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The CAMS-COI Joint webinar entitled “COVID Without Borders-A collaboration to Investigate A New Disease III – Diagnostic and Treatment for COVID-19” was held online on the morning of November 17th 2020.
Acidosis attenuates the hypoxic stabilization of HIF-1α by activating lysosomal degradation
Hypoxia-inducible factors (HIFs) mediate cellular responses to low oxygen, notably enhanced fermentation that acidifies poorly perfused tissues and may eventually become more damaging than adaptive. How pH feeds back on hypoxic signaling is unclear but critical to investigate because acidosis and hypoxia are mechanistically coupled in diffusion-limited settings, such as tumors. Here, we examined the pH sensitivity of hypoxic signaling in colorectal cancer cells that can survive acidosis. HIF-1α stabilization under acidotic hypoxia was transient, declining over 48 h. Proteomic analyses identified responses that followed HIF-1α, including canonical HIF targets (e.g., CA9, PDK1), but these did not reflect a proteome-wide downregulation. Enrichment analyses suggested a role for lysosomal degradation. Indeed, HIF-1α destabilization was blocked by inactivating lysosomes, but not proteasome inhibitors. Acidotic hypoxia stimulated lysosomal activity and autophagy via mammalian target of rapamycin complex I (mTORC1), resulting in HIF-1α degradation. This response protects cells from excessive acidification by unchecked fermentation. Thus, alkaline conditions are permissive for at least some aspects of HIF-1α signaling.
Immune–epithelial–stromal networks define the cellular ecosystem of the small intestine in celiac disease
Abstract The immune–epithelial–stromal interactions underpinning intestinal damage in celiac disease (CD) are incompletely understood. To address this, we performed single-cell transcriptomics (RNA sequencing; 86,442 immune, parenchymal and epithelial cells; 35 participants) and spatial transcriptomics (20 participants) on CD intestinal biopsy samples. Here we show that in CD, epithelial populations shifted toward a progenitor state, with interferon-driven transcriptional responses, and perturbation of secretory and enteroendocrine populations. Mucosal T cells showed numeric and functional changes in regulatory and follicular helper-like CD4+ T cells, intraepithelial lymphocytes, CD8+ and γδ T cell subsets, with skewed T cell antigen receptor repertoires. Mucosal changes remained detectable despite treatment, representing a persistent immune–epithelial ‘scar’. Spatial transcriptomics defined transcriptional niches beyond those captured in conventional histological scores, including CD-specific lymphoid aggregates containing T cell–B cell interactions. Receptor–ligand spatial analyses integrated with disease susceptibility gene expression defined networks of altered chemokine and morphogen signaling, and provide potential therapeutic targets for CD prevention and treatment.
Vaccine effects on in-hospital COVID-19 outcomes.
Here, we posit that studies comparing outcomes of patients hospitalized with COVID-19 by vaccination status are important descriptive epidemiologic studies, but contrast two groups that are not comparable with regard to causal analyses. We use the principal stratification framework to show that these studies can estimate a causal vaccine effect only for the subgroup of individuals who would be hospitalized with or without vaccination. Further, we describe the methodology for, and present sensitivity analyses of, this effect. Using this approach can change the interpretation of studies only reporting the standard analyses that condition on observed hospital admission status - that is, analyses comparing outcomes for all hospitalised COVID-19 patients by vaccination status.
External validation of the LENT and PROMISE prognostic scores for malignant pleural effusion.
BACKGROUND: Accurate survival estimation in malignant pleural effusion is essential to guide clinical management strategies and inform patient discussion. The LENT and PROMISE scores were developed to aid prognostication in malignant pleural effusion; however their uptake in practice has been limited. We aimed to conduct a detailed external validation of the LENT and PROMISE scores to develop recommendations regarding clinical utility, and to highlight factors limiting performance. METHODS: Medical records of patients diagnosed with malignant pleural effusion between 2015-2023 at Oxford University Hospitals were retrospectively reviewed to determine length of survival and the LENT and PROMISE scores at diagnosis. Performance of the scores in predicting overall survival and chance of survival at 3, 6 and 12 months was assessed using measures of discrimination, calibration and overall model performance. Kaplan-Meier analysis and Cox models were utilised to further investigate individual score variables. RESULTS: 773 patients with malignant pleural effusion were included. Both scores showed predictive ability for overall survival; however median survival estimates lacked precision. Score performance in predicting survival at 3, 6 and 12 months was stronger, with C-indices around 0.8 for both at each time point, and the models appearing well calibrated. Limited stratification of tumour types and lack of consideration of sensitising mutations were demonstrated to be potential factors restricting performance. CONCLUSIONS: Both scores have the ability to prognosticate in malignant pleural effusion, and greater use in practice should be considered. However, areas to improve score performance were also highlighted, and these may aid future model development.
