Anion- and Water-facilitated Oxidative Carbon-Carbon Bond Cleavage and Diketonate Carboxylation in Cu(II) Chlorodiketonate Complexes.

The O2-dependent carbon-carbon (C-C) bond cleavage reactions of the mononuclear Cu(II) chlorodiketonate complexes [(6-Ph2TPA)Cu(PhC(O)CClC(O)Ph)]ClO4 (1-ClO 4 ) and [(bpy)Cu(PhC(O)CClC(O)Ph)(ClO4)] (3-ClO 4 ) have been further examined in terms of their anion and water dependence. The bpy-ligated Cu(II) chlorodiketonate complex 3-ClO 4 is inherently more reactive with O2 than the 6-Ph2TPA-ligated analog 1-ClO 4 . Added chloride is needed to facilitate O2 reactivity for 1-ClO 4 but not for 3-ClO 4 at 25(1) °C. Evaluation of k obs for the reaction of 1-ClO 4 with O2 under pseudo first-order conditions as a function of the amount of added chloride ion produced saturation type behavior. The bpy-ligated 3-ClO 4 exhibits different behavior, with rate enhancement resulting from both the addition of chloride ion and water. Computational studies indicate that the presence of water lowers the barrier for O2 activation for 3-ClO 4 by ~12 kcal/mol whereas changing the anion from perchlorate to chloride has a smaller effect (lowering of the barrier by ~3 kcal/mol). Notably, the effect of water for 3-ClO 4 is of similar magnitude to the barrier-lowering chloride effect found in the O2 activation pathway for 1-ClO 4 . Thus, both systems involve lower energy O2 activation pathways available, albeit resulting from different ligand effects. Probing the effect of added benzoate anion, it was found that the chloro substituent in the diketonate moiety of 1-ClO 4 and 3-ClO 4 will undergo displacement upon treatment of each complex with tetrabutyl ammonium benzoate to give Cu(II) benzoyloxydiketonate complexes (4 and 5). Complexes 4 and 5 exhibit slow O2-dependent C-C cleavage in the presence of added chloride ion. These results are discussed in the context of the chemistry identified for various divalent metal chlorodiketonate complexes, which have relevance to catalytic systems and metalloenzymes that mediate O2-dependent C-C cleavage within diketonate substrates.

The Effect of Nitriding Temperature of AISI 316L Steel on Sub-Zero Corrosion Resistance in C2H5OH.

In this paper, glow nitriding processes at cathode potential are used at various temperatures to investigate how they affect the corrosion resistance of 316L steel in ethanol at temperatures of 22 °C and -30 °C. Lowering the test temperature reduces the corrosion rate of the nitrided layers. Conversely, glow nitriding at 450 °C improves the corrosion resistance of the tested steel. Increasing the nitriding temperature to 520 °C increases the corrosion rate. It should be noted that the ethyl alcohol solution, due to the lack of aggressive ions, does not cause significant changes in the corrosion rate of the steel. The value of the corrosion current varies in the range of 10-2-10-3 µA/cm2. Nitrided layers increase the contact angle measured for water and are entirely wettable for ethanol. The objective of this study is to evaluate the effect of the nitriding temperature of AISI 316L steel on its corrosion resistance in an ethanol solution at room temperature and at -30 °C.

Structure and Properties of Bioactive Titanium Dioxide Surface Layers Produced on NiTi Shape Memory Alloy in Low-Temperature Plasma.

