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Marine bacterium Vibrio natriegensis a novel host platform for different applications in molecular biology and biotechnology. It has one of the fastest growth rates of any known microorganisms and its extremely short doubling time indicates a high level of proteosynthetic activity. Regarding the necessity of developing new high-level protein expression systems it represents an extremely interesting subject. V. natriegens fulfills many important features for a suitable host including non- pathogenicity, easy scale-up process, potential for using alternative carbon sources (compared to E. coli), growth media and potential for further genetic and metabolic engineering with employment of a wide range of genetic tools. This work compares V. natriegens as an expression host for production of recombinant human growth hormone (hGH), yeast alcohol dehydrogenase (ADH) and archaeal catalase-peroxidase (AfKatG) to E. coliand establishes the basis for future development of this platform. The selected proteins are of different origins, sizes and intended applications. Our results have shown that cultures of V. natriegens using sucrose as a main carbon source can be used for the production of industrially applicable proteins, where it offers higher biomass productions compared to E. coli. In case of human growth hormone production, produced amounts were lower compared to those of E. coli (38 % of total cell protein (TCP) for V. natriegens vs. 58 % of TCP for E. coli, with similar solubility of around 40 % in both cases). In case of yeast alcohol dehydrogenase, V. natriegens produced 26 % of TCP vs. 42 % of TCP in E. coli, but with severely decreased solubility in case of V. natriegens cultures. Finally V. natriegens cultures were able to produce catalase-peroxidase AfKatG at the level of 33 % of TCP compared to 26 % of TCP in E. coli. Obtained results suggest that there are still significant differences in reliability and ease of use between E. coli and V. natriegens, with latter being more susceptible to condition changes and producing inconsistent results.

This paper investigates the Angle of Repose (AoR) of powder materials with respect to their morphological and rheological properties. Glass beads, sand, flour and semolina of different particle sizes were used as the experimental materials. The investigated material was analysed with respect to particle shape and size. The rheological properties of the material were obtained by a shear cell test. The AoR was analysed in terms of cohesion, bulk density, particle size and circularity. More cohesive materials such as the flour samples exhibited the largest AoR > 40°, indicating their poor flowability. Glass bead samples with a high circularity value had significantly lower AoR than the flour. The Angle of Internal Friction values were not dependent on those of the AoR. Using a dimensional analysis, a mathematical model was developed to determine the AoR values based on the material properties. By the application of this model, highly accurate calculation of the value of AoR is made possible.

The bromate-aniline oscillatory reaction was discovered 4 decades ago, but neither the detailed mechanism nor the key products or intermediates of the reaction were described. We report herein a detailed study of this reaction, which yielded new insights. We found that oscillatory oxidation of aniline by acidic bromate proceeds, to a significant extent, via a novel reaction pathway with the periodic release of carbon dioxide. Several products were isolated, and their structures, not described so far, were justified on the basis of MS and NMR. One of the main products of the reaction associated with the CO2 release route can be assigned to 2,2-dibromo-5-(phenylimino)cyclopent-3-en-1-one. A number of known compounds produced in the studied reaction, including unexpected brominated 1-phenylpyrroles and 1-phenylmaleimides, were identified by comparison with standards. A mechanism is suggested to explain the appearance of the detected compounds, based on coupling of the anilino radical with the produced 1,4-benzoquinone. We assume that the radical adduct reacts with bromine to form a cyclopropanone intermediate that undergoes a Favorskii-type rearrangement. Further oxidation and bromination steps including decarboxylation lead to the found brominated phenyliminocyclopentenones. The detected derivatives of 1-phenylpyrrole could be produced by a one-electron oxidation of a proposed intermediate 2-phenylamino-5-bromocyclopenta-1,3-dien-1-ol followed by β-scission with the abstraction of carbon monoxide. Such a mechanism is known from the combustion chemistry of cyclopentadiene. The proposed mechanism of this reaction provides a framework for understanding the observed oscillatory kinetics.

The aim of this systematic study was to analyze the granulometric and rheological behavior of tableting mixtures in relation to tabletability by single tablet and lab-scale batch compression with an eccentric tablet machine. Three mixtures containing 33, 50, and 66% of the cohesive drug paracetamol were prepared. The high compressibility of the powder mixtures caused problems with overcompaction or lamination in the single tablet compression method; due to jamming of the material during the filling of the die, the lab-scale batch compression was impossible. Using high shear granulation, the flow properties and tabletability were adjusted. A linear relationship between the span of granules and the specific energy measured by FT4 powder rheometer was detected. In parallel, a linear relationship between conditioned bulk density and the tensile strength of the tablets at lab-scale batch tableting was noted. The combination of dynamic image analysis and powder rheometry was useful for predicting the tabletability of pharmaceutical mixtures during the single tablet (design) compression and the lab-scale batch compression.

