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Pleural fluid proteomics from patients with pleural infection shows signatures of diverse neutrophilic responses: The Oxford Pleural Infection Endotyping Study (TORPIDS-2).
BACKGROUND: Pleural infection is a complex disease with poor clinical outcomes and increasing incidence worldwide, yet its biological endotypes remain unknown. METHODS: We analysed 80 pleural fluid samples from the PILOT study, a prospective study on pleural infection, using unlabelled mass spectrometry. A total of 449 proteins were retained after filtering. Unsupervised hierarchical clustering and UMAP analyses were used to cluster samples and pathway analysis was performed to identify the biological processes. Protein signatures as identified by the pathway analysis were compared to microbiology as defined by 16S rRNA next generation sequencing. Spearman and exact Fischer's methods were used for correlation assessment. RESULTS: Higher neutrophil degranulation was correlated with increased glycolysis (OR=281, p<2.2E-16) and pentose phosphate activation (OR=371.45, p<2.2E-16). Samples dominated by Streptococcus pneumoniae exhibited higher neutrophil degranulation (OR=12.08, p=0.005), glycolysis (OR=11.4, p=0.006), and pentose phosphate activity (OR=12.82, p=0.004). On the other hand, samples dominated by anaerobes and Gram-negative bacteria exhibited lower neutrophil degranulation (OR=0.15, p=0.01, glycolysis (OR=0.14, p=0.01), and pentose phosphate activity (OR=0.07, p=0.001). Increased activity of the liver and retinoid X receptors (LXR-RXR) pathway was associated with lower risk of one-year mortality (OR=0.24, p=0.04). CONCLUSIONS: These findings suggest that pleural infection patients exhibit diverse responses of neutrophil mediated immunity, glycolysis, and pentose phosphate activation which are associated with microbiology. Therapeutic targeting of the LXR-RXR pathway with agonists is a possible treatment approach.
Bacterial pathogen deploys the iminosugar glycosyrin to manipulate plant glycobiology.
The extracellular space (apoplast) in plants is a key battleground during microbial infections. To avoid recognition, the bacterial model phytopathogen Pseudomonas syringae pv. tomato DC3000 produces glycosyrin. Glycosyrin inhibits the plant-secreted β-galactosidase BGAL1, which would otherwise initiate the release of immunogenic peptides from bacterial flagellin. Here, we report the structure, biosynthesis, and multifunctional roles of glycosyrin. High-resolution cryo-electron microscopy and chemical synthesis revealed that glycosyrin is an iminosugar with a five-membered pyrrolidine ring and a hydrated aldehyde that mimics monosaccharides. Glycosyrin biosynthesis was controlled by virulence regulators, and its production is common in bacteria and prevents flagellin recognition and alters the extracellular glycoproteome and metabolome of infected plants. These findings highlight a potentially wider role for glycobiology manipulation by plant pathogens across the plant kingdom.
Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium
The ubiquitin-proteasome system (UPS) is essential for Plasmodium falciparum survival and represents a potential target for antimalarial therapies. We utilised a ubiquitin- activity based probe (Ub-Dha) to capture active components of the ubiquitin conjugating machinery during asexual blood-stage development. Several E2 ubiquitin-conjugating enzymes, the E1 activating enzyme, and the HECT E3 ligase PfHEUL were identified and validated through in vitro ubiquitination assays. We also demonstrate selective functional interactions between PfHEUL and a subset of both human and P. falciparum E2s. Additionally, the Ub-Dha probe captured an uncharacterized protein, PF3D7_0811400 (C0H4U0) with no known homology to ubiquitin-pathway enzymes in other organisms. Through structural and biochemical analysis, we validate it as a novel E2 enzyme, capable of binding ubiquitin in a cysteine-specific manner. These findings contribute to our understanding of the P. falciparum UPS, identifying promising novel drug targets and highlighting the evolutionary uniqueness of the Ub-proteasome system in this parasite.
