Composition of Bacterial Assemblages in Different Components of Reed Warbler Nests and a Possible Role of Egg Incubation in Pathogen Regulation

Bacteria play a central role in animal health. Yet, little is known about the acquisition of bacteria and the extent to which bacteria are acquired from different environmental sources. For example, bird nests host diverse bacteria associated with the eggs, nestlings and nesting material, but previous research has typically focussed on only a limited number of nest components at a time. It therefore remains unknown to what extent bacteria are transmitted between these components. Using both molecular and culture techniques, we characterised nest-associated bacterial assemblages throughout the entire nesting cycle of reed warblers by sampling bacteria on eggs before and during incubation, within nestling faeces, and on the nesting material of post-breeding nests. We found that bacterial assemblages clustered by nest component. Yet some overlap existed between nest components, suggesting that bacterial transmission across components is likely to occur. Eggs and nestlings from the same nest harboured more similar bacteria than expected by chance, suggesting an influence of environment or genetics on bacterial assemblages. Bacterial loads were not lower on incubated eggs. Instead, incubation was associated with a change in the structure of assemblages, including a decrease in potentially-harmful Gram-negative bacteria. In addition we show for the first time, that incubation is associated with the complete extinction of harmful haemolytic bacteria. Overall, our study appears to be the first to demonstrate differences in bacterial assemblages between bird nest components. In addition, we highlight the complexity of nest bacterial assemblages and provide new insights into the benefits of incubation.     

The Combination of Salt Induced Peptide Formation Reaction and Clay Catalysis: A Way to Higher Peptides under Primitive Earth Conditions

Two reactions with suggested prebiotic relevance for peptide evolution, the saltinduced peptide formation reaction and the peptide chain elongation/stabilization on clay minerals have been combined in experimental series starting from dipeptides and dipeptide/amino acid mixtures. The results show that both reactions can take place simultaneously in the same reaction environment and that the presence of mineral catalysts favours the formation of higher oligopeptides. These findings lend further support to the relevance of these reactions for peptide evolution on the primitive earth. The detailed effects of the specific clay mineral depend both on the nature of the mineral and the reactants in solution.     

Sensitivity of different endpoints for in vitro measurement of genotoxicity of extractable organic matter associated with ambient airborne particles (PM10)

Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM₁₀ (particulate matter < 10 μm) collected in three European cities (Prague, Sofia, Košice) during winter and summer period. To compare the sensitivity of particular endpoints for in vitro measurement of complex mixture genotoxicity, the metabolically competent human hepatoma cell line Hep G2 was treated with equivalent EOM concentration of 50 μg/ml. Cell exposure to EOMs resulted in significant DNA adduct formation and DNA strand break induction, however, a lack of protein p53 up-regulation over the steady-state level was found. While the maximum of DNA strand breaks was determined after 2 h cell exposure to EOMs, 24 h treatment interval was optimal for DNA adduct determination.No substantial location- and season-related differences in EOM genotoxicity were detected using DNA strand break assessment. In agreement with these results no significant variation in DNA adduct levels were found in relation to the locality and season except for the monitoring site in Prague. The Prague EOM sample collected during summer period produced nearly three-fold lower DNA adduct level in comparison to the winter EOM sample.Comparable results were obtained when the ambient air genotoxicity, based on the concentration of carcinogenic PAHs in cubic meter of air (ng c-PAHs/m³), was elicited using either DNA adduct or strand break determination. In general, at least six-fold higher genotoxicity of the winter air in comparison to the summer air was estimated by each particular endpoint. Moreover, the genotoxic potential of winter air revealed by DNA adduct assessment and DNA strand break measurement increased in the same order: Košice  Prague < Sofia.Based on these data we suppose that two endpoints DNA breakage and DNA adduction are sensitive in vitro biomarkers for estimation of genotoxic activity of organic complex mixture associated with airborne particles. On the other hand, the measurement of protein p53 up-regulation manifested some limitations; therefore it cannot be used as a reliable endpoint for in vitro genotoxicity assessment.     

