Characterization of Tryptophanase from Vibrio cholerae O1

AbstractTryptophanase (Trpase) encoded by the tnaA gene catalyzes the conversion of tryptophan to indole, which is an extracellular signaling molecule detected in various bacteria including Vibrio cholerae. Indole has been demonstrated to regulate biofilm formation, drug resistance, plasmid maintenance and spore formation of bacteria. In the present study, the tnaA gene from V. cholerae O1 (VcTrpase) was cloned and expressed in E. coli BL21(DE3) tn5:tnaA (a Trpase-deficient competent). VcTrpase was purified by Ni2+-NTA chromatography. The obtained VcTrpase had a molecular mass of approximately 49 kDa, a specific activity of 3 U/mg protein, and absorption peaks at 330 and 435nm. Using a site-directed mutagenesis technique, replacement of Arg419 by Val resulted in a VcTrpase completely devoid of activity. Thus, this site can be a target for drug design for controlling V. cholerae

High performance cryogen-free microkelvin platform

Improved accessibility to the microkelvin temperature regime is important for future research in quantum materials; for quantum information science; and for applications of quantum sensors. Here we report the design and performance of a microkelvin platform based on a nuclear demagnetization stage, engineered and well optimized for operation on a standard cryogen-free dilution refrigerator. PrNi5 is used as the dominant refrigerant. The platform provides a large area for mounting experiments in an ultralow temperature, low electromagnetic noise environment. The performance is characterized using current sensing noise thermometry. Temperatures as low as 395 $\mu$K have been reached, and a protocol has been established in which it is possible to operate experiments below 1 mK for 95% of the time, providing an efficient cryogen-free microkelvin environment for a wide range of science applicationsComment: 8 pages, 5 figure

The immune response to lumpy skin disease virus in cattle is influenced by inoculation route

Lumpy skin disease virus (LSDV) causes severe disease in cattle and water buffalo and is transmitted by hematophagous arthropod vectors. Detailed information of the adaptive and innate immune response to LSDV is limited, hampering the development of tools to control the disease. This study provides an in-depth analysis of the immune responses of calves experimentally inoculated with LSDV via either needle-inoculation or arthropod-inoculation using virus-positive Stomoxys calcitrans and Aedes aegypti vectors. Seven out of seventeen needle-inoculated calves (41%) developed clinical disease characterised by multifocal necrotic cutaneous nodules. In comparison 8/10 (80%) of the arthropod-inoculated calves developed clinical disease. A variable LSDV-specific IFN-γ immune response was detected in the needle-inoculated calves from 5 days post inoculation (dpi) onwards, with no difference between clinical calves (developed cutaneous lesions) and nonclinical calves (did not develop cutaneous lesions). In contrast a robust and uniform cell-mediated immune response was detected in all eight clinical arthropod-inoculated calves, with little response detected in the two nonclinical arthropod-inoculated calves. Neutralising antibodies against LSDV were detected in all inoculated cattle from 5-7 dpi. Comparison of the production of anti-LSDV IgM and IgG antibodies revealed no difference between clinical and nonclinical needle-inoculated calves, however a strong IgM response was evident in the nonclinical arthropod-inoculated calves but absent in the clinical arthropod-inoculated calves. This suggests that early IgM production is a correlate of protection in LSD. This study presents the first evidence of differences in the immune response between clinical and nonclinical cattle and highlights the importance of using a relevant transmission model when studying LSD

Effectiveness and cost-effectiveness of early assisted discharge for Chronic Obstructive Pulmonary Disease exacerbations: the design of a randomised controlled trial

Background: Exacerbations of Chronic Obstructive Pulmonary Disease (COPD) are the main cause for hospitalisation. These hospitalisations result in a high pressure on hospital beds and high health care costs. Because of the increasing prevalence of COPD this will only become worse. Hospital at home is one of the alternatives that has been proved to be a safe alternative for hospitalisation in COPD. Most schemes are early assisted discharge schemes with specialised respiratory nurses providing care at home. Whether this type of service is cost-effective depends on the setting in which it is delivered and the way in which it is organised. Methods/Design: GO AHEAD (Assessment Of Going Home under Early Assisted Discharge) is a 3-months, randomised controlled, multi-centre clinical trial. Patients admitted to hospital for a COPD exacerbation are either discharged on the fourth day of admission and further treated at home, or receive usual inpatient hospital care. Home treatment is supervised by general nurses. Primary outcome is the effectiveness and cost effectiveness of an early assisted discharge intervention in comparison with usual inpatient hospital care for patients hospitalised with a COPD exacerbation. Secondary outcomes include effects on quality of life, primary informal caregiver burden and patient and primary caregiver satisfaction. Additionally, a discrete choice experiment is performed to provide insight in patient and informal caregiver preferences for different treatment characteristics. Measurements are performed on the first day of admission and 3 days, 7 days, 1 month and 3 months thereafter. Ethical approval has been obtained and the study has been registered. Discussion: This article describes the study protocol of the GO AHEAD study. Early assisted discharge could be an effective and cost-effective method to reduce length of hospital stay in the Netherlands which is beneficial for patients and society. If effectiveness and cost-effectiveness can be proven, implementation in the Dutch health care system should be considered. Trial registration: Netherlands Trial Register NTR1129

