A novel framework for engineering protein loops exploring length and compositional variation

Insertions and deletions (indels) are known to affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear significance, this class of mutation remains an underexploited tool in protein engineering with few available platforms capable of systematically generating and analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction digestion and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 β-lactamase Ω-loop, involved in substrate specificity, identifying multiple novel extended spectrum β-lactamases with loops of modified length and composition—areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efficient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters

Using psychological theory to understand the clinical management of type 2 diabetes in Primary Care : a comparison across two European countries

Peer reviewedPublisher PD

The Terneuzen Birth Cohort:BMI change between 2 and 6 years is most predictive of adult cardiometabolic risk

BACKGROUND: We recently reported the age interval 2–6y being the earliest and most critical for adult overweight. We now aim to determine which age intervals are predictive of cardiometabolic risk at young adulthood. METHODS AND FINDINGS: We analyzed data from 642 18–28 years olds from the Terneuzen Birth Cohort. Individual BMI SDS trajectories were fitted by a piecewise linear model. By multiple regression analyses relationships were assessed between subsequent conditional BMI SDS changes and components of the metabolic syndrome (MetS), skinfold thickness and hsCRP at young adulthood. Results were adjusted for gender and age, and other confounders. Gender was studied as an effect modifier. All BMI SDS changes throughout childhood were related to waist circumference and skinfold thickness. No other significant relationship was found before the age of 2 years, except between the BMI SDS change 0–1y and hsCRP. Fasting blood glucose was not predicted by any BMI SDS change. BMI SDS change 2–6y was strongly related to most outcome variables, especially to waist circumference (ß 0.47, SE 0.02), systolic and diastolic blood pressure (ß 0.20 SE 0.04 and ß 0.19 SE 0.03), and hsCRP (ß 0.16 SE 0.04). The BMI SDS change 10–18y was most strongly related to HDL cholesterol (ß -0.10, SE 0.03), and triglycerides (ß 0.21, SE 0.03). To a lesser degree, the BMI SDS change 6–10y was related to most outcome variables. BMI SDS changes 2–6y and 10–18y were significantly related to MetS: the OR was respectively 3.39 (95%CI 2.33–4.94) and 2.84 (95%CI 1.94–4.15). CONCLUSION: BMI SDS changes from 2y onwards were related to cardiometabolic risk at young adulthood, the age interval 2–6y being the most predictive. Monitoring and stabilizing the BMI SDS of children as young as 2–6y may not only reverse the progression towards adult overweight, but it may also safeguard cardiometabolic status

'Correction:'Peer chart audits: A tool to meet Accreditation Council on Graduate Medical Education (ACGME) competency in practice-based learning and improvement

<p>Abstract</p> <p>Background</p> <p>The Accreditation Council on Graduate Medical Education (ACGME) supports chart audit as a method to track competency in Practice-Based Learning and Improvement. We examined whether peer chart audits performed by internal medicine residents were associated with improved documentation of foot care in patients with diabetes mellitus.</p> <p>Methods</p> <p>A retrospective electronic chart review was performed on 347 patients with diabetes mellitus cared for by internal medicine residents in a university-based continuity clinic from May 2003 to September 2004. Residents abstracted information pertaining to documentation of foot examinations (neurological, vascular, and skin) from the charts of patients followed by their physician peers. No formal feedback or education was provided.</p> <p>Results</p> <p>Significant improvement in the documentation of foot exams was observed over the course of the study. The percentage of patients receiving neurological, vascular, and skin exams increased by 20% (from 13% to 33%) (p = 0.001), 26% (from 45% to 71%) (p < 0.001), and 18% (51%–72%) (p = 0.005), respectively. Similarly, the proportion of patients receiving a well-documented exam which includes all three components – neurological, vascular and skin foot exam – increased over time (6% to 24%, p < 0.001).</p> <p>Conclusion</p> <p>Peer chart audits performed by residents in the absence of formal feedback were associated with improved documentation of the foot exam in patients with diabetes mellitus. Although this study suggests that peer chart audits may be an effective tool to improve practice-based learning and documentation of foot care in diabetic patients, evaluating the actual performance of clinical care was beyond the scope of this study and would be better addressed by a randomized controlled trial.</p

Inequalities in energy-balance related behaviours and family environmental determinants in European children: changes and sustainability within the EPHE evaluation study

BACKGROUND: Increasing social inequalities in health across Europe are widening the gap between low and high socio-economic groups, notably in the prevalence of obesity. Public health interventions may result in differential effects across population groups. Therefore, the EPHE (EPODE for the Promotion of Health Equity) project analysed the added value of community-based programmes, based on the EPODE (Ensemble Prévenons l'Obésité Des Enfants-Together Let's Prevent Obesity) model, to reduce socio-economic inequalities in energy balance-related behaviours of children and their family-environmental related determinants in seven European communities. This study presents the changes between baseline and follow-up after the one-year interventions and their sustainability one year after. METHODS: This is a prospective study with a one school-year intervention, followed by one year of follow-up. In all, 1266 children (age 6-8 years) and their families from different socio-economic backgrounds were recruited at baseline. For 1062 children, information was available after one year (T1) and for 921 children after two years (T2). A self-reported questionnaire was completed by the parents to examine the children's energy balance-related behaviours and family- environmental determinants. Socio-economic status was defined by the educational level of the mother. The Wilcoxon signed-rank test for paired data was used to test the differences between baseline and intermediate, and between intermediate and final, measurements for each of the socio-economic status groups. RESULTS: Post-intervention effects in energy-balance related behaviours showed the following improvements among the low socio-economic status groups: increased fruit consumption (Netherlands), decreased fruit juices amount consumed (Romania) and decreased TV time on weekdays (Belgium). Whereas in only the latter case the behavioural change was accompanied with an improvement in a family-environmental determinant (monitoring the time the child watches TV), other improvements in parental rules and practices related to soft drinks/fruit juices and TV exposure were observed. A few of those effects were sustainable, notably in the case of Belgium. CONCLUSIONS: Inequalities in obesity-related behaviours could be potentially reduced when implementing community-based interventions, tailored to inequality gaps and using the EPODE methodology. Within-group changes varied widely, whereas monitoring of interventions and process evaluation are crucial to understand the observed results

Application of Building Typologies for Modelling the Energy Balance of the Residential Building Stock.

