The humidity level matters during the desiccation of Norway spruce somatic embryos.

In Norway spruce, as in many other conifers, the germination capacity of somatic embryos is strongly influenced by the desiccation phase inserted after maturation. The intensity of drying during desiccation eminently affected the formation of emblings (i.e., seedlings developed from somatic embryos). Compared to non-desiccated embryos, the germination capacity of embryos desiccated at 100% relative humidity was about three times higher, but the reduction of relative humidity to 95 and 90% had a negative effect on the subsequent embryo development. The water loss observed in these embryos did not lead to an increase in lipid peroxidation, as shown by malondialdehyde levels. Another metabolic pathway in plants that mediates a response to abiotic stresses is directed toward the biosynthesis of polyamines (PAs). The activities of PA biosynthetic enzymes increased steadily in embryos during desiccation at 100% relative humidity, whereas they decreased at lower humidity. The total content of free PAs in the embryos gradually decreased throughout desiccation. The increase in free putrescine (Put) and perchloric acid-insoluble Put conjugates was observed in embryos desiccated at lower humidity. These changes were accompanied to some extent by the transcription of the genes for the PA biosynthesis enzymes. Desiccation at 100% relative humidity increased the activity of the cell wall-modifying enzymes ß-1,3-glucanases and chitinases; the activities of these enzymes were also significantly suppressed at reduced humidity. The same pattern was observed in the transcription of some ß-1,3-glucanase and chitinase genes. Desiccation treatments triggered metabolic processes that responded to water availability, suggesting an active response of the embryo to the reduction in humidity. A positive effect was demonstrated only for desiccation at high relative humidity. Some of the physiological characteristics described can be used as markers of inappropriate relative humidity during somatic embryo desiccation.

To be or not to be vaccinated: The risk of MS or NMOSD relapse after COVID-19 vaccination and infection.

BACKGROUND: COVID-19 vaccination and infection are speculated to increase the activity of immune-mediated diseases, including multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). The aim of this study was to evaluate a short-term risk of relapse after COVID-19 vaccination and COVID-19 infection in patients with these demyelinating disorders of the central nervous system and to determine disease exacerbation risk factors. METHODS: Data in this retrospective, observational cohort study was collected via the Czech nationwide registry ReMuS from March 1, 2020, to October 30, 2021. We compared the proportion of patients with at least one clinical relapse in the 90 days following vaccination or infection to the 90-day intervals during the year before. For the evaluation of the risk factors of relapse, a comparison between groups with and without relapses after COVID-19 vaccination or infection was made. RESULTS: We identified 1661 vaccinated (90.11% BNT162b2) patients with MS without a history of COVID-19 and 495 unvaccinated patients with MS who experienced COVID-19. A mild increase in the proportion of patients with at least one clinical relapse (-360 to -270 days: 4.46%; -270 to -180: 4.27%; -180 to -90: 3.85%; -90 to 0: 3.79% vs. 0 to +90 days: 5.30%) after vaccination in patients with MS was observed, as well as a rise in the proportion of patients with at least one clinical relapse after COVID-19. Lower age was associated with MS relapse after vaccination or infection. Although there were only 17 vaccinated and eight post-COVID-19 patients with NMOSD, the results were broadly consistent with those of patients with MS. CONCLUSION: There is a mild increase in the relapse incidence after the COVID-19 vaccination. The risks, however, need to be balanced against the risks of COVID-19 itself, also leading to the rise in relapse rate and particularly to morbidity and mortality.

Role of cytochromes P450 in metabolism of carcinogenic aristolochic acid I: evidence of their contribution to aristolochic acid I detoxication and activation in rat liver.

OBJECTIVE: The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Therefore, the present study has been designed to evaluate the cytochrome P450 (CYP)-mediated oxidative detoxification and reductive activation of AAI in a rat model. METHODS: DNA adduct formation was investigated by the nuclease P1 version of the 32P-postlabeling method. The CYP-mediated formation of a detoxication metabolite of AAI, 8-hydroxyaristolochic acid I (AAIa), in vitro in rat hepatic microsomes was determined by HPLC. RESULTS: Rat hepatic CYPs both detoxicate AAI by its oxidation to AAIa and reductively activate this carcinogen to a cyclic N-acylnitrenium ion forming AAI-DNA adducts in vitro. To define the role of hepatic CYPs in AAI demethylation and activation, the modulation of AAIa and AAI-DNA adduct formation by CYP inducers and selective CYP inhibitors was investigated. Based on these studies, we attribute the major role of CYP1A1 and 1A2 in AAI detoxication by its demethylation to AAIa, and, under hypoxic conditions also to AAI activation to species forming DNA adducts. Using microsomes of Baculovirus transfected insect cells (Supersomes™) containing recombinantly expressed rat CYPs, NADPH:CYP reductase and/or cytochrome b5, a major role of CYP1A1 and 1A2 in both reactions in vitro was confirmed. CONCLUSION: Based on the results found in this and former studies we propose that AAI activation and detoxication in rats are dictated mainly by AAI binding affinity to CYP1A1/2 or NADPH(P)H:quinone oxidoreductase, by their turnover and by the balance between oxidation and reduction of AAI by CYP1A.

Uptake of thallium from naturally-contaminated soils into vegetables.

Thallium transfer from naturally (pedogeochemically) contaminated soils into vegetables was studied. Three different types of top-soil (heavy, medium, and light) were used for pot experiments. The soils were collected from areas with low, medium, and high levels of pedogeochemical thallium (0.3, 1.5 and 3.3 mg kg(-1)). The samples of vegetables were collected and analysed. The total content of thallium in soil and the type of soil (heavy, medium and light), plant species and plant variety were found to be the main factors influencing thallium uptake by plants. The uptake of thallium from soils with naturally high pedogeochemical content of this element can be high enough to seriously endanger the food chain. These findings are very important because of the high toxicity of thallium and the absence of threshold limits for thallium in soils, agricultural products, feedstuffs and foodstuffs in most countries, including the Czech Republic.

Liquid chromatographic--cold vapour atomic fluorescence spectrometric determination of mercury species.

Solvent extraction, sonication, and microwave-assisted extractions in the presence of extraction agents (thioacetic acid, citric acid, cysteine, 2-mercaptoethanol, HCl + NaCl, etc.) were tested for the isolation of mercury species. A mixture of 6 M HCl and 0.1 M NaCl was selected as the most suitable extraction agent. The extraction efficiency was about 10% higher and the RSD below 3.3% when microwave-assisted extraction was applied instead of sonication. The liquid chromatography-cold vapour atomic fluorescence spectrometry (LC/CV-AFS) method was optimised and used for separation and determination of inorganic mercury cations and alkylated and arylated mercury species. Isocratic elution at a flow rate of 0.15 mL/min (with a mobile phase containing 0.05% 2-mercaptoethanol (pH = 5) and 7% methanol and with a stepwise increase of methanol content up to 100% MeOH in the 15th min) was used for separation of mercury species on a Hypersil BDS C18 RP column. The limits of detection of the LC/CV-AFS system were estimated as 0.2 microg/L (3%) for MeHg+, 0.07 microg/L (5.3%) for inorganic Hg, 0.06 microg/L (3.4%) for PhHg+, and 0.12 microg/L (4.4%) for EtHg with the corresponding RSDs at 5 microg/L (n = 10) given in parentheses. The concentrations (2-10 mg/kg fresh weight) of total mercury and methylmercury (90-99% of the total mercury) in selected fish obtained by HPLC/CV-AFS were in good agreement (absolute deviations 0.05 mg/kg) but more precise (RSDs <5.4% at 5 mg/L, n = 10) than those determined by GC coupled to an electron capture detector. The RSDs (3.1-8.2% and 4.1-9.0%) of the overall analytical procedure for the determination of total mercury (AMA 254) and methylmercury (HPLC/CV-AFS) were determined for intra-day and inter-day assays, respectively.

Uptake of thallium from artificially and naturally contaminated soils into rape (Brassica napus L.).

An inductively coupled plasma mass spectrometry (ICP/MS) method was used for the evaluation of thallium transfer from naturally (pedogeochemically) and artificially contaminated soils into rape. Two sets of three different types of top soils (heavy, medium, and light) were used for pot experiments. The first set was collected from areas with high levels of pedogeochemical thallium (0.3, 1.5, and 3.3 mg kg(-1) DM). The second set of three soils with naturally low content of thallium was artificially contaminated with thallium sulfate to achieve five levels of contamination (0, 0.4, 2, 4, and 6 mg kg(-1) DM Tl). The soil samples and the samples of winter and spring rape (straw, seeds) from both sets were collected and analyzed. Plant and soil samples from fields were collected at 42 selected sites situated in South Bohemia and in Czech-Moravian Highlands where higher pedogeochemical content of thallium was expected. More intensive transport (better availability) of Tl was observed in the case of artificially contaminated soils. The physicochemical form and the total content of Tl in soil were found to be the main factors influencing its uptake by plants. The concentration of Tl in rapeseeds in the field samplings was mostly 45% of its content in the particular soil. Nevertheless the uptake of Tl from soils with naturally high pedogeochemical content can be high enough to seriously endanger food chains. These findings are very important because of the high toxicity of Tl and the absence of threshold limits for Tl in soils, agricultural products, feedstuffs, and foodstuffs in most countries including the Czech Republic.