Processing of TOP2-DNA covalent complexes by the ubiquitin-proteasome system

PhD ThesisDNA topoisomerase II (TOP2) poisons are widely used anticancer drugs which induce cytotoxic protein-DNA crosslinks termed TOP2-DNA covalent complexes. The TOP2-DNA covalent complex is a normally transient intermediate of the TOP2 reaction mechanism, whereby an intact DNA duplex is passed through an enzymemediated double strand break (DSB) in another DNA molecule. A covalent linkage between the TOP2 active site tyrosine and each DNA end conceals the break and prevents its recognition by DNA damage response proteins. However, in the presence of a TOP2 poison the TOP2-DNA complex is stabilised, leading to the accumulation of enzyme-linked DSBs which ultimately lead to cell death. Repair of the TOP2-DNA covalent complex first requires the removal of TOP2 from DNA ends, leading to the liberation of a protein-free DSB. While a number of pathways are available for the removal of the TOP2 adduct, one mechanism involves the proteasomal degradation of TOP2. The proteasomal degradation of proteins is largely regulated through the conjugation of ubiquitin to target proteins (ubiquitination). However, the requirement for ubiquitination in the processing of TOP2-DNA covalent complexes is unclear, with conflicting studies reporting both ubiquitin-dependent and -independent mechanisms. In the current study, inhibition of ubiquitination slowed the removal of TOP2 adducts from DNA and reduced the appearance of protein-free DSBs following etoposide treatment. Inhibition of the ubiquitin-dependent AAA ATPase VCP/p97 also prevented the processing of TOP2- DNA complexes to protein-free DSBs, indicating a previously unreported role for VCP/p97 in the repair of TOP2 poison-induced DNA damage. Inhibition of the ubiquitin-proteasome system increased the growth-inhibitory effects of four clinically relevant TOP2 poisons, and may be a viable strategy for the improvement of therapy with TOP2 poisons. This work confirms a ubiquitin-dependent mechanism of TOP2- DNA complex processing by the proteasome, which may be facilitated by the unfolding and extraction of TOP2-DNA complexes by VCP/p97.Bloodwise for providing this opportunity and funding the project (Gordon Piller Studentship, 13063

Narratives Countering the Democratising Ideal of Discourse in an Online Forum of a Higher Education Institution

This paper describes power inequalities among participants in an online forum at a higher education institution in South Africa. Critical poststructuralist theory informs the study as it investigates how hegemony influences the strategic interaction of participants. An interpretive analysis uncovered elements of a cyclic process of intensified exclusion, inequality and oppression. This took place within a virtual space which is theoretically idealized as an equalizer and promoter of freedom of speech. The process involved in the eliciting of voices is described and the interpretation of subjective accounts tells of the disillusioned experiences of a potential liberating form of technology. Instead of alleviating conflict, the potential of the online forum is subverted and intensifies the alienation of and animosity between participants. Proposals for moderation are made to change the forum to a democratic, inclusive space

The complex interplay between kidney injury and inflammation

Abstract Acute kidney injury (AKI) has gained significant attention following patient safety alerts about the increased risk of harm to patients, including increased mortality and hospitalization. Common causes of AKI include hypovolaemia, nephrotoxic medications, ischaemia and acute glomerulonephritis, although in reality it may be undetermined or multifactorial. A period of inflammation either as a contributor to the kidney injury or resulting from the injury is almost universally seen. This article was compiled following a workshop exploring the interplay between injury and inflammation. AKI is characterized by some degree of renal cell death through either apoptosis or necrosis, together with a strong inflammatory response. Studies interrogating the resolution of renal inflammation identify a whole range of molecules that are upregulated and confirm that the kidneys are able to intrinsically regenerate after an episode of AKI, provided the threshold of damage is not too high. Kidneys are unable to generate new nephrons, and dysfunctional or repeated episodes will lead to further nephron loss that is ultimately associated with the development of renal fibrosis and chronic kidney disease (CKD). The AKI to CKD transition is a complex process mainly facilitated by maladaptive repair mechanisms. Early biomarkers mapping out this process would allow a personalized approach to identifying patients with AKI who are at high risk of developing fibrosis and subsequent CKD. This review article highlights this process and explains how laboratory models of renal inflammation and injury assist with understanding the underlying disease process and allow interrogation of medications aimed at targeting the mechanistic interplay.</jats:p

