NIHRDataInsights/HRCSData huggingface dataset:
Title: The roles of PACT and TRBP in microRNA processing and viral sensings
Funder: Wellcome Trust

Abstract: The homologous proteins PACT and TRBP play roles in microRNA biogenesis and innate viral response, through their interactions with DICER, RIG-I and PKR. There is little structural information available about either PACT or TRBP, which has led to their functions remaining poorly understood. This project hastwo aims: to determine how PACT and TRBP bind DICER and RIG-I at the molecularlevel; and to characterise the function of these interactions in microRNA biogenesis and the interferon response to viral double stranded RNA. To achieve this, I will identify residues involved in the interaction surfaces using NMR spectroscopy, characterise the thermodynamics of binding, determine whether binding is affected by phosphorylation, and to study the functional consequences for human cells. In the context of RNA silencing, this work couldhelp develop structural models of the DICER and RISC-loading complexes. In relation to viral sensing, findings from this project would contribute towardsunderstanding how RIG-I distinguishes between viral and self RNA. Importantly,these studies will determine at the molecular level how RNA silencing and viral sensing are concurrently controlled by TRBP and PACT during an immune response


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: A Translational Engine for Biomedical Discoveries .
Funder: Wellcome Trust

Abstract: The Division of Biological Chemistry and Drug Discovery (BCDD) is aiming to be a hub for early phase drug discovery at the national and international level. We aim to develop chemical tools to help understand emerging and complex biology and where possible to develop these chemical tools to yield new drug candidates for human and animal health. In order to carry out this objective we need basic infrastructure to support our on-going research; specifically high performance computing to manage com plex computer networks, protein production to allow us to rapidly obtain initial data on new biology, access to scientific databases, a compound manager to facilitate interactions with collaborators and funding for scientific networking to develop interactions. This infrastructure is not provided by the normal grant funding mechanisms We have particular focuses in neglected diseases and bacterial and fungal infections and novel targets emerging from fundamental biology studies funded by the Wel lcome Trust and others in the UK University sector.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Mass spectrometry infrastructure underpinning research into the molecular basis and biological mechanisms in health and disease
Funder: Wellcome Trust

Abstract: Funding of £715k is requested as a 50% contribution to the purchase of three mass spectrometers in Leeds: 1) Orbitrap Q-Exactive Plus-EMR (collaboration with Thermo Scientific; high-mass modified for membrane protein/lipid studies and top-down protein characterization); 2) Tofwerk high-resolution ion-mobility platform, allowing acquisition of unprecedented conformational detail of dynamic protein structure and ligand interactions; 3) Orbitrap Q-Exactive Plus for providing a major upgrade to the proteomics capabilities. The instruments will include associated nano-LC systems, software, processors and extended maintenance contracts. This equipment will revolutionise current capabilities in biomolecular characterization and quantification using mass spectrometry (MS)-based technologies, impacting on translational medicine through mechanistic studies, biomarker discovery and drug development. The new state-of-the-art platforms will extend analysis capabilities in membrane protein structure, “top-down” analysis of post-translational modification and ligand binding, and conformational and (mis-)folding studies. The replacement of an aged unreliable Orbitrap MS will allow considerably faster proteomic data acquisition with higher resolution and sensitivity and lower sample requirements, and exploratory metabolomics. Forming part of a virtual, Leeds-wide facility for MS with complementary centres of expertise at the Astbury Centre on the main campus and at the St James’s University hospital (SJUH) campus, beneficiaries span the Life, Medical and Physical Sciences.

Lay abstract: Mass spectrometry is one of the key tools which scientists in chemistry, biology and medicine use to identify the exact molecular composition of samples, including the amount and structure of different small molecules as well as (bio-)polymers such as proteins, DNA molecules, carbohydrates and lipids. In this proposal, we ask for an investment to update and transform this crucial infrastructure in Leeds so that we can address important biochemical and biomedical questions and meet the needs of the research community, both in the University, the University hospital, and also external collaborators. The cutting-edge equipment requested here will complement the research strategy at Leeds which has seen a recent £17 million investment in the Astbury Biostructure labs, making it one of the leading centers worldwide for the study of biomolecular structure and function, supporting cross-Faculty collaborations to provide patient benefit through better diagnosis, new treatments and curing disease.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;CANCER AND NEOPLASMS
Title: DNA repair and genetic stability: Elucidating the effects of cell physiology in Escherichia coli
Funder: Wellcome Trust

