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Copper-enriched automotive brake wear particles perturb human alveolar cellular homeostasis.
No full textBACKGROUND: Airborne fine particulate matter with diameter < 2.5 μm (PM2.5), can reach the alveolar regions of the lungs, and is associated with over 4 million premature deaths per year worldwide. However, the source-specific consequences of PM2.5 exposure remain poorly understood. A major, but unregulated source is car brake wear, which exhaust emission reduction measures have not diminished. METHODS: We used an interdisciplinary approach to investigate the consequences of brake-wear PM2.5 exposure upon lung alveolar cellular homeostasis using diesel exhaust PM as a comparator. This involved RNA-Seq to analyse global transcriptomic changes, metabolic analyses to investigate glycolytic reprogramming, mass spectrometry to determine PM composition, and reporter assays to provide mechanistic insight into differential effects. RESULTS: We identified brake-wear PM from copper-enriched non-asbestos organic, and ceramic brake pads as inducing the greatest oxidative stress, inflammation, and pseudohypoxic HIF activation (a pathway implicated in diseases associated with air pollution exposure, including cancer, and pulmonary fibrosis), as well as perturbation of metabolism, and metal homeostasis compared with brake wear PM from low- or semi-metallic pads, and also, importantly, diesel exhaust PM. Compositional and metal chelator analyses identified that differential effects were driven by copper. CONCLUSIONS: We demonstrate here that brake-wear PM may perturb cellular homeostasis more than diesel exhaust PM. Our findings demonstrate the potential differences in effects, not only for non-exhaust vs exhaust PM, but also amongst different sources of non-exhaust PM. This has implications for our understanding of the potential health effects of road vehicle-associated PM. More broadly, our findings illustrate the importance of PM composition on potential health effects, highlighting the need for targeted legislation to protect public health
An in vivo CRISPR screen in chick embryos reveals a role for MLLT3 in specification of neural cells from the caudal epiblast.
No full textTissue development relies on the coordinated differentiation of stem cells in dynamically changing environments. The formation of the vertebrate neural tube from stem cells in the caudal lateral epiblast (CLE) is a well characterized example. Despite an understanding of the signalling pathways involved, the gene regulatory mechanisms remain poorly defined. To address this, we developed a multiplexed in vivo CRISPR screening approach in chick embryos targeting genes expressed in the caudal epiblast and neural tube. This revealed a role for MLLT3, a component of the super elongation complex, in the specification of neural fate. Perturbation of MLLT3 disrupted neural tube morphology and reduced neural fate acquisition. Mutant forms of Retinoic Acid Receptor A lacking the MLLT3 binding domain similarly reduced neural fate acquisition. Together, these findings validate an in vivo CRISPR screen strategy in chick embryos and identify a previously unreported role for MLLT3 in caudal neural tissue specification
Train the Trainers in Bioimage Analysis: A Community Initiative
No full textThe Royal Microscopical Society Data Analysis in IMaging (RMS DAIM), with the support of the Crick and Partners Networking Fund, has organised a "Train the Trainers in Bioimage Analysis" workshop in June 2025 at University College London. The Train the Trainers initiative aimed to establish and disseminate guidelines on how to organise courses and train researchers in bioimage analysis. Attendees of the Train the Trainer session became part of a network that provides local expertise and creates efficient channels for the dissemination of knowledge and good practices in bioimage analysis. The event was free, and travel and accommodation support was available for attendees outside the London area (max €100 pp). Participants were selected based on their expertise and role as trainers in bioimaging facilities or research groups. The target audience included researchers with knowledge of bioimage analysis principles who wished to improve their skills in providing training. Here, we present our experience in organising such an event, our take-home messages and feedback from the attendees. 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
Spatiotemporal orchestration of mitosis by cyclin-dependent kinase.
No full textMitotic onset is a critical transition for eukaryotic cell proliferation. The commonly held view of mitotic control is that the master regulator, cyclin-dependent kinase (CDK), is first activated in the cytoplasm, at the centrosome, initiating mitosis1-3. Bistability in CDK activation ensures that the transition is irreversible, but how this unfolds in a spatially compartmentalized cell is unknown4-8. Here, using fission yeast, we show that CDK is first activated in the nucleus, and that the bistable responses differ markedly between the nucleus and the cytoplasm, with a stronger response in the nucleus driving mitotic signal propagation from there to the cytoplasm. Abolishing cyclin-CDK localization to the centrosome led to activation occurring only in the nucleus, spatially uncoupling the nucleus and cytoplasm mitotically, suggesting that centrosomal cyclin-CDK acts as a 'signal relayer'. We propose that the key mitotic regulatory system operates in the nucleus in proximity to DNA, which enables incomplete DNA replication and DNA damage to be effectively monitored to preserve genome integrity and to integrate ploidy within the CDK control network. This spatiotemporal regulatory framework establishes core principles for control of the onset of mitosis and highlights that the CDK control system operates within distinct regulatory domains in the nucleus and cytoplasm
Functional liver genomics identifies hepatokines promoting wasting in cancer cachexia.
No full textIn cancer cachexia, the presence of a tumor triggers systemic metabolic disruption that leads to involuntary body weight loss and accelerated mortality in affected patients. Here, we conducted transcriptomic and epigenomic profiling of the liver in various weight-stable cancer and cancer cachexia models. An integrative multilevel analysis approach identified a distinct gene expression signature that included hepatocyte-secreted factors and the circadian clock component REV-ERBα as key modulator of hepatic transcriptional reprogramming in cancer cachexia. Notably, hepatocyte-specific genetic reconstitution of REV-ERBα in cachexia ameliorated peripheral tissue wasting. This improvement was associated with decreased levels of specific cachexia-controlled hepatocyte-secreted factors. These hepatokines promoted catabolism in multiple cell types and were elevated in cachectic cancer patients. Our findings reveal a mechanism by which the liver contributes to peripheral tissue wasting in cancer cachexia, offering perspectives for future therapeutic interventions
Identification of RING E3 pseudoligases in the TRIM protein family.
No full textTRIpartite Motif (TRIM) family proteins have diverse roles across a broad variety of cellular functions, which are largely presumed to depend on their ubiquitin E3 ligase activity, conferred by a RING domain. However, recent reports have shown that some TRIMs lack detectable ubiquitination activity in isolation, despite containing a RING domain. Here, we present parallel in cellulo, in vitro, and in silico structure-function analyses of the ubiquitin E3 ligase activity and RING domain structural characteristics of the whole TRIM protein family. In-depth follow-up studies of this comprehensive dataset reveals a number of 'pseudoligases', whose RING domains have structurally diverged at either the homodimerisation or E2~ubiquitin interfaces, thereby disrupting their ability to catalyse ubiquitin transfer. Together, these data raise intriguing open questions regarding the unknown TRIM functions in physiology and disease
Life and death of Yfh1: how cool is cold denaturation.
No full textYeast frataxin (Yfh1) is a small natural protein from yeast that has the unusual property of undergoing cold denaturation at temperatures above the freezing point of water when under conditions of low ionic strength. This peculiarity, together with remarkable resilience, allows the determination, for the whole protein as well as for individual residues, of the stability curve, that is the temperature dependence of the free energy difference between the unfolded and folded forms. The ease of measuring stability curves without the need to add denaturants or introduce ad hoc destabilizing mutations makes this protein an ideal 'tool' for investigating the influence of many environmental factors on protein stability. The present review aims at recapitulating all the open questions that Yfh1 has helped to address, including understanding the differences and commonalities of the cold, heat and pressure unfolded states. This protein thus offers a unique tool for studying aspects of protein stability so far been considered difficult to assess and provides important guidelines that could allow the identification of other similar systems
Cell nuclei segmentation in mm-scale x-ray holographic nanotomography images of mouse brain tissue
No full textBiological soft tissues are functional agglomerates of cells. They constitute the microenvironment where intercellular communication occurs. In turn, their woven structure underlies mechanical properties that contribute to their roles in the context of the organs and the organisms that contain them. Therefore, determining the density and spatial distribution of cells within the tissue offers key information for understanding its physiological properties and its state. X-ray holographic nanotomography is a non-destructive imaging technique capable of resolving subcellular details in biological tissues that has shown promising advantages to study the structure of neuronal circuits. However, the dimensions of the datasets required – covering volume landscapes of ~mm3 – make manual annotation of individual nuclei an unrealistic task. We developed and trained an automated image segmentation classifier that accurately detects and segments cell nuclei in mouse brain tissue imaged with X-ray holographic nanotomography, and that generalises to similar datasets obtained from biological replicates with minimal additional ground truth. It provides the spatial locations and morphologies of the ~80k nuclei per dataset with a high recall. It harnesses the strengths of a high-performance computing cluster and embeds the curated results in two main simplified outcomes: a data table and explorable image segmentations and meshes associated with the original dataset, in a browser-compatible format that simplifies proofreading by multiple users. The classifier we present here can be readily integrated into an automated analytical pipeline for histological datasets obtained with synchrotron X-ray holographic nanotomography in the context of systems neuroscience as well as broader tissue life science studies
Clone copy number diversity is linked to survival in lung cancer.
No full textBoth single nucleotide variants (SNVs) and somatic copy number alterations (SCNAs) accumulate in cancer cells during tumour development, fuelling clonal evolution. However, accurate estimation of clone-specific copy numbers from bulk DNA-sequencing data is challenging. Here we present allele-specific phylogenetic analysis of copy number alterations (ALPACA), a method to infer SNV and SCNA coevolution by leveraging phylogenetic trees reconstructed from multi-sample bulk tumour sequencing data using SNV frequencies. ALPACA estimates the SCNA evolution of simulated tumours with a higher accuracy than current state-of-the-art methods1-4. ALPACA uncovers loss-of-heterozygosity and amplification events in minor clones that may be missed using standard approaches and reveals the temporal order of somatic alterations. Analysing clone-specific copy numbers in TRACERx421 lung tumours5,6, we find evidence of increased chromosomal instability in metastasis-seeding clones and enrichment for losses affecting tumour suppressor genes and amplification affecting CCND1. Furthermore, we identify increased SCNA rates in both tumours with polyclonal metastatic dissemination and tumours with extrathoracic metastases, and an association between higher clone copy number diversity and reduced disease-free survival in patients with lung cancer