Development and validation of an LC-MS/MS method for determination of B vitamins and some its derivatives in whole blood.

Obligate symbiotic bacteria associated with the insects feeding exclusively on vertebrate blood are supposed to complement B vitamins presumably lacking in their diet. Recent genomic analyses revealed considerable differences in biosynthetic capacities across different symbionts, suggesting that levels of B vitamins may 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 can provide valuable informative background and understanding of general principles of insect symbiosis. In this work, a chromatographic separation of eight B vitamins (thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folic acid, and cyanocobalamine), four B vitamin derivatives (niacinamide, pyridoxal-5-phosphate, 4-pyridoxic acid, and tetrahydrofolic acid), and 3 stable isotope labelled internal standards was developed. Detection was carried out using dual-pressure linear ion trap mass spectrometer in FullScan MS/MS and SIM mode. Except for vitamin B9 (tetrahydrofolic acid), the 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. The best results were obtained for zinc sulphate in methanol, but only nine analytes 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%. The content of B vitamins in whole blood samples from human and various vertebrates is presented as an application example of this newly developed method.

Photodegradation of fluoroquinolones in aqueous solution under light conditions relevant to surface waters, toxicity assessment of photoproduct mixtures.

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 was especially high with regard to alkaline pH photoproducts.

Activation of a Cryptic Manumycin-Type Biosynthetic Gene Cluster of Saccharothrix espanaensis DSM44229 by Series of Genetic Manipulations.

(1) Background: Manumycins are small actinomycete polyketides with prominent cancerostatic and immunosuppressive activities via inhibition of various eukaryotic enzymes. Their overall activity towards human cells depends on the structural variability of both their polyketide chains, mainly the upper one. In our genetic screening project to find novel producers of anti-inflammatory manumycins, the strain Saccharothrix espanaensis DSM44229 was identified as containing a novel manumycin-type biosynthetic gene cluster (BGC). (2) Methods: The biosynthetic genes appeared to be silent under all assayed laboratory conditions. Several techniques were used to activate the BGC, including: (i) heterologous expression in various hosts, (ii) overexpression of putative pathway-specific regulatory genes, and (iii) overexpression of a bottleneck cyclizing aminolevulinate synthase gene in both natural and heterologous producers. (3) Results: Multiple novel manumycin-type compounds were produced at various levels by genetically-modified strains, sharing a tetraene lower chain structure with a colabomycin subgroup of manumycins, but possessing much shorter and saturated upper chains. (4) Conclusions: A cryptic manumycin-type BGC was successfully activated by genetic means to gain production of novel manumycin-type compounds for future comparative activity assays. Heterologously produced compounds were identical to those found after final activation of the BGC in the original strain, proving the intactness of the cloned BGC.

Comprehensive Identification of Glycosphingolipids in Human Plasma Using Hydrophilic Interaction Liquid Chromatography-Electrospray Ionization Mass Spectrometry.

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 glycosphingolipid 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.

Toxicity assessment of verapamil and its photodegradation products.

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.

Inhibition of Pro-Inflammatory Cytokines by Metabolites of Streptomycetes-A Potential Alternative to Current Anti-Inflammatory Drugs?

Current treatment of chronic diseases includes, among others, application of cytokines, monoclonal antibodies, cellular therapies, and immunostimulants. As all the underlying mechanisms of a particular diseases are not always fully clarified, treatment can be inefficient and associated with various, sometimes serious, side effects. Small secondary metabolites produced by various microbes represent an attractive alternative as future anti-inflammatory drug leads. Compared to current drugs, they are cheaper, can often be administered orally, but still can keep a high target-specificity. Some compounds produced by actinomycetes or fungi have already been used as immunomodulators-tacrolimus, sirolimus, and cyclosporine. This work documents strong anti-inflammatory features of another secondary metabolite of streptomycetes-manumycin-type polyketides. We compared the effect of four related compounds: manumycin A, manumycin B, asukamycin, and colabomycin E on activation and survival of human monocyte/macrophage cell line THP-1. The anti-cancer effect of manucycine A has been demonstrated; the immunomodulatory capacities of manumycin A are obvious when using micromolar concentrations. The application of all four compounds in 0.25-5 µM concentrations leads to efficient, concentration-dependent inhibition of IL-1ß and TNF expression in THP-1 upon LPS stimulation, while the three latter compounds show a significantly lower pro-apoptotic effect than manumycin A. We have demonstrated the anti-inflammatory capacity of selected manumycin-type polyketides.

Toxicity of atrazine and the products of its homogeneous photocatalytic degradation on the aquatic organisms Lemna minor and Daphnia magna.

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.

The S. cerevisiae cation translocation protein Trk1 is functional without its "long hydrophilic loop" but LHL regulates cation translocation activity and selectivity.

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 (M2A) which can switch between bent and straight conformation, thereby directly modifying the radius of the pore and selectivity filter.

Biosynthesis of poly-3-hydroxybutyrate from grass silage by a two-stage fermentation process based on an integrated biorefinery concept.

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.

Effect of heat treatment and storage conditions on mead composition.

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.

Determination of phenolic compounds and hydroxymethylfurfural in meads using high performance liquid chromatography with coulometric-array and UV detection.

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.