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Structure-function analysis of SLX4 and its scaffolded nucleases
No full textThe ever-expanding utility of cryoelectron microscopy (cryo-EM) has enabled resolving architectures of protein complexes that were once considered beyond reach. However, DNA damage-associated protein complexes pose unique challenges due to their pronounced flexibility and intrinsic disorder, which render them poorly defined even by advanced in silico machine-learning-based structural predictions. Such complexes denote high- priority but structurally elusive targets, necessitating careful purification and experimental structure determination.One such complex is the SLX4-scaffolded nuclease complex (SMX) comprising the DNA nucleases XPF-ERCC1, MUS81-EME1 and SLX1. This complex is profoundly implicated in cancer, ageing, and Fanconi anaemia (FA) - diseases intricately linked to DNA damage and repair mechanisms. SLX4 serves as a platform for recruiting these nucleases and coordinates the resolution of diverse DNA lesions, particularly interstrand crosslinks (ICLs), which are among the most cytotoxic forms of DNA damage. The structural characterisation of the SMX complex remains a critical and ambitious objective of this project. Furthermore, endogenous causative agents generating ICLs in cells remain an area of active investigation.This work contributes to the growing evidence implicating formaldehyde as a key endogenous source of ICLs, supporting its intrinsic connection to aldehyde toxicity and the Fanconi anaemia pathway. This study further proposes that the accumulation of formaldehyde during epigenetic reprogramming may represent a significant source of ICLs, potentially contributing to the differentiation defects observed in the blood lineages of patients with FA. In parallel, CRISPR-Cas9-targeted disruption of SLX1 and XPF was undertaken to determine biological roles and genetic interactions underlying their loss. Moreover, significant efforts were made toward the homogeneous purification of SMX, employing multi-pronged expression systems to address obstacles in achieving stability and functionality. Given challenges associated with purifying the entire complex, functional studies on sub-complexes and investigations into novel interactions of SLX4 provide valuable insights into the post-translational modifications regulating SLX4’s association with MUS81-EME1 and other DNA repair factors. The ultimate goal of these efforts is to obtain high-quality SMX suitable for structural characterisation by cryo-EM, advancing understanding of its molecular architecture and biological roles
Developing a high-integrity nature-based carbon sequestration strategy for Oxfordshire
Get PDFThis report assesses options for developing an integrated nature and climate strategy in Oxfordshire, based on Oxfordshire’s Local Nature Recovery Strategy (LNRS). By combining the LNRS map with the high-resolution Agile Opportunity Map of Oxfordshire developed by the University of Oxford, we developed a detailed spatial scenario for habitat restoration within the LNRS network, removing constraints such as buildings, roads, water, existing woodland and other priority habitats. We prioritised potential measures to maximise carbon sequestration while still creating a balanced mix of habitats in line with the LNRS priorities. The scenario included restoration of woodland, grassland, wetlands, heathland and tree-grass-scrub mosaics, as well as an increase of 40% in hedgerow length, widespread adoption of silvopasture and silvoarable agroforestry on farmland, and measures to improve soil health and enhance soil carbon on arable land. Urban habitats and ponds were out of scope for this study, and peatland was not considered as there is only 17 hectares in Oxfordshire. We show that a high-integrity nature-based carbon strategy could increase carbon storage in Oxfordshire by up to 28% (6 million tonnes of carbon) once habitats are mature (e.g. 100 years for woodlands). Over the next 30 years, this strategy could sequester over 180,000 tonnes of carbon annually, offsetting 18% of Oxfordshire’s 2023 emissions. However, only 38,000 tonnes of this total estimated sequestration is covered by existing or emerging market mechanisms (Wilder Carbon, the Woodland Carbon Code, and estimated units under future Soil, Agroforestry and Hedgerow carbon codes), offsetting 4% of emissions. This is because these market schemes set aside risk buffers of up to 40% of the estimated carbon savings, and do not issue carbon units in advance for all habitat transitions. The most notable missing marketable transition is restoration of intensively cultivated grassland to semi-natural grassland, as well as restoration of wetland and heathland on mineral soils. However, these can be claimed in retrospect under the Wilder Carbon scheme, following implementation and verification of the actual carbon gain, provided good baseline measurements have been taken. Extending hedgerow and soil carbon restoration to the whole county rather than just within the LNRS network could increase sequestration to offset 20% of emissions, of which 7% are currently marketable. These broad estimates are subject to high levels of uncertainty and represent upper limits, as they assume almost complete conversion or enhancement of low-grade farmland within the LNRS network over the next few decades, which is unlikely to occur in practice. If emissions continue to decline as planned, the share of residual emissions that could be offset through nature-based solutions could increase significantly, although the sequestration rate will decline as restored habitats mature. Sequestration from nature-based solutions can therefore play an important role, especially in the next 100 years, but this must be accompanied by continued strong measures to reduce emissions from fossil fuels and land-use. As well as playing a key role in delivering both nature recovery and net zero objectives, this strategy would also deliver multiple benefits for climate change adaptation, helping to reduce the impacts of floods, droughts and heatwaves, and would help to support human health and well-being through improving air quality and providing nature-rich green spaces for recreation and relaxation. It can help to support more resilient food production, by boosting populations of pollinators and pest predators, and protecting from soil erosion. In contrast, other carbon sequestration options such as fast-growing non-native plantations generally deliver little or no benefit for biodiversity, while the impacts of biochar are highly dependent on the biomass source. However, this strategy would have trade-offs for food production, as farmland is converted to woodland or less productive grassland options. This is minimised through avoiding conversion of Grade 1 and 2 land to non-agricultural use, but it would still lead to leakage of emissions due to food production in other areas, unless policies also encourage a shift to more plant-rich diets that require less land area. Delivery of this strategy depends on strong national and local support mechanisms, including adequate carbon prices and payments or incentives for wider benefits, such as through agri-environment schemes. Restored habitats must be protected from development and other threats, and managed sensitively to deliver long-term benefits for carbon, nature and people. Alongside restoring habitats, it is also vital to protect existing habitats for their stored carbon as well as biodiversity and ecosystem services. With good support from both national and local policy mechanisms, this high-integrity policy can play a key role in delivering multiple benefits for carbon, nature and people in Oxfordshire
Changes in platelet count as a marker of myocardial iron uptake after administration of ferric carboxymaltose in patients with heart failure
Get PDFIron deficiency (ID) treated with ferric carboxymaltose (FCM) previously showed reduced platelet count; however, no studies have evaluated its biological significance in heart failure. In the current study, we aimed to: (a) assess the changes in platelet count at 7 and 30 days post-FCM, and (b) explore its association with non-invasive surrogates of myocardial iron uptake and left systolic function in patients with ID and heart failure with left ventricular ejection fraction < 50% (HFrEF and HFmrEF). This post-hoc analysis of a randomized, double-blind, FCM vs. placebo clinical trial (Myocardial-IRON Trail) involved 45 outpatients with HFrEF and HFmrEF and ID in which the platelet count value was available. Platelet count, cardiac magnetic resonance T1-mapping and 3D-global longitudinal strain (CMR-GLS) were assessed at baseline, 7, and 30 days. Linear regression models were used to evaluate the between-treatment differences and endpoints. The mean (SD) age was 71 ± 8 years, and 32 (71%) were men. At 30 days, we found a significant reduction in platelet count in those treated with FCM (p-value = 0.027). In those treated with FCM, the greater 30-day decrease in platelets showed lower 30-day changes in T1-mapping (p = 0.024) and CMR-GLS (p = 0.028). After administration of FCM, we found a significant 30-day reduction in platelet count. The greater platelet count reduction was related to lower myocardial iron repletion and a smaller improvement in left ventricular systolic function
Mutant TDP-43 drives impairments in axonal transport and glycolysis in a mouse stem-cell-derived motor neuron model of amyotrophic lateral sclerosis (ALS)
