Evaluation of exudates by solid phase microextraction-gas chromatography

Head-space solid phase microextraction combined with gas chromatography (SPME-GC) was used for the determination of bacterial volatile fatty acid (VFA) patterns. The method was validated with cultures of reference bacterial strains. It was confirmed that VFA production depends on the composition of the cultivation medium, which limits accurate characterisation of particular bacterial species. A set of 195 clinical exudates of various origin and consistence was analysed using SPME-GC and compared with 73 samples extracted using tert-butyl methyl ether. Approximate agreement of VFA profiles with cultivation findings was found in most cases. However, 20.5% of clinical exudates with distinct VFA profiles appeared to be false-negative by cultivation. Using SPME-GC of exudates, the frequency of false-negative cultivations was higher than that of solvent extraction of exudates or blood cultures found previously. The described method is suitable for preliminary detection of bacteria, particularly non-sporulating anaerobes, in clinical samples. It can reveal false-negative findings due to cultivation. Analysis can be performed in 30 min without the need for cultivation.     

Paleoenvironmental model for Eocene foraminiferal limestones of the Adriatic carbonate platform (Istrian Peninsula)

The foraminiferal limestones from the Middle Eocene Central Istrian region illustrate progressive deepening of depositional gradients. Shifting of Lower Cuisian to Upper Lutetian microfacies can be described in terms of a ramp model. The Orthophragminae-bearing parts of the foraminiferal limestones are interpreted in terms of larger foraminiferal faunal associations, planktonic foraminiferal relative abundance, limitations of algal endosymbionts, foraminiferal lamellar thickness and flattening of test shapes. Microfacies I contains the most diverse larger foraminiferal association with a predominance of large, thick nummulitids, assilinids, and asterocyclinids. Microfacies II is characterized by a gradual increase of Orthophragminae diversity and abundance. Nummulitids, equally abundant, are dominated by lenticular and subspherical specimens. The reduction in number of nummulitid specimens with characteristic biconical radiate morphologies, and relative abundance of flattened orthophragminids, characterizes Microfacies III. Scattered biodestructed orthophragminid tests and planktonic foraminifera constitute Microfacies IV, indicating the end of a long-lasting, shallow-marine Adriatic Carbonate Platform regime.     

Interpretation of the last‐glacial vegetation of eastern‐central Europe using modern analogues from southern Siberia

Aim Interpretation of fossil pollen assemblages may benefit greatly from comparisons with modern palynological and vegetation analogues. To interpret the full‐ and late‐glacial vegetation in eastern‐central Europe we compared fossil pollen assemblages from this region with modern pollen assemblages from various vegetation types in southern Siberia, which presumably include the closest modern analogues of the last‐glacial vegetation of central Europe. Location Czech and Slovak Republics (fossil pollen assemblages); Western Sayan Mountains, southern Siberia (modern pollen assemblages). Methods Eighty‐eight modern pollen spectra were sampled in 14 vegetation types of Siberian forest, tundra and steppe, and compared with the last‐glacial pollen spectra from seven central European localities using principal components analysis. Results Both full‐ and late‐glacial pollen spectra from the valleys of the Western Carpathians (altitudes 350–610 m) are similar to modern pollen spectra from southern Siberian taiga, hemiboreal forest and dwarf‐birch tundra. The full‐glacial and early late‐glacial pollen spectra from lowland river valleys in the Bohemian Massif (altitudes 185–190 m) also indicate the presence of patches of hemiboreal forest or taiga. Other late‐glacial pollen spectra from the Bohemian Massif suggest an open landscape with steppe or tundra or a mosaic of both, possibly with small patches of hemiboreal forest. Main conclusions Our results are consistent with the hypothesis that during the full glacial and late glacial, the mountain valleys of the north‐western Carpathians supported taiga or hemiboreal forest dominated by Larix, Pinus cembra, Pinus sylvestris and Picea, along with some steppic or tundra formations. Forests tended to be increasingly open or patchy towards the west (Moravian lowlands), gradually passing into the generally treeless landscape of Bohemia, with possible woodland patches in locally favourable sites.     

The potential pitfalls of using 1,1-diphenyl-2-picrylhydrazyl to characterize antioxidants in mixed water solvents

Approaching living systems, aqueous solutions are appropriate to characterize antioxidants, whereas the frequently used standard 1,1-diphenyl-2-picrylhydrazyl (DPPH) is insoluble in water. Therefore, mixed water-ethanol solvents were investigated using the electron paramagnetic resonance (EPR) spectroscopy. Two forms of DPPH were identified: at higher ethanol ratios a quintet spectrum characteristic of solutions, and at lower ratios, a singlet spectrum typical for solid DPPH, were found. Mixed solvents with 0-50% (v/v) water reproduced the same antioxidant equivalent points well and the reaction rate between DPPH and the antioxidant may increase considerably with increasing water ratios, as demonstrated using vitamin E as an antioxidant. But at still higher water ratios (70-90% (v/v)) the antioxidant activities dropped, since a part of the DPPH in the aggregated form does not react sufficiently with the antioxidants. Characteristics of the most common antioxidants were determined in ethanol or its 50% (v/v) aqueous solution.     

