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In-cell chromatin structure by Cryo-FIB and Cryo-ET.
Chromatin, the complex of DNA and proteins that organises genetic material in eukaryotic cells, has been a focal point of biological research for over a century. Its structure determines critical functions such as gene regulation, DNA replication and chromosome segregation. Early models of chromatin were limited by technological constraints, but advancements in imaging, particularly X-ray and electron microscopy (EM), gradually unveiled its hierarchical organisation. The recent emergence of cryo-electron tomography (cryo-ET) coupled with cryo-focused ion beam (cryo-FIB) milling has revolutionised our understanding of chromatin organisation by providing native, three-dimensional (3D) views of various macromolecules and architectures of chromatin at unprecedented resolution. This review traces the historical progression of chromatin structural studies, from early EM and fluorescence microscopy to the transformative insights offered by cryo-ET, culminating in a synthesis of current knowledge and future directions.
Dominant variants in major spliceosome U4 and U5 small nuclear RNA genes cause neurodevelopmental disorders through splicing disruption.
The major spliceosome contains five small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) essential for splicing. Variants in RNU4-2, encoding U4, cause a neurodevelopmental disorder called ReNU syndrome. We investigated de novo variants in 50 snRNA-encoding genes in a French cohort of 23,649 individuals with rare disorders and gathered additional cases through international collaborations. Altogether, we identified 145 previously unreported probands with (likely) pathogenic variants in RNU4-2 and 21 individuals with de novo and/or recurrent variants in RNU5B-1 and RNU5A-1, encoding U5. Pathogenic variants typically arose de novo on the maternal allele and cluster in regions critical for splicing. RNU4-2 variants mainly localize to two structures, the stem III and T-loop/quasi-pseudoknot, which position the U6 ACAGAGA box for 5' splice site recognition and associate with different phenotypic severity. RNU4-2 variants result in specific defects in alternative 5' splice site usage and methylation patterns (episignatures) that correlate with variant location and clinical severity. This study establishes RNU5B-1 as a neurodevelopmental disorder gene, suggests RNU5A-1 as a strong candidate and highlights the role of de novo variants in snRNAs.
DeepCA: Deep Learning-Based 3D Coronary Artery Tree Reconstruction from Two 2D Non-Simultaneous X-Ray Angiography Projections
Cardiovascular diseases (CVDs) are the most common cause of death worldwide. Invasive x-ray coronary angiography (ICA) is one of the most important imaging modalities for the diagnosis of CVDs. ICA typically acquires only two 2D projections, which makes the 3D geometry of coronary vessels difficult to interpret, thus requiring 3D coronary artery tree reconstruction from two projections. State-of-the-art approaches require significant manual interactions and cannot correct the non-rigid cardiac and respiratory motions between non-simultaneous projections. In this study, we propose a novel deep learning pipeline named DeepCA. We leverage the Wasserstein conditional generative adversarial network with gradient penalty, latent convolutional transformer layers, and a dynamic snake convolutional critic to implicitly compensate for the non-rigid motion and provide 3D coronary artery tree reconstruction. Through simulating projections from coronary computed tomography angiography (CCTA), we achieve the generalisation of 3D coronary tree reconstruction on real non-simultaneous ICA projections. We incorporate an application-specific evaluation metric to validate our proposed model on both a CCTA dataset and a real ICA dataset, together with Chamfer £2 distance. The results demonstrate promising performance of our DeepCA model in vessel topology preservation, recovery of missing features, and generalisation ability to real ICA data. To the best of our knowledge, this is the first study that leverages deep learning to achieve 3D coronary tree reconstruction from two real non-simultaneous x-ray angiographic projections. The implementation of this work is available at: https//github.com/WangStephen/DeepCA.
Systematic identification of disease-causing promoter and untranslated region variants in 8040 undiagnosed individuals with rare disease
Abstract Background Both promoters and untranslated regions (UTRs) have critical regulatory roles, yet variants in these regions are largely excluded from clinical genetic testing due to difficulty in interpreting pathogenicity. The extent to which these regions may harbour diagnoses for individuals with rare disease is currently unknown. Methods We present a framework for the identification and annotation of potentially deleterious proximal promoter and UTR variants in known dominant disease genes. We use this framework to annotate de novo variants (DNVs) in 8040 undiagnosed individuals in the Genomics England 100,000 genomes project, which were subject to strict region-based filtering, clinical review, and validation studies where possible. In addition, we performed region and variant annotation-based burden testing in 7862 unrelated probands against matched unaffected controls. Results We prioritised eleven DNVs and identified an additional variant overlapping one of the eleven. Ten of these twelve variants (82%) are in genes that are a strong match to the individual’s phenotype and six had not previously been identified. Through burden testing, we did not observe a significant enrichment of potentially deleterious promoter and/or UTR variants in individuals with rare disease collectively across any of our region or variant annotations. Conclusions Whilst screening promoters and UTRs can uncover additional diagnoses for individuals with rare disease, including these regions in diagnostic pipelines is not likely to dramatically increase diagnostic yield. Nevertheless, we provide a framework to aid identification of these variants.
