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Pathways of photochemical degradation of a cardiovascular drug verapamil under conditions relevant to natural waters and the toxicity of the photoproducts to Daphnia magna were investigated. Photodegradation was shown to proceed via photocatalysed mechanism. Two main photodegradation pathways were recognised: the first leading to hydroxylation at the methylamino position followed by splitting of verapamil molecule into two fragments, and the second providing the main active metabolite of verapamil, norverapamil, and a series of norverapamil isomers, followed again by their splitting at the amino group position. Twenty-two products of photodegradation were identified. Toxicity assays in sublethal concentrations of the parental drug, of the photoproduct mixture, and of norverapamil revealed no direct negative response in Daphnia magna to verapamil. On the other hand, photochemical products significantly lowered the number of juveniles, number of clutches, and body size of Daphnia. The exposition of Daphnia to norverapamil showed the same but even more pronounced effects than its exposition to the mixture of photoproducts, which leads to the conclusion that norverapamil is mainly responsible for the toxicity of photoproduct mixture and represents a noteworthy threat to aquatic invertebrates.

In this study we investigate the ability of RF magnetron sputtered nylon 6,6 films (C:H:N:O) to store and release two model antibiotics, ampicillin and ciprofloxacin, that differ significantly in their chemical structure. We demonstrate that the addition of nitrogen to the deposition process leads to a substantial enhancement in the abundance of nitrogen-containing functional groups in the resulting coatings as well as to the significant changes in their swelling and dissolution. These variations were found to result in different impregnation and release capabilities of the produced coatings: an almost 5 times higher amount of released antibiotics was observed for the samples prepared in nitrogen-rich atmosphere when compared to films deposited in pure argon. It was also found that the amount of released antibiotics strongly depends on the natural character of the antibiotics used. However, it is demonstrated that minimum inhibitory concentrations against S. epidermidis and E. coli can be exceeded for both antibiotics.

Photochemical degradation of fluoroquinolones ciprofloxacin, enrofloxacin and norfloxacin in aqueous solution under light conditions relevant to surface waters at neutral and alkaline pH was found to proceed readily with half-lives between 0.9 and 2.7 min. The products of photochemical degradation identified by HPLC-MS included defluorinated, hydroxylated, and decarboxylated structures as well as structures with opened cyclic structures. For all of the studied substances, the reaction pathways were influenced significantly by the pH of the reaction system, with more products formed at alkaline pH than at neutral pH: the ratios of products in neutral and alkaline pH were 16/26, 9/19, 15/23 for ciprofloxacin, enrofloxacin, and norfloxacin, respectively. The structures of photoproducts and pathways of photochemical degradation are proposed. The antibacterial activities of photoproduct mixtures tested on E. coli and S. epidermidis were significantly higher in comparison to parental antibiotics in the case of both ciprofloxacin and enrofloxacin with p-values less than 0.0001 in most cases. The effect of the photoproducts was shown to be dependent on the pH value of the original antibiotic solutions before photodegradation: for ciprofloxacin, antibacterial activity against E. coli was more notably pronounced with regard to neutral pH photoproducts, while a less significant, or in one case not significant, effect of pH was observed against S. epidermidis ; for norfloxacin, antibacterial activity against both E. coli and S. epidermidis were especially high with regard to alkaline pH photoproducts

