The functional assembly of the mammalian minichromosome maintenance complex

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and is essential for DNA replication. Mis-regulation of its assembly and function can lead to genomic instability, replication stress and genome re-replication, which are powerful drivers toward the acquisition of mutations and the formation of cancer. I have produced and purified recombinant human MCM2-7 (hMCM) in Escherichia coli (E. coli). Recombinant hMCM has ATP hydrolysis and helicase activity, when assayed in the absence of post-translational modifications or accessory proteins. Using electron microscopy asymmetric single particle reconstruction, I have produced 23 Å resolution structures. The structures reveal that recombinant hMCM forms an asymmetric complex that changes conformation in the presence of a forked DNA substrate. By applying serial extractions to mammalian cells synchronised by release from quiescence, I have revealed dynamic changes in the sub-nuclear compartmentalisation of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear matrix. This suggests that functional MCM2-7 loading takes place at the nuclear matrix. Using this information I have developed a system to study the regulated assembly of recombinant hMCM, to support molecular dissection of the loading process. Using cellular extracts, I show regulated binding of recombinant hMCM, which translates into initiation of DNA replication and is stimulated by cyclin E/CDK2. Furthermore, I have demonstrated that both cyclin A/CDK2 and Dbf4/Drf1-dependant kinase (DDK) are required for inducing a conformational change in hMCM that is associated with S phase of the cell cycle. Understanding of the assembly of hMCM will serve as a foundation for analysis of corruption of the process, its effect on genome instability and gene expression, and how these events lead to the ‘birth’ of cancer cells. Very early events in the development of cancer cells are most likely to yield new and effective opportunities for detection and treatment of a broad range of cancers

Vitamins D2 and D3 Have Overlapping But Different Effects on the Human Immune System Revealed Through Analysis of the Blood Transcriptome

Vitamin D is best known for its role in maintaining bone health and calcium homeostasis. However, it also exerts a broad range of extra-skeletal effects on cellular physiology and on the immune system. Vitamins D(2) and D(3) share a high degree of structural similarity. Functional equivalence in their vitamin D-dependent effects on human physiology is usually assumed but has in fact not been well defined experimentally. In this study we seek to redress the gap in knowledge by undertaking an in-depth examination of changes in the human blood transcriptome following supplementation with physiological doses of vitamin D(2) and D(3). Our work extends a previously published randomized placebo-controlled trial that recruited healthy white European and South Asian women who were given 15 µg of vitamin D(2) or D(3) daily over 12 weeks in wintertime in the UK (Nov-Mar) by additionally determining changes in the blood transcriptome over the intervention period using microarrays. An integrated comparison of the results defines both the effect of vitamin D(3) or D(2) on gene expression, and any influence of ethnic background. An important aspect of this analysis was the focus on the changes in expression from baseline to the 12-week endpoint of treatment within each individual, harnessing the longitudinal design of the study. Whilst overlap in the repertoire of differentially expressed genes was present in the D(2) or D(3)-dependent effects identified, most changes were specific to either one vitamin or the other. The data also pointed to the possibility of ethnic differences in the responses. Notably, following vitamin D(3) supplementation, the majority of changes in gene expression reflected a down-regulation in the activity of genes, many encoding pathways of the innate and adaptive immune systems, potentially shifting the immune system to a more tolerogenic status. Surprisingly, gene expression associated with type I and type II interferon activity, critical to the innate response to bacterial and viral infections, differed following supplementation with either vitamin D(2) or vitamin D(3), with only vitamin D(3) having a stimulatory effect. This study suggests that further investigation of the respective physiological roles of vitamin D(2) and vitamin D(3) is warranted

Results From Australia’s 2016 Report Card on Physical Activity for Children and Youth

Background: Two years on from the inaugural Active Healthy Kids Australia (AHKA) Physical Activity Report Card, there has been little to no change with the majority of Australian children still insufficiently active. Methods: The 2016 AHKA Report Card was developed using the best available national- and state-based physical activity data, which were evaluated by the AHKA Research Working Group using predetermined weighting criteria and benchmarks to assign letter grades to the 12 Report Card indicators. Results: In comparison with 2014, Overall Physical Activity Levels was again assigned a D- with Organized Sport and Physical Activity Participation increasing to a B (was B-) and Active Transport declining to a C- (was C). The settings and sources of influence again performed well (A- to a C+), however Government Strategies and Investments saw a decline (C+ to a D). The traits associated with physical activity were also graded poorly (C- to a D). Conclusions: Australian youth are insufficiently active and engage in high levels of screen-based sedentary behaviors. While a range of support structures exist, Australia lacks an overarching National Physical Activity Plan that would unify the country and encourage the cultural shift needed to face the inactivity crisis head on