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.
Temporal correlations between RBD-ACE2 blocking and binding antibodies to SARS-CoV-2 variants in CoronaVac-vaccinated individuals and their persistence in COVID-19 patients.
Antibodies play a crucial role in protection against SARS-CoV-2. Understanding the correlation between binding and functional antibodies is essential to determine whether binding antibody levels can reliably predict neutralizing activity. We assessed antibody responses in 111 individuals vaccinated with the inactivated vaccine CoronaVac and 111 COVID-19 patients in Thailand. Plasma levels of ACE2-blocking antibodies targeting the receptor-binding domain (RBD) of SARS-Co-V2 variants were measured before vaccination and at 14 and 28 days after the second dose using a multiplex surrogate virus neutralization test. Anti-spike and anti-nucleocapsid antibodies were quantified by electrochemiluminescence immunoassay, and anti-RBD IgG by ELISA. After vaccination, blocking, anti-spike, and IgG antibody levels increased but declined rapidly within a month, whereas antibody levels in COVID-19 patients increased and persisted. Blocking and anti-spike antibody correlated at day 14 post-vaccination but not at day 28. In COVID-19 patients, correlations were moderate at day 14, and stronger at day 28. Correlations were weaker for Omicron subvariants than for the ancestral strain and non-Omicron variants. The weak correlation between blocking and anti-RBD IgG suggests binding antibodies might not predict neutralizing activity. These findings highlight the temporal nature of CoronaVac-induced immunity and the need for booster doses and variant-adapted vaccine.
British Society of Gastroenterology guidelines on colorectal surveillance in inflammatory bowel disease
Patients with inflammatory bowel disease (IBD) remain at increased risk for colorectal cancer and death from colorectal cancer compared with the general population despite improvements in inflammation control with advanced therapies, colonoscopic surveillance and reductions in environmental risk factors. This guideline update from 2010 for colorectal surveillance of patients over 16 years with colonic inflammatory bowel disease was developed by stakeholders representing UK physicians, endoscopists, surgeons, specialist nurses and patients with GRADE (Grading of Recommendations Assessment, Development and Evaluation) methodological support.An a priori protocol was published describing the approach to three levels of statement: GRADE recommendations, good practice statements or expert opinion statements. A systematic review of 7599 publications, with appraisal and GRADE analysis of trials and network meta-analysis, where appropriate, was performed. Risk thresholding guided GRADE judgements.We made 73 statements for the delivery of an IBD colorectal surveillance service, including outcome standards for service and endoscopist audit, and the importance of shared decision-making with patients.Core areas include: risk of colorectal cancer, IBD-related post-colonoscopy colorectal cancer; service organisation and supporting patient concordance; starting and stopping surveillance, who should or should not receive surveillance; risk stratification, including web-based multivariate risk calculation of surveillance intervals; colonoscopic modalities, bowel preparation, biomarkers and artificial intelligence aided detection; chemoprevention; the role of non-conventional dysplasia, serrated lesions and non-targeted biopsies; management of dysplasia, both endoscopic and surgical, and the structure and role of the multidisciplinary team in IBD dysplasia management; training in IBD colonoscopic surveillance, sustainability (green endoscopy), cost-effectiveness and patient experience. Sixteen research priorities are suggested.
