Influence of delta-hexachlorocyclohexane (δ-HCH) to Phytophthora ×alni resistant Alnus glutinosa genotypes - Evaluation of physiological parameters and remediation potential.

Hexachlorocyclohexanes (HCHs) are persistent organochlorine pesticides with the adverse effects on human health and the environment. The effect of delta-isomer of hexachlorocyclohexane (δ-HCH) on germination, growth parameters and physiological parameters was studied in different Alnus glutinosa (L.) Gaertn. progeny of resistant genotypes to pathogen Phytophthora ×alni. Two experiments were performed: a short-term experiment to determine the effect of δ-HCH on total germination (GT), germination energy (GE), speed of germination (SG), shoot length and biomass of seedlings, and a long-term experiment devoted to remediation aspects. In addition, changes in the hormonal system of alders were monitored in both cases. Significant differences were found between the treated and control group in most of the evaluated characteristics. Also, the content of studied phytohormones differs between groups. Furthermore, the obtained results indicate genetically determined variability in response to δ-HCH. Of the six tested, the Brezové and Turany progeny seem to be suitable candidates for phytoremediation because of the adaptation to stress conditions or high remediation efficiency. The rest of tested progeny seems to be unsuitable due to higher mortality, lower remediation efficiency and higher levels of stress hormones resulting in significant decrease in biomass and plant height. Moreover, results indicate the role of the plant as a remediation accelerator, probably through released exudates, and a positive effect on the soil microbiome as the presence of plants increased the remediation efficiency by 20.85 - 35.89%. The obtained research findings may be helpful in better understanding the processes involved in removing these pesticides from the soil. Further research should be focused on rhizosphere microbiome, mechanism of in-plant isomerization and metabolites identification.

Benefits and Pitfalls of HPLC Coupled to Diode-Array, Charged Aerosol, and Coulometric Detections: Effect of Detection on Screening of Bioactive Compounds in Apples.

The new screening method for rapid evaluation of major phenolic compounds in apples has been developed. Suitability of coupling HPLC/UHPLC separation with the diode-array detection and universal charged aerosol detection with respect to the presence of interfering substances was tested. Characteristics of both detection techniques were compared and method linearity, limits of detection and quantitation, and selectivity of them determined. Student t-test based on slopes of calibration plots was applied for the detailed comparison. The diode-array detection provided the best results regarding sensitivity and selectivity of the developed method in terms of evaluation of phenolics profiles. The response of the charged aerosol detector was negatively affected by co-eluting substances during rapid-screening analyses. Coulometric detection was used for advanced characterization of extracts in terms of antioxidant content and strength to obtain more complex information concerning sample composition. This detection also allowed evaluation of unidentified compounds with antioxidant activity. HPLC/UHPLC separation using a combination of diode-array and coulometric detectors thus represented the best approach enabling quick, yet complex characterization of bioactive compounds in apples.

Mutation frequency dynamics in HPRT locus in culture-adapted human embryonic stem cells and induced pluripotent stem cells correspond to their differentiated counterparts.

The genomic destabilization associated with the adaptation of human embryonic stem cells (hESCs) to culture conditions or the reprogramming of induced pluripotent stem cells (iPSCs) increases the risk of tumorigenesis upon the clinical use of these cells and decreases their value as a model for cell biology studies. Base excision repair (BER), a major genomic integrity maintenance mechanism, has been shown to fail during hESC adaptation. Here, we show that the increase in the mutation frequency (MF) caused by the inhibition of BER was similar to that caused by the hESC adaptation process. The increase in MF reflected the failure of DNA maintenance mechanisms and the subsequent increase in MF rather than being due solely to the accumulation of mutants over a prolonged period, as was previously suggested. The increase in the ionizing-radiation-induced MF in adapted hESCs exceeded the induced MF in nonadapted hESCs and differentiated cells. Unlike hESCs, the overall DNA maintenance in iPSCs, which was reflected by the MF, was similar to that in differentiated cells regardless of the time spent in culture and despite the upregulation of several genes responsible for genome maintenance during the reprogramming process. Taken together, our results suggest that the changes in BER activity during the long-term cultivation of hESCs increase the mutagenic burden, whereas neither reprogramming nor long-term propagation in culture changes the MF in iPSCs.

Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells.

Although the detection of several components of the fibroblast growth factor (FGF) signaling pathway in human embryonic stem cells (hESCs) has been reported, the functionality of that pathway and effects on cell fate decisions are yet to be established. In this study we characterized expression of FGF-2, the prototypic member of the FGF family, and its receptors (FGFRs) in undifferentiated and differentiating hESCs; subsequently, we analyzed the effects of FGF-2 on hESCs, acting as both exogenous and endogenous factors. We have determined that undifferentiated hESCs are abundant in several molecular-mass isoforms of FGF-2 and that expression pattern of these isoforms remains unchanged under conditions that induce hESC differentiation. Significantly, FGF-2 is released by hESCs into the medium, suggesting an autocrine activity. Expression of FGFRs in undifferentiated hESCs follows a specific pattern, with FGFR1 being the most abundant species and other receptors showing lower expression in the following order: FGFR1 --> FGFR3 --> FGFR4 --> FGFR2. Initiation of differentiation is accompanied by profound changes in FGFR expression, particularly the upregulation of FGFR1. When hESCs are exposed to exogenous FGF-2, extracellular signal-regulated kinases are phosphorylated and thereby activated. However, the presence or absence of exogenous FGF-2 does not significantly affect the proliferation of hESCs. Instead, increased concentration of exogenous FGF-2 leads to reduced outgrowth of hESC colonies with time in culture. Finally, the inhibitor of FGFRs, SU5402, was used to ascertain whether FGF-2 that is released by hESCs exerts its activities via autocrine pathways. Strikingly, the resultant inhibition of FGFR suppresses activation of downstream protein kinases and causes rapid cell differentiation, suggesting an involvement of autocrine FGF signals in the maintenance of proliferating hESCs in the undifferentiated state. In conclusion from our data, we propose that this endogenous FGF signaling pathway can be implicated in self-renewal or differentiation of hESCs.