Glycosphingolipids (GSL) represent a highly heterogeneous class of lipids with many cellular functions, implicated in a wide spectrum of human diseases. Their isolation, detection, and comprehensive structural analysis is a challenging task due to the structural diversity of GSL molecules. In this work, GSL subclasses are isolated from human plasma using an optimized monophasic ethanol–water solvent system capable to recover a broad range of GSL species. Obtained deproteinized plasma is subsequently purified and concentrated by C18-based solid-phase extraction (SPE). The hydrophilic interaction liquid chromatography coupled to electrospray ionization linear ion trap tandem mass spectrometry (HILIC-ESI-LIT-MS/MS) is used for GSL analysis in the human plasma extract. Our results provide an in-depth profiling and structural characterization of glycosph- ingolipid and some phospholipid subclasses identified in the human plasma based on their retention times and the interpretation of tandem mass spectra. The structural composition of particular lipid species is readily characterized based on the detailed interpretation of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) spectra and further confirmed by specific fragmentation behavior following predictable patterns, which yields to the unambiguous identification of 154 GSL species within 7 lipid subclasses and 77 phospholipids representing the highest number of GSL species ever reported in the human plasma. The developed HILIC-ESI-MS/MS method can be used for further clinical and biological research of GSL in the human blood or other biological samples. Glykosfingolipidy (GSL) představují vysoce heterogenní třídu lipidů s mnoha buněčnými funkcemi, implikovanými do širokého spektra lidských onemocnění. Jejich izolace, detekce a komplexní strukturní analýza je náročným úkolem vzhledem ke strukturní rozmanitosti molekul GSL. V této práci jsou podtřídy GLS izolovány z lidské plazmy pomocí optimalizovaného monofázického systému rozpouštědla ethanol-voda schopného obnovit širokou škálu GSL. Získaná deproteinizovaná plazma se následně čistí a koncentruje extrakcí na pevné fázi na bázi C18 (SPE). Hydrofilní interakční kapalinová chromatografie spojená s tandemovou hmotnostní spektrometrií s elektrosprejovou ionizací a lineání iontovou pastí (HILIC-ESI-LIT-MS/MS) se používá pro analýzu GSL v lidském extraktu plazmy. Naše výsledky poskytují hloubkové profilování a strukturní charakterizaci glykosfingolipidu a některých fosfolipidových podtříd identifikovaných v lidské plazmě na základě jejich retenčních časů a interpretace tandemových hmotnostních spekter. Strukturní složení jednotlivých lipidů je snadno charakterizováno na základě podrobné interpretace hmotnostně spektrometrických (MS) a tandemových hmotnostně spektrometrických (MS/MS) spekter a dále potvrzeno specifickým fragmentačním chováním podle předvídatelných vzorců, což vede k jednoznačné identifikaci 77 fosfolipidů a 154 GSL v rámci 7 lipidových podtříd představujících nejvyšší počet GSL, který byl kdy v lidské plazmě hlášen. Vyvinutá metoda HILIC-ESI-MS/MS může být použita pro další klinický a biologický výzkum GSL v lidské krvi nebo jiných biologických vzorcích.

Obligate symbiotic bacteria associated with the insects feeding exclusively on vertebrate blood are supposed to complement B vitamins presumably lacking in their diet, vertebrate blood. Recent genomic analyses revealed considerable differences in biosynthetic capacities across different symbionts, indicating that levels of B vitamins vary across different vertebrate hosts. However, a rigorous determination of B vitamins content in blood of various vertebrates has not yet been approached. A reliable analytical method focused on B vitamin complex in blood and hemolymph can provide valuable informative background and understanding of general principles of insect symbiosis.In this work chromatographic separation of a mixture of eight B vitamins (B1 – thiamine, B2 – riboflavin, B3 – niacin, B5 – pantothenic acid, B6 – pyridoxine, B7 – biotin, B9 – folic acid and B12 – cyanocobalamine), four B vitamin derivatives (B3 – niacinamide, B6 – pyridoxal-5-phosphate, B6 – 4-pyridoxic acid and B9 – tetrahydrofolic acid) and 3 stable isotope labelled internal standards (B3 – niacin-13C6, B5 – pantothenic acid (di-β-alanine-13C6,15N2) and B7 – biotin-(ring-6,6-d2)) on C30 column was developed. Detection was carried out using dual-pressure linear ion trap mass spectrometer in FullScan MS/MS and SIM mode. Matching internal standards with analytes was done according to the results of linearity, accuracy and precision. Except for vitamin B9 (tetrahydrofolic acid) instrument quantitation limits of all analytes were ranging from 0.42 to 5.0 μg/L, correlation coefficients from 0.9997 to 1.0000 and QC coefficients from 0.53 to 3.2 %.Optimization of whole blood sample preparation step was focused especially on evaluation of two types of protein-precipitation agents: trichloroacetic acid and zinc sulphate in methanol. Samples of whole blood prepared in six independent replicates were spiked at 10 μg/L and 100 μg/L level. The best results were obtained for zinc sulphate in methanol, but only nine analytes (B1 – thiamine, B2 – riboflavin, B3 – niacin, B3 – niacinamide, B5 – pantothenic acid, B6 – pyridoxine, B6 – 4-pyridoxic acid, B7 – biotin and B12 – cyanocobalamine) were successfully validated. Accuracy of the procedure using this protein-precipitating agent was ranging from 89 to 120 %, precision from 0.5 to 13 % and process efficiency from 65 to 108 %.HighlightsLC-MS/MS method for quantitation of eight B vitamins and four B vitamin derivatives was developed.Deproteinization agents trichloroacetic acid and ZnSO4/methanol were tested for protein precipitation of whole blood.Accuracy, precision and process efficiency were evaluated.Successful method validation for seven B vitamins and two B vitamin derivatives in whole blood.

