Phylogenetic relationships and status of taxa of Pulsatilla uralensis and P. patens s.str. (Ranunculaceae) in north-eastern European Russia.

We studied the allopatric complex Pulsatilla patens (L.) Mill. s.lat. (Ranunculaceae) in north-eastern European Russia and the Urals. In this region, there are two kinds of P. patens with different perianth colours in monochrome and polychrome populations. To clarify their taxonomic boundaries, we used the sequences of chloroplast DNA (rbcL and matK) and nuclear DNA (ITS2), in addition to morphological characteristics. The combination of three markers (rbcL+matK+ITS2) was found to be the most effective for phylogenetic resolution. The samples of two morphologically-different taxa P. uralensis and P. patents s.str. were shown to form a single clade on the phylogenetic tree. Based on the molecular phylogenetic analysis, we were not able to unequivocally prove the independent existence of P. uralensis.

Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1.

Molecular responses to genotoxic stress, such as ionizing radiation, are intricately complex and involve hundreds of genes. Whether targeted overexpression of an endogenous gene can enhance resistance to ionizing radiation remains to be explored. In the present study we take an advantage of the CRISPR/dCas9 technology to moderately overexpress the RPA1 gene that encodes a key functional subunit of the replication protein A (RPA). RPA is a highly conserved heterotrimeric single-stranded DNA-binding protein complex involved in DNA replication, recombination, and repair. Dysfunction of RPA1 is detrimental for cells and organisms and can lead to diminished resistance to many stress factors. We demonstrate that HEK293T cells overexpressing RPA1 exhibit enhanced resistance to cell killing by gamma-radiation. Using the alkali comet assay, we show a remarkable acceleration of DNA breaks rejoining after gamma-irradiation in RPA1 overexpressing cells. However, the spontaneous rate of DNA damage was also higher in the presence of RPA1 overexpression, suggesting alterations in the processing of replication errors due to elevated activity of the RPA protein. Additionally, the analysis of the distributions of cells with different levels of DNA damage showed a link between the RPA1 overexpression and the kinetics of DNA repair within differentially damaged cell subpopulations. Our results provide knew knowledge on DNA damage stress responses and indicate that the concept of enhancing radioresistance by targeted alteration of the expression of a single gene is feasible, however undesired consequences should be considered and evaluated.

Genetic analysis in earthworm population from area contaminated with radionuclides and heavy metals.

This study assessed the effects of environmental contamination by naturally occurring radionuclides and heavy metals on the genetic structure of a population of the earthworm Aporrectodea caliginosa. A. caliginosa were collected from four sites and characterized by amplified fragment length polymorphism (AFLP) analyses. No differences in genetic structure and diversity were found between sites that differed greatly in soil contamination levels of radionuclides and metals. However, when the genetic structure of the A. caliginosa population was analyzed without considering information about the sampling site, a complex intraspecific genetic structure was identified. At least three highly divergent lineages were found, in unequal proportions, of each genetically isolated group from each study site. No associations were found between the distribution of the detected genetic clusters and the geographical origin of the samples. Thus, no noticeable adaptive changes or signs of directional selection were detected, despite the long history of genotoxic waste disposal at the sampling site. These results suggest a combined effect of three factors on the genetic structure and diversity of A. caliginosa in soils: the complexity of the contaminant composition, the heterogeneous spatial distribution of the pollutants, and the complexity of the intraspecific genetic structures of A. caliginosa.

Selective binding of a bioactive porphyrin-based photosensitizer to the G-quadruplex from the KRAS oncogene promoter.

BACKGROUND: The G-quadruplex-forming sequence within the KRAS proto-oncogene P1 promoter is a promising target for anticancer therapy. Porphyrin derivatives are among the most rewarding G-quadruplex binders. They can also behave as photosensitizers. METHODS: Three water-soluble, positively charged porphyrin-like compounds were synthesized and tested for their interaction with the KRAS G-quadruplex by circular dichroism, fluorescence, and molecular docking calculations. For a comparison of ligands binding affinity and selectivity, TMPyP4 was taken as a reference. RESULTS: One out of the three tested compounds proved biological activity and selectivity for G-quadruplex over duplex DNA. It also showed to discriminate between different G-quadruplex topologies, with a preference for the parallel over antiparallel conformation. Molecular docking studies suggested a preferential binding to the 3'-end of the KRAS G-quadruplex driven through π-π stacking interactions. Biological assays also revealed a good photodynamic-induced cytotoxicity on HeLa cells. CONCLUSIONS: The reported results show that these porphyrin-like compounds could actually give the basis for the development of G-quadruplex ligands with effective photodynamic-induced cytotoxicity on cancer cells. GENERAL SIGNIFICANCE: The possibility of obtaining photosensitizers with improved physico-chemical features and able to selectively target G-quadruplexes is a very interesting perspective to develop new therapeutic agents.

Novel hydrophilic galactose-conjugated chlorin e6 derivatives for photodynamic therapy and fluorescence imaging.

We synthesized new hydrophilic chlorin e6 derivatives with two and four galactose fragments conjugated to the macrocycle via carbon atom in position 6 of the galactose fragment. Galactose fragments were inserted by alkylation of the amino groups of chlorin e6 amides with one and two ethylene diamine fragments on the macrocycle periphery with triflate of diacetone galactose, followed by removal of diisopropylidene protection by 70% aqueous trifluoroacetic acid. The synthesized compounds were shown to be capable of penetrating the membrane of HeLa cells; they have intense red fluorescence inside the cell and have phototoxic properties towards HeLa cells (upon LED irradiation at 660 nm and light exposure value of 12 J/cm2). These properties, along with water solubility, allow us to consider the synthesized compounds to be promising as potential antitumor PSs and diagnostic compounds for visualizing malignant tumors and creating on their basis preparations for simultaneous diagnostics and therapy of oncological diseases.

Programming of Cell Resistance to Genotoxic and Oxidative Stress.

Different organisms, cell types, and even similar cell lines can dramatically differ in resistance to genotoxic stress. This testifies to the wide opportunities for genetic and epigenetic regulation of stress resistance. These opportunities could be used to increase the effectiveness of cancer therapy, develop new varieties of plants and animals, and search for new pharmacological targets to enhance human radioresistance, which can be used for manned deep space expeditions. Based on the comparison of transcriptomic studies in cancer cells, in this review, we propose that there is a high diversity of genetic mechanisms of development of genotoxic stress resistance. This review focused on possibilities and limitations of the regulation of the resistance of normal cells and whole organisms to genotoxic and oxidative stress by the overexpressing of stress-response genes. Moreover, the existing experimental data on the effect of such overexpression on the resistance of cells and organisms to various genotoxic agents has been analyzed and systematized. We suggest that the recent advances in the development of multiplex and highly customizable gene overexpression technology that utilizes the mutant Cas9 protein and the abundance of available data on gene functions and their signal networks open new opportunities for research in this field.

Dark and Photoinduced Cytotoxic Activity of the New Chlorophyll-a Derivatives with Oligoethylene Glycol Substituents on the Periphery of Their Macrocycles.

In the present work, we investigated the dark and photoinduced cytotoxic activity of the new chlorophyll-a derivatives which contain the substituents of oligoethylene glycol on the periphery of their macrocycles. These compounds were tested using human cell lines to estimate their potential as photosensitizers for photodynamic therapy of cancer. It was shown that all the tested compounds have expressed photoinduced cytotoxic activity in vitro. Detailed study of the biological activity of one of the most perspective compound in this series-pyropheophorbide-a 17-diethylene glycol ester (Compound 21) was performed. This new compound is characterized by lower dark cytotoxicity and higher photoinduced cytotoxicity than previously described in a similar compound (DH-I-180-3) and clinically used PhotolonTM. Using fluorescent microscopy, it was shown that Compound 21 quickly penetrates the cells. Analysis of caspase-3 activity indicated an apoptosis induction 40 min after exposure to red light (λ = 660 nm). The induction of DNA damages and apoptosis was shown using Comet assay. The results of expression analysis of the stress-response genes indicate an activation of the genes which control the cell cycle and detoxification of the free radicals after an exposure of HeLa cells to Compound 21 and to red light. High photodynamic activity of this compound and the ability to oxidize biomolecules was demonstrated on nuclear-free mice erythrocytes. In addition, it was shown that Compound 21 is effectively activated with low energy 700 nm light, which can penetrate deep into the tissue. Thus, Compound 21 is a prospective substance for development of the new drugs for photodynamic therapy of cancer.

Differential Molecular Stress Responses to Low Compared to High Doses of Ionizing Radiation in Normal Human Fibroblasts.

Understanding the mechanisms producing low dose ionizing radiation specific biological effects represents one of the major challenges of radiation biology. Although experimental evidence does suggest that various molecular stress response pathways may be involved in the production of low dose effects, much of the detail of those mechanisms remains elusive. We hypothesized that the regulation of various stress response pathways upon irradiation may differ from one another in complex dose-response manners, causing the specific and subtle low dose radiation effects. In the present study, the transcription level of 22 genes involved in stress responses were analyzed using RT-qPCR in normal human fibroblasts exposed to a range of gamma-doses from 1 to 200 cGy. Using the alkali comet assay, we also measured the level of DNA damages in dose-response and time-course experiments. We found non-linear dose responses for the repair of DNA damage after exposure to gamma-radiation. Alterations in gene expression were also not linear with dose for several of the genes examined and did not follow a single pattern. Rather, several patterns could be seen. Our results suggest a complex interplay of various stress response pathways triggered by low radiation doses, with various low dose thresholds for different genes.