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Suppression of ERK signalling promotes pluripotent epiblast in the human blastocyst.
No full textStudies in the mouse demonstrate the importance of fibroblast growth factor (FGF) and extra-cellular receptor tyrosine kinase (ERK) in specification of embryo-fated epiblast and yolk-sac-fated hypoblast cells from uncommitted inner cell mass (ICM) cells prior to implantation. Molecular mechanisms regulating specification of early lineages in human development are comparatively unclear. Here we show that exogenous FGF stimulation leads to expanded hypoblast molecular marker expression, at the expense of the epiblast. Conversely, we show that specifically inhibiting ERK activity leads to expansion of epiblast cells functionally capable of giving rise to naïve human pluripotent stem cells. Single-cell transcriptomic analysis indicates that these epiblast cells downregulate FGF signalling and maintain molecular markers of the epiblast. Our functional study demonstrates the molecular mechanisms governing ICM specification in human development, whereby segregation of the epiblast and hypoblast lineages occurs during maturation of the mammalian embryo in an ERK signal-dependent manner
Mycobacterium tuberculosis overcomes phosphate starvation by extensively remodelling its lipidome with phosphorus-free lipids.
No full textTuberculosis (TB) is the biggest cause of death from infectious disease worldwide. The causative agent, Mycobacterium tuberculosis (Mtb), possesses a complex cell envelope comprised of multiple classes of unique lipids. The macrophage phagosome is a key reservoir of infection in pulmonary TB and multiple studies have shown that inorganic phosphate (Pi) is limiting in this environment. Here, we show that during Pi restriction the Mtb lipidome markedly remodels such that phospholipids are replaced with multiple classes of phosphorus-free lipids. This envelope lipidome remodelling suggests that standard Mtb culture conditions that use media with high concentrations of Pi do not reflect the physiologic environment during infection, thereby undermining vaccine and drug development for tuberculosis. Further, we discover that Mtb can metabolise phospholipid polar heads abundant in host pulmonary surfactant as an alternative phosphate source. Therefore, we present two mechanisms where Mtb manipulates lipid metabolism to overcome host restriction
Third exposure to COVID-19 infection or vaccination differentially impacts T cell responses.
No full textBACKGROUND: In 2021, rapid rollout of two doses of SARS-CoV-2 vaccines reduced COVID-19 severity and mortality. However, further vaccine doses as a prime-boost schedule were limited, and lifting of public health restrictions by late 2021 frequently led to infection, rather than vaccine, as a third exposure. OBJECTIVE: To compare how third exposure through mRNA booster or SARS-CoV-2 infection shapes humoral and cellular immunity following two vaccine doses. METHODS: We compared immune responses after third exposure in healthy adults enrolled in the UCLH-Crick Legacy cohort study (NCT04750356) between those receiving ancestral spike-encoded mRNA booster (vaccine immunity, n = 38) or COVID-19 infection (hybrid immunity, n = 13) following two vaccine doses. Immune profiles were evaluated using live virus neutralization assays, IFN-γ ELISpot, Luminex assay, flow cytometry and mass cytometry. RESULTS: Both total anti-Spike IgG and variant-specific neutralising antibodies were comparable following infection or vaccine as a third exposure. Overall, T cell populations were similar, but functionally different. CD8⁺ Effector Memory (TEM) cells in the vaccine group showed higher expression of CD69 and Granzyme B following stimulation with SARS-CoV-2 Spike peptides. In contrast, the hybrid group produced higher levels of innate immune associated cytokines IL-10 and IL-34, as well as the T cell homing chemokine CCL25, after stimulation. CONCLUSIONS: While both exposures generated comparable breadth of protection against SARS-CoV-2 variants, our findings suggest that the route of third exposure influences different aspects of the immune response, warranting further investigation into long-term immunity at both systemic and mucosal sites
Genetic basis of non-syndromic childhood glaucoma associated with anterior segment dysgenesis: A narrative review
No full textTwenty causative genes have been reported that cause non-syndromic childhood glaucoma associated with anterior segment dysgenesis. FOXC1, PAX6 and PITX2 are the most well-known, but cases linked to SLC4A11, PITX3 and SOX11 have also been reported. As genetic testing becomes increasingly widespread and rates of molecular diagnosis rise, the extent of phenotypic overlap between the different genetic causes of non-syndromic glaucoma associated with anterior segment dysgenesis is becoming more evident. Taking aniridia as an example, whilst PAX6 mutations remain the predominant cause, variants in CYP1B1, FOXC1, PXDN and SOX11 have also been reported in patients with childhood glaucoma and aniridia. Developments in molecular-based therapies for retinal and corneal disease are advancing rapidly, and pre-clinical studies of gene-based treatments for glaucoma and aniridia are showing promising results. Use of adeno-associated viral vectors for gene delivery is most common, with improvements in intraocular pressure and retinal ganglion cell survival in Tg-MYOCY437H mouse models of glaucoma, and successful correction of a germline PAX6G194X nonsense variant in mice using CRISPR-Cas9 gene editing. This review will explore the actions and interactions of the genetic causes of non-syndromic glaucoma associated with anterior segment dysgenesis and discuss the current developments in molecular therapies for these patients
CLEM-Reg: an automated point cloud-based registration algorithm for volume correlative light and electron microscopy.
