NIHRDataInsights/HRCSData huggingface dataset:
Title: Flow Cytometry for Investigating HIV Infection of Living Cells
Funder: Wellcome Trust

Abstract: Funding is requested to purchase a Becton Dickinson FACSMelody Benchtop Cell Sorter System that will be dedicated to analysing and/or purifying living cells either obtained from HIV-1 infected patients, or purposefully infected with HIV-1 in culture. The sorter will be located in the KCL containment level 3 (CL3) laboratories at Guy’s Hospital. Flow cytometry is a recognised strength at KCL, but none of our facilities offer CL3 capability, thus prohibiting any flow cytometry-based analysis or purification of living cells infected with HIV-1, or experimental workflow that requires downstream steps involving living cells. The acquisition of the FACSMelody will be transformative for our HIV/AIDS research community, and for our collaborators, by enabling the pursuit of new projects as well as the application of additional technologies such as single cell cloning and analytics to existing projects. Our work will address key areas relevant to understanding HIV-1 immunopathogenesis, including: HIV-1’s ability to manipulate normal cell function, the role(s) of cell-encoded factors in productive HIV-1 replication, anti-viral innate immunity and mechanisms of viral evasion, the molecular basis for HIV-1 persistence/ latency and its reversal, adaptive T and B cell immune responses in infants and adults, and mechanisms of HIV-1 induced immune cell dysfunction.

Lay abstract: HIV-1 is a deadly virus that causes AIDS, with the ongoing global epidemic remaining a critical health and humanitarian concern. Scientists at KCL investigate a number of fundamental research questions aiming to understand how HIV-1 grows, persists in an infected patient, and causes disease and death. The use of flow cytometers is central to this type of work as it enables the purification and analysis of individual virus infected cells and cell populations. However, laboratory investigations with infectious/ living HIV-1 must be undertaken under specialised biological containment conditions called CL3. We are requesting the funds to install a flow cytometer in the CL3 laboratories at KCL, as this will significantly extend the scope and impact of the HIV/AIDS research that we and our collaborators undertake. Novel areas of experimentation will provide new information central to the development of approaches for preventing, treating or curing HIV/AIDS, including vaccination.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;INFLAMMATORY AND IMMUNE SYSTEM
Title: Deciphering the role of phagosomal ubiquitylation in innate immunity
Funder: Wellcome Trust

Abstract: Phagosomes are key organelles in innate and adaptive immunity. They are membranous compartments formed by phagocytosis of large particles by specialised cells such as macrophages. Phagosomes mature by fusion with endosomal and lysosomal populations to form phagolysosomes where the foreign matter within is degraded. Several human pathogens, including Mycobacterium tuberculosis, are able to survive and replicate within phagosomes by inhibiting their maturation, highlighting the importance of understanding phagosome functions. As they are in direct contact with the prey, phagosomes are hubs for innate immune signalling, which is distinct from signalling at the plasma membrane. However, the mechanistic basis of phagosome mediated cell signalling is poorly understood. Our recent data show extensive and complex polyubiquitylation of phagosomal proteins which regulate vesicle trafficking and serve as scaffolds for the recruitment of pro-inflammatory signalling complexes. Using a combination of proteomics, cell biology and biochemistry, my team will define the role of phagosomal protein ubiquitylation in general and identify how key ubiquitin regulatory enzymes control phagosomal innate immune responses to bacterial infection. For the first time I will establish at a molecular level how ubiquitylation regulates phagosomal innate immune responses and additionally provide details about the general role of ubiquitylation in the endo-lysosomal system.

Lay abstract: In the human body immune cells kill invading microbes by ‘eating’ them in a process called phagocytosis. After uptake, the microbes reside in a specialised compartment called the phagosome where they are normally killed and degraded. Some bacteria, however, such as tuberculosis-inducing Mycobacteria, have evolved to survive within the phagosome. Immune cells have receptor proteins that help identify the cargo that they have just phagocytosed. In order to signal this information and initiate the appropriate immune response, they use a protein modification called ubiquitylation. In this proposal, we aim to identify how ubiquitylation changes the activity and signalling from the phagosome and how this process helps to regulate the microbe’s destruction and to alert the immune system. Our groundbreaking research will help understanding molecular mechanisms of infection which is particularly important at a time when many bacteria are becoming resistant to antibiotics.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;INFLAMMATORY AND IMMUNE SYSTEM
Title: Use of the cytoskeleton to control Shigella infection
Funder: Wellcome Trust