Mycobacterium tuberculosis, one of the deadliest pathogens in human history, is distinguished by a unique, multilayered cell wall, which offers the bacterium a high level of protection from the attacks of the host immune system. The primary structure of the cell wall core, composed of covalently linked peptidoglycan, branched heteropolysaccharide arabinogalactan, and mycolic acids, is well known, and numerous enzymes involved in the biosynthesis of its components are characterized. The cell wall biogenesis takes place at both cytoplasmic and periplasmic faces of the plasma membrane, and only recently some of the specific transport systems translocating the metabolic intermediates between these two compartments have been characterized [M. Jackson, C. M. Stevens, L. Zhang, H. I. Zgurskaya, M. Niederweis, Chem. Rev., 10.1021/acs.chemrev.0c00869 (2020)]. In this work, we use CRISPR interference methodology in Mycobacterium smegmatis to functionally characterize an ATP-binding cassette (ABC) transporter involved in the translocation of galactan precursors across the plasma membrane. We show that genetic knockdown of the transmembrane subunit of the transporter results in severe morphological changes and the accumulation of an aberrantly long galactan precursor. Based on similarities with structures and functions of specific O-antigen ABC transporters of gram-negative bacteria [C. Whitfield, D. M. Williams, S. D. Kelly, J. Biol. Chem. 295, 10593-10609 (2020)], we propose a model for coupled synthesis and export of the galactan polymer precursor in mycobacteria.

Thymol is a substance with a great therapeutic potential possessing antibacterial and antifungal activity, with a characteristic odour that remains long after application but is not pleasant at higher concentrations. In this study, attention has been focused on describing the chemical and biological properties of the simply prepared trimethylsilyl ether of thymol (kubicin). Interestingly, kubicin has similar volatility as thymol, undergoes hydrolysis in the water (moisture; forming thymol and trimethylsilanol) and can be used at 6,000 times higher concentration than thymol without any negative and irritating odour. Kubicin showed diverse fungistatic and fungicidal activities when tested by direct contact assay, or in vapour phase. The volatile vapour of kubicin was effective on all tested fungal strains. These results suggest that vapours of kubicin might provide an alternative way to fight against fungal contamination.

This work deals with the creation of a mathematical model which will predict with sufficient accuracy the size of granules in the high-shear granulation process. The aim of the paper was to create ...

The presented work focused on determination of derivatives of flavonols presented in American Cranberry (Vaccinium macrocarpon), more specifically glycoside derivatives of quercetin: quercetin-3-O-β-d-galactoside (Q-gal), quercetin-3-O-β-d-glucopyranoside (Q-glu), and quercetin-3-O-α-d-arabinoside (Q-ara), due to their anti-inflammatory, antitumor, antiviral, and antibacterial effects, by high temperature gas chromatography with mass spectrometry detector (HTGC–MS). For this purpose, two-step derivatization was applied to transfer the chosen glycoside derivatives of quercetin into a form suitable for further HTGC–MS analysis. The quality and quantity of chosen derivatives were determined in several nutritional supplements and teas with extract of American cranberry, cranberry juice, cranberry jam, cranberry syrup, dried cranberries, and fresh cranberries. The newly developed analytical procedure can serve as an alternative tool for the analysis of other polar and low-volatility chemicals present in nutritional supplement and herbal products. LOD values were in the range of 0.5 mg kg−1 for Q-glu to 0.6 mg kg−1 for Q-ara, while the LOQ ranged from 1.7 mg kg−1 for Q-glu to 1.9 mg kg−1 for Q-ara.

This work is focused on the detection of butter adulteration by sunflower and rapeseed oils using low pressure gas chromatography mass spectrometry (LPGC-MS). The method is based on simple dissolution of triglycerides from butter samples in chloroform and their direct analysis with LPGC-MS. The method allows the detection of characteristic triglycerides as butter adulteration markers and their quantification in different samples, with the possibility to quantify plant oil addition to butter at 0.45% level (using m/z = 600.4 for rapeseed oil) and 0.15% for sunflower oil (using m/z = 598.4). Ten different commercial butters were tested for the presence of sunflower and rapeseed oil. Interestingly, the addition of rapeseed oil up to 1.2% was recorded in one butter brand.

