NIHRDataInsights/HRCSData huggingface dataset:
Title: Electron microscope for screening of grids for cryo-electron microscopy
Funder: Wellcome Trust

Abstract: As cryo-electron microscopy and tomography (cryoEM, cryoET) methods develop, groups at MRC-LMB are studying increasingly ambitious samples. Identifying and optimizing the appropriate sample requires preparing and screening large numbers of cryoEM grids prior to final data collection. The capacity, quality, and properties of screening microscopes at the LMB currently limit how much screening is possible. We require an electron microscope capable of high-quality imaging for screening vitrified samples. It will be run in a highly automated, high-throughput manner, and allow selected grids to be transferred for data collection on a Titan Krios microscope. The microscope will overcome our severe bottleneck in screening, and allow us to develop new screening approaches that will permit new experimental directions and will be shared with the community. The microscope should have similar capabilities to the ThermoFisher Glacios CryoTEM for Life Sciences. It should be equipped with Field Emission Gun and Direct Electron Detector to obtain high quality images, a multi-specimen automatic grid transfer system to allow multiple samples to be screened in a single run, and the ability to run and adapt scripts for computer-controlled screening. The specimen holder must allow selected samples to be saved and directly transferred into existing Titan Krios microscopes.

Lay abstract: Cryo-electron microscopy and tomography allow us to determine the structures of protein complexes, to image proteins and membranes interacting in simplified model systems, and even to image biological machines directly within cells. We study systems including cellular machines that malfunction in disease, pathogenic viruses, bacteria, and mammalian cells. We use powerful, automated microscopes to collect many images of the sample that are then computationally processed. Prior to collecting these images, however, we spend long periods working out how to make and purify our proteins, how to put these molecules back together in a working form, and how to prepare specimens without damaging or losing them. In an iterative approach, we check the sample in a smaller electron microscope, and use this information to inform the next experiment. Here, we are proposing to buy a microscope which will allow us to check our samples in an efficient, automated manner.

Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: State of the art direct electron detection at the Astbury Biostructure Laboratory
Funder: Wellcome Trust

Abstract: The Astbury Biostructure Laboratory, was established in 2015 with University of Leeds investment, and a Wellcome multi-user equipment award, allowing us to install two Titan Krios microscopes (2016). In just three years, we have established an international reputation for excellence in cryoEM, and a buoyant user base that means our facility is already operating at capacity. We request funds to upgrade our microscopes, aiming to increase data acquisition rates, whilst improving data quality, allowing us to shorten session lengths, increase sample throughput, and introduce new methodologies. Upgrades will include: 1. A ThermoFisher Falcon-4EC camera on Krios#1 2. A Gatan Bioquantum K3 on Krios#2 3. Aberration-Free/Fringe Free Imaging Systems (both microscopes) 4. EPU-D software for electron diffraction (Krios#1) 5. Whole-system maintenance contract extensions to 5 years from installation. We anticipate a 5-8x improvement in data acquisition, empowering users to drive more challenging biological and biomedical discovery. We will be able to determine new high-resolution structures and integrate them into their cellular/tissue context, helping us to understand the molecular basis of diseases such as infections, cardiovascular disease, degeneration and cancer. We request £1m from Wellcome, with a University capital contribution of £325k, plus salary costs, Giving a total project cost of ~£2.13m.

Lay abstract: Structural Biology aims to understand life in molecular detail, and is a major focus in biomedical discovery. Cryo-electron microscopy (cryoEM) is a key tool in structural biology, and we run a major cryoEM facility at the University of Leeds (UoL). We currently run at full capacity, supporting a wide range of scientists from Leeds, across the UK and beyond. To enhance our capacity to support this biomedical discovery science, we are requesting £1m in funding from Wellcome, together with £1.125m of cash and in-kind contributions from UoL, to upgrade the cameras and controls systems on our microscopes. These upgrades will mean that ~5x as many images can be recorded, and crucially, each will be of higher quality. In turn this will allow more users to collect more data, reducing the time and cost to solve 3D structures that are important in areas such as immunology, cancer biology, degeneration and virology.

Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Establishment of Cryo-EM screening facility at University of Dundee
Funder: Wellcome Trust

Abstract: Cryo-electron microscopy (Cryo-EM) has become a powerful method to determine three-dimensional (3D) structures of macromolecular complexes. The technological developments to electron microscopes, detectors and processing software have enabled researchers to achieve high-resolution structures. Hence Cryo-EM has revolutionized structural biology to provide unprecedented insights into protein structure and function. Substantial investment has established national centres for data collection at the eBIC facility in Harwell and at the SCMI facility in Glasgow. To ensure that only the highest quality samples are selected for ultimate resolution data collection at the national facilities, it is equally important that the institutes that feed into them have excellent instrumentation for sample screening. To meet growing demand for Cryo-EM in Dundee this proposal requests support for the purchase of a Glacios Cryo-TEM with 200 kV X-FEG optics with an Autoloader for cryogenic sample manipulation and Falcon 3EC detector (or similar). In addition to efficient sample screening, this system supports high resolution data acquisition for single particle analysis, sample quality assessment, tomography, and other applications. This microscope will provide high quality screening for optimizing cryo-EM samples prior to data collection at National facilities, but also can determine structures at sub-3 Å resolution in house.

Lay abstract: Many research groups across Dundee and St Andrews investigate how cells grow and respond to their environments. This involves the action of molecular machines including assemblies of proteins that until recently have been difficult to observe. Recent advances to cryo-electron microscopy make it possible to see these molecular machines with unprecedented definition. To make full use of cryo-electron microscopy researchers need access to local facilities to optimise samples prior to sending them to national facilities for characterisation in greatest possible detail. We request funds to establish a state of the art local screening facility to enable researchers from across Dundee and St Andrews to apply cryo-electron microscopy to their research projects. This will transform these research projects providing new insight into the molecular basis of life with the detail required to generate new treatments for diseases.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Super-resolving the physical mechanisms of bacterial cell division
Funder: Wellcome Trust

Abstract: I aim to reveal the elusive basic principles of bacterial cell division by super-resolution microscopy. This is the next step in my long term vision to discover fundamental principles of how protein nanomachines spatially organize cells. The bacterial cell division machinery is a crucial cellular module essential for bacterial survival, and a mechanistic understanding of division is essential to understanding mode of action of cell-division-targeting antibiotics. Cell division is also a fascinating puzzle: bacteria and animal cells both use a cytoskeleton to constrict the cell; but how do bacteria, unlike animal cells, manage to do so without using any motor proteins? The mechanistic principles of bacterial cell division have escaped elucidation for decades because the cell division machinery is organized on a scale below the diffraction limit of conventional microscopy. I will overcome this problem by pushing the limits of super-resolution microscopy to reveal the organization and motion of the Bacillus subtilis cell division machinery at unprecedented resolution. This will reveal the physical mechanisms of cell division by answering three critical questions: • How is the bacterial cell division machinery organized? • How does it generate constrictive force? • How is force generation coordinated with remodelling of the bacterial cell wall?

Lay abstract: Antibiotic-resistant bacteria are a grievous challenge to our society, threatening to return us to the pre-antibiotic era where even a minor scratch could kill. In order to identify genuinely new strategies for antibiotic development, we need to better understand bacteria at a fundamental level. I will use cutting edge microscopy to discover the basic principles of how bacteria divide. Bacterial cell division is an utterly essential process, and an excellent target for new antibiotics. Bacteria divide against high internal pressure, which is like trying to cut an inflated balloon in two without bursting it. For decades, this process has been mysterious, because cell division occurs on the nanoscale, invisible to a conventional microscope. I will solve this problem using super-resolution microscopy, a recently developed technique which can observe single protein molecules in living cells at a resolution of tens of nanometres. I will use super-resolution microscopy to reveal the organization and motion of individual cell division proteins in live bacteria. This will reveal how the cell division proteins work together as a single nanoscale machine to cut the bacterial balloon skin (cell wall) in two. In the longer term this will help find new antibiotics to target cell division.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: The role of CpG dinucleotides in regulating virus replication kinetics
Funder: Wellcome Trust