Abstract: The cytoskeleton occupies a central role in innate immunity by promoting bacterial sensing and executing antibacterial functions. We recently discovered that mitochondria promote septin assembly into cages that entrap Shigella for antibacterial autophagy, highlighting new roles for the cytoskeleton in host defence. However, the processes underlying septin cage assembly, and the breadth of roles for septins in bacterial infection control, remain to be established. To investigate the cell biology of Shigella infection in vivo we developed zebrafish infection models which provide unprecedented opportunities to follow innate immunity at a resolution that cannot be achieved using any other animal model. I will now exploit the novelty of septin biology, and its direct link to host defence, as the foundation for this research programme. Using Shigella I will: (1) Discover new roles for the cytoskeleton in host defence against bacterial infection, and (2) Investigate the role of septin-mediated host defence mechanisms in vivo using zebrafish models of infection. The results generated from this research programme will provide fundamental advances in understanding septin biology and cellular immunity. They could also suggest the development of new strategies aimed at combating infectious diseases, and possibly other human diseases in which septins have been implicated.

Lay abstract: The intracellular bacterium Shigella flexneri has emerged as an exceptional model pathogen to address key issues in biology, including how bacteria can move inside host cells or be recognized by the immune system. I discovered that host cells employ septins, a poorly understood component of the cytoskeleton, to restrict the motility of Shigella and target them for destruction by autophagy, an important mechanism of innate immune defence. We recently established the antibacterial activity of septin caging, and discovered a fundamental link between mitochondria and the assembly of septin cages around Shigella. A major issue is now to fully decipher the underlying molecular and cellular mechanisms, and to validate these events in vivo using relevant animal models. I developed zebrafish infection models to study the cell biology of Shigella infection in vivo and to discover new roles for septins in host defence against bacterial infection. This approach has enabled a cutting edge platform for in vivo studies both at the single cell and whole animal level. Collectively, the results generated from my proposed research will provide fundamental advances in understanding cellular immunity. This information should provide vital clues towards understanding bacterial disease and for illuminating new therapeutic strategies.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;INFLAMMATORY AND IMMUNE SYSTEM
Title: Studying lentiviruses to understand mechanisms, regulation and consequences of nucleic acid sensing
Funder: Wellcome Trust

Abstract: I propose a new overarching hypothesis that innate immune sensing of nucleic acids, cell cycle regulation, and a hitherto unappreciated function of endogenous viral elements, contribute to an integrated system of regulating inflammatory responses to infection. Encouraged by preliminary data we will investigate the relationship between cell cycle and innate immune sensing by manipulating cell cycle and measuring responses to infection and relating expression of endogenous viral elements to cell cycle and their activation of nucleic acid sensing. We will also study the mechanisms of lentiviral Vpx/Vpr proteins taking molecular genetic and structural approaches to examine their enhancement of viral expression, manipulation of endogenous element expression and inhibition of innate immune signaling. We will also investigate the role of G3BP1 stress granules and their role in coordinating nucleic acid sensing with regulation of translation by PKR, mediated through cyclophilins. Overall our approach is to manipulate cellular pathways, for example cell division, and test sensing responses or manipulate virus and measure activation of sensing or viral inhibition of sensing. We expect to reveal mechanisms of nucleic acid sensing that can be tractably inhibited therapeutically and provide new mechanistic knowledge of inflammatory pathways broadly relevant to infectious and inflammatory diseases.

Lay abstract: Activation of immune responses against viruses depends on detection in a process called sensing. We have discovered that cells briefly turn off sensing as they divide. We propose to discover how cell division regulates sensing, if this creates a window of susceptibility to infection and how viruses manipulate this to evade detection. Half of our DNA genome encodes viral sequences from ancient infections. We suspect that they play an important role in amplifying host defences because when viruses infect cells, we see these old sequences being switched on. We will determine how they are activated and whether they contribute to sensing infection. We will also determine the role of proteins called cyclophilins, which regulate sensing in interesting ways that we don’t understand. Overall our work promises to reveal new ways that cells regulate sensing, how viruses escape them, and how we might manipulate sensing to treat infection and associated diseases.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;INFLAMMATORY AND IMMUNE SYSTEM
Title: Importance of N-glycosylation at the Neuromuscular Junction
Funder: Medical Research Council