This work is focused on the development of a new analytical method based on mass spectrometry that allows analysis of multi-component mixtures of substances with high boiling point. The principle of this method is the introduction of a sample into a gas chromatograph without its initial modification (derivatization) and determination of analytes based on the ion fragmentation (m/z) without chromatographic separation. The advantages of this newly developed method compared to the conventional methods (gas chromatography with flame ionization detector) are shorter analysis time and reduced background at lower temperatures. The presented method was successfully applied for evaluation of beeswax adulteration with paraffin and stearin at the 2.1 and 3.5% level, respectively.

The newly developed gas chromatography–tandem mass spectrometry method allows the analysis of non-volatile, polar substances in cigarette ash. Only a few mg of ash, derivatized by two-step silanization with hexamethyldisilazane and N,O-bis(trimethylsilyl)trifluoroacetamide, are necessary for the analysis. Samples are directly analyzed after derivatization, without the need of extraction as a next purification step. Differences in chromatographic profiles reflect the composition uniqueness of each matrix and can be used for product authentication by chromatographic fingerprint method. The method was used to differentiate between cigarette ash samples of three cigarette brands (Marlboro, Davidoff, and West) and can be useful for forensic purposes.

This work focuses on the monitoring of changes in the time and incident light dependent ratio of endogenous analytes, squalene, and pentadecanoic acid, in fingerprint samples by gas chromatography with tandem mass spectrometry. The method can be used for fingerprint age determination of two age different samples on a glass surface from the same donor or allows identity determination from fingerprints of different persons imprinted at the same time, even in case of their blurring on a glass surface.

The bromate-aniline oscillatory reaction was discovered 4 decades ago, but neither the detailed mechanism nor the key products or intermediates of the reaction were described. We report herein a detailed study of this reaction, which yielded new insights. We found that oscillatory oxidation of aniline by acidic bromate proceeds, to a significant extent, via a novel reaction pathway with the periodic release of carbon dioxide. Several products were isolated, and their structures, not described so far, were justified on the basis of MS and NMR. One of the main products of the reaction associated with the CO

Work was focused on the development of INCAT, a solventless microextraction technique with adsorbents inside a steel needle. The device was used for the extraction and preconcentration of volatile compounds, and subsequently analyzed by gas chromatographic methods. The sorption characteristics of INCAT devices with different sorbents were compared, and the device with sorbents Chromosorb W + 20 % SE-54, Carbopack X, and Carboxen 1000 (in 1:1:1 v/v ratio) was applied for the analysis of volatile organic compounds in urine samples of patients with metabolic diseases.

This work focuses on the simultaneous analysis of creatinine and fructose with the gas chromatographic tandem mass spectrometric (GC–MS/MS) method after silylation with hexamethyldisilazane and N,O-bis(trimethylsilyl)trifluoroacetamide and the comparison of GC–MS/MS and enzyme-catalyzed photometric analysis of creatinine. The GC–MS/MS method allows the direct silylation of urine samples without sample pretreatment before derivatization. Due to this option, the analysis and diagnostics of inborn metabolic diseases are faster than in the case of a separate determination of both analytes.

This paper aims to develop a non-solvent microextraction technique using INCAT (Inside Needle Capillary Adsorption Trap) for GC-MS analysis of volatile metabolites in urine. INCATs with different sorbents were tested, resulting in an INCAT with 3 sorbent layers Chromosorb W+ 20% SE-54, Carbopack X and Carboxen 1000 (1 : 1 : 1), which proved to be the most suitable for differentiating and identifying volatile metabolites in urine samples of patients from those of healthy people.

Direct injection mass spectrometry was amended with chromatographic methods in analysis of membrane phospholipids of Bacillus subtilis mutant strain, which contains four polar headgroups only (phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid). Phospholipid classes were determined by thin layer chromatography as the basic method. Analysis of fatty acids by gas chromatography determined relative abundances of saturated, unsaturated, linear, and branched C14 to C18 fatty acids in phospholipids. Experimental data measured by direct injection mass spectrometry in the m/z range of 590–790 were used to estimate theoretical phospholipid composition which was compared with the data of chromatographic methods. The phospholipid class relative abundances measured by mass spectrometry did not agree fully with those of thin layer chromatography and the probable explanation was proposed. The direct injection mass spectrometry approach seems to be fast and easy procedure suitable for analyses of proportional changes in phospholipid composition and can be used as a complementary method for trend analyses and in process monitoring.

