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Placental small extracellular vesicles from normal pregnancy and gestational diabetes increase insulin gene transcription and content in β cells
Abstract Insulin secretion increases progressively during pregnancy to maintain normal maternal blood glucose levels. The placenta plays a crucial role in this process by releasing hormones and extracellular vesicles into the maternal circulation, which drive significant changes in pregnancy physiology. Placental extracellular vesicles, which are detectable in the plasma of pregnant women, have been shown to signal peripheral tissues and contribute to pregnancy-related conditions. While studies using murine models have demonstrated that extracellular vesicles can modulate insulin secretion in pancreatic islets, it remains unclear whether these effects translate to human biology. Understanding how placental signals enhance insulin synthesis and secretion from β cells could be pivotal in developing new therapies for diabetes. In our study, we isolated placental small extracellular vesicles from human placentae and utilised the human β cell line, EndoC-βH3, to investigate their effects on β-cell function in vitro. Our results indicate that human β cells internalise placental small extracellular vesicles, leading to enhanced insulin gene expression and increased insulin content within the β cells. Moreover, these vesicles up-regulated the expression of Annexin A1, a protein known to increase insulin content. This up-regulation of Annexin A1 holds promise as a potential mechanism by which placental small extracellular vesicles enhance insulin biosynthesis.
Tau filaments are tethered within brain extracellular vesicles in Alzheimer’s disease
AbstractThe abnormal assembly of tau protein in neurons is a pathological hallmark of multiple neurodegenerative diseases, including Alzheimer’s disease (AD). Assembled tau associates with extracellular vesicles (EVs) in the central nervous system of individuals with AD, which is linked to its clearance and prion-like propagation. However, the identities of the assembled tau species and EVs, as well as how they associate, are not known. Here, we combined quantitative mass spectrometry, cryo-electron tomography and single-particle cryo-electron microscopy to study brain EVs from individuals with AD. We found tau filaments composed mainly of truncated tau that were enclosed within EVs enriched in endo-lysosomal proteins. We observed multiple filament interactions, including with molecules that tethered filaments to the EV limiting membrane, suggesting selective packaging. Our findings will guide studies into the molecular mechanisms of EV-mediated secretion of assembled tau and inform the targeting of EV-associated tau as potential therapeutic and biomarker strategies for AD.
Prospective cohort for early detection of liver cancer (Pearl): a study protocol
IntroductionHepatocellular carcinoma (HCC) is the fastest-rising and fourth most common cause of cancer death worldwide. Liver cirrhosis is the largest underlying risk factor for HCC. Therefore, patients with cirrhosis should have regular ultrasound and biochemical screening to pick up early HCC. Early HCC can be cured; more advanced HCCs have limited treatment options and poor prognosis. Current screening methods are suboptimal with poor sensitivity in picking up early disease. In this study, the investigators aim to recruit people with liver cirrhosis into a Prospective cohort for early detection of liver cancer—the Pearl cohort. The investigators believe that by using state-of-the-art tests we can improve the detection of early HCC.Methods and analysisThis is a UK-based prospective, longitudinal, diagnostic, prognostic, multicentre, non-CTIMP study. Aiming to recruit 3000 patients with liver cirrhosis without a HCC diagnosis, the Pearl cohort will be followed actively for 3 years from recruitment and then passively via registry data for ten years thereafter. Blood and urine samples will be taken and information from routine care will be gathered. These will be used to assess novel diagnostic approaches for the detection early HCC and to develop models to identify those most at risk for developing HCC.Participants will be linked to national UK health registries to ensure long-term capture of HCC incidence and other relevant endpoints. Approximately 75 patients are predicted to develop de novo HCC within the 3-year follow up period. After this period, the study teams will obtain data on participants for at least 10 years after the last contact. This cohort will help develop an understanding of the incidence of HCC in a UK population stratified by underlying cirrhosis aetiology.Ethics and disseminationEthical approval has been granted by REC and the trial is registered on ClinicalTrials.gov. The results will be published in peer-reviewed journals and presented at relevant meetings.Trial registration numberNCT05541601.
Covalent Inhibitors of KEAP1 with Exquisite Selectivity.