Abstract: ssRNA viruses infecting vertebrates almost universally suppress CpG dinucleotides in their genomes. Recently, an RNA-CpG binding protein, zinc finger antiviral protein (ZAP) was identified, which elicits an innate immune response. I propose three interlinked aims that integrate bioinformatic study with technical laboratory process to explore this, using influenza A virus (IAV) as a tractable system: 1. For IAV, I show that spliced transcripts have more CpGs than their unspliced counterparts. I will investigate this by undertaking pulldowns to determine the repertoire of IAV-CpG motif binding proteins. I will use CpG modified viruses to characterise the importance of these interacting proteins, and investigate whether CpGs regulate splicing. 2. How does ZAP regulate IAV replication? I will determine the effect of knockdown of two ZAP isoforms on IAV replication kinetics and assess the importance of ZAP for RNA turnover and splicing efficiency. ZAP interacts with several cellular pathways, including interferon, splicing and stress response. I will investigate which pathways are important during IAV infection using a knockout cell library. 3. There appear to be fundamental differences between avian and mammalian ZAP, which I will characterise. I will identify CpG binding protein(s) in avian cells and investigate how CpGs evolve when IAV jumps between host species.

Lay abstract: Studying the genetic code reveals patterns made by the four building blocks of our genetic makeup (A, C, G and T). For example, in the human genome, G follows C at a low frequency. This CG avoidance is mimicked in the genomes of viruses infecting us because too many CGs activate the immune response, via a protein called ZAP. My project has three aims: Firstly, in two discrete genome regions of influenza A virus (IAV) there are high numbers of CG. These two regions exclusively are edited by the host cell machinery. I will identify which proteins of an infected cell interact with these regions. Secondly, I will characterise how ZAP impacts IAV replication, by making cells which don't have ZAP and determining how viruses replicate in them. Thirdly, I will characterise the differences between human and chicken ZAP to understand better how IAV jumps between these hosts.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: A pragmatic randomised study to optimise screening, prevention and care for tuberculosis in Malawi (PROSPeCT Study)
Funder: Wellcome Trust

Abstract: Ambitious global targets have been set to eliminate tuberculosis as a public health problem by 2035. However, in Africa, where HIV has driven extremely high incidence rates, progress in reducing new infections and TB deaths remains too slow. My previous research has shown that adults seeking diagnosis and treatment for TB and HIV face considerable barriers, and have high pre-treatment mortality. Efforts to reduce TB mortality have been hampered by limitations in TB diagnostics, with considerable uncertainty about how available and new tests can be best implemented. The key goals of this Fellowship are: 1. To develop expertise in pragmatic trial design, statistical analysis and policy-translation 2. In a pragmatic randomised trial among adults with TB symptoms in Blantyre, investigate the effects of optimised TB/HIV diagnosis and treatment linkage on case detection and treatment initiation 3. In a nested sub-study, evaluate the accuracy of computer-aided digital chest x-ray triage classification (normal vs. any abnormality) compared to expert radiologist classification and bacteriological diagnosis. This research will provide urgently-needed evidence to inform policy decisions on how best to improve TB/HIV diagnosis and treatment in Africa. The training opportunities will help me develop internationally-recognised expertise in pragmatic randomised trials.

Lay abstract: Tuberculosis is the leading cause of death from an infection worldwide. In Africa, HIV has led to very high TB rates, and progress towards reducing TB deaths has been too slow. People attending clinics often don’t get the tests they need, must make many visits to health facilities at personal expense, and have high risk of dying before they start treatment. Critical knowledge gaps that need to be answered include: how can people with TB symptoms be most-effectively diagnosed and linked to TB and HIV treatment? This research will investigate a new strategy to answer this question. We will take chest x-rays from adults with TB symptoms in Malawi, and use a new computer tool to identify x-rays that show any abnormality. We will then confirm TB disease in the clinic using a new rapid sputum test. We will test how good this strategy is at improving the timeliness of TB/HIV diagnosis and treatment uptake by comparing it against what clinicians usually do to diagnose TB and HIV in the clinic. This sort of study (a “pragmatic trial”) will provide urgently needed information to patients and policymakers about how TB can be best diagnosed in poor-settings where HIV is common.

