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Evidence for enhancer activity in intron 1 of TNFRSF1A using CRISPR/Cas9 in human induced pluripotent stem cell-derived macrophages.
No full textTNFα is a common drug target in the treatment of autoimmune diseases, with pro-inflammatory functions that are primarily mediated through its receptor, TNFRSF1A. TNFRSF1A has been genetically associated with many immune-mediated diseases including ankylosing spondylitis, multiple sclerosis, and inflammatory bowel disease. Many of the genetic variants within or near TNFRSF1A that have been associated with disease through genome-wide association studies (GWAS) lie in non-coding regions of the genome. Understanding the functional consequences of these genetic variants is limited by incomplete understanding of TNFRSF1A gene regulation, including for specific cellular contexts relevant to inflammation and immunity such as macrophages. This work used CRISPR/Cas9 in human induced pluripotent stem cells followed by differentiation into macrophages to investigate putative regulatory elements in the TNFRSF1A gene locus. Through gene editing, with functional genomic readouts including the assay for transposase-accessible chromatin using sequencing (ATAC-Seq), chromatin immunoprecipitation with sequencing (ChIP-Seq), and RNA-Seq to assess the consequences of these edits, we present evidence for an enhancer of TNFRSF1A contained within an intron of the gene. Understanding gene regulation and the genomic context in which GWAS variants lie could bring us closer to deconvoluting the genetic basis of common disease aetiology and uncover effective drug targets
muvis-align: building a registration pipeline for large data & next gen file format
No full textWe present a new registration pipeline called muvis-align, which is based on the multiview-stitcher toolbox. Multiview-stitcher is an open-source modular toolbox developed for distributed and tiled stitching of2-3D data. This modular, powerful toolbox is used to develop a flexible registration pipeline including pre-and post-processing steps as well as custom functions allowing x-y stitching and z reconstruction, for different image modalities. Importantly muvis-align overcomes limitations in existing tools used commonly, in particular handling large datasets (TBs) and fully supports the Next Generation File Format OME-Zarr. Preliminary registration tests show results of various LM and EM datasets equal or better compared to existing tools using affine transformations. This tool is further being developed with a napari user interface allowing easy configuration, exploring responsive dynamic configuration and providing visual feedback on preliminary and final results.</p
Exploiting time to phenotype cell morphology
No full text• The morphology and appearance of cells in time-lapse microscopy can be diagnostic for biological state.• Classical measures of morphology offer an incomplete and poorly-aligned read-out for these presumed states.• In computer vision, deep neural networks can produce rich representations of natural images that align with language models, but it is unclear how to align these representations to the underlying biology.• Many biological states of interest vary only slowly in time, so we hypothesised that neural networks trained for time-consistency could better align with such states by learning to ignore short-term change like reorientation of organelles or drift in the focal plane.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
Scaling up automated cell tracking
No full textGlioblastoma (GBM) cells are a highly motile, meaning they can infiltrate healthy tissue far from the original tumour site, contributing to the low survival rates post diagnosis [1]. At the CRUK Scotland Institute, we are investigating treatments to impede GBM motility to improve patient outcomes post diagnosis [2]. Manually tracking cell motility is a labour-intensive task, requiring researchers to evaluate their datasets by hand to extract key information about cell speed [3]. As such, a subset of cells are usually chosen for analysis (usually 10-20 cells per image region), which can lead to researcher bias influencing the results of the analysis [4]. We present a workflow here for the imaging and analysis of cells that have been treated with a nuclear dye prior to imaging. With the addition of the nuclear dye, and imaging in the fluorescence channel, we take advantage of the improved signal to noise ratio for cell nuclei segmentation using Cellpose 3 [5] and track the subsequent cell masks using the automated cell tracking package Btracks [6]. This process has been automated using python and is packaged together in a Jupyter Notebook to create a workflow that has been custom built with non-coders in mind.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
TDP-43 loss induces cryptic polyadenylation in ALS/FTD.
