Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action

Is health-related quality of life associated with the risk of low-energy wrist fracture: a case-control study

<p>Abstract</p> <p>Background</p> <p>Some risk factors for low-energy wrist fracture have been identified. However, self-reported measures such as health-related quality of life (HRQOL) have not been examined as potential risk factors for wrist fracture. The aims of this study were to compare HRQOL prior to a low-energy wrist fracture in elderly patients (≥ 50 years) with HRQOL in age- and sex-matched controls, and to explore the association between HRQOL and wrist fracture after adjusting for known risk factors for fracture such as age, weight, osteoporosis and falls.</p> <p>Methods</p> <p>Patients with a low-energy wrist fracture (n = 181) and age- and sex-matched controls (n = 181) were studied. Shortly after fracture (median 10 days), patients assessed their HRQOL before fracture using the Short Form 36 (SF-36). Statistical tests included <it>t </it>tests and multivariate logistic regression analysis.</p> <p>Results</p> <p>Several dimensions of HRQOL were significantly associated with wrist fracture. The direction of the associations with wrist fracture varied between the different sub-dimensions of the SF-36. After controlling for demographic and clinical variables, higher scores on <it>general health </it>(odds ratio (OR) = 1.31, 95% confidence interval (CI) = 1.10–1.56), <it>bodily pain </it>(OR = 1.18, 95% CI = 1.03–1.34) and <it>mental health </it>(OR = 1.39, 95% CI = 1.09–1.79) were related to an increased chance of being a wrist fracture patient rather than a control. In contrast, higher scores on <it>physical role limitation </it>(OR = 0.87, 95% CI = 0.79–0.95) and <it>social function </it>(OR = 0.65, 95% CI 0.53–0.80) decreased this chance. Significant associations with wrist fracture were also found for living alone (OR = 1.91, 95% CI 1.07–3.4), low body mass index (BMI) (OR = 0.92, 95% CI 0.86–0.98), osteoporosis (OR = 3.30, 95% CI 1.67–6.50) and previous falls (OR = 2.01, 95% CI 1.16–3.49).</p> <p>Conclusion</p> <p>Wrist fracture patients perceive themselves to be as healthy as the controls before fracture. Our data indicate that patients with favourable and unfavourable HRQOL measures may be at increased risk of wrist fracture.</p

Disparate oxidant gene expression of airway epithelium compared to alveolar macrophages in smokers

<p>Abstract</p> <p>Background</p> <p>The small airway epithelium and alveolar macrophages are exposed to oxidants in cigarette smoke leading to epithelial dysfunction and macrophage activation. In this context, we asked: what is the transcriptome of oxidant-related genes in small airway epithelium and alveolar macrophages, and does their response differ substantially to inhaled cigarette smoke?</p> <p>Methods</p> <p>Using microarray analysis, with TaqMan RT-PCR confirmation, we assessed oxidant-related gene expression in small airway epithelium and alveolar macrophages from the same healthy nonsmoker and smoker individuals.</p> <p>Results</p> <p>Of 155 genes surveyed, 87 (56%) were expressed in both cell populations in nonsmokers, with higher expression in alveolar macrophages (43%) compared to airway epithelium (24%). In smokers, there were 15 genes (10%) up-regulated and 7 genes (5%) down-regulated in airway epithelium, but only 3 (2%) up-regulated and 2 (1%) down-regulated in alveolar macrophages. Pathway analysis of airway epithelium showed oxidant pathways dominated, but in alveolar macrophages immune pathways dominated.</p> <p>Conclusion</p> <p>Thus, the response of different cell-types with an identical genome exposed to the same stress of smoking is different; responses of alveolar macrophages are more subdued than those of airway epithelium. These findings are consistent with the observation that, while the small airway epithelium is vulnerable, alveolar macrophages are not "diseased" in response to smoking.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov ID: NCT00224185 and NCT00224198</p

