Reliability of systolic blood pressure measured by parents in young children at home using a hand held doppler device and aneroid sphygmomanometer.

OBJECTIVE: We report data regarding systolic BP monitoring in children aged <5 years performed over a 2-week period by parents at home using a hand-held doppler device and aneroid sphygmomanometer for SBP measurements (HDBPM). Our objectives were to compare health professional measured office systolic BP by doppler device (Office-SBP Doppler ) with parent measured home systolic BP using the same doppler device (Home-SBP Doppler ). We also report data evaluating reliability and optimal number of days of measurement required. DESIGN AND METHODS: We taught parents to measure systolic BP and assessed their technique using a hand-held doppler device and aneroid sphygmomanometer. We requested parents to perform three consecutive BP measurements twice daily (ideally morning and evening around similar times) when the child was awake, settled and cooperative. RESULTS: Over a 3-year period, data from 48 of 62 children who underwent HDBPM measurements were evaluated with median (IQR) age of 1.9 (0.9, 3.6) years, 27 (56%) boys and 14 (29%) on antihypertensive medication. Office-SBP Doppler was 2.9 ±â€Š8.9 mmHg [95% confidence interval (CI), -14.4 to 20.4, P  = 0.026] higher than Home-SBP Doppler . Mean Home-SBP Doppler between Week-1 and Week-2 monitoring was similar -0.45 ±â€Š3.5 mmHg (95% CI, -7.35 to 6.45, P  = 0.41). Morning HDBPM measurements were lower than evening with a mean difference of -2.77 ±â€Š3.92 mmHg, P  < 0.001). Over Week-1, mean Home-SBP Doppler was closer to mean Office-SBP Doppler with increasing cumulative days of monitoring and with smaller standard deviations suggesting that readings become more reliable from day 4 onwards. CONCLUSIONS: HDBPM is a reliable method for measuring systolic BP in young children with BP levels measured by parents comparable to those performed by health professional in clinic. HDBPM technique described here and performed by parents over a 7-day period with a minimum of 4-days, offers a reliable and reproducible technique to measure blood pressure at home.

Ataluren and similar compounds (specific therapies for premature termination codon class I mutations) for cystic fibrosis.

BACKGROUND: Cystic fibrosis is a common life-shortening genetic disorder in the Caucasian population (less common in other ethnic groups) caused by the mutation of a single gene that codes for the production of the cystic fibrosis transmembrane conductance regulator protein. This protein coordinates the transport of salt (and bicarbonate) across cell surfaces and the mutation most notably affects the airways. In the lungs of people with cystic fibrosis, defective protein results in a dehydrated surface liquid and compromised mucociliary clearance. The resulting thick mucus makes the airway prone to chronic infection and inflammation, which consequently damages the structure of the airways, eventually leading to respiratory failure. Additionally, abnormalities in the cystic fibrosis transmembrane conductance regulator protein lead to other systemic complications including malnutrition, diabetes and subfertility.Five classes of mutation have been described, depending on the impact of the mutation on the processing of the cystic fibrosis transmembrane conductance regulator protein in the cell. In class I mutations, the presence of premature termination codons prevents the production of any functional protein resulting in a severe cystic fibrosis phenotype. Advances in the understanding of the molecular genetics of cystic fibrosis has led to the development of novel mutation-specific therapies. Therapies targeting class I mutations (premature termination codons) aim to mask the abnormal gene sequence and enable the normal cellular mechanism to read through the mutation, potentially restoring the production of the cystic fibrosis transmembrane conductance regulator protein. This could in turn make salt transport in the cells function more normally and may decrease the chronic infection and inflammation that characterises lung disease in people with cystic fibrosis. OBJECTIVES: To evaluate the benefits and harms of ataluren and similar compounds on clinically important outcomes in people with cystic fibrosis with class I mutations (premature termination codons). SEARCH METHODS: We searched the Cochrane Cystic Fibrosis Trials Register which is compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched the reference lists of relevant articles. Last search of Group's register: 24 October 2016.We searched clinical trial registries maintained by the European Medicines Agency, the US National Institutes of Health and the WHO. Last search of clinical trials registries: 28 November 2016. SELECTION CRITERIA: Randomised controlled trials of parallel design comparing ataluren and similar compounds (specific therapies for class I mutations) with placebo in people with cystic fibrosis who have at least one class I mutation. Cross-over trials were reviewed individually to evaluate whether data from the first treatment arm could be included. We excluded trials that combined therapies for premature termination codon class I mutations with other mutation-specific therapies. DATA COLLECTION AND ANALYSIS: The authors independently assessed the risk of bias and extracted data from the included trial; they contacted trial authors for additional data. MAIN RESULTS: Our searches identified 28 references to eight trials; five trials were excluded (three were cross-over and one was not randomised and one did not have relevant outcomes), one cross-over trial is awaiting classification pending provision of data and one trial is ongoing. The included parallel randomised controlled trial compared ataluren to placebo for a duration of 48 weeks in 238 participants (age range 6 to 53 years) with cystic fibrosis who had at least one nonsense mutation (a type of class I mutation).The quality of evidence and risk of bias assessments for the trial were moderate overall. Random sequence generation, allocation concealment and blinding of trial personnel were well-documented; participant blinding was less clear. Some participant data were excluded from the analysis. The trial was assessed as high risk of bias for selective outcome reporting, especially when reporting on the trial's post hoc subgroup of participants by chronic inhaled antibiotic use.The trial was sponsored by PTC Therapeutics Incorporated with grant support by the Cystic Fibrosis Foundation, the Food and Drug Administration's Office of Orphan Products Development and the National Institutes of Health (NIH).The trial reported no significant difference between treatment groups in quality of life, assessed by the Cystic Fibrosis Questionnaire-Revised respiratory domain score and no improvement in respiratory function measures (mean difference of relative change in forced expiratory volume at one second 2.97% (95% confidence interval -0.58 to 6.52)). Ataluren was associated with a significantly higher rate of episodes of renal impairment, risk ratio 17.70 (99% confidence interval 1.28 to 244.40). The trial reported no significant treatment effect for ataluren for the review's secondary outcomes: pulmonary exacerbation; computerised tomography score; weight; body mass index; and sweat chloride. No deaths were reported in the trial.A post hoc subgroup analysis of participants not receiving chronic inhaled tobramycin (n = 146) demonstrated favourable results for ataluren (n = 72) for relative change in % predicted forced expiratory volume at one second and pulmonary exacerbation rate. Participants receiving chronic inhaled tobramycin appeared to have a reduced rate of pulmonary exacerbation compared to those not receiving chronic inhaled tobramycin. This drug interaction was not anticipated and may affect the interpretation of the trial results. AUTHORS' CONCLUSIONS: There is currently insufficient evidence to determine the effect of ataluren as a therapy for people with cystic fibrosis with class I mutations. Future trials should carefully assess for adverse events, notably renal impairment and consider the possibility of drug interactions. Cross-over trials should be avoided given the potential for the treatment to change the natural history of cystic fibrosis.

Real-time monitoring of tyrosine hydroxylase activity using a plate reader assay.

Tyrosine hydroxylase (TH) is the rate-limiting step in dopamine (DA) synthesis, oxidizing tyrosine to l-DOPA, which is further metabolized to DA. Current assays for monitoring activity of this enzyme require extensive work-up, require long analysis time, and measure end points, thereby lacking real-time kinetics. This work presents the development of the first real-time colorimetric assay for determining the activity of TH using a plate reader. The production of l-DOPA is followed using sodium periodate to oxidize l-DOPA to the chromophore dopachrome, which can be monitored at 475 nm. Advantages to this method include decreased sample analysis time, shorter assay work-up, and the ability to run a large number of samples at one time. Furthermore, the assay was adapted for high-throughput screening and demonstrated an excellent Z-factor (> 0.8), indicating suitability of this assay for high-throughput analysis. Overall, this novel assay reduces analysis time, increases sample number, and allows for the study of activity using real-time kinetics.

Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli.

Tyrosine hydroxylase is the rate-limiting step in the synthesis of dopamine and is tightly regulated. Previous studies have shown it to be covalently modified and potently inhibited by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an endogenous neurotoxin via dopamine catabolism which is relevant to Parkinson's disease. In order to elucidate the mechanism of enzyme inhibition, a source of pure, active tyrosine hydroxylase was necessary. The cloning and novel purification of human recombinant TH from Escherichia coli is described here. This procedure led to the recovery of ~23 mg of pure, active and stable enzyme exhibiting a specific activity of ~17 nmol/min/mg. The enzyme produced with this procedure can be used to delineate the tyrosine hydroxylase inhibition by DOPAL and its relationship to Parkinson's disease. This procedure improves upon previous methods because the fusion protein gives rise to high expression and convenient affinity-capture, and the cleaved and highly purified hTH makes the product useful for a wider variety of applications.

Specificity determinants of allosteric modulation in the neuronal nicotinic acetylcholine receptor: a fine line between inhibition and potentiation.

We are interested in the allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs). We have postulated that the anthelmintic morantel (Mor) positively modulates (potentiates) rat α3ß2 receptors through a site located at the ß(+)/α(-) interface that is homologous to the canonical agonist site (J Neurosci 29:8734-8742, 2009). On this basis, we aimed to determine the site specificity by studying differences in modulation between α3ß2 and α4ß2 receptors. We also compared modulation by Mor with that of the related compound oxantel (Oxa). Whereas Mor and Oxa each potentiated α3ß2 receptors 2-fold at saturating acetylcholine (ACh) concentrations, Mor had no effect on α4ß2 receptors, and Oxa inhibited ACh-evoked responses. The inhibition was noncompetitive, but not due to open channel block. Furthermore, the nature and extent of modulation did not depend on subunit stoichiometry. We studied six positions at the α(-) interface that differ between α3 and α4. Two positions (α3Ile57 and α3Thr115) help mediate the effects of the modulators but do not seem to contribute to specificity. Mutations in two others (α3Leu107 and α3Ile117) yielded receptors with appreciable α4-character; that is, Mor potentiation was reduced compared with wild-type α3ß2 control and Oxa inhibition was evident. A fifth position (α3Glu113) was unique in that it discriminated between the two compounds, showing no change in Mor potentiation from control but substantial Oxa inhibition. Our work has implications for rational drug design for nicotinic receptors and sheds light on mechanisms of allosteric modulation in nAChRs, especially the subtle differences between potentiation and inhibition.