Pathways of photochemical degradation of a cardiovascular drug verapamil under conditions relevant to natural waters and the toxicity of the photoproducts to Daphnia magna were investigated. Photodegradation was shown to proceed via photocatalysed mechanism. Two main photodegradation pathways were recognised: the first leading to hydroxylation at the methylamino position followed by splitting of verapamil molecule into two fragments, and the second providing the main active metabolite of verapamil, norverapamil, and a series of norverapamil isomers, followed again by their splitting at the amino group position. Twenty-two products of photodegradation were identified. Toxicity assays in sublethal concentrations of the parental drug, of the photoproduct mixture, and of norverapamil revealed no direct negative response in Daphnia magna to verapamil. On the other hand, photochemical products significantly lowered the number of juveniles, number of clutches, and body size of Daphnia. The exposition of Daphnia to norverapamil showed the same but even more pronounced effects than its exposition to the mixture of photoproducts, which leads to the conclusion that norverapamil is mainly responsible for the toxicity of photoproduct mixture and represents a noteworthy threat to aquatic invertebrates.

The effects of heat treatment and storage conditions on the composition of pure mead (honey wine) made from only honey and water were investigated. Heat treatment experiments were performed at 7 different temperatures ranging from 40°C to100°C with 10°C increments for 60min. Storage condition experiments were performed at room temperature (20-25°C) in daylight without direct sunlight and in darkness in a refrigerator at 4°C for 1, 2, 4 and 12weeks. The parameters evaluated were phenolic compounds, peak area of unidentified compounds, 5-hydroxymethylfurfural content and antioxidant capacity. Significant changes in compound content were observed in the case of 6 identified compounds and 9 unidentified compounds. However, the antioxidant activity was not affected by the heat treatments or storage at room temperature.

Photocatalytic degradation of pharmaceuticals (hydrocortisone, estradiol, and verapamil) and personal care product additives (parabens-methyl, ethyl, and propyl derivatives) was investigated in the homogeneous phase (with ferric ions as the catalyst) and on TiO2. Ferric ions in concentrations corresponding to concentrations in natural water bodies were shown to be a significant accelerator of the degradation in homogeneous reaction mixtures. In heterogeneous photocatalytic reactions on TiO2, lower reaction rates, but mineralisation to higher extents, were observed.

In Saccharomyces cerevisiae , K + -uptake under K + -limiting conditions is largely mediated by the cation translocation systems Trk1 and Trk2 belonging to the family of SKT proteins. They are related to two-transmembrane-domain (inward rectifying K-) channels but unlike the symmetrical tetrameric structure of K-channels, a single Trk contains four pore-forming domains (A-D) encoded on one polypeptide chain. Between domains A and B Trks contain large cytosolic regions dubbed “long hydrophilic loop” (LHL). LHLs are not homologous/similar to any other identified protein (domain) and also show little similarity between Trk1 and Trk2. Here we demonstrate that Trk1 is functional without LHL. However, in growth experiments NaCl sensitivity of Trk1[ΔLHL] expressing cells is increased under K + -limiting conditions compared to full-length Trk1. Non-invasive ion flux measurements showed that K + -influx through Trk1 and Trk1[ΔLHL] is decreased in the presence of surplus Na + due to permeability of the proteins for both cations and competition between them. Trk1[ΔLHL] is less affected than full-length Trk1 because it is more selective for K + over Na + . Furthermore, K + re-uptake after starvation is delayed and decreased in Trk1[ΔLHL]. Thus, a role of LHL is regulating cation fluxes through Trk1 by (i) allowing also Na + to pass if monovalent cations (mainly K + ) are limiting and (ii) by accelerating/enhancing a switch from low to high affinity ion translocation. We propose that LHL could modulate Trk1 transport properties via direct influence on a transmembrane helix (M2 A ) which can switch between bent and straight conformation, thereby directly modifying the radius of the pore and selectivity filter.

Usage of atrazine, a widely used herbicide, is now banned in many countries. Although forbidden to use, significant concentration of this herbicide is still present in the environment. The study focused not only on the toxicity of atrazine itself but also on products of homogeneous photocatalytic degradation. Such degradation was very fast in given conditions (sufficient amount of Fe(III) in the reaction system)-more than 95% of the initial amount of atrazine was eliminated after 30 min of irradiation. The toxicity of atrazine and its photodegradation products were examined on the aquatic plant Lemna minor and microcrustacean Daphnia magna in both acute and chronic tests. While the growth inhibition assay of atrazine for Lemna minor revealed EC50 value of 128.4 μg dm-3, the herbicide did not affect Daphnia in the acute toxicity assay. A degradation product, desethyl-atrazine, has been demonstrated to have a pronounced negative effect on the plant growth. Both atrazine and desethyl-atrazine affect negatively the number of juveniles and number of clutches of Daphnia magna in the chronic toxicity assay. Photocatalytic degradation lowers the negative effect of atrazine in Daphnia magna while photodegradation products still negatively affect Lemna growth.

