[Novel miRNAs as Potential Regulators of PD-1/PD-L1 Immune Checkpoint, and Prognostic Value of MIR9-1 and MIR124-2 Methylation in Ovarian Cancer].

Ovarian cancer (OC) is mostly detected at late stages weighed down with metastasis, and the five-year survival rate of patients is only 30%, which dictates the necessity to develop gentler and more selectively targeted drugs that current chemotherapeutic agents. The search for factors that can influence on the activity of the PD-1/PD-L1 immune checkpoint signaling pathway in tumors is relevant, and micro RNAs (miRNAs) play an important role in it. Over the past 5 years, only a few miRNAs (miR-34a, miR-145, and miR-424), which have a regulatory effect on the PD-1/PD-L1 system in OC patients, have been discovered. In present work, the methylation levels of 13 miRNA genes in 26 primary tumors and 19 peritoneal metastases of OC patients were determined and compared with the level of the soluble form of PD-L1 (sPD-L1) in the blood plasma of the same patients. It was shown that the methylation levels of five miRNA genes (MIR124-2, MIR34B/C, MIR9-1, MIR9-3, and MIR339) in tumors are in direct correlation with the sPD-L1 level in the blood plasma. In addition, when analyzing these five genes, a significant association of the methylation level of the MIR9-1 gene with a decrease in the three-year relapse-free survival, and a trend for decrease in the three-year survival rate with the methylation level of the MIR124-2 gene of OC patients were determined. Thus, the first data suggesting the role of inhibitors of the sPD-L1 immune checkpoint for five miRNAs (miR-124, miR-34b, miR-34c, miR-9, miR-339) and the possibility of using hypermethylated MIR9-1 and, presumably, MIR124-2 genes as independent prognostic markers of poor disease-free survival in OC patients were obtained.

[Method for predicting perinatal hypoxic lesions of the central nervous system in newborns.]

Perinatal lesions of the Central nervous system (CNS) in newborns occupy a leading place in the structure of perinatal morbidity and subsequent disability of children. To identify the features of the content of sRAGE in pregnant women with threatening preterm labor (UPR) in the period of 22-27 weeks, who subsequently gave birth to children with perinatal CNS lesion. Serum of venous blood of pregnant women with UPR at the term of 22-27 weeks was determined by ELISA once the content of sRAGE. If the value of sRAGE in pregnant women is 659.5 PG/ml or less, perinatal hypoxic lesions of the Central nervous system in newborns are predicted with an accuracy of 75.8% (sensitivity of 82.6%, specificity of 66.7%).

Biodistribution of Alpha-Fetoprotein-Containing Noncovalent Complex Aimpila with Antitumor Activity.

Biodistribution of [125I]Aimpila (20 mg/kg) in the tumor and normal tissues, including the mammary gland tissue, after single oral dose was studied in BALB/c nude mice with T47D/ReCAF+++ human breast tumor sensitive to this drug and in closely related BALB/c nude+mice without tumors. The maximum concentration of [125I]Aimpila was in fact the same in the tumor and in the mammary gland, while the time course of its accumulation/elimination differed. The time of the maximum accumulation of the drug in the tumor was shorter and its persistence longer than in normal tissue. After 24 h, label concentration in the tumor was 4.5 times higher (p=0.002). Differences in the time course of label accumulation in the tumor were detected. The maximum ratio of tumor/blood concentrations of the preparation was recorded in 1 h after administration. [125I]Aimpila and [125I]alpha-fetoprotein accumulated in the tumor in comparable concentrations and were eliminated simultaneously at the same rate. The results of comparative analysis of accumulation of the labeled compounds in Aimpila-sensitive T47D/RECAF+++ tumor from 0.5 to 9.0 h after drug administration could be interpreted as a result of possible receptor-mediated binding of the complex with the tumor at the expense of the alpha-fetoprotein transporting part. Differences in the parameters of [125I]Aimpila biodistribution in the tumor and normal mammary tissue indirectly attested to selective antiproliferative activity of the complex.