BACKGROUND: The NiTi alloy, known for its shape memory and superelasticity, is increasingly used in medicine. However, its high nickel content requires enhanced biocompatibility for long-term implants. Low-temperature plasma treatments under glow-discharge conditions can improve surface properties without compromising mechanical integrity. METHODS: This study explores the surface modification of a NiTi alloy by oxidizing it in low-temperature plasma. We examine the impact of process temperatures and sample preparation (mechanical grinding and polishing) on the structure of the produced titanium oxide layers. Surface properties, including topography, morphology, chemical composition, and bioactivity, were analyzed using TEM, SEM, EDS, and an optical profilometer. Bioactivity was assessed through the deposition of calcium phosphate in simulated body fluid (SBF). RESULTS: The low-temperature plasma oxidization produced titanium dioxide layers (29-55 nm thick) with a predominantly nanocrystalline rutile structure. Layer thickness increased with extended processing time and higher temperatures (up to 390 °C), though the relationship was not linear. Higher temperatures led to thicker layers with more precipitates and inhomogeneities. The oxidized layers showed increased bioactivity after 14 and 30 days in SBF. CONCLUSIONS: Low-temperature plasma oxidation produces bioactive titanium oxide layers on NiTi alloys, with a structure and properties that can be tuned through process parameters. This method could enhance the biocompatibility of NiTi alloys for medical implants.

Surface Modification of Zr48Cu36Al8Ag8 Bulk Metallic Glass through Glow Discharge Plasma Nitriding.

Bulk metallic glasses are modern engineering materials with unique functional properties. Zr-based alloys are particularly attractive as they exhibit high glass forming ability as well as good mechanical properties. Due to their relatively high thermal stability, reaching as much as 400 °C, they can be surface-treated in low-temperature plasma to further improve their mechanical properties. The subject of this study was to determine the influence of the technological parameters of nitriding in low-temperature plasma on the structure and mechanical properties of Zr48Cu36Al8Ag8 bulk metallic glass. In the course of this study, the influence of the ion accelerating voltage on the structure and micromechanical properties of the bulk metallic glass was analyzed. The produced samples were characterized in terms of nanohardness, layer adhesion by using the scratch test, and wear resistance by using the ball-on-disc method. As a result of low-temperature plasma nitriding, a significant increase in the surface nanohardness of the Zr48Cu36Al8Ag8 bulk metallic glass was obtained. The produced layers exhibited high adhesion to the substrate and they improved the wear resistance of the glass. The present study indicates the possibility of modifying the surface properties of bulk metallic glasses by using diffusion processes in low-temperature plasma without substrate crystallization.

Reaction Mechanism Catalyzed by the Dissimilatory Sulfite Reductase - The Role of the Siroheme-[4FeS4] Cofactor.

The present work is another part of our investigation on the pathway of dissimilatory sulfate reduction and covers a theoretical study on the reaction catalyzed by dissimilatory sulfite reductase (dSIR). dSIR is the terminal enzyme involved in this metabolic pathway, which uses the siroheme-[4Fe4S] cofactor for six-electron reduction of sulfite to sulfide. In this study we use a large cluster model containing siroheme-[4Fe4S] cofactor and protein residues involved in the direct interactions with the substrate, to get insight into the most feasible reaction mechanism and to understand the role of each considered active site component. In combination with earlier studies reported in the literature, our results lead to several interesting insights. One of the most important conclusions is that the reaction mechanism consists of three steps of two-electron reduction of sulfur and the probable role of the siroheme-[4Fe4S] cofactor is to ensure the delivery of packages of two electrons to the reactant.

Bacteria at Work - Experimental and Theoretical Studies Reveal the Catalytic Mechanism of Ectoine Synthase.

Ectoine synthase (EctC) catalyses the ultimate step of ectoine biosynthesis, a kosmotropic compound produced as compatible solute by many bacteria and some archaea or eukaryotes. EctC is an Fe2+-dependent homodimeric cytoplasmic protein. Using Mössbauer spectroscopy, molecular dynamics simulations and QM/MM calculations, we determined the most likely coordination number and geometry of the Fe2+ ion and proposed a mechanism of the EctC-catalysed reaction. Most notably, we show that apart from the three amino acids binding to the iron ion (Glu57, Tyr84 and His92), one water molecule and one hydroxide ion are required as additional ligands for the reaction to occur. They fill the first coordination sphere of the Fe2+-cofactor and act as critical proton donors and acceptors during the cyclization reaction.

Structural and functional studies reveal the molecular basis of substrate promiscuity of a glycosyltransferase originating from a major agricultural pest.