An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family

A mutant of the high fidelity family-B DNA polymerase from the archaeon Thermococcus gorgonarius (Tgo-Pol), able to replicate past DNA lesions, is described. Gain of function requires replacement of the three amino acid loop region in the fingers domain of Tgo-Pol with a longer version, found naturally in eukaryotic Pol zeta (a family-B translesion synthesis polymerase). Inactivation of the 3'–5' proofreading exonuclease activity is also necessary. The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers. Tgo-Pol Z1 is also proficient at extending from primers that terminate opposite aberrant bases. The fidelity of Tgo-Pol Z1 is reduced, with amarked tendency tomake changes at G:C base pairs. Together, these results suggest that the loop region of the fingers domain may play a critical role in determining whether a family-B enzyme falls into the accurate genome-replicating category or is an errorprone translesion synthesis polymerase. Tgo-Pol Z1 may also be useful for amplification of damaged DNA

HIDDen: Hospice Inpatient Deep vein thrombosis Detection prospective longitudinal observational study to explore the prevalence, symptom burden and natural history of venous thromboembolism in people with advanced cancer

BACKGROUND: The prevalence of deep venous thrombosis in patients with advanced cancer is unconfirmed and it is unknown whether current international thromboprophylaxis guidance is applicable to this population. We aimed to determine prevalence and predictors of femoral deep vein thrombosis in patients admitted to specialist palliative care units (SPCUs). METHODS: We did this prospective longitudinal observational study in five SPCUs in England, Wales, and Northern Ireland (four hospices and one palliative care unit). Consecutive adults with cancer underwent bilateral femoral vein ultrasonography on admission and weekly until death or discharge for a maximum of 3 weeks. Data were collected on performance status, attributable symptoms, and variables known to be associated with venous thromboembolism. Patients with a short estimated prognosis (<5 days) were ineligible. The primary endpoint of the study was the prevalence of femoral deep vein thrombosis within 48 h of SPCU admission, analysed by intention to treat. This study is registered with the ISRCTN registry, number ISRCTN97567719. FINDINGS: Between June 20, 2016, and Oct 16, 2017, 343 participants were enrolled (mean age 68·2 years [SD 12·8; range 25-102]; 179 [52%] male; mean Australian-modified Karnofsky performance status 49 [SD 16·6; range 20-90]). Of 273 patients with evaluable scans, 92 (34%, 95% CI 28-40) had femoral deep vein thrombosis. Four participants with a scan showing no deep vein thrombosis on admission developed a deep vein thrombosis on repeat scanning over 21 days. Previous venous thromboembolism (p=0·014), being bedbound in the past 12 weeks for any reason (p=0·003), and lower limb oedema (p=0·009) independently predicted deep vein thrombosis. Serum albumin concentration (p=0·43), thromboprophylaxis (p=0·17), and survival (p=0·45) were unrelated to deep vein thrombosis. INTERPRETATION: About a third of patients with advanced cancer admitted to SPCUs had a femoral deep vein thrombosis. Deep vein thrombosis was not associated with thromboprophylaxis, survival, or symptoms other than leg oedema. These findings are consistent with venous thromboembolism being a manifestation of advanced disease rather than a cause of premature death. Thromboprophylaxis for SPCU inpatients with poor performance status seems to be of little benefit. FUNDING: National Institute for Health Research (Research for Patient Benefit programme)

Structure of Human DNA Polymerase κ Inserting dATP Opposite an 8-OxoG DNA Lesion

Background: Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase κ (Polκ) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polκ\u27s preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polκ in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide. Methodology and Principal Findings: We show that the Polκ active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP. Conclusions and Significance: The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polκ is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polκ is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases

IQuaD dental trial; improving the quality of dentistry : a multicentre randomised controlled trial comparing oral hygiene advice and periodontal instrumentation for the prevention and management of periodontal disease in dentate adults attending dental primary care

Peer reviewedPublisher PD

Phenotypic effects of genetic variants associated with autism

While over 100 genes have been associated with autism, little is known about the prevalence of variants affecting them in individuals without a diagnosis of autism. Nor do we fully appreciate the phenotypic diversity beyond the formal autism diagnosis. Based on data from more than 13,000 individuals with autism and 210,000 undiagnosed individuals, we estimated the odds ratios for autism associated to rare loss-of-function (LoF) variants in 185 genes associated with autism, alongside 2,492 genes displaying intolerance to LoF variants. In contrast to autism-centric approaches, we investigated the correlates of these variants in individuals without a diagnosis of autism. We show that these variants are associated with a small but significant decrease in fluid intelligence, qualification level and income and an increase in metrics related to material deprivation. These effects were larger for autism-associated genes than in other LoF-intolerant genes. Using brain imaging data from 21,040 individuals from the UK Biobank, we could not detect significant differences in the overall brain anatomy between LoF carriers and non-carriers. Our results highlight the importance of studying the effect of the genetic variants beyond categorical diagnosis and the need for more research to understand the association between these variants and sociodemographic factors, to best support individuals carrying these variants