Abstract: This Proposal has 3 aims: Aim 1: study the structure and function of proteins involved in the N-glycosylation pathway, and how mutations found in patients affect their function and interactions. Methods: use suspension adapted insect and mammalian cells for large scale protein production, LIC methods to produce DNA vectors, and high throughput purification and crystallisation methods to obtain protein crystals. X-ray diffraction data will be collected at Diamond Light Source. Enzyme kinetics will be assessed using colorimetric assays, rapidfire-QTOF, and TLC based assays using radioisotope labelled substrates. Protein-protein interactions will be assessed in vitro using co-affinity purification, SEC-MALS, and SPR, and in cells using super resolution microscopy. Scientific opportunities: create protein structural and biochemical tools for studying the fundamental properties of these proteins, as well as the effect of disease genotypes. Aim 2: examine how mutations identified in CMS patients lead to changes in the glycosylation of key glycoproteins involved in NMJ development and function. Methods: immortalised muscles cells derived from CMS patients and controls will be differentiated into myotubes. Agrin will be added to stimulate AChR clustering. NMJ glycoproteins will be purified from these patient and control cells using cell fractionation, affinity purification, SEC, and IEC. Glycomics analysis of purified glycoproteins to determine the differences between the glycosylation of patient and control cells. Scientific opportunities: create a tissue platform for studying glycosylation-associated CMS. Aim3: Test methods of treatment. Methods: Use protein and cell platforms developed in 1) and 2) to test different methods of altering the enzymatic flux of the N-glycosylation pathway, and see if they can rescue the effect of disease causing genotypes. Medical opportunities: produce potential therapeutic methods for treating glycosylation-associated CMS.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;MUSCULOSKELETAL;NEUROLOGICAL
Title: Ultrastructural imaging
Funder: Wellcome Trust

Abstract: This application is for funds to purchase a Transmission Electron Microscope (TEM) and necessary accessories to enable post-embedding immuno-electron microcopy to be performed. We also request funds for salary support for a full-time specialist technician to oversee tissue preparation, sectioning and microscope operation. The microscope will be located within the imaging facility within Capella (due to open in November 2018), a new research-dedicated building on the Cambridge Biomedical Campus (CBC), which will house the Wellcome Trust-MRC Cambridge Stem Cell Institute (CSCI) and the Cambridge Institute of Therapeutic Immunology and Infectious Disease (CITIID), and will be available for use by other research groups across the CBC. Access to ultrastructural imaging is essential for many research groups working with Capella and across the CBS. This investment will establish a core EM facility on the CBC, the importance of which is reflected by both the large number of PIs who have expressed their support for this application and the wide range of research questions for which ultrastructural imaging data will play a key role. The establishment of a core EM research facility for the CBC will provide the basis for subsequent expansion, enabling, for example, addition of equipment for 3D ultrastructural reconstruction.

Lay abstract: The Biomedical Campus at the University of Cambridge’s School of Clinical Medicine undertakes a very wide range of research programmes aimed at achieving a better understanding of the healthy and diseased body with a view to developing new ways of treating and managing some of the most devastating diseases. These investigations involve a very wide variety of approaches from the whole body down to the individual molecules of which it is composed. One of the most powerful approaches is to examine cells and their components of which they are comprised at extremely high magnification. This is possible using a microscope called a transmission electron microscope (TEM). This is a very widely used approach throughout the campus. However, the existing microscope are now obsolete. This application is to provide funds for purchasing a state-of-the-art new TEM that will be extensively used and is of central importance to our research endeavours.

Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;NEUROLOGICAL
Title: Integration of kinases with membrane trafficking machinery
Funder: Wellcome Trust

Abstract: Endocytosis is a highly dynamic process that determines the composition of the plasma membrane (PM). The dysfunction of endocytic pathways is a causal factor behind a number of human genetic diseases, and its dys-regulation can contribute to neurodegeneration. Despite the critical role that endocytosis plays in health and disease, surprisingly little is known about how it is regulated. A major potential regulation point is the dynamic phosphorylation of endocytic components by kinases. However, it is poorly understood which kinases (and phosphatases) control endocytosis and the PM proteome, even though they represent potential targets for disease-modifying therapies. My long-term vision is to understand the integration of protein kinases within membrane-trafficking machinery. My starting focus is on the Numb-associated kinase family (NAK). NAKs were identified as a susceptibility factor in Parkinson’s disease and neuropathic pain but the molecular mechanisms behind this are not clear. I will elucidate the key cellular functions of NAKs: (1) which cellular processes and substrates are associated with NAKs (2) the consequences of their activity for cellular physiology and (3) the regulatory processes and interaction partners which govern NAK activity states.

