Background: Many plant secondary metabolites (PSMs) were shown to intercalate into DNA helix or interact with DNA grooves. This may influence histone-DNA interactions changeing chromatin structure and genome functioning. Methods: Nucleosome stability and linker histone H1.2, H1.4 and H1.5 localizations were studied in HeLa cells after the treatment with 15 PSMs, which are DNA-binders and possess anticancer activity according to published data. Chromatin remodeler CBL0137 was used as a control. Effects of PSMs were studied using fluorescent microscopy, flowcytometry, quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), western-blotting. Results: We showed that 1-hour treatment with CBL0137 strongly inhibited DNA synthesis and caused intensive linker histone depletion consistent with nucleosome destabilization. None of PSMs caused nucleosome destabilization, while most of them demonstrated significant influence on linker histone localizations. In particular, cell treatment with 11 PSMs at non-toxic concentrations induced significant translocation of the histone H1.5 to nucleoli and most of PSMs caused depletion of the histones H1.2 and H1.4 from chromatin fraction. Curcumin, resveratrol, berberine, naringenin, and quercetin caused significant redistribution of all three variants of the studied linker histones showing some overlap of PSM effects on linker histone DNA-binding. We demonstrated that PSMs, which induced the most significant redistribution of the histone H1.5 (berberine, curcumin and naringenin), influence the proportion of cells synthesizing DNA, expressing or non-expressing cyclin B and influence cell cycle distribution. Berberine induction of H1.5 translocations to nucleoli was shown to occur independently on the phases of cell cycle (metaphase was not analyzed). Conclusions: For the first time we revealed PSM influence on linker histone location in cell nuclei that opens a new direction of PSM research as anticancer agents.

Background. The most important task in the field of renal-cell cancer (RCC) treatment results improvement is the search and validation of the markers for its early diagnostics still absent in the clinical practice. It was established that even before the onset and/or detection of RCC the level of kidney injury molecule-1 (KIM-1) in blood plasma did increase.Objective of the study — comparative evaluation of KIM-1 levels in blood plasma of practically healthy persons, RCC cancer, benign kidney tumor patients, patients with non-oncological renal pathologies, and analysis of its role in RCC diagnostics and prognosis.Materials and methods. 125 RCC (age 33—81 years), 14 — benign kidney neoplasms (29—84 years) patients, 90patients with chronic nephritis (28—82 years) and 68 practically healthy persons (18—71 years) were included in the study. Plasma KIM-1 content was measured using Human Serum TIM-1/KIM-1/HAVCR Quantikine® ELISA kit (RD Systems Biotechne®, USA).Results. KIM-1 level in blood plasma of RCC and chronic nephritis patients was significantly higher than in control (medians 305, 282 and 37.8pg/ml respectively, p 0.0001). The rate of KIM-1 elevation over cut-offvalue 90pg/ml corresponding to the upper 95 % confidence interval of control in RCC patients comprised 79.2 %, in patients with nephritis — 83 %, in those with benign renal tumors — 50 %. Specificity in relation to healthy control was 96 %. KIM-1 level highly significantly increased with RCC progression, and already at stage I was 4.3-fold higher by median than in control (p 0.0001). Sensitivity of stage I—IIRCC detection at cut-off 90pg/ml comprised 75 %; stage III—IV — 94 %. The highest plasma KIM-1 levels were detected in papillary cancer patients (median 644pg/ml), that was more than 2-fold higher than in clear-cell and 32-fold higher than in chromophobic RCC. Plasma KIM-1 median level was 7-fold higher in patients with G3 4RCC than in those with G12 (p 0.0001). At the cut-off KIM-1 value of 163pg/ml, corresponding to the median at stage I, significant differences in 3.5-years overall survival both in the total group: 49 % at high, 95 % at low marker level (p 0.01), and at stage I RCC: 62 % and 100 % respectively (p 0.05) — were revealed.Conclusion. Plasma KIM-1 may become the first highly sensitive marker for the early detection of RCC, but it does not allow differentiating between oncologic and non-oncologic renal pathologies. Increased basal plasma KIM-1 is an unfavorable prognostic factor irrespective of the stage of tumor progression.

Introns are widely used in the assembly of genetic constructions expressing transgenic proteins in eukaryotic cells for the enhancement of this expression. However, the choice of introns that can be applied for such purposes is limited by the excessively large size of the majority of natural introns (several thousand nucleotides) and therefore they cannot be cloned in a genetic construction. With the help of site-directed mutagenesis we have generated a library of short (99 nucleotides long) introns. The efficiency of these introns in the enhancement of gene expression was analyzed. As a result, a set of 12 introns was selected. The generated intros can be used for genetic constructions with high expression level of recombinant proteins.

The isothermal amplification of nucleic acids refers to processes that quickly increase the amount of DNA at a constant temperature. These methods are mainly developed as alternatives to PCR for cases in which the application of a thermal cycler is not possible or the assay method must be as rapid as possible. We have developed a new method of isothermal amplification based on the formation of hairpins at the ends of DNA fragments containing palindromic sequences and increased by the hydrolysis of one or both DNA strands by restriction endonuclease, known as hairpin-assisted isothermal reaction (HAIR). The key steps in HAIR are the formation of a self-complementary hairpin and the DNA breakage introduced by nickase. The end hairpins facilitate primer-free amplification, the amplicon strand cleavage by nickase produces additional 3′ ends that serve as new amplification points, and the amount of DNA can increase exponentially. The rate of amplification in HAIR is more than five times the rate of loop-mediated isothermal amplification (LAMP), and the total amount of DNA product of HAIR is more than double the amount of the LAMP product.