Mutationen im PTS-Gen und mögliche Auswirkungen auf Funktion und Struktur der 6-Pyruvoyl-Tetrahydropterin-Synthase

Background: Research on Juvenile Idiopathic Arthritis (JIA) should support patients, caregivers/parents (carers) and clinicians to make important decisions in the consulting room and eventually to improve the lives of patients with JIA. Thus far these end-users of JIA-research have rarely been involved in the prioritisation of future research. Main body: Dutch organisations of patients, carers and clinicians will collaboratively develop a research agenda for JIA, following the James Lind Alliance (JLA) methodology. In a 'Priority Setting Partnership' (PSP), they will gradually establish a top 10 list of the most important unanswered research questions for JIA. In this process the input from clinicians, patients and their carers will be equally valued. Additionally, focus groups will be organised to involve young people with JIA. The involvement of all contributors will be monitored and evaluated. In this manner, the project will contribute to the growing body of literature on how to involve young people in agenda setting in a meaningful way. Conclusion: A JIA research agenda established through the JLA method and thus co-created by patients, carers and clinicians will inform researchers and research funders about the most important research questions for JIA. This will lead to research that really matters.</p

Reasons of general practitioners for not prescribing lipid-lowering medication to patients with diabetes: a qualitative study

Background: Lipid-lowering medication remains underused, even in high-risk populations. The objective of this study was to determine factors underlying general practitioners' decisions not to prescribe such drugs to patients with type 2 diabetes. Methods: A qualitative study with semi-structured interviews using real cases was conducted to explore reasons for not prescribing lipid-lowering medication after a guideline was distributed that recommended the use of statins in most patients with type 2 diabetes. Seven interviews were conducted with general practitioners (GPs) in The Netherlands, and analysed using an analytic inductive approach. Results: Reasons for not-prescribing could be divided into patient and physician-attributed factors. According to the GPs, some patients do not follow-up on agreed medication and others object to taking lipid-lowering medication, partly for legitimate reasons such as expected or perceived side effects. Furthermore, the GPs themselves perceived reservations for prescribing lipid-lowering medication in patients with short life expectancy, expected compliance problems or near goal lipid levels. GPs sometimes postponed the start of treatment because of other priorities. Finally, barriers were seen in the GPs' practice organisation, and at the primary-secondary care interface. Conclusion: Some of the barriers mentioned by GPs seem to be valid reasons, showing that guideline non-adherence can be quite rational. On the other hand, treatment quality could improve by addressing issues, such as lack of knowledge or motivation of both the patient and the GP. More structured management in general practice may also lead to better treatment

Effect of Homocysteine-Lowering Nutrients on Blood Lipids: Results from Four Randomised, Placebo-Controlled Studies in Healthy Humans

BACKGROUND: Betaine (trimethylglycine) lowers plasma homocysteine, a possible risk factor for cardiovascular disease. However, studies in renal patients and in obese individuals who are on a weight-loss diet suggest that betaine supplementation raises blood cholesterol; data in healthy individuals are lacking. Such an effect on cholesterol would counteract any favourable effect on homocysteine. We therefore investigated the effect of betaine, of its precursor choline in the form of phosphatidylcholine, and of the classical homocysteine-lowering vitamin folic acid on blood lipid concentrations in healthy humans. METHODS AND FINDINGS: We measured blood lipids in four placebo-controlled, randomised intervention studies that examined the effect of betaine (three studies, n = 151), folic acid (two studies, n = 75), and phosphatidylcholine (one study, n = 26) on plasma homocysteine concentrations. We combined blood lipid data from the individual studies and calculated a weighted mean change in blood lipid concentrations relative to placebo. Betaine supplementation (6 g/d) for 6 wk increased blood LDL cholesterol concentrations by 0.36 mmol/l (95% confidence interval: 0.25–0.46), and triacylglycerol concentrations by 0.14 mmol/l (0.04–0.23) relative to placebo. The ratio of total to HDL cholesterol increased by 0.23 (0.14–0.32). Concentrations of HDL cholesterol were not affected. Doses of betaine lower than 6 g/d also raised LDL cholesterol, but these changes were not statistically significant. Further, the effect of betaine on LDL cholesterol was already evident after 2 wk of intervention. Phosphatidylcholine supplementation (providing approximately 2.6 g/d of choline) for 2 wk increased triacylglycerol concentrations by 0.14 mmol/l (0.06–0.21), but did not affect cholesterol concentrations. Folic acid supplementation (0.8 mg/d) had no effect on lipid concentrations. CONCLUSIONS: Betaine supplementation increased blood LDL cholesterol and triacylglycerol concentrations in healthy humans, which agrees with the limited previous data. The adverse effects on blood lipids may undo the potential benefits for cardiovascular health of betaine supplementation through homocysteine lowering. In our study phosphatidylcholine supplementation slightly increased triacylglycerol concentrations in healthy humans. Previous studies of phosphatidylcholine and blood lipids showed no clear effect. Thus the effect of phosphatidylcholine supplementation on blood lipids remains inconclusive, but is probably not large. Folic acid supplementation does not seem to affect blood lipids and therefore remains the preferred treatment for lowering of blood homocysteine concentrations