No full textNuclear depletion and cytoplasmic aggregation of the RNA-binding protein TDP-43 are cellular hallmarks of amyotrophic lateral sclerosis (ALS). TDP-43 nuclear loss causes de-repression of cryptic exons, yet cryptic alternative polyadenylation (APA) events have been largely overlooked. In this study, we developed a bioinformatic pipeline to reliably identify alternative last exons, 3' untranslated region (3'UTR) extensions and intronic polyadenylation APA event types, and we identified cryptic APA sites induced by TDP-43 loss in induced pluripotent stem cell (iPSC)-derived neurons. TDP-43 binding sites are enriched at sites of these cryptic events, and TDP-43 can both repress and enhance APA. All categories of cryptic APA were also identified in ALS and frontotemporal dementia (FTD) postmortem brain tissue. RNA sequencing (RNA-seq), thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) and ribosome profiling (Ribo-seq) revealed that distinct cryptic APA categories have different downstream effects on transcript levels and that cryptic 3'UTR extensions can increase RNA stability, leading to increased translation. In summary, we demonstrate that TDP-43 nuclear depletion induces cryptic APA, expanding the palette of known consequences of TDP-43
Mechanochemical coupling of cell shape and organ function optimizes heart size and contractile efficiency in zebrafish.
No full textHow simple tissue primordia sculpt complex functional organs, robustly and reproducibly, remains elusive. During zebrafish development, the embryonic myocardial wall matures into an intricate 3D architecture, composed of an outer compact layer enveloping an inner layer of multicellular trabecular ridges. How these tissue layers acquire their characteristic form suited for their function remains an open question. Here, we find that multiscale mechanochemical coupling and an emergent tissue-scale morphological transition steer functional maturation of the developing zebrafish heart. Single-celled trabecular seeds recruit outer compact layer cells to mature into clonally heterogeneous multicellular ridges, thereby amplifying cardiac contractile forces. In response, the remaining compact layer cells are stretched, which impedes their further recruitment, thereby constraining trabecular ridge density. Concomitantly, Notch-dependent actomyosin dampening triggers a sharp transition in myocardial tissue area, activating rapid organ growth that expands blood-filling capacity. Thus, multiscale self-organizing interactions optimize heart size and contractile efficiency to support embryonic life
A genetic circuit that extends the useful range of a BMP morphogen arose alongside insect wing evolution.
No full textThe range over which a morphogen gradient provides reliable positional information is limited by intrinsic noise. We identify a regulatory circuit that counteracts this constraint for Dpp, a BMP that organizes the anterior/posterior axis of Drosophila wings. The transcriptional repressor Brinker (Brk), a Dpp target, enhances positional precision by repressing Dad, an inhibitory Smad, thereby extending Dpp's effective range. Thus, Brk mediates a feedback circuit that selectively amplifies low-level Dpp signals as would a logarithmic amplifier. This circuit also achieves temporal integration, mitigating the inevitable noise penalty associated with amplification. Although a core component of BMP signaling in flies, Brk is found exclusively in insects. Phylogenetic and expression analyses in the apterygote insect Thermobia domestica suggest that Brk originated in insects and was incorporated into the BMP network in pterygotes, possibly to permit long-range signaling in wing primordia. Brk exemplifies how gene regulatory network (GRN) evolution can enhance developmental precision, thus opening the door to increased morphological complexity
Data associated with "Effects of M. tuberculosis and HIV-1 infection on in vitro blood-brain barrier function" paper
