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.

New records of Lumbricidae and Collembola in anthropogenic soils of East European tundra.

The terrestrial environment of the East European tundra consists of a mosaic of habitat types. In addition to the natural habitat diversity, various human-influenced types may occur. In the town of Vorkuta, Komi Republic, Russia the manure-enriched soils near hydrogen sulfide springs were observed. This site represents an unusually nutrient-rich location with considerable development of organic soils, in contrast to the naturally forming soils in East European tundra which are typically thin and nutrient poor. In these organic soils, two species of Lumbricidae and two species of Collembola previously not recorded from the natural ecosystems in the study area of research territory were found. One earthworm species, Dendrodrilus rubidus tenuis, is likely to have been introduced. The presence of the three other species (Eiseniella tetraedra, Folsomia fimetaria, and Proisotoma minuta) is quite natural in East European tundra and such anthropogenic soils with high organic content may be a good habitat for them.

Population and molecular datasets for Gymnadenia conopsea (Orchidaceae).

The paper presents data on the ecological and phytocoenotic conditions of habitats of the G. conopsea (L.) R. Br. orchid in the Northeast of European Russia (Komi Republic, Russia). The data include characteristics of the populations of this species on the northern border of its range (size and ontogenetic structure of the populations, density of specimens, phenology), as well as information demonstrating the genetic variability of the species by ISSR-markers that was not included in the main publication. The data presented here supplement our earlier published results O.E.Valuyskikh et al., 2019 and are useful for more detailed analysis of population biology and genetic variability of this rare orchid species. G. conopsea is the species of terrestrial orchids widespread in Europe and Asia and characterized by the widest ecological-cenotic amplitude and occurrence in different types of ecotopes. The size of G. conopsea populations in different parts of its range is usually small, 25-100 ind. but sometimes increases to 200-1000 ind. Hansen et al., 1999 to Travnichek et al., 2012. In the many regions of the Russian Federation, the G. conopsea are subject to protection due to the small number of habitats, long stages of ontogenesis, low population sizes and anthropogenic impact. The complex of G. conopsea s.l. included in the Red Data Book of the Komi Republic Taskaev, 2009.

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.