Currently, a large number of reagent kits are commercially available for the isolation of highly purified plasmid DNA for subsequent transfection of human cell lines. However, due to high cost and logistical problems, it may be necessary to isolate plasmid DNA using only the simplest reagents and materials. We present one of the possible methods for such DNA isolation, suitable for routine laboratory use. It is based on well-known principles and methods for plasmid DNA purification, has minimal cost, does not require special skills, and is easily scalable. The technique includes the steps of alkaline lysis, purification with silica particles and gel filtration. It was shown that plasmids isolated using the proposed method transfect human embryonic kidney Expi293F cells no less efficiently than plasmids purified using a specialized Qiagen plasmid maxi kit («Qiagen», USA).

Five new bifunctional conjugates of pyropheophorbide a with 17-substituted testosterone, dihydrotestosterone and epitestosterone differing in the length of linker (1 – 5) and two new complex conjugates 6 and 7 (containing three functional units: pyropheophorbide a, 17α-substituted testosterone, and lipophylic hexadecyl chain, connected with L-lysine joining block) were synthesized. Mutual influence of steroidal and macrocyclic fragments in conjugates (1 – 7) was established by analysis of 1H NMR spectra and molecular models of conjugates. Studies of interaction of conjugates 1 – 5 with prostate carcinoma cells revealed that their uptake and internalization were dependent on the structure of conjugates, particularly on the stereochemical configuration of 17-hydroxyl group in steroidal moiety, and the length of linker connecting pyropheophorbide a with steroid fragments. Conjugates 1 – 5 significantly decreased the growth and proliferation of LNCaP and PC-3 cells. The highest anti-proliferative activity demonstrated by epitestosterone derivative 3, comprising short linker. Irradiation of labeled cells with light (λ = 660 nm) was significantly increased cytotoxicity. Trifunctional conjugates 6 and 7 easily formed mixed micells with phosphatidyl choline and pluronic F68; these mixed micelles efficiently internalized by human hepatocarcinoma Hep G2 cells. The binding of conjugates 6 and 7 in the form of mixed micelles to Hep G2 cells depended on the conjugate structure, rather than on the method of solubilization.

The aim of this work is to present the experimental results in the form of an algorithm for analyzing the modification of screen printed electrodes, including the possibility of its regeneration for irreversibly oxidizing biologically active compounds (drugs, DNA and proteins). A protocol was developed for quantitative analysis and study of the mechanism of drug-DNA interaction by differential pulse voltammetry, including the following parameters: complex binding constant, Gibbs free energy, and electrochemical coefficients of the toxic effect.

The electroanalytical characteristics of double-stranded DNA (dsDNA) and the complex of dsDNA and the antitumor drug abiraterone acetate (AA) were studied by differential pulse voltammetry. The effect of abiraterone acetate on dsDNA was shown, which was registered by alteration the intensity of electrochemical oxidation of purine heterocyclic bases guanine and adenine using screen printed electrodes modified with functionalized carbon nanotubes. The binding constants (Kb) of the [dsDNA-AA] complex for guanine and adenine were 1.63×10⁴ M^-1 and 1.93×10⁴ M^-1, respectively. The electrochemical coefficients of the toxic effect were calculated as the ratio of the intensity of the electrochemical oxidation signals of guanine and adenine, in the presence of abiraterone acetate to the intensity of the electrooxidation signals of these nucleobases without drug (%). At concentrations of abiraterone acetate exceeding 60 μM, a decrease in the currents of electrochemical oxidation of guanine and adenine by 50% or more is recorded. Based on the analysis of electrochemical parameters and values ​​of binding constants, an assumption was made about the mechanism of interaction of abiraterone acetate with DNA, mainly due to the formation of hydrogen bonds with the minor groove. An electrochemical DNA biosensor was first used to study the mechanism of interaction of the anticancer drug abiraterone acetate with dsDNA.

The paper describes an original technique for detecting the collateral activity of CRISPR/Cas 13a ribonuclease based on the assessment of ribosomal RNA degradation. The Agilent 2100 bioanalyzer is used as an analyzing device. This approach is an alternative to existing detection methods and has a number of advantages over them in the case when a quantitative assessment of activity is not required. On the example of the test sample, the optimal concentrations and ratios of the components of the reaction mixture, which are necessary to obtain the most indicative result, were determined. The proposed technique can be used for qualitative assessment of the activity of recombinant ribonuclease Cas13a preparations obtained under different conditions of heterologous protein expression and purification, as well as for testing guide RNAs.

Renalase (RNLS) is a protein that performs various protective functions both inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase (EC 1.6.3.5). Extracellular RNLS lacking an N-terminal peptide does not interact with FAD and exhibits various protective effects on the cell through interaction with receptor proteins. The mechanisms and factors responsible for RNLS transport out of the cell are not fully understood. It is well known that the signal sequence plays a key role in the classical mechanism of protein transport outside cells. One of the approaches to study the secretion of RNLS from the cell can be the creation of chimeric forms of the protein with a modified N-terminal amino acid signal sequence. Bioinformatics analysis showed that the signal sequence of the prolactin gene (PRL), connected to the template sequence of the RNLS gene, gave the classic signal characteristic of secretory proteins. On this basis, this paper describes: (i) a method for constructing the human RNLS gene in which the N-terminal sequence encoded by the RNLS gene was replaced by the N-terminal sequence encoded by the PRL gene; (ii) expression of this chimeric genetic construct.