The NRF2-KEAP1 interaction is central for cytoprotection against stresses, giving it high clinical significance. Covalent modification of KEAP1 is an efficient approach, but the covalent inhibitors used in the clinic carry undesired side effects originating in their moderate selectivity. Starting with a phenotypic screen, we identified a new covalent inhibitor chemotype that was optimized to deliver a series of potent and highly selective KEAP1 binders. While the developed compounds showed both cellular and in vivo activity, upregulating antioxidant response element-dependent target genes, they showed no genotoxicity in vitro. The lead compound exhibited broad selectivity in activity-based protein profiling and showed no significant interaction with a panel of commonly studied receptors nor with a broad panel of kinases. The nature of its interaction with KEAP1 and the origin of its selectivity were revealed by X-ray crystallography.
Elevated Cerebrospinal Fluid Ubiquitin Carboxyl‐Terminal Hydrolase Isozyme L1 in Asymptomatic C9orf72 Hexanucleotide Repeat Expansion Carriers
ObjectiveTo identify biochemical changes in individuals at higher risk of developing amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) via C9orf72 hexanucleotide repeat expansion (HRE) heterozygosity.MethodsCross‐sectional observational study of 48 asymptomatic C9orf72 HRE carriers, 39 asymptomatic non‐carrier controls, 19 people with sporadic ALS, 10 with C9orf72 ALS, 14 with sporadic FTD, and 10 with C9orf72 FTD. Relative abundance of 30 pre‐defined cerebrospinal fluid biomarkers of ALS and FTD were compared in asymptomatic C9orf72 HRE carriers and age‐matched non‐carrier controls. Differential abundance of these proteins was quantified using data independent acquisition mass spectrometry or electro chemiluminescent assay for neurofilament light chain. Unbiased analysis of the entire cerebrospinal fluid proteome was then carried out.ResultsUbiquitin carboxyl‐hydrolase isozyme L1 levels were higher in asymptomatic C9orf72 HRE carriers compared with age‐matched non‐carriers (log2fold change 0.20, FDR‐adjusted p‐value = 0.034), whereas neurofilament light chain levels did not significantly differ. Ubiquitin carboxyl‐hydrolase isozyme L1 levels remained elevated after matching of groups by neurofilament levels (p = 0.011), and after adjusting for age, sex, and neurofilament levels. A significant difference was also observed when restricting analysis to younger participants (<37) matched by neurofilament level (p = 0.007).InterpretationElevated cerebrospinal fluid ubiquitin carboxyl‐hydrolase isozyme L1 levels in C9orf72 HRE carriers can occur in the absence of increased neurofilament levels, potentially reflecting either compensatory or pathogenic mechanisms preceding rapid neuronal loss. This brings forward the window on changes associated with the C9orf72 HRE carrier state, with potential to inform understanding of penetrance and approaches to prevention. ANN NEUROL 2024
The Chemical Probes Portal – 2024: update on this public resource to support best-practice selection and use of small molecules in biomedical research
Abstract The Chemical Probes Portal (www.chemicalprobes.org) is a free, public resource, based on expert-reviews, that supports the assessment, selection and use of small-molecule compounds that qualify as chemical probes. These high-quality reagents are essential for exploring the function of individual proteins in complex biological systems, such as cells and organisms, and for validating proteins as potential therapeutic targets. The use of reliable chemical probes accelerates protein annotation in basic biological studies and informs drug discovery. However, the use of low-quality compounds has historically led to erroneous conclusions in biomedical research, and experience shows that failure to follow best practice continues, an issue which the Portal aims to address. Here, we describe the latest updates to the Chemical Probes Portal in both content and functionality. The number of chemical probes and human protein targets covered has increased significantly, with improvements in the processes for obtaining expert reviews and user engagement. Moreover, new functionalities and enhanced tools have been introduced to better support biological researchers in selecting and using the best chemical probes for their studies.
