completion of replication, with associated mitotic defects, which result in cell death or aneuploidy. In addition, metabolic imbalance in dNTP pools leads to increased DNA mutation, altered telomere homeostatis and a range of disease phenotypes. In collaboration with Prof. Samie Jaffrey, who developed the original single cell biosensor system for bacteria, and AptaSol, a commercial Aptamer-based company, we will develop biosensor for analysis of replciation precursor pools in single human cells


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: The Spatiotemporal Regulation of the Keap1/Nrf2 pathway
Funder: Biotechnology and Biological Sciences Research Council

Abstract: The transcription factor Nrf2 regulates the expression of more than 1% of the human genome, including detoxification, anti-inflammatory and antioxidant cytoprotective genes. Nrf2 dysregulation is causally-associated with the development of chronic diseases and ageing, and Nrf2 is considered a drug target for disease prevention and healthy ageing. Nrf2 is normally targeted for degradation by its major negative regulator, Keap1. In response to electrophiles and oxidants (termed inducers), which modify specific cysteines within Keap1, Nrf2 is stabilized, accumulates in the nucleus, and activates transcription of cytoprotective genes. Recently, using live cell imaging, we discovered that the Keap1 : Nrf2 interaction follows a cycle whereby the complex sequentially adopts two distinct conformations: "open", in which Nrf2 interacts with one molecule of Keap1, followed by "closed", in which Nrf2 binds to both members of the Keap1 dimer and allows Nrf2 ubiquitination. This project will test the hypothesis that the conformational cycling of the Keap1 : Nrf2 protein complex endows the Keap1/Nrf2-mediated cytoprotective response with regulatory flexibility with respect to both small molecule inducers as well as protein regulators. Specifically, we will determine the effect on the conformational cycling of the Keap1 : Nrf2 complex of: (i) the cysteines of Keap1 which are most commonly modified by electrophiles, (ii) non-electrophilic inducers which directly disrupt the interaction between Keap1 and Nrf2, and (iii) modulating the levels of p21 and p62, two critical protein regulators of Nrf2 activity. Together, these experiments will provide comprehensive understanding of the mechanisms controlling the interaction between Keap1 and Nrf2, leading to new strategies for targeting Nrf2 for life-long health and wellbeing.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Novel BRET approaches to unravel the molecular pharmacology of VEGFR2 receptors: Insights into ligand binding, allosterism and signalling bias
Funder: Biotechnology and Biological Sciences Research Council

Abstract: The novel bioluminescent protein NanoLuc will be fused to the N-terminus of VEGFR2 or its co-receptor neuropilin-1 (NRP1) to provide the basis for a unique and exquisitly sensitive live cell binding assay utilizing bioluminescence energy transfer (BRET) to monitor the direct binding of fluorescent VEGFR isoforms Competition binding between fluorescent VEGF (VEGF-165 or VEGF-121; red fluorophores) and non-labelled VEGF isoforms will allow a detailed molecular pharmacology (in terms of binding affinity) to be deduced for the full VEGF isoform family. Fluorescent VEGF-121 will be used as a specific probe for VEGFR2 since it does not bind to NRP1. A non-VEGF binding mutant of NRP1 (Y297A) will be used to define non-specific binding to NRP1. Co-expression of wild-type NRP1 and NanoLuc-VEGFR2 will also be used to evaluate the impact of NRP1 on the ligand binding characteristics of fluorescent VEGF-121 and the consequent intracellular signalling (PI3K, ERK, Calcium, NFAT or SRE reporter genes). N- or C-terminal NanoLuc VEGFR2 constructs will also be used in combination with N- or C-terminal HaloTag (red fluorophore) fusions of VEGFR2, VEGFR1, VEGFR2 or NRP1 to evaluate the ability of specific VEGF isoforms to induce the formation of VEGFR2 homodimers, heterodimers or VEGFR2-NRP1 protein complexes. The BRET technology will also be used to monitor the impact of co-expression with wild-type VEGFR1, VEGFR3 and NRP1 on the ability of Halo-tagged signalling and adapter proteins to bind to C-terminal NLuc VEGFR2 in response to different VEGF isoforms This will provide some insight into the propensity of different VEGFR2-protein complexes to induce signalling bias. However, NanoLuc bimolecular luminescence complementation will also be used in combination with fluorescent ligands or fluorescent signalling proteins to evaluate directing by BRET VEGF binding, efficacy and signalling bias from identified VEGFR2 protein complexes (homodimers, heterodimers, NRP1 complexes).


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Overactivity in growth and its effects on rates of ageing
Funder: Biotechnology and Biological Sciences Research Council

Abstract: The comparison of organisms that have naturally different ageing rates may lead to new insights into the causes of ageing. This research will make use of a nematode that shows plasticity in ageing. This nematode, referred by its strain name SB347, produces hermaphrodites that live a third longer than females. The advantage of this system is that we can convert one gender into another, thus being able to disentangle developmental trajectory and mode of reproduction from lifespan. SB347 has many of the features that made C. elegans a prominent model in ageing research: it can be easily cultured in the laboratory, has a short lifespan, and rapid reproduction with large progeny size. In our first aim, we will contrast the ageing process between hermaphrodites and females, and tease apart the possible developmental and physiological causes underlying the differences in ageing process. We will use microscopy and loss-of-function mutations to characterize the morphological and physiological changes that occur during ageing. In this aim, we will also investigate if the ageing process is compatible with the theory that ageing is the result of hyperfunction of developmental processes. We will study in details three types of changes that occur during ageing and that are related to reproduction (yolk, oocyte and pheromone production). In the second aim, we will test if the different modes of reproduction and different rates of calorie intake can explain differences in lifespan and ageing between the two genders. We will perform RT-qPCR, laser microsurgery and diet manipulations to investigate the role of the reproductive mode and diet in the lifespan.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Life-long telomere dynamics, health and fitness in a long-lived mammal
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Telomere attrition is thought to play an important role in the ageing process, and leukocyte telomere length (LTL) measured in adult humans predicts various age-related diseases. We will use an exceptionally detailed longitudinal study of unmanaged Soay sheep on St Kilda, in which around 3,000 individuals have been closely monitored and regularly blood sampled all the way from birth to death. Importantly, sheep have a similar telomere biology to humans and Soay sheep live long, natural lives on St Kilda and show clear signs of ageing in later life. We will use a high-throughput qPCR-based system to measure average LTL from >8000 samples collected from these individuals, and use this data to test how genes, parental and early-life environment effects influence LTL at birth and telomere attrition rates thereafter, and determine how this, in turn, influences adult health and lifespan. We will also continue our annual fieldwork programme over the course of the grant to collect samples suitable for more detailed, high-resolution telomeric and immunological assays. We will develop the high-resolution STELA technique for use in sheep and use it to compare the importance of longitudinal changes in average LTL versus the presence of critically short telomeres in predicting ill-health and mortality in adulthood. We will also use flow cytometry and cell proliferation assays to test how such changes are associated with changes in composition of leukocytes and their proliferative potential, and how these in turn are associated with an individual's ability to resist infection. Taken together, this will provide the most complete study to date of the causes and consequences of life-life variation in LTL in a long-lived mammal.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE