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Several protein ensembles facilitate crossover recombination and the associated assembly of synaptonemal complex (SC) during meiosis. In yeast, meiosis-specific factors including the DNA helicase Mer3, the “ZZS” complex consisting of Zip4, Zip2, and Spo16, the RING-domain protein Zip3, and the MutSγ heterodimer collaborate with crossover-promoting activity of the SC component, Zip1, to generate crossover-designated recombination intermediates. These ensembles also promote SC formation ‐ the organized assembly of Zip1 with other structural proteins between aligned chromosome axes. We used proximity labeling to investigate spatial relationships between meiotic recombination and SC proteins in S . cerevisiae . We find that recombination initiation and SC factors are dispensable for proximity labeling of Zip3 by ZZS components, but proteins associated with early steps in recombination are required for Zip3 proximity labeling by MutSγ, suggesting that MutSγ joins Zip3 only after a recombination intermediate has been generated. We also find that zip1 separation-of-function mutants that are crossover deficient but still assemble SC fail to generate protein ensembles where Zip3 can engage ZZS and/or MutSγ. The SC structural protein Ecm11 is proximity labeled by ZZS proteins in a Zip4-dependent and Zip1-independent manner, but labeling of Ecm11 by Zip3 and MutSγ requires, at least in part, Zip1. Finally, mass spectrometry analysis of biotinylated proteins in eleven proximity labeling strains uncovered shared proximity targets of SC and crossover-associated proteins, some of which have not previously been implicated in meiotic recombination or SC formation, highlighting the potential of proximity labeling as a discovery tool.

BackgroundThe challenges of measuring socioeconomic position in older populations were first set out two decades ago. However, the question of how best to measure older people’s socioeconomic position remains pertinent as populations age and health inequalities widen.MethodsA scoping review aimed to identify and appraise measures of socioeconomic position used in studies of health inequalities in older populations in high-income countries. Medline, Scopus, EMBASE, HMIC and references lists of systematic reviews were searched for observational studies of socioeconomic health inequalities in adults aged 60 years and over, published between 2000 and 2020. A narrative synthesis was conducted.FindingsOne-hundred and thirty-eight studies were included; 20 approaches to measuring socioeconomic position were identified. Few studies considered which pathways the chosen measures of socioeconomic position intended to capture. The validity of subjective socioeconomic position measures, and measures that assume shared income and educational capital, should be verified in older populations. Incomplete financial data risk under-representation of some older groups when missing data are socially patterned. Older study samples were largely homogeneous on measures of housing tenure, and to a lesser extent, measures of educational attainment. Measures that use only two response categories risk missing subtle differences in older people’s socioeconomic circumstances.ConclusionPoor choice of measures of socioeconomic position risk underestimating the size of health inequalities in older populations. Choice of measures should be shaped by considerations of theory, context and response categories that detect subtle, yet important, inequalities. Further evidence is required to ascertain the validity of some measures identified in this review.

Background Giant cell arteritis (GCA), if untreated, can lead to blindness and stroke. The study’s objectives were to (1) determine a new evidence-based benchmark of the extent of diagnostic delay for GCA and (2) examine the role of GCA-specific characteristics on diagnostic delay. Methods Medical literature databases were searched from inception to November 2015. Articles were included if reporting a time-period of diagnostic delay between onset of GCA symptoms and diagnosis. Two reviewers assessed the quality of the final articles and extracted data from these. Random-effects meta-analysis was used to pool the mean time-period (95% confidence interval (CI)) between GCA symptom onset and diagnosis, and the delay observed for GCA-specific characteristics. Heterogeneity was assessed by I 2 and by 95% prediction interval (PI). Results Of 4128 articles initially identified, 16 provided data for meta-analysis. Mean diagnostic delay was 9.0 weeks (95% CI, 6.5 to 11.4) between symptom onset and GCA diagnosis ( I 2  = 96.0%; P  < 0.001; 95% PI, 0 to 19.2 weeks). Patients with a cranial presentation of GCA received a diagnosis after 7.7 (95% CI, 2.7 to 12.8) weeks ( I 2  = 98.4%; P  < 0.001; 95% PI, 0 to 27.6 weeks) and those with non-cranial GCA after 17.6 (95% CI, 9.7 to 25.5) weeks ( I 2  = 96.6%; P  < 0.001; 95% PI, 0 to 46.1 weeks). Conclusions The mean delay from symptom onset to GCA diagnosis was 9 weeks, or longer when cranial symptoms were absent. Our research provides an evidence-based benchmark for diagnostic delay of GCA and supports the need for improved public awareness and fast-track diagnostic pathways.

