Vol 14, No 4 (2022)

DNA methylation is the most important epigenetic modification involved in the regulation of transcription, imprinting, establishment of X-inactivation, and the formation of a chromatin structure. DNA methylation in the genome is often associated with transcriptional repression and the formation of closed heterochromatin. However, the results of genome-wide studies of the DNA methylation pattern and transcriptional activity of genes have nudged us toward reconsidering this paradigm, since the promoters of many genes remain active despite their methylation. The differences in the DNA methylation distribution in normal and pathological conditions allow us to consider methylation as a diagnostic marker or a therapy target. In this regard, the need to investigate the factors affecting DNA methylation and those involved in its interpretation becomes pressing. Recently, a large number of protein factors have been uncovered, whose ability to bind to DNA depends on their methylation. Many of these proteins act not only as transcriptional activators or repressors, but also affect the level of DNA methylation. These factors are considered potential therapeutic targets for the treatment of diseases resulting from either a change in DNA methylation or a change in the interpretation of its methylation level. In addition to protein factors, a secondary DNA structure can also affect its methylation and can be considered as a therapy target. In this review, the latest research into the DNA methylation landscape in the genome has been summarized to discuss why some DNA regions avoid methylation and what factors can affect its level or interpretation and, therefore, can be considered a therapy target.

The nucleotide excision repair (NER) system removes a wide range of bulky DNA lesions that cause significant distortions of the regular double helix structure. These lesions, mainly bulky covalent DNA adducts, are induced by ultraviolet and ionizing radiation or the interaction between exogenous/endogenous chemically active substances and nitrogenous DNA bases. As the number of DNA lesions increases, e.g., due to intensive chemotherapy and combination therapy of various diseases or DNA repair impairment, clustered lesions containing bulky adducts may occur. Clustered lesions are two or more lesions located within one or two turns of the DNA helix. Despite the fact that repair of single DNA lesions by the NER system in eukaryotic cells has been studied quite thoroughly, the repair mechanism of these lesions in clusters remains obscure. Identification of the structural features of the DNA regions containing irreparable clustered lesions is of considerable interest, in particular due to a relationship between the efficiency of some antitumor drugs and the activity of cellular repair systems. In this review, we analyzed data on the induction of clustered lesions containing bulky adducts, the potential biological significance of these lesions, and methods for quantification of DNA lesions and considered the causes for the inhibition of NER-catalyzed excision of clustered bulky lesions.

D-amino acid oxidase (DAAO, EC 1.2.1.2) plays an important role in the functioning of prokaryotes as well as of lower (yeast and fungi) and higher eukaryotes (mammals). DAAO genes have not yet been found in archaean genomes. D-amino acid oxidase is increasingly used in various fields, which requires the development of new variants of the enzyme with specific properties. However, even within one related group (bacteria, yeasts and fungi, mammals), DAAOs show very low homology between amino acid sequences. In particular, this fact is clearly observed in the case of DAAO from bacteria. The high variability in the primary structures of DAAO severely limits the search for new enzymes in known genomes. As a result, many (if not most) DAAO genes remain either unannotated or incorrectly annotated. We propose an approach that uses bioinformatic methods in combination with general 3D structure and active center structure analysis to confirm that the gene found encodes D-amino acid oxidase and to predict the possible type of its substrate specificity. Using a homology search, we obtained a set of candidate sequences, modelled the tertiary structure of the selected enzymes, and compared them with experimental and model structures of known DAAOs. The effectiveness of the proposed approach for discrimination of DAAOs and glycine oxidases is shown. Using this approach, new DAAO genes were found in the genomes of six strains of extremophilic bacteria, and for the first time in the world, one gene was identified in the genome of halophilic archaea. Preliminary experiments confirmed the predicted specificity of DAAO from Natronosporangium hydrolyticum ACPA39 with D-Leu and D-Phe.

A comprehensive analysis of the cell phenotype of the inflammatory infiltrate of the tumor stroma represents a promising area of molecular oncology. The study of not only soluble forms of various immunoregulatory molecules, but also their membrane-bound forms is also considered highly relevant. We performed a comprehensive analysis of tissue and circulating forms of the PD-1 and PD-L1 proteins, as well as macrophage and B-cell markers in the tumor stroma of gastric cancer, to assess their clinical and prognostic significance. The tumor and blood plasma samples from 63 gastric cancer patients were studied using ELISA and immunohistochemistry. Malignant gastric tumors were shown to be strongly infiltrated by B-cells, and their number was comparable to that of macrophages. For PU.1 expression, an association with tumor size was observed; i.e., larger tumors were characterized by fewer PU.1+ infiltrating cells (p = 0.005). No clinical significance was found for CD20 and CD163, but their numbers were higher at earlier stages of the disease and in the absence of metastases. It was also demonstrated that the PD-L1 content in tumor cells was not associated with the clinical and morphological characteristics of GC. At the same time, PD-L1 expression in tumor stromal cells was associated with the presence of distant metastases. The analysis of the prognostic significance of all the markers studied demonstrated that CD163 was statistically significantly associated with a poor prognosis for the disease (p = 0.019). In addition, PD-L1 expression in tumor cells tended to indicate a favorable prognosis (p = 0.122). The results obtained in this work indicate that the study of soluble and