- Total grants
- Total funders
- Total recipients
- Earliest award date
- 21 Jan 2017
- Latest award date
- 11 Dec 2017
- Total GBP grants
- Total GBP awarded
- Largest GBP award
- Smallest GBP award
- Total Non-GBP grants
We request funds to purchase equipment to perform correlative light and electron microscopy (CLEM) and electron microscopy of cells & tissues at the University of Leeds. This will include: a high-pressure freezer to allow rapid freezing of large and/or labile specimens whilst minimizing structural deformation; a freeze-substitution unit for low-temperature substitution of water by dehydrating agents and fixatives, avoiding the damaging effects of room-temperature dehydration; a modern cryo-ultramicrotome, for room-temperature and cryo-sectioning of samples for (cryo-)EM imaging; and a cryo-CLEM system, to image cryo-fixed samples by fluorescent microscopy, identifying interesting biological events for further analysis at high-resolution by cryo-EM. This equipment will build upon the outstanding infrastructure for super-resolution light microscopy and cryo-EM in Leeds. The combination of new and existing equipment will give us an unprecedented ability to integrate high-resolution structural information into its cellular/tissue context. This will help us to tackle the most challenging projects in biomedical discovery, and understand the molecular basis of diseases such as infections, cardiovascular disease, neurodegeneration and cancer. We request £429k of Wellcome Trust funds, complementing the University’s contribution of ~£192k in capital and support salary costs, towards a total project cost of ~£621k.
Mass spectrometry infrastructure underpinning research into the molecular basis and biological mechanisms in health and disease 06 Jul 2017
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.
Light sheet microscopy in biomedical imaging 06 Jul 2017
We want to purchase a LaVision Ultramicroscope II, the only commercially available light sheet microscope capable of imaging immunofluorescence or endogenous fluorescence in tissues cleared with novel organic solvent-based techniques, including iDISCO (Cell 2014; 159(4); 896-910) and uDISCO (Nature Methods 2016; 13(10): 859-867). The system uses triple sheet optics to create a focused plane of light from a white laser, used in conjunction with appropriate excitation/emission filters. The sample is imaged with specially protected dipping objectives (2x and 20x), which allow rapid 3-dimensional visualisation of multi-colour fluorescence in tissues exceeding 10mm in each dimension, achieving 1-2 micron resolution in all axes. Imaging is conducted using a high-specification workstation, with a separate workstation requested for processing-intensive offline analyses. In view of the large datasets produced, we have applied for 50 terabytes of server storage capacity, managed by IT staff already running the Wellcome Trust funded Astbury cryo-EM facility. We also request support for a half-time grade 5 technician to train new users in the tissue preparation and imaging approaches required to maximise the potential of this equipment. We will use this technological advance to better understand disease mechanisms, test novel therapeutics, and define the potential of 3-dimensional imaging in clinical diagnostics.
Structure-based drug design has relied heavily upon X-ray crystallography to yield atomic resolution structures of protein targets, subsequently used for inhibitor design. However, the structures obtained are in a static state which may not be a true representation of how the protein exists under physiological conditions. The advances in Electron Microscopy (EM) has led to an increase in the number of near-atomic resolution structures solved and enabled conformational information to be obtained for dynamic systems. Therefore, the project’s ultimate goal is to use EM structures to aid the design of novel therapeutic agents against two different proteins which could be used to treat toxoplasmosis; vacuolar ATPase (V-ATPase) and cytochrome bc1 complex. In silico drug design, including virtual high throughput screening (vHTS) and de novo design, will be conducted to produce putative inhibitors. The compounds will be designed against the most populated functional state of the protein and will be synthesised and screened for their biological activity using in-cell and enzymatic assays. EM can then be used to probe the mode of inhibitor binding to the complex leading to one of the first examples of EM structures being used as a tool in drug discovery.
BK polyomavirus is ≥90% seroprevalent in the global population, causing nephropathies and ultimately acute rejection in renal transplant recipients. Previous work has shown that the agnoprotein of BK polyomavirus is a critical egress/release factor during the viral life cycle. The proposed project aims to further our understanding of the putative viroporin function of BK agnoprotein, and determine whether the agnoprotein would be a suitable target for anti-viral therapeutics. These aims will be achieved using a combination of structural and biochemical techniques to provide the first structural information about BK agnoprotein, characterisation of agnoprotein pore formation in in vitro assays, and screening of small molecule compound to identify potential agnoprotein inhibitors. In addition, the project will take advantage of existing primary kidney cell culture systems to study viroporin function during the BK life cycle. Should inhibitors of viroporin be identified, their efficacy will be tested against a number of clinical isolates of BK, obtained from our collaborators in the kidney transplant unit at Leeds. Together, these studies will provide new insight into the structure and function of agnoprotein and may validate a novel target for anti-viral therapeutic intervention.
