The tomato cytochrome P450 CYP712G1 catalyses the double oxidation of orobanchol en route to the rhizosphere signalling strigolactone, solanacol.

Strigolactones (SLs) are rhizosphere signalling molecules and phytohormones. The biosynthetic pathway of SLs in tomato has been partially elucidated, but the structural diversity in tomato SLs predicts that additional biosynthetic steps are required. Here, root RNA-seq data and co-expression analysis were used for SL biosynthetic gene discovery. This strategy resulted in a candidate gene list containing several cytochrome P450s. Heterologous expression in Nicotiana benthamiana and yeast showed that one of these, CYP712G1, can catalyse the double oxidation of orobanchol, resulting in the formation of three didehydro-orobanchol (DDH) isomers. Virus-induced gene silencing and heterologous expression in yeast showed that one of these DDH isomers is converted to solanacol, one of the most abundant SLs in tomato root exudate. Protein modelling and substrate docking analysis suggest that hydroxy-orbanchol is the likely intermediate in the conversion from orobanchol to the DDH isomers. Phylogenetic analysis demonstrated the occurrence of CYP712G1 homologues in the Eudicots only, which fits with the reports on DDH isomers in that clade. Protein modelling and orobanchol docking of the putative tobacco CYP712G1 homologue suggest that it can convert orobanchol to similar DDH isomers as tomato.

UPLC-MS/MS analysis and biological activity of the potato cyst nematode hatching stimulant, solanoeclepin A, in the root exudate of Solanum spp.

MAIN CONCLUSION: Solanoeclepin A is a hatching stimulant for potato cyst nematode in very low (pM) concentrations. We report a highly sensitive method for the analysis of SolA in plant root exudates using UHPLC-MS/MS and show that there is considerable natural variation in SolA production in Solanum spp. corresponding with their hatching inducing activity. Potato cyst nematode (PCN) is a plant root sedentary endoparasite, specialized in the infection of solanaceous species such as potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Earlier reports (Mulder et al. in Hatching agent for the potato cyst nematode, Patent application No. PCT/NL92/00126, 1996; Schenk et al. in Croat Chem Acta 72:593-606, 1999) showed that solanoeclepin A (SolA), a triterpenoid metabolite that was isolated from the root exudate of potato, induces the hatching of PCN. Its low concentration in potato root exudate has hindered progress in fully understanding its hatching inducing activity and exploitation in the control of PCN. To further investigate the role of SolA in hatching of PCN, the establishment of a highly sensitive analytical method is a prerequisite. Here we present the efficient single-step extraction and UHPLC-MS/MS based analysis for rapid determination of SolA in sub-nanomolar concentrations in tomato root exudate. This method was used to analyze SolA production in different tomato cultivars and related solanaceous species, including the trap crop Solanum sisymbriifolium. Hatching assays with PCN, Globodera pallida, with root exudates of tomato genotypes revealed a significant positive correlation between SolA concentration and hatching activity. Our results demonstrate that there is natural variation in SolA production within solanaceous species and that this has an effect on PCN hatching. The analytical method we have developed can potentially be used to support breeding for crop genotypes that induce less hatching and may therefore display reduced infection by PCN.

Long-term effect of one-time nutritional education in school on nutritional knowledge of early school-aged children.

BACKGROUND: In the group of school-aged children nutritional education (NE) enables an early development of healthy eating habits, which can be transferred to the adult life. OBJECTIVE: The aim of the study was to assess the long-term effect of a one-time nutritional education, including also culinary workshops, on the level of nutritional knowledge (NK) of early school-aged children as well as to analyse the nutritional content of the children's books. MATERIAL AND METHODS: The study was conducted among second- and third-grade pupils from a primary school in Warsaw (n=76). The level of NK was determined using the same, self-administrated questionnaire at each of the three stages of the study. RESULTS: The level of NK was assumed as very good in 47% of respondents before the education, in 91% immediately after the education, and in 74% six months after it. Out of all 23.0 points (max.) on average 18.1 points were obtained at the first stage, 21.1 points at the second stage and 19.7 points at the third stage (p<0.001). Immediately after the NE and six months after it the level of NK increased significantly (comparing to the first stage) especially on these topics: the daily water requirements and the recommendations on physical activity (p<0.001 for both questions). CONCLUSIONS: The results of the study confirmed the effectiveness of a one-time nutritional education on the level of nutritional knowledge. Moreover, results show that in the research group nutritional knowledge about importance of physical activity, adequate nutrition, eating varicoloured vegetables and fruits, screen time limits and distinguishing between healthy and unhealthy products was satisfactory, even at the baseline, but in the main source of fats in a diet, the role of milk products, the effect of having too little water in a diet and the sugar content in food children's knowledge was not enough.

Detailed study of polystyrene solubility using pyrolysis-gas chromatography-mass spectrometry and combination with size-exclusion chromatography.

Measuring polymer solubility accurately and precisely is challenging. This is especially true at unfavourable solvent compositions, when only very small amounts of polymer dissolve. In this paper, pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) is demonstrated to be much more informative and sensitive than conventional methods, such as ultraviolet spectroscopy. By using a programmed-temperature-vapourisation injector as the pyrolysis chamber, we demonstrate that Py-GC-MS can cover up to five orders of magnitude in dissolved polymer concentrations. For polystyrene, a detection limit of 1 ng mL(-1) is attained. Dissolution in poor solvents is demonstrated to be discriminating in terms of the analyte molecular weight. Py-GC-MS additionally can yield information on polymer composition (e.g. in case of copolymers). In combination with size-exclusion chromatography, Py-GC-MS allows us to estimate the molecular weight distributions of minute amounts of a dissolved polymer and variations therein as a function of time.

Novel AlkB dioxygenases--alternative models for in silico and in vivo studies.

BACKGROUND: ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1-8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes. METHODOLOGY AND FINDINGS: Bioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB(-) mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment. CONCLUSIONS: Our in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis.

Pyrolysis-gas chromatography-mass spectrometry for studying N-vinyl-2-pyrrolidone-co-vinyl acetate copolymers and their dissolution behaviour.

Knowledge on the solubility behaviour and dissolution rate of speciality and commodity polymers is very important for the use of such materials in high-tech applications. We have developed methods for the quantification and characterization of dissolved copolymers of N-vinyl-2-pyrrolidone (VP) and vinyl acetate (VA) during dissolution in water. The methods are based on pyrolysis (Py) performed in a programmed-temperature vaporization injector with subsequent identification and quantification of the components in the pyrolysate using capillary gas chromatography-mass spectrometry (GC-MS). By injecting large volumes and applying cryo-focussing at the top of the column, low detection limits could be achieved. The monomer ratio was found to have the greatest effect on the dissolution rate of the PVP-co-VA copolymers. The material with the highest amount of VA (50%) dissolves significantly slower than the other grades. Size-exclusion chromatography (SEC) and Py-GC-MS were used to measure molecular weights and average chemical compositions, respectively. Combined off-line SEC//Py-GC-MS was used to determine the copolymer composition (VP/VA ratio), as a function of the molecular weight for the pure polymers. In the dissolution experiments, a constant VP/VA ratio across the dissolution curve was observed for all copolymers analysed. This suggests a random distribution of the two monomers over the molecules.

[Arsenic, cadmium, lead and mercury in bay bolete Xerocomus badius and tolerance limits]. / Arsen, kadm, olów i rtec w podgrzybku brunatnym Xerocomus badius a tolerancje.

In the article are reviewed available data on arsenic, cadmium, lead and mercury contents of the fruiting bodies of Bay Bolete in Europe. Cadmium and lead contents of Bay Bolete at the sites unpolluted with those elements reaches up to 5 and 20 mg/kg dry matters, respectively (up to 0.50 and 2.0 mg/kg fresh weight), i.e. is a much more greater when compared to the tolerance limit of 1,0 and 2,0 mg/kg dry weight in force earlier in Poland. In the light of available analytical data a suggested tolerance limit for cadmium in Bay Bolete should be 5 mg per kg of dried fruiting bodies, and 20 mg per kg of dried fruiting bodies for lead.

[Arsenic, cadmium, lead and mercury in king bolete Boletus edulis and tolerance limits]. / Arsen, kadm, olów i rtec w borowiku szlachetnym Boletus edulis a tolerancje.

In the article are reviewed and discussed available data on arsenic, cadmium, lead and mercury content of the fruiting bodies of king bolete. The values of cadmuim concentration of the fruiting bodies of king bolete collected from the areas unpoluted with metals and metaloids in Poland and other European countries usually are greater than an actual tolerance limits set by E.U. law for 0.2 mg/kg wet weight in cultivated mushrooms. Analogically, as is for cadmium also content of lead to be sometimes greater than tolerance limit of 0.3 mg/kg w.w. set in EU for cultivated mushrooms. The fruiting bodies of king bolete usually are also relatively rich in mercury at concentration much greater when compared to tolerance limit set earlier in Poland (in E.U. there is no tolerance limit for mercury in mushrooms). In the case of cadmium it can bee agreed, that this element content of pooled samples of the fruiting bodies of king bollete collected from unpolluted regions usually will not exceed a value of 20 mg/kg d.m., while at polluted areas will exceed a value of 20 mg/kg d.m. In the case of mercury it can bee agreed, that this element content of pooled samples of the fruiting bodies of king bollete collected from unpolluted regions usually will not exceed a value of 5 mg/kg d.m., while at polluted areas will exceed a value of 5 mg/kg d.m. In the case of lead it can be agreed, that this element content of some percent of pooled samples of the fruiting bodies of king bolete collected from unpoluted regions will sometimes exceed an actual the EU tolerance limit of 0.3 mg/kg w.w. as set only or cultivated mushrooms. In the case of arsenic it can be agreed, that total arsenic contant of some percent of pooled samples of the fruiting bodies of king bolete collected from unpoluted regions will exceed value of 0.50 mg/kg d.m.