A phase 1/2a clinical trial to assess safety and immunogenicity of an adenoviral-vectored capsular group B meningococcal vaccine
Capsular group B meningococcus (MenB) remains an important cause of disease globally, and additional vaccines against MenB would aid in reducing the incidence of infection. Previous work has demonstrated that a MenB adenoviral-vectored vaccine, ChAdOx1 MenB.1, elicited high serum bactericidal responses in preclinical models after a single dose, supporting further clinical development of this vaccine. Here, we report the results of a trial designed to assess the safety and immunogenicity of ChAdOx1 MenB.1 in healthy adults aged 18 to 50. In this phase 1/2a, single-center trial, participants received one or two doses of ChAdOx1 MenB.1 at days 0 and 180. One dose of ChAdOx1 MenB.1 was also given at day 180 to some individuals primed with one dose of 4CMenB at day 0. Participants recorded their symptoms in an electronic diary after vaccination, and safety blood readouts were monitored. Serum bactericidal antibody (SBA) assays were performed against a panel of MenB target strains. ChAdOx1 MenB.1 was well tolerated with no safety concerns and elicited protective SBA titers against a MenB strain expressing a homologous factor H–binding protein (fHbp) variant in 100% of participants after two doses. However, cross-reactivity analysis indicated a low SBA response to strains expressing heterologous fHbp, suggesting that a multivalent vaccine may be needed. In sum, ChAdOx1 MenB.1 is immunogenic in humans, and addition of another fHbp variant or of another antigen in this platform could provide an approach to extend protection against endemic MenB disease.
Structural maturation of the matrix lattice is not required for HIV-1 particle infectivity.
During HIV-1 maturation, the matrix (MA) lattice underlying the viral membrane undergoes a structural rearrangement, and the newly released capsid (CA) protein forms a mature CA. While it is well established that CA formation is essential for particle infectivity, the functional role of MA structural maturation remains unclear. Here, we examine maturation of an MA triple mutant, L20K/E73K/A82T, which, despite replicating similarly to wild-type (WT) in some cell lines, exhibits distinct biochemical behaviors that suggest altered MA-MA interactions. Cryo-electron tomography with subtomogram averaging reveals that, although the MA lattice in immature L20K/E73K/A82T virions closely resembles that of the WT, mature L20K/E73K/A82T virions lack a detectable MA lattice. All-atom molecular dynamics simulations suggest that this absence results from destabilized inter-trimer MA interactions in mature L20K/E73K/A82T mutant virions. These findings suggest that an ordered, membrane-associated mature MA lattice is not essential for HIV-1 infectivity, providing insights into the structural requirements for HIV-1 particle maturation and generation of infectious particles.
T cell memory response to MPXV infection exhibits greater effector function and migratory potential compared to MVA-BN vaccination.
In 2022, a global mpox outbreak occurred, and remains a concern today. The T cell memory response to MPXV (monkeypox virus) infection has not been fully investigated. In this study, we evaluate this response in convalescent and MVA-BN (Modified Vaccinia Ankara - Bavarian Nordic) vaccinated individuals using VACV-infected cells. Strong CD8+ and CD4+ T cell responses are observed, and T cell responses are biased towards viral early expressed proteins. We identify seven immunodominant HLA-A*02:01 restricted MPXV-specific epitopes and focus our detailed phenotypic and scRNAseq analysis on the immunodominant HLA-A*02:01-G5R18-26-specific CD8+ T cell response. While tetramer+CD8+ T cells share similar differentiation and activation phenotypes, T cells from convalescent individuals show greater cytotoxicity, migratory potential to site of infection and TCR clonal expansion. Our data suggest that effective functional profiles of MPXV-specific memory T cells induced by Mpox infection may have an implication on the long-term protective responses to future infection.
Assessments and developments in constructing a National Health Index for policy-making, in the UK
Composite indicators are useful for summarizing and comparing changes among different communities. The UK Office for National Statistics has created an annual England Health Index (2015-2018) comprised of three main health domains - lives, places, and people - to monitor health over time and across different geographical areas and evaluate the nation's health. We reviewed the conceptual coherence and statistical requirements, focusing on three main steps: correlation analysis at different levels, comparison of the implemented weights, and a sensitivity and uncertainty analysis. Based on the results, we have highlighted features that have improved the statistical requirements of the forthcoming UK Health Index.
Delineating Mpl-dependent and -independent phenotypes of Jak2 V617F- positive MPNs in vivo.