IMMUNOMODULATORY EFFECT OF ALLOGENEIC MESENCHYMAL STEM CELLS OF RATS.

INTRODUCTION: Recently, more and more attracted the attention of cell therapy, which requires a study of the efficacy and safety of allogeneic MSCs transplantation in acute and chronic inflammatory reactions. The aim of our study was to examine the effectiveness of transplantation of allogeneic mesenchymal bone marrow stromal cells for the healing of surgical wounds the glandular stomach in rats. MATERIAL AND METHODS: Using white Wistar rats. Producing cell transplantation mononuclear fraction derived from rat bone marrow aspirate. Injected cells 8 and 9 th passage. The dose of cells administered to 3-th and / or 7-th days 3,5h106 cells / ml twice or 5,0h106 cells / ml dose. Autopsy on day 10-th and 17-th. The serum ELISA determined the content of the pro- and anti-inflammatory cytokines IL1ß, TNFα, IFNy, IL-4. RESULTS AND DISCUSSION: Introduction MSCs contributed to the decline of the immune mediators of inflammation IL1P, TNFa, IFNy, increase anti-inflammatory IL4. Quality improved healing. CONCLUSION: Rapid curative effect of stem cells may be associated with the formation of blood immune cells (macrophages) that produce substances that restore damaged tissue. They restore the balance between Th1 and Th2.

Effects of cadmium chloride on the functional state of human intestinal cells.

Toxic effects of cadmium chloride in concentration range from 1 to 300 µM on differentiated human intestinal epithelial Caco-2 cells after three hours of exposure were investigated. Processes of disorganization of the actin cytoskeleton associated with the toxic effects of cadmium were characterized by fluorescent microscopy. The cadmium-induced activation of cellular stress response processes (changes in the mRNA expression of caspase-3, heat-shock and oxidative stress genes) has been demonstrated. The study revealed dose-dependent changes in mRNA expression levels of proteins involved in the formation of adherens (E-Cadherin and p120 catenin) and tight intercellular junction contacts (Claudin 4 and ZO1). The time- and concentration-dependent trend of cell monolayer transepithelial resistance lowering, characterizing the loss of intercellular contacts density with prolongation of cell exposure cadmium chloride was estimated. Results indicates that proteins associated with tight and adhesion junctions are primary targets of cadmium. Amongst genes involved in cell junction formation, the genes encoding E-Cadherin and p120-catenin proved to be the most sensitive to cadmium influence.

Reactive oxygen species are involved in regulation of pollen wall cytomechanics.

Production and scavenging of reactive oxygen species (ROS) in somatic plant cells is developmentally regulated and plays an important role in the modification of cell wall mechanical properties. Here we show that H2O2 and the hydroxyl radical ((•)OH) can regulate germination of tobacco pollen by modifying the mechanical properties of the pollen intine (inner layer of the pollen wall). Pollen germination was affected by addition of exogenous H2O2, (•)OH, and by antioxidants scavenging endogenous ROS: superoxide dismutase, superoxide dismutase/catalase mimic Mn-5,10,15,20-tetrakis(1-methyl-4-pyridyl)21H, 23H-porphin, or a spin-trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone, which eliminates (•)OH. The inhibiting concentrations of exogenous H2O2 and (•)OH did not decrease pollen viability, but influenced the mechanical properties of the wall. The latter were estimated by studying the resistance of pollen to hypo-osmotic shock. (•)OH caused excess loosening of the intine all over the surface of the pollen grain, disrupting polar growth induction. In contrast, H2O2, as well as partial removal of endogenous (•)OH, over-tightened the wall, impeding pollen tube emergence. Feruloyl esterase (FAE) was used as a tool to examine whether H2O2-inducible inter-polymer cross-linking is involved in the intine tightening. FAE treatment caused loosening of the intine and stimulated pollen germination and pollen tube growth, revealing ferulate cross-links in the intine. Taken together, the data suggest that pollen intine properties can be regulated differentially by ROS. (•)OH is involved in local loosening of the intine in the germination pore region, while H2O2 is necessary for intine strengthening in the rest of the wall through oxidative coupling of feruloyl polysaccharides.