Understanding control of protein concentration and cell fate in the absence of extracellular stimulation 31 Jan 2017
Intricate cellular signaling pathways are essential for maintaining cellular homeostasis and enabling the cell to respond appropriately to extracellular stimuli. Aberrant cell signaling has been implicated in many diseases in particular cancer. The non-stimulated state is particularly important for cancer research as it mimics the early stages of tumourgenesis, when cells at the centre of a growing tumour are unable to receive any stimulus due to surrounding cells. In the absence of extracellular stimulation aberrant signaling can be caused by fluctuations in protein concentrations. Grb2 is a ubiquitously expressed adaptor protein, essential to many signaling pathways. Preliminary data suggests that Grb3-3 a naturally occurring isoform of Grb2 exerts dominant negative control over Grb2. Therefore the balance between the intrinsic levels of these two proteins will influence key signaling pathways. Grb3-3 expression was observed in non-cancerous breast cells but was reduced in breast cancer cells suggesting a potential role of Grb3-3 in rescuing deregulated Grb2 mediated signaling. The aims of this project are to determine structurally and mechanistically how Grb3-3 binds and inhibits Grb2, to investigate their expression in normal and cancer cells and to understand what controls the levels of these two proteins in the cell.
Investigating the Structure and Regulation of Fibroblast Growth Factor Receptor 3 (FGFR3) 31 Jan 2017
Receptor tyrosine kinases (RTKs) are cell surface receptors which play a critical role in cell proliferation, homeostasis and survival, mediated by initiation of intracellular signalling cascades in response to extracellular ligand binding. The fundamental importance of RTKs is underlined by the prevalence of pathologies associated with their dysregulation. Despite extensive research in the RTK field, there remain many unanswered questions with regards to the structural and dynamic mechanisms of activation and regulation, both in physiological and pathological settings. Crucially, the key question of how the binding of growth factor to the extracellular domain results in choreographed conformational transitions at the intracellular kinase domain to trigger downstream signalling remains an enigma. To elucidate this question, I will use cryo-electron microscopy to determine structures of full-length fibroblast growth factor receptor 3 (FGFR3), an RTK. Hsp90, a member of the cellular protein-folding machinery, is thought to regulate kinases and protect oncogenic variants from proteasomal degradation. Though glimpses of the mechanisms underlying kinase-(co)chaperone interactions are beginning to appear, the structural determinants of chaperone-dependence of kinases are unclear, especially for RTKs. Thus, in parallel, I will use NMR spectroscopy to investigate the dynamic interactions of the FGFR3 kinase domain with the Cdc37-Hsp90 chaperone system.
Discovery of small molecules that modulate transient protein-protein interactions leading to amyloid formation 31 Jan 2017
The development of anti-amyloid drugs has been hampered by the mechanistic complexity of amyloid formation pathways. In order to gain a greater understanding of the conformational changes and dynamic motions associated with fibrillogenesis, this project aims to develop and use small molecules to study an archetypical amyloid protein, beta2-microglobulin (beta2m) – specifically, the amyloidogenic truncation variant, DeltaN6. Small molecule fragments which are compatible with site-directed screening methods will be synthesised, so as to allow amyloid-modulating regions of DeltaN6 to be targeted. Hits which are identified in site-directed screens will be optimised as necessary, in order to produce small molecules which are capable of perturbing amyloid formation pathways upon binding. Understanding the link between small molecule-induced changes in DeltaN6 dynamics (to be studied using nuclear magnetic resonance spectroscopy), and changes in the rate and outcome of fibrillogenesis, will provide both beta2m-specific and generic insights into amyloid formation pathways.
Harnessing the molecular-scale resolution of DNA-PAINT to study the structural basis of electrical signals of the healthy and arrhythmic hearts 12 May 2017
Single molecule localisation microscopy (SMLM) has revolutionised our understanding of signalling microdomains in many biological systems. Among such molecular systems, are arrays of gap junction (GJ) channels paving the fundamental electrical signalling pathways of the heart. Conventional SMLMs are yet, limited by resolution, unable to visualise individual GJ channels within their native tissue environment. One of the newest SMLM techniques, called DNA-PAINT, promises resolution of ~ 5 nm, sufficient to resolve single proteins within tightly packed arrays or clusters and to visually observe their interactions with other molecules. However, by design, this technique is not suitable for imaging complex tissues like the myocardium. This project aims to implement three possible modifications to the DNA-PAINT protocol to enable in situ optical mapping of GJ isoforms at an unprecedented single-channel-level resolution. DNA-PAINT will be further harnessed to quantify the changes in the phosphorylation states of GJ channels within the ventricular cardiac tissue exhibiting pathological electrical disturbances known as arrhythmias. Both the improved DNA-PAINT protocol and the pilot measurements on GJ remodelling will seed a larger investigation pioneering a direct, in situ correlation of the GJ nanostructure and the disturbed electrical patterns within hearts undergoing life-threatening arrhythmias.
