NIHRDataInsights/HRCSData huggingface dataset:
Title: FUNGAL NEIGHBOURHOOD WATCH: UNDERSTANDING HOW COMBINATORIAL SIGNALS FROM THE HOST NICHE DRIVE PATHOGENESIS IN CANDIDA ALBICANS
Funder: Medical Research Council

Abstract: Candida albicans is the most common fungus isolated from patients. This fungus infects the oral and vaginal mucosa of healthy individuals causing local inflammation and increased morbidity. In immune compromised hosts, it infects major organs, resulting in life threatening infections. Our understanding of the response the fungus has to the niche environment is based largely on the analysis of single environmental stimuli, not the multifactorial signals that occur in the niche. I will addresses this gap in our knowledge by studying combinations of stimuli that more accurately represent in vivo environments. In phase 1, I will employ large scale screening to deduce how combinatorial environmental stimuli (CO2, pH, quorum sensing molecules etc.) impact on the growth, biofilm formation, and morphology of the fungus. I will identify core environment-regulated genes by RNA-Seq and compare in vitro transcript profiles to those from in vivo samples. I will determine the role of environment-regulated genes in fungal virulence using the concurrent oral-vaginal mucosal and disseminated candidiasis infection models. In phase 2, I will determine how combinatorial signaling affects the fungal cell wall. I will use state of the art proteomics to identify proteins in the cell wall, NMR to determine the structure of the cell wall glycans, and TEM to visualise the cell wall architecture. In phase 3, I will characterise how these cell wall perturbations affect the host's immune response, focusing on epithelial cells, and neutrophil and macrophage phagocytosis. I will quantify epithelial damage by histology, cytokine secretion by ELISA and phagosome maturation by live cell imaging. In phase 4, I will develop fungal reporter strains that respond to changes in specific environmental conditions. These strains will then be use as an indicator of the environmental parameters experienced in the host niche. This proposal has the potential to identify novel diagnostics and therapeutic targets.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Molecular and Structural Basis of Cell Entry by Emerging and Zoonotic RNA Viruses
Funder: Medical Research Council

Abstract: Aims: I will elucidate the molecular mechanism by which by emergent negative-sense, single-stranded viruses undergo attachment and fusion with host cells. This investigation is limited to emergent viruses from three families: Paramyxoviridae (e.g. African Henipaviruses), Arenaviridae (e.g. Guanarito virus), and Bunyaviridae (e.g. Sin nombre virus; a list of targets is provided in the Case for Support, Table 1). Objectives: This will be addressed in the context of both intact virus and the individual structural glycoproteins responsible for viral entry. As a result, this work will integrate techniques in (1) protein chemistry and virology, (2) X-ray crystallography, (3) electron microscopy, and (4) molecular biology. These techniques are detailed below: 1. Recombinant expression and purification of viral attachment glycoproteins alone in complex with functional cellular receptors, preparation of model non-pathogenic viral orthologues and VLPs, and the acquisition/development of neutralising antibodies (nAbs) against these glycoproteins. 2. Crystallographic analysis of viral glycoproteins alone and in complex with cellular receptors and nAbs to define the molecular specificity which underlies virus-host interactions. 3. Cryo-electron microscopic analysis of intact, non-pathogenic viral orthologues into visualise the ultrastructure of these viruses and the higher order organization of their subunit glycoproteins as they extend from the virus envelope. 4. Mutagenesis of the viral genome by reverse genetics to complement structure predicted determinants for virus host cell entry. Scientific and Medical opportunities: Through the concerted effort from my laboratory, this multidisciplinary work will offer a rational description of host cell entry and enhance our ability to design therapeutics against the targeted emergent pathogens.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFECTION
Title: Prevalence of, and harm caused by medication error in antiretroviral programs in Uganda
Funder: Medical Research Council