Mycobacterium tuberculosis possesses a thick and highly hydrophobic cell wall principally composed of a mycolyl-arabinogalactan-peptidoglycan complex, which is critical for survival and virulence. DprE1 is a well-characterized component of decaprenyl-phospho-ribose epimerase, which produces decaprenyl-phospho-arabinose (DPA) for the biosynthesis of mycobacterial arabinans. Upstream of dprE1 lies rv3789 , which encodes a short transmembrane protein of the GtrA family, whose members are often involved in the synthesis of cell surface polysaccharides. We demonstrate that rv3789 and dprE1 are cotranscribed from a common transcription start site situated 64 bp upstream of rv3789 . Topology mapping revealed four transmembrane domains in Rv3789 and a cytoplasmic C terminus consistent with structural models built using analysis of sequence coevolution. To investigate its role, we generated an unmarked rv3789 deletion mutant in M. tuberculosis . The mutant was characterized by impaired growth and abnormal cell morphology, since the cells were shorter and more swollen than wild-type cells. This phenotype likely stems from the decreased incorporation of arabinan into arabinogalactan and was accompanied by an accumulation of DPA. A role for Rv3789 in arabinan biosynthesis was further supported by its interaction with the priming arabinosyltransferase AftA, as demonstrated by a two-hybrid approach. Taken together, the data suggest that Rv3789 does not act as a DPA flippase but, rather, recruits AftA for arabinogalactan biosynthesis. IMPORTANCE Upstream of the essential dprE1 gene, encoding a key enzyme of the decaprenyl phospho-arabinose (DPA) pathway, lies rv3789 , coding for a short transmembrane protein of unknown function. In this study, we demonstrated that rv3789 and dprE1 are cotranscribed from a common transcription start site located 64 bp upstream of rv3789 in M. tuberculosis . Furthermore, the deletion of rv3789 led to a reduction in arabinan content and to an accumulation of DPA, confirming that Rv3789 plays a role in arabinan biosynthesis. Topology mapping, structural modeling, and protein interaction studies suggest that Rv3789 acts as an anchor protein recruiting AftA, the first arabinosyl transferase. This investigation provides deeper insight into the mechanism of arabinan biosynthesis in mycobacteria.

The effects of adding sunflower and linseed oils to a standard winter ration with a lower concentrate content on the milk fatty acid composition in lactating ewes were investigated. Eighteen dairy ewes randomly chosen from the ewe flock were divided into three groups: the first group was fed a winter ration, the second one a winter ration supplemented with sunflower oil, and the third a winter ration supplemented with linseed oil for a period of 12 days. In the treatment groups, the concentrate was partially replaced by 3.0 g/100 g sunflower or linseed oils. Milk samples were taken daily from morning milking for the analysis of fatty acid composition to determine their temporal daily variations. The responses to sunflower oil compared with linseed oil addition after the end of experi - ment were slightly higher for conjugated linoleic acid (CLA) ( P < 0.05), linoleic acid ( P < 0.001), trans-10 18:1 (P < 0.001), 6:0 to 16:0 ( P ≈ 0.05 - P < 0.001), whereas a higher content of α-linolenic acid (ALA) (3-fold) ( P < 0.001), oleic acid ( < 0.001), and 18:0 (P < 0.001) was found in milk after linseed oil addition. The responses to both oil additions were relatively sustainable with regard to CLA, trans-11 18:1 (vaccenic acid, VA), and ALA content after the last 6 days of supplementation. The winter ration supplementation with sunflower or linseed oil led to a 3-fold increase in CLA milk fat content (0.6-2.0 or 1.8 g/100 g fatty acid methyl esters (FAME), P < 0.001) and a 3-fold increase in VA milk fat content (1.2-3.8 or 4.1 g/100 g FAME, P < 0.001), however the content of trans-10 18:1 was 5-6-fold higher, compared with unsupplemented winter ration. Plant oil supplementation enhanced the total content of CLA, VA, ALA by 5.0 and 3.9 g/100 g FAME ( P < 0.001) for linseed and sunflower oil supplementation, and decreased the total content of 12:0, 14:0, and 16:0 by 9.3 and 5.8 g/100 g FAME ( P < 0.001) compared to winter diet, respectively.

A new arrangement of the INCAT (inside needle capillary adsorption trap) device with Carbopack X and Carboxen 1000 as sorbent materials was applied for sampling, preconcentration and injection of C6 C19 n-alkanes and their monomethyl analogs in exhaled breath samples. For the analysis both GC–MS/MS and GC × GC-FID techniques were used. Identification of the analytes was based on standards, measured retention indices and selective SRM transitions of the individual isomers. The GC–MS/MS detection limits were in the range from 2.1 pg for n-tetradecane to 86 pg for 5-methyloctadecane. The GC × GC-FID detection limits ranged from 19 pg for n-dodecane to 110 pg for 3-methyloctane.

