Comparative analysis of CE‐SSCP to standard RFLP‐CE‐FLA method in quantification of known viral variants within an RNA virus quasispecies

RNA viruses display the highest replication error rate in our biosphere, leading to highly diverse viral populations termed quasispecies. The gold standard method for detection and quantification of variants in a quasispecies is cloning and sequencing, but it is expensive, laborious and time consuming. Therefore, other mutation detection approaches, including SSCP, are often used. In this study, we demonstrate development and the usage of a CE‐SSCP method for quantification of two nearly identical viral variants in heterogenic population of a mumps virus strain and its comparison to RFLP‐CE‐fragment length analysis (RFLP‐CE‐FLA). Analyzed PCR fragments were of the same size (245 bp) with one difference in their nucleotide sequence. The limit of detection of both methods was at 5% of the minor variant. When PCR amplicons of the two variants were pooled, methods' results were very similar. On the contrary, the quantification results of samples in which variants were mixed prior to PCR showed substantial difference between the two methods. Our results indicate that although both methods can be used for detection and monitoring of a specific mutation within a viral population, caution should be taken when quantitative analysis of complex samples is based solely on results of one method.     

Isopiestic Determination of the Osmotic and Activity Coefficients of K<Subscript>2</Subscript>HPO<Subscript>4</Subscript>(aq), Including Saturated and Supersaturated Solutions,at <Emphasis Type="Italic">T</Emphasis>=298.15 K

The osmotic coefficients of K₂HPO₄(aq) have been measured at T=298.15 K by the isopiestic vapor pressure method over the range of molalities from 1.3846 mol⋅kg⁻¹ to 13.939 mol⋅kg⁻¹ (oversaturation) with CaCl₂(aq) as the reference solution. The molalities and osmotic coefficients of saturated solutions in equilibrium with K₂HPO₄⋅xH₂O(cr) were measured simultaneously by the same method. Available literature osmotic coefficients of K₂HPO₄(aq) at T=298.15 K, and our new experimental data, were combined and modeled using an extended form of Pitzer’s equation and the Clegg-Pitzer-Brimblecombe equation based on the mole-fraction-composition scale. These equations were used to calculate the activity coefficients of K₂HPO₄(aq) at T=298.15 K.     

Simultaneous Determination of Pb and Cd Traces in Water Samples by Anodic Stripping Voltammetry Using a Modified GC Electrode

The determination of Pb and Cd with a Nafion‐modified glassy carbon electrode and Cu‐DPABA complex (Cu‐DPABA–NA/GCE; DPABA is methyl 3,5‐bis{bis‐[(pyridin‐2‐yl)methyl]amino}methyl‐benzoate) as an alternative electrode for anodic stripping voltammetry was described. Pb and Cd were accumulated in acetate buffer pH 4 at a potential of ?1.4 V (vs. Ag/AgCl electrode) for 120 s followed by a DPASV scan from ?1.2 to ?0.2 V. Under optimum conditions the calibration curves were linear in the range of 4.8×10^?9–5.0×10^?5 and 5.0×10^?9–5×10^?5 mol L^?1 for Pb and Cd, respectively. Detection limits were 1.8×10^?9 and 1.2×10^?9 mol L^?1 for Pb and Cd, respectively. Different parameters and conditions, such as membrane ingredients, accumulation time, potential and pH value were optimized. A study of interfering substances was also performed. A significant increase in current was achieved at the modified electrode in comparison with the bare glassy carbon electrode. The validation of the proposed method was made by Pb and Cd determination in the certified reference material Groundwater CRM 610 (BCR, Community Bureau of Reference, Brussels, Belgium). The electrode was successfully applied for determination of Pb and Cd in river water with a high content of organic contaminants without any pretreatment.     

Structural identification of SAR-943 metabolites generated by human liver microsomes in vitro using mass spectrometry in combination with analysis of fragmentation patterns

SAR-943 (32-deoxo rapamycin) is a proliferation signal inhibitor via interaction with the mammalian target of rapamycin (mTOR). Most importantly, SAR-943 has improved chemical stability compared to rapamycin (sirolimus) and is currently under investigation as a drug coated on coronary stents. It was the goal of this study to identify the SAR-943 metabolites generated after incubation with human liver microsomes using high-resolution mass spectrometry (MS) and MS/iontrap (MS(n)) and comparison of fragmentation patterns of the metabolites with those of SAR-943 and other known rapamycin derivatives. Our study showed that SAR-943 is mainly hydroxylated and/or demethylated by human liver microsomes. The structures of the following metabolites were identified: O-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl and 27-O-desmethyl SAR-943; hydroxylated metabolites: hydroxy piperidine SAR-943, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 46-hydroxy and 49-hydroxy SAR-943; didemethylated metabolites: 16,39-O-didesmethyl and 27,39-O-didesmethyl SAR-943; demethylated-hydroxylated metabolites: 39-O-desmethyl, 23- or 24-hydroxy and 39-O-desmethyl, hydroxy piperidine SAR-943 and dihydroxylated metabolites: 12-,23- or 24-dihydroxy SAR-943. In addition, several other demethylated-hydroxylated and dihydroxylated metabolites were detected. However, their exact structures could not be identified.     

