New separation-free assay technique for SNPs using two-photon excitation fluorometry

A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia™ TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot™ assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.     

Quantitative detection of cell surface protein expression by time-resolved fluorimetry.

A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.     

The effects of iron-containing superoxide dismutases on haemodynamic parameters in spontaneously hypertensive rats

The product of FeSOD activity is hydrogen peroxide (H2O2). Furthermore, FeSOD can modify the chemical versatility of NO into its redox-active forms: nitrosonium cation (NO+) and nitroxyl anion (NO-). All of these low molecular weight species are vasoactive and, in particular, NO- induces calcitonin gene-related peptide (CGRP) synthesis (known to be the most potent relaxation-promoting peptide). In this study the effects of bolus infusions of iron-containing superoxide dismutase (FeSOD) and of superoxide dismutase containing both iron and manganese (FeMnSOD) on the arterial blood pressure (MAP), the arterial blood pressure (CO) and the total vascular resistance (TVR) in spontaneously hypertensive (SH) rats were determined. Bolus infusion of FeSOD induced a biphasic response in the MAP (an initial increase was followed by a significant decrease). At the end of the experiment the MAP returned to its basal value. FeMnSOD (the enzymatically inactive form of FeSOD) had no effect on the MAP in these experiments. Bolus infusions of FeSOD and of FeMnSOD had no effect either on the both the CO or on the TVR in SH rats. Our results indicate that arterial relaxation changes mediated by NO- may be important for regulation of blood pressure in SH rats.     

A Simple and Efficient DNA Isolation Method for Salvia officinalis

We report an efficient, simple, and cost-effective protocol for the isolation of genomic DNA from an aromatic medicinal plant, common sage (Salvia officinalis L.). Our modification of the standard CTAB protocol includes two polyphenol adsorbents (PVP 10 and activated charcoal), high NaCl concentrations (4?M) for removing polysaccharides, and repeated Sevag treatment to remove proteins and other carbohydrate contaminants. The mean DNA yield obtained with our Protocol 2 was 330.6?μg DNA g^?1 of dry leaf tissue, and the absorbance ratios 260/280 and 260/230?nm averaged 1.909 and 1.894, respectively, revealing lack of contamination. PCR amplifications of one nuclear (26S rDNA) and one chloroplast (rps16-trnK) locus indicated that our DNA isolation protocol may be used in common sage and other aromatic and medicinal plants containing essential oil for molecular biologic and biotechnological studies and for population genetics, phylogeographic, and conservation surveys in which nuclear or chloroplast genomes would be studied in large numbers of individuals.     

Birds on the move in the face of climate change: High species turnover in northern Europe

Species richness is predicted to increase in the northern latitudes in the warming climate due to ranges of many southern species expanding northwards. We studied changes in the composition of the whole avifauna and in bird species richness in a period of already warming climate in Finland (in northern Europe) covering 1,100 km in south–north gradient across the boreal zone (over 300,000 km²). We compared bird species richness and species‐specific changes (for all 235 bird species that occur in Finland) in range size (number of squares occupied) and range shifts (measured as median of area of occupancy) based on bird atlas studies between 1974–1989 and 2006–2010. In addition, we tested how the habitat preference and migration strategy of species explain species‐specific variation in the change of the range size. The study was carried out in 10 km squares with similar research intensity in both time periods. The species richness did not change significantly between the two time periods. The composition of the bird fauna, however, changed considerably with 37.0% of species showing an increase and 34.9% a decrease in the numbers of occupied squares, that is, about equal number of species gained and lost their range. Altogether 95.7% of all species (225/235) showed changes either in the numbers of occupied squares or they experienced a range shift (or both). The range size of archipelago birds increased and long‐distance migrants declined significantly. Range loss observed in long‐distance migrants is in line with the observed population declines of long‐distance migrants in the whole Europe. The results show that there is an ongoing considerable species turnover due to climate change and due to land use and other direct human influence. High bird species turnover observed in northern Europe may also affect the functional diversity of species communities.     

Climate-driven synchrony in seed production of masting deciduous and conifer tree species

Aims Understanding fluctuations in plant reproductive investment can constitute a key challenge in ecology, conservation and management. Masting events of trees (i.e. the intermittent and synchronous production of abundant seeding material) is an extreme example of such fluctuations. Our objective was to establish the degree of spatial and temporal synchrony in common four masting tree species in boreal Finland and account for potential causal drivers of these patterns.Methods We investigated the spatial intraspecific and temporal interspecific fluctuations in annual seed production of four tree species in Finland, silver birch Betula pendula Roth, downy birch Betula pubescens Ehrh., Norway spruce Picea abies (L.) H.Karst. and rowanberry Sorbus aucuparia L. We also tested to see whether variations in seed production were linked to annual weather conditions. Seeding abundance data were derived from tens of stands per species across large spatial scales within Finland during 1979 to 2014 (for rowanberries only 1986 to 2014).Important findings All species showed spatial synchrony in seed production at scales up to 1000 km. Annual estimates of seed production were strongly correlated between species. Spring and summer temperatures explained most variation in crop sizes of tree species with 0-to 2-year time lags, whereas rainfall had relatively little influence. Warm weather during flowering (May temperature) in the flowering year (Year t) and 2 years before (t ?2) were correlated with seed production. However, high May temperatures during the previous year (t^-1) adversely affected seed production. Summer temperatures in Year t^-1 was positively correlated with seed production, likely because this parameter enhances the development of flower primordials, but the effect was negative with a time lag of 2 years. The negative feedback in temperature coefficients is also likely due to patterns of resource allocation, as abundant flowering and seed production in these species is thought to reduce the subsequent initiation of potential new flower buds. Since the most important weather variables also showed spatial correlation up to 1000 km, weather parameters likely explain much of the spatial and temporal synchrony in seed production of these four studied tree species.     

Adult predation risk drives shifts in parental care strategies: a long-term study

1. Grouping provides antipredatory benefits, and therefore aggregation tendencies increase under heightened predation risk. In socially breeding groups, however, conflicts over reproductive shares or safety tend to disintegrate groups. Group formation thereby involves a balance between the antipredatory benefits of aggregation and the destabilizing effect of reproductive conflict. 2. We study the grouping behaviour of a facultatively social precocial sea duck with uniparental female care, the eider (Somateria mollissima Linnaeus). Females tend their young solitarily or in groups of 2-5 females. Here, we focus on the effect predation on adults has on group-formation decisions of brood-caring females. 3. By modifying an existing bidding game over care, we model the effects of predation risk on the width of the window of selfishness, which delimits the reproductive sharing allowing cooperation within brood-rearing coalitions, and generate predictions about the relative frequencies of solitary versus cooperative parental care modes. Furthermore, we model the dilution effect as a function of female group size and predation risk. 4. The window of selfishness widens with increasing predation risk, and the dilution of predation risk increases with both female group size and increasing predation risk, yielding the following predictions: (i) cooperative brood care becomes more prevalent and, conversely, solitary brood care less prevalent under heightened predation risk and (ii) group sizes increase concomitantly. 5. We tested these predictions using 13 years of data on female grouping decisions and annual predation rates, while controlling for the potentially confounding effect of female body condition. 6. Our data supported both predictions, where heightened predation risk of nesting females, but not changes in their condition, increased the relative frequency of cooperative brood care. Increased female nesting mortality also resulted in larger groups of cooperative females. 7. The predation risk of incubating females has long-term implications for later parental care decisions. We discuss the potential consequences of predation-induced shifts in group size on per capita fitness and population-wide productivity.