A simple method for the simultaneous derivatization of carbohydrates, polyols, amines and amino acids using hexamethyldisilazane and N,O-bis(trimethylsilyl)trifluoroacetamide was developed. This method allows the direct derivatization of urine samples without sample pretreatment before derivatization. The method was successfully used for analysis of the selected metabolites in urine samples of healthy individuals and neonates suffering from galactosemia. The limits of detection by positive chemical ionization gas chromatography with tandem mass spectrometry analysis were in the range of 1.0 mgL-1 for mannitol to 4.7 mgL-1 for glucose. This article is protected by copyright. All rights reserved

A simple two-step method for the derivatization of polar compounds (lactate, alanine, glycerol, succinate and glucose) using hexamethyldisilazane (HMDS) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was developed. This method allows direct derivatization of aqueous samples wihout sample pretreatment. The method was used for the analysis of the metabolites of the unicellular organism Trypanosoma brucei. The limits of detection by GC–MS/MS analysis were in the range of 0.02 mgL−1 for glucose to 0.85 mgL−1 for lactate.

The experimental flock of 328 ewes belonging to three breeds (148 Tsigai, 124 Improved Valachian, and 56 Lacaune) with ewes’ parity 1–8 was grazed on natural pasture and milk was sampled on the same day in the morning. Fatty acid (FA) milk fat content in particular the content of conjugated linoleic acid (CLA), oleic acid (OA) and 12:0–16:0 saturated FA (SFA) was examined with regard to variation among individual ewes, breed, parity and milk yield. Individual ewes showed the most significant effect on the FA milk fat profile. The CLA milk fat content varied in the wide range of 5–26 mg g −1 FA, thus 5-fold among individual ewes. Similarly, the values of Δ9-desaturase indexes varied within a triple range. The ewes’ breed and parity had only little effect on some milk FA and atherogenicity index, however systematic trends were found. The ewes’ milk yield varied within a 12-fold range, and that of Lacaune ewes was significantly higher ( P P ≈ 0.05) between the milk yield and the CLA milk fat content were found in Tsigai and Lacaune breeds, and significant for Improved Valachian ( P P P P P P

The development of resistance to azole antifungals used in the treatment of fungal infections can be a serious medical problem. Here, we investigate the molecular mechanisms associated with reduced susceptibility to fluconazole in clinical isolates of Candida dubliniensis , showing evidence of the trailing growth phenomenon. The changes in membrane sterol composition were studied in the presence of subinhibitory fluconazole concentrations. Despite lanosterol and eburicol accumulating as the most prevalent sterols after fluconazole treatment, these ergosterol precursors still support growth of Candida isolates. The overexpression of ABC transporters was demonstrated by immunoblotting employing specific antibodies against Cdr1p and Cdr2p. The presence of a full-length 170 kDa protein Cdr1p was detected in two isolates, while a truncated form of Cdr1p with the molecular mass of 85 kDa was observed in isolate 966/3(2). Notably, Cdr2p was detected in this isolate, and the expression of this transporter was modulated by subinhibitory concentrations of fluconazole. These results suggest that C. dubliniensis can display the trailing growth phenomenon, and such isolates express similar molecular mechanisms like that of fluconazole-resistant isolates and can therefore be associated with recurrent infections.

Isomer mixtures of monomethyl branched saturated C7–C23 fatty acid methyl esters (FAME) were prepared by performing a methylene insertion reaction to the straight chain FAME and this study model was completed by using commercially available standards of C4–C7 FAME. The equivalent chain lengths (ECL) of all 220 C4–C23 monomethyl branched FAME on OV-1 stationary phase were measured, achieving an average repeatability of ±0.0004 ECL units. The monomethyl branched FAME was identified by GC on the basis of regularity of the fractional chain lengths (FCL) dependence on the number of carbon atoms (Cz) of individual homologous series of methyl 2-, 3-, …, 21-FAME. The prediction of retention of the first homologues, having the new position of methyl group beginning at higher carbon atoms number, and analogously for the second, third, fourth, and other members of the homologous series, allowed the dependence FCL = f(Cz) for the first and subsequent members of beginning homologous of monomethyl derivatives of FAME. The identification was confirmed by mass spectrometry. All of the methyl isomers of FAME, which could not be completely separated by gas chromatography due to having a methyl group in surroundings of the middle of the carbon chain, were resolved by mass spectrometry using deconvolution in a SIM-mode. Measured gas chromatographic and mass spectrometric data were applied for identification of the monomethyl branched saturated FAME in tongue coating.