The two-spotted spider mite, Tetranychus urticae, is a major cosmopolitan pest that feeds on more than 1100 plant species. Its genome contains an unprecedentedly large number of genes involved in detoxifying and transporting xenobiotics, including 80 genes that code for UDP glycosyltransferases (UGTs). These enzymes were acquired via horizontal gene transfer from bacteria after loss in the Chelicerata lineage. UGTs are well-known for their role in phase II metabolism; however, their contribution to host adaptation and acaricide resistance in arthropods, such as T. urticae, is not yet resolved. TuUGT202A2 (Tetur22g00270) has been linked to the ability of this pest to adapt to tomato plants. Moreover, it was shown that this enzyme can glycosylate a wide range of flavonoids. To understand this relationship at the molecular level, structural, functional, and computational studies were performed. Structural studies provided specific snapshots of the enzyme in different catalytically relevant stages. The crystal structure of TuUGT202A2 in complex with UDP-glucose was obtained and site-directed mutagenesis paired with molecular dynamic simulations revealed a novel lid-like mechanism involved in the binding of the activated sugar donor. Two additional TuUGT202A2 crystal complexes, UDP-(S)-naringenin and UDP-naringin, demonstrated that this enzyme has a highly plastic and open-ended acceptor-binding site. Overall, this work reveals the molecular basis of substrate promiscuity of TuUGT202A2 and provides novel insights into the structural mechanism of UGTs catalysis.

Influence of Magnetron Sputtering-Deposited Niobium Nitride Coating and Its Thermal Oxidation on the Properties of AISI 316L Steel in Terms of Its Medical Applications.

An NbN coating was produced on AISI 316L steel using reactive DC magnetron sputtering. The effects of oxidation of the NbN coating in air on the microstructure, mechanical properties, corrosion resistance, contact angle and bioactivity were investigated. Phase composition was determined using X-ray diffraction (XRD), the coatings' cross-sectional microstructure and thickness including surface morphology using a scanning electron microscope (SEM), microhardness via the Vickers method, corrosion by means of a potentiodynamic polarisation test in Ringer's solution and bioactivity by observation in an SBF solution, while the contact angle was studied using a goniometer. The NbN coating and the oxidised coating were shown to demonstrate a Ca/P ratio close to that of hydroxyapatite, as well as increased microhardness and corrosion resistance. The best combination of mechanical, corrosion, bioactivity and hydrophilic properties was demonstrated by the air oxidised NbN coating, which featured an orthorhombic Nb2O5 structure in the top, surface layer.

Influence of Low Temperature Plasma Oxidizing on the Bioactivity of NiTi Shape Memory Alloy for Medical Applications.

The present study elucidates the impact of glow discharge oxidation within a low-temperature plasma environment on the bioactivity characteristics of an NiTi shape memory alloy. The properties of the produced surface layers, such as structure (TEM observations), surface morphology (SEM observations), chemical and phase composition (EDS and XRD measurements), wettability (optical gonimeter), and the biological response of osteoblasts and platelets to the oxidized surface compared with the NiTi alloy without a surface layer are presented. The presented surface modification of the NiTi shape memory alloy, achieved through oxidizing in a low-temperature plasma environment, led to the creation of a continuous surface layer composed of nanocrystalline titanium oxide TiO2 (rutile). The findings obtained from this study provide evidence that the oxidized layer augments the bioactivity of the shape memory alloy. This augmentation was substantiated through the spontaneous biomimetic deposition of apatite from a simulated body fluid (SBF) solution. Furthermore, the modified surface exhibited improved osteoblast proliferation, and enhanced platelet adhesion and activation. This proposed surface modification strategy holds promise as a prospective solution to enhance the biocompatibility and bioactivity of NiTi shape memory alloy intended for prolonged use in bone implant applications.

Experimental and Computational Studies Reveal Novel Interaction of Lymphocytes Antigen 6K to TGF-ß Receptor Complex.

TGF-ß signaling promotes migration, invasion, and distant colonization of cancer cells in advanced metastatic cancers. TGF-ß signaling suppresses the anti-tumor immune response in a tumor microenvironment, allowing sustained tumor growth. TGF-ß plays an important role in normal physiology; thus it is no surprise that the clinical development of effective and safe TGF-ß inhibitors has been hampered due to their high toxicity. We discovered that increased expression of LY6K in cancer cells led to increased TGF-ß signaling and that inhibition of LY6K could lead to reduced TGF-ß signaling and reduced in vivo tumor growth. LY6K is a highly cancer-specific protein, and it is not expressed in normal organs except in the testes. Thus, LY6K is a valid target for developing therapeutic strategies to inhibit TGF-ß signaling in cancer cells. We employed in vitro pull-down assays and molecular dynamics simulations to understand the structural determinants of the TGF-ß receptor complex with LY6K. This combined approach allowed us to identify the critical residues and dynamics of the LY6K interaction with the TGF-ß receptor complex. These data are critical in designing novel drugs for the inhibition of TGF-ß in LY6K expressing cancer, induction of anti-tumor immune response, and inhibition of tumor growth and metastatic spread.

Abrasive Wear Resistance of Ultrafine Ausferritic Ductile Iron Intended for the Manufacture of Gears for Mining Machinery.

The purpose of this study was to experimentally determine the abrasion wear properties of ausferritic ductile iron austempered at 250 °C in order to obtain cast iron of class EN-GJS-1400-1. It has been found that such a cast iron grade makes it possible to create structures for material conveyors used for short-distance transport purposes, required to perform in terms of abrasion resistance under extreme conditions. The wear tests addressed in the paper were conducted at a ring-on-ring type of test rig. The test samples were examined under the conditions of slide mating, where the main destructive process was surface microcutting via loose corundum grains. The mass loss of the examined samples was measured as a parameter characteristic of the wear. The volume loss values thus obtained were plotted as a function of initial hardness. Based on these results, it has been found that prolonged heat treatment (of more than 6 h) causes only an insignificant increase in the resistance to abrasive wear.

Inactivation and aggregation of R-specific 1-(4-hydroxyphenyl)-ethanol dehydrogenase from Aromatoleum aromaticum.

R-specific 1-(4-hydroxyphenyl)-ethanol dehydrogenase (R-HPED) is a promising biotool for stereoselective synthesis of chiral aromatic alcohols. This work focused on the evaluation of its stability under storage and in-process conditions in the pH range from 5.5 to 8.5. The relationship between the dynamics of aggregation and activity loss under various pH conditions and in the presence of glucose, serving as a stabilizer, was analysed using spectrophotometric techniques and dynamic light scattering. pH 8.5 was indicated as a representative environment in which the enzyme, despite relatively low activity, shows high stability and the highest total product yield. Based on a series of inactivation experiments, the mechanism of thermal inactivation at pH 8.5 was modelled. The irreversible first-order mechanism of R-HPED inactivation in the temperature range of 47.5-60 °C was verified by isothermal and multi-temperature evaluation of data, confirming that in the alkaline pH 8.5, R-HPED aggregation is the secondary process occurring at already inactivated protein molecules. The rate constants were from 0.029 min-1 to 0.380 min-1 for a buffer solution but they decreased to 0.011 min-1 and 0.161 min-1, respectively, when 1.5 M glucose was added as a stabilizer. The activation energy was however about 200 kJ mol-1 in both cases.

Lymphocyte antigen 6K signaling to aurora kinase promotes advancement of the cell cycle and the growth of cancer cells, which is inhibited by LY6K-NSC243928 interaction.

Lymphocyte antigen 6K (LY6K) is a small GPI-linked protein that is normally expressed in testes. Increased expression of LY6K is significantly associated with poor survival outcomes in many solid cancers, including cancers of the breast, ovary, gastrointestinal tract, head and neck, brain, bladder, and lung. LY6K is required for ERK-AKT and TGF-ß pathways in cancer cells and is required for in vivo tumor growth. In this report, we describe a novel role for LY6K in mitosis and cytokinesis through aurora B kinase and its substrate histone H3 signaling axis. Further, we describe the structural basis of the molecular interaction of small molecule NSC243928 with LY6K protein and the disruption of LY6K-aurora B signaling in cell cycle progression due to LY6K-NSC243928 interaction. Overall, disruption of LY6K function via NSC243928 led to failed cytokinesis, multinucleated cells, DNA damage, senescence, and apoptosis of cancer cells. LY6K is not required for vital organ function, thus inhibition of LY6K signaling is an ideal therapeutic approach for hard-to-treat cancers that lack targeted therapy such as triple-negative breast cancer.

Development of fluorophore labeled or biotinylated anticancer small molecule NSC243928.

Small molecule NSC243928 binds with LY6K, a potential target for the treatment of triple-negative breast cancer, and induces cancer cell death with an unclear mechanism. We have developed chemical tools to identify the molecular mechanisms of NSC243928-LY6K interaction. Herein, we report on the development and synthesis of biotinylated and fluorophore-tethered derivatives of NSC243928 guided by docking studies and molecular dynamics. Surface plasmon resonance assay indicates that these derivatives retained a direct binding with LY6K protein. Confocal analysis revealed that nitrobenzoxadiazole (NBD) fluorophore tagged NSC243928 is retained in LY6K expressing cancer cells. These novel modified compounds will be employed in future in vitro and in vivo studies to understand the molecular mechanisms of NSC243928 mediated cancer cell death. These studies will pave the path for developing novel targeted therapeutics and understanding any potential side-effects of these treatments for hard-to-treat cancers such as triple-negative breast cancer or other cancers with high expression of LY6K.

Effect of pine essential oil and rotating magnetic field on antimicrobial performance.

This work presents the results ofa study which concerns the influence of rotating magnetic field (RMF) on the antibacterial performance of commercial pine essential oil. A suspension of essential oil in saline solution and Escherichia coli were exposed to the rotating magnetic Afield (the frequency of electrical current supplied by a RMF generator f = 1-50 Hz; the averaged values of magnetic induction in the cross-section of the RMF generator B = 13.13 to - 19.92 mT, time of exposure t = 160 min, temperature of incubation 37 °C). The chemical composition of pine (Pinus sylvestris L.) essential oil was determined by gas chromatography coupled with mass spectrometry (GC-MS). The main constituents were α-pinene (28.58%), ß-pinene (17.79%), δ-3-carene (14.17%) and limonene (11.58%). The present study indicates the exposition to the RMF, as compared to the unexposed controls causing an increase in the efficacy of antibacterial properties of pine oil. We have shown that rotating magnetic fields (RMF) at a frequency, f, between 25 Hz to and 50 Hz increased the antimicrobial efficiency of oil a concentration lower than 50%.

Mechanistic studies of visible light-induced CO release from a 3-hydroxybenzo[g]quinolone.

Organic compounds that can be triggered using light to release CO in biological environments are of significant current interest to probe the role of CO in biology and as potential therapeutics. We recently reported that a 3-hydroxybenzo[g]quinolone (5) can be used as a CO delivery molecule to produce anticancer and potent anti-inflammatory effects. Herein we report mechanistic studies of the visible light-induced CO release reaction of this compound. In wet CH3CN under aerobic conditions, 5 releases 0.90(2) equivalents of CO upon illumination with visible light (419 nm) to give a single depside product. Performing the same reaction under an 18O2 atmosphere results in quantitative incorporation of two labeled oxygen atoms in the depside product. Monitoring via 1H NMR and UV-vis during the illumination of 5 in CH3CN using 419 nm light revealed the substoichiometric formation of a diketone (6) in the reaction mixture. H2O2 formation was detected in the same reaction mixtures. DFT studies indicate that upon light absorption an efficient pathway exists for the formation of a triplet excited state species (5b) that can undergo reaction with 3O2 resulting in CO release. DFT investigations also provide insight into diketone (6) and H2O2 formation and subsequent reactivity. The presence of water and exposure to visible light play an important role in lowering activation barriers in the reaction between 6 and H2O2 to give CO. Overall, two reaction pathways have been identified for CO release from a 3-hydroxybenzo[g]quinolone.

Properties of the Reactants and Their Interactions within and with the Enzyme Binding Cavity Determine Reaction Selectivities. The Case of Fe(II)/2-Oxoglutarate Dependent Enzymes.

Fe(II)/2-oxoglutarate dependent dioxygenases (ODDs) share a double stranded beta helix (DSBH) fold and utilise a common reactive intermediate, ferryl species, to catalyse oxidative transformations of substrates. Despite the structural similarities, ODDs accept a variety of substrates and facilitate a wide range of reactions, that is hydroxylations, desaturations, (oxa)cyclisations and ring rearrangements. In this review we present and discuss the factors contributing to the observed (regio)selectivities of ODDs. They span from inherent properties of the reactants, that is, substrate molecule and iron cofactor, to the interactions between the substrate and the enzyme's binding cavity; the latter can counterbalance the effect of the former. Based on results of both experimental and computational studies dedicated to ODDs, we also line out the properties of the reactants which promote reaction outcomes other than the "default" hydroxylation. It turns out that the reaction selectivity depends on a delicate balance of interactions between the components of the investigated system.

Model Setup and Procedures for Prediction of Enzyme Reaction Kinetics with QM-Only and QM:MM Approaches.

The enzyme-catalyzed reactions are traditionally studied with experimental kinetic assays. The modern theoretical modeling techniques provide a complementary way to investigate these catalytic reactions. Experimental assay frequently does not allow an unequivocal answer to the factors controlling the reaction mechanism. On the other hand, the theoretical experiments provide a precise understanding of the molecular-level steps involved in catalytic reactions. However, modeling requires at least structural data on the enzyme and reactant, and the complexity of the enzyme systems can still be a challenge.In this chapter, we are going to describe how to apply theoretical modeling methods, such as MD simulation, QM-only cluster models of enzyme active site, or QM:MM multiscale modeling to study enzyme kinetics and even to predict kinetic isotope effect (KIE). We present a full protocol that starts from the PDB structure of the enzyme, through MD simulation of enzyme: substrate complex and statistical analysis of MD trajectory, selection of a model of the active site, and study of reaction pathways. We show how theoretical predictions basing on QM-only cluster models, QM:MM model, or multiple QM:MM models derived from QM:MM:MD simulations can be correlated with experimental kinetic results. Finally, we show how one can calculate intrinsic KIE associated with an individual molecular step.

Seven quick tips for beginners in protein crystallography.

The aim of this brief review is to provide a roadmap for beginning crystallographers who have little or no experience in structural biology and yet are keen to produce protein crystals and analyze their 3D structures to understand their biological roles. To achieve this goal it is crucial to perform crystallization, structure determination, visualization and analysis of the protein's structural features related to its biological function. Keeping that objective in mind, tips presented herein cover the most important steps in a crystallographic endeavor and present a selection of databases and software which can aid and accelerate the whole process. We hope that this short overview will help novices coming from different disciplines to navigate a protein crystallography project and, hopefully, allow avoiding some costly mistakes, even though being a crystallographer means learning by trial and error.

Enhancing the Corrosion Resistance of Austenitic Steel Using Active Screen Plasma Nitriding and Nitrocarburising.

AISI 316L steel was subjected to active screen plasma nitriding and nitrocarburising. The processes were carried out at 440 °C for 6 h. The nitriding process employed an atmosphere of nitrogen and hydrogen, while nitrocarburising was carried out in nitrogen, hydrogen and methane. The processes yielded structures consisting of nitrogen and nitro-carbon expanded austenite, respectively. Microhardness was measured via the Vickers method, surface roughness using an optical profilometer, microstructure by means of light microscopy, while a scanning electron microscope (SEM) served to determine surface topography. Phase composition, lattice parameter and lattice deformation tests were carried out using the X-ray diffraction (XRD) method. Corrosion resistance measurements were performed in a 0.5 M NaCl solution using the potentiodynamic method. The produced layers showed very high resistance to pitting corrosion, while the pitting potential reached 1.5 V, a value that has not yet been recorded in a chloride environment. After the passive layer was broken down, there was a clear deceleration of pitting in the nitrocarburised layer. It was found that in the case of nitro-carbon expanded austenite, pits are formed much slower compared to the nitrogen austenite layer.