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Nuclear and cellular foci detection using Fiji and QuPath
No full textNuclear and cellular foci are ubiquitous in fluorescence microscopy images of cells and tissue (e.g. DNA damage response, protein aggregation, receptor clustering, vesicles, centromeres, FISH probes). Foci Analyzer is a comprehensive user-friendly Fiji macro to detect, visualize and quantify foci in 2D and 3D fluorescence microscopy images of cells and tissue. Accurate cell/nuclei segmentation is achieved by utilizing StarDist and Cellpose. CLIJ2 and MorpholobJ are employed for efficient and reliable foci detection across different imaging modalities and resolutions. The output data include detailed foci statistics, segmentations overlay images and colocalization overlap maps. We recently combined Foci Analyzer with QuPath, enabling foci analysis on very large images. Image tiles from annotated regions together with cell segmentations are exported from QuPath and analyzed in Fiji. Foci results can be imported into QuPath, allowing cells to be classified on their foci characteristics. Installation is straightforward via the Fiji Update Site 'Foci-Analyzer'. QuPath scripts are on GitHub. </p
Kropotkin and the anarchist case for penal abolition
No full textThis chapter examines the anarchist case for abolition by analysing the Peter Kropotkin’s In French and Russian Prisons (1887), perhaps the most influential historical critique. Using themes of environment, culture and social relationships, I discuss his account, explain his scepticism about reform and explain why he concluded that the only sensible answer to the question ‘are prisons necessary?’ was ‘no’. The final section follows the trajectory of two lines of Kropotkin’s abolitionist thesis in anarchist thought. The first ‘environmental’ strand focuses on the systemic injustices that incentivise wrongdoing and the second ‘ethical’ thread emphasises the faultiness of the concept of crime. The argument is that both underwrite the anarchist case for prison abolition.</p
Prevalence of drug resistance associated substitutions in persons with chronic hepatitis C infection and virological failure following initial or re-treatment with pan-genotypic direct-acting antivirals: A systematic review and meta-analysis.
No full textBACKGROUND: The advent of short-course, curative treatment with direct-acting antivirals (DAA) has given promise for the global elimination of hepatitis C virus (HCV) infections by 2030. Virological failure occurs in 2%-12% of persons receiving curative DAA treatment and may be presaged by pre-existing polymorphisms or result from selection of drug resistant variants during therapy. METHODS: We conducted a systematic review to assess the prevalence of HCV resistance associated substitutions (RAS) among individuals with chronic hepatitis C infection who had virological failure following initial or re-treatment with pan-genotypic DAA regimens. We included 34 and 22 studies assessing RAS in people with virological failure published between January 2014 and July 2023. Pooled RAS prevalence was estimated using random-effects meta-analysis. RESULTS: The pooled prevalence of RAS in people with virological failure following initial DAA treatment was 78.0% (95% confidence interval [CI]: 62.0-92.0) for sofosbuvir/velpatasvir, 81.0% (95% CI: 67.0-93.0) for sofosbuvir/daclatasvir, and 79.0% (95% CI: 70.0-87.0) for glecaprevir/pibrentasvir, with a high prevalence of resistance to the NS5A inhibitors. Among those with virological failure following re-treatment regimens, RAS were present in 93.0% (95% CI: 83.0-99.0) for sofosbuvir/velpatasvir/voxilepravir and in 100% (95% CI: 92.0-100) for glecaprevir/pibrentasvir, with resistance driven by RAS to NS5A inhibitors. DISCUSSION: At least 1 RAS is present in a high proportion of the few individuals with virological failure following initial or re-treatment with pan-genotypic DAA regimens. There is a need for ongoing surveillance for DAA-associated resistance, to assess risk factors for their development and clinical impact to inform best practice strategies for re-treatment
Clearance of beta-amyloid and tau aggregates is size dependent and altered by an inflammatory challenge.
No full textExtracellular beta-amyloid aggregation and inflammation are in a complex and not fully understood interplay during hyperphosphorylated tau aggregation and pathogenesis of Alzheimer's disease. Our group has previously shown that an immune challenge with tumour necrosis factor alpha can alter extracellular beta-sheet containing aggregates in human-induced pluripotent stem cell-derived cortical neurons carrying familial Alzheimer's disease-related presenilin 1 mutations. Here, using single-molecule detection and super-resolution imaging techniques, we quantified and characterized the intra- and extracellular beta-amyloid and AT8-positive tau aggregates. Our results indicate a pre-existing Alzheimer's disease-like pathology caused by the presenilin 1 mutation, with increased beta-amyloid aggregates in both the cell lysate and conditioned media compared to isogenic controls and also increased intracellular tau aggregates. The main effect of tumour necrosis factor alpha treatment on presenilin 1 neurons was the formation of larger intracellular beta-amyloid aggregates. In contrast, isogenic controls showed more significant changes with tumour necrosis factor alpha treatment with an increase in beta-amyloid aggregates in the media but not intracellularly and an increase in tau aggregates in both the media and cell lysate, suggesting a chronic inflammation-driven mechanism for the development of sporadic Alzheimer's disease. Remarkably, we also found significant morphological differences between intra- and extracellular beta-amyloid and tau aggregates in human-induced pluripotent stem cell-derived cortical neurons, suggesting these neurons can only clear aggregates when small, and that larger aggregates stay inside the neurons. While majority of the beta-amyloid aggregates were cleared into the media, a greater portion of the tau aggregates remained intracellular. This size-dependent aggregate clearance was also shown to be conserved in vivo, using soaked and homogenized mouse and human post-mortem Alzheimer's disease brain samples. As such, our results are proposing a previously unknown, size-dependent aggregate clearance mechanism, which can possibly explain the intracellular aggregation of tau and extracellular aggregation of beta-amyloid
Semisynthesis of isomerized histone H4 reveals robustness and vulnerability of chromatin toward molecular aging.
No full textProteins are subject to aging in the form of spontaneous, nonenzymatic post-translational modifications (PTMs). One such PTM is the formation of the β-linked isomer l-isoaspartic acid (isoAsp) from aspartic acid (Asp) or asparagine residues, which tends to occur in long-lived proteins. Histones can exhibit half-lives on the order of 100 days, and unsurprisingly, isoAsp formation has been observed in nearly every histone family. Delineating the molecular consequences of isoAsp formation in histones is challenging due to the multitude of processes that occur on such time scales. To isolate the effects of a specific isoAsp modification thus necessitates precise in vitro characterization with well-defined substrates. Here, we adapt a protein semisynthesis approach to generate full-length variants of histone H4 in which the canonical Asp at position 24 is replaced by its isoAsp isomer (H4isoD24). This variant was incorporated into chromatin templates, and the resulting constructs were used to interrogate key parameters of chromatin integrity and maintenance in vitro: compaction, nucleosome remodeling, and methylation of H4 lysine 20 (H4K20). Remarkably, despite its disruptive changes to the backbone's spacing and direction, isoD24 did not dramatically disrupt Mg2+-mediated chromatin self-association or nucleosome repositioning by the remodeler Chd1. In contrast, H4isoD24 significantly inhibited both Set8- and Suv4-20h1-catalyzed methylation at H4K20. These results suggest that H4isoD24 gives rise to a complex reorganization of the chromatin functional landscape, in which macroscopic processes show robustness and local mechanisms exhibit vulnerability to the presence of this mark
Molecular basis of FIGNL1 in dissociating RAD51 from DNA and chromatin
No full textMaintaining genome integrity is an essential and challenging process. RAD51 recombinase, the central player of several crucial processes in repairing DNA and protecting genome integrity, forms filaments on DNA, which are tightly regulated. One of these RAD51 regulators is FIGNL1, that prevents persistent RAD51 foci without or after DNA damage and genotoxic chromatin association in cells. The cryogenic electron microscopy structure of FIGNL1 in complex with RAD51 reveals that FIGNL1 forms a non-planar hexamer and RAD51 N terminus enclosure in the FIGNL1 hexamer pore. Mutations in pore loop or catalytic residues of FIGNL1 render it defective in filament disassembly and are lethal in mouse embryonic stem cells. Our study reveals a unique mechanism for removing RAD51 from bound substrates and provides the molecular basis for FIGNL1 in maintaining genome stability
<b>ELASTOGRAPHY AND CAP ASSESSMENT FOR LIVER DISEASE: </b><b>Training events and feedback representing clinical research</b><b> </b><b>programmes in South Africa, Uganda and Kenya</b>
No full textWe utilized data collected from three clinical research settings in Africa, where FibroScan™ devices were made available through research funding: the MRC funded a device for use in Uganda and Kenya, while the Francis Crick Institute, UK, provided core funding for a device in South Africa. Our objective was to train clinical and research practitioners on the use of FibroScan, assess its perceived usefulness and application in both healthcare and research contexts, and gather insights to improve and enhance future training efforts. In this report, we present the training sessions conducted across our study sites and analyze the feedback received from participants. This results were presented at the Global Hepatitis Summit (GHS) conference in Los Angeles, USA (March 2025) https://doi.org/10.6084/m9.figshare.27249723.v2.You can also take the survey: Scan QR in the report. </p
Modelling co-development between the somites and neural tube in human trunk-like structures.
No full textHuman stem cell-based embryo models provide experimentally amenable in vitro systems for developmental research. A key feature of embryo models is their multi-lineage differentiation, which allows for the study of tissue co-development. Here we develop human trunk-like structures that have morphologically organized somites and a neural tube that form through self-organized, endogenous signalling. Transcriptomic comparison with human embryo datasets suggests that human trunk-like structure cells approximate Carnegie stage 13-14 (28-35 days after fertilization). The absence of a notochord leads to a dorsal identity, but exogenous Sonic Hedgehog signalling activation ventralizes both the somites and the neural tube in a dose-dependent manner. We further identify reciprocal signalling: neural tube-derived cues induce medial ALDH1A2 in somites, which in turn generate retinoic acid signals that drive spontaneous neural-tube patterning. Together, our data highlight the value of modularity in embryo models, which we leverage to explore human trunk co-development
Network analysis of cross-income-level collaboration on multiple myeloma in sub-Saharan Africa
No full textCross-income-level collaboration (CILC) is crucial for developing global health approaches that benefit low- and middle-income countries (LMICs). Multiple myeloma (MM) is a representative example of a complex, understudied disease in sub-Saharan Africa (SSA). Based on publications, we developed a network analysis tool to assess scientific collaborations. Here, we present findings from a systematic analysis of publications retrieved from PubMed between January 2002 and June 2022. We evaluated individual institutional contributions and collaboration patterns using undirected weighted networks. Our findings reveal that intra-income-level collaborations dominate MM research in SSA, with high-income countries (HICs) primarily engaging with a few local institutions, mainly in South Africa and Nigeria. Increasing CILC is essential to advance research in this area. Our analysis tool provides insights into the collaboration strength, highlights gaps in the field and identifies leading institutions, ultimately aiming to support the development of more effective international collaboration and research strategies
Identification of undetected SARS-CoV-2 infections by clustering of Nucleocapsid antibody trajectories
No full textDuring the COVID-19 pandemic, numerous SARS-CoV-2 infections remained undetected. We combined results from routine monthly nose and throat swabs, and self-reported positive swab tests, from a UK household survey, linked to national swab testing programme data from England and Wales, together with Nucleocapsid (N-)antibody trajectories clustered using a longitudinal variation of K-means (N = 185,646) to estimate the number of infections undetected by either approach. Using N-antibody (hypothetical) infections and swab-positivity, we estimated that 7.4% (95%CI: 7.0–7.8%) of all true infections (detected and undetected) were undetected by both approaches, 25.8% (25.5–26.1%) by swab-positivity-only and 28.6% (28.4–28.9%) by trajectory-based N-antibody-classifications-only. Congruence with swab-positivity was respectively much poorer and slightly better with N-antibody classifications based on fixed thresholds or fourfold increases. Using multivariable logistic regression N-antibody seroconversion was more likely as age increased between 30–60 years, in non-white participants, those less (recently/frequently) vaccinated, for lower cycle threshold values in the range above 30, and in symptomatic and Delta (vs. BA.1) infections. Comparing swab-positivity data sources showed that routine monthly swabs were insufficient to detect infections and incorporating national testing programme/self-reported data substantially increased detection. Overall, whilst N-antibody serosurveillance can identify infections undetected by swab-positivity, optimal use requires fourfold-increase-based or trajectory-based analysis