Gas chromatographic analysis was used for the investigation of temporal variations of approximately 60 C4-C24 fatty acids (FA) in colostrum of individual ewes consuming a winter diet within 26 h after lambing. Attention was focused on variations in individual straight-chain saturated fatty acids (SSFA), branch-chain saturated fatty acids (BSFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA). The colostrum of individual ewes kept under identical living and feeding conditions displays high variability mainly in the content of individual SSFA and MUFA. At most a 50–110% increase in the content of 4:0, 6:0, 8:0, 10:0, 18:0 and trans -11 18:1, contrary to 17–91% decrease in the content of 12:0, 14:0, and 16:0 in individual ewes were determined during the first colostrum day. For some ewes, even reverse trends in temporal dependence of individual FA content were observed. For the most abundant MUFA cis -9 18:1 reverse trends in temporal dependences of Δ-9 desaturase index and those of content during first colostrum day were found.

Presented method for determination of sum of phthalates is based on their alkaline hydrolysis to phthalic acid at 80 °C for 20 h, followed by the selective extraction of lipophilic interferents from acidified hydrolysate at pH 1 with n -hexane. Phthalic acid is derivatized to dimethyl phthalate (DMP) with diazomethane in aqueous-chloroform two-phase system. Resulting DMP is absorbed in chloroform and determined by GC-FID. Method calibration resulted in LOD and LOQ of 0.4 (2.1) and 1.2 (6.2) μg g −1 (nmol g −1 ) DMP, respectively. Real samples of Baltic herring and codfish, butter, pork, goose and duck fats, sunflower, olive, rapeseed and linseed oils were analysed and the background corrected total phthalates content was found in the range from not detected level in duck fat to 12.5 (64.3) μg g −1 (nmol g −1 ) in butter, respectively.

Temporal content variations of approximately 70 C4–C24 fatty acids (FA) in colostrum and milk of ewes consuming winter diet were determined by gas chromatography. The content of monounsaturated, polyunsaturated and straight-chain saturated FA 14:0–16:0 in colostrum was higher whereas the content of 4:0–12:0, 18:0 and branched-chain saturated FA 15:0–17:0 was lower than that in mature milk. The effect of ewe breed on the FA profiles was not significant. The composition of FA changed most significantly 1–2 days after lambing in agreement with the time schedule of colostrum formation. Nevertheless, the content of FA further successively changed up to approximately the 6th day of lactation, whereas further changes up to the 60th day of lactation were smaller. This is consistent with the recommendation that ewes' milk is suitable for human consumption after six-to-eight days of lactation. The higher contents of palmitic and myristic acids in colostrum compared with mature milk suggest that FA composition in colostrum matches the changing needs of the growing lambs.

Chronic obstructive pulmonary disease (COPD) is a major cause of death worldwide. Acute exacerbations COPD (AECOPD), caused by infectious and non-infectious agents, contribute to an increase in mortality. The diagnostic procedure of AECOPD is mainly based on clinical features. The aim of this pilot study was to identify whether volatile organic compounds (VOCs) in breath could be used to discriminate for acute exacerbated COPD. Three patient groups were included in this controlled study: AECOPD patients (n = 14, age mean ± SD: 71.4 ± 7.46), stable COPD patients (n = 16, age mean ± SD: 66.9 ± 9.05) and healthy volunteers (n = 24, age mean ± SD: 28 ± 6.08). Breath samples were collected by optimizing a sampling strategy developed by us. These samples were then analyzed using a thermal desorption-gas chromatography-time of flight-mass spectrometer (TD-GC-ToF-MS). A total of 105 VOCs were identified in the breath samples. Relevant substances were subsequently selected by overall occurrence rate, the frequency of positive alveolar gradient (AG) (i.e. the difference in exhaled and inhaled VOCs concentration), exclusion of 'smoking related' VOCs and significant differences in AGs between the three groups. These steps dramatically reduced the number of relevant analytes and resulted in 12 key VOCs having discriminative values. The performance of patients' classification described by the Receiver Operating Characteristic (ROC) curve using all 12 substances delineates an area under the curve (AUC) of 0.97. A further reduction to four VOCs (AGs only different between AECOPD and COPD) delineates an AUC of 0.92. These results indicate that breath analysis with TD-GC-ToF-MS holds promise for an accurate and easy to perform differential diagnosis between AECOPD and COPD. In this regard, ketones were observed at the highest levels in exhaled breath of AECOPD, some of which are also related to potential bacterial pathogens. Using a set of VOCs that can discriminate for AECOPD, the calculated AUCs in ROC curve analysis show far superior results in comparison to serum AECOPD biomarkers, such as C-reactive protein. The identified VOCs should be further investigated in translational studies addressing their potential for developing highly specific nanosensors for breath gas analysis which would give clinicians a tool for non-invasive diagnosis of AECOPD at the point of care.