Abstract: The project aims to quantify the prevalence of and harm caused by medication error and unmanaged drug-drug interactions (DDIs) in antiretroviral (ARV) programs in Uganda. In an outpatient study, a medication history and adherence assessment will be taken for 1000 patients across diverse sites. Medication lists will be screened for DDIs aided by an offline Android application. Information will be recorded in the clinical notes prior to scheduled clinic appointments. Prescribing and dispensing errors will be assessed. Inpatient studies will identify the prevalence, type, and severity of prescribing errors and DDIs in hospital settings. Hospital admissions will be evaluated for adverse drug events and causality in relation to medication errors and unmanaged DDIs. Primary endpoints are: the prevalence, type and severity of medication errors and DDIs; the patients most at risk of medication error in terms of CD4 count, WHO stage, number of medicines, age, gender (via multivariate analysis); and health systems factors precipitating or protecting from errors. Also, the extent of clinical harm arising from medication error and DDIs in patients taking ARVs admitted to hospital; the proportion of patients taking medication that their HIV doctors are not aware of; and the extent of DDI recognition by prescribers. Semi-structured interviews and focus groups will explore attitudes of healthcare workers to the causes of and barriers to medication errors and the acceptability of potential interventions. healthcare utilisation resulting from medication error and unmanaged DDIs will be described and reported. Recommendations to reduce and prevent harm from errors will be developed. Studies of medication error in African ARV programs are lacking, and no data exist on the extent of harm caused by error and unmanaged DDIs, although impact on patients and programs is likely to be significant.


Research activity codes: 8.1 ORGANISATION AND DELIVERY OF SERVICES
Health categories: INFECTION
Title: The MRC Consortium for Medical Microbial Bioinformatics
Funder: Medical Research Council

Abstract: The MRC CONSORTIUM FOR MEDICAL MICROBIAL BIOINFORMATICS (CMMB) will enhance UK capability and infrastructure in microbial bioinformatics. Led by Mark Pallen and Sam Sheppard, the CMMB will recruit THREE HIGHLY TALENTED INDIVIDUALS into UK medical microbial bioinformatics from careers outside the discipline or the country. We will develop a DISTRIBUTED COMPUTING INFRASTRUCTURE that will operate together to provide: 1. AN AGILE, SCALABLE SYSTEM available to consortium members that can be dynamically provisioned to handle different workloads/projects as needed; this will include: -FOUR CLUSTERS (one on each site) providing a heterogeneous mix of high-memory/low CPU and low-memory/high CPU servers suitable for both memory-intensive tasks (e.g. metagenomic assembly) and CPU-intensive activities. -SUBSTANTIAL STORAGE (>2 petabytes). -sufficient NETWORK CAPACITY for each site to operate at 10 gigabit/second connectivity. 2. a web-based instance of the GALAXY PLATFORM customised for medical microbial research. 3. a freely accessible DATABASE of relevant workflows, pipelines, scripts, programs, virtual machine images built on Galaxy/Github and mirrored across our sites. 4. a DATA ARCHIVE of relevant microbial (meta)genomes 5. a computational infrastructure for linking PATIENT METADATA with microbial (meta)genomic data. We will develop an ambitious and exciting TRAINING PROGRAMME that will include bootcamps, hackathons, workshops, modules and courses, suitable for a wide range of users from professional bioinformaticians to undergraduate students. We will strengthen national microbial bioinformatics research through COMMUNITY-BUILDING ACTIVITIES, encouraging knowledge transfer and dissemination of best practice. We will exploit pump-priming funds together with externally funded research activities to "stress our systems", confirming that the facilities that we have created work as planned and/or priming ITERATIVE REFINEMENTS TO OUR INFRA-STRUCTURE


Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: INFECTION
Title: A structural and functional characterisation of the L-polymerase replication complex of the Monanegaviruses
Funder: Medical Research Council

Abstract: The Mononegavirales order of negative-sense single-stranded RNA viruses comprises many significant human pathogens. The L-polymerase (MW ~250kDa) is key to the replication of these viruses, and is an ideal drug target. No structures of L-pol or domains from Mononegaviruses have been published and molecular mechanisms are not understood. We recently solved a C-terminal domain of L-pol of hMPV (CR-VI+), and showed that it is responsible for cap synthesis. We propose to capitalise on this success, by structural and activity studies on L-pols of other, medically important mononegaviruses, whilst also carrying out more in-depth analysis of CR-VI+ of hMPV (and other members of the pneumovirinae subfamily). Bioinformatics will inform the decision on which representatives from a large pool of L-pol genes are likely to express. Synthetic genes will be used, optimised for our eukaryotic expression systems (often crucial in the expression of "difficult to express" proteins). Small-scale expression tests will fine-tune the construct boundaries, and select the most suitable expression systems, prior to/in preparation of large-scale protein expression, purification and crystallisation. Crystals will be analysed at Diamond Light Source (Harwell, UK). Once suitably diffracting crystals are obtained, standard methods will be employed to solve the structures. Recombinant protein will also be used for relevant activity studies (in vitro polymerase-, phosphohydrolase-, guanylyltransferase-, methyltransferase- and polyadenylation assays), in collaboration with Dr E. Decroly at the AFMB ( http://www.afmb.univ-mrs.fr). Testing of structure based hypothesises and the effect on virus replication, using cell-based reverse genetic systems, will be done in collaboration with Dr B. van den Hoogen (Erasmus MC) and Dr H. Bourhy (Pasteur Institute). Cryo-EM will be utilized for imaging of L-pol, and crystal structures will be fitted into 3D EM reconstructions


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;1.3 CHEMICAL AND PHYSICAL SCIENCES
Health categories: INFECTION
Title: The role of small regulatory RNAs in Mycobacterium tuberculosis pathogenesis
Funder: Medical Research Council

Abstract: The proposed project aims at investigating various aspects associated with M. tuberculosis small RNAs (Mtb sRNAs). I propose to characterize stimuli and regulators of expression, the targets that the sRNAs regulate, the role of one of the main RNases and whether the sRNAs require chaperones to facilitate interactions with their targets. Finally, I want to investigate the role that these sRNAs play in Mtb pathogenesis. In order to investigate stimuli and regulators of expression I will subject cultures of Mtb to infection related stresses (e.g. oxidative stress, NO stress, hypoxia, iron depletion) and quantify the expression of each sRNA using northern blotting and qRT-PCR. In order to define regulons of the sRNAs, I will employ a panel of Mtb strains in which the expression of each sRNA can be manipulated (deletion mutants/complemented/over-expression strains). The strains will be subjected to different growth conditions in vitro and used to infect macrophages and mice via low-dose aerosol. RNA will be extracted and differential gene expression will be characterized using microarrays. To further identify targets I have devised a strategy for isolating proteins and (m)RNAs that interact with the sRNAs. Aptamer-tagged versions of each sRNA will expressed in vitro and used to isolate interacting molecules from Mtb cell extracts using commercially available streptavidin beads. The tagged sRNAs will subsequently be introduced into their cognate knockout background, cultures will be grown under appropriate conditions and sRNA bound to interaction partners isolated from cell extracts. Weak interactions can be stabilized with an optional UV irradiation step before cell disruption. Isolated proteins will be identified by mass spectroscopy. Isolated RNA will be characterized by RNAseq.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Is targeting vascular remodelling by filarial parasites a viable anti-morbidity solution?
Funder: Medical Research Council