Abstract: All living organisms rely on the correct replication and repair of their chromosomes to transmit genetic information. In bacteria, failures in those processes occur during exposure to antibiotics, resulting in cell death but also in increased genetic instability. Therefore, understanding DNA maintenance is crucial for efficient use of antibiotics and to limit the emergence of resistant strains. Recent new concepts in cell physiology have revolutionized our understanding of gene regulation, cell size and cell division control. However, how emergent properties of cell physiology couple cell fitness with genetic stability has yet to be unravelled. I plan to quantify how growth conditions determine bacterial susceptibility to DNA damage and their mutations rates. Building on my current experimental and theoretical work, I will first use cutting-edge microscopy to quantify how variable growth conditions impact DNA damage and repair. Next, I will show how cell physiology affects the expression of the DNA damage response and identify the underlying mechanisms. Finally, I will measure the impact on antibiotic resistance. This research will lead to a molecular understanding of the relationship between cellular fitness and genomic stability with the ultimate goal of controlling cell physiology for potential applications in antibiotic therapy.

Lay abstract: In all domains of life, cells rely on the correct replication and repair of their chromosomes to transmit genetic information. In bacteria, the importance of these processes is highlighted by the many clinically relevant antibiotics that cause DNA damage resulting in cell death but also in mutations leading to antibiotic resistance. Bacteria can proliferate at very different speeds depending on their environment; some infections are very rapid whilst other will take much more time to develop. How fast bacteria grow affects all the molecular processes necessary for life, yet the connection between bacterial growth and sensitivity to DNA damaging agents has so far been overlooked. For example, it has been observed that slow growing bacteria are less sensitive to DNA damaging antibiotics, but the reasons underlying this observation are not known. Combining experimental and theoretical methods, I aim to elucidate the molecular mechanisms that explain this important phenomenon and to quantify how it impacts the acquisition of drug resistance. My ultimate goal is to discover new ways of manipulating bacterial growth for novel applications in antibiotic therapies.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;CANCER AND NEOPLASMS
Title: Regulation of fission yeast cell polarity by stress-signalling pathways
Funder: Wellcome Trust

Abstract: Cell polarity is essential to eukaryotic cell organisation and function and important in cell/tissue homeostasis and disease. Cdc42 and other Rho-family GTPases have widely-conserved, central roles in cell polarity. Using fission yeast Schizosaccharomyces pombe, we discovered that Cdc42-mediated cell polarity is regulated by the stress-activated protein kinase (SAPK) pathway, involving MAP kinase Sty1, homologous to mammalian p38. Although the SAPK pathway is known to regulate gene expression in response to stress, our work suggests that stress signalling regulates cell polarity post-translationally, via novel pathways. Our overall goal is to understand, at a molecular level, how SAPK activation leads to Cdc42-module regulation, through combined proteomics, genetics, imaging, and biochemistry approaches. We will identify polarity-specific substrates of Sty1 and determine how they influence Cdc42 and its interactors. We will also study the two different Cdc42 guanine-nucleotide exchange factors, as we have shown that they have distinct roles in polarity regulation and are oppositely affected by stress. We will also determine how SAPK-regulation of cell-polarity is integrated with other signalling pathways, including nutrient stress/TOR signalling, cell-cycle control, and the cytoskeleton. This work in a model eukaryote will provide insights into potential roles of stress signalling in cell polarity in humans and pathogenic fungi.

Lay abstract: Cell polarity is important for the spatial organisation and function of nearly all eukaryotic cells, including migrating cells, neurons, epithelial cells, and stem cells. A key player in cell polarity is the Rho-family GTPase Cdc42, which interacts with multiple effector proteins to help execute cell-polarity programmes in response to internal and/or external cues. Using the fission yeast Schizosaccharomyces pombe, we discovered that the cell-polarity module associated with Cdc42 is regulated by stress-signalling pathways. We aim to understand at a detailed molecular level how activation of stress signalling regulates the Cdc42 polarity-module. We will take a multidisciplinary approach, involving proteomics, genetics, biochemistry, and live-cell imaging. We will also investigate how stress-regulation of cell polarity is integrated with other cell-physiological pathways. By determining the detailed mechanisms of stress-regulation of cell polarity in a relatively simple model eukaryote, we will provide insights into how similar regulation may occur in more complex organisms.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;CANCER AND NEOPLASMS
Title: Strategic endgames – modulating transcription termination to control gene expression
Funder: Wellcome Trust

