NIHRDataInsights/HRCSData huggingface dataset:
Title: Structure, dynamics and function of OMPs in physiological-like membranes
Funder: Wellcome Trust

Abstract: The outer membrane (OM) of gram-negative bacteria is the first line of defence against exogenous threats, and helps confer antibiotic resistance and enable biofilm formation. It is an asymmetric bilayer, consisting of an outer leaflet of lipopolysaccharide (LPS) and an inner leaflet of phospholipids and cardiolipin, and is crowded with outer membrane proteins (OMPs). While much is known about OMPs in symmetric phospholipid membranes, the effects of LPS, membrane asymmetry and protein crowding remain largely unclear – although they have been shown to be important. I will explore the structure, dynamics and function of OMPs in native-like membranes through an interdisciplinary approach combining molecular dynamics simulations, cryo-electron microscopy and biochemical/biophysical approaches. Specifically, I will develop protocols to generate OM-like asymmetric vesicles. Exploiting cryo-electron microscopy and molecular dynamics simulation methodology, I will elucidate the effects of the physiological OM components on FusA (a model OMP) in a broad range of asymmetric membrane contexts. In addition, substrate binding and in vivo function of FusA will be determined. Together, this work will elucidate details of how the OM modulates OMPs, providing clearer pathways to understand gram-negative bacterial biology, the role of the OM in antibiotic resistance and to develop better therapeutics.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Determining the structure of Pseudomonas aeruginosa Lipopolysaccharide across spatial scales
Funder: Wellcome Trust

Abstract: Pseudomonas aeruginosa (P. aeruginosa) is a major human pathogen, needing urgent development of novel antimicrobials. It lives in bacterial communities called biofilms and severe infections are typical within hospital settings. Due to increased antibiotic tolerance of P. aeruginosa in biofilms, treatment is lacking and infections can be fatal, especially for those who are immunocompromised. Lipopolysaccharide (LPS) is the main component on the outer membrane of the bacteria, which protects the cell and provides rigidity. LPS is important for bacterial survival as it interacts with our immune signals, protecting bacteria from being killed. LPS also contributes to antibiotic resistance, due to variations of LPS in different bacterial strains. Additionally, it forms part of the physical barrier within the membrane, preventing antibiotics from entering. Using biochemical and structural techniques, including CryoEM and CryoET, I will determine what P. aeruginosa LPS looks like in vitro purified, on the outer membrane of single P. aeruginosa cells and within the biofilm environment. Accomplishing this will further our understanding of how LPS functions and reveal the structure of this essential drug target. Ultimately, this information will help us understand how to treat P. aeruginosa antibiotic resistant infections more effectively and provide insight for novel antibiotic development.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Combining computational structural biology methods to visualise the dynamics of cell-virus interactions at molecular resolution
Funder: Wellcome Trust

Abstract: Archaea are single-celled organisms distinct from bacteria, but that share a number of traits. Like some bacteria, many archaea have a surface layer (S-layer) surrounding the cell, which is a proteinaceous lattice that helps protect the cell from attack from viruses. Understanding how the viruses bind to the S-layer will help us to understand how they infect the cells.Using cryo-electron microscopy (cryo-EM), an increasingly popular technique for obtaining structural information about proteins, we will study a variety of archaeal S-layers and some of the viruses that infect them. This data will then be combined with molecular dynamics (MD) for a multiscale investigation of how these S-layer proteins move and interact over time. By solving the structures of the S-layers using cryo-EM, then simulating the interaction of the proteins in the lattice using MD, we push method development to lead the way in cellular structural biology.With this work we will develop the understanding of viral entry in archaea, and the structure-function relationship of S-layers which has implications on the evolution of life. Tomography will provide cellular context to structures, and direct the large-scale MD simulations, built up from smaller simulations of atomic structures solved by cryo-EM.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Examining the use of infectious disease modelling in international outbreak response
Funder: Wellcome Trust

Abstract: Infectious disease modelling (IDM) is a branch of epidemiology that uses mathematical models to predict the spread of disease, often through a population. It is especially useful for estimating the impact of prevention and control measures. After attending recent international conferences, the clear take-home message is that IDM research does not always reach healthcare decision-makers in a usable format, in useful time. ‘Healthcare decision-makers’ include such organisations as the WHO; Centres for Disease Control; International health NGOs; and country-specific Governments and Departments of Health. The bridge between researchers and decision-makers is especially challenging in emergency epidemic-response situations, where crucial action must be taken immediately. In applied epidemiological research, the goal should be to inform evidence-based healthcare decision-making - answering the policy questions that need an answer. How do we ensure this can be achieved effectively? This PhD seeks to analyse the use of infectious disease modelling in pandemics and large epidemics, by scrutinizing existing models and working closely with both influential modellers and global healthcare decision-makers on a broad range of outbreak case-studies. We will identify existing challenges on the interface of these two fields and work to develop a document of best practice, implementing change at the international level.


Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Drug transport through human peptide transporters
Funder: Wellcome Trust

Abstract: The human proton-coupled oligopeptide transporters PepT1 and PepT2 play a crucial role not only in the absorption of dietary nitrogen in the form of di- and tri-peptides, but also in absorption and retention of structurally similar drugs, such as beta-lactam antibiotics. Recent structural studies on bacterial homologues have highlighted the roles for key protein residues involved in transport, and molecular dynamics (MD) simulations have been used to gain insight into the free energy landscape of the alternating access mechanism of conformational changes during transport. The molecular basis of the transporter’s substrate promiscuity, that is why some molecules are transported but not others, remains poorly understood, however. In this project, we aim in the first instance to use MD to probe how binding of transported drugs affects the transporter on a molecular level. This will enable us to construct a pharmacophore model for transport to cover the wider chemical space of drugs, by helping us understand key substrate–transporter interactions which initiate transport. We will test these computational predictions in transport assays, and collaborate with synthetic chemists to design better substrates for transport. These studies will help the design of drugs and prodrugs with better pharmacokinetic properties.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Coronaviridae’s ‘Utility Belt’: Identification, Characterization and Evolutionary Analysis of the Accessory Proteome of SARS-CoV-2 and other Coronaviruses
Funder: Wellcome Trust

Abstract: Despite the great efforts of international vaccination campaigns, the COVID-19 pandemic remains a major global health threat, as no antiviral drugs are available for severe COVID-19 patients, and the emergence of mutant viral strains risks to nullify vaccinations’ efforts. SARS-CoV-2’s genome has been found to encode for several strain-specific, non-essential accessory proteins with diverse functions. Some of these are known to be expressed from overlapping sequences within structural genes (e.g. 9b/9c within the Nucleocapsid (N) gene), but other overlapping sequences’ potential for protein production remain unascertained. To identify new targets for drug development and help assess the threat variants pose to public health, it is crucial to identify these protein-coding sequences, study their products’ functions and understand their origins. Our goals are to: • Identify additional potential accessory genes encoded within SARS-CoV-2 N gene through in vitro translations and cell viability assays. • Assess their role in in vitro infection models through generation of mutant SARS-CoV-2 strains. • Dissect the dynamics of accessory gene birth/death in SARS-CoV-2 and related coronaviruses through phylogenetic analyses and dN/dS calculations. • Test the hypothesis that coronaviruses adapt to changing host environments by acquiring/losing accessory genes by expressing a functional library of coronaviruses’ accessory genes in multiple cell systems.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Structural characterisation of Tol-mediated bacteriocin translocation by electron cryotomography
Funder: Wellcome Trust

Abstract: One of the primary barriers to successful antibiotics is the bacterial cell envelope, a dense and impenetrable coat structure which protects bacteria from environmental toxins. In bacteria such as Escherichia coli, membrane-bound proteins such as Tol and Pal crosslink and stabilise the cell envelope. However, certain predatory bacterial proteins (bacteriocins) exploit these Tol-Pal proteins to bypass the cell envelope. Here we will examine: • The role of Tol-Pal proteins in stabilising the E. coli cell envelope • How bacteriocins exploit Tol-Pal to enter target cells We will investigate these questions with electron cryotomography (cryo-ET), a specialised form of electron microscopy used to image protein complexes within their native environment. We will apply this technique to bacteriocins trapped in E. coli cells, to physically observe the process of bacteriocin uptake. We will combine these data with supplementary approaches including protein mass-spectrometry, folding prediction software, and computational modelling to identify the molecular mechanisms of bacteriocin uptake. This will further our fundamental understanding of both symbiotic and pathogenic bacteria, and the mechanisms by which foreign proteins bypass the bacterial cell envelope. This may be applied to either creating synthetic bacteriocins, or fusing bacteriocins to existing antibiotics to assist cell entry.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Investigating the structure and regulation of microtubule lumenal actin
Funder: Wellcome Trust