Abstract: This proposal seeks to address cellular and molecular mechanisms of the filarial pathologies lymphoedema and ocular kerititis. The project is focussed on the role of blood and lymphatic endothelial remodelling events (including neovascularisation; NV, angiogenesis, dilation, contusion and fibrosis), the molecular processes that initiate or prevent remodelling during filarial inflammation and the pathological ramifications of their occurrence. A second major aim of the proposal will be to test whether these putative pathogenic processes can be successfully targeted by registered small molecule drugs, novel small molecule drugs or novel biologic therapeutics. The proposal will utilise preclinical murine models of dermal, lymphatic and ocular filarial pathogenesis to examine these hypothesised disease pathways. Canonical pro-angiogenic (VEGFR2) or pro-lymphangiogenic (VEGFR3) pathways will be manipulated using a combination of specific murine reagents including antagonistic or neutralising antibodies, Fc-fusion receptors (Traps), agonistic Fc-fusions or transgenics over-expressing agonists. Mouse lines with specific fluorescent protein gene knock-ins into lymphatic endothelium will be exploited in addition to standard immuno-staining to examine morphological changes in blood and lymphatic vasculature by confocal microscopy. Lymphatic reporter mice will also be used in a lymph node transplant model to explore a putative immune cellular mechanism of lymphatic repair by multi-photon confocal microscopy. A non-invasive, optical imaging-based technique will be applied to study effect of filarial parasitism on the development of lymphatic insufficiency and how specific cellular or biologic treatments may improve this pathology. Antiangiogenic drugs or drugs with possible antiangiogenic properties will be tested for anti-pathology efficacy. Finally a novel VEGFTrap molecule will be engineered to examine whether this offers an improvement on standard treatment for ocular NV.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: The impact of IFTIM3 genetic variation on Influenza virus infection, immune responses and disease outcome
Funder: Medical Research Council

Abstract: 1. The effect of the C variant on IFITM3: virus entry and intracellular location. We will employ optical microscopy including different fluorescent labeling approaches such as protein-specific antibodies or live-cell tagging technologies such as GFP or SNAP- and HALO-tags. Optical super-resolution microscopes (or nanoscopes), including the STED/RESOLFT or PALM/STORM technologies will enable us to explore the subtle changes in the spatial organization of IFITM3 between variants. 2. IFITM3 genotype impact on innate sensing of influenza A virus and virus entry. Primary fibroblasts will be grown from skin biopsies. Subsequently, these cells will be infected with IAV. The induction of chemokines and cytokines will be measured by ELISA, bioassays or RT Q-PCR. The accumulation of RIG-I stimulatory RNA in cells of different genotypes will be assessed by RNA extraction from infected cells and re-transfection into RIG-I reporter cells. JR has experience with this approach. In these experiments primary human cells will be used and complemented by human cell lines overexpressing or depleted of IFITM3, as well as IFITM3-deficient mouse fibroblasts. 3. Immune responses in IFITM3 CC/TT patients who do badly compared to those who do well. a. Trascriptome profiles will be determined using Illumina Human HT-12 V3 Beadchip arrays, which contain more than 48,000 probes. A gene expression analysis software program, Genespring, version 7.1.3 (Agilent), will be used to perform statistical analysis, and hierarchical clustering of samples. A modular analysis strategy will be used as described, enabling the discrimination of clinical groups without reducing the dimensionality of the data. b. Detailed analysis of influenza specific T cell responses will be performed using overlapping peptides in the ELISPOT assay. In some cases, T cell line and clones will be grown and antiviral efficacy will be evaluated using target cells infected with different viral strains


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Global Approaches to Elucidate the Function of Post-transcriptional Networks in HIV-1 Infection
Funder: Medical Research Council

Abstract: HIV-1 encodes only 15 distinct polypeptides, so it relies heavily on host proteins for replication. After reverse transcription, HIV-1 proviral DNA is integrated into the cellular chromosome. This allows viral RNAs to be synthesized, processed, exported and translated by the host cell machinery. This proposal aims at comprehensively identifying the catalog of host RNA-binding proteins (RBPs) involved in the posttranscriptional regulation of HIV-1 gene expression by applying novel system-wide methodologies built on protocols developed during my postdoctoral work. HIV-1-infected cells will be irradiated with ultraviolet light to crosslink proteins to RNA. HIV-1 RNAs will be isolated through stringent washes using specific locked nucleic acid probes. Co-isolated proteins will be identified by mass spectrometry. I will also analyze the RBPs activated and inactivated by HIV-1 infection using standard "mRNA interactome capture" protocol. Functionally relevant RBPs will be selected by applying sophisticated data analysis and their effects in HIV-1 infection will be evaluated in functional assays. Those RBPs causing strong phenotypes in viral replication will be characterized at the molecular level to understand their role in HIV-1 gene expression. The impact of one selected RBP in systemic HIV-1 infection will be determined using mouse transgenic models. This proposal may reveal the host circuits controlling HIV-1 gene expression, representing an important resource to expand our knowledge in HIV-1 biology.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Mitochondrial biogenesis and function in trypanosomes
Funder: Medical Research Council