Abstract: Viruses employ various approaches to avoid host cell defence-mechanisms. In Herpes Simplex Virus 1 (HSV1) and Influenza A Virus (IAV) these include strategies to inhibit transcription termination. This leads to non-terminated readthrough transcripts, likely nuclear retained and untranslated. Exactly how viruses inhibit transcription termination, and how this promotes their survival and efficacy is currently unclear. Using my experience and expertise in transcription termination, I propose to systematically analyse the mechanism by which IAV alters 3’ end processing transcription termination and determine how this affects host gene expression. I will use biochemistry, molecular biology and structural biology, to identify the termination components affected by IAV, and reverse genetics to identify the IAV-component effecting termination inhibition. I will also employ sequencing of fractionated, cellular and ribosome-associated RNA to examine how virally-induced termination defects perturb mRNA-export and translation, as well as cell biology and genetics to identify the mechanisms and signalling cascades through which this change is realised. Finally, I will perform orthogonal studies in budding yeast to identify molecular mechanisms that can modulate the activity of transcription termination and RNA processing factors. Overall, my programme will scrutinize an under-explored yet fascinating mechanism to alter gene expression through modulation of transcription termination.

Lay abstract: Virally infected cells quickly mount defence strategies to restrict viral growth. Cells and viruses therefore continuously coevolve to outplay each other. Genome-wide studies have recently shown that influenza A virus (IAV), which causes common flu, impedes gene expression through an unexpected strategy: rather than inhibiting transcriptional activation, viruses interfere with gene-end recognition, otherwise known as transcription termination. I will use a multidisciplinary approach to determine how IAV impedes transcription termination. I will also examine the consequences this has for the infected cell and use this to learn about mechanisms that control gene expression in healthy cells. My studies may help to find novel therapies against flu or other viruses employing similar strategies. They will also help to explain general mechanisms of gene expression.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;CANCER AND NEOPLASMS
Title: Metabolic cooperation in eukaryotes: When metabolic networks operate non-cell autonomously
Funder: Wellcome Trust

Abstract: Living cells possess a system of biochemical reactions that produces the molecular building blocks of life, such as amino acids and lipids. It has recently become clear that cells are not producing metabolites only for themselves, but intensively exchange them between each other, and in this way, share the metabolic ‘workload’. Exchanging metabolites is however not without consequence, as it renders the cells metabolically different to each other: a cell that receives a metabolite from another cell, does not need to synthesize this metabolite. Metabolism is connected to many biological systems, inparticular those that mediate protection against toxic substances that are a problem during ageing and in several disorders, including cancer. We found that cells which exchange metabolites have a different survival chance in the presence of toxic substances, and that the way they share the metabolites is influencing it. In this proposal, we will elaborate the biological basis of why and how cells exchange which metabolites. We do this, as we think that interrupting this exchange of metabolites can open new ways of manipulating pathogens and cancer cells, and to render them more susceptible to therapies, and for the same reason, improve production of biofuels and food.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;CANCER AND NEOPLASMS
Title: Childhood evolution of B cell responses to COVID-19 vaccination
Funder: Medical Research Council