Evaluation of Mucociliary Clearance by Three Dimension Micro-CT-SPECT in Guinea Pig: Role of Bitter Taste Agonists

Different image techniques have been used to analyze mucociliary clearance (MCC) in humans, but current small animal MCC analysis using in vivo imaging has not been well defined. Bitter taste receptor (T2R) agonists increase ciliary beat frequency (CBF) and cause bronchodilation but their effects in vivo are not well understood. This work analyzes in vivo nasal and bronchial MCC in guinea pig animals using three dimension (3D) micro-CT-SPECT images and evaluates the effect of T2R agonists. Intranasal macroaggreggates of albumin-Technetium 99 metastable (MAA-Tc99m) and lung nebulized Tc99m albumin nanocolloids were used to analyze the effect of T2R agonists on nasal and bronchial MCC respectively, using 3D micro-CT-SPECT in guinea pig. MAA-Tc99m showed a nasal mucociliary transport rate of 0.36 mm/min that was increased in presence of T2R agonist to 0.66 mm/min. Tc99m albumin nanocolloids were homogeneously distributed in the lung of guinea pig and cleared with time-dependence through the bronchi and trachea of guinea pig. T2R agonist increased bronchial MCC of Tc99m albumin nanocolloids. T2R agonists increased CBF in human nasal ciliated cells in vitro and induced bronchodilation in human bronchi ex vivo. In summary, T2R agonists increase MCC in vivo as assessed by 3D micro-CT-SPECT analysis

Two-year changes in quality of life in elderly patients with low-energy hip fractures. A case-control study

<p>Abstract</p> <p>Background</p> <p>The long-term effect of hip fracture on health-related quality of life (HRQOL) and global quality of life (GQOL) has not been thoroughly studied in prospective case-control studies.</p> <p>Aims</p> <p>a) to explore whether patients with low-energy hip fracture regain their pre-fracture levels in HRQOL and GQOL compared with changes in age- and sex-matched controls over a two year period; b) to identify predictors of changes in HRQOL and GQOL after two years.</p> <p>Methods</p> <p>We examined 61 patients (mean age = 74 years, <it>SD </it>= 10) and 61 matched controls (mean age = 73 years, <it>SD </it>= 8). The Short Form 36 assessed HRQOL and the Quality of Life Scale assessed GQOL. Paired samples <it>t </it>tests and multiple linear regression analyses were applied.</p> <p>Results</p> <p>HRQOL decreased significantly between baseline and one-year follow-up in patients with hip fractures, within all the SF-36 domains (<it>p </it>< 0.04), except for social functioning (<it>p </it>= 0.091). There were no significant decreases within the SF-36 domains in the controls. Significantly decreased GQOL scores (<it>p </it>< 0.001) were observed both within patients and within controls between baseline and one-year follow-up. The same pattern persisted between baseline and two-year follow-up, except for the HRQOL domain mental health (<it>p </it>= 0.193). The patients with hip fractures did not regain their HRQOL and GQOL. Worsened physical health after two years was predicted by being a patient with hip fracture (B = -5.8, <it>p </it>< 0.001) and old age (B = -1.0, <it>p </it>= 0.015), while worsened mental health was predicted by co-morbidity (B = -2.2, <it>p </it>= 0.029). No significant predictors of differential changes in GQOL were identified.</p> <p>Conclusion</p> <p>A hip fracture has a long-term impact on HRQOL and is a strong predictor of worsened physical health. Our data emphasize the importance of preventing hip fracture in the elderly to maintain physical health. This knowledge should be included in decision-making and health care plans.</p

Eligibility for interventions, co-occurrence and risk factors for unhealthy behaviours in patients consulting for routine primary care: results from the Pre-Empt study