The profile variations of approximately 70 fatty acids (FAs) in milk of ewes consuming total mixed rations (TMR) and grazing natural pasture were determined. Milk of ewes grazing pasture in May and September in the Slovak Republic contained 3-times more conjugated linoleic acid (CLA) (P 0.05). No significant differences in the FA composition in milk of ewes grazing on summer pasture at higher altitude were observed.

A generally known problem of GC separation of trans-7 ; cis-9 ; cis-9 , trans-11 ; and trans-8 , cis-10 CLA (conjugated linoleic acid) isomers was studied by GC–MS on 100 m capillary column coated with cyanopropyl silicone phase at isothermal column temperatures in a range of 140–170 °C. The resolution of these CLA isomers obtained at given conditions was not high enough for direct quantitative analysis, but it was, however, sufficient for the determination of their peak areas by commercial deconvolution software. Resolution factors of overlapped CLA isomers determined by the separation of a model CLA mixture prepared by mixing of a commercial CLA mixture and CLA isomer fraction obtained by the HPLC semi-preparative separation of milk fatty acids methyl esters were used to validate the deconvolution procedure. Developed deconvolution procedure allowed the determination of the content of studied CLA isomers in ewes’ and cows’ milk samples, where dominant isomer cis-9 , trans-11 is eluted between two small isomers trans-7 , cis-9 and trans-8 , cis-10 (in the ratio up to 1:100).

The relations between fatty acids (FAs) composition of pasture forage plants and the content of conjugated linoleic acid (CLA) as a total content of cis -9, trans -11 + trans -7, cis -9 + trans -8, cis- 10 CLA isomers in ewes’ milk fat during natural pasture season (April–September) were investigated. The extracts of ewes’ milk fat samples as well as the pasture samples were analyzed for fatty acid composition by capillary gas chromatography with flame ionization and mass spectrometric detection. α-Linolenic, linoleic, and palmitic acids were predominant in pasture plants, and their contents varied during pasture season. The most abundant and most varied fatty acid compound in pasture plants was α-linolenic acid. Its content significantly decreased from 62% to 39% (of total FA) ( P P P P P P

A GC/MS procedure has been developed, optimized, and applied to characterization of oil binders in paintings. The procedure involves hydrolysis of lipids to fatty acids (FAs) and derivatization of FAs to fatty acid methyl esters (FAMEs) by a solution of sodium methanolate in methanol at an elevated temperature. FAMEs are analyzed by temperature-programed GC followed by full-scan MS. Old and dried samples are subjected to extraction of nonpolymerized FAMEs into dichloromethane prior to hydrolysis. The method provides a good repeatability of results and has been applied to the characterization of common plant oils used in paintings, to commercial oil and tempera paints, to model painting samples, and to samples taken from real paintings. The fresh oils and binders can readily be identified and characterized. The ratio of the methyl esters of palmitic and stearic acids can be used to characterize oil binders in old works of art.

Summary To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target., Graphical Abstract, Highlights • Two compounds activated by EthA kill M. tuberculosis through PyrG inhibition • EthA metabolite is active against PyrG and M. tuberculosis growth • Definition of the mechanism of activation and validation of PyrG as a new drug target, CTP synthetase PyrG, essential in Mycobacterium tuberculosis, could represent a new potential drug target. With a multidisciplinary approach, Mori et al. identify two compounds killing growing and dormant mycobacteria through PyrG inhibition, and define their mechanism of action.