Identifying perinatal depression with case-finding instruments : a mixed-methods study (BaBY PaNDA – Born and Bred in Yorkshire PeriNatal Depression Diagnostic Accuracy)

Background: Perinatal depression is well recognised as a mental health condition but < 50% of cases are identified in routine practice. A case-finding strategy using the Whooley questions is currently recommended by the National Institute for Health and Care Excellence. Objectives: To determine the diagnostic accuracy, acceptability and cost-effectiveness of the Whooley questions and the Edinburgh Postnatal Depression Scale (EPDS) to identify perinatal depression. Design: A prospective diagnostic accuracy cohort study, with concurrent qualitative and economic evaluations. Setting: Maternity services in England. Participants: A total of 391 pregnant women. Main outcome measures: Women completed the Whooley questions, EPDS and a diagnostic reference standard (Clinical Interview Schedule – Revised) during pregnancy (20 weeks) and postnatally (3–4 months). Qualitative interviews were conducted with health professionals (HPs) and a subsample of women. Results: Diagnostic accuracy results: depression prevalence rates were 10.3% during pregnancy and 10.5% postnatally. The Whooley questions and EPDS (cut-off point of ≥ 10) performed reasonably well, with comparable sensitivity [pregnancy: Whooley questions 85.0%, 95% confidence interval (CI) 70.2% to 94.3%; EPDS 82.5%, 95% CI 67.2% to 92.7%; postnatally: Whooley questions 85.7%, 95% CI 69.7% to 95.2%; EPDS 82.9%, 95% CI 66.4% to 93.4%] and specificity (pregnancy: Whooley questions 83.7%, 95% CI 79.4% to 87.4%; EPDS 86.6%, 95% CI 82.5% to 90.0%; postnatally: Whooley questions 80.6%, 95% CI 75.7% to 84.9%; EPDS 87.6%, 95% CI 83.3% to 91.1%). Diagnostic accuracy of the EPDS (cut-off point of ≥ 13) was poor at both time points (pregnancy: sensitivity 45%, 95% CI 29.3% to 61.5%, and specificity 95.7%, 95% CI 93.0% to 97.6%; postnatally: sensitivity 62.9%, 95% CI 44.9% to 78.5%, and specificity 95.7%, 95% CI 92.7% to 97.7%). Qualitative evaluation: women and HPs were supportive of screening/case-finding for perinatal depression. The EPDS was preferred to the Whooley questions by women and HPs, mainly because of its ‘softer’ wording. Whooley question 1 was thought to be less acceptable, largely because of the terms ‘depressed’ and ‘hopeless’, leading to women not revealing their depressive symptoms. HPs identified a ‘patient-centred’ environment that focused on the mother and baby to promote discussion about mental health. Cost-effectiveness results: screening/case-finding using the Whooley questions or the EPDS alone was not the most cost-effective strategy. A two-stage strategy, ‘Whooley questions followed by the Patient Health Questionnaire’ (a measure assessing depression symptomatology), was the most cost-effective strategy in the range between £20,000 and £30,000 per quality-adjusted life-year in both the prenatal and postnatal decision models. Limitations: Perinatal depression diagnosis was not cross-referenced with women’s medical records so the proportion of new cases identified is unknown. The clinical effectiveness and cost-effectiveness of screening/case-finding strategies was not assessed as part of a randomised controlled trial. Conclusions: The Whooley questions and EPDS had acceptable sensitivity and specificity, but their use in practice might be limited by low predictive value and variation in their acceptability. A two-stage strategy was more cost-effective than single-stage strategies. Neither case-finding instrument met National Screening Committee criteria. Future work: The yield of screening/case-finding should be established with reference to health-care records. The clinical effectiveness and cost-effectiveness of screening/case-finding for perinatal depression needs to be tested in a randomised controlled trial. Funding: The National Institute for Health Research Health Services and Delivery Research programme

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes