PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication

DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) lightdamaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol h-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells

PLK1 regulates the PrimPol damage tolerance pathway during the cell cycle

Replication stress and DNA damage stall replication forks and impede genome synthesis. During S phase, damage tolerance pathways allow lesion bypass to ensure efficient genome duplication. One such pathway is repriming, mediated by Primase-Polymerase (PrimPol) in human cells. However, the mechanisms by which PrimPol is regulated are poorly understood. Here, we demonstrate that PrimPol is phosphorylated by Polo-like kinase 1 (PLK1) at a conserved residue between PrimPol’s RPA binding motifs. This phosphorylation is differentially modified throughout the cell cycle, which prevents aberrant recruitment of PrimPol to chromatin. Phosphorylation can also be delayed and reversed in response to replication stress. The absence of PLK1-dependent regulation of PrimPol induces phenotypes including chromosome breaks, micronuclei, and decreased survival after treatment with camptothecin, olaparib, and UV-C. Together, these findings establish that deregulated repriming leads to genomic instability, highlighting the importance of regulating this damage tolerance pathway following fork stalling and throughout the cell cycle

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

EXD2 governs germ stem cell homeostasis and lifespan by promoting mitoribosome integrity and translation

Mitochondria are subcellular organelles critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, as well as their coordinated translation, import and respiratory complex assembly. Here we describe the characterization of exonuclease domain like 2 (EXD2), a nuclear encoded gene that we show is targeted to the mitochondria and prevents the aberrant association of mRNAs with the mitochondrial ribosome. The loss of EXD2 resulted in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of EXD2/CG6744 in D.melanogaster caused developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that could be reversed with an anti-oxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and aging

Increasing frailty is associated with higher prevalence and reduced recognition of delirium in older hospitalised inpatients: results of a multi-centre study

Purpose: Delirium is a neuropsychiatric disorder delineated by an acute change in cognition, attention, and consciousness. It is common, particularly in older adults, but poorly recognised. Frailty is the accumulation of deficits conferring an increased risk of adverse outcomes. We set out to determine how severity of frailty, as measured using the CFS, affected delirium rates, and recognition in hospitalised older people in the United Kingdom. Methods: Adults over 65 years were included in an observational multi-centre audit across UK hospitals, two prospective rounds, and one retrospective note review. Clinical Frailty Scale (CFS), delirium status, and 30-day outcomes were recorded. Results: The overall prevalence of delirium was 16.3% (483). Patients with delirium were more frail than patients without delirium (median CFS 6 vs 4). The risk of delirium was greater with increasing frailty [OR 2.9 (1.8–4.6) in CFS 4 vs 1–3; OR 12.4 (6.2–24.5) in CFS 8 vs 1–3]. Higher CFS was associated with reduced recognition of delirium (OR of 0.7 (0.3–1.9) in CFS 4 compared to 0.2 (0.1–0.7) in CFS 8). These risks were both independent of age and dementia. Conclusion: We have demonstrated an incremental increase in risk of delirium with increasing frailty. This has important clinical implications, suggesting that frailty may provide a more nuanced measure of vulnerability to delirium and poor outcomes. However, the most frail patients are least likely to have their delirium diagnosed and there is a significant lack of research into the underlying pathophysiology of both of these common geriatric syndromes

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

A heterotrimeric PCNA in the hyperthermophilic archaeon Sulfolobus solfataricus

The sliding clamp, PCNA, of the archaeon Sulfolobus solfataricus P2 is a heterotrimer of three distinct subunits (PCNA1, 2, and 3) that assembles in a defined manner. The PCNA heterotrimer, but not individual subunits, stimulates the activities of the DNA polymerase, DNA ligase I, and the flap endonuclease (FEN1) of S. solfataricus. Distinct PCNA subunits contact DNA polymerase, DNA ligase, or FEN1, imposing a defined architecture at the lagging strand fork and suggesting the existence of a preformed scanning complex at the fork. This provides a mechanism to tightly couple DNA synthesis and Okazaki fragment maturation. Additionally, unique subunit-specific interactions between components of the clamp loader, RFC, suggest a model for clamp loading of PCNA

Comparative evolutionary analysis of VPS33 homologues: genetic and functional insights

VPS33B protein is a homologue of the yeast class C vacuolar protein sorting protein Vps33p that is involved in the biogenesis and function of vacuoles. Vps33p homologues contain a Sec1 domain and belong to the family of Sec1/Munc18 (SM) proteins that regulate fusion of membrane-bound organelles and interact with other vps proteins and also SNARE proteins that execute membrane fusion in all cells. We demonstrated recently that mutations in VPS33B cause ARC syndrome (MIM 208085), a lethal multisystem disease. In contrast, mutations in other Vps33p homologues result in different phenotypes, e.g. a mutation in Drosophila melanogaster car gene causes the carnation eye colour mutant and inactivation of mouse Vps33a causes buff hypopigmentation phenotype. In mammals two Vps33p homologues (e.g. VPS33A and VPS33B in humans) have been identified. As comparative genome analysis can provide novel insights into gene evolution and function, we performed nucleotide and protein sequence comparisons of Vps33 homologues in different species to define their inter-relationships and evolution. In silico analysis (a) identified two homologues of yeast Vps33p in the worm, fly, zebrafish, rodent and human genomes, (b) suggested that Carnation is an orthologue of VPS33A rather than VPS33B and (c) identified conserved candidate functional domains within VPS33B. We have shown previously that wild-type VPS33B induced perinuclear clustering of late endosomes and lysosomes in human renal cells. Consistent with the predictions of comparative analysis: (a) VPS33B induced significantly more clustering than VPS33A in a renal cell line, (b) a putative fly VPS33B homologue but not Carnation protein also induced clustering and (c) the ability to induce clustering in renal cells was linked to two evolutionary conserved domains within VPS33B. One domain was present in VPS33B but not VPS33A homologues and the other was one of three regions predicted to form a t-SNARE binding site in VPS33B. In contrast, VPS33A induced significantly more clustering of melanosomes in melanoma cells than VPS33B. These investigations are consistent with the hypothesis that there are two functional classes of Vps33p homologues in all multicellular organisms and that the two classes reflect the evolution of organelle/tissue-specific functions