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Targeted CRISPR screens reveal genes essential for Cryptosporidium survival in the host intestine.
No full textThe Cryptosporidium parasite is one of the leading causes of diarrheal morbidity and mortality in children, and adolescent infections are associated with chronic malnutrition. There are no vaccines available for protection and only one drug approved for treatment that has limited efficacy. A major barrier to developing new therapeutics is a lack of foundational knowledge of Cryptosporidium biology, including which parasite genes are essential for survival and virulence. Here, we iteratively improve the tools for genetically manipulating Cryptosporidium and develop a targeted CRISPR-based screening method to rapidly assess how the loss of individual parasite genes influence survival in vivo. Using this method, we examine the parasite's pyrimidine salvage pathway and a set of leading Cryptosporidium vaccine candidates. From this latter group, using inducible knockout, we determined the parasite gene known as Cp23 to be essential for survival in vivo. Parasites deficient in Cp23 were able to replicate within and emerge from infected epithelial cells, yet unable to initiate gliding motility which is required for the reinfection of neighbouring cells. The targeted screening method presented here is highly versatile and will enable researchers to more rapidly expand the knowledge base for Cryptosporidium infection biology, paving the way for new therapeutics
Adults in Ghana generate higher and more durable neutralising antibody titres following primary course COVID-19 vaccination than matched UK adults: The HERITAGE Study.
No full textBACKGROUND: Little data exist on the COVID-19 vaccine response in African countries who despite having high disease burden, have low COVID-19 mortality rates. We investigated the longitudinal immune response in a West-African urban population upon COVID-19 vaccination, two years after the start of the pandemic. METHODS: The HERITAGE study is a prospective cohort study of 301 residents of Accra, Ghana. Participants received two doses of a COVID-19 vaccine (AZD1222 or BNT162b2) from December 2021 and were followed-up for 12 months. COVID-19 status was determined by RT-PCR at seven time points. Serological responses, including anti-Nucleocapsid IgG, anti-Spike IgG and live-virus neutralisation were determined at four time points during the 12 months follow-up. RESULTS: COVID-19 positivity was 19.3% at baseline and reduced rapidly upon vaccination. Serological analyses indicated previous exposure to SARS-CoV-2 in 80.5% of the HERITAGE participants. After vaccination, neutralising antibody titres (NAbTs) against six different SARS-CoV-2 variants significantly (p < 0.001) increased, with fold changes (FC) ranging from 1.87 to 4.59. Highest NAbTs were recorded in the previously exposed group. Participants without prior exposure showed a continues increase in NAbTs between months 3 and 12 for circulating variants (Omicron B.A2 (FC 2.44, p < 0.001) and XBB.1.5 (FC 1.91, p = 0.05)). By comparison a matched cohort from the UK-based LEGACY study showed generally lower NAbTs at baseline (HERITAGE vs LEGACY for Wild-type: 250.3 vs 141.3, p < 0.0001, for A.27 84.6 vs 43.2, p = 0.0129, for Eta 159.7 vs 118.1, p = 0.3428, for Delta 158.6 vs 10.0, p < 0.0001, for Omicron B.A2 153.7 vs 10.0, p < 0.0001) and after receiving the vaccine (HERITAGE vs LEGACY for Wild-type: 882.6 vs 337.7, p < 0.0001, for A.27 552.0 vs 227.7, p = 0.0001, for Eta 682.2 vs 295.3, p < 0.0001, for Delta 557.6 vs 165.1, p < 0.0001, for Omicron B.A2 283.3 vs 124.2, p < 0.0001). NAbTs kinetics between the two cohorts were more similar when analysis was restricted to previously unexposed participants when adjusted for circulating variants during the sampling period. CONCLUSIONS: Two doses of AZD1222 or BNT162b2 significantly increased existing NAbTs against SARS-CoV-2 in a highly exposed population, showing durable boosting of pre-existing infection-induced immunity. This indicates the importance of considering local population exposure in vaccination design and deployment
Human DNA replication initiation sites are specified epigenetically by oxidation of 5-methyl-deoxycytidine.