Conformational selection by small molecules expands inhibitory possibilities for protein kinases. Nuclear magnetic resonance (NMR) measurements of the mitogen-activated protein (MAP) kinase ERK2 have shown that activation by dual phosphorylation induces global motions involving exchange between two states, L and R. We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analog. Thus, the L→R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding interactions for Vertex-11e, GDC-0994, and AMP-PNP with active vs. inactive ERK2, where the extent of HX protection correlates with R state formation. Furthermore, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity toward 2P-ERK2. We conclude that global motions in ERK2 reflect conformational changes at the active site that promote productive nucleotide binding and couple with changes at the activation loop to allow control of dephosphorylation by conformationally selective inhibitors.

A relatively unique feature of papillomaviruses is that the viral genome is associated with host histones inside the virion. However, little is known about the nature of the epigenome within papillomavirions or its biological relevance to the infectious viral cycle. An unusual feature of papillomaviruses is that their genomes are packaged into virions along with host histones. Viral minichromosomes were visualized as “beads on a string” by electron microscopy in the 1970s but, to date, little is known about the posttranslational modifications of these histones. To investigate this, we analyzed the histone modifications in HPV16/18 quasivirions, wart-derived bovine papillomavirus (BPV1), and wart-derived human papillomavirus type 1 (HPV1) using quantitative mass spectrometry. The chromatin from all three virion samples had abundant posttranslational modifications (acetylation, methylation, and phosphorylation). These histone modifications were verified by acid urea polyacrylamide electrophoresis and immunoblot analysis. Compared to matched host cell controls, the virion minichromosome was enriched in histone modifications associated with active chromatin and depleted for those commonly found in repressed chromatin. We propose that the viral minichromosome acquires specific histone modifications late in infection that are coupled to the mechanisms of viral replication, late gene expression, and encapsidation. We predict that, in turn, these same modifications benefit early stages of infection by helping to evade detection, promoting localization of the viral chromosome to beneficial regions of the nucleus, and promoting early transcription and replication. IMPORTANCE A relatively unique feature of papillomaviruses is that the viral genome is associated with host histones inside the virion. However, little is known about the nature of the epigenome within papillomavirions or its biological relevance to the infectious viral cycle. Here, we define the epigenetic signature of the H3 and H4 histones from HPV16 virions generated in cell culture and native human papillomavirus type 1 (HPV1) and bovine papillomavirus 1 (BPV1) virions isolated from bovine and human wart tissue. We show that native virions are enriched in posttranslational modifications associated with active chromatin and depleted with those associated with repressed chromatin compared to cellular chromatin. Native virions were also enriched in the histone variant H3.3 compared to the canonical histone H3.1. We propose that the composition of virion-packaged chromatin reflects the late stages of the viral life cycle and promotes the early stages of infection by being primed for viral transcription.

