148 research outputs found
Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure
Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member of the catalytic triad, was studied by site-directed mutagenesis. Mutation of Asp260 to asparagine resulted in a catalytically inactive D260N mutant, which demonstrates that the triad acid Asp260 is essential for dehalogenase activity. Furthermore, Asp260 has an important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor product could bind in the active-site cavity. Activity for brominated substrates was restored to D260N by replacing Asn148 with an aspartic or glutamic acid. Both double mutants D260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-fold higher Km values for 1,2-dibromoethane compared to the wild-type enzyme. Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in kcat was mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate. On the other hand, bromide was released 12-fold faster and via a different pathway than in the wild-type enzyme. Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charge distribution in the tunnel region that connects the active site with the solvent. On the basis of primary structure similarity between DhlA and other Ξ±/Ξ²-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.
Atomic layer deposition of high-k dielectric layers on Ge and III-V MOS channels
Ge and III-V semiconductors are potential high performance channel materials for future CMOS devices. In this work, we have studied At. Layer Deposition (ALD) of high-k dielec. layers on Ge and GaAs substrates. We focus at the effect of the oxidant (H2O, O3, O2, O2 plasma) during gate stack formation. GeO2, obtained by Ge oxidn. in O2 or O3, is a promising passivation layer. The germanium oxide thickness can be scaled down below 1 nm, but such thin layers contain Ge in oxidn. states lower than 4+. Still, elec. results indicate that small amts. of Ge in oxidn. states lower than 4+ are not detrimental for device performance. Partial intermixing was obsd. for high-k dielec. and GeO2 or GaAsOx, suggesting possible correlations in the ALD growth mechanisms on Ge and GaAs substrates. [on SciFinder (R)
Blood and Imaging Biomarkers in the Long-term Follow-up of Bicuspid Aortic Valve Patients
Background:Β Bicuspid aortic valve (BAV) is a common congenital heart defect. Patients with BAV are at risk for long-term complications such as valve stenosis and regurgitation. This study aimed to investigate sex differences in blood and imaging biomarkers and to describe the long-term prognostic value of blood and echocardiographic biomarkers.Β Methods:Patients were included from 2 prospective observational cohort studies; they underwent venous blood sampling and transthoracic echocardiography including speckle tracking. Analyzed blood biomarkers were red-cell distribution width (RDW), creatinine, C-reactive protein (CRP), troponin T, N-terminal pro B-type natriuretic peptide (NT-proBNP), and transforming growth factor-beta (TGF-Ξ²). Sex differences were analyzed at baseline. Associations between biomarkers and arrhythmia-free and intervention-free survival were determined by Cox regression, adjusted for age and sex.Β Results:A total of 182 patients with BAV were included: median age 34; interquartile range [IQR]: 23-46 years; 55.5% male. CRP, NT-proBNP, and RDW were higher in women, whereas creatinine, troponin T and TGF-Ξ² were higher among men. After a median follow-up time of 6.9 (IQR: 6.5-9.9) years, arrhythmia-free and intervention-free survival was, 81.0% and 73.1%, respectively. NT-proBNP was associated with both arrhythmia-free and intervention-free survival (hazard ratio [HR], 1.94, P = 0.005 and HR, 2.06, P = 0.002, respectively). On echocardiography higher left atrial (LA) size, left ventricular end-diastolic diameter (LVEDD), left ventricular (LV) mass index and E/eβ ratio were associated with lower arrhythmia-free survival, whereas higher LA size, LV mass index, aortic valve peak velocity, and aortic regurgitation were associated with lower intervention-free survival.Β Conclusions:Β Differences were observed in blood biomarkers between men and women with BAV. Besides LV systolic parameters, diastolic LV function and NT-proBNP should have a more prominent role as prognostic markers in clinical care.</p
Celiac disease: how complicated can it get?
In the small intestine of celiac disease patients, dietary wheat gluten and similar proteins in barley and rye trigger an inflammatory response. While strict adherence to a gluten-free diet induces full recovery in most patients, a small percentage of patients fail to recover. In a subset of these refractory celiac disease patients, an (aberrant) oligoclonal intraepithelial lymphocyte population develops into overt lymphoma. Celiac disease is strongly associated with HLA-DQ2 and/or HLA-DQ8, as both genotypes predispose for disease development. This association can be explained by the fact that gluten peptides can be presented in HLA-DQ2 and HLA-DQ8 molecules on antigen presenting cells. Gluten-specific CD4+ T cells in the lamina propria respond to these peptides, and this likely enhances cytotoxicity of intraepithelial lymphocytes against the intestinal epithelium. We propose a threshold model for the development of celiac disease, in which the efficiency of gluten presentation to CD4+ T cells determines the likelihood of developing celiac disease and its complications. Key factors that influence the efficiency of gluten presentation include: (1) the level of gluten intake, (2) the enzyme tissue transglutaminase 2 which modifies gluten into high affinity binding peptides for HLA-DQ2 and HLA-DQ8, (3) the HLA-DQ type, as HLA-DQ2 binds a wider range of gluten peptides than HLA-DQ8, (4) the gene dose of HLA-DQ2 and HLA-DQ8, and finally,(5) additional genetic polymorphisms that may influence T cell reactivity. This threshold model might also help to understand the development of refractory celiac disease and lymphoma
In Search of the Optimal Surgical Treatment for Velopharyngeal Dysfunction in 22q11.2 Deletion Syndrome: A Systematic Review
<div><h3>Background</h3><p>Patients with the 22q11.2 deletion syndrome (22qDS) and velopharyngeal dysfunction (VPD) tend to have residual VPD following surgery. This systematic review seeks to determine whether a particular surgical procedure results in superior speech outcome or less morbidity.</p> <h3>Methodology/ Principal Findings</h3><p>A combined computerized and hand-search yielded 70 studies, of which 27 were deemed relevant for this review, reporting on a total of 525 patients with 22qDS and VPD undergoing surgery for VPD. All studies were levels 2c or 4 evidence. The methodological quality of these studies was assessed using criteria based on the Cochrane Collaboration's tool for assessing risk of bias. Heterogeneous groups of patients were reported on in the studies. The surgical procedure was often tailored to findings on preoperative imaging. Overall, 50% of patients attained normal resonance, 48% attained normal nasal emissions scores, and 83% had understandable speech postoperatively. However, 5% became hyponasal, 1% had obstructive sleep apnea (OSA), and 17% required further surgery. There were no significant differences in speech outcome between patients who underwent a fat injection, Furlow or intravelar veloplasty, pharyngeal flap pharyngoplasty, Honig pharyngoplasty, or sphincter pharyngoplasty or Hynes procedures. There was a trend that a lower percentage of patients attained normal resonance after a fat injection or palatoplasty than after the more obstructive pharyngoplasties (11β18% versus 44β62%, pβ=β0.08). Only patients who underwent pharyngeal flaps or sphincter pharyngoplasties incurred OSA, yet this was not statistically significantly more often than after other procedures (pβ=β0.25). More patients who underwent a palatoplasty needed further surgery than those who underwent a pharyngoplasty (50% versus 7β13%, pβ=β0.03).</p> <h3>Conclusions/ Significance</h3><p>In the heterogeneous group of patients with 22qDS and VPD, a grade C recommendation can be made to minimize the morbidity of further surgery by choosing to perform a pharyngoplasty directly instead of only a palatoplasty.</p> </div
The Dutch Review Process for Evaluating the Quality of Psychological Tests: History, Procedure, and Results
The Impact of Tax and Expenditure Policies on Income Distribution: Evidence from a Large Panel of Countries
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