Healthcare utilisation and clinical outcomes among survivors of acute myocardial infarction: a national electronic health records cohort study 19 Apr 2017
Although cardiovascular disease remains the leading cause of death worldwide, survival following acute myocardial infarction (AMI) has improved dramatically in recent decades. AMI patients are predominantly elderly and multi-morbid, yet little is known about the impact of their multi-morbidity upon receipt of guideline recommended care. Moreover, increased survivorship is associated with an increased risk of recurrent events and future co-morbidities. To date, data about the healthcare burden following AMI have been limited to either the acute hospital setting only, or restricted to cardiovascular related events. Therefore, I aim to investigate the total cardiovascular and non-cardiovascular healthcare burden among AMI survivors using large scale electronic healthcare databases covering in-hospital and primary care patient pathways. Quantifying the association between the receipt of guideline care and existing multi-morbidities for patients with AMI will help target therapeutic strategies to specific groups of patients to further reduce premature mortality from AMI. In addition, characterising the incidence and nature of primary and secondary care visits following AMI across a spectrum of outcomes will lead to increased understanding of disease progression and can be used to inform the selection of new clinical trial endpoints as well as aid data driven medicine for the development of guidelines.
Gamma knife®(GK) is a stereotactic radiosurgery that can be used to treat trigeminal neuralgia(TN), without the need for pharmacological medication, and has been shown to result in patients being pain free without medication from as early as 6 months after surgery (Loescher et al, 2012). TN is a serious health issue that causes short, reoccurring sessions of intense, sharp facial pain, which has been compared to the feeling of an electric shock (Headache Classification Subcommittee of the International Headache Society, 2013). The cost effectiveness of GK has yet to be fully elucidated. Qualitative work has evaluated the effects of the drug of choice, Carbamazepine, used to treat TN, and found that patients report motor and cognitive difficulties (Zakrzewska et al, 2017). However, a more objective, quantitative investigation into the effects of Carbamazepine and GK treatment on patients with TN would allow for standardised assessment of risks and benefits and therefore make cost-effectiveness discussions more informed. This study will investigate the effects of different treatment regimens on manual dexterity tasks, where participants use a handheld stylus to interact with visual stimuli and postural stability tasks, where participants are exposed to a cognitive load and their gross motor control is measured.
The electrophysiological correlates of action and selection errors in human decision-making 27 Apr 2017
If you lose a point playing tennis, how should you approach your next shot? To respond optimally, we must solve a "credit assignment problem" - was the lack of reward due to wrong shot selection or poor execution? Understanding how the brain responds to these distinct sources of error is important because this information could be used to predict subsequent behavioural responses. To this end, this project will use scalp-recorded EEG and the ERP analysis technique to examine the neural correlates of processing outcomes related to execution and selection error. Specifically, participants will complete a decision-making task involving arm reaches towards targets on a screen with three possible outcomes: (1) Success- when a target is successfully hit and provides a reward; (2) Execution error- when the target is missed; and (3) Selection error- when the movement is accurate but no reward is given. The specific goal is to examine variation in a putative neural marker of reinforcement learning processes in the human brain- the feedback-related negativity (FRN) following selection and execution errors. This will allow us to uncover how the brain discriminates errors of action and selection for reinforcement learning.
Lead identification of a novel anticoagulant agent targeted against activated factor XII for the prevention of thrombosis without the risk of bleeding associated with current antithrombotics 01 Oct 2017
At the University of Leeds, Dr Helen Philippou (PI) and Prof Robert Ariens (Faculty of Medicine and Health), with colleagues (Faculty of Mathematics and Physical Sciences) Dr Richard Foster and Prof Colin Fishwick with Prof Gregory Lip (University of Birmingham) have secured a £3,021,002 Wellcome Trust Seeding Drug Discovery award to develop new safer anticoagulants with minimal risk of bleeding. The medicines available to treat patients who suffer from an increased risk of blood clotting are effective but carry a significant risk of causing bleeding. Current treatments therefore involve a balance between reducing blood clots with inducing bleeding. The objective of this proposal is to discover highly specific compounds which block a protein, Factor XIIa, which is involved in clot formation, and for which there is good evidence to suggest inhibition will not cause bleeding. This will allow patients to be treated more safely with minimal risk of bleeding without needing therapeutic monitoring. The aim is to produce an agent which will be given by mouth daily.
To support the development of the WASSUP project.
Beyond Transitions 21 Jan 2017
Volunteering Matters will use the grant to support younger people with disabilities in Blaenau Gwent by providing them with skills and empowering them during the transition from education to further education, employment or training. This grant for Â£5,000 will fund volunteer expenses, mentor training, stationery and telephone costs, promotional materials, evaluation, and administration support.