Activity-Based Protein Profiling (ABPP) of Cellular DeISGylating Enzymes and Inhibitor Screening.
A detailed methodology platform is described for activity-based protein profiling (ABPP) of cellular deISGylating enzymes using a specific activity-based interferon-stimulated gene 15 (ISG15) probe. Manual and semi-automated workflows for medium- to high-throughput applications are outlined in this chapter, with western blotting and proteomics-based techniques as the main readouts. This methodology informs us of endogenous deISGylating enzyme expression and activity in a cellular context, including USP18, the type I interferon (IFN-I)-inducible deISGylase, and several constitutively expressed deubiquitinases (DUBs), such as USP5, USP14, USP16, and USP36, that exert cross-reactivity to ISG15. ISG15-ABPP also enables the identification and characterization of potent and selective deISGylating enzyme modulators.
Virion Structure
Picornaviruses were the first animal viruses whose structure was determined in atomic detail and, as of October 2009, the Protein Data Bank (PDB) registered 53 structure depositions for picornaviruses. These data have contributed significantly to the understanding of picornavirus evolution, assembly, host-cell interaction, host adaptation, and antigenic variation and are providing the basis for novel therapeutic strategies. Subsequently classified as a picornavirus, the general morphology of FMDV could not be visualized until the advent of the electron microscope, when negative-stained images to a resolution of 4 to 5 nm revealed rather smooth round particles of ˜30 nm diameter. The current classification of picornaviruses is based on genome and protein sequence properties which are derived from the interplay of the error-prone replication mechanism of the virus with the process of natural selection. Differences in physical properties, such as buoyant density in cesium chloride and pH stability, underpinned the early classification of picornaviruses. Virus capsids recognize susceptible cells by attachment to specific receptors on the host cell membrane, thereby determining the host range and tropism of infection. The majority of antibodies are weak neutralizers that appear to operate by using the two arms of the antibody to cross-link different virus particles, causing aggregation.
Ethnic Disparities in Severe Maternal Morbidity and the Contribution of Deprivation: A Population-Based Causal Analysis.
ObjectiveTo investigate the association between ethnicity and severe maternal morbidity (SMM) in England and the mediating effects of neighbourhood-level socio-economic deprivation across detailed ethnic groups.DesignPopulation-based nationwide cohort study using English Hospital Episode Statistics Admitted Patient Care (HES APC) data.SettingAll hospital births in NHS facilities in England between 1 January 2013 and 31 March 2023.PopulationA cohort of 3 839 156 women aged 10- 55 years with births of ≥ 20 weeks' gestation.MethodsMultivariable fixed and mixed-effects Poisson regression models were used to estimate adjusted risk ratios (RR) for SMM across 10 ethnic groups compared to White women and for each ethnic group in different deprivation quintiles compared to White women in the least deprived neighbourhoods, respectively. Causal mediation analysis was used to calculate the proportion of the association mediated by deprivation.Main outcome measuresThe modified English Maternal Morbidity Outcome Indicator (EMMOI), a composite outcome of SMM.ResultsMinoritised ethnic groups experienced higher SMM risks than White women, with the highest risk for Black African women (RR 1.96, 95% CI: 1.82-2.02) and Bangladeshi women (RR 1.97, 95% CI: 1.88-2.07) compared to White women. The strength of the association varied across ethnic subgroups. Most of the effect of ethnicity on SMM was not mediated by deprivation (11%-29%).ConclusionsDeprivation plays a minor role in ethnic disparities in SMM. Policies must address the unique challenges faced by minoritised ethnic women.
