Biology subjects

Owen, D. R.

Publications and source records attributed to Owen, D. R..

8 recordsLinked to original sources

bioRxiv2025

Characterising the microbial and antimicrobial resistance signatures of hospital-acquired pneumonia using nanopore metagenomic sequencing

Hospital-acquired pneumonia (HAP) is a significant burden in nosocomial settings, yet its microbial underpinnings remain poorly understood. Here, we leverage shotgun nanopore sequencing to characterise the respiratory microbiomes of 250 HAP patients in a UK multi-site cohort, validating these using paired PCR and culture assays. Sequencing identified the dominant microbes implicated in HAP, including detection of probable pathogens in 49 PCR- and culture-negative cases. We found a high prevalence of fungi in 81/239 (34%) in HAP patients, of whom 26/81 (32%) were PCR/culture-negative, suggesting that fungi may represent an under-investigated component of HAP, whether as colonists or pathogens. Although HAP is clinically sub-categorised based on the use and duration of ventilation before disease onset, we found that the microbial profiles of these sub-groups were indistinguishable. We also found a concerningly high proportion of multi-drug-resistant microbes in HAP patients, with 21% of assembled bacterial genomes harbouring acquired antimicrobial resistance (AMR) genes that confer resistance to at least three classes of antimicrobials. This included high AMR gene carriage associated to Staphylococcus epidermidis, which may be an important reservoir of AMR, though typically viewed as a commensal. Our work provides extensive metagenomic characterisation of HAP, underscores the value of metagenomics in describing its complex aetiology, and further prompts its potential role for pathogen detection, resistance profiling and treatment.

microbiology↗

bioRxiv2023

Chemical Tools for the Gid4 Subunit of the Human E3 Ligase C-terminal to LisH (CTLH) Degradation Complex

We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.

biophysics↗

bioRxiv2023

Altered astrocytic and microglial homeostasis characterizes a decreased proinflammatory state in bipolar disorder

Multiple lines of evidence point to peripheral immune alterations in bipolar disorder (BD) although the activity of brain immune mechanisms remain largely unexplored. To identify the cell type-specific immune alterations in the BD brain, we performed a proteomic and single nuclear transcriptomic analysis of postmortem cingulate cortex samples from BD and control subjects. Our results showed that genes associated to the genetic risk for BD are enriched in microglia and astrocytes. Transcriptomic alterations in microglia point to a reduced proinflammatory phenotype, associated to reduced resistance to oxidative stress and apoptosis, which was confirmed with immunohistochemical quantification of IBA1 density. Astrocytes show transcriptomic evidence of an imbalance of multiple metabolic pathways, extracellular matrix composition and downregulated immune signalling. These alterations are associated to ADCY2 and NCAN, two GWAS genes upregulated in astrocytes. Finally, cell-cell communication analysis prioritized upregulated SPP1-CD44 signalling to astrocytes as a potential regulator of the transcriptomic alterations in BD. Our results indicate that microglia and astrocytes are characterized by downregulated immune responses associated to a dysfunction of core mechanisms via which these cells contribute to brain homeostasis.

neuroscience↗

bioRxiv2023

Etifoxine inhibits NLRP3 inflammasome activity in human and murine myeloid cells

BackgroundMultiple sclerosis (MS) is a chronic neuroinflammatory disease that is characterized by immune-mediated demyelination within the central nervous system. NLRP3 inflammasome activation has been previously reported as a possible pathophysiological contributor to microglial activation and oligodendroglial loss in MS, particularly in progressive forms of the disease. MethodsUsing both in vivo and in vitro approaches, this report investigated the use of a previously described ligand of the 18kDa translocator protein (TSPO), etifoxine, as an immunomodulator that inhibits inflammasome activation in primary human and murine macrophages and microglia. To further elucidate pathologic relevance in the MS context, investigations were also performed ex vivo using peripheral blood mononuclear cells and purified CD14+ monocytes derived from secondary progressive MS patients. ResultsHerein, it is demonstrated that etifoxine attenuated clinical symptoms in a mouse model of MS and significantly inhibited NLRP3 inflammasome activation in human and murine myeloid-derived cells in vitro by decreasing inflammasome-associated genes and inflammatory cytokine production. These anti-inflammatory effects of etifoxine were mediated independently of its previously described mechanisms related to engagement with TSPO and the GABAA receptor. Furthermore, we observed a similar anti-inflammatory effect of etifoxine on MS patient-derived monocytes, which provides clinical relevance for the investigation of etifoxine as a potential therapeutic in progressive MS. Lastly, through the use of a gene array, we identified multiple signalling pathways in order to elucidate a novel mechanism whereby etifoxine may be inhibiting NLRP3 inflammasome activation. ConclusionsOur results suggest that the anti-inflammatory effects of etifoxine were mediated independently of its previously described mechanisms related to engagement with TSPO and the GABAA receptor. Furthermore, we observed an anti-inflammatory effect in murine- and human-derived myeloid cells, as well as in MS patient-derived monocytes, which provides clinically relevant evidence to support the exploration of etifoxine as a possible form of therapy for secondary progressive MS.

neuroscience↗

bioRxiv2023

A chemical probe to modulate human GID4 Pro/N-degron interactions

The CTLH complex is a multi-subunit ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via the substrate receptor GID4. Recently, focus has turned to this complex as a potential mediator of targeted protein degradation, but the role GID4-mediated substrate ubiquitylation and proteasomal degradation plays in humans has thus far remained unclear. Here, we report PFI-7, a potent, selective, and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation enabled the identification of dozens of endogenous GID4-interacting proteins that bind via the GID4 substrate binding pocket, only a subset of which possess canonical Pro/N-degron sequences. GID4 interactors are enriched for nuclear and nucleolar proteins including RNA helicases. GID4 antagonism by PFI-7 altered protein levels of several proteins including RNA helicases as measured by label-free quantitative proteomics, defining proteins that are regulated by GID4 and the CTLH complex in humans. Interactions with GID4 via Pro/N-degron pathway did not result in proteasomal degradation, demonstrating that CTLH interactors are regulated through a combination of degradative and non-degradative functions. The lack of degradation of GID4 interactors highlights potential challenges in utilizing GID4-recruiting bifunctional molecules for targeted protein degradation. Going forward, PFI-7 will be a valuable research tool for defining CTLH complex biology and honing targeted protein degradation strategies.

molecular biology↗

bioRxiv2022

Translocator protein is a marker of activated microglia in rodent models but not human neurodegenerative diseases

Microglial activation plays central roles in neuro-inflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. We show that TSPO expression increases in activated microglia in mouse brain disease models but does not change in a non-human primate disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter, consistent with the hypothesis that the increase in TSPO expression in activated myeloid cells is unique to a subset of species within the Muroidea superfamily of rodents. We show that TSPO is mechanistically linked to classical pro-inflammatory myeloid cell function in rodents but not humans. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO PET reflects density of inflammatory cells rather than activation state.

neuroscience↗

bioRxiv2021

Single nuclear transcriptional signatures of dysfunctional brain vascular homeostasis in Alzheimer's disease

Brain perfusion and normal blood brain barrier integrity are reduced early in Alzheimers disease (AD). We performed single nucleus RNA sequencing of vascular cells isolated from AD and control brains to characterise pathological transcriptional signatures. We found that endothelial cells (EC) are enriched for expression of genes associated with susceptibility to AD. EC transcriptional signatures identified mechanisms for impaired {beta}-amyloid clearance. Evidence for immune activation was found with upregulation of interferon signalling genes in EC and in pericytes (PC). Transcriptional signatures suggested dysregulation of vascular homeostasis and angiogenesis with upregulation of pro-angiogenic signals (HIF1A) and metabolism in EC, but downregulation of homeostatic growth factor pathways (VEGF, EGF, insulin) in EC and PC and of extracellular matrix genes in fibroblasts (FB). Our genomic dissection of vascular cell risk gene enrichment suggests a potentially causal role for EC and defines transcriptional signatures associated with microvascular dysfunction in AD.

neuroscience↗

bioRxiv2021

Structure and inhibitor binding characterization of oncogenic MLLT1 mutants

Dysfunction of YEATS-domain-containing MLLT1, an acetyl/acyl-lysine dependent epigenetic reader domain, has been implicated in the development of aggressive cancers. Mutations in the YEATS domain have been recently reported as a cause of MLLT1 aberrant reader function. However, structural basis for the reported alterations in affinity for acetyled/acylated histone has remained elusive. Here, we report the crystal structures of both insertion and substitution present in cancer, revealing significant conformational changes of the YEATS-domain loop 8. Structural comparison demonstrates that such alteration not only altered the binding interface for acetylated/acylated histones, but the sequence alterations in the T1 loop may enable dimeric assembly consistent inducing self-association behavior. Nevertheless, we show that also the MLLT1 mutants can be targeted by developed acetyllysine mimetic inhibitors with affinities similarly to wild type. Our report provides a structural basis for the altered behaviors and potential strategy for targeting oncogenic MLLT1 mutants.

biochemistry↗