Perturbing LSD1 and WNT rewires transcription to synergistically induce AML differentiation
Abstract Impaired differentiation is a hallmark of myeloid malignancies1,2. Therapies that enable cells to circumvent the differentiation block, such as all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), are by and large curative in acute promyelocytic leukaemia3, but whether ‘differentiation therapy’ is a generalizable therapeutic approach for acute myeloid leukaemia (AML) and beyond remains incompletely understood. Here we demonstrate that simultaneous inhibition of the histone demethylase LSD1 (LSD1i) and the WNT pathway antagonist GSK3 kinase4 (GSK3i) robustly promotes therapeutic differentiation of established AML cell lines and primary human AML cells, as well as reducing tumour burden and significantly extending survival in a patient-derived xenograft mouse model. Mechanistically, this combination promotes differentiation by activating genes in the type I interferon pathway via inducing expression of transcription factors such as IRF7 (LSD1i) and the co-activator β-catenin (GSK3i), and their selective co-occupancy at targets such as STAT1, which is necessary for combination-induced differentiation. Combination treatment also suppresses the canonical, pro-oncogenic WNT pathway and cell cycle genes. Analysis of datasets from patients with AML suggests a correlation between the combination-induced transcription signature and better prognosis, highlighting clinical potential of this strategy. Collectively, this combination strategy rewires transcriptional programs to suppress stemness and to promote differentiation, which may have important therapeutic implications for AML and WNT-driven cancers beyond AML.
Management and outcomes of women with placenta accreta spectrum grade 3: an INOSS multicountry multiperiod population-based study.
BackgroundManagement and outcomes in women with placenta accreta spectrum grade 3 are rarely reported from population-based studies. The objective of this study is to describe profiles, management, and outcomes, of women with placenta accreta spectrum (PAS) grade 3 from three multiperiod studies.MethodsThis analysis used data from three multiperiod population-based cohort studies from the United Kingdom (UK) (May 2010-April 2011), France (November 2013-October 2015), and Italy (September 2014-August 2016) to compare the management and outcomes of women with grade 3 PAS. The main outcome measures were postpartum hemorrhage (PPH) ≥ 3000 mL, blood transfusion ≥ 4 units, and other severe maternal complications (death, damage to bowel or urinary tract).ResultsThis study included 39 women with PAS grade 3 in the UK, 51 in France, and 34 in Italy, a total of 124 women. PAS was suspected before birth in 59% of the UK cases, 88% in France, and 82% in Italy (P ConclusionMaternal outcomes in women with grade 3 PAS varied between the three periods and countries, alongside the evolution in prenatal screening and peri-operative management.Trial registrationFor the UK: reference number: RP-PG-0608-10038. For France: reference number: AOR12156. For Italy: reference number: Port. PRE-839/13.
Reducing inequalities through greater diversity in clinical trials – As important for medical devices as for drugs and therapeutics
In medicine and public health, the randomised controlled trial (RCT) is generally considered the key generator of ‘gold standard’ evidence. However, basic and clinical research and trials are often unrepresentative of real-world populations. Recruiting insufficiently diverse cohorts of participants in trials (e.g. in terms of socioeconomic status, racial and ethnic background, or sex and gender) may not only overstate the general effectiveness of a technology; it may also actively increase health inequalities. We highlight some general issues in this domain, before discussing several specific illustrative examples in the context of medical devices. High quality evidence on factors that would improve trial recruitment is extremely limited. There is a clear need for research on candidate strategies for improving recruitment of under-represented groups in RCTs. These could include, for example, offering various forms of financial incentives; non-monetary incentives, such as preferential access to the technologies that are being tested if they are found to be effective; and various types of informational messages and nudges; as well as involvement of community partners and champions in the recruitment process. Ideally, recruitment practices should ultimately be based on evidence generated from RCTs. Studies Within a Trial (SWAT), where randomised experiments are built into the actual recruitment processes in RCTs, are an ideal way to gain this evidence. SWAT studies are seeing an increase in traction, as indicated by funding streams in bodies such as the UK-based NIHR. Making greater funding available for studies of this kind is needed to improve the evidence base on how best to improve diversity in trial recruitment.
Severe, uncontrolled epilepsy in pregnancy: A population-based case-control study.
BackgroundEpilepsy affects one percent of the UK population and is the most common serious neurological condition experienced during pregnancy. We compared the characteristics, clinical management, and pregnancy outcomes in women with severe, uncontrolled epilepsy to those of women with well controlled disease.MethodsWe conducted a population-based case-control study in all UK consultant-led maternity units. Cases of severe uncontrolled epilepsy during pregnancy were identified prospectively and reported via the UK Obstetric Surveillance System (UKOSS). Severe epilepsy was defined a-priori as ≥1 of the following: admission to hospital during pregnancy to manage seizures; prescribed ≥3 antiepileptic medications; or died from epilepsy. Controls comprised women with epilepsy not meeting the case definition, identified within the same centres as cases. Pre-pregnancy epilepsy control and pregnancy outcomes were compared between groups using multivariable logistic regression.ResultsWe identified 94 cases between 1 October 2015 and 31 March 2017 and compared these with 186 controls. Cases were significantly more likely to be admitted to manage seizures in the year preceding pregnancy (42/94 cases vs 10/186 controls, adjusted odds ratio [aOR]=7.38 [95% CI 2.70-20.2]), and to report their most recent seizure within 3 months of pregnancy (51/94 cases vs 18/186 controls, aOR=5.86 [95% CI 2.30-15.0]). Cases were significantly more likely to deliver before 37 weeks (20/94 cases vs 8/186 controls, aOR=7.61 [95% CI 2.87-20.2]).ConclusionsWomen admitted for seizure management in the year before pregnancy are at higher risk of severe epilepsy during pregnancy and of preterm birth. These women should be prioritised for discussion about pregnancy and contraception. When pregnant, they should be reviewed as early as possible by specialists in the management of epilepsy during pregnancy. Delivering messages about the importance of pregnancy planning and contraception to all women with epilepsy should be viewed as the responsibility of all clinicians involved their care.