Dysfunctional atrial fibroblast processing of filamin A in atrial fibrillation
Abstract Background/Introduction Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, is associated with atrial structural remodelling, hallmarked by fibrosis. Atrial fibrosis is instigated by atrial fibroblasts (AFBs). It was previously uncovered the hormone calcitonin (CT) is produced in the heart and, via binding to the CT-receptors (CTR), inhibits atrial fibrogenesis and fibroblast profibrotic activity. [1] In AF, abnormally increased CTR internalisation causes loss of physiological surface CTR and prevents fibroblast activation by CT. The molecular mechanisms of this phenomenon in ACFs are unknown. Aim To explore the role of filamin A (FLNa), an actin cross-linking protein that was previously shown to interact with CTR, [2] in the regulation of CTR trafficking. Methods AFBs were enzymatically isolated from left atrial appendage biopsies collected from 30 patients in persistent AF or normal sinus rhythm (SR, controls) undergoing elective heart surgery. Immunostaining assessed the localisation of CTR and its overlap with the nucleus (stained with DAPI) and FLNa in AFBs. Co-localisation was quantified by the JACoP BIOP plugin for ImageJ. Target mRNA expression was assessed by RT-qPCR and protein levels by immunoblot. Immunoprecipitation (IP) then immunoblot (IB) validated protein-protein interactions. Calpain activity was assessed with a fluorescence-based assay. The effects of calpains on FLNa proteolytic processing was examined with calpain inhibitor calpeptin (10nM–100nM vs DMSO loading control). Results Immunostaining revealed aberrant, intracellular localisation of CTR in AF-AFBs (A) with increased nuclear localisation (p=0.03) and reduced co-localisation with FLNa (p=0.03). FLNa gene expression showed a trend towards reduction in AF (by 36%, p=0.06; B). Meanwhile, although full-length FLNa protein was unaltered in AF, there was a striking 67% reduction (p=0.02) in the expression of FLNa’s c-terminal fragment (FLNaCT), which contains the CTR binding domain (C). Co-immunoprecipitation confirmed a physical interaction between CTR and FLNaCT (D) in both SR and AF AFBs. FLNaCT is typically generated when FLNa is cleaved by the protease calpain 1, whose protein (F) but not mRNA (E), was significantly downregulated (71%, p<0.001) in AF. Puzzlingly, a ~70% loss of calpain 1 protein was associated with unchanged calpain activity (G) and FLNaCT levels in AFBs treated with calpain inhibitor calpeptin (H). Conclusions Persistent AF is associated with an aberrant CTR localisation and FLNa proteolytic processing, not attributable to altered calpain activity. Further characterisation of the molecular determinants of the altered CTR localisation may uncover novel and potentially druggable facets of structural remodelling in AF.
Aging-associated mechanisms of atrial fibrillation progression and their therapeutic potential
Atrial fibrillation (AF) is the most common sustained arrhythmia, with a particularly high prevalence in the elderly. As the global aging population rapidly expands, it is increasingly important to examine how alterations to the aging heart contribute to an increased AF susceptibility. This work critically reviews the key molecular mechanisms that may underpin the complex association between aging and AF. Moreover, we identify emerging novel opportunities for therapeutic intervention that may be able to prevent and/or improve the current treatment paradigms for age-related AF. This review contributes to a holistic understanding of the intricate relationship between aging and AF.
Spirometry services in England post-pandemic and the potential role of AI support software: a qualitative study of challenges and opportunities
BackgroundSpirometry services to diagnose and monitor lung disease in primary care were identified as a priority in the NHS Long Term Plan, and are restarting post-COVID-19 pandemic in England; however, evidence regarding best practice is limited.AimTo explore perspectives on spirometry provision in primary care, and the potential for artificial intelligence (AI) decision support software to aid quality and interpretation.Design and settingSemi-structured interviews with stakeholders in spirometry services across England.MethodParticipants were recruited by snowball sampling. Interviews explored the pre- pandemic delivery of spirometry, restarting of services, and perceptions of the role of AI. Transcripts were analysed thematically.ResultsIn total, 28 participants (mean years’ clinical experience = 21.6 [standard deviation 9.4, range 3–40]) were interviewed between April and June 2022. Participants included clinicians (n= 25) and commissioners (n= 3); eight held regional and/or national respiratory network advisory roles. Four themes were identified: 1) historical challenges in provision of spirometry services; 2) inequity in post- pandemic spirometry provision and challenges to restarting spirometry in primary care; 3) future delivery closer to patients’ homes by appropriately trained staff; and 4) the potential for AI to have supportive roles in spirometry.ConclusionStakeholders highlighted historic challenges and the damaging effects of the pandemic contributing to inequity in provision of spirometry, which must be addressed. Overall, stakeholders were positive about the potential of AI to support clinicians in quality assessment and interpretation of spirometry. However, it was evident that validation of the software must be sufficiently robust for clinicians and healthcare commissioners to have trust in the process.