Abstract: Paediatric COVID-19 vaccine development has lagged behind that for adults despite paediatric vaccination being important in curtailing the pandemic. Logistical and ethical issues present barriers to rapid set-up of paediatric vaccine trials. Levels of neutralising antibody, produced by antibody secreting cells of the B cell lineage, are now a recognised correlate of protection in the evaluation of COVID-19 vaccination in adult. Immunobridging studies are now permitted to infer vaccine efficacy in children. However, much of our understanding of B cell and antibody development comes from previous studies in infants and younger children under 2 years, focusing on the ages during which they receive their childhood vaccines through the Extended Programme for Immunisation (EPI). Recently developed enable us to interrogate both Fv and Fc antibody functions, and children have been shown to produce antibodies with distinct antibody-dependent mediated immune functions (i.e. Fc functions) compared with adults following SARS-CoV-2 infection. Whether this is also true for vaccination is unknown. In addition, Fv characteristics, including antibody affinity, following SARS-CoV-2 infection demonstrate age-dependent variation in older children but whether this is associated with differences in vaccine response is also unknown. It is important to investigate these differences to enable us to be confident that immunobridging studies are valid in the context of COVID-19 vaccine development and identify potential adjustments which may need to be made in such studies. Here, we propose to perform systems serology assays on plasma, together with multiomic analysis of B cells from peripheral blood mononuclear cell samples taken from participants of a paediatric COVID-19 vaccine trial aged between 6-17 years of age. The results will provide a detailed picture of how antibody responses to COVID-19 vaccination evolve in late childhood and relate to adult vaccine responses.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;3.4 VACCINES
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;INFLAMMATORY AND IMMUNE SYSTEM
Title: Structural and functional characterisation of E3 ligase complexes involved in inflammation
Funder: Wellcome Trust

Abstract: Tissue damage and infection can elicit a specific response in the bone marrow called emergency haematopoiesis, which results in the production of myeloid-type blood cells. This response is mediated by a protein complex signalling hub called the Myddosome. After damage or infection resolution, the termination of emergency haematopoiesis is crucial, as prolonged emergency haematopoiesis may result in chronic inflammation, autoimmune disease and cancer. As such, termination of emergency haematopoiesis is carefully regulated by orchestrating a series of signalling events.Using a multidisciplinary approach involving structural and cellular biology, this project aims to understand the molecular and functional basis of Myddosome-regulating complexes that participate in termination of emergency haematopoiesis. To achieve this, I will use cryo-electron microscopy to gain high resolution structures and use mutational analysis to probe protein-protein interactions and their functions. Results from this project will provide structural and functional information which will enable us to design potential therapeutics against immune-mediated diseases and hematopoietic cell proliferation.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;INFLAMMATORY AND IMMUNE SYSTEM
Title: Differential immune transcription in health and disease
Funder: Wellcome Trust

Abstract: In immune conditions such as sepsis, treatment options can be limited by a lack of understanding of the causes of the condition, as well as the vast differences in how the immune system of the patient can respond. While expression differences between genes explain some of the variance between patient outcomes, there is still a lot of variability unaccounted for, and how this relates to the genome remains to be studied. RNA sequencing performed on samples from large existing cohorts of patients will give transcript level information, revealing not only the expression levels but also information such as how the genes are differentially spliced between individuals. With paired medical records I will relate these observed transcript differences with patient outcomes, as well as infer how innate genotypes can modulate these effects. Provided microbiological information on the cause of the condition or infection will allow me to also understand the differences in human responses to infection type. This knowledge will lead to a more personalised treatment regime for patients based on the course of their illness and innate characteristics, as well as giving the option to better predict and address the cause of their illness.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;INFLAMMATORY AND IMMUNE SYSTEM
Title: Spatiotemporal host and viral transcriptomics, host restriction and DNA sensing at single cell and single molecule resolution
Funder: Wellcome Trust

Abstract: During the earliest stages of infection, the initiation of transcription from an infecting virus genome is a critical event and essential for subsequent virus replication, progeny assembly and onward transmission. However, the same virus genome is simultaneously sensed by the host cell as a “pathogen-associated molecular pattern”. This results in the activation of numerous cellular restriction processes to limit virus transcription and replication, as well as, stimulating anti-viral processes in surrounding cells. To understand the critical dynamics of these opposing processes, I will exploit innovative new techniques in bio-orthogonal chemistry and single molecule RNA analysis to investigate a model DNA virus, herpes simplex virus (HSV), at single cell and single genome level. Using these techniques in relevant cell models, together with knock-outs in key cell DNA sensors and virus mutants, I aim to understand the processes that operate at the earliest stages of infection, the precise importance and role(s) of various host DNA sensors and viral antagonists, and identify novel restriction factors and associated mechanisms of viral suppression. From high resolution and quantitative investigation of these events, I aim to establish new insight into the early innate immune response and contribute to the development of novel anti-viral therapies.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;INFLAMMATORY AND IMMUNE SYSTEM