Background Intervening to modify frailty trajectories may be critical to maintain health and independence in later life. The Active Ageing Programme (AAP) is a 16-week community-based intervention for older people with mild frailty that combines physical activity and social interaction. The programme aims to positively impact resilience and wellbeing, changing the physical, mental and social factors that impact on frailty trajectories. We conducted a rapid qualitative study with the first cohort to understand the acceptability and feasibility of the AAP. Methods Purposive sampling was used to identify and recruit staff and participants involved in the AAP for semi-structured interviews. The topic guides covered experiences and perceptions of training and referral, delivery and participation in activities, and benefits and challenges. Data from 20 interviews were analysed using a reflexive thematic approach with inductive coding. The Theoretical Domains Framework prompted consideration of potential individual, social and environmental factors influencing changes in behaviour and practice. Results Sixteen AAP staff (10) and participants (6) completed interviews. Two themes were developed: combining motivating components; addressing what matters. The AAP brought together a number of components that supported its acceptability and successful implementation by motivating older people and staff and sustaining their engagement. These included the convenient and familiar location, the opportunity to improve physical health (participants) and to gain experience of an activity intervention, training and confidence (staff), and the variety of activities on offer. The programme was perceived to have potential to improve physical and mental health and increase the personal resources (e.g. knowledge, confidence, motivation) of those taking part. Areas identified as important for the AAP’s future sustainability and success were: tailoring recruitment practices; enhancing organisational communication; and strengthening support for participants to achieve long-term increased physical and social activity and resilience. Conclusions Our findings suggest that community-based physical and social activity interventions targeting frailty may be acceptable, feasible and useful. Further work is needed to investigate the impact of such programmes on health and service utilisation. Efforts should be targeted at improving the potential for long-term sustainability of programmes and their impacts.

DNA methylation is the most stable type of epigenetic modification modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation profiles of human chromosomes 6, 20 and 22, providing a resource of about 1.9 million CpG methylation values derived from 12 different tissues. Analysis of six annotation categories showed that evolutionarily conserved regions are the predominant sites for differential DNA methylation and that a core region surrounding the transcriptional start site is an informative surrogate for promoter methylation. We find that 17% of the 873 analyzed genes are differentially methylated in their 5′ UTRs and that about one-third of the differentially methylated 5′ UTRs are inversely correlated with transcription. Despite the fact that our study controlled for factors reported to affect DNA methylation such as sex and age, we did not find any significant attributable effects. Our data suggest DNA methylation to be ontogenetically more stable than previously thought.

BackgroundThe COVID-19 pandemic has taken a heavy toll on the care home sector, with residents accounting for up to half of all deaths in Europe. The response to acute illness in care homes plays a particularly important role in the care of residents during a pandemic. Digital recording of a National Early Warning Score (NEWS), which involves the measurement of physical observations, started in care homes in one area of England in 2016. Implementation of a NEWS intervention (including equipment, training and support) was accelerated early in the pandemic, despite limited evidence for its use in the care home setting.ObjectivesTo understand how a NEWS intervention has been used in care homes in one area of North-East England during the COVID-19 pandemic, and how it has influenced resident care, from the perspective of stakeholders involved in care delivery and commissioning.MethodsA qualitative interview study with care home (n=10) and National Health Service (n=7) staff. Data were analysed using thematic analysis.ResultsUse of the NEWS intervention in care homes in this area accelerated during the COVID-19 pandemic. Stakeholders felt that NEWS, and its associated education and support package, improved the response of care homes and healthcare professionals to deterioration in residents’ health during the pandemic. Healthcare professionals valued the ability to remotely monitor resident observations, which facilitated triage and treatment decisions. Care home staff felt empowered by NEWS, providing a common clinical language to communicate concerns with external services, acting as an adjunct to staff intuition of resident deterioration.ConclusionsThe NEWS intervention formed an important part of the care home response to COVID-19 in the study area. Positive staff perceptions now need to be supplemented with data on the impact on resident health and well-being, workload, and service utilisation, during the pandemic and beyond.

