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Image Analysis Challenges Within a Centralized Interdisciplinary Core Imaging Facility
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
Structural and functional characterization of the extended-diKH domain from the antiviral endoribonuclease KHNYN.
No full textZinc finger antiviral protein (ZAP) binds CpG dinucleotides in viral RNA and targets them for decay. ZAP interacts with several cofactors to form the ZAP antiviral system, including KHNYN, a multidomain endoribonuclease required for ZAP-mediated RNA decay. However, it is unclear how the individual domains in KHNYN contribute to its activity. Here, we demonstrate that the KHNYN amino terminal extended-diKH (ex-diKH) domain is required for antiviral activity and present its crystal structure. The structure belongs to a rare group of KH-containing domains, characterized by a non-canonical arrangement between two type-1 KH modules, with an additional helical bundle. N4BP1 is a KHNYN paralog with an ex-diKH domain that functionally complements the KHNYN ex-diKH domain. Interestingly, the ex-diKH domain structure is present in N4BP1-like proteins in lancelets, which are basal chordates, indicating that it is evolutionarily ancient. While many KH domains demonstrate RNA binding activity, biolayer interferometry and electrophoretic mobility shift assays indicate that the KHNYN ex-diKH domain does not bind RNA. Furthermore, residues required for canonical KH domains to bind RNA are not required for KHNYN antiviral activity. By contrast, an inter-KH domain cleft in KHNYN is a potential protein-protein interaction site and mutations that eliminate arginine salt bridges at the edge of this cleft decrease KHNYN antiviral activity. This suggests that this domain could be a binding site for an unknown KHNYN cofactor
Enhancing research through image analysis workshops: Experiences and best practices.
No full textModern microscopy systems allow researchers to generate large volumes of image data with relative ease. However, the challenge of analyzing these data effectively is often hindered by a lack of computational skills. This bottleneck negatively impacts both research reproducibility and efficiency, as researchers frequently rely on manual or semi-automated analysis methods. Interactive image analysis workshops offer a valuable solution, equipping researchers with the skills and tools needed to automate image processing tasks. In this paper, we share our experiences and best practices from conducting such workshops, which emphasize the use of open-source software like ImageJ, FIJI, and Python-based tools such as JupyterLab and napari. We discuss key considerations for workshop design, logistics, and outcomes, while highlighting common pitfalls to avoid. Using two recent workshops as case studies, we also present strategies for optimizing participant engagement and learning. Our insights offer practical guidance for planning and conducting image analysis workshops and serve as a starting point for researchers looking to establish similar training initiatives and enrich their local imaging communities
Researchers and research data: Improving and incentivising sharing and archiving
No full textThere has been a lot of discussion within the scientific community around the issues of reproducibility in research, with questions being raised about the integrity of research due to failure to reproduce or confirm the findings of some of the studies. Researchers need to adhere to the FAIR (findable, accessible, interoperable, and reusable) principles to contribute to collaborative and open science, but these open data principles can also support reproducibility and issues around ensuring data integrity. This article uses observations and metrics from data sharing and research integrity related activities, undertaken by a Research Integrity and Data Specialist at the Francis Crick Institute, to discuss potential reasons behind a slow uptake of FAIR data practices. We then suggest solutions undertaken at the Francis Crick institute which can be followed by institutes and universities to improve the integrity of research from a data perspective. One major solution discussed is the implementation of a data archive system at the Francis Crick Institute to ensure the integrity of data long term, comply with our funders’ data management requirements, and to safeguard our researchers against any potential research integrity allegations in the future
Dominant RDH12-retinitis pigmentosa impairs photoreceptor development and implicates cone involvement in retinal organoids.