Abstract: Trypanosomatid parasites cause human and animal diseases with devastating health and economic consequences. Their survival crucially depends on mitochondrial (mt) function and expression of the organellar genome. Mitochondrial activity is highly regulated during the life cycle of the sleeping sickness parasite Trypanosoma brucei. Aim 1 uses an RNAi screen to identify genes that are important for mtDNA maintenance and expression in T. brucei. Some of these genes will be trypanosomatid-specific genes and potential drug targets. Characterisation of the corresponding proteins will involve localisation by light and electron microscopy and functional categorisation using a panel of assays that interrogate the various steps along the maintenance/expression pathway. Some proteins will be characterised further by pull-down of tagged versions and identification of interaction partners by mass spectroscopy. Aim 2 identifies mtDNA-encoded functions required for differentiation from mammalian bloodstream stage to tsetse midgut stage, and dissects the mechanism of the compensatory ATPase mutation that permits bloodstream stage survival in the absence of mtDNA. Pleomorphic T. brucei parasites that lack mtDNA or key nuclearly encoded complex I subunits will be tested for their ability to differentiate in vitro and in mice and flies. The cause of any differentiation defects will be investigated by analysis of mt and cellular metabolism. The composition and function of wild type ATPase will be compared with that of mutated enzyme in the presence and absence of mtDNA-encoded subunits. Aim 3 identifies the complete T. brucei mtDNA minicircle and guide RNA repertoire by deep sequencing. Guide RNAs and their target mRNAs will be verified by in vivo cross-linking and deep sequencing. The fidelity of minicircle distribution to daughter cells will be determined by measuring changes in complexity over time and feeding the data into a mathematical model.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFECTION
Title: A structure analysis of the intact virion and replicative complexes of human respiratory syncytial virus
Funder: Medical Research Council

Abstract: Respiratory syncytial virus (RSV) is a pneumovirus in the family Paramyxoviridae. The viral genome is a negative sense RNA that is encapsidated by a viral protein N, to form a helical ribonucleoprotein (RNP) called the nucleocapsid (NC). The NC is the template for RNA synthesis by the viral RNA dependent RNA polymerase (RdRp - comprised of three viral proteins, L, P and M2-1), together the NC and RdRp make up the holonucleocapsid (hNC). RSV is an enveloped virus and assembly is directed by the M protein that binds the hNC, trafficking the complex to the plasma membrane, where interactions with the envelope glycoproteins F and G promote virion morphogenesis. The aim of this project is to use cryo-electron microscopy and tomography (CEM and CET) to characterise RSV at intermediate (10-30 angstroms) to high (6-10 angstroms) resolution. We have established methods for the propagation of cells on TEM grids for CEM. RSV infected cells produce long filamentous virions which we imaged by CEM and CET without incurring preparation artefacts. Previous CET of purified paramyxoviruses has shown poorly ordered bag-like particles with tangled NCs inside. Our data show that RSV filaments are highly ordered. CET and Fourier analysis of CEM images shows that RSV exhibits helical symmetry, possibly extending throughout the envelope from the M layer to the glycoproteins. Helical reconstruction will give insights into M oligomerisation and interactions with the viral envelope and F/G proteins. Sub-tomogram averaging of CET data will build on our X-ray and CET studies of the NC by providing data on the terminal structures and possibly the RdRp. Purification of recombinant RdRp and hNC will allow higher resolution CEM and single-particle studies. Finally cryo-sectioning of RSV infected cells using focussed ion beam milling (FIB) will allow CET of virus assembly at the plasma membrane, potentially providing structural insights into the involvement of host-cell factors such as actin.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION