Exploring the Role of L10 Loop in New Delhi Metallo-β-lactamase (NDM-1): Kinetic and Dynamic Studies

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some β-lactams. Significant reduction of k_cat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of T_mB and T_mD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some β-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.     

Visible Light Reductive Photocatalysis of Azo-Dyes with n–n Junctions Based on Chemically Deposited CdS

New composite photocatalysts have been obtained by chemical bath deposition of CdS on top of either nanostructured crystalline ZrO₂ or TiO₂ films previously deposited on conductive glass FTO. Their morphological, photoelectrochemical and photochemical properties have been investigated and compared. Time resolved spectroscopic, techniques show that in FTO/TiO₂/CdS films the radiative recombination of charges, separated by visible illumination of CdS, is faster than in FTO/ZrO₂/CdS, evidencing that carrier dynamics in the two systems is different. Photoelectrochemical investigation evidence a suppression of electron collection in ZrO₂/CdS network, whereas electron injection from CdS to TiO₂ is very efficient since trap states of TiO₂ act as a reservoir for long lived electrons storage. This ability of FTO/TiO₂/CdS films is used in the reductive cleavage of N=N bonds of some azo-dyes by visible light irradiation, with formation and accumulation of reduced aminic intermediates, identified by ESI-MS analysis. Needed protons are provided by sodium formate, a good hole scavenger that leaves no residue upon oxidation. FTO/TiO₂/CdS has an approximately 100 meV driving force larger than FTO/ZrO₂/CdS under illumination for azo-dye reduction and it is always about 10% more active than the seconds. The films showed very high stability and recyclability, ease of handling and recovering.     

UV-visible,IR, and 13C NMR studies on CT complexes between some thiohydantoins and molecular diiodine

The reaction of some 5,5-dimethyl-2-thiohydantoin derivatives (X = O, S; R, R′ = H, Me) with molecular diiodine has been studied in CH₂Cl₂ solution by different spectroscopic techniques. The formation constants (K) of the 1 : 1 molecular adducts and their thermodynamic parameters have been measured by UV-visible spectroscopy. The results allow us to point out the different donor properties of C(2) = S thioketonic sulfur between the two series of compounds (X = O, S) and the influence of N(1) and N(3) methylation on the K's. From the analysis of the ν(NH) frequencies, it has been possible to show hydrogen bond interactions between the NH's and the S-bonded iodine; this seems to be an important factor in determining the K values.     

Hydrophobic Eutectic Solvent-Based Dispersive Liquid-Liquid Microextraction Applied to the Analysis of Pesticides in Wine

A green solvent-based DLLME/HPLC-MS method for the determination of 19 pesticides in wine samples has been developed. The extractant solvent is a hydrophobic eutectic mixture composed of L-menthol and butylated hydroxytoluene in a molar ratio of 3:1. The endogenous ethanol of wine has been used as dispersive solvent, in order to avoid the solidification of the extracts under 19 °C. The mobile phase composition, the elution gradient and the sample injection volume were optimized in order to make this hydrophobic mixture compatible with conventional reversed phase chromatography and electrospray ionization. The method was validated in matrix, using a wine free from the target compounds. Average recovery as high as 80%, precision between 3 and 14%, and limits of detection and quantification much lower than the maximum residue levels (MRLs) for grapes and wines fixed by the EU regulation, make this multiresidue method fitted for the purpose, with the further advantages of being quick, cheap and in compliance with the green analytical chemistry. From the analysis of 11 commercial wines it was found that just in a bio sample the target compounds were not detectable or lower than quantification limit; as for the other samples, the most widespread and abundant pesticides were methoxyfenozide and boscalid, but their levels were much lower than the relative MRLs.     

Natural Deep Eutectic Solvents as a Key Metal Extractant for Fractionation in Speciation Analysis

The research aimed to use natural deep eutectic solvents (NADES) as an extractant for fractionation of compounds of selected elements from young barley and to compare it with the fractionation of elements from certified element materials. The use of such a comparison made it possible to prove the possibility of extracting the same forms of elements (species) from different materials, which confirmed the option of using NADES as extractants in speciation analysis. The research was conducted with hyphenated techniques—separation by high-performance chromatography coupled to an isotope-specific detector, mass spectrometry (MS) with ionization in inductively coupled plasma (ICP)—which are widely used in speciation analysis. Natural deep eutectic solvents also help introduce Green Analytical Chemistry principles (GAC). According to the results of our studies, the use of different NADES permit the extraction of various metals from a single sample. Moreover, using other natural solvents of eutectic properties helps extract different species of a given metal.     

New Biological and Chemical Evidences of Two Lamiaceae Species (Thymbra capitata and Thymus sipyleus subsp. rosulans): In Vitro,In Silico and Ex Vivo Approaches

In this study, the methanolic and infusion extracts of two species, Thymbra capitata and Thymus sipyleus subsp. rosulans, were tested for their chemical composition and biological abilities (antioxidant, enzyme inhibitory and anti-inflammatory effects). The extracts yielded total phenolic and flavonoid contents in the range of 83.43–127.52 mg GAE/g and 9.41–46.34 mg RE/g, respectively. HPLC analysis revealed rosmarinic acid to be a major component of the studied extracts (15.85–26.43%). The best ABTS radical scavenging ability was observed in the methanol extract of T. capitata with 379.11 mg TE/g, followed by in the methanol extract of T. sipylus (360.93 mg TE/g). In the CUPRAC assay, the highest reducing ability was also found in the methanol extract of T. capitata with 802.22 mg TE/g. The phosphomolybdenum ability ranged from 2.39 to 3.61 mmol TE/g. In terms of tyrosinase inhibitory effects, the tested methanol extracts (83.18–89.66 mg KAE/g) were higher than the tested water extracts (18.74–19.11 mg KAE/g). Regarding the BChE inhibitory effects, the methanol extracts were active on the enzyme while the water extracts showed no inhibitory effect on it. Overall, the methanolic extracts showed better enzyme inhibition compared to the infusion extracts. Molecular docking also showed the selected exhibited potential binding affinities with all enzymes, with a preference for cholinesterases. Additionally, the extracts were effective in attenuating the LPS-induced increase in COX-2 and IL-6 gene expression in isolated colon, thus indicating promising anti-inflammatory effects. The preliminary results of this study suggest that these species are good natural sources of antioxidants and also provide some scope as enzyme inhibitors, most likely due to their bioactive contents such as phenolic acids, and thus can be exploited for different applications related to health promotion and disease prevention.     

Hybrid materials with an increased resistance to hard X‐rays using fullerenes as radical sponges

The protection of organic and hybrid organic–inorganic materials from X‐ray damage is a fundamental technological issue for broadening the range of applications of these materials. In the present article it is shown that doping hybrid films with fullerenes C₆₀ gives a significant reduction of damage upon exposure to hard X‐rays generated by a synchrotron source. At low X‐ray dose the fullerene molecules act as `radical scavengers', considerably reducing the degradation of organic species triggered by radical formation. At higher doses the gradual hydroxylation of the fullerenes converts C₆₀ into fullerol and a bleaching of the radical sinking properties is observed.