Neurological complications following obstetric neuraxial anaesthesia: a four-year United Kingdom population-based study of epidural haematoma and epidural abscess (2014-2017).
BackgroundEpidural haematoma and epidural abscess are rare but serious complications of obstetric neuraxial anaesthesia. We investigated their incidence, diagnosis, management, and outcomes in the UK.MethodsThis prospective, population-based study used the UK Obstetric Surveillance System to collect data on all cases of epidural haematoma or abscess occurring in obstetrician-led UK maternity units between January 2014 and December 2017.ResultsSix cases of epidural haematoma and 11 of epidural abscess were confirmed, giving estimated incidences of 1.97 (95% CI 0.72 to 4.29) and 3.61 (95% CI 1.80 to 6.46) per 1,000,000 maternities, respectively. Women with haematoma commonly reported pain or tenderness (3/6) and fever (2/6). All women with abscess presented with pain; eight had tenderness, six fever, and four lower limb motor block. Diagnosis was confirmed by MRI in all cases. Risk factors for haematoma included perinatal anticoagulant therapy (1/6) and multiple catheter attempts in one woman. Among women with abscess, aseptic precautions were potentially suboptimal in around 70% of cases. No immunodeficiency was reported. Two women with haematoma and six with abscess underwent surgery, with antibiotics used in one and four of these cases, respectively. Three women with abscess received antibiotics alone. Expectant management was used in two-thirds of haematoma cases and one abscess case. There were no maternal deaths, ICU admissions, stillbirths, or neonatal deaths, though three women experienced major maternal morbidity.ConclusionsEpidural abscess and haematoma were rare complications of obstetric neuraxial anaesthesia in the UK, with most women recovering well, though a minority experienced major morbidity.
Continuous Indexing of Fibrosis (CIF): improving the assessment and classification of MPN patients.
The grading of fibrosis in myeloproliferative neoplasms (MPN) is an important component of disease classification, prognostication and monitoring. However, current fibrosis grading systems are only semi-quantitative and fail to fully capture sample heterogeneity. To improve the quantitation of reticulin fibrosis, we developed a machine learning approach using bone marrow trephine (BMT) samples (n = 107) from patients diagnosed with MPN or a reactive marrow. The resulting Continuous Indexing of Fibrosis (CIF) enhances the detection and monitoring of fibrosis within BMTs, and aids MPN subtyping. When combined with megakaryocyte feature analysis, CIF discriminates between the frequently challenging differential diagnosis of essential thrombocythemia (ET) and pre-fibrotic myelofibrosis with high predictive accuracy [area under the curve = 0.94]. CIF also shows promise in the identification of MPN patients at risk of disease progression; analysis of samples from 35 patients diagnosed with ET and enrolled in the Primary Thrombocythemia-1 trial identified features predictive of post-ET myelofibrosis (area under the curve = 0.77). In addition to these clinical applications, automated analysis of fibrosis has clear potential to further refine disease classification boundaries and inform future studies of the micro-environmental factors driving disease initiation and progression in MPN and other stem cell disorders.
The effect of D-cycloserine on brain connectivity over a course of pulmonary rehabilitation - A randomised control trial with neuroimaging endpoints.
Combining traditional therapies such as pulmonary rehabilitation with brain-targeted drugs may offer new therapeutic opportunities for the treatment of chronic breathlessness. Recently, we asked whether D-cycloserine, a partial NMDA-receptor agonist which may enhance behavioural therapies, modifies the relationship between breathlessness related brain activity and breathlessness anxiety over pulmonary rehabilitation. However, whether any changes are supported by alterations to underlying brain structure remains unknown. Here we examine the effect of D-cycloserine over a course of pulmonary rehabilitation on the connectivity between key brain regions associated with the processing of breathlessness anxiety. 72 participants with mild-to-moderate COPD took part in a longitudinal study in parallel to their pulmonary rehabilitation course. Diffusion tensor brain imaging and clinical measures of respiratory function were collected at three time points (before, during and after pulmonary rehabilitation). Participants were assigned to 250mg of D-cycloserine or placebo, which they were administered with on four occasions in a randomised, double-blind procedure. Following the first four sessions of pulmonary rehabilitation (visit 2), during which D-cycloserine was administered, improvements in breathlessness anxiety were linked with increased insula-hippocampal structural connectivity in the D-cycloserine group when compared to the placebo group. No differences were found between the two groups following the completion of the full pulmonary rehabilitation course 4-6 weeks later (visit 3). The action of D-cycloserine on brain connectivity appears to be restricted to within a short time-window of its administration. This temporary boost of the brain connectivity of two key regions associated with the evaluation of how unpleasant an experience is may support the re-evaluation of breathlessness cues, illustrated improvements in breathlessness anxiety. Trial registration ClinicalTrials.gov (NCT01985750).