[Sporopollenin accumulation in Nicotiana tabacum L. microspore wall during its development].

Accumulation of sporopollenin components in microspore wall, its polymerization dynamics and possible participation of reactive oxygen species (ROS) in this process has been studied. For this purpose fluorescent and electron microscopy (TEM) was used. It has been determined that phenylpropanoid components of sporopollenin that form the exine accumulate in the microspore cell wall at the middle and late tetrad stages. At the late tetrad stage, they fully cover the microspore surface and accumulate abundantly in aperture areas. In accordance with this, numerous thick sporopollenin lamellae, electron-dense and acetolysis-resistant, emerge in aperture areas. Exine in the areas between apertures includes both acetolysis-resistant sporopollenin and washout components. These particular parts of the wall are intensively stained with fluorescent dye MitoSOX, which detects the presence of ROS. The staining disappeared after the treatment of microspore with superoxide dismutase, demonstrating the presence of superoxide in the exine. Superoxide easily converts to hydrogen peroxide, which can cause oxidative polymerization of sporopollenin components, leading to the formation of chemically stable biopolymer. The data obtained favor the hypothesis of ROS involvement in the formation of sporopollenin.

[Antioxidant properties of the pollen exine polymer matrix].

The antioxidant properties of exine polymer matrix which forms the outer layer of pollen grain wall were studied. The main component of this matrix is sporopollenin - a unique biopolymer resistant to mechanical and chemical damage. The samples of isolated exine, purified from soluble compounds, were studied with EPR using stable nitroxyl radical TEMPO and DMPO spin trap. At the same time, we analyzed changes in fluorescence of DCFH which detected ROS in the solution. It has been established that exine effectively reduces TEMPO radical and eliminates hydroxyl radical. Also, the fluorometric analysis demonstrated that the exine eliminated H2O2, and this ability significantly decreased after treatment of exine with feruloyl esterase or mild alkaline hydrolysis (1M NaOH), i.e. after hydrolysis of hydroxycinnamic acid esters. After harsh hydrolysis (4M NaOH, 170 degrees C) of ethers bonds a large amount of hydroxycinnamic acids has been released, and exines have lost their antioxidant capacity almost completely. The obtained results point to the ability of extracellular polymer matrix of the exine to eliminate free radicals and H2O2 during crucial periods of male gametophyte development. The participation of ferulic acid and, possibly, of other hydroxycinnamic acids of sporopollenin in these processes has been demonstrated.

[Another discussion of membrane voltage alterations in growing pollen tube].

Here we give a critical analysis of the opinion of Andreev (2011) on membrane potential distribution along the pollen tube plasmalemma. He assumes that a lateral gradient of dipole potential exists, but suggests a lateral gradient of transmembrane potential impossible. We demonstrate by concrete examples that the argumentation of the initiator of discussion is based on inaccurate citation of our experimental data (Breygina et al., 2009) and incomplete analysis of previously published articles. Speaking about transmembrane potential, he doesn't consider numerous facts demonstrating the uneven distribution of transmembrane ion fluxes and ion-transport proteins in the pollen tube plasmalemma, as well as data obtained by modeling of transmembrane potential distribution in objects of different shape. In addition, the assumption on the uneven distribution of dipole potential doesn't have an experimental basis neither in studies of the pollen tube, nor in the practice of using fluorescent voltage-sensitive dyes DiBAC4(3) and Di-4-ANEPPS. We are expecting the author to obtain experimental data in support of his position.

[Antiproliferative activity of diclofenac at tumor cell cultures].

It is known that for risk group's importance of early diagnostics and prevention of a cancer of a thick section of the intestines. The view was expressed that the use of nonsteroidal anti-inflammatory drugs prospectively. The article presents the data obtained after the determination of the effect on the domestic drug diclofenac on the ability of the lines of tumor cells of the human colon multiply this antiproliferativoe action on the dose of the drug. Used photometric method for determination antiproliferativoe action. In vitro determined the effect of the national drug diclofenac on the ability of the lines of tumor cells of the human colon multiply, and is set in a dose-dependent effect. It is established that under the action of NSAIDS metabolic activity of cells can both strengthen and weaken that depends on the line of tumor cells and the concentration of the drug.

[Effects of anion channel blockers NPPB and DIDS on tobacco pollen tube growth and its mitochondria state].

Influence of anion channel blockers NPPB and DIDS on pollen tube growth and its mitochondria functioning was studied by means of fluorescence microscopy and flow cytometry. NPPB (40 microM) blocked pollen tube growth completely, but did not change its diameter. DIDS (20-80 microM) caused pollen tube swelling and bursting, suggesting that DIDS-sensitive channels take part in the regulation of pollen tube osmotic balance. The osmotic effect of low DIDS concentration (20 (Mkappa)M) was not accompanied by changes in the tube growth rate. The mapping of membrane potential on the pollen tube plasmalemma using Di-4-ANEPPS revealed the involvement of NPPB-sensitive but not DIDS-sensitive anion channels in the maintenance of the longitudinal membrane potential gradient along the tube surface. The study of isolated pollen mitochondria showed that DIDS increased their capacity to take up potential-dependent dye DiOC5(3), i. e. caused hyperpolarization of mitochondrial membranes. At the same time DIDS influenced on intramitochondrial ROS content and excretion of ROS from mitochondria. Thus, NPPB and DIDS differently influenced on transmembrane potential distribution along pollen tube plasmalemma, on its osmotic balance, and on mitochondria functioning. This set of data suggests that pollen tube growth is dependent on activity of anion channels that differ in localization and functions.

[Membrane potential changes during pollen germination and tube growth].

We applied quantitative fluorescent microscopy to study membrane potential alterations during pollen germination and in growing pollen tube. Two voltage-sensitive dyes were applied: DiBAC4(3) was used to detect average membrane potential values in pollen grains and isolated protoplasts; Di-4-ANEPPS gave an option of membrane potential mapping on pollen protoplast and pollen tube surfaces. We have found out that tobacco pollen grain activation is accompanied by hyperpolarization of the vegetative cell plasma membrane by about 8 mV. Lily pollen protoplasts were significantly hyperpolarized (-108 mV) with respect to the pollen grains (-23 mV) from which they were isolated. We found polar distribution of the membrane potential along the protoplast surface, and longitudinal potential gradient along the pollen tube. In the presence of plasma membrane H(+)-ATPase inhibitor, sodium orthovanadate (1 mM) or its activator fusicoccin (1 microM), the longitudinal voltage gradient altered but did not disappear. Anion channel blocker, NPPB (40 microM), fully discarded the gradient in pollen tubes. Obtained results give evidence of the plasma membrane hyperpolarization during pollen germination and uneven potential distribution on pollen grain and tube surfaces. Inhibitory analysis showed involvement of the plasma membrane H(+)-ATPase and anion channels in membrane potential regulation.

[Formation of reactive oxygen species during pollen grain germination].

The formation of reactive oxygen species in pollen at the early germination stage, which precedes the formation of the pollen tube, was studied. During this period, pollen grain is being hydrated, abruptly increasing its volume, and it passes from the resting state to active metabolism. Fluorescent methods have made it possible to reveal reactive oxygen species in the cytoplasm and inner layer of the pollen wall, intine. The cytoplasmic reactive oxygen species were mostly found in mitochondria, while extracellular ones were localized in aperture zones of intine, as well as in the solution surrounding pollen grains in vitro. The content of extracellular reactive oxygen species decreased after superoxide dismutase (100 units per ml) and diphenylene iodonium (100 microM), which indicates NADPH oxidase as one of possible producent of them. In conditions of suppression of extracellular reactive oxygen species production (100 microM diphenilene iodonium) or their promoted removal (after addition of 10 to 100 microM ascorbic acid), the number of germinating pollen grains increased. This effect disappeared after further increase in the concentration of the listed reagents. The result is evidence of the significance of processes of generation/removal of extracellular reactive oxygen species for pollen germination.