Get PDFTDP-43 dysfunction is thought to be central to ALS pathogenesis. Studying mutations in the gene which encodes TDP-43, TARDBP, provides a valuable opportunity to gain insight into how TDP-43 dysfunction alters cellular homoeostasis. Our group has previously developed a TDP-43M337V mouse embryonic stem cell-derived motor neuron (mESC-MN) model, which expresses a single copy of the human TARDBP gene expressing the pathogenic M337V mutation at low levels. Here, we perform extensive phenotypic characterisation of this model, and show that TDP-43M337V leads to reduced MN viability, impaired axonal transport and reduced basal glycolysis compared to TDP-43WT controls. Altered neuronal viability and function occurs in the absence of TDP-43 mislocalisation or aggregation, suggesting ‘proteinopathy’ is downstream of these ALS-relevant phenotypes. These findings provide further support for a link between TDP-43 dyshomeostasis, cellular bioenergetics and axonal transport and suggest these pathways warrant further investigation as targets for therapeutic intervention
Dynamins maintain nuclear envelope homeostasis and genome stability
Get PDFThe nuclear envelope is a protective barrier for the genome and a mechanotransduction interface between cytoplasm and nucleus, whose malfunction disrupts nucleocytoplasmic transport, compromises DNA repair, accelerates telomere shortening, and promotes genomic instability. Mechanisms governing nuclear envelope remodeling and maintenance in interphase and post-mitotic cells remain poorly understood. Here, we report a role for dynamins, a family of essential brain-enriched membrane- and microtubule-binding GTPases, in preserving nuclear envelope and genomic homeostasis. Cells lacking dynamins exhibit nuclear envelope dysmorphisms, including buds with long narrow necks where damaged DNA frequently accumulates. These cells also show impaired autophagic clearance, reduced levels of key DNA repair proteins, and aberrant microtubules. Nocodazole treatment restores nuclear morphology and reduces DNA damage. Collectively, the data reveal that dynamins promote nuclear envelope homeostasis and removal of damaged DNA via their GTPase activity and interaction with microtubules, providing insights into mechanisms that uphold genome stability and counteract aging-related pathologies
Development of a micropatterned hydrogel scaffold for the delivery of polarised photoreceptor cells in retinal regeneration therapies
No full textPhotoreceptor degeneration is a leading cause of irreversible sight loss worldwide. In patients affected by conditions such as age-related macular degeneration or retinitis pigmentosa, progressive degeneration of the outer nuclear layer (ONL) is followed by progressive vision decline, ultimately leading to blindness. In end stage disease, despite the loss of the ONL, the remainder of the neuroretina structures remain largely intact. Consequently, cell therapies aiming to recreate the ONL could restore the visual circuitry. Recent preclinical studies in mouse models have shown great promise in restoring visually mediated behaviour following cone photoreceptor cell transplantation into the degenerated subretinal space. However, delivery of cells in a bolus injection leads to dissipation of the transplanted cell mass away from the intended target, effectively reducing the density of the transplanted cells. Furthermore, in healthy retinae, the polarity of photoreceptor cells enables effective support from the retinal pigment epithelium and functional interactions with the inner nuclear layer. In this work, hyaluronic acid-derived hydrogel scaffolds are micropatterned to achieve polarisation of photoreceptor cells in vitro ahead of transplant. The hydrogel scaffolds also act as a protective niche for the cells, effectively increasing transplanted cell density. This thesis first describes the development of the manufacturing process for the patterned hydrogel scaffold. By combining two-photon polymerisation printing and casting techniques, microscale topological features were patterned into the hydrogel to achieve basal-apical polarisation of photoreceptor cells in the z-direction. Secondly, biocompatibility of the optimised material formulation is established both in vitro and in vivo, including in murine models of degeneration. Finally, murine and hESC-derived photoreceptor cells were successfully cultured in the patterned hydrogel scaffold. Taken together, these findings highlight the potential of the developed hydrogel scaffold in augmenting established cell therapies by improving photoreceptor cell density and polarisation prior to transplant into the subretinal space of degenerated retinae
Immune dysregulation through longitudinal lymphocyte trajectories and their clinical determinants in hospitalized COVID-19 patients
Get PDFObjective: Immune dysregulation plays a pivotal role in the pathophysiology of sepsis and COVID-19, with lymphopenia emerging as a consistent marker of severity and poor prognosis. However, most existing studies have assessed lymphocyte counts at isolated time points, limiting insights into their temporal behavior and prognostic value. The dynamics of lymphocyte recovery or persistence of lymphopenia remain largely unexplored in large populations, as well as the impact of adjunctive therapies such as corticosteroids. We hypothesized that the persistence or recovery of lymphopenia may be key to understanding disease progression and predicting outcomes. Using the multinational ISARIC cohort, we investigated longitudinal lymphocyte trajectories in hospitalized patients and the clinical determinants associated with their evolution over time. Methods: We conducted a multinational prospective observational cohort study using data from the ISARIC-WHO Clinical Characterization Protocol. Patients with confirmed SARS-CoV-2 infection and at least four lymphocyte measurements during the first 28 days of hospitalization were included. We analyzed lymphocyte trajectories, Cox regression survival analyses and multivariable linear regression modelling. We also applied multistate models and joint modeling to assess the association between lymphocyte trajectories and 28-day mortality, incorporating corticosteroid use as a time-varying covariate. Results: Of 945,317 screened patients, 231,933 hospitalized adults with confirmed COVID-19 and sufficient lymphocyte data were included, with 56.6% classified as lymphopenic. Lymphopenia was independently associated with higher rates of ICU admission, organ support, and in-hospital mortality (OR = 1.52, 95% CI 1.48–1.55), and lower absolute lymphocyte counts were strongly linked to worse survival in adjusted Cox models (HR = 1.33 per 1 × 10⁹ cells/L decrease, 95% CI 1.28–1.38). Multistate modeling revealed that lymphopenic patients had a significantly higher daily transition rate to death and a shorter duration in that immune state, while corticosteroid exposure was associated with an increased likelihood of entering and remaining in lymphopenia. Joint modeling identified age, sex, and corticosteroid use as significant predictors of lower lymphocyte trajectories over time, with distinct dynamics between survivors and non-survivors. Conclusion: Lymphopenia was common and strongly associated with worse outcomes in hospitalized COVID-19 patients, with impaired recovery particularly evident in those receiving corticosteroids. These findings highlight the value of lymphocyte monitoring to inform tailored immunomodulatory strategies in sepsis and severe viral infections
Confirmation of Charges in Absentia at the International Criminal Court and the Right to be Present
Get PDFIt is a core principle of international human rights law that criminal defendants have the right to be present in proceedings against them. This article examines the conformity of confirmation of charges proceedings in absentia at the International Criminal Court (ICC) under Article 61(2) ICC Statute with the right to be present in international human rights law. Drawing on the jurisprudence of regional human rights courts and the United Nations Human Rights Committee, it first establishes that the right to be present is not confined to trials, but applies with equal force to certain pre-trial procedures, including confirmation of charges before the ICC. The paper then proceeds to argue, in light of the jurisprudence of human rights bodies and the Special Tribunal for Lebanon, that the absence of a right to de novo confirmation of charges proceedings — a ‘retrial’ — should a suspect subsequently appear before the Court after charges are confirmed against them in absentia renders the framework of Article 61(2)(b) incompatible with international human rights law. On this basis, it concludes Article 21(3) ICC Statute precludes the confirmation of charges in absentia at the ICC under the Court’s current legal regime. It then addresses the possible procedural remedies under the Rome Statute framework, ultimately concluding that they are insufficient to mitigate against the violation of an absent suspect’s right to be present in confirmation proceedings. Finally, it suggests that an amendment to the ICC Rules of Procedure and Evidence would be necessary to hold confirmation proceedings in absentia in conformity with international human rights law
Beyond good and bad: rethinking solidarity and coercion in public health
Get PDFWe often use certain terms as if, in using them, they contain a decided moral judgement of an action. Especially in public health ethics, this is not always the case, as shown most starkly by recent (mis)use of the terms ‘solidarity’ and ‘coercion’ to label, and thereby judge, public health actions responding to the COVID-19 pandemic. We analyse the terms solidarity and coercion, and argue that they cannot be used alone as moral judgements of public health actions. Rather, they are better considered as descriptive terms that are merely frequent proxies for normative terms such as justice or utility. We illustrate our argument by reference to three case studies: school reopenings in the USA, mandatory isolation measures in the UK, and vaccine distribution within the EU