No full textINTRODUCTION: Retinal dehydrogenase 12 (RDH12) is a photoreceptor NADPH-dependent retinal reductase enzyme, converting all-trans-retinal to all-trans-retinol. Heterozygous variants in RDH12 cause a rare autosomal dominant (AD) retinitis pigmentosa. METHODS: As no disease models exist, we generated human induced pluripotent stem cell-derived retinal organoids (RO) from an RDH12-AD patient (with pathogenic variant c.759del, p.Phe254Leufs*24) alongside a healthy, unaffected control. RESULTS: RDH12-AD RO exhibited correct localization of RDH12 to the photoreceptor inner segments up to week 44; however, transmission electron microscopy at week 37 showed that photoreceptors were less abundant and shorter in length. Visual cone function, retinol biosynthesis, and the vitamin A pathway were also highly disrupted at week 44. DISCUSSION: Our study is the first to describe an RDH12-AD disease model with pathology at later stages of photoreceptor differentiation, in keeping with the milder disease course seen in humans. It provides insights into the etiology and possible targets for future therapeutic development
Mutations in PSEN1 predispose inflammation in an astrocyte model of familial Alzheimer's disease through disrupted regulated intramembrane proteolysis.
No full textBACKGROUND: Mutations in PSEN1 cause familial Alzheimer's disease with almost complete penetrance. Age at onset is highly variable between different PSEN1 mutations and even within families with the same mutation. Current research into late onset Alzheimer's disease implicates inflammation in both disease onset and progression. PSEN1 is the catalytic subunit of γ-secretase, responsible for regulated intramembrane proteolysis of numerous substrates that include cytokine receptors. For this reason, we tested the hypothesis that mutations in PSEN1 impact inflammatory responses in astrocytes, thereby contributing to disease progression. METHODS: We developed patient-derived models of iPSC-astrocytes, representing three lines harbouring PSEN1 mutations and six control lines (including two isogenic controls). Transcriptomic and biochemical assays were used to investigate differential inflammatory responses to TNFα, IL1α and C1Q. RESULTS: We show that PSEN1 is upregulated in response to inflammatory stimuli, and this upregulation is disrupted by pathological PSEN1 mutations. Using transcriptomic analyses, we demonstrate that PSEN1 mutant astrocytes have an augmented inflammatory profile in their basal state, concomitant with gene expression signatures revealing dysregulated intramembrane proteolysis and JAK-STAT signalling. Detailed investigation of the JAK-STAT2 signalling pathway showed reduced cell surface expression of IFNAR2, lower STAT2 phosphorylation cascades and delayed NFκB nuclear localisation in PSEN1 mutant astrocytes in response to inflammatory stimuli, thereby implicating the notion of altered cytokine signalling cascades. Finally, we use small molecule modulators of γ-secretase to confirm a role for PSEN1/γ-secretase in regulating the astrocytic response to inflammatory stimuli. CONCLUSIONS: Together, these data suggest that mutations in PSEN1 enhance cytokine signalling via impaired regulated intramembrane proteolysis, thereby predisposing astrocytic inflammatory profiles. These findings support a two-hit contribution of PSEN1 mutations to fAD pathogenesis, not only impacting APP and Aβ processing but also altering the cellular response to inflammation
Retroelement co-option disrupts the cancer transcriptional programme.
No full textBACKGROUND: Transcriptional activation of otherwise repressed retrotransposable elements (RTEs) is a hallmark of cancer, shaping tumour progression and immunogenicity by multifaceted, yet incompletely understood, mechanisms. METHODS: We used an extended pan-cancer transcriptome assembly to identify potential effects of RTEs on the genes within which they have integrated or those in proximity. These were subsequently verified in test cases by further analysis of transcriptional profiles in cancer patient data, and by in vitro studies involving restoration of gene activity, and proliferation and migration assays in cancer cell lines. RESULTS: We report that cancer-specific transcriptional activation of RTEs causes frequent reduction or loss of gene function. Exonisation and alternative splicing of RTEs creates non-functional RNA and protein isoforms and derepressed RTE promoter activity initiates antisense transcription, both at the expense of the canonical isoforms. Contrary to theoretical expectation, transcriptionally activated RTEs affect genes with established tumour-promoting functions, including the common essential RNGTT and the lung cancer-promoting CHRNA5 genes. Furthermore, the disruptive effect of RTE activation on adjacent tumour-promoting genes is associated with slower disease progression in clinical data, whereas experimental restoration of gene activity enhances tumour cell growth and invasiveness in vitro. CONCLUSIONS: These findings underscore the gene-disruptive potential of seemingly innocuous germline RTE integrations, unleashed only by their transcriptional utilisation in cancer. They further suggest that such metastable RTE integrations are co-opted as sensors of the epigenetic and transcriptional changes occurring during cellular transformation and as executors that disrupt the function of tumour-promoting genes
Single-cell RNA-seq identifies protracted mouse germline X chromosome reactivation dynamics directed by a PRC2-dependent mechanism.
No full textFemale primordial germ cells (PGCs) undergo X chromosome reactivation (XCR) during genome-wide reprogramming. XCR kinetics and dynamics are poorly understood at a molecular level. Here, we apply single-cell RNA sequencing and chromatin profiling on germ cells from F1 mouse embryos, performing a precise appraisal of XCR spanning from migratory-stage PGCs to gonadal germ cells. Establishment of germ cell sexual dimorphism and X chromosome dosage compensation states in vivo are temporally linked to XCR. Allele-specific analysis evidence that the reactivating X chromosome is minimally active in embryonic day (E)9.5 female PGCs, reactivates gradually, and reaches parity to the active X chromosome in E16.5 oogonia. While Xist is repressed from E10.5 onward, epigenetic memory of X inactivation persists from self-sustained polycomb repressive complex 2 (PRC2) activity. The reactivating X is asymmetrically enriched for histone 3-lysine-27-trimethylation (H3K27me3) at E13.5, which is later reversed, permitting germline gene expression. Our findings relate XCR with PRC2 function in promoting female meiosis
The molecular dissection of TRIM25's RNA-binding mechanism provides key insights into its antiviral activity.
No full textTRIM25 is an RNA-binding ubiquitin E3 ligase with central but poorly understood roles in the innate immune response to RNA viruses. The link between TRIM25's RNA binding and its role in innate immunity has not been established. Thus, we utilized a multitude of biophysical techniques to identify key RNA-binding residues of TRIM25 and developed an RNA-binding deficient mutant (TRIM25-m9). Using iCLIP2 in virus-infected and uninfected cells, we identified TRIM25's RNA sequence and structure specificity, that it binds specifically to viral RNA, and that the interaction with RNA is critical for its antiviral activity
Plasmodium falciparum protein phosphatase PP7 is required for early ring-stage development.
No full textWe previously reported that the Plasmodium falciparum putative serine/threonine protein phosphatase 7 (PP7) is a high-confidence substrate of the cAMP-dependent protein kinase (PKA). Here we explore the function of PP7 in asexual P. falciparum blood stage parasites. We show that conditional disruption of PP7 leads to a severe growth arrest. We show that PP7 is a calcium-dependent phosphatase that interacts with calmodulin and calcium-dependent protein kinase 1 (CDPK1), consistent with a role in calcium signaling. Notably, PP7 was found to be dispensable for erythrocyte invasion, but was crucial for ring-stage development, with PP7-null parasites arresting shortly following invasion and showing no transition to ameboid forms. Phosphoproteomic analysis revealed that PP7 may regulate certain PKAc substrates. Its interaction with calmodulin and CDPK1 further emphasizes a role in calcium signaling, while its impact on early ring development and PKAc substrate phosphorylation underscores its importance in parasite development. IMPORTANCE: Plasmodium falciparum causes malaria and is responsible for more than 600,000 deaths each year. Although effective drugs are available to treat disease, the spread of drug-resistant parasites endangers their future efficacy. It is hoped that a better understanding of the biology of malaria parasites will help us to discover new drugs to tackle the resistance problem. Our work is focused on the cell signaling mechanisms that control the development of the parasite throughout its complex life cycle. All signal transduction pathways are ultimately regulated by reversible protein phosphorylation by protein kinase and protein phosphatase enzymes. In this study, we investigate the function of calcium-dependent protein phosphatase PP7 and show that it is essential for the development of ring-stage parasites following the invasion of human erythrocytes. Our results contribute to the understanding of the erythrocytic stages of the parasite life cycle that cause malaria pathology.</p