Lay abstract: All living cells are surrounded by cell membranes. These are essential to keep the inside of the cell separate from its outside surroundings, but are also very dynamic, controlled structures to make sure that the cell remains healthy and functioning within its outside environment. Problems in the functioning of cell membranes can result in disease. It is therefore important to understand the complex processes which control cell membrane structure and function. I am studying the process of ‘endocytosis’ – an important way for cell membranes to transport materials from outside to inside. My research is focused on a group of proteins called ‘NAKs’. Using cells grown in dishes, I will measure the exact mechanisms of NAKs to control endocytosis. NAK proteins may also have a role in some diseases of the nervous system, and so my research will inform efforts to discover and develop new treatments targeting NAKs.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;NEUROLOGICAL
Title: Cargo transport by dynein/dynactin
Funder: Wellcome Trust

Abstract: Eukaryotic cells rely on motor proteins for their internal organization and movement. I want to ask how one cytoplasmic dynein, together with its cofactor dynactin, can be responsible for almost all of the minus-end directed microtubule transport in our cells. My group will focus on endosome transport as a model system to understand which adaptor proteins are needed to link dynein to cargos. We will use in vitro reconstitution and cryo-EM to ask how different candidate adaptors interact with dynein/dynactin. We will also use structural approaches to understand how the different adaptors are themselves linked to membrane surfaces. To complement this approach we will purify endosomes and use cross-linking and mass spectrometry to search for any missing components required for dynein binding. We will take advantage of recent advances in cryo-electron tomography (cryoET) to ask how dynein/dynactin complexes and their associated connections are arranged on endosomes, both in vitro and in axons of cultured neurons. These studies will uncover the principles governing dynein/dynactin interactions with cargos and will allow us to ask how the process can be hijacked by pathogens such as herpes or rabies viruses.

Lay abstract: The components within our cells are arranged and moved by motor proteins walking along microtubules. The dynein motor and its cofactor dynactin form a transport machine that travels toward the center of the cell. It carries many different cargos, from organelles such as mitochondria to toxic aggregated proteins, and is hijacked by disease causing viruses and bacteria. Many different proteins have been proposed to act as adaptors to link dynein/dynactin to its cargos. In this proposal we ask which of these candidates are crucial for transport and search for any previously unidentified components. We use cryo-EM and X-ray crystallography to understand the structural basis by which the adaptors link dynein/dynactin to cargos. We take advantage of recent advances in cryo-electron tomography to observe how motors are arranged on moving organelles both in vitro and in neurons. Our work will lay the framework for understanding how dynein connects to its cargos.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION;NEUROLOGICAL
Title: Structure and Mechanism of Key Nonsense-Mediated mRNA Decay Factor Complexes
Funder: Wellcome Trust

Abstract: Nonsense-mediated mRNA decay (NMD) is an essential eukaryotic surveillance mechanism to eliminate aberrant and potentially harmful mRNAs that contain a premature termination codon (PTC). The discrimination of a PTC from a correct stop codon during active translation is key, but the underlying molecular mechanisms remain elusive. We recently discovered a role for the NMD factor UPF3B in delay of translation termination in vitro, mediated by new interactions of UPF3B with ribosome, release factors and UPF1. This puts UPF3B at centre stage of the events at the terminating ribosome. To validate our discovery and to discover novel interactions of NMD factors, I will establish in vivo cross-linking/mass spectrometry. I will explore the impact of disease-conferring mutations in NMD factors UPF3B and SMG1 kinase on functionally important protein-protein interactions, and characterise novel functions of UPF1 and UPF3B at near-atomic resolution in an integrative approach, combining biochemistry, biophysics, electron cryo-microscopy and crystallography or NMR. This highly interdisciplinary research aims at a step-change in our molecular-level understanding of the role of the individual NMD factors in a paramount step of human translational control; a vital prerequisite for the development of new intervention strategies to treat NMD-related disease.

Lay abstract: The genetic information in our bodies is transcribed into a molecule called messenger-RNA. This mRNA message is translated into proteins which catalyse biological function. At times, these mRNAs contain a translation termination signal at the wrong place (a PTC), giving rise to truncated proteins, some of which are toxic and cause severe disease. The cell has devised a vital quality-control mechanism, called nonsense-mediated mRNA decay (NMD), to recognise and eliminate such faulty mRNAs. Despite its paramount importance, our understanding of key NMD mechanisms remain elusive. We do not know how the NMD factors orchestrate the vital step of recognising faulty mRNAs. Recently, we discovered novel NMD factor interactions which may unlock this enigma, which we will now test in living cells and in vitro, e.g. using electron cryo-microscopy to visualise how these proteins function and thus reveal the interplay of the protein-synthesis and the quality-control machinery, in health and disease.

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