Simultaneous determination and differentiation of glycidyl esters and 3-monochloropropane-1,2-diol (MCPD) esters in different foodstuffs by GC-MS

The aim of this study was the development of a method for the simultaneous determination and differentiation of fatty acid esters of 3-monochloropropane-1,2-diol (3-MCPD esters) and glycidol (glycidyl esters) in different foodstuffs. The esters were isolated from fat-rich food samples using a single extraction step and separated from interfering substances. For differentiation of 3-MCPD esters and glycidyl esters the glycidol moiety was converted into 3-methoxypropane-1,2-diol (3-MPD) by acidic alcoholysis. Subsequent determination was achieved by isotope dilution GC-MS after transesterification using an isotope-labeled 3-MCPD ester as internal standard. During optimization of the procedure, critical parameters affecting simultaneous determination and differentiation of these analytes were investigated. Rapid ester cleavage and derivatization at ambient temperature proved to be essential for the simultaneous determination of these analytes. The method was validated for various fat-rich foodstuffs such as bakery products, sweets, gravy, and soup powders as well as edible fats and oils. LODs of 8 and 15 μg/kg (fat-rich foodstuffs) as well as 50 and 65 μg/kg (edible oils and fats) were obtained for 3-MCPD esters and glycidyl esters, respectively. Recoveries for 3-MCPD esters and glycidyl esters ranged within 98 ± 4 and 88 ± 2% in all tested foodstuffs (0.05-2.5 mg/kg) and within 99 ± 16 and 93 ± 13% for edible oils and fats (0.15-3 mg/kg) over a wide concentration range. These results proved an accurate and differentiated determination of 3-MCPD esters and glycidyl esters with successful application to the fast screening of samples, avoiding tedious and laborious sample preparation.     

Anthocyanin-rich blackberry extract suppresses the DNA-damaging properties of topoisomerase I and II poisons in colon carcinoma cells

In the present study, we addressed the question whether cyanidin-3-glucoside (C3G) or complex C3G-rich blackberry extracts affect human topoisomerases with special emphasis on the contribution of the potential degradation products phloroglucinol aldehyde (PGA) and protocatechuic acid (PCA). In HT29 colon carcinoma cells a C3G-rich blackberry extract suppressed camptothecin- (CPT-) or doxorubicin- (DOX-) induced stabilization of the covalent DNA-topoisomerase intermediate, thus antagonizing the effects of these classical topoisomerase poisons on DNA integrity. As a single compound, C3G (100 μM) decreased the DNA-damaging effects of CPT as well, but did not significantly affect those induced by DOX. At the highest applied concentration (100 μM), cyanidin protected DNA from CPT- and DOX-induced damage. Earlier reports on DNA-damaging properties of cyanidin were found to result most likely from the formation of hydrogen peroxide as an artifact in the cell culture medium when the incubation was performed in the absence of catalase. The suppression of hydrogen peroxide accumulation, achieved by the addition of catalase, demonstrated that cyanidin does not exhibit DNA-damaging properties in HT29 cells (up to 100 μM). The observed effects on topoisomerase interference and DNA protection against CPT or DOX were clearly limited to the parent compound and were not observed for the potential cyanidin degradation products PGA and PCA.     

Endothelial–platelet interactions in influenza-induced pneumonia: A potential therapeutic target

Every year, influenza viruses spread around the world, infecting the respiratory systems of countless humans and animals, causing illness and even death. Severe influenza infection is associated with pulmonary epithelial damage and endothelial dysfunction leading to acute lung injury (ALI). There is evidence that an aggressive cytokine storm and cell damage in lung capillaries as well as endothelial/platelet interactions contribute to vascular leakage, pro-thrombotic milieu and infiltration of immune effector cells. To date, treatments for ALI caused by influenza are limited to antiviral drugs, active ventilation or further symptomatic treatments. In this review, we summarize the mechanisms of influenza-mediated pathogenesis, permissive animal models and histopathological changes of lung tissue in both mice and men and compare it with histological and electron microscopic data from our own group. We highlight the molecular and cellular interactions between pulmonary endothelium and platelets in homeostasis and influenza-induced pathogenesis. Finally, we discuss novel therapeutic targets on platelets/endothelial interaction to reduce or resolve ALI.     

Effects of pollination distance on reproduction and offspring performance in Hypochoeris radicata: Experiments with plants from three European regions

Fragmentation of habitats has resulted in increased inbreeding for many plant species, while the introduction of foreign seed material for ecological restoration has resulted in crosses between plants from distant populations. Both processes may reduce plant fitness and increase the risk of extinction. Variation in the expression of inbreeding and outbreeding depression has been found among different genotypes and among populations, but little is known about large scale geographical patterns within species. We studied the effects of cross-proximity on seed production and offspring performance in the perennial meadow plant Hypochoeris radicata (Asteraceae) from five populations in each of three European regions (Bohemia in northwest Czechia, Northern Hesse in central Germany, and Salland in the central Netherlands). Five artificial cross types were conducted with varying proximity of mates: selfing (self), within family crosses (WF), within population crosses (WP), between population crosses (BP), and between region crosses (BR), and the offspring were grown in a common garden. Independent of the region of origin of the maternal plant, selfing, WF and BP crosses resulted in lower seed set and germination than WP crosses, indicating partial self-incompatibility, inbreeding depression and reduced performance in the F1 progeny resulting from outbreeding. However, crosses between regions resulted in similar seed set and germination as within population crosses. For late traits, the effects of inbreeding and interpopulation crosses differed among regions. WP crosses exhibited the highest survival, flowering and multiplicative fitness only in progeny from Czech maternal plants. Our results suggest that the sensitivity of populations to introgression may vary among regions and that outbreeding depression does not necessarily increase with interpopulation distance. However, the current study investigated only effects in the F1 in a common garden and outbreeding depression may be stronger in the F2 and in field populations.     