Research activity codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Structural cell biology of bacterial biofilm formation
Funder: Wellcome Trust

Abstract: Recent developments in microscopic imaging methods that use electrons rather than light have provided an important alternative method to researchers for investigating biological material. It is my goal to apply the latest electron microscopy imaging technology to study the important biomedical problem of bacterial biofilms. Bacterial cells can attach to surfaces and form large communities known as biofilms. Cells in a biofilm community are tolerant to a wide variety of environmental stresses. Most notably they can become tolerant to antibiotics. Two such bacteria are Escherichia coli and Pseudomonas aeruginosa. Both of these bacteria can enter the human body, form a biofilm in the infected tissue and lead to disease. Understanding how bacterial biofilms are built is therefore important to understand the infection process of these pathogenic bacteria. I will use electron microscopy imaging to produce high-resolution pictures of bacterial biofilms in three dimensions. Using these pictures, I will work out where key molecules that mediate biofilm formation are located. I will then use electron microscopy methods to solve the atomic structures of these key molecules to understand how they influence biofilm formation. Together, these experiments may lead to ideas on how to disrupt bacterial biofilms, to help infected individuals.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Toxic DNA: a model for all domains of life
Funder: Wellcome Trust

Abstract: The instructions for life are encoded by DNA. As such, it is counter intuitive that certain DNA sequences can be intrinsically toxic to the cell. The problem is acute for DNA where the frequency of adenine and thymine exceeds what is normal for a given organism. Toxic effects of such "AT-rich" DNA have been reported for species as diverse as E. coli and humans. Working with bacteria, I have shown that misdirection of transcription is closely linked to the toxicity of AT-rich DNA. This happens because promoters, the DNA sequences that instigate transcription, are also AT-rich. Since promoters from all cell types have a high AT-content, I argue this may be a universal phenomenon. If true, the implications are far reaching; AT-rich DNA impacts processes as diverse as antibiotic resistance in bacteria and cancer in humans. My application probes the mechanistic details underlying the toxicity of AT-rich DNA for both prokaryotic and eukaryotic cell types. Hypothesis: The toxicity of AT-rich DNA is a consequence of spurious transcription Aim 1. Understand molecular basis Aim 2. Understand toxic mechanisms Aim 3. Understand evolutionary prevalence

Lay abstract: Soon after the emergence of life on our planet DNA became the method of choice for passing information between generations; DNA is now found in all cell types. The universal nature of DNA means that certain biological principles are true throughout life. We argue that the tendency of certain DNA sequences to poison cells is one such principle. Based on studies of simple unicellular organisms, we suggest that toxic DNA exerts its effect by corrupting a central biological process; accurate reading of the DNA code. I propose to understand consequences of this, and the adaptations that cells undergo in response, for all forms of life. Our findings will have implications for processes as diverse as the acquisition of antibiotic resistance in bacteria and the development of cancers in humans.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Targeting Protein-Protein Interactions in Salmonella Persisters: An Innovative Approach to Tackling Recurrent Infections
Funder: Wellcome Trust

Abstract: Antimicrobial resistance and the recalcitrance of bacterial infections have been identified as one of the most pressing challenges facing human health. One lesser known mechanism by which bacteria are able to survive in the presence of antibiotics is that of bacterial persistence. Persistence has evolved as a mechanism by which bacteria enter a non-growing state when confronted with environmental stress. The generation of these populations of persisters is controlled by protein-protein interactions between toxin and antitoxin binding partners. The activity of the toxin is regulated by the antitoxin, which acts to prevent the formation of persisters. By using a series of chemical biology strategies to mimic the natural activity of the antitoxin I will aim to revert bacteria back to their proliferating antibiotic sensitive state. Initially, stapled peptide fragments based on the interacting region of the antitoxin will be used as first generation toxin inhibitors. The key antitoxin amino acid residues will also be presented as side chains on a synthetic scaffold in order to identify more enzymatically resistant mimetics. Finally, a target-directed synthesis approach will be employed in order to more efficiently select helix mimetic side chains based on their individual affinity for the toxin.

Lay abstract: The resistance and tolerance of bacteria to antibiotics is one of the biggest challenges facing the modern world. One way in which bacteria survive in the presence of antibiotics is to generate small populations of non-growing cells called persisters. This process is triggered by proteins called toxins whose activities are tightly controlled by antitoxin proteins. In the presence of an environmental stimulus, such as an antibiotic, the antitoxin is degraded allowing the toxin to initiate a dormant, antibiotic tolerant persister state. Importantly, once this external stimulus is removed the bacteria can return to their original prolific state directly resulting in the recurrence of problematic infections such as typhoid and tuberculosis which affect thousands of people worldwide each year. This project aims to reverse the formation of persisters in bacteria and restore their sensitivity to antibiotics. I will do this by making molecules which mimic the natural activity of antitoxin proteins.

Research activity codes: 5.1 PHARMACEUTICALS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION