On the specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein as a probe for nitric oxide

The specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) for nitric oxide was evaluated in in vitro systems. The probe was found fairly specific for nitric oxide. Potential sources of artifacts include the autoxidation of DAF-FM, potentiated by light, and its oxidation by sources of superoxide and peroxyl radicals, leading to fluorescence spectra indistinguishable from those of the nitric oxide adduct. Although DAF-FM reacts with peroxynitrite, this reaction seems to be of secondary importance under quasi-physiological conditions. On the other hand, a simultaneous presence of a nitric oxide source and a superoxide or hydrogen peroxide decreases or increases the fluorescence of DAF-FM, respectively, resulting in biased estimates of nitric oxide production.     

Macrophyte and macroinvertebrate patterns in unimpacted mountain rivers of two European ecoregions

Hydrobiologia - The aim of the study was to compare the patterns of development of macrophytes and macroinvertebrates in different types of reference mountain rivers. The study is based on...     

Compositional turnover and variation in Eemian pollen sequences in Europe

Vegetation History and Archaeobotany - The Eemian interglacial represents a natural experiment on how past vegetation with negligible human impact responded to amplified temperature changes...     

Current standards and pitfalls associated with the transfection of primary fibroblast cells

Cultured fibroblast cells, especially dermal cells, are used for various types of scientific research, particularly within the medical field. Desirable features of the cells include their ease of isolation, rapid cellular growth, and high degree of robustness. Currently, fibroblasts are mainly used to obtain pluripotent cells via a reprogramming process. Dermal fibroblasts, are particularly useful for gene therapies used for promoting wound healing or minimizing skin aging. In recent years, fibroblast transfection efficiencies have significantly improved. In order to introduce molecules (most often DNA or RNA) into cells, viral-based systems (transduction) or non-viral methods (transfection) that include physical/mechanical processes or lipid reagents may be used. In this article, we describe critical points that should be considered when selecting a method for transfecting fibroblasts. The most effective methods used for the transfection of fibroblasts include both viral-based and non-viral nucleofection systems. These methods result in a high level of transgene expression and are superior in terms of transfection efficacy and viability.     

Structural damages in adsorbed vaccines affected by freezing

This study was planned to evaluate structural damages in adsorbed vaccines affected by freezing using scanning electron microscopy and X-ray analysis of the elements. Randomly selected 42 vials of eight different types of WHO pre-qualified adsorbed freeze-sensitive vaccines from 10 manufacturers were included in the study. Vaccines were kept at 5 °C. Selected numbers of vials from each type were then exposed to ?25 °C for 24 h periods. All samples were evaluated for their structure using scanning electron microscopy, X-ray analysis of the elements and precipitation time. Scanning electron microscopy of vaccines affected by freezing showed either smooth or rough surfaced conglomerates associated with phosphate content of the precipitate. These vaccines precipitated 2–15 times faster compared to non-frozen samples. Non-frozen samples showed uniform flocculent structure either dense or dispersed. X-ray analysis of precipitates in frozen samples confirmed that the precipitate is mainly aluminium clutters. Scanning electron microscopy confirmed that the lattice structure of bonds between adsorbent and the antigen is broken and aluminium forms conglomerates that grow in size and weight. The precipitation time of vaccines affected by freezing is 4.5 times faster on average compared to non-frozen samples. These facts form the basis of the "shake test".     

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25°C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of K_i* = 4.5 × 10^{? 7} mM. The respective inhibition constant of TCoBQ was equal to: K_i* = 2.4 × 10^{? 6} mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.     

Ecological causes of morphological evolution in the three‐spined stickleback

The central assumption of evolutionary theory is that natural selection drives the adaptation of populations to local environmental conditions, resulting in the evolution of adaptive phenotypes. The three‐spined stickleback (Gasterosteus aculeatus) displays remarkable phenotypic variation, offering an unusually tractable model for understanding the ecological mechanisms underpinning adaptive evolutionary change. Using populations on North Uist, Scotland we investigated the role of predation pressure and calcium limitation on the adaptive evolution of stickleback morphology and behavior. Dissolved calcium was a significant predictor of plate and spine morph, while predator abundance was not. Stickleback latency to emerge from a refuge varied with morph, with populations with highly reduced plates and spines and high predation risk less bold. Our findings support strong directional selection in three‐spined stickleback evolution, driven by multiple selective agents.     

TLR9 -1486T/C and 2848C/T SNPs Are Associated with Human Cytomegalovirus Infection in Infants

Toll-like receptor 9 (TLR9) recognizes non-methylated viral CpG-containing DNA and serves as a pattern recognition receptor that signals the presence of human cytomegalovirus (HCMV). Here, we present the genotype distribution of single-nucleotide polymorphisms (SNPs) of the TLR9 gene in infants and the relationship between TLR9 polymorphisms and HCMV infection. Four polymorphisms (-1237T/C, rs5743836; -1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) in the TLR9 gene were genotyped in 72 infants with symptomatic HCMV infection and 70 healthy individuals. SNP genotyping was performed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digested fragments were separated and identified by capillary electrophoresis. The HCMV DNA copy number was measured by a quantitative real-time PCR assay. We found an increased frequency of heterozygous genotypes TLR9 -1486T/C and 2848C/T in infants with HCMV infection compared with uninfected cases. Heterozygous variants of these two SNPs increased the risk of HCMV disease in children (P = 0.044 and P = 0.029, respectively). In infants with a mutation present in at least one allele of -1486T/C and 2848C/T SNPs, a trend towards increased risk of cytomegaly was confirmed after Bonferroni’s correction for multiple testing (Pc = 0.063). The rs352139 GG genotype showed a significantly reduced relative risk for HCMV infection (Pc = 0.006). In contrast, the -1237T/C SNP was not related to viral infection. We found no evidence for linkage disequilibrium with the four examined TLR9 SNPs. The findings suggest that the TLR9 -1486T/C and 2848C/T polymorphisms could be a genetic risk factor for the development of HCMV disease.     

The role of selected tree species in industrial sewage sludge/flotation tailing management

The aim of the study was to estimate the ability of ten tree and bush species to tolerate and accumulate Cd, Cu, Pb, Zn, and As species [As(III), As(V), and total organic arsenic] in industrial sewage sludge extremely contaminated with arsenic (almost 27.5 g kg^?1) in a pot experiment. The premise being that it will then be possible to select the most promising tree/bush species, able to grow in the vicinity of dams where sewage sludge/flotation tailings are used as landfill. Six of the ten tested tree species were able to grow on the sludge. The highest content of total As was observed in Betula pendula roots (30.0 ± 1.3 mg kg^?1 DW), where the dominant As species was the toxic As(V). The highest biomass of Quercus Q1 robur (77.3 § 2.6 g) and Acer platanoides (76.0 § 4.9 g) was observed. A proper planting of selected tree species that are able to thrive on sewage sludge/flotation tailings could be an interesting and promising way to protect dams. By utilizing differences in their root systems and water needs, we will be able to reduce the risk of fatal environmental disasters.