Building typologies can serve as a basis for analysing the national housing sector. During the TABULA project which was introducing or further developing building typologies in thirteen EU countries, six of the European partners have carried out model calculations which aim at imaging the energy consumption and estimating the energy saving potentials of their national residential building stocks (IWU / Germany, NOA / Greece, POLITO / Italy, VITO / Belgium, STU-K / Czech Republic, SBi / Denmark). The results show that the model calculations can provide plausible projections of the energy consumption of the national residential buildings stock. The fit of model calculations and national energy statistics is satisfactory, deviations can often be explained and corrected by adapting standard boundary conditions of the applied calculation models to more realistic values. In general, the analysis shows that building typologies can be a helpful tool for modelling the energy consumption of national building stocks and for carrying out scenario analysis beyond the TABULA project. The consideration of a set of representative buildings makes it possible to have a detailed view on various packages of measures for the complete buildings stock or for its sub-categories. The effects of different insulation measures at the respective construction elements as well as different heat supply measures including renewable energies can be considered in detail. The quality of future model calculations will depend very much on the availability of statistical data. For reliable scenario analysis information is necessary about the current state of the building stock (How many buildings and heating systems have been refurbished until now?) and about the current trends (How many buildings and heating systems are being refurbished every year?). The availability and regular update of the relevant statistical data will be an important basis for the development and evaluation of national climate protection strategies in the building sector

A cluster randomized controlled trial of the effectiveness and cost-effectiveness of Intermediate Care Clinics for Diabetes (ICCD) : study protocol for a randomized controlled trial

Background World-wide healthcare systems are faced with an epidemic of type 2 diabetes. In the United Kingdom, clinical care is primarily provided by general practitioners (GPs) rather than hospital specialists. Intermediate care clinics for diabetes (ICCD) potentially provide a model for supporting GPs in their care of people with poorly controlled type 2 diabetes and in their management of cardiovascular risk factors. This study aims to (1) compare patients with type 2 diabetes registered with practices that have access to an ICCD service with those that have access only to usual hospital care; (2) assess the cost-effectiveness of the intervention; and (3) explore the views and experiences of patients, health professionals and other stakeholders. Methods/Design This two-arm cluster randomized controlled trial (with integral economic evaluation and qualitative study) is set in general practices in three UK Primary Care Trusts. Practices are randomized to one of two groups with patients referred to either an ICCD (intervention) or to hospital care (control). Intervention group: GP practices in the intervention arm have the opportunity to refer patients to an ICCD - a multidisciplinary team led by a specialist nurse and a diabetologist. Patients are reviewed and managed in the ICCD for a short period with a goal of improving diabetes and cardiovascular risk factor control and are then referred back to practice. or Control group: Standard GP care, with referral to secondary care as required, but no access to ICCD. Participants are adults aged 18 years or older who have type 2 diabetes that is difficult for their GPs to control. The primary outcome is the proportion of participants reaching three risk factor targets: HbA1c (≤7.0%); blood pressure (<140/80); and cholesterol (<4 mmol/l), at the end of the 18-month intervention period. The main secondary outcomes are the proportion of participants reaching individual risk factor targets and the overall 10-year risks for coronary heart disease(CHD) and stroke assessed by the United Kingdom Prospective Diabetes Study (UKPDS) risk engine. Other secondary outcomes include body mass index and waist circumference, use of medication, reported smoking, emotional adjustment, patient satisfaction and views on continuity, costs and health related quality of life. We aimed to randomize 50 practices and recruit 2,555 patients

Identification of DNA methylation changes at cis-regulatory elements during early steps of HSC differentiation using tagmentation-based whole genome bisulfite sequencing

Epigenetic alterations during cellular differentiation are a key molecular mechanism which both instructs and reinforces the process of lineage commitment. Within the haematopoietic system, progressive changes in the DNA methylome of haematopoietic stem cells (HSCs) are essential for the effective production of mature blood cells. Inhibition or loss of function of the cellular DNA methylation machinery has been shown to lead to a severe perturbation in blood production and is also an important driver of malignant transformation. HSCs constitute a very rare cell population in the bone marrow, capable of life-long self-renewal and multi-lineage differentiation. The low abundance of HSCs has been a major technological barrier to the global analysis of the CpG methylation status within both HSCs and their immediate progeny, the multipotent progenitors (MPPs). Within this Extra View article, we review the current understanding of how the DNA methylome regulates normal and malignant hematopoiesis. We also discuss the current methodologies that are available for interrogating the DNA methylation status of HSCs and MPPs and describe a new data set that was generated using tagmentation-based whole genome bisulfite sequencing (TWGBS) in order to comprehensively map methylated cytosines using the limited amount of genomic DNA that can be harvested from rare cell populations. Extended analysis of this data set clearly demonstrates the added value of genome-wide sequencing of methylated cytosines and identifies novel important cis-acting regulatory regions that are dynamically remodeled during the first steps of haematopoietic differentiation