Compliance with Australian stroke guideline recommendations for outdoor mobility and transport training by post-inpatient rehabilitation services: an observational cohort study

Background: Community participation is often restricted after stroke, due to reduced confidence and outdoor mobility. Australian clinical guidelines recommend that specific evidence-based interventions be delivered to target these restrictions, such as multiple escorted outdoor journeys. The aim of this study was to describe post-inpatient outdoor mobility and transport training delivered to stroke survivors in New South Wales, Australia and whether therapy differed according to type, sector or location of service provider. Methods: Using an observational retrospective cohort study design, 24 rehabilitation service providers were audited. Provider types included outpatient (n = 8), day therapy (n = 9), home-based rehabilitation (n = 5) and transitional aged care services (TAC, n = 2). Records of 15 stroke survivors who had received post-hospital rehabilitation were audited per service, for wait time, duration, amount of therapy and outdoor-related therapy. Results: A total of 311 records were audited. Median wait time for post-hospital therapy was 13 days (IQR, 5–35). Median duration of therapy was 68 days (IQR, 35–109), consisting of 11 sessions (IQR 4–19). Overall, a median of one session (IQR 0–3) was conducted outdoors per person. Outdoor-related therapy was similar across service providers,except that TAC delivered an average of 5.4 more outdoor-related sessions (95 % CI 4.4 to 6.4), and 3.5 more outings into public streets (95 % CI 2.8 to 4.3) per person, compared to outpatient services. Conclusion: The majority of service providers in the sample delivered little evidence-based outdoor mobility and travel training per stroke participant, as recommended in national stroke guidelines

The Early Prevention of Obesity in CHildren (EPOCH) Collaboration - an Individual Patient Data Prospective Meta-Analysis

BackgroundEfforts to prevent the development of overweight and obesity have increasingly focused early in the life course as we recognise that both metabolic and behavioural patterns are often established within the first few years of life. Randomised controlled trials (RCTs) of interventions are even more powerful when, with forethought, they are synthesised into an individual patient data (IPD) prospective meta-analysis (PMA). An IPD PMA is a unique research design where several trials are identified for inclusion in an analysis before any of the individual trial results become known and the data are provided for each randomised patient. This methodology minimises the publication and selection bias often associated with a retrospective meta-analysis by allowing hypotheses, analysis methods and selection criteria to be specified a priori.Methods/DesignThe Early Prevention of Obesity in CHildren (EPOCH) Collaboration was formed in 2009. The main objective of the EPOCH Collaboration is to determine if early intervention for childhood obesity impacts on body mass index (BMI) z scores at age 18-24 months. Additional research questions will focus on whether early intervention has an impact on children\u27s dietary quality, TV viewing time, duration of breastfeeding and parenting styles. This protocol includes the hypotheses, inclusion criteria and outcome measures to be used in the IPD PMA. The sample size of the combined dataset at final outcome assessment (approximately 1800 infants) will allow greater precision when exploring differences in the effect of early intervention with respect to pre-specified participant- and intervention-level characteristics.DiscussionFinalisation of the data collection procedures and analysis plans will be complete by the end of 2010. Data collection and analysis will occur during 2011-2012 and results should be available by 2013.<br /

Using surveillance data to determine treatment rates and outcomes for patients with chronic hepatitis C virus infection

The aim of this work was to develop and validate an algorithm to monitor rates of, and response to, treatment of patients infected with hepatitis C virus (HCV) across England using routine laboratory HCV RNA testing data. HCV testing activity between January 2002 and December 2011 was extracted from the local laboratory information systems of a sentinel network of 23 laboratories across England. An algorithm based on frequency of HCV RNA testing within a defined time period was designed to identify treated patients. Validation of the algorithm was undertaken for one center by comparison with treatment data recorded in a clinical database managed by the Trent HCV Study Group. In total, 267,887 HCV RNA test results from 100,640 individuals were extracted. Of these, 78.9% (79,360) tested positive for viral RNA, indicating an active infection, 20.8% (16,538) of whom had a repeat pattern of HCV RNA testing suggestive of treatment monitoring. Annual numbers of individuals treated increased rapidly from 468 in 2002 to 3,295 in 2009, but decreased to 3,110 in 2010. Approximately two thirds (63.3%; 10,468) of those treated had results consistent with a sustained virological response, including 55.3% and 67.1% of those with a genotype 1 and non-1 virus, respectively. Validation against the Trent clinical database demonstrated that the algorithm was 95% sensitive and 93% specific in detecting treatment and 100% sensitive and 93% specific for detecting treatment outcome. Conclusions: Laboratory testing activity, collected through a sentinel surveillance program, has enabled the first country-wide analysis of treatment and response among HCV-infected individuals. Our approach provides a sensitive, robust, and sustainable method for monitoring service provision across Englan