Evidence of focusing the MHC class I immunopeptidome by tapasin.
Major Histocompatibility Complex class I (MHC-I) molecules bind and present peptides to cytotoxic T cells, protecting against pathogens and cancer. MHC-I is highly polymorphic and each allotype is promiscuous, and capable of binding a unique and diverse repertoire of peptide ligands. The peptide editing chaperone tapasin optimizes this allotype specific repertoire of peptides, resulting in the selection of high affinity peptides. MHC-I allotypes differ in the extent they engage tapasin. This suggests that tapasin-dependent MHC-I allotypes should present a less diverse repertoire that is enriched in higher-affinity peptides, and which are present in higher abundance, than tapasin independent MHC-I allotypes, which should present a broader repertoire containing peptides with a lower average affinity. Experimental verification of this hypothesis has been confounded by the different peptide binding specificities of MHC-I allotypes. Here, we independently investigated the peptide focusing function of tapasin by introducing a point mutation into a tapasin independent MHC-I allotype that dramatically increased its tapasin dependence without substantially altering its peptide binding specificity. This allowed us to demonstrate ligand focusing by tapasin at both the repertoire level in cellulo, and by using an in vitro system in which tapasin was artificially tethered to MHC-I, at the individual peptide level. We found that tapasin had a greater influence on tapasin dependent MHC-I molecules, and that tapasin modulated peptide selection according to peptide-MHC-I complex stability, disfavoring short-lived peptide-MHC-I complexes. Thus, tapasin dependent MHC-I molecules experience greater tapasin filtering, resulting in less diverse MHC-I immunopeptidomes that are enriched in high affinity peptide-MHC-I complexes.
Strict advantage of complex quantum theory in a communication task
Standard formulations of quantum theory are based on complex numbers: Quantum states can be in superpositions, with weights given by complex probability amplitudes. Motivated by quantum theory promising a range of practical advantages over classical for a multitude of tasks, we investigate how the presence of complex amplitudes in quantum theory can yield operational advantages over counterpart real formulations. We identify a straightforward communication task for which complex quantum theory exhibits a provably lower communication cost than not just any classical approach, but also any approach based on real quantum theory. We certify the necessity of complex quantum theory for optimal approaches to the task through geometric properties of quantum state ensembles that witness the presence of basis-independent complexity. This substantiates a strict operational advantage of complex quantum theory. We discuss the relevance of this finding for quantum advantages in stochastic simulation.
Deep palaeoproteomic profiling of archaeological human brains.
Palaeoproteomics leverages the persistence, diversity, and biological import of ancient proteins to explore the past, and answer fundamental questions about phylogeny, environment, diet, and disease. These insights are largely gleaned from hard tissues like bone and teeth, as well-established protocols exist for extracting ancient proteins from mineralised tissues. No such method, however, exists for the soft tissues, which are underexplored in palaeoproteomics given permission for destructive analysis routinely depends on a proven methodology. Considering less than one-tenth of all human proteins are expressed in bone, compared to three-quarters in the internal organs, the amount of biological information presently inaccessible is substantial. We address this omission with an optimised LC-FAIMS-MS/MS workflow yielding the largest, most diverse palaeoproteome yet described. Using archaeological human brains, we test ten protocols with varied chemistries and find that urea lysis effectively disrupts preserved membrane regions to expose low-abundant, intracellular analytes. Further, we show that ion mobility spectrometry improves unique protein identification by as much as 40%, and represents a means of "cleaning" dirty archaeological samples. Our methodology will be useful for improving protein recovery from a range of ancient tissues and depositional environments.