Grass silage as a renewable feedstock for an integrated biorefinery includes nutrients and carbon sources directly available in the press juice (PJ) and in lignocellulosic saccharides from the plant framework. Here, a novel two-stage fed-batch fermentation process for biosynthesis of poly-3-hydroxybutyrate (PHB) by Cupriavidus necator DSM 531 is presented. For bacterial growth, nutrient-rich PJ was employed as a fermentation medium, without any supplements. Saccharides derived from the mechano-enzymatic hydrolysis of the press cake (PC) were subjected to a lactic acid fermentation process, before the fermentation products were fed into the polymer accumulation phase. By combination of pH-stat feeding and cell recycling, the PHB content in 22 g L−1 total-dry cells reached 39% after 32 h of cultivation. Using mimicked hydrolyzate of diluted PJ artificially supplemented with glucose and xylose, the resulting cell dry weight of 21 g L−1 contained 42% PHB.

Toxicity in reaction mixtures of atrazine irradiated on immobilized TiO2 at varying irradiation times was studied using an algal growth test on unicellular green alga Raphidocelis subcapitata and a cytotoxicity test with a RTgill-W1 cell line. The toxicity of atrazine samples to algae decreased exponentially with the time of irradiation on TiO2 up to 3 h for both IC50 and IC20 values calculated for growth rate inhibition and yield. A trend to increasing variability between replicates in atrazine samples irradiated on TiO2 for longer time periods was observed; the trend was particularly pronounced in samples irradiated for 3 and 5 hours, where the atrazine samples caused moderate stimulation in lower concentration treatments (up to 550 μg/l of the initial atrazine concentration). None of the atrazine samples showed significant cytotoxicity to the rainbow trout gill cell line (RTgill-W1).

A slurry sampling technique has been utilised for elemental analysis of multivitamins preparations using inductively coupled plasma-emission spectrometry (ICP-OES). For results comparison, samples were mineralised. Slurry concentration 0.1–0.2% m/v in 6% v/v HNO 3 , was used. The calibration by water standard solutions, slurry standards and standard additions were tested for determination above-mentioned elements in slurries. The method offers good precision for macro elements (RSD ranged from 5% to 10%). For in-home control sample, the measured concentrations are in satisfactory agreement with independent laboratories. For the analysed multivitamin preparations, the found element concentration is compared to amount declared by producer. The concentrations of Ca, Mg, P, K, Fe, Mn, Zn, Cu and Cr, Ni, V were determined in the range 1000–100,000 and 5–50 μg g −1 , respectively. The slurry ICP-OES analysis was found to be suitable for quality control monitoring of multivitamin preparations and could be useful as a routine procedure.

Thin films, which are able to deliver antibiotics in a controlled way, are considered as a promising approach to combat bacterial infections. A novel drug delivery method based on reservoir/diffusion barrier is introduced in this paper. As reservoir serves a film of sputtered nylon 6,6 impregnated with ampicillin, which is then covered by a diffusion barrier from the same material. It is demonstrated that the impregnation process does not affect either the morphology or the surface chemical structure. The amount of immobilized antibiotics can be tailored by changing the reservoir film thickness, while the release kinetics can be controlled by the thickness of the diffusion barrier. Finally, it is shown that the ampicillin impregnated films have an antibacterial effect against Staphylococcus epidermidis.

The objective of this study was the determination of 25 phenolic compounds in different mead samples (honeywines) using high performance liquid chromatography (HPLC) with coulometric-array detection and in case of hydroxymethylfurfural with UV detection. Our method was optimized in respect to both the separation selectivity of individual phenolic compounds and the maximum sensitivity with the electrochemical detection. The method development included the optimization of mobile phase composition, the pH value, conditions of the gradient elution and the flow rate using a window-diagram approach. The developed method was used for the determination of limits of detection and limits of quantitation for individual compounds. The linearity of calibration curves, accuracy and precision (intra- and inter-day) at three concentration levels (low, middle and high concentration range) were verified. Mead samples were diluted with the mobile phase at 1:1 to 1:50 ratio depending on the concentration and filtered through a PTFE filter without any other sample pre-treatment. Phenolic compounds concentration was determined in 50 real samples of meads and correlated with meads composition and hydroxymethylfurfural concentration. The most frequently occurred compounds were protocatechuic acid and vanillic acid (both of them were present in 98% samples), the least occurred compounds were (+)-catechin (10% samples) and sinapic acid (12% samples). Vanillin and ethylvanillin, which are used as artificial additives for the taste improvement, were found in 60% and 42% samples, respectively. Hydroxymethylfurfural concentration, as an indicator of honey quality, was in the range from 2.47 to 158 mg/L. Our method is applicable for the determination of 25 phenolic compounds in mead, honey and related natural samples.