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2,4,6-trichloroanisole and 2,4,6-tribromoanisole were investigated by means of electron transmission spectroscopy and two different types of dissociative electron attachment spectrometers. The results obtained were interpreted with the support of density functional theory calculations. The dominant dissociative decay channels of the temporary molecular negative ions lead to the formation of Cl- and Br-in the low electron energy region. Formation of long-lived parent anions is observed at thermal electron energies. Their relative intensity depends on the experimental time window, ∼36 μs in the case of the static magnet mass analyzer and ∼200 μs for the quadrupole mass analyzer employed. The results obtained may be useful for rapid detection of these compounds in wine and pharmaceutical industries, as well as other branches connected to the food industry, e.g., packaging. [ABSTRACT FROM AUTHOR]

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Spinal cord injury (SCI) resulting from trauma decreases the quality of human life. Numerous clues indicate that the limited endogenous regenerative potential is a result of the interplay between the inhibitory nature of mature nervous tissue and the inflammatory actions of immune and glial cells. Knowledge gained from comparing regeneration in adult and juvenile animals could draw attention to factors that should be removed or added for effective therapy in adults. Therefore, we generated a minimal SCI (mSCI) model with a comparable impact on the spinal cord of Wistar rats during adulthood, preadolescence, and the neonatal period. The mechanism of injury is based on unilateral incision with a 20 ga needle tip according to stereotaxic coordinates into the dorsal horn of the L4 lumbar spinal segment. The incision should harm a similar amount of gray matter on a coronal section in each group of experimental animals. According to our results, the impact causes mild injury with minimal adverse effects on the neurological functions of animals but still has a remarkable effect on nervous tissue and its cellular and humoral components. Testing the mSCI model in adults, preadolescents, and neonates revealed a rather anti-inflammatory response of immune cells and astrocytes at the lesion site, as well as increased proliferation in the central canal lining in neonates compared with adult animals. Our results indicate that developing nervous tissue could possess superior reparative potential and confirm the importance of comparative studies to advance in the field of neuroregeneration., (© 2024 Wiley Periodicals LLC.)

In the rat, tail nerves are the longest peripheral nerves in their body. We suggest that ventral caudal nerve (VCN) may serve as a model for studying nerve injury and long distance regeneration. For this purpose, we have studied the anatomy and morphometry of the VCN in control animals. 10 cm long segment of the VCN was removed, and transversal sections were collected at 10 mm distances. The myelinated axons were counted, and the series of data were used to characterize the craniocaudal tapering of the nerve. In a separate group of animals, retrograde tracing with Fluorogold was used to localize and quantitate the spinal neurons projecting their axons into the VCN. After complete nerve transection, the time course of histopathological changes in the distal segment was studied. The primary goal was to define the time needed for axonal disintegration. In later periods, axonal debris removal and rearrangement of tissue elements was documented. After compression injury (axonotmesis), Wallerian degeneration was followed by spontaneous regeneration of axons. We show that the growing axons will span the 10 cm distance within 4-8 weeks. After different survival periods, the numbers of regenerating axons were counted at 10 mm distances. These data were used to characterize the dynamics of axonal regeneration during 4 months' survival period. In the present study we show that axonal regeneration across 10 cm distance can be studied and quantitatively analyzed in a small laboratory animal., Competing Interests: None., (© 2024 The Authors.)

In this study, the bone marrow mesenchymal stem cells conditioned media (BMMSC-CM) obtained by conditioning for 24(CM24), 48(CM48) and 72(CM72) hours was characterised. In vitro, the impact of BMMSC-CM on the astrocyte migratory response and oligodendrocyte density was evaluated using the scratch model. The proteomic profiles of individual secretomes were analysed by mass spectrometry and the concentrations of four selected neurotrophins (BDNF, NGF, GDNF and VEGF) were determined by ELISA. Our results revealed an increased number of proteins at CM72, many of which are involved in neuroregenerative processes. ELISA documented a gradual increase in the concentration of two neurotrophins (NGF, VEGF), peaking at CM72. In vitro , the different effect of individual BMMSC-CM on astrocyte migration response and oligodendrocyte density was observed, most pronounced with CM72. The outcomes demonstrate that the prolonged conditioning results in increased release of detectable proteins, neurotrophic factors concentration and stronger effect on reparative processes in neural cell cultures.