CysTRAQ - A combination of iTRAQ and enrichment of cysteinyl peptides for uncovering and quantifying hidden proteomes

Shotgun proteomics is capable of characterizing differences in both protein quality and quantity, and has been applied in various biomedical applications. Unfortunately, the high complexity and dynamic range of proteins in studied samples, clinical in particular, often hinders the identification of relevant proteins. Indeed, information-rich, low abundance proteins often remain undetected, whereas repeatedly reported altered concentrations in high abundance proteins are often ambiguous and insignificant. Several techniques have therefore been developed to overcome this obstacle and provide a deeper insight into the proteome. Here we report a novel approach, which enables iTRAQ reagent quantitation of peptides fractionated based on presence of a cysteine residue (thus CysTRAQ). For the first time, we prove that iTRAQ quantitation is fully compatible with cysteinyl peptide enrichment and is not influenced by the fractionation process. Moreover, the employment of the method combined with high-resolution TripleTOF 5600 mass spectrometer for very fast MS/MS acquisition in human amniotic fluid analysis significantly increased the number of identified proteins, which were simultaneously quantified owing to the introduction of iTRAQ labeling. We herein show that CysTRAQ is a robust and straightforward method with potential application in quantitative proteomics experiments, i.e. as an alternative to the ICAT reagent approach.     

Nuclear distribution and chromatin association of DNA polymerase α-primase is affected by TEV protease cleavage of Cdc23 (Mcm10) in fission yeast

Background  

Cdc23/Mcm10 is required for the initiation and elongation steps of DNA replication but its biochemical function is unclear. Here, we probe its function using a novel approach in fission yeast, involving Cdc23 cleavage by the TEV protease.     

Induced cell death of preimplantation mouse embryos cultured in vitro evaluated by comet assay

The occurrence of apoptosis in mouse preimplantation embryos was analyzed using DNA staining (Hoechst 33342, PI) for the visualization of nuclear changes and by the comet assay, a single-cell gel electrophoresis assay, modified for the analysis of blastocysts. Mouse preimplantation embryos isolated 56 h after superovulation were cultured in vitro for 64 h. Apoptosis was induced by treatment with camptothecin and actinomycin D during the first 15 h of culture. After culture in vitro, a number of embryos were stained and analyzed using morphological criteria. The remaining embryos were examined using the comet assay for the detection of DNA fragmentation. The proportion of damaged embryos in experimental groups, in comparison to controls, was dependent on the dose of apoptosis inductor. At high doses (camptothecin, microg/ml and actinomycin D, 0.05 microg/ml) over 90% (chi-square test, P<0.001) of embryos had apoptotic comets, at medium doses (camptothecin, 0.01 microg/ml and actinomycin D, 0.005 microg/ml) comets appeared only in 30-70% of embryos (camptothecin, P<0.01 and actinomycin D, P<0.001). At low doses (camptothecin, 0.001 microg/ml and actinomycin D, 0.0005 microg/ml) the increase in damaged embryos was not statistically significant. Hoechst/PI staining showed a higher percentage of damaged blastomeres at high doses. Morphological changes correlated with the outcome of the comet assay. Our results show that comet assay is an appropriate method for studying apoptosis in preimplantation embryos, and it appears to be more sensitive than the classically used morphological analyses.     

Measurement of the blood flow velocity in the pericallosal artery of children with hydrocephalus by transcranial Doppler ultrasonography--preliminary results

AIM: The goal of this study was to evaluate selected parameters of the Doppler curve of the pericallosal artery at children with hydrocephalus. METHODS: 12 patients with hydrocephalus were divided into two groups. Group 1 comprised children needing cerebrospinal fluid drainage, and group 2 comprised children without any indication for drainage or with an already inserted well-functioning drainage system. Dilatation of the cerebral ventricles was determined by transcranial ultrasonography. Following parameters of a blood flow of the pericallosal branch of the anterior cerebral artery: peak systolic blood flow velocity (PSFV), end-diastolic blood flow velocity (EDFV) and resistive index (RI) were observed by transcranial Doppler ultrasonography. Parameters of The Doppler curve were measured without pressure (baseline parameters) and during compression of the anterior fontanelle (pressure provocation test). RESULTS: Group 1: baseline parameters: PSFV 68.9 +/- 13.52 cm/s, EDFV 18.26 +/- 10.39 cm/s, RI 0.76 +/- 0.12; parameters during pressure provocation test: PSFV 66.92 +/- 19.75 cm/s, EDFV 10.88 +/- 11.18 cm/s, RI 0.86 +/- 0.14. Group 2: baseline parameters: PSFV 59.95 +/- 19.38 cm/s, EDFV 20.65 +/- 8 cm/s, RI 0.65 +/- 0.04; parameters during the pressure provocation test: PSFV 57.14 +/- 18.91 cm/s, EDFV 17.7 +/- 8.3 cm/s, RI 0.68 +/- 0.05. CONCLUSION: The results show increased baseline and postcompressive values of RI of pericallosal artery in infants with hydrocephalus before drainage procedure and normal values of RI at children without the need for cerebrospinal fluid drainage or with a well-functioning drainage system.