Active regulatory elements recruit cohesin to establish cell specific chromatin domains.
As the 3D structure of the genome is analysed at ever increasing resolution it is clear that there is considerable variation in the 3D chromatin architecture across different cell types. It has been proposed that this may, in part, be due to increased recruitment of cohesin to activated cis-elements (enhancers and promoters) leading to cell-type specific loop extrusion underlying the formation of new sub-TADs. Here we show that cohesin correlates well with the presence of active enhancers and that this varies in an allele-specific manner with the presence or absence of polymorphic enhancers which vary from one individual to another. Using the alpha globin cluster as a model, we show that when all enhancers are removed, peaks of cohesin disappear from these regions and the erythroid specific sub-TAD is no longer formed. Re-insertion of the major alpha globin enhancer (R2) is associated with re-establishment of recruitment and increased interactions. In complementary experiments insertion of the R2 enhancer element into a "neutral" region of the genome recruits cohesin, induces transcription and creates a new large (75 kb) erythroid-specific domain. Together these findings support the proposal that active enhancers recruit cohesin, stimulate loop extrusion and promote the formation of cell specific sub-TADs.
Production of an immunogenic trivalent poliovirus virus-like particle vaccine candidate in yeast using controlled fermentation.
The success of the poliovirus (PV) vaccines has enabled the near-eradication of wild PV, however, their continued use post-eradication poses concerns, due to the potential for virus escape during vaccine manufacture. Recombinant virus-like particles (VLPs) that lack the viral genome remove this risk. Here, we demonstrate the production of PV VLPs for all three serotypes by controlled fermentation using Pichia pastoris. We determined the cryo-EM structure of a new PV2 mutant, termed SC5a, in comparison to PV2-SC6b VLPs described previously and investigated the immunogenicity of PV2-SC5a VLPs. Finally, a trivalent immunogenicity trial using bioreactor-derived VLPs of all three serotypes in the presence of Alhydrogel adjuvant, showed that these VLPs outperform the current IPV vaccine in the standard vaccine potency assay, offering the potential for dose-sparing. Overall, these results provide further evidence that yeast-produced VLPs have the potential to be a next-generation polio vaccine in a post-eradication world.
Tumor-Infiltrating Lymphocyte Scoring Improves Progression Risk Prediction in Stage II Melanoma: A Retrospective Cohort Study.
BackgroundAJCC 8th edition substaging might be suboptimal for predicting melanoma progression. Using it to select stage II patients for adjuvant immunotherapy risks overtreating low-risk stage IIB/IIC patients and undertreating high-risk stage IIA patients. Prognostic capability of tumor-infiltrating lymphocytes (TILs) is unclear in stage II melanoma.ObjectiveTo evaluate AJCC substaging and TIL scoring as predictors of progression in stage II melanoma.MethodsRetrospective cohort study of 366 SLN(-) stage II melanoma patients from four UK hospitals (2004-2017), with long-term follow-up.Results23% of melanomas progressed (median 9.5-year follow-up). Among those, 41.5% were stage IIA, 41.5% IIB, and 17.1% IIC. TIL scoring independently predicted progression risk (non-brisk vs brisk: OR 0.298,p=0.009; non-brisk vs absent: OR 0.436,p=0.049) and PFS. Non-brisk TILs, present in 80% of progressing tumors, denoted high risk. TIL scoring split patients into high and low risk across substages: stage IIA patients with non-brisk TILs had similar 5-year PFS to stage IIB/IIC patients with absent/brisk TILs.LimitationsRetrospective study design and unknown generalizability.ConclusionStage II melanoma progression is poorly predicted by AJCC 8 substage. TIL scoring offers improved risk stratification across substages and could serve as a cost-effective method to better identify patients who may benefit from adjuvant immunotherapies.