A number of simple filters formulated from general considerations that take into account the peculiarities of the experiments as well as results obtained in 2D electrophoresis experiments are considered. These filters can be used for automated dataset formation and verification of learning of system for predicting protein isoelectric point values. These include: (i) filtering obvious errors introduced during initial database formation; (ii) selection of a known plausible range of values; (iii) selection of a single variant among various proteoforms; (iv) selection within a preset value of electrophoretic shift deviation, etc. Using a dataset combining data from 8 maps of Homo sapiens, Mus musculus, and Rattus norvegicus, the application of this set of filters improved the R² value of predictions from 0.44 to 0.67.

Despite the large number of studies on the new coronavirus infection SARS-Cov-2 characterized by vascular damage on the impact of the virus on the mother-placenta-fetus system remains unknown. The umbilical cord, according to its functional purpose, is a complex of vessels protected from external influences by the embryonic connective tissue –Wharton's jelly. The aim of the study was to detect the structural components of SARS-Cov-2 in the histological structures of umbilicals cord in patients with COVID-19 in the tissues of the umbilical cord of women with COVID-19. The main group included 40 pregnant women who were treated at the academician V. I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology (March-April 2020) with a confirmed diagnosis of COVID-19 (according to the positive PCR test of a nasopharyngeal swab) and 40 pregnant women of the comparison group, without clinical symptoms and laboratory tests, including the negative PCR test. As a result of the immunohistochemical study of the umbilical cord with primary antibodies to the SARS-Cov-2 N-protein in the COVID-19 group, staining of the cytoplasm of fibroblast-like cells and single macrophages Wharton's jelly was detected (p<0.05). In the group of women without coronavirus infection no staining in Wharton's jelly cells was detected. The probable persistence of viral particles in mesenchymal cells and macrophages of the Wharton's jelly in the umbilical cord can be important for establishment new aspects of influence COVID-19 on the mother-placenta-fetus system. Our results suggesting persistence of COVID-19 viral proteins in mesenchymal cells of the umbilical cord could be an important in establishing new pathogenic mechanisms for the fetal protection against viruses.

Renalase (RNLS) is a flavoprotein; its N-terminal peptide (amino acid residues 1-17) performs various important functions. Inside cells, it is involved in the Rossmann fold formation (residues 2-35), which is necessary for the binding of the FAD cofactor and the manifestation of the enzymatic activity of RNLS as a FAD-dependent oxidoreductase (EC 1.6.3.5). When RNLS is secreted into the extracellular space, this peptide is cleaved off, and the resulting truncated extracellular RNLS can no longer bind FAD and, therefore, numerous effects described in the literature are carried out by non-catalytic mechanisms. In this work, we have investigated the sensitivity to trypsinolysis of two recombinant forms of human RNLS expressed in prokaryotic cells: (a) full-length RNLS containing the FAD cofactor; (b) a truncated RNLS lacking the 1-17 N-terminal peptide (truncatedRNLS, tRNLS) unable to bind the FAD cofactor. Trypsin (1 unit/20 μL of medium) effectively cleaved both forms of renalase (RNLS and tRNLS). When exposed to a lower concentration of trypsin (0.1 U/20 μL of medium), full length RNLS was more trypsin resistant than tRNLS. We suggest that the different sensitivity of RNLS and tRNLS is apparently determined by the presence of the FAD cofactor in the full-length recombinant protein, which contributes to the formation of a spatial structure that is more resistant to the action of certain proteases.

Prostate cancer is hormone-dependent and the androgen receptor (AR) is involved in its development. AR is a transcription factor that is activated by ligand binding, result in its translocation into the nucleus, where it initiates gene transcription. In an inactive state in cytoplasm AR exists as a complex with heat shock protein 90 (HSP90) and some other proteins. When the agonist binds, a conformational change in AR occurs, resulting in HSP90 and other chaperones dissociating. Recently it has been shown that for the dissociation of the HSP90-AR complex and the translocation of the latter into the nucleus, phosphorylation of the Thr-90 residue of the N-terminal domain of HSP90 is necessary. In this work, the effect of the HSP90 inhibitor, geldanamycin, interacting with the ATP-binding site, on the Thr90 phosphorylation site was investigated by molecular modeling methods. It has been shown that inhibitor binding slightly affects the size and mobility of cavity around Thr90. It is suggested that inhibitor binding to HSP90 does not result in changing the protein structure and does not influence on protein phosphorylation, and partially explains low effectiveness of such type of drugs in the therapy of prostate cancer.