Methods based on the blank signal as proposed by IUPAC procedure and on the signal to noise ratio (S/N) as listed in the ISO-11843-1 norm for determination of the limit of detection (LOD) and quantitation (LOQ) in one-dimensional capillary gas chromatography (1D-GC) and comprehensive two-dimensional capillary gas chromatography (CG×GC) are described in detail and compared for both techniques. Flame ionization detection was applied and variables were the data acquisition frequency and, for CG×GC, also the modulation time. It has been stated that LOD and LOQ estimated according to IUPAC might be successfully used for 1D-GC-FID method. Moreover, LOD and LOQ decrease with decrease of data acquisition frequency (DAF). For GC×GC-FID, estimation of LOD by IUPAC gave poor reproducibility of results while for LOQ reproducibility was acceptable (within ±10% rel.). The LOD and LOQ determined by the S/N concept both for 1D-GC-FID and GC×GC-FID methods are ca. three times higher than those values estimated by the standard deviation of the blank. Since the distribution pattern of modulated peaks for any analyte separated by GC×GC is random and cannot be predicted, LOQ and LOD may vary within 30% for 3s modulation time. Concerning sensitivity, 1D-GC-FID at 2Hz and of GC×GC-FID at 50Hz shows a ca. 5 times enhancement of sensitivity in the modulated signal output.

Solvent-free chlorination of biphenyl and naphthalene was performed under mechanochemical stressing by high-energy ball milling (HEM) of a mixture of CuCl2 (95 wt %) and the hydrocarbon (5 wt %). The reactivity of the selected hydrocarbons towards CuCl2 during HEM partially correlates with their ionisation potentials (IP): hexadecane (9.91 eV) > biphenyl (8.27 eV) > naphthalene (8.14 eV).

The stereoselective syntheses of heptaprenylphosphoryl β- d -arabinofuranose and heptaprenylphosphoryl β- d -ribofuranose are described. In the synthesis of the d -arabino product, the stereoselectivity was achieved by the coupling of a suitably protected β- d -arabinofuranosyl phosphate intermediate with an activated form of heptaprenol and subsequent deprotection. In the case of the ribo-analog, the desired β-anomer could be obtained by the more convenient phosphoramidite method. The products were successfully employed in the mycobacterial epimerase assay.

The role of archaeal membrane and its lipid constituents was investigated in bioenergetic functions of Methanothermobacter thermautotrophicus. The effects were determined of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, pravastatin, on lipid composition, and its impact on some bioenergetic functions of treated cells. Pravastatin remarkably inhibited the growth of M. thermautotrophicus. On membrane level, pravastatin treatment modulated the composition of the mixture of squalene and hydrosqualene derivatives as well as the activities of ATPase, A1Ao-ATP synthase and Na+/H+ antiporter. SDS-PAGE of chloroform-methanol extracts of membranes from control and pravastatin-treated cells revealed changes in the amount of AtpK proteolipids, which suggests that pravastatin modifies cell-membrane composition, hereby modulating the properties of some membrane-bound enzymes participating in energy transformation in methanoarchaea.

A newly designed needle trap device with Carbopack X as a sorbent material is used for sampling, preconcentration and injection of volatile analytes benzene, toluene, ethylbenzene and xylenes (BTEX) into gas chromatograph. The closed system of stripping the analytes from water samples was used. An injection port with a modified metal liner was used to desorb analytes trapped in needle trap device. The main advantage of needle trap device consists in the simple methodology and easiness and rapidity of the analysis. Needle trap device is suitable for sampling in field. The experimental parameters as breakthrough volume of stripping gas, linearity, repeatability and limit of detection (LOD) and quantification (LOQ) were investigated. LOD ranges from 0.05 to 0.07 μg L −1 and relative standard deviation ranges from 0.5% to 11.6% at concentrations 5 and 0.1 μg L −1 , respectively.

An overview of the existing methods for minimization of the analysis time in gas chromatography (GC) is presented and a new system for fast temperature programming and very fast cooling down is evaluated. In this study, a system of coaxial tubes, a heating/cooling module (HC-M), was developed and studied with a capillary column placed inside the HC-M. The module itself was heated by a GC oven and cooled down by an external cooling medium. The HC-M was heated at rates of up to 330 °C min−1 and cooled at the rate of 6000 °C min−1. The GC system was prepared for the next run within a few seconds. The HC-M permits good separation reproducibility, comparable with that of a conventional GC, expressed in terms of relative retention times and peak areas of analytes reproducibilities. The HC-M can be used within any commercial gas chromatograph.

The biochemical basis of a defective bioenergetic system was attempted to be determined in N,N′-dicyclohexylcarbodiimide (DCCD)-resistant mutant of Methanothermobacter thermautotrophicus. Components participating in the maintenance of methanoarchaeal membrane structure and function, such as the composition of the mixture of squalene and its hydrosqualene derivatives and also properties of membrane-associated proteins were compared in wild-type and mutant cells. The impairment of the bioenergetic system in DCCD-resistant mutant was detectable in the membrane-protein profile; it was also accompanied by changes in proportions of squalene-hydrosqualenes.