Development and validation of a new algorithm for improved cardiovascular risk prediction
AbstractQRISK algorithms use data from millions of people to help clinicians identify individuals at high risk of cardiovascular disease (CVD). Here, we derive and externally validate a new algorithm, which we have named QR4, that incorporates novel risk factors to estimate 10-year CVD risk separately for men and women. Health data from 9.98 million and 6.79 million adults from the United Kingdom were used for derivation and validation of the algorithm, respectively. Cause-specific Cox models were used to develop models to predict CVD risk, and the performance of QR4 was compared with version 3 of QRISK, Systematic Coronary Risk Evaluation 2 (SCORE2) and atherosclerotic cardiovascular disease (ASCVD) risk scores. We identified seven novel risk factors in models for both men and women (brain cancer, lung cancer, Down syndrome, blood cancer, chronic obstructive pulmonary disease, oral cancer and learning disability) and two additional novel risk factors in women (pre-eclampsia and postnatal depression). On external validation, QR4 had a higher C statistic than QRISK3 in both women (0.835 (95% confidence interval (CI), 0.833–0.837) and 0.831 (95% CI, 0.829–0.832) for QR4 and QRISK3, respectively) and men (0.814 (95% CI, 0.812–0.816) and 0.812 (95% CI, 0.810–0.814) for QR4 and QRISK3, respectively). QR4 was also more accurate than the ASCVD and SCORE2 risk scores in both men and women. The QR4 risk score identifies new risk groups and provides superior CVD risk prediction in the United Kingdom compared with other international scoring systems for CVD risk.
A High-Throughput Drug Repurposing Strategy to Treat TBX2 and/or TBX3 Dependent Cancers.
BackgroundThe highly homologous T-box transcription factors TBX2 and TBX3 are critical for embryonic development, and their overexpression in postnatal tissues contributes to a wide range of malignancies, including melanoma and rhabdomyosarcoma. Importantly, when TBX2 and TBX3 are depleted in cancers where they are overexpressed, the malignant phenotype is inhibited, and they have therefore been regarded as druggable targets. However, the time and costs associated with de novo drug development are challenging and result in drugs that are costly, especially for patients in low- and middle-income countries. In the current study, we therefore combined a targeted and drug repurposing approach to identify drugs that are expected to be more efficacious and cost-effective with significantly reduced side effects.MethodsA high-throughput cell-based immunofluorescence screen was performed to identify drugs in the Pharmakon 1600 drug library that can negatively regulate TBX2 and/or TBX3 levels. "Hit" drugs were validated for their effect on TBX2/TBX3 levels and cytotoxicity in TBX2/TBX3-dependent melanoma and rhabdomyosarcoma cells. To this end, immunofluorescence, western blotting, quantitative real-time PCR, and MTT cell viability assays were performed.ResultsNiclosamide, piroctone olamine, and pyrvinium pamoate, were identified as TBX2 and/or TBX3-targeting drugs, and they exhibited cytotoxicity in a TBX2/TBX3-dependent manner. Furthermore, these "Hit" drugs were shown to induce senescence and/or apoptosis.ConclusionsNiclosamide, piroctone olamine, and pyrvinium pamoate are promising, cost-effective therapeutic agents for the treatment of TBX2/TBX3-dependent cancers.
Inhaled corticosteroids for the treatment of COVID-19.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused severe illness and mortality for millions worldwide. Despite the development, approval and rollout of vaccination programmes globally to prevent infection by SARS-CoV-2 and the development of coronavirus disease 2019 (COVID-19), treatments are still urgently needed to improve outcomes. Early in the pandemic it was observed that patients with pre-existing asthma or COPD were underrepresented among those with COVID-19. Evidence from clinical studies indicates that the inhaled corticosteroids (ICS) routinely taken for asthma and COPD could have had a protective role in preventing severe COVID-19 and, therefore, may be a promising treatment for COVID-19. This review summarises the evidence supporting the beneficial effects of ICS on outcomes in patients with COVID-19 and explores the potential protective mechanisms.