Cost-effectiveness of a stepped-care intervention to prevent major depression in patients with type 2 diabetes mellitus and/or coronary heart disease and subthreshold depression: design of a cluster-randomized controlled trial

Background: Co-morbid major depression is a significant problem among patients with type 2 diabetes mellitus and/or coronary heart disease and this negatively impacts quality of life. Subthreshold depression is the most important risk factor for the development of major depression. Given the highly significant association between depression and adverse health outcomes and the limited capacity for depression treatment in primary care, there is an urgent need for interventions that successfully prevent the transition from subthreshold depression into a major depressive disorder. Nurse led stepped-care is a promising way to accomplish this. The aim of this study is to evaluate the cost-effectiveness of a nurse-led indicated stepped-care program to prevent major depression among patients with type 2 diabetes mellitus and/or coronary heart disease in primary care who also have subthreshold depressive symptoms.Methods/design: An economic evaluation will be conducted alongside a cluster-randomized controlled trial in approximately thirty general practices in the Netherlands. Randomization takes place at the level of participating practice nurses. We aim to include 236 participants who will either receive a nurse-led indicated stepped-care program for depressive symptoms or care as usual. The stepped-care program consists of four sequential but flexible treatment steps: 1) watchful waiting, 2) guided self-help treatment, 3) problem solving treatment and 4) referral to the general practitioner. The primary clinical outcome measure is the cumulative incidence of major depressive disorder as measured with the Mini International Neuropsychiatric Interview. Secondary outcomes include severity of depressive symptoms, quality of life, anxiety and physical outcomes. Costs will be measured from a societal perspective and include health care utilization, medication and lost productivity costs. Measurements will be performed at baseline and 3, 6, 9 and 12 months.Discussion: The intervention being investigated is expected to prevent new cases of depression among people with type 2 diabetes mellitus and/or coronary heart disease and subthreshold depression, with subsequent beneficial effects on quality of life, clinical outcomes and health care costs. When proven cost-effective, the program provides a viable treatment option in the Dutch primary care system.Trial registration: Dutch Trial Register NTR3715. © 2013 van Dijk et al.; licensee BioMed Central Ltd

Polymorphisms of the TUB Gene Are Associated with Body Composition and Eating Behavior in Middle-Aged Women

BACKGROUND: The TUB gene, encoding an evolutionary conserved protein, is highly expressed in the hypothalamus and might act as a transcription factor. Mutations in TUB cause late-onset obesity, insulin-resistance and neurosensory deficits in mice. An association of common variants in the TUB gene with body weight in humans has been reported. METHODS/FINDINGS: The aim was to investigate the relationship of single nucleotide polymorphisms (SNPs) of the TUB gene (rs2272382, rs2272383 and rs1528133) with both anthropometry and self-reported macronutrient intake from a validated food frequency questionnaire. These associations were studied in a population-based, cross-sectional study of 1680 middle-aged Dutch women, using linear regression analysis. The minor allele C of the rs1528133 SNP was significantly associated with increased weight (+1.88 kg, P = 0.022) and BMI (+0.56 units, P = 0.05). Compared with non-carriers, both AG heterozygotes and AA homozygotes of the rs2272382 SNP derived less energy from fat (AG: -0.55+/-0.28%, P = 0.05, AA: -0.95+/-0.48%, P = 0.047). However, both genotypes were associated with an increased energy intake from carbohydrates (0.69+/-0.33%, P = 0.04 and 1.68+/-0.56%, P = 0.003, respectively), mainly because of a higher consumption of mono- and disaccharides. Both these SNPs, rs2272382 and rs1528133, were also associated with a higher glycemic load in the diet. The glycemic load was higher among those with AG and AA genotypes for the variant rs2272382 than among the wild types (+1.49 (95% CI: -0.27-3.24) and +3.89 (95% CI: 0.94-6.85) units, respectively). Carriers of the minor allele C of rs1528133 were associated with an increased glycemic load of 1.85 units compared with non-carriers. CONCLUSIONS: Genetic variation of the TUB gene was associated with both body composition and macronutrient intake, suggesting that TUB might influence eating behavior