Abstract: Microtubules and actin are core components of the cell cytoskeleton and critical for many cell processes from ranging from intracellular transport, to cell migration and division. Microtubules are helical polymers of tubulin monomers that form a hollow cylinder. Actin monomers assemble to form a helical filament. They have been understood as distinct structures but recently, actin filaments have been found in the microtubule lumen (microtubule lumenal (ML-)actin), revealing an unexpected versatility in cytoskeletal form. These ML-actin filaments were found to exist in two conformational classes, both of which have a higher degree of helical twist than canonical actin filaments. It is unclear how these structures form and how they influence microtubule properties. I will address this question using cryo-EM techniques in model HAP1 cells where ML-actin was first discovered. I will integrate these findings with biochemical experiments to reveal its regulation. I will also be relating my findings to a physiological system, platelets, where lumenal filaments have been previously observed. Understanding microtubule lumenal actin will uncover new aspects and functions of the cell cytoskeleton and reveal how they could be targeted in disease states.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Understanding the Influence of Increasing Persecution and Hostility on Delivery of and Access to HIV services among Men who have Sex with Men in Tanzania
Funder: Wellcome Trust

Abstract: Qualitative research is proposed to examine how increasing persecution and hostility towards gay, bisexual and men who have sex with men (MSM) influence delivery of and access to HIV prevention and care services in Tanzania. By situating delivery and access to HIV services among MSM in a larger context, the study builds an appreciation of how the legal and policy environment influence the supply and demand sides of healthcare as well as the implementation of global health strategies to curb the HIV epidemic. Data collection techniques will involve focus group discussion and in-depth interviews with MSM, and key informant interviews with regional AIDS control coordinators in participating regions, members of district health management teams, and healthcare providers in the non-governmental organizations and community-based organizations providing services to MSM. The study setting will be in Dar es Salaam and Shinyanga regions. Results from this study can be used to inform various service delivery strategies and increasing access to HIV prevention and care services among MSM in contexts where homosexuality is criminalised. A workshop with relevant stakeholders will be arranged to discuss how the research findings can be translated into practical policy and health service delivery regarding HIV prevention and care.

Lay abstract: This study seeks to examine the influence of increasing persecution and hostility towards men who have sex with men (MSM) on the delivery of and access to HIV prevention and care services. The setting for this study will be in Dar es Salaam and Shinyanga regions in Tanzania. I will conduct focus group discussions and in-depth interviews with MSM, and key informant interviews with health decision makers at regional and district levels as well as HIV service providers to MSM. The results of this study will inform strategies to deliver HIV prevention and care services to MSM and ways to increase their access to available HIV services in contexts where same-sex sexual practices are criminalised.

Research activity codes: 8.1 ORGANISATION AND DELIVERY OF SERVICES
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Live cell imaging capability for research on pathogenic viruses
Funder: Wellcome Trust

Abstract: We request a 75% contribution to purchase two IncuCyte S3 Live-Cell Analysis Systems, together with archive data storage capacity (£189,485). These instruments are designed to acquire real-time growth, migration, morphology, phenotypic and gene expression data from virus-infected cells in microplates. They will be located in newly-opened Biological Safety Level 3 (BSL3) and BSL2 laboratories, transforming existing capacity for real-time analysis of virus-infected cells and enabling ambitious plans for expansion of virology research. The overall aims of the research supported by these instruments are: 1) to understand the molecular mechanisms by which viruses enter cells, replicate their genomes and assemble into infectious virions, and 2) to drive early-stage development of new anti-viral therapeutics (eg screening compound libraries). The IncuCyte instruments will facilitate both work streams. They will be housed in standard cell culture incubators and can be remote controlled allowing the analysis of cell parameters over time safely without disturbance. In order to effectively train users, provide technical advice, oversee usage and manage the instruments, we also request funding for 10% of the salary of the current BSL2/BSL3 laboratory manager, and 5% of the BioImaging facility support scientist over 5 years (£31,390).

