Efficacy of Different Root Canal Irrigants on Smear Layer Removal after Post Space Preparation: A Scanning Electron Microscopy Evaluation

Introduction: Effective durable adhesion between post material and dentine using resin cements is essential for longevity of restoration. The aim of this in vitro study was to compare the effect of different irrigants on smear layer removal after post space preparation. Methods and Materials: A total of 75 extracted anterior human teeth were selected. The canals were instrumented by rotary system and then were filled. After preparing the post space, teeth were divided into 5 groups according to irrigants: 17% EDTA; 17% EDTA+2% CHX; 5.25% NaOCl; 17% EDTA+5.25% NaOCl; and saline. The canals were irrigated with 5 cc of each irrigants for 1 min. Specimens were examined with scanning electron microscopy (SEM). Hulsmann’s score was used for marking of smear layer removal at coronal, middle and apical thirds of post space. The data were analyzed using the Kruskal-Wallis and Mann-Whitney U tests. Results: The results revealed that subsequent use of 17% EDTA+5.25% NaOCl was more effective than the other groups in smear layer removal. No statistical difference was found among different levels of root canal within each group. Conclusion: It can be concluded that 17% EDTA+5.25% NaOCl could be an effective irrigant for smear layer removal after post space preparation.Keywords: Post Space; Root Canal Irrigant; Scanning Electron Microscopy; Smear Laye

KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding

The functionalization of polymer surfaces by polymer-binding peptides offers tremendous opportunities for directed immobilization of enzymes, bioactive peptides, and antigens. The application of polymer-binding peptides as adhesion promoters requires reliable and stable binding under process conditions. Molecular modes of interactions between material surfaces, peptides, and solvent are often not understood to an extent that enables (semi-) rational design of polymer-binding peptides, hindering the full exploitation of their potential. Knowledge-gaining directed evolution (KnowVolution) is an efficient protein engineering strategy that facilitates tailoring protein properties to application demands through a combination of directed evolution and computational guided protein design. A single round of KnowVolution was performed to gain molecular insights into liquid chromatography peak I peptide, 47 aa (LCI)-binding to polypropylene (PP) in the presence of the competing surfactant Triton X-100. KnowVolution yielded a total of 8 key positions (D19, S27, Y29, D31, G35, I40, E42, and D45), which govern PP-binding in the presence of Triton X-100. The recombination of two of the identified amino acid substitutions (Y29R and G35R; variant KR-2) yielded a 5.4 ± 0.5-fold stronger PP-binding peptide compared to LCI WT in the presence of Triton X-100 (1 mM). The LCI variant KR-2 shows a maximum binding capacity of 8.8 ± 0.1 pmol/cm2 on PP in the presence of Triton X-100 (up to 1 mM). The KnowVolution approach enables the development of polymer-binding peptides, which efficiently coat and functionalize PP surfaces and withstand surfactant concentrations that are commonly used, such as in household detergents

An Analysis of the Cultural Landscape and Settlement Pattern of the Kashafrud Basin (Mashhad Plain) in the Iron Age

The Khorasan region, especially the Kashafrud basin, has culturally retained a strategic position by virtue of its location between three major cultural spheres of southwestern Central Asia, the Central Plateau of Iran, and northeastern Iran. The Iron Age still remains a mainly unknown period in this region. In northeast Iran, particularly in the Greater Khorasan, the period is characterized by cultural attributes utterly different from those of the other parts of the Iranian Plateau. Coeval archaeological evidence from Khorasan shows affinities with the Yaz and Dahistan cultures of Central Asia. The major topics considered in this study are: the distribution pattern and major influential factors in the formation of the Iron Age settlements in the Kashafrud basin (Mashhad Plain), the nature of Iron Age cultural material in the region, and the characteristics shared between the contemporary settlements in Mashhad and the adjacent regions. Further major research objectives include proposing a comparative chronology, analyzing the regional cultural landscape, and specifying the Iron Age settlement patterns in the region. Thus, attempts have been made to answer the research questions through ArcGIS maps, analysis of surface ceramics, and the landscape archaeology approach. The results indicate the impressive role of environmental factors, especially the Kashafrud River as the leading regional resource, in the advent of Iron Age settlements. Moreover, the regional material culture exhibits strong affinities with the Iron Age culture of “Yaz” in Central Asia, and indicates relations and population movements between the different regions that were under the influence of this culture

Investigation of intercritical heat treatment temperature effect on microstructure and mechanical properties of dual phase (DP) steel

In the present study, the effect of intercritical heat treatment temperature on the tensile properties and work hardening behavior of ferritic-martensitic dual-phase steel have been investigated utilizing tensile test, microhardness measurement and microscopic observation. Plain carbon steel sheet with a thickness of 2 mm was heat treated at 760, 780, 800, 820 and 840 °C intercritical temperatures. The results showed that martensite volume fraction (Vm) increases from 32 to 81%with increasing temperature from 760 to 840 °C. The mechanical properties of samples were examined by tensile and microhardness tests. The results revealed that yield strength was increased linearly with the increase in Vm, but the ultimate strength was increased up to 55% Vm and then decreased afterward. Analyzing the work hardening behavior in term of Hollomon equation showed that in samples with less than 55% Vm, the work hardening took place in one stage and the work hardening exponent increased with increasing Vm. More than one stage was observed in the work hardening behavior when Vm was increased. The results of microhardness test showed that microhardness of the martensite is decreased by increase in heat treatment temperature while the ferrite microhardness is nearly constant for all heat-treated samples

In Silico and Experimental ADAM17 Kinetic Modeling as Basis for Future Screening System for Modulators

Understanding the mechanisms of modulators’ action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regu lated mainly by a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM170 s broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New ‘exosite’ (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modula tors as therapeutic drugs. By combining experimental and in silico methods (structural modeling and docking), we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibi tion mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity

Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-β-glucanase

Cellulases are industrially important enzymes, e.g., in the production of bioethanol, in pulp and paper industry, feedstock, and textile. Thermostability is often a prerequisite for high process stability and improving thermostability without affecting specific activities at lower temperatures is challenging and often time-consuming. Protein engineering strategies that combine experimental and computational are emerging in order to reduce experimental screening efforts and speed up enzyme engineering campaigns. Constraint Network Analysis (CNA) is a promising computational method that identifies beneficial positions in enzymes to improve thermostability. In this study, we compare CNA and directed evolution in the identification of beneficial positions in order to evaluate the potential of CNA in protein engineering campaigns (e.g., in the identification phase of KnowVolution). We engineered the industrially relevant endoglucanase EGLII from Penicillium verruculosum towards increased thermostability. From the CNA approach, six variants were obtained with an up to 2-fold improvement in thermostability. The overall experimental burden was reduced to 40% utilizing the CNA method in comparison to directed evolution. On a variant level, the success rate was similar for both strategies, with 0.27% and 0.18% improved variants in the epPCR and CNA-guided library, respectively. In essence, CNA is an effective method for identification of positions that improve thermostability

How to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study

Expanding synthetic capabilities to routinely employ enzymes in organic solvents (OSs) is a dream for protein engineers and synthetic chemists. Despite significant advances in the field of protein engineering, general and transferable design principles to improve the OS resistance of enzymes are poorly understood. Herein, we report a combined computational and directed evolution study of Bacillus subtlis lipase A (BSLA) in three OSs (i. e., 1,4-dioxane, dimethyl sulfoxide, 2,2,2-trifluoroethanol) to devise a rational strategy to guide engineering OS resistant enzymes. Molecular dynamics simulations showed that OSs reduce BSLA activity and resistance in OSs by (i) stripping off essential water molecules from the BLSA surface mainly through H-bonds binding; and (ii) penetrating the substrate binding cleft leading to inhibition and conformational change. Interestingly, integration of computational results with “BSLA-SSM” variant library (3439 variants; all natural diversity with amino acid exchange) revealed two complementary rational design strategies: (i) surface charge engineering, and (ii) substrate binding cleft engineering. These strategies are most likely applicable to stabilize other lipases and enzymes and assist experimentalists to design organic solvent resistant enzymes with reduced time and screening effort in lab experiments. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGa

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles

The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min−1; R255L: 1168 ± 163 min−1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation

Disulfide-induced self-assembled targets:A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol-modified probes, each of which specifically binds at one half of the target introduced SH groups at both ends of dsDNA. Continuous disulfide bond formation at 3' and 5' terminals of targets leads to the self-assembly of dsDNAs into the sulfur-rich and flexible products with different lengths. These products have a high affinity for the surface of Au-NPs and efficiently protect the surface from salt induced aggregation. To evaluate the assay efficacy, a small part of the citrus tristeza virus (CTV) genome was targeted, leading to a detection limit of about 5 x 10(-9) mol. L-1 over a linear ranged from 20 x 10(-9) to 10 x 10(-7) mol. L-1. This approach also exhibits good reproducibility and recovery levels in the presence of plant total RNA or human plasma total circulating RNA extracts. Self-assembled targets can be then sensitively distinguished from non-assembled or mismatched targets after gel electrophoresis. The disulfide reaction method and integrating self-assembled DNAs/RNAs targets with bare AuNPs as a sensitive indicator provide us a powerful and simple visual detection tool for a wide range of applications

Rational reprogramming of the sesquiterpene synthase BcBOT2 yields new terpenes with presilphiperfolane skeleton

Computer-aided rational design of the substrate binding pocket of sesquiterpene synthases BcBOT2 from Botrytis cinerea yielded FPP cyclization products with presilphiperfolane backbone other than the naturally formed sesquiterpene presilphiperfolan-8β-ol. Particularly, amino acids W118 and F138 were found to strongly control the stability and conformation of key cationic intermediates. The W118Q variant forms only presilphiperfolan-9β-ol, whereas the exchange of amino acids at position 138, such as F138V, has a fundamental effect on the course of the cationic cascade. Here, the 1,3-hydride shift en route to presilphiperfolan-8β-ol is suppressed and substituted by a so far unknown 1,2-hydride shift that leads to presilphiperfol-1-ene and presilphiperfolan-1α-ol along with β-caryophyllene and the so far unknown caryophyllene-8β-ol