The aim of this paper was to describe the influence of high-shear wet granulation process parameters on tablet tensile strength and compaction behavior of a powder mixture and granules containing hydralazine. The hydralazine powder mixture and eight types of granules were compacted into tablets and evaluated using the Heckel, Kawakita and Adams analyses. The granules were created using two types of granulation liquid (distilled water and aqueous solution of polyvinylpyrrolidone), at different impeller speeds (500 and 700 rpm) and with different wet massing times (without wet massing and for 2 min). Granulation resulted in improved compressibility, reduced dustiness and narrower particle-size distribution. A significant influence of wet massing time on parameters from the Kawakita and Adams analysis was found. Wet massing time had an equally significant effect on tablet tensile strength, regardless of the granulation liquid used. Granules formed with the same wet massing time showed the same trends in tabletability graphs. Tablets created using a single-tablet press (batch compaction) and an eccentric tablet press showed opposite values of tensile strength. Tablets from granules with a higher bulk density showed lower strength during batch compaction and, conversely, higher strength during eccentric tableting., Competing Interests: The authors declare no conflict of interest.

Chemotherapy-induced peripheral neuropathy is one of the most frequent dose-limiting side effects, observed in patients receiving antineoplastic agents, persisting for up to two years after completing treatment, greatly affecting both the course of chemotherapy and patients' quality of life. Approximately 20 to 85% of patients treated with neurotoxic chemotherapy will develop peripheral neuropathy and there is considerable variability in its severity among patients. The main symptoms are numbness, paresthesia, and burning pain in a "glove and stocking" distribution. The prevalence of chemotherapy-induced peripheral neuropathy will likely increase as cancer survival rates continue to improve. Currently, there are only a few therapeutic options available for the prevention or successful therapy because the mechanisms of chemotherapy-induced peripheral neuropathy remain unclear. A better understanding of the risk factors and underlying mechanisms of chemotherapy-induced peripheral neuropathy is needed to develop effective preventive and therapeutic strategies.

Thymol affects various types of tumor cell lines, including colorectal cancer cells. However, the hydrophobic properties of thymol prevent its wider use. Therefore, new derivatives (acetic acid thymol ester, thymol β-D-glucoside) have been synthesized with respect to hydrophilic properties. The cytotoxic effect of the new derivatives on the colorectal cancer cell lines HT-29 and HCT-116 was assessed via MTT assay. The genotoxic effect was determined by comet assay and micronucleus analysis. ROS production was evaluated using ROS-Glo™ H 2 O 2 Assay. We confirmed that one of the thymol derivatives (acetic acid thymol ester) has the potential to have a cyto/genotoxic effect on colorectal cancer cells, even at much lower (IC 50 ~0.08 μg/mL) concentrations than standard thymol (IC 50 ~60 μg/mL) after 24 h of treatment. On the other side, the genotoxic effect of the second studied derivative-thymol β-D-glucoside was observed at a concentration of about 1000 μg/mL. The antiproliferative effect of studied derivatives of thymol on the colorectal cancer cell lines was found to be both dose- and time-dependent at 100 h. Moreover, thymol derivative-treated cells did not show any significantly increased rate of micronuclei formation. New derivatives of thymol significantly increased ROS production too. The results confirmed that the effect of the derivative on tumor cells depends on its chemical structure, but further detailed research is needed. However, thymol and its derivatives have great potential in the prevention and treatment of colorectal cancer, which remains one of the most common cancers in the world.

In this paper, we describe a protocol for a non-penetrating embedding matrix that can be used for frozen or vibratome sectioning of various formaldehyde-fixed tissue specimens. In our experiments, we wanted to prepare thin frozen sections from miniature specimens for fluorescent staining. As we could not achieve satisfactory results with any of the previously published methods, we have tried to modify the existing protocols, and systematically evaluated the effect of these modifications on the properties of the embedding matrix. The resulting protocol is simple, the matrix gets firmly attached to the tissues, does not cause autofluorescence and enables preparing extremely thin frozen sections. The matrix can be used for 1, embedding miniature specimens from problematic tissues to enable cutting very thin frozen sections, 2, grouping multiple specimens into one large block for simultaneous processing, and 3, dispersing single cells and preparing cell blocks for frozen sectioning., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The aim of this study was to develop immobilized enzyme systems that reduce carbonyl compounds to their corresponding alcohols. The demand for natural aromas and food additives has been constantly growing in recent years. However, it can no longer be met by extraction and isolation from natural materials. One way to increase the availability of natural aromas is to prepare them by the enzymatic transformation of suitable precursors. Recombinant enzymes are currently being used for this purpose. We investigated trans- 2-hexenal bioreduction by recombinant Saccharomyces cerevisiae alcohol dehydrogenase (ScADH1) with simultaneous NADH regeneration by recombinant Candida boidinii formate dehydrogenase (FDH). In a laboratory bioreactor with two immobilized enzymes, 88% of the trans- 2-hexenal was transformed to trans- 2-hexenol. The initial substrate concentration was 3.7 mM. The aldehyde destabilized ScADH1 by eluting Zn 2+ ions from the enzyme. A fed-batch operation was used and the trans- 2-hexenal concentration was maintained at a low level to limit the negative effect of Zn 2+ ion elution from the immobilized ScADH1. Another immobilized two-enzyme system was used to reduce acetophenone to (S)-1-phenylethanol. To this end, the recombinant alcohol dehydrogenase (RrADH) from Rhodococcus ruber was used. This biocatalytic system converted 61% of the acetophenone to (S)-1-phenylethanol. The initial substrate concentration was 8.3 mM. All enzymes were immobilized by poly-His tag to Ni 2+ , which formed strong but reversible bonds that enabled carrier reuse after the loss of enzyme activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Varga, Štefuca, Mihálová, Levarski, Struhárňanská, Blaško, Kubinec, Farkaš, Sitkey, Turňa, Rosenberg and Stuchlík.)

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., Competing Interests: The authors declare no competing interest.

We present a method that allows preparing histological sections from large blocks of nervous tissue embedded in epoxy resin. Resin-embedding provides excellent resolution especially for the myelin-rich white matter and is often being used for visualizing the myelinated axons in peripheral nerves. However, because of the limited penetration of the reagents, only very small tissue specimens can be processed in this way. Here, we describe a method that enables to embed large specimens and their sectioning on a standard sliding microtome. To process the large specimens, modifications in several steps of the processing technique had to be made. In this paper we demonstrate, that with this technique 1-3 μm thick transversal sections can be prepared from the resin-embedded specimens as large as rat brain hemisphere. Such a large section allows simultaneously: 1.) overviewing and delineating the gross anatomical structures, and 2.) observing the subcellular details at the highest possible optical magnifications. Such a large section with excellent resolution allows application of unbiased stereological methods and reliable quantification of very small objects within the area of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare there are no competing interests., (©2020 Kubinec et al.)

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 2 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.

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.

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 mg/L for glucose., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Despite strong efforts in the field, spinal cord trauma still belongs among the untreatable neurological conditions at present. Given the complexity of the nervous system, an effective therapy leading to complete recovery has still not been found. One of the potential tools for supporting tissue regeneration may be found in mesenchymal stem cells, which possess anti‑inflammatory and trophic factor‑producing properties. In the context of transplantations, application of degradable biomaterials which could form a supportive environment and scaffold to bridge the lesion area represents another attractive strategy. In the present study, through a combination of these two approaches we applied both alginate hydrogel biomaterial alone or allogenic transplants of MSCs isolated from bone marrow seeded in alginate biomaterial into injured rat spinal cord at three weeks after spinal cord compression performed at Th8‑9 level. Following three‑week survival, using immunohistochemistry we studied axonal growth (GAP‑43 expression) and both microglia (Iba‑1) and astrocyte (GFAP) reactions at the lesion site and in the segments below and above the lesion. To detect functional improvement, during whole survival period we performed behavioral analyses of locomotor abilities using a classical open field test (BBB score) and a Catwalk automated gait analyzing device (Noldus). We found that despite the absence of locomotor improvement, application of both alginate and MSCs caused significant increase in the number of GAP‑43 positive axons.