Forage quality and yield increments of intensive managed grassland in response to combined sulphur-nitrogen fertilization Dedicated to Prof. em.Dr. Alois Kornher on his 75th birthday.

Abstract

A three-year experiment was carried out at three different sites in northern Germany to investigate the effects of combined sulphur (S, up to 50 kg S ha^?1 year^?1) and nitrogen (N, up to 300 kg N ha^?1 year^?1) fertilization on dry matter (DM) yield and forage quality. There was an interaction effect of site, year, S and N fertilization. The greatest DM yield increment relative to yield at the start of the experiment (1997) with no S and N applied was 10.2 t DM ha^?1 at Ostenfeld (arable grassland). Cattle slurry when applied to provide 50 kg N ha^?1 and 10 kg S ha^?1 did not noticeably increase yield. The S content in forage decreased significantly over the years without S fertilization. At 300 kg N ha^?1 and 0 kg S ha^?1, crude protein (CP) contents achieved 173 g kg^?1 DM and were diluted due to higher DM yields with S fertilization. The true protein content (TP% of CP) differed significantly at 300 kg N ha^?1. TP achieved 93% with 50 and 87% with 0 kg S ha^?1 year^?1, respectively. In conclusion, with N fertilizer intensities in the range of 300 kg N ha^?1, it is necessary to apply 25 kg S ha^?1 to improve forage yield and quality. On the other hand, with N fertilization levels below 300 kg N ha^?1, S fertilization could be omitted.     

Diversity of nonribosomal peptide synthetase genes in the microbial metagenomes of marine sponges

Genomic mining revealed one major nonribosomal peptide synthetase (NRPS) phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis.     

Evaluation of diagnostic tests in the absence of a gold standard using an Anaplasma marginale field data set

The evaluation of newly developed diagnostic tests (tests) commonly involves the comparison of the test outcomes (pos/neg.) of a sample of animals to those of a reference test (gold standard) in order to derive sensitivity and specificity estimates. Often, however, new tests have to be evaluated against an imperfect reference test since a true gold standard test is either too expensive or too costly to apply. This results in bias in the test characteristic estimates. To solve this problem, latent class and Bayesian models can be used to estimate sensitivity and specificity when evaluating a diagnostic test in the absence of a gold standard. They require at least two imperfect reference tests applied to all individuals in the study. In our approach we used a two-test two-population scenario. Both the gold standard and these modelling approaches rely on various assumptions. When violated, biased results will be obtained. The analysis of field data from an Anaplasma marginale outbreak in cattle in Switzerland with four diagnostic procedures (detection of the agent, serology, PCR and hematocrit measurements) was used as a practical example to demonstrate and critically discuss the approaches taken. In this relatively small data set (n = 275) the estimates for the test characteristics obtained by the different methods were quite similar. Overall, the bias in the point estimates depended mainly on the chosen estimation approach. All tests showed a non-negligible correlation mainly in the test sensitivities. This emphasizes the importance of taking into account test dependence even if it seems not biologically plausible at first thought.     

Intracellular survival of Salmonella strains in Caco-2 cells

In the in vitro model using Caco-2 cells at different stages of differentiation the invasion and intracellular survival of virulent (predominant infection strains) and less virulent (predominant attenuated mutant strains) Salmonella strains were studied. The statistical evaluation of experimental data has shown that the logarithmized colony forming unit after 18 hours of incubation in differentiated cells (14 days old) is a suitable parameter for the determination of intracellular survival. Using this parameter a relationship between intracellular survival and Salmonella virulence (LD50 mouse) was demonstrated and quantified. The model presented could be suitable for the replacement of animal experiments after further investigations.     

Morphological and immunohistochemical studies in testes of normal and cryptorchid boars

Scrotal (n = 66), inguinal (n = 7) and abdominal (n = 36) testes from pigs differing in age (from 1 day to 4 years) were collected after slaughtering or by castration, fixed in 4% formalin and examined by light microscopy. The expression of vimentin, desmin, alpha-actin and PCNA was detected by immunohistochemical technique. Histologically no differences between scrotal and cryptorchid testes are obvious during the prepubertal period, until the age of 10-12 weeks, but with the onset of puberty degenerative alterations (Sertoli-cell-only morphology, giant cells) in dystopic testes occur. Additionally the differentiation of the interstitial cells is disturbed in abdominal testes resulting in a deviating expression pattern of alpha-actin. The Sertoli cells show different distribution patterns (basal, perinuclear, apical) of vimentin depending on the position of the testis and the age of the animal.