A multilevel intervention to increase physical activity and improve healthy eating and physical literacy among young children (ages 3-5) attending early childcare centres: the Healthy Start-Départ Santé cluster randomised controlled trial study protocol

Abstract: Background: Childhood obesity is a growing concern for public health. Given a majority of children in many countries spend approximately 30 h per week in early childcare centers, this environment represents a promising setting for implementing strategies to foster healthy behaviours for preventing and controlling childhood obesity. Healthy Start-Départ Santé was designed to promote physical activity, physical literacy, and healthy eating among preschoolers. The objectives of this study are to assess the effectiveness of the Healthy Start-Départ Santé intervention in improving physical activity levels, physical literacy, and healthy eating among preschoolers attending early childcare centers. Methods/Design: This study follows a cluster randomized controlled trial design in which the childcare centers are randomly assigned to receive the intervention or serve as usual care controls. The Healthy Start-Départ Santé intervention is comprised of interlinked components aiming to enable families and educators to integrate physical activity and healthy eating in the daily lives of young children by influencing factors at the intrapersonal, interpersonal, organizational, community, physical environment and policy levels. The intervention period, spanning 6-8 months, is preceded and followed by data collections. Participants are recruited from 61 childcare centers in two Canadian provinces, New Brunswick and Saskatchewan. Centers eligible for this study have to prepare and provide meals for lunch and have at least 20 children between the ages of 3 and 5. Centers are excluded if they have previously received a physical activity or nutrition promoting intervention. Eligible centers are stratified by province, geographical location (urban or rural) and language (English or French), then recruited and randomized using a one to one protocol for each stratum. Data collection is ongoing. The primary study outcomes are assessed using accelerometers (physical activity levels), the Test of Gross Motor Development-II (physical literacy), and digital photography-assisted weighted plate waste (food intake). Discussion: The multifaceted approach of Healthy Start-Départ Santé positions it well to improve the physical literacy and both dietary and physical activity behaviors of children attending early childcare centers. The results of this study will be of relevance given the overwhelming prevalence of overweight and obesity in children worldwide. Trial registration: NCT02375490 (ClinicalTrials.gov registry)

Unpacking the behavioural components and delivery features of early childhood obesity prevention interventions in the TOPCHILD Collaboration: a systematic review and intervention coding protocol.

INTRODUCTION: Little is known about how early (eg, commencing antenatally or in the first 12 months after birth) obesity prevention interventions seek to change behaviour and which components are or are not effective. This study aims to (1) characterise early obesity prevention interventions in terms of target behaviours, delivery features and behaviour change techniques (BCTs), (2) explore similarities and differences in BCTs used to target behaviours and (3) explore effectiveness of intervention components in preventing childhood obesity. METHODS AND ANALYSIS: Annual comprehensive systematic searches will be performed in Epub Ahead of Print/MEDLINE, Embase, Cochrane (CENTRAL), CINAHL, PsycINFO, as well as clinical trial registries. Eligible randomised controlled trials of behavioural interventions to prevent childhood obesity commencing antenatally or in the first year after birth will be invited to join the Transforming Obesity in CHILDren Collaboration. Standard ontologies will be used to code target behaviours, delivery features and BCTs in both published and unpublished intervention materials provided by trialists. Narrative syntheses will be performed to summarise intervention components and compare applied BCTs by types of target behaviours. Exploratory analyses will be undertaken to assess effectiveness of intervention components. ETHICS AND DISSEMINATION: The study has been approved by The University of Sydney Human Research Ethics Committee (project no. 2020/273) and Flinders University Social and Behavioural Research Ethics Committee (project no. HREC CIA2133-1). The study's findings will be disseminated through peer-reviewed publications, conference presentations and targeted communication with key stakeholders. PROSPERO REGISTRATION NUMBER: CRD42020177408