No full textDNA replication initiates at tens of thousands of sites on the human genome during each S phase. However, no consensus DNA sequence has been found that specifies the locations of these replication origins. Here, we investigate modifications of human genomic DNA by density equilibrium centrifugation and DNA sequencing. We identified short discrete sites with increased density during quiescence and G1 phase that overlap with DNA replication origins before their activation in S phase. The increased density is due to the oxidation of 5-methyl-deoxycytidines by ten-eleven-translocation DNA dioxygenase (TET) enzymes at GC-rich domains. Reversible inhibition of de novo methylation and of subsequent oxidation of deoxycytidines results in a reversible inhibition of DNA replication and of cell proliferation. Our findings suggest a mechanism for the epigenetic specification and semiconservative inheritance of DNA replication origin sites in human cells that also provides a stable integral DNA replication licence to support once-per-cell cycle control of origin activation
CARM1/PRMT4 facilitates XPF-ERCC1 heterodimer assembly and maintains nucleotide excision repair activity.
No full textThe structure-specific endonuclease, XPF-ERCC1, plays a central role in DNA damage repair. This nuclease is known to be important for nucleotide excision repair, interstrand crosslink repair, and DNA double-strand repair. We found that the arginine methyltransferase, CARM1/PRMT4, is essential for XPF stabilization and maintenance of intracellular protein levels. Loss of CARM1 results in a decrease in XPF protein levels and a concomitant decrease in ERCC1 protein. A similar destabilization of XPF protein was observed in cells expressing a mutant in which XPF arginine 568 was replaced by lysine. Loss of CARM1 impaired XPF-ERCC1 accumulation at the site of damage and delayed removal of cyclobutane pyrimidine dimers by UV. As a result, CARM1-deficient cells showed increased UV sensitivity. Our results provide insight into the importance of CARM1 not only in the mechanism of XPF-ERCC1 complex stabilization but also in the maintenance of genome stability
Unidirectional MCM translocation away from ORC drives origin licensing.
No full textThe MCM motor of the eukaryotic replicative helicase is loaded as a double hexamer onto DNA by the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ATP binding supports formation of the ORC-Cdc6-Cdt1-MCM (OCCM) helicase-recruitment complex where ORC-Cdc6 and one MCM hexamer form two juxtaposed rings around duplex DNA. ATP hydrolysis by MCM completes MCM loading but the mechanism is unknown. Here, we used cryo-EM to characterise helicase loading with ATPase-dead Arginine Finger variants of the six MCM subunits. We report the structure of two MCM complexes with different DNA grips, stalled as they mature to loaded MCM. The Mcm2 Arginine Finger-variant stabilises DNA binding by Mcm2 away from ORC/Cdc6. The Arginine Finger-variant of the neighbouring Mcm5 subunit stabilises DNA engagement by Mcm5 downstream of the Mcm2 binding site. Cdc6 and Orc1 progressively disengage from ORC as MCM translocates along DNA. We observe that duplex DNA translocation by MCM involves a set of leading-strand contacts by the pre-sensor 1 ATPase hairpins and lagging-strand contacts by the helix-2-insert hairpins. Mutating any of the MCM residues involved impairs high-salt resistant DNA binding in vitro and double-hexamer formation assessed by electron microscopy. Thus, ATPase-powered duplex DNA translocation away from ORC underlies MCM loading
Integrated Mathematical Modelling of Image Analysis, Deep Learning, and Data Analysis Using Category Theory Tools
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
Heart of the matter: Neutrophils, cancer, and cardiovascular disease.