No full textREAD ME---Below is all the information you will need to understand the raw data associated with the paper:**Effects of M. tuberculosis and HIV-1 infection on in vitro blood-brain barrier function***Alizé Proust, Katalin A. Wilkinson, Robert J. Wilkinson* ---**Figure 1 and Additional file 1:**This folder contains all data used for Figure 1 and Additional file 1.The file names indicate the purpose of the experiment (HIV infection, Mtb entry of Mtb growth) and the cell type (astrocytes, hCMEC/d3, HBVP or HMC3).Each file contains all the data organised per experiment (TB-HIV#1 to TB-HIV#7).**Figure 2:**This folder contains all data used for Figure 2.- Figure 2A-B - Dextran-Rhodamine : it includes fluorescence readings of dextran rhodamine B per experiment at 2 and 6 days post-infection (TB-HIV#4 to TB-HIV#12)- Figure 2C-D - CFU: it includes CFUs count per experiment at 2 and 6 days post-infection (TB-HIV#4 to TB-HIV#12).**Figure 3:**This folder contains all data used for Figure 3.The file names indicate the cell type (astrocytes, hCMEC/d3, HBVP or HMC3).Each file contains all the data organised per experiment (TB-HIV#1 to TB-HIV#15).**Figure 4 and Additional file 5:**This folder contains all data used for Figure 4 and Supplementary Figure 5.- Subfolder Figure 4A-D : it includes the absorbance readings per cell type (TB-HIV#1 to TB-HIV#7).- Subfolder Figure 4E-H (and Additional file 5): it includes the luminescence readings per cell type. Each file contains all the data organised per experiment (TB-HIV#1 to TB-HIV#7).The file names indicate the type of experiment (MTS assay or ROS assay) and the cell type.**Figure 5:**This folder contains all data used for Figure 5.- Figure 5A-B - Astrogliosis: it contains all the qPCR data organised per experiment (TB-HIV#1 to TB-HIV#7).- Subfolder Figure 5C-H: it includes the fluorescence readings of glutamate organised per cell type (astrocytes, hCMEC/d3, HBVP or HMC3). Each file contains all the data organised per experiment (TB-HIV#1 to TB-HIV#12).**Figure 6 and Additional file 7:**This folder contains all Luminex data in pg/mL and stats used for Figure 6 and Additional file 7.The file names indicate the cell type (astrocytes, hCMEC/d3, HBVP or HMC3).Each file contains all the data organised per cytokine.**Additional file 4:**This folder contains all data used for Additional file 4.it contains all the qPCR data organised per experiment (TB-HIV#1 to TB-HIV#6).**Additional file 6:**This folder contains all data used for Additional file 6. it contains all the qPCR data organised per cell type (astrocytes, hCMEC/d3, HBVP or HMC3).Each files contains all the qPCR data organised per experiment (TB-HIV#1 to TB-HIV#6).</p
Cryptosporidium modifies intestinal microvilli through an exported virulence factor.
No full textCryptosporidium is a common intestinal infection of vertebrates and a significant threat to public health. Within the epithelial layer of the intestine, the parasite invades and replicates. Infected cells are readily detected under a microscope by the presence of elongated microvilli, particularly around the vacuole where the parasite resides. Here, we identify a family of Cryptosporidium virulence factors that are exported into the host cell during infection and localize to the microvilli. We examine the trafficking and function of the most highly expressed family member, Microvilli protein 1 (MVP1), which appears to control the elongation of microvilli through engagement of host EBP50 and CDC42. Remarkably, this mechanism closely mirrors that of an enteropathogenic Escherichia coli virulence factor, MAP, which is also known to drive host microvilli elongation during infection. This highlights a unique instance where eukaryotic and prokaryotic virulence factors have convergently evolved to modulate host actin structures through a similar mechanism
Macrocyclic peptide probes for immunomodulatory protein CD59: Potent modulators of bacterial toxin activity and antibody-dependent cytotoxicity.
No full textCD59 is an immunomodulatory cell surface receptor associated with human disease. Despite its importance in complement regulation and bacterial pathogenesis, CD59 remains a challenging therapeutic target. Research to date has focused on antibody or protein-based strategies. Here we present a new approach to target CD59 using macrocyclic peptides with low nanomolar affinity for CD59. Through X-ray crystallographic studies and structure-activity relationship studies we identify key interactions which are essential for binding and activity. We find that the macrocyclic peptide CP-06 adopts a beta-hairpin structure and binds CD59 through an intermolecular beta-sheet, mimicking protein-protein interactions of biologically relevant CD59 interaction partners. We create dimeric and lipidated macrocyclic peptide conjugates as enhanced cell-active CD59 inhibitors and show that these probes can be used to modulate both complement-mediated killing of human cells and lytic activity of bacterial virulence factors. Together, our data provide a starting point for future development of macrocyclic peptides to target CD59 activity in diverse cellular contexts