Exhaustive mutagenesis of six secondary active-site residues in Escherichia coli chorismate mutase shows the importance of hydrophobic side chains and a helix N-capping position for stability and catalysis

Secondary active-site residues in enzymes, including hydrophobic amino acids, may contribute to catalysis through critical interactions that position the reacting molecule, organize hydrogen-bonding residues, and define the electrostatic environment of the active site. To ascertain the tolerance of an important model enzyme to mutation of active-site residues that do not directly hydrogen bond with the reacting molecule, all 19 possible amino acid substitutions were investigated in six positions of the engineered chorismate mutase domain of the Escherichia coli chorismate mutase-prephenate dehydratase. The six secondary active-site residues were selected to clarify results of a previous test of computational enzyme design procedures. Five of the positions encode hydrophobic side chains in the wild-type enzyme, and one forms a helix N-capping interaction as well as a salt bridge with a catalytically essential residue. Each mutant was evaluated for its ability to complement an auxotrophic chorismate mutase deletion strain. Kinetic parameters and thermal stabilities were measured for variants with in vivo activity. Altogether, we find that the enzyme tolerated 34% of the 114 possible substitutions, with a few mutations leading to increases in the catalytic efficiency of the enzyme. The results show the importance of secondary amino acid residues in determining enzymatic activity, and they point to strengths and weaknesses in current computational enzyme design procedures

Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity

Computational protein design methods were used to predict five variants of monofunctional Escherichia coli chorismate mutase expected to maintain catalytic activity. The variants were tested experimentally and three active site mutants exhibited catalytic activity similar to or greater than the wild-type enzyme. One mutant, Ala32Ser, showed increased catalytic efficiency

Evaluation of bis-GMA/MMA resin adhesion to silica-coated and silanized titanium

The effects of pH value and alcohol solvent type of a silane solution on the bonding of an experimental resin to the silica-coated titanium (Ti) surface were studied. First, Ti surfaces underwent tribochemical Rocatec ™ treatment followed by silanization of the surface with 3-methacryloxypropyltrimethoxysilane (MPS). Then, resin stubs based on a mixture of bisphenol-A-glycidyl dimethacrylate and methyl methacrylate were bonded and light-cured onto each silica-coated Ti surface (n = 6 per group). Two different solvents for MPS, namely iso-propanol (i-PrOH)/H2O and ethanol (EtOH)/H2O were used, at pH values of 4.5, 5.0, and 5.5, and shear bond strengths were tested both under dry storage conditions and after water sorption induced by accelerated aging (i.e. thermo-cycling). The shear bond strengths were also re-determined after the silane solutions had been stored at 4°C for 15 weeks before the silanization step. For dry samples, the shear bond strengths ranged from 7.5 to 10.6 MPa (ANOVA, p < 0.05) when the Ti surface had been silanized with MPS in i-PrOH/H2O, and from 6.5 to 12.4 MPa (ANOVA, p < 0.05) when the Ti surface had been silanized with MPS in EtOH/H2O at pH 4.5. Fifteen weeks of storage of the silane solution increased the shear bond strength of dry samples by ca. 1-4 MPa per test group. In contrast, thermo-cycling reduced the shear bond strength in both solvent systems. The weight of the test sample stubs increased by ca. 3.5 wt% after 187 days of being subjected to the water sorption test. © 2009 VSP.postprin

Accurate solution of Bayesian inverse uncertainty quantification problems combining reduced basis methods and reduction error models

Computational inverse problems related to partial differential equations (PDEs) often contain nuisance parameters that cannot be effectively identified but still need to be considered as part of the problem. The objective of this work is to show how to take advantage of a reduced order framework to speed up Bayesian inversion on the identifiable parameters of the system, while marginalizing away the (potentially large number of) nuisance parameters. The key ingredients are twofold. On the one hand, we rely on a reduced basis (RB) method, equipped with computable a posteriori error bounds, to speed up the solution of the forward problem. On the other hand, we develop suitable reduction error models (REMs) to quantify in an inexpensive way the error between the full-order and the reduced-order approximation of the forward problem, in order to gauge the effect of this error on the posterior distribution of the identifiable parameters. Numerical results dealing with inverse problems governed by elliptic PDEs in the case of both scalar parameters and parametric fields highlight the combined role played by RB accuracy and REM effectivity

The effect of chlorhexidine and dimethyl sulfoxide on long-term sealing ability of two calcium silicate cements in root canal

Objectives. To evaluate the long-term effect of chlorhexidine (CHX) and dimethyl sulfoxide (DMSO) on the sealing ability and biomineralization of two different calcium silicate cements (CSC) in root canal. Methods. Sixty human third molar root canals were obturated with ProRoot MTA or Biodentine. Before obturation the canals were irrigated with saline (control), 2% CHX or 5% DMSO. Microleakage was tested after three days and after six months. After additional six months (12 months after root filling) the roots were cut into 2 mm thick dentine discs. The discs were stored in artificial saliva for one year. The bond strength was measured with the push-out method, and the failure mode was evaluated with a stereomicroscope. The most apical disc of each tooth was used for Vickers hardness test. Results. No significant differences between the groups was found in initial microleakage. The leakage increased significantly during the 6-month storage in all groups except in Biodentine-CHX group and Biodentine-DMSO group. CHX and DMSO irrigation significantly increased the leakage with ProRoot MTA with time, but there was no statistically significant difference compared to the ProRoot MTA-control group at six months' time point. CHX significantly reduced the push-out bond strength of ProRoot MTA. With Biodentine irrigation with CHX or DMSO resulted with significantly higher push-out strength compared to the Biodentine control group. Fracture analysis showed statistically significant difference in the distribution of the fractures between the groups, but neither CHX nor DMSO change the fracture pattern statistically significantly. With Vickers hardness test ProRoot MTA with and without DMSO as the final irrigant showed significantly higher dentin hardness than any Biodentine-group. Significance. Considering that aging increased the leakage in all groups except with Biodentine-DMSO and the differences in the push-out strength and surface microhardness data, it appears that the time-related biomineralizing effect of MTA and Biodentine does not improve sealing to dentin. CHX significantly reduced ProRoot MTA bond strength and increased pure adhesive failures with both cements. (C) 2020 Published by Elsevier Inc. on behalf of The Academy of Dental Materials.Peer reviewe

The effect of chlorhexidine and dimethyl sulfoxide on long-term sealing ability of two calcium silicate cements in root canal

Objectives. To evaluate the long-term effect of chlorhexidine (CHX) and dimethyl sulfoxide (DMSO) on the sealing ability and biomineralization of two different calcium silicate cements (CSC) in root canal. Methods. Sixty human third molar root canals were obturated with ProRoot MTA or Biodentine. Before obturation the canals were irrigated with saline (control), 2% CHX or 5% DMSO. Microleakage was tested after three days and after six months. After additional six months (12 months after root filling) the roots were cut into 2 mm thick dentine discs. The discs were stored in artificial saliva for one year. The bond strength was measured with the push-out method, and the failure mode was evaluated with a stereomicroscope. The most apical disc of each tooth was used for Vickers hardness test. Results. No significant differences between the groups was found in initial microleakage. The leakage increased significantly during the 6-month storage in all groups except in Biodentine-CHX group and Biodentine-DMSO group. CHX and DMSO irrigation significantly increased the leakage with ProRoot MTA with time, but there was no statistically significant difference compared to the ProRoot MTA-control group at six months' time point. CHX significantly reduced the push-out bond strength of ProRoot MTA. With Biodentine irrigation with CHX or DMSO resulted with significantly higher push-out strength compared to the Biodentine control group. Fracture analysis showed statistically significant difference in the distribution of the fractures between the groups, but neither CHX nor DMSO change the fracture pattern statistically significantly. With Vickers hardness test ProRoot MTA with and without DMSO as the final irrigant showed significantly higher dentin hardness than any Biodentine-group. Significance. Considering that aging increased the leakage in all groups except with Biodentine-DMSO and the differences in the push-out strength and surface microhardness data, it appears that the time-related biomineralizing effect of MTA and Biodentine does not improve sealing to dentin. CHX significantly reduced ProRoot MTA bond strength and increased pure adhesive failures with both cements. (C) 2020 Published by Elsevier Inc. on behalf of The Academy of Dental Materials.Peer reviewe

Cellulose Fibre-Reinforced Biofoam for Structural Applications

Traditionally, polymers and macromolecular components used in the foam industry are mostly derived from petroleum. The current transition to a bio-economy creates demand for the use of more renewable feedstocks. Soybean oil is a vegetable oil, composed mainly of triglycerides, that is suitable material for foam production. In this study, acrylated epoxidized soybean oil and variable amounts of cellulose fibres were used in the production of bio-based foam. The developed macroporous bio-based architectures were characterised by several techniques, including porosity measurements, nanoindentation testing, scanning electron microscopy, and thermogravimetric analysis. It was found that the introduction of cellulose fibres during the foaming process was necessary to create the three-dimensional polymer foams. Using cellulose fibres has potential as a foam stabiliser because it obstructs the drainage of liquid from the film region in these gas-oil interfaces while simultaneously acting as a reinforcing agent in the polymer foam. The resulting foams possessed a porosity of approximately 56%, and the incorporation of cellulose fibres did not affect thermal behaviour. Scanning electron micrographs showed randomly oriented pores with irregular shapes and non-uniform pore size throughout the samples

Dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic with shear-dependent effects on platelet-collagen binding and thrombin generation

Heparin proteoglycans (HEP-PGs) carry standard heparin-mediated anticoagulant properties as well as novel antiplatelet functions, a combination that may be significant for targeting multiple pathways in a single therapy. Recent work developing semisynthetic HEP-PG mimetics has shown promising results also in vivo, however flow conditions in vitro that replicate in vivo hemodynamics have not been reported. In this work, we present several assays (platelet calcium mobilization, aggregometry, microfluidic tests at venous and arterial hemodynamics) to characterize specific mechanistic effects of dual antiplatelet and anticoagulant (APAC) constructs as mimetics of HEP-PGs. Three APACs with different conjugation levels of heparin chains (CL10, CL18, HICL) were shown to decrease platelet deposition to collagen surfaces in PPACK-treated whole blood at venous shear rate (200 s(-1)). FXIIa-inhibited whole blood (CTI: corn trypsin inhibitor, 40 mu g/mL) perfused over collagen/tissue factor showed reduced both platelet and fibrin deposition when treated with APACs. IC50 values for platelet and fibrin inhibition were calculated for each molecule at venous shear rate. Increasing the shear rate to arterial flows (1000 s(-1)) and using APAC as the sole anticoagulant, resulted in a more potent antiplatelet effect of APAC, suggesting an added effect on von Willebrand Factor (vWF) function. Additionally, APAC caused an inhibition of calcium mobilization specific to thrombin and collagen stimulation and a dose-dependent reduction in collagen-mediated platelet aggregation. Understanding the sensitivity of APAC activity to shear rate, platelet signaling and procoagulant pathways is important for applications in which APAC administration may have beneficial therapeutic effects.Peer reviewe

Experimental and Theoretical Results for Weak Charge Current Backward Proton Production

In this paper, we do three things in the study of deuteron break-up by high energy neutrino beams. (1) We present previously unpublished data on neutrino induced backward protons from deuteron targets; (2) we calculate the contributions from both the two-nucleon (2N) and six-quark (6q) deuteron components, which depend upon the overall normalization of the part that is 6q; and (3) we suggest other signatures for distinguishing the 2N and 6q clusters. We conclude that the 6q cluster easily explains the shape of the high momentum backward proton spectrum, and its size is nicely explained if the amount of 6q is one or a few percent by normalization of the deuteron. There is a crossover, above which the 6q contribution is important or dominant, at 300--400 MeV/c backward proton momentum.Comment: 8 pages, 5 figure

Load-bearing capacity of Fibre-reinforced Fixed Dental Prostheses with CAD-CAM Pontic

Poster Presentation: 49. Late-breaking News - Dental Materials II: no. 393OBJECTIVES: The aim of this study was to evaluate the load-bearing capacity of three-unit fiber reinforced composite (FRC) fixed dental prostheses (FDP) with three different types of pontics. METHODS: Inlay preparations for retaining FDP were made to lower second premolar and second molar of a phantom model. We aimed to replace the lower left first molar using a pontic. Twenty four FDPs with fiber-reinforced composite frameworks (everStick, StickTech-GC) were fabricated. Two continuous unidirectional fiber reinforcements were attached with flowable composite (Stick®FLOW), and composite (G-ӕnial, GC), between inlays of the abutment. One inlay preparation model with the FRC framework was scanned by CEREC. An artificial tooth of a denture was scanned by CEREC to multiply its form to ...postprin