Smoking, excessive drinking, lack of exercise and a poor diet remain key causes of premature morbidity and mortality globally, yet it is not clear what proportion of patients attending for routine primary care are eligible for interventions about these behaviours, the extent to which they co-occur within individuals, and which individuals are at greatest risk for multiple unhealthy behaviours. The aim of the trial was to examine 'intervention eligibility' and co-occurrence of the 'big four' risky health behaviours - lack of exercise, smoking, an unhealthy diet and excessive drinking - in a primary care population. Data were collected from adult patients consulting routinely in general practice across South Wales as part of the Pre-Empt study; a cluster randomised controlled trial. After giving consent, participants completed screening instruments, which included the following to assess eligibility for an intervention based on set thresholds: AUDIT-C (for alcohol), HSI (for smoking), IPAQ (for exercise) and a subset of DINE (for diet). The intervention following screening was based on which combination of risky behaviours the patient had. Descriptive statistics, χ2 tests for association and ordinal regressions were undertaken. Two thousand sixty seven patients were screened: mean age of 48.6 years, 61.9 % female and 42.8 % in a managerial or professional occupation. In terms of numbers of risky behaviours screened eligible for, two was the most common (43.6 %), with diet and exercise (27.2 %) being the most common combination. Insufficient exercise was the most common single risky behaviour (12.0 %). 21.8 % of patients would have been eligible for an intervention for three behaviours and 5.9 % for all four behaviours. Just 4.5 % of patients did not identify any risky behaviours. Women, older age groups and those in managerial or professional occupations were more likely to exhibit all four risky behaviours. Very few patients consulting for routine primary care screen ineligible for interventions about common unhealthy behaviours, and most engage in more than one of the major common unhealthy behaviours. Clinicians should be particularly alert to opportunities to engaging younger, non professional men and those with multi-morbidity about risky health behaviour. ISRCTN22495456. BACKGROUND METHODS RESULTS CONCLUSION TRIAL REGISTRATIO

Identification of ORC1/CDC6-Interacting Factors in Trypanosoma brucei Reveals Critical Features of Origin Recognition Complex Architecture

DNA Replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing

Preventing disease through opportunistic, rapid engagement by primary care teams using behaviour change counselling (PRE-EMPT): protocol for a general practice-based cluster randomised trial

BACKGROUND: Smoking, excessive alcohol consumption, lack of exercise and an unhealthy diet are the key modifiable factors contributing to premature morbidity and mortality in the developed world. Brief interventions in health care consultations can be effective in changing single health behaviours. General Practice holds considerable potential for primary prevention through modifying patients' multiple risk behaviours, but feasible, acceptable and effective interventions are poorly developed, and uptake by practitioners is low. Through a process of theoretical development, modeling and exploratory trials, we have developed an intervention called Behaviour Change Counselling (BCC) derived from Motivational Interviewing (MI). This paper describes the protocol for an evaluation of a training intervention (the Talking Lifestyles Programme) which will enable practitioners to routinely use BCC during consultations for the above four risk behaviours. METHODS/DESIGN: This cluster randomised controlled efficacy trial (RCT) will evaluate the outcomes and costs of this training intervention for General Practitioners (GPs) and nurses. Training methods will include: a practice-based seminar, online self-directed learning, and reflecting on video recorded and simulated consultations. The intervention will be evaluated in 29 practices in Wales, UK; two clinicians will take part (one GP and one nurse) from each practice. In intervention practices both clinicians will receive training. The aim is to recruit 2000 patients into the study with an expected 30% drop out. The primary outcome will be the proportion of patients making changes in one or more of the four behaviours at three months. Results will be compared for patients seeing clinicians trained in BCC with patients seeing non-BCC trained clinicians. Economic and process evaluations will also be conducted. DISCUSSION: Opportunistic engagement by health professionals potentially represents a cost effective medical intervention. This study integrates an existing, innovative intervention method with an innovative training model to enable clinicians to routinely use BCC, providing them with new tools to encourage and support people to make healthier choices. This trial will evaluate effectiveness in primary care and determine costs of the intervention