No full textVolume correlative light and electron microscopy (vCLEM) is a powerful imaging technique that enables the visualization of fluorescently labeled proteins within their ultrastructural context. Currently, vCLEM alignment relies on time-consuming and subjective manual methods. This paper presents CLEM-Reg, an algorithm that automates the three-dimensional alignment of vCLEM datasets by leveraging probabilistic point cloud registration techniques. Point clouds are derived from segmentations of common structures in each modality, created by state-of-the-art open-source methods. CLEM-Reg drastically reduces the registration time of vCLEM datasets to a few minutes and achieves correlation of fluorescent signal to submicron target structures in electron microscopy on three newly acquired vCLEM benchmark datasets. CLEM-Reg was then used to automatically obtain vCLEM overlays to unambiguously identify TGN46-positive transport carriers involved in protein trafficking between the trans-Golgi network and plasma membrane. Datasets are available on EMPIAR and BioStudies, and a napari plugin is provided to aid end-user adoption
Conformational dynamics of the bacterial E3 ligase SspH1.
No full textThe SspH/IpaH family of novel E3 ligases (NELs) are found in a number of Gram-negative bacteria and are used to target host enzymes for degradation to support pathogenesis. These E3 enzymes are autoinhibited in the absence of substrate and different models for release of autoinhibition have been suggested. However, many of the molecular details of individual steps during the ubiquitin transfer reaction remain unknown. Here, we present the crystal structure of Salmonella SspH1 and an analysis of the solution properties of SspH1 on its own and in complex with substrate and ubiquitin. Our data show that SspH1 exists in a conformational equilibrium between open and closed states and that substrate binding only modulates the distribution of these states but does not induce major conformational changes. This suggests that additional mechanisms must exist to bring the substrates close to the active site to mediate transfer of ubiquitin from the E3∼Ub conjugate
Imaging NRF2 activation in non-small cell lung cancer with positron emission tomography.
No full textMutations in the NRF2-KEAP1 pathway are common in non-small cell lung cancer (NSCLC) and confer broad-spectrum therapeutic resistance, leading to poor outcomes. Currently, there is no means to non-invasively identify NRF2 activation in living subjects. Here, we show that positron emission tomography imaging with the system xc- radiotracer, [18F]FSPG, provides a sensitive and specific marker of NRF2 activation in orthotopic, patient-derived, and genetically engineered mouse models of NSCLC. We found a NRF2-related gene expression signature in a large cohort of NSCLC patients, suggesting an opportunity to preselect patients prior to [18F]FSPG imaging. Furthermore, we reveal that system xc- is a metabolic vulnerability that can be therapeutically targeted with an antibody-drug conjugate for sustained tumour growth suppression. Overall, our results establish [18F]FSPG as a predictive marker of therapy resistance in NSCLC and provide the basis for the clinical evaluation of both imaging and therapeutic agents that target this important antioxidant pathway
CCP-volumeEM: Making modern computational methods accessible for volume electron microscopy
No full textPoster presented as part of the Crick BioImage Analysis Symposium 2025.Permission has been given by authors to upload to Crick Figshare.Copyright remains with the original authors.</p
Automated analysis of multi-photon calcium imaging datasetswith photon-mosaic
No full textModern calcium imaging datasets are growing rapidly in size and complexity. Researchers often rely on a patchwork of custom scripts and manual steps to process and analyze their data. This fragmentation makes it difficult to reproduce results, scale analyses across experiments, or share workflows with others. photon-mosaic addresses this challenge by providing a fully automated and modular analysis pipeline that connects widely used community tools under a consistent and reproducible project structure, from raw imaging data to analysis-ready results.Poster presented as part of the Crick BioImage Analysis Symposium 2025.Permission has been given by authors to upload to Crick Figshare.Copyright remains with the original authors.</p
HIFα isoform specific activities drive cell-type specificity of VHL-associated oncogenesis.
No full textCancers arising from dysregulation of generally operative signaling pathways are often tissue specific, but the mechanisms underlying this paradox are poorly understood. Based on striking cell-type specificity, we postulated that these mechanisms must operate early in cancer development and set out to study them in a model of von Hippel Lindau (VHL) disease. Biallelic mutation of the VHL ubiquitin ligase leads to constitutive activation of hypoxia inducible factors HIF1A and HIF2A and is generally a truncal event in clear cell renal carcinoma. We used an oncogenic tagging strategy in which VHL-mutant cells are marked by tdTomato, enabling their observation, retrieval, and analysis early after VHL-inactivation. Here, we reveal markedly different consequences of HIF1A and HIF2A activation, but that both contribute to renal cell-type specific consequences of VHL-inactivation in the kidney. Early involvement of HIF2A in promoting proliferation within the proximal tubular epithelium supports therapeutic targeting of HIF2A early in VHL disease