ObjectivesTo identify and map evidence about the consequences of unpaid caring for all carers of older people, and effective interventions to support this carer population.DesignA rapid review of systematic reviews, focused on the consequences for carers of unpaid caring for older people, and interventions to support this heterogeneous group of carers. Reviews of carers of all ages were eligible, with any outcome measures relating to carers’ health, and social and financial well-being. Searches were conducted in MEDLINE, PsycInfo and Epistemonikos (January 2000 to January 2020). Records were screened, and included systematic reviews were quality appraised. Summary data were extracted and a narrative synthesis produced.ResultsTwelve systematic reviews reporting evidence about the consequences of caring for carers (n=6) and assessing the effectiveness of carer interventions (n=6) were included. The review evidence typically focused on mental health outcomes, with little information identified about carers’ physical, social and financial well-being. Clear estimates of the prevalence and severity of carer outcomes, and how these differ between carers and non-carers, were absent. A range of interventions were identified, but there was no strong evidence of effectiveness. In some studies, the choice of outcome measure may underestimate the full impact of an intervention.ConclusionsCurrent evidence fails to fully quantify the impacts that caring for older people has on carers’ health and well-being. Information on social patterning of the consequences of caring is absent. Systematic measurement of a broad range of outcomes, with comparison to the general population, is needed to better understand the true consequences of caring. Classification of unpaid caring as a social determinant of health could be an effective lever to bring greater focus and support to this population. Further work is needed to develop and identify suitable interventions in order to support evidence-based policymaking and practice.

The histone chaperone Chromatin Assembly Factor 1 (CAF-1) deposits tetrameric (H3/H4)2 histones onto newly-synthesized DNA during DNA replication. To understand the mechanism of the tri-subunit CAF-1 complex in this process, we investigated the protein-protein interactions within the CAF-1-H3/H4 architecture using biophysical and biochemical approaches. Hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry reveal interactions that are essential for CAF-1 function in budding yeast, and importantly indicate that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex. Cac1 alone not only binds H3/H4 with high affinity, but also promotes histone tetramerization independent of the other subunits. Moreover, we identify a minimal region in the C-terminus of Cac1, including the structured winged helix domain and glutamate/aspartate-rich domain, which is sufficient to induce (H3/H4)2 tetramerization. These findings reveal a key role of Cac1 in histone tetramerization, providing a new model for CAF-1-H3/H4 architecture and function during eukaryotic replication. The DNA of a human, yeast or other eukaryotic cell is bound to proteins called histones to form repeating units called nucleosomes. Every time a eukaryotic cell divides, it must duplicate its DNA. Old histones are first removed from the nucleosomes before being re-assembled onto the newly duplicated DNA along with new histone proteins, producing a full complement of nucleosomes. A group of proteins called the chromatin assembly factor 1 (or CAF-1 for short) helps to assemble the histones onto the DNA. CAF-1 is made up of three proteins, and binds to two copies of each of the histones known as H3 and H4. These are the first histones to be assembled onto the nucleosomes. It was not clear how the components of CAF-1 are organized, or how CAF-1 recognizes histones. Liu et al. have now investigated the structure of CAF-1 and its interactions with the H3 and H4 histones by studying yeast proteins and cells. Yeast is a good model system because yeast CAF-1 is smaller and easier to isolate than human CAF-1, yet still performs the same essential activities. Using a combination of biochemical and biophysical techniques, Liu et al. found that one of the three proteins that makes up yeast CAF-1 – called Cac1 – forms a scaffold that supports the other CAF-1 proteins and histones H3 and H4. Moreover, a specific part of Cac1 is able to bind to these histones and assemble two copies of each of them to prepare for efficient nucleosome assembly. Further experiments revealed the specific areas where the CAF-1 proteins interact with each other and with the histones, determined how strong those interactions are, and confirmed that these interactions play important roles in yeast. Overall, the results presented by Liu et al. provide new insights into the structure of CAF-1 bound to H3 and H4. In order to understand in detail how CAF-1 helps to assemble histones onto DNA, future work needs to capture three-dimensional snapshots of the different steps in this process. Further investigation is also needed to discover how CAF-1 cooperates with other factors that promote DNA duplication.