A Novel Toolkit of SARS-CoV-2 Sub-Genomic Replicons for Efficient Antiviral Screening.
SARS-CoV-2 is classified as a containment level 3 (CL3) pathogen, limiting research access and antiviral testing. To address this, we developed a non-infectious viral surrogate system using reverse genetics to generate sub-genomic replicons. These replicons contained the nsp1 mutations K164A and H165A and had the spike, membrane, ORF6, and ORF7a coding sequences replaced with various reporter and selectable marker genes. Replicons based on the ancestral Wuhan Hu-1 strain and the Delta variant of concern were replication-competent in multiple cell lines, as assessed by Renilla luciferase activity, fluorescence, immunofluorescence staining, and single-molecule fluorescent in situ hybridization. Antiviral assays using transient replicon expression showed that remdesivir effectively inhibited both replicon and viral replication. Ritonavir and cobicistat inhibited Delta variant replicons similarly to wild-type virus but did not inhibit Wuhan Hu-1 replicon replication. To further investigate the impact of nsp1 mutations, we generated a recombinant SARS-CoV-2 virus carrying the K164A and H165A mutations. The virus exhibited attenuated replication across a range of mammalian cell lines, was restricted by the type I interferon response, and showed reduced cytopathic effects. These findings highlight the utility of sub-genomic replicons as reliable CL2-compatible surrogates for studying SARS-CoV-2 replication and drug activity mechanisms.
RIFINs displayed on malaria-infected erythrocytes bind KIR2DL1 and KIR2DS1.
Natural killer (NK) cells use inhibitory and activating immune receptors to differentiate between human cells and pathogens. Signalling by these receptors determines whether an NK cell becomes activated and destroys a target cell. In some cases, such as killer immunoglobulin-like receptors, immune receptors are found in pairs, with inhibitory and activating receptors containing nearly identical extracellular ligand-binding domains coupled to different intracellular signalling domains1. Previous studies showed that repetitive interspersed family (RIFIN) proteins, displayed on the surfaces of Plasmodium falciparum-infected erythrocytes, can bind to inhibitory immune receptors and dampen NK cell activation2,3, reducing parasite killing. However, no pathogen-derived ligand has been identified for any human activating receptor. Here we identified a clade of RIFINs that bind to inhibitory immune receptor KIR2DL1 more strongly than KIR2DL1 binds to the human ligand (MHC class I). This interaction mediates inhibitory signalling and suppresses the activation of KIR2DL1-expressing NK cells. We show that KIR2DL1-binding RIFINs are abundant in field-isolated strains from both Africa and Asia and reveal how the two RIFINs bind to KIR2DL1. The RIFIN binding surface of KIR2DL1 is conserved in the cognate activating immune receptor KIR2DS1. We find that KIR2DL1-binding RIFINs can also bind to KIR2DS1, resulting in the activation of KIR2DS1-expressing NK cells. This study demonstrates that activating killer immunoglobulin-like receptors can recruit NK cells to target a pathogen and reveals a potential role for activating immune receptors in controlling malaria infection.
Chromothripsis-associated chromosome 21 amplification orchestrates transformation to blast-phase MPN through targetable overexpression of DYRK1A
Abstract Chromothripsis, the chaotic shattering and repair of chromosomes, is common in cancer. Whether chromothripsis generates actionable therapeutic targets remains an open question. In a cohort of 64 patients in blast phase of a myeloproliferative neoplasm (BP-MPN), we describe recurrent amplification of a region of chromosome 21q (‘chr. 21amp’) in 25%, driven by chromothripsis in a third of these cases. We report that chr. 21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. DYRK1A, a serine threonine kinase, is the only gene in the 2.7-megabase minimally amplified region that showed both increased expression and chromatin accessibility compared with non-chr. 21amp BP-MPN controls. DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development and is essential for BP-MPN cell proliferation in vitro and in vivo, and represents a druggable axis. Collectively, these findings define chr. 21amp as a prognostic biomarker in BP-MPN, and link chromothripsis to a therapeutic target.