The Jak2 V617F mutation stands as the main driver of myeloproliferative neoplasms (MPNs) by constitutively activating signaling of several type I cytokine receptors, namely those for erythropoietin (EpoR), thrombopoietin (TpoR), and Granulocyte Colony Stimulating Factor (G-CSFR). Among these, TpoR assumes a pivotal role in hematopoietic stem cell renewal and differentiation, being positioned as a key driver of MPNs alongside mutated Jak2. However, the impact of TpoR/MPL absence in the context of Jak2 V617F in vivo has been explored only through a transgenic Jak2 V617F mouse model, where regulation of Jak2 expression does not depend on its natural promoter. In this study, we use a novel mouse model expressing Jak2 V617F under its endogenous promoter at the heterozygous state within a Mpl knock-out background. Our findings indicate that erythrocytosis, leukocytosis and moderate splenomegaly with mild spleen peri-vascular fibrosis persist even in the absence of Mpl expression. Notably, the inherent growth-stimulating effect induced by Jak2 V617F remains consistent across diverse early hematopoietic progenitor populations in the absence of Mpl but is reduced at the stem cell level and does not allow clonal expansion in competitive transplantation. Our results delineate Mpl-dependent and -independent phenotypes induced by Jak2 V617F and confirm that inhibiting Mpl expression at the stem cell level negates the long-term advantage of the mutant clone. Consequently, while MPL emerges as a major player in Jak2 V617F positive MPNs, our study underscores that it is not the exclusive contributor, broadening the spectrum for therapeutic intervention.
Disease-specific B cell clones are shared between patients with Crohn's disease.
B cells have important functions in gut homeostasis, and dysregulated B cell populations are frequently observed in patients with inflammatory bowel diseases, including both ulcerative colitis (UC) and Crohn's disease (CD). How these B cell perturbations contribute to disease remains largely unknown. Here, we perform deep sequencing of the B cell receptor (BCR) repertoire in four cohorts of patients with CD, together with healthy controls and patients with UC. We identify BCR clones that are shared between patients with CD but not found in healthy individuals nor in patients with UC, indicating CD-associated B cell immune responses. Shared clones are present in the inflamed gut mucosa, draining intestinal lymph nodes and blood, suggesting the presence of common CD-associated antigens that drive B cell responses in CD patients.
Amyotrophic lateral sclerosis caused by TARDBP mutations: from genetics to TDP-43 proteinopathy
Mutations in the TARDBP gene, which encodes the TDP-43 protein, account for only 3–5% of familial cases of amyotrophic lateral sclerosis and less than 1% of cases that are apparently idiopathic. However, the discovery of neuronal inclusions of TDP-43 as the neuropathological hallmark in the majority of cases of amyotrophic lateral sclerosis has transformed our understanding of the pathomechanisms underlying neurodegeneration. An individual TARDBP mutation can cause phenotypic heterogeneity. Most mutations lie within the C-terminus of the TDP-43 protein. In pathological conditions, TDP-43 is mislocalised from the nucleus to the cytoplasm, where it can be phosphorylated, cleaved, and form insoluble aggregates. This mislocalisation leads to dysfunction of downstream pathways of RNA metabolism, proteostasis, mitochondrial function, oxidative stress, axonal transport, and local translation. Biomarkers for TDP-43 dysfunction and targeted therapies are being developed, justifying cautious optimism for personalised medicine approaches that could rescue the downstream effects of TDP-43 pathology.
Quantifying prevalence and risk factors of HIV multiple infection in Uganda from population-based deep-sequence data.
People living with HIV can acquire secondary infections through a process called superinfection, giving rise to simultaneous infection with genetically distinct variants (multiple infection). Multiple infection provides the necessary conditions for the generation of novel recombinant forms of HIV and may worsen clinical outcomes and increase the rate of transmission to HIV seronegative sexual partners. To date, studies of HIV multiple infection have relied on insensitive bulk-sequencing, labor intensive single genome amplification protocols, or deep-sequencing of short genome regions. Here, we identified multiple infections in whole-genome or near whole-genome HIV RNA deep-sequence data generated from plasma samples of 2,029 people living with viremic HIV who participated in the population-based Rakai Community Cohort Study (RCCS). We estimated individual- and population-level probabilities of being multiply infected and assessed epidemiological risk factors using the novel Bayesian deep-phylogenetic multiple infection model (deep - phyloMI) which accounts for bias due to partial sequencing success and false-negative and false-positive detection rates. We estimated that between 2010 and 2020, 4.09% (95% highest posterior density interval (HPD) 2.95%-5.45%) of RCCS participants with viremic HIV multiple infection at time of sampling. Participants living in high-HIV prevalence communities along Lake Victoria were 2.33-fold (95% HPD 1.3-3.7) more likely to harbor a multiple infection compared to individuals in lower prevalence neighboring communities. This work introduces a high-throughput surveillance framework for identifying people with multiple HIV infections and quantifying population-level prevalence and risk factors of multiple infection for clinical and epidemiological investigations.