Unlabelled: 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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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 C6C19n-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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A combination of chemical genetic and biochemical assays was applied to investigate the mechanism of action of the anticancer drug 5-fluorouracil (5-FU), against Mycobacterium tuberculosis (Mtb). 5-FU resistance was associated with mutations in upp or pyrR. Upp-catalyzed conversion of 5-FU to FUMP was shown to constitute the first step in the mechanism of action, and resistance conferred by nonsynonymous SNPs in pyrR shown to be due to derepression of the pyr operon and rescue from the toxic effects of FUMP and downstream antimetabolites through de novo production of UMP. 5-FU-derived metabolites identified in Mtb were consistent with the observed incorporation of 5-FU into RNA and DNA and the reduced amount of mycolyl arabinogalactan peptidoglycan in 5-FU-treated cells. Conditional depletion of the essential thymidylate synthase ThyX resulted in modest hypersensitivity to 5-FU, implicating inhibition of ThyX by fluorodeoxyuridylate as a further component of the mechanism of antimycobacterial action of this drug., (Copyright © 2015 Elsevier Ltd. 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 mg L(-1) for glucose to 0.85 mg L(-1) for lactate., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Previously it has been demonstrated that processes of postnatal neurogenesis in the olfactory system neurogenic region-the subventricular zone (SVZ), rostral migratory stream (RMS), and olfactory bulb (OB) can be significantly altered by different factors of an environment. However, the mechanisms involved in regulation of neurogenesis by exogenous factors in the olfactory system remain unclear. The purpose of the present study was to contribute to the understanding of these mechanisms by immunohistochemical assessment of Fos protein induction in areas of adult neurogenesis. To evaluate the coordinate activation of Fos production in neurons of the olfactory system neurogenic region, a brief exposure to artificial odor (eau de Cologne) or naturalistic odor (cat odor) has been used in alert rats. Our results revealed that the effects of these odors are easily distinguishable at both the behavioral and the morphological level. Cat odor induced greater changes in anxiety level, and produced typical pattern of Fos activation in the accessory olfactory bulb (AOB), a brain region associated with defensive behavior. An important finding is, that next to distinct Fos expression in the OB and the AOB, Fos positive cells have been found also within the SVZ/RMS of the odor stimulated rats. Interestingly, Fos expression in the RMS was detected only after exposure to artificial odor stimulus. These results provide new evidence that some SVZ/RMS cells have complete prerequisites necessary for the Fos signal transduction cascade., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The secondary damage that follows central nervous system (CNS) injury is a target for neuroprotective agents aimed at tissue and function sparing. FK506, a clinically used immunosuppressant, acts neuroprotectively in rat models of brain and spinal cord injury and ischemia. Evidence of in vivo experimental studies highlights the neuroprotective role of FK506 by its direct impact on various cell populations within the CNS. The participation of FK506 in modulation of post-traumatic inflammatory processes is a further potential aspect involved in CNS neuroprotection. In this review we provide an overview of the current laboratory research focusing on the multiple effects of FK506 on neuroprotection following CNS injury., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The developed method for trace analysis of volatile components in plasma allows direct injection of up to 150 samples to the GC-MS/MS system without injector cleaning. This method requires no modification of plasma and the working environment does not interfere with the determination of these analytes. The method allows simultaneous quantification of non-polar sevoflurane and its polar metabolite hexafluoroisopropanol (free, unconjugated form). It is characterized by high repeatability and sensitivity with the detection limit of 0.009 mg L(-1) for sevoflurane and 0.018 mg L(-1) for hexafluoroisopropanol and the linear range 0.050-150 mg L(-1). The method was used to determine the concentration of sevoflurane and hexafluoroisopropanol in plasma samples of 7 patients undergoing general anesthesia with sevoflurane. The average concentration of sevoflurane and free hexafluoroisopropanol was 57.2 mg L(-1) and 0.39 mg L(-1), respectively. The method can be applied for clinical monitoring, as well as for analytical toxicology., (Copyright © 2011. Published by Elsevier B.V.)

The immediate effects of whole body electromagnetic radiation (EMR) were used to study postnatal neurogenesis in the subventricular zone (SVZ) and rostral migratory stream (RMS) of Wistar rats of both sexes. Newborn postnatal day 7 (P7) and young adult rats (P28) were exposed to pulsed electromagnetic fields (EMF) at a frequency of 2.45 GHz and mean power density of 2.8 mW/cm(2) for 2 h. Post-irradiation changes were studied using immunohistochemical localization of Fos and NADPH-d. We found that short-duration exposure induces increased Fos immunoreactivity selectively in cells of the SVZ of P7 and P28 rats. There were no Fos positive cells visible within the RMS of irradiated rats. These findings indicate that some differences exist in prerequisites of proliferating cells between the SVZ and RMS regardless of the age of the rats. Short-duration exposure also caused praecox maturation of NADPH-d positive cells within the RMS of P7 rats. The NADPH-d positive cells appeared several days earlier than in age-matched controls, and their number and morphology showed characteristics of adult rats. On the other hand, in the young adult P28 rats, EMR induced morphological signs typical of early postnatal age. These findings indicate that EMR causes age-related changes in the production of nitric oxide (NO), which may lead to different courses of the proliferation cascade in newborn and young adult neurogenesis., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

The olfactory bulb is one of a few brain structures characterized by high plasticity due to the fact that new neurons are continually integrated into the olfactory bulb circuit throughout life. The new cells originate from the subventricular zone of the forebrain and migrate through the rostral migratory stream (RMS) to the olfactory bulb that also represents the first synaptic relay of the olfactory system. Data accumulating in recent years have confirmed that sensory inputs can influence the level of postnatal neurogenesis in the olfactory bulb. In this study, we studied neurogenesis in the rostral migratory stream of Wistar albino rat pups after exposure to an odor-enriched environment. The rats were olfactory stimulated twice daily with different odorants from the day of their birth up to 1, 2 or 3 weeks, respectively. Using bromodeoxyuridine, a marker of cell proliferation, we found an increased number of proliferating cells in the rostral migratory stream of rat pups submitted to olfactory stimulation. Conversely, the number of dying cells, labeled with the fluorescent dye Fluoro Jade-C, was down-regulated in groups of rats exposed to an odor-enriched environment., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

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 (C(z)) 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(C(z)) 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., (Copyright © 2011 Elsevier B.V. All rights reserved.)