Comparison of UPLC and HPLC for Analysis of Dietary Folates

Ultra performance liquid chromatography (UPLC) using small sub-2 ??m particles and high performance liquid chromatography (HPLC) were compared for separation and determination of the most common dietary folates; 5-methyltetrahydrofolate, tetrahydrofolate, 5-formyltetrahydrofolate, 10-formylfolic acid and folic acid. Two UPLC columns??Acquity BEH C₁₈ and Acquity HSS T3, and two HPLC columns with similar surface chemistry??Xbridge C₁₈ and Atlantis d18 were tested. When using UPLC, the signal-to-noise ratio could be improved by a factor of 2?C50 for different folate derivatives and the run time could be reduced fourfold without sacrificing separation efficiency. The applicability of UPLC to real food samples was demonstrated.     

Short- and long-term stability of lyophilised melatonin-loaded lecithin/chitosan nanoparticles

The aim of this study was to establish a freeze-drying process for melatonin-loaded lecithin/chitosan nanoparticles (NPs) to preserve their chemical and physical stability for a longer time period that what is possible in an aqueous suspension. Glucose and trehalose were investigated as potential excipients during freeze-drying of NP suspensions. Lecithin/chitosan NPs were characterised by mean diameter and zeta potential, ranging between 117.4 and 328.5 nm and 6.7 and 30.2 mV, respectively, depending on the lecithin type and chitosan content in the preparation. Melatonin loadings were up to 7.1%. For all lecithin/chitosan NPs, no notable differences in the mean particle size, size distribution, zeta potential or melatonin content were observed before or immediately after the lyophilisation process or after 7 months of storage at 4 °C. The residual moisture contents of lyophilisates with glucose and trehalose immediately after the lyophilisation process varied between 4.0-4.8% and 2.4-3.0%, respectively. All lecithin/chitosan NPs had a fully amorphous nature after the freeze-drying process, as indicated by modulated differential scanning calorimetry. NP lyophilisates with glucose had a low glass transition temperature (ca. 5 °C), confirming that lyophilisation with glucose as a cryoprotectant was not appropriate. All lyophilisates with trehalose had a glass transition temperature above the room temperature, allowing formation of the cake without a collapse of the structure, which was capable of preserving its characteristics and appearance following 7 months of storage at 4 °C.     

Novel polycarbonate-based polyurethane elastomers: Composition–property relationship

Novel all-aliphatic polycarbonate-based polyurethane (PC-PU) elastomers, as well as PC-PU nanocomposites filled with organic-modified clays were synthesized, characterized and studied. It was found that they have very attractive mechanical properties (e.g., elongation at break between 600% and 800%). The prepared PC-PUs possess a distinctly segmented structure, which is the key prerequisite for their behavior as strong physical rubbery networks. All synthesized materials melt at elevated temperatures (between 110 and 200 °C) and hence can be processed like normal thermoplastics. The dispersion of the clay nanofiller was achieved by its one day swelling in the alcohol and a brief successive stirring. This procedure is very successful and leads to a partial exfoliation of the clay (documented by X-ray diffraction and TEM). The best nanocomposites with very good tensile properties, particularly with significantly increased moduli were obtained using the bentonite nanofiller. The study shows that the nanofiller interacts strongly with the hard domains and influences their melting temperature (DMTA and DSC), but it does not affect the glass transition temperature of soft domains. While Cloisite 15A was found to interact preferentially with the hard domains, the organic modified bentonite shows a strong interaction with both soft and hard segments, behaving as a blending agent. Hard domains in neat matrices, formed by hydrogen bonding of hard segments, were practically invisible by X-ray or TEM, but were successfully detected by AFM. Besides excellent mechanical properties, the prepared elastomers and their nanocomposites showed an interesting phase behavior (which was studied by combining DMTA and modulated DSC).     

catena‐Poly[[[(di‐2‐pyridylamine‐κ2N2,N2′)copper(II)]‐μ‐benzene‐1,3‐dicarboxylato‐κ3O1,O1′:O3] monohydrate], a zigzag coordination polymer with strong π–π interactions

The novel title coordination polymer, {[Cu(C₈H₄O₄)(C₁₀H₉N₃)]·H₂O}_n, synthesized by the slow‐diffusion method, takes the form of one‐dimensional zigzag chains built up of Cu^II cations linked by benzene‐1,3‐dicarboxylate (ipht) anions. An exceptional characteristic of this structure is that it belongs to a small group of metal–organic polymers where ipht is coordinated as a bridging tridentate ligand with monodentate and chelate coordination of individual carboxylate groups. The Cu^II cation has a highly distorted square‐pyramidal geometry formed by three O atoms from two ipht anions and two N atoms from a di‐2‐pyridylamine (dipya) ligand. The zigzag chains, which run along the b axis, further construct a three‐dimensional metal–organic framework via strong face‐to‐face π–π interactions and hydrogen bonds. A solvent water molecule is linked to the different carboxylate groups via hydrogen bonds. Thermogravimetric and differential scanning calorimetric analyses confirm the strong hydrogen bonding.