No full textCancer and cardiovascular disease together are leading causes of death worldwide, and cancer patients display an abnormally elevated burden of cardiovascular disease. Neutrophils-key immune cells known primarily by their roles in inflammation and infection-can link these two pathological conditions. Neutrophils contribute to cancer progression and cardiovascular complications through various mechanisms, including their ability to promote inflammation, thrombosis, and vascular damage by interacting with vascular endothelial cells, platelets, and other immune cells, or by forming NETs. In cancer, neutrophils contribute to a hypercoagulability state, which promotes tumor growth and metastasis, and can also lead to thrombotic events, myocardial infarction, and stroke. Cancer affects neutrophil numbers and functional properties, induces the appearance of several neutrophil subtypes, and can alter hematopoiesis. Here we summarize the links between cancer and cardiovascular disease, focusing on the role of neutrophils and cancer-elicited changes to their function in connecting these two disease states and highlighting the neutrophils' dynamic interaction with both diseases
Capturing nematic order on tissue surfaces of arbitrary geometry.
No full textA leading paradigm for understanding the large-scale behavior of tissues is via generalizations of liquid crystal physics; much like liquid crystals, tissues combine fluid-like, viscoelastic behaviors with local orientational order, such as nematic symmetry. Whilst aspects of quantitative agreement have been achieved for flat monolayers, the most striking features of tissue morphogenesis-including symmetry breaking, folding and invagination-concern surfaces with complex curved geometries in three dimensions. As yet, however, characterizing such behaviors has been frustrated due to the absence of proper image analysis methods; current state-of-the-art methods almost exclusively rely on two-dimensional intensity projections of multiple image planes, which superimpose data and lose geometric information that can be crucial. Here, we describe an analysis pipeline that properly captures the nematic order and topological defects associated with tissue surfaces of arbitrary geometry, which we demonstrate in the context of in vitro multicellular aggregates, and in vivo zebrafish hearts.</p
A role for human senataxin in contending with pausing and backtracking during transcript elongation.
No full textSenataxin (SETX) regulates RNA polymerase II (RNAPII) transcription and helps maintain genome stability, at least partly by suppressing R-loops. However, despite its importance in human disease, the precise function of SETX has remained unclear. Employing the degradation tag system for acute protein depletion, we demonstrate that SETX loss perturbs RNAPII elongation but does not markedly influence transcription termination at the end of genes. Through in vitro reconstitution of elongation, we show that SETX uses ATP-dependent RNA translocation to drive RNAPII forward across challenging DNA sequences, reminiscent of how bacterial ribosomes help mitigate RNAP pausing. In vivo, SETX depletion accordingly results in increased RNAPII pausing or backtracking, particularly during early elongation, with a corresponding, time-dependent local increase in R-loop formation. Together, these findings redefine our understanding of SETX's role in transcription and provide a mechanistic framework for interpreting R-loops and the causes of neurological disorders associated with SETX mutation
Force-triggered thermodynamically uphill disulfide reduction through sulfur oxidation state control.
No full textIn addition to thermal energy, current, and light, mechanical forces activate chemical reactions, often steering reaction pathways that result in products different from those obtained under thermodynamic control. Single-molecule mechanochemistry experiments have probed how the forced activation of a single covalent bond results in accelerated scission of both homolytic and heterolytic bonds, and the ring-opening of strained mechanophores in long polymers. Due to its mechanistic simplicity, the concerted SN2 thiol-disulfide nucleophilic substitution has been successfully used as a model system to interrogate how the nucleophilicity of an attacking organic, low-oxidation state thiol determines the force dependency of the thiol/disulfide exchange rate. Inorganic sulfur-oxyanions are comparatively much less reactive. Whether mechanical forces can activate the rupture of a protein disulfide by sulfur-oxyanions featuring higher oxidation states remains unknown. Here we employ single-molecule force-clamp spectroscopy, complemented by density functional theory (DFT) calculations and colorimetric assay measurements, to show that the thermodynamically nonfavored reduction of a disulfide bond by inorganic oxyanions can be activated by mechanical force. Occurring within the core of a protein with a physiological mechanical role, the force-unlocked reactivity has a direct impact on protein elasticity