Lay abstract: Viruses must infect living cells in order to multiply. Virus multiplication cycles take many hours, during which time they hijack cellular machinery and change the inner workings of the cell to favour their own growth. To better understand how these processes occur, it is important to study the events that occur in virus-infected cells throughout the complete multiplication cycle, and the requested IncuCyte instruments will allow us to do this. Additionally, when studying viruses that cause human or animal disease we need to work safely. At Leeds, we have just invested in state-of-the-art laboratories designed for safe work with pathogenic viruses, this is where the IncuCyte instruments will be located. As well as enabling us to better understand virus growth and effects on cells, the IncuCyte instruments will allow us to establish efficient screening programmes for the identification of new anti-viral drugs, with potential long-term benefits for patients.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: The ethics of tracking infectious disease transmission
Funder: Wellcome Trust

Abstract: New ‘pathogen sequencing technologies’, which have the power to identify and describe infectious disease transmission networks, are transforming public health practice. They also raise practically important ethical questions (for example regarding privacy protection), as-well-as conceptual and normative questions concerning responsibility for disease transmission, blame and the obligations of various actors. This empirical bioethics study will combine qualitative research with normative ethical analysis to develop an account of ethical practice in the use of technologies that can identify and describe infectious disease transmission networks. This will be achieved by answering the following research question: 1. What are expert and lay views on the potential social consequences and ethical risks of identifying transmission networks through the use of pathogen sequencing technologies? 2. What ethical norms concerning transmission of infectious disease underpin these views? 3. How do the concepts of personal responsibility, blame and obligation bear on the ethics of using pathogen technologies to identify and describe infectious disease transmission networks? 4. What contextual factors influence ethical norms and ethical practice in this area?

Lay abstract: What if I could tell you, which of your healthcare-providers infected your child with the bacteria that killed them? Or which of your sexual partners gave you HIV? Would you blame them? Should I have told you? New ‘pathogen sequencing’ technologies can track and understand the spread of infectious disease more accurately than ever before. This means we can stop the spread of infection more effectively. However, this also raises important ethical questions. I will speak to members of the public and experts to identify what harms they think might arise from the use of this technology, and what ethical policy and practice should look like. I will investigate how people characterise their responsibilities and associate blame with regards to the transmission of infectious disease. I will then develop an account of ethical practice to ensure these technologies are used ethically, and can be effective in combating rising disease threats.

Research activity codes: 8.3 POLICY, ETHICS AND RESEARCH GOVERNANCE
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: An Ethics Account of Global Health Security (DPhil proposal)
Funder: Wellcome Trust

Abstract: Global health security (GHS) comprises of the activities required to reduce vulnerability to acute public health events, commonly infectious disease outbreaks, that affect populations across international boundaries. One such public health threat was the 2014 West African Ebola outbreak, which revealed failings in the ability of the World Health Organization to adequately respond to outbreaks of this severity. A consequence of this was the expansion of state-led GHS programmes in countries including the US and UK. These programmes comprise of states’ actions abroad to improve national health security. As the key aims of these programmes are not just to aid the primarily low and lower-middle income countries in which they act, but to protect the states from infectious disease outbreaks that threaten their own populations, their activities can be ethically, socioculturally and politically challenging. The research objective of this DPhil is to undertake a normative and empirical ethics account of state-led GHS programmes, focusing on the actions and stakeholders from the US and UK in the period after the 2014 West African Ebola outbreak. The project has three stages: A critical interpretative synthesis of literature, semi-structured interviews with key informants, and an empirical bioethics analysis and formation of recommendations.

Lay abstract: Infectious disease outbreaks, like the 2014 Ebola outbreak, can have devastating effects on communities. When these outbreaks spread through a region, they require international responses. Commonly we may think of the World Health Organization and medical charities as responding to outbreaks, but countries, including the UK and US, also have roles. Their global health security programmes aim to help those affected, but also to maintain the security of the UK and US by preventing outbreaks affecting their own people. Ethical issues can arise when these aims conflict. For example, stopping flights to the UK from a country with an outbreak may be a good security precaution, but can also hamper getting aid to the country. My research will look at ethical issues in UK and US programmes, by both interviewing key persons involved and analysing ethical arguments, in order to form recommendations on how these programmes should be run.

Research activity codes: 8.3 POLICY, ETHICS AND RESEARCH GOVERNANCE
Health categories: GENERIC HEALTH RELEVANCE;INFECTION