Hyperthermophilic Aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines

The placement of the extreme thermophile Aquifex aeolicus in the bacterial phylogenetic tree has evoked much controversy. We investigated whether adaptations for growth at high temperatures would alter a key functional component of the replication machinery, specifically DnaG primase. Although the structure of bacterial primases is conserved, the trinucleotide initiation specificity for A. aeolicus was hypothesized to differ from other microbes as an adaptation to a geothermal milieu. To determine the full range of A. aeolicus primase activity, two oligonucleotides were designed that comprised all potential trinucleotide initiation sequences. One of the screening templates supported primer synthesis and the lengths of the resulting primers were used to predict possible initiation trinucleotides. Use of trinucleotide-specific templates demonstrated that the preferred initiation trinucleotide sequence for A. aeolicus primase was 5′-d(CCC)-3′. Two other sequences, 5′-d(GCC)-3′ and d(CGC)-3′, were also capable of supporting initiation, but to a much lesser degree. None of these trinucleotides were known to be recognition sequences used by other microbial primases. These results suggest that the initiation specificity of A. aeolicus primase may represent an adaptation to a thermophilic environment

Three-Dimensional Structure of N-Terminal Domain of DnaB Helicase and Helicase-Primase Interactions in Helicobacter pylori

Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD) of H. pylori DnaB (HpDnaB) helicase at 2.2 Å resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria

Inequalities, harm reduction and non-combustible nicotine products:A meta-ethnography of qualitative evidence

BACKGROUND: We sought to review qualitative evidence on how smokers in different socioeconomic groups engage with non-combustible nicotine products (NCNP), including electronic cigarettes and nicotine replacement therapies, in order to provide insight into how these products might impact on smoking inequalities. METHODS: We searched ten electronic databases in February 2017 using terms relating to NCNP and socioeconomic status. We included qualitative studies that were published since 1980 and were available in English. We used guidelines adapted from the Critical Appraisal Skills Programme for appraising qualitative research. RESULTS: The review only identified studies exploring the attitudes of socioeconomically disadvantaged smokers towards NCNP for harm reduction or cessation purposes (i.e. we did not identify any relevant studies of more advantaged socioeconomic groups). Using a lines-of-argument meta-ethnographic approach, we identified a predominantly pessimistic attitude to NCNP for harm reduction or cessation of smoking due to: wider circumstances of socioeconomic disadvantage; lack of a perceived advantage of alternative products over smoking; and a perceived lack of information about relative harms of NCNP compared to smoking. Optimistic findings, although fewer, suggested the potential of NCNP being taken up among smokers experiencing socioeconomic disadvantage. CONCLUSIONS: Overall, our review highlights the importance of considering the social, cultural and economic circumstances that influence experiences of smoking and of alternative product use

Potential for non-combustible nicotine products to reduce socioeconomic inequalities in smoking: a systematic review and synthesis of best available evidence

While some experts have emphasised the potential for e-cigarettes to facilitate cessation among smokers with low socioeconomic status (SES), there is limited evidence of their likely equity impact. We assessed the potential for electronic cigarettes and other non-combustible nicotine-containing products (NCNPs) to reduce inequalities in smoking by systematically reviewing evidence on their use by SES in countries at stage IV of the cigarette epidemic

Between history and values: A study on the nature of interpretation in international law

My thesis discusses the place of evaluative judgements in the interpretation of general international law. It concentrates on two questions. First, whether it is possible to interpret international legal practices without making an evaluative judgement about the point or value that provides the best justification of these practices. Second, whether the use of evaluative judgements in international legal interpretation threatens to undermine the objectivity of international law, the neutrality of international lawyers or the consensual and voluntary basis of the international legal system. I answer both questions in the negative. As regards the first, I argue that international legal practice has an interpretive structure, which combines appeals to the history of international practice with appeals to the principles and values that these practices are best understood as promoting. This interpretive structure is apparent not only in the claims of international lawyers about particular rules of international law (here I use the rule of estoppel as an example) but also in the most basic intuitions of international theorists about the theory and sources of general international law. I then argue that some popular concerns to the effect that the exercise of evaluation in the interpretation of international law will undermine the coherence or the usefulness of the discipline are generally unwarranted. The fact that international legal practice has an interpretive structure does not entail that propositions of international law are only subjectively true, that the interpreter enjoys license to manipulate their meaning for self-serving purposes, or that international law will collapse under the weight of irresolvable disagreements, divisions and conflicts about its proper interpretation

DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings

Loading of the replicative ring helicase onto the origin of replication (oriC) is the final outcome of a well coordinated series of events that collectively constitute a primosomal cascade. Once the ring helicase is loaded, it recruits the primase and signals the switch to the polymerization mode. The transient nature of the helicase-primase (DnaB-DnaG) interaction in the Escherichia coli system has hindered our efforts to elucidate its structure and function. Taking advantage of the stable DnaB-DnaG complex in Bacillus stearothermophilus, we have reviewed conflicting mutagenic data from other bacterial systems and shown that DnaG interacts with the flexible linker that connects the N- and C-terminal domains of DnaB. Furthermore, atomic force microscopy (AFM) imaging experiments show that binding of the primase to the helicase induces predominantly a 3-fold symmetric morphology to the hexameric ring. Overall, three DnaG molecules appear to interact with the hexameric ring helicase but a small number of complexes with two and even one DnaG molecule bound to DnaB were also detected. The structural/functional significance of these data is discussed and a speculative structural model for this complex is suggested

Interim evaluation of an integrated approach to improving health and wellbeing in County Durham, UK

Background: The County Durham Wellbeing for Life (WFL) service was developed in recognition that the previous ‘silo’ approach to provision of single-issue lifestyle services had made little impact on inequalities. It involves targeting the most disadvantaged communities locally, as well as specialist populations at increased risk of poor health. The model draws on evidence relating to the clustering of unhealthy behaviours, coupled with calls for behaviour change to be addressed in a more integrated and holistic manner. The service launched on 1st April 2015 and this paper reports interim findings from the academic evaluation. Methods: The impact of the WFL service is being evaluated via secondary analysis of routinely collected monitoring data, which includes EQ-5DTM and the short Warwick Edinburgh Mental Wellbeing Scale (SWEMWBS). Data have been analysed descriptively to examine key characteristics of the WFL user population (n = 1345). Appropriate statistical analyses have been performed to assess changes amongst those with data available at pre- and post-intervention periods (n = 224). Results: WFL users are predominantly female (73.4%), white British (97.6%) and living in the least affluent areas (80.4%). Mean BMI at baseline was 32.9kg/m2 (s.d. 7.7). Pre/post analysis of the intervention data revealed significant improvements across all outcome measures. These were: BMI, physical activity, alcohol intake, self-efficacy, self-rated health, quality of life and mental wellbeing. Furthermore, the largest changes were observed in those with the least positive results at baseline. Conclusions: The interim evaluation findings are being used to inform commissioning decisions and future WFL service delivery in County Durham. They also add to the evidence base around complex, community-based interventions that aim to tackle multiple health-related behaviours using an integrated wellbeing approach. Key messages: An integrated wellbeing approach can be used to reach those living in disadvantaged areas Improvements in health, quality of life and wellbeing were observed amongst service users