Study of the oxidative stress in a rat model of chronic brain hypoperfusion

A multiple analysis of the cerebral oxidative stress was performed on a physiological model of dementia accomplished by three-vessel occlusion in aged rats. The forward rate constant of creatine kinase, k_for, was studied by saturation transfer ³¹P magnetic resonance spectroscopy in adult and aged rat brain during chronic hypoperfusion. In addition, free radicals in aging rat brain homogenates before and/or after occlusion were investigated by spin-trapping electron paramagnetic resonance spectroscopy (EPR). Finally, biochemical measurements of oxidative phosphorylation parameters in the above physiological model were performed. The significant reduction of k_for in rat brain compared to controls 2 and 10 weeks after occlusion indicates a disorder in brain energy metabolism. This result is consistent with the decrease of the coefficient of oxidative phosphorylation (ADP:O), and the oxidative phosphorylation rate measured in vitro on brain mitochondria. The EPR study showed a significant increase of the ascorbyl free radical concentration in this animal model. Application of -phenyl-N-tert-butylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin traps revealed formation of highly reactive hydroxyl radical (OH) trapped in DMSO as the CH₃ adduct. It was concluded that the ascorbate as a major antioxidant in brain seems to be useful in monitoring chronic cerebral hypoperfusion.     

Efficacy of S-adenosylhomocysteine hydrolase inhibitors, D-eritadenine and (S)-DHPA, against the growth of Cryptosporidium parvum in vitro

D-eritadenine and (S)-DHPA are aliphatic adenosine analogues known to target S-adenosylhomocysteine hydrolase (SAHH) and potent antiviral compounds. In the present study, we demonstrate that these two compounds also display efficacy against recombinant SAHH enzyme of the protozoan parasite Cryptosporidium parvum, as well as inhibition of parasite growth in vitro. Our data confirm that SAHH could serve as a rational drug target in cryptosporidial infection and antiviral adenosine analogues are potential candidates for drug development against cryptosporidiosis.     

Bronchoalveolar lavage examined by solid phase microextraction, gas chromatography--mass spectrometry and selected ion flow tube mass spectrometry

Samples (210 in total) of broncholaveolar lavages (BALs), obtained from patients hospitalized with pneumonia in various departments of two hospitals, were analysed using the method of solid phase microextraction-gas chromatography (SPME-GC) with FID detection. Up to 20% (9% unequivocally, 11% probably) of these samples was found to contain volatile fatty acids (VFAs) in the series from acetic acid to heptanoic acid. Importantly, the presence of these acids indicates the presence of fermenting anaerobic bacteria, which were not detected by the conventional microbiological examination. Other compounds, namely the heptanol and cyclohexanone, were also detected by this method in some samples. Cyclohexanone occurred almost exclusively in samples from patients receiving intensive care with mechanical ventilation, and is suspected to originate from plastic parts of ventilators. Selected representative samples were also analysed using further methods, namely gas chromatography-mass spectrometry (GC-MS) of native and silylated samples, and selected ion flow tube mass spectrometry (SIFT-MS). These methods confirmed the identities of above mentioned compounds, and detected numerous other compounds tentatively identified as various alcohols, aldehydes, ketones, esters and hydrogen cyanide, HCN. Most of these compounds occurred in small amounts and their origin and diagnostic significance remains uncertain, except, that is, for the HCN, which indicates the presence of Pseudomonas aeruginosa.     

Condom use and its consistency among metropolitan high school students in Croatia, 1997-2001: has anything changed?

Sexually active adolescents face serious health risks associated with unprotected sexual intercourse, such as HIV infection and other sexually transmitted diseases (STDs), as well as unwanted pregnancy. To assess protective behavior among urban adolescents in Croatia we surveyed over 4000 high-school students, 15-19 years of age, in two waves: 2070 in 1997 and 1972 in 2001. The survey was carried out in various types of secondary schools in the capital city. The analyses suggest statistically significant increase in HIVIAIDS related knowledge in the observed period. A slight but significant increase was also observed in the acceptance of condom use. Both changes were more substantial among female students. In regards to protective behavior, we found significant increase in condom use, as well as in its consistency. Still, a large proportion of students did not use condom at last sexual intercourse (40% in 2001). In addition to generational effect, number of sexual partners, sexual victimization and mother's education (only in the case of young women) were identified as correlates of condom use consistency. While mother's education had positive effect, having 4 or more partners and having survived sexual violence decreased the likelihood of consistent condom use. In conclusion, the authors discuss the relevance of systematic school-based prevention of sexual risk taking among Croatian adolescents.     

Comparison of the effect of individual saturated and unsaturated fatty acids on cell growth and death induction in the human pancreatic beta-cell line NES2Y

We tested the effects of various types of fatty acids, differing in the degree of saturation and in the cis/trans configuration of the double bond, on the growth and viability of NES2Y cells (a human pancreatic beta-cell line). We found that during a 48-hour incubation period, saturated fatty acids, i.e. palmitic and stearic acids, at a physiologically relevant concentration of 1 mM and higher concentrations induced death of the beta-cells while their counterpart unsaturated fatty acids, i.e. palmitoleic and oleic acids, did not induce cell death at concentrations up to 3 mM. We also found that unsaturated elaidic acid with a trans double bond exerted significant inhibition of growth of the beta-cells at a concentration approximately ten times lower, i.e. 0.1 mM vs. 1 mM, than counterpart oleic acid with a cis double bond. This is the first direct evidence that a trans unsaturated fatty acid is significantly more effective in inhibiting beta-cell growth than a counterpart cis unsaturated fatty acid. Furthermore, we newly demonstrated that beta-cell death induced by saturated fatty acids is related to significant increase of caspase-2 activity (2 to 5-fold increase) but not to caspase-3 activation. The growth-inhibiting effect of saturated fatty acids at concentrations lower than death-inducing concentrations correlates with certain increase of caspase-2 activity.