Genetic mapping of sulfur assimilation genes reveals a QTL for onion bulb pungency

Onion exhibits wide genetic and environmental variation in bioactive organosulfur compounds that impart pungency and health benefits. A PCR-based molecular marker map that included candidate genes for sulfur assimilation was used to identify genomic regions affecting pungency in the cross 'W202A' × 'Texas Grano 438'. Linkage mapping revealed that genes encoding plastidic ferredoxin-sulfite reductase (SiR) and plastidic ATP sulfurylase (ATPS) are closely linked (1–2 cM) on chromosome 3. Inbred F₃ families derived from the F₂ population used to construct the genetic map were grown in replicated trials in two environments and bulb pungency was evaluated as pyruvic acid or lachrymatory factor. Broad-sense heritability of pungency was estimated to be 0.78–0.80. QTL analysis revealed significant associations of both pungency and bulb soluble solids content with marker intervals on chromosomes 3 and 5, which have previously been reported to condition pleiotropic effects on bulb carbohydrate composition. Highly significant associations (LOD 3.7–8.7) were observed between ATPS and SiR Loci and bulb pungency but not with bulb solids content. This association was confirmed in two larger, independently derived F₂ families from the same cross. Single-locus models suggested that the partially dominant locus associated with these candidate genes controls 30–50% of genetic variation in pungency in these pedigrees. These markers may provide a practical means to select for lower pungency without correlated selection for lowered solids.     

Seroprevalence of Toxocara antibodies among patients suspected of ocular toxocariasis in Slovenia

Ocular toxocariasis named also ocular larva migrans is caused by larvae of the roundworm Toxocara spp. The purpose of this study was to find out the seroprevalence of Toxocara antibodies in patients suspected of ocular toxocariasis. Between January 2001 and December 2003, sera from 239 ocular patients, aged 3 to 80 years, were examined by ELISA and confirmed by Western blot test. Out of the 239 patients, 172 (72%) were seronegative and 67 (28%) were Toxocara seropositive; 95% CI (22-34%). The median age of Toxocara seropositive patients was 37.6 years. There was no significant difference in the number of Toxocara positive sera between the younger age group (< or = 14 years) and the older age group (> 14 years), p > 0.05. A high rate of Toxocara seropositivity in ocular patients should alert the ophthalmologists in Slovenia to include toxocariasis in the differential diagnosis of eye diseases more frequently.     

How fast monoamine oxidases decompose adrenaline? Kinetics of isoenzymes A and B evaluated by empirical valence bond simulation

This work scrutinizes kinetics of decomposition of adrenaline catalyzed by monoamine oxidase (MAO) A and B enzymes, a process controlling the levels of adrenaline in the central nervous system and other tissues. Experimental kinetic data for MAO A and B catalyzed decomposition of adrenaline are reported only in the form of the maximum reaction rate. Therefore, we estimated the experimental free energy barriers form the kinetic data of closely related systems using regression method, as was done in our previous study. By using multiscale simulation on the Empirical Valence Bond (EVB) level, we studied the chemical reactivity of the MAO A catalyzed decomposition of adrenaline and we obtained a value of activation free energy of 17.3 ± 0.4 kcal/mol. The corresponding value for MAO B is 15.7 ± 0.7 kcal/mol. Both values are in good agreement with the estimated experimental barriers of 16.6 and 16.0 kcal/mol for MAO A and MAO B, respectively. The fact that we reproduced the kinetic data and preferential catalytic effect of MAO B over MAO A gives additional support to the validity of the proposed hydride transfer mechanism. Furthermore, we demonstrate that adrenaline is preferably involved in the reaction in a neutral rather than in a protonated form due to considerably higher barriers computed for the protonated adrenaline substrate. The results are discussed in the context of chemical mechanism of MAO enzymes and possible applications of multiscale simulation to rationalize the effects of MAO activity on adrenaline level.     

High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin

Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC–UHPLC–FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC–UHPLC–FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.     

A simple and reliable method for monitoring platelet serotonin levels in rats

A simple and reliable method for individual monitoring of platelet serotonin in rats is developed. Platelet-rich plasma is prepared under standardized conditions from 1 mL of venous blood and the platelets are quantitatively separated by a highly reproducible procedure. Platelet serotonin content is determined spectrophotofluorometrically and the results are comparatively expressed per standardized platelet rich plasma sample (1.01 +/- 0.18 microgram), per mg of platelet protein (1.57 +/- 0.15 microgram) and per 10(9) platelets (2.16 +/- 0.38 micrograms). Normal distribution of platelet serotonin levels in a sample of 338 animals is shown. By use of the described method, the intraindividual stability of platelet serotonin concentration in rats is demonstrated for the first time.     

Cholesterol-mediated membrane surface area dynamics in neuroendocrine cells

How cholesterol, a key membrane constituent, affects membrane surface area dynamics in secretory cells is unclear. Using methyl-β-cyclodextrin (MβCD) to deplete cholesterol, we imaged melanotrophs from male Wistar rats in real-time and monitored membrane capacitance (C_m), fluctuations of which reflect exocytosis and endocytosis. Treatment with MβCD reduced cellular cholesterol and caused a dose-dependent attenuation of the Ca²⁺-evoked increase in C_m (IC₅₀ = 5.3 mM) vs. untreated cells. Cytosol dialysis of MβCD enhanced the attenuation of C_m increase (IC₅₀ = 3.3 mM), suggesting cholesterol depletion at intracellular membrane sites was involved in attenuating exocytosis. Acute extracellular application of MβCD resulted in an immediate C_m decline, which correlated well with the cellular surface area decrease, indicating the involvement of cholesterol in the regulation of membrane surface area dynamics. This decline in C_m was three-fold slower than MβCD-mediated fluorescent cholesterol decay, implying that exocytosis is the likely physiological means for plasma membrane cholesterol replenishment. MβCD had no effect on the specific C_m and the blockade of endocytosis by Dyngo 4a, confirmed by inhibition of dextran uptake, also had no effect on the time-course of MβCD-induced C_m decline. Thus acute exposure to MβCD evokes a C_m decline linked to the removal of membrane cholesterol, which cannot be compensated for by exocytosis. We propose that the primary contribution of cholesterol to surface area dynamics is via its role in regulated exocytosis.     

Markov random field model for segmenting large populations of lipid vesicles from micrographs

Giant unilamellar lipid vesicles, artificial replacements for cell membranes, are a promising tool for in vitro assessment of interactions between products of nanotechnologies and biological membranes. However, the effect of nanoparticles can not be derived from observations on a single specimen, vesicle populations should be observed instead. We propose an adaptation of the Markov random field image segmentation model which allows detection and segmentation of numerous vesicles in micrographs. The reliability of this model with different lighting, blur, and noise characteristics of micrographs is examined and discussed. Moreover, the automatic segmentation is tested on micrographs with thousands of vesicles and the result is compared to that of manual segmentation. The segmentation step presented is part of a methodology we are developing for bio-nano interaction assessment studies on lipid vesicles.     

Influence of abandonment on syrphid assemblages in mountainous meadows

Grasslands are often managed with different intensities in the European Alps. Studies have shown that a medium management can benefit plant and animal biodiversity in these ecosystems. However, in recent decades abandonment of extensively managed meadows is an ongoing global challenge. Syrphids (hoverflies) have been recognized as a threatened group due to environmental drivers, and these managed grasslands provide preferred habitats for them. Yet, at what extent syrphids respond to grassland management strategies is little known. We investigated whether abandonment of managed mountain meadows (mown once a year, no fertilizer use) affects syrphid abundance, richness and species assemblages. Moreover, the influence of plant richness, flower frequency and surrounding landscape on syrphids were assessed. Four managed meadows were compared with four abandoned meadows in a mountainous region in Austria. Adult syrphids were sampled over two consecutive years (June and August 2015–2016) by sweep netting using line transects and observation plots. Syrphid abundance was significantly higher in managed than abandoned meadows and increased with increasing plant richness and flower frequency across management types. Management and sampling time also significantly affected syrphid species assemblages. Syrphid richness and abundance were not influenced by surrounding landscape. We conclude that both abandoned and managed meadows provide important habitat types for syrphids. Therefore, efforts should be made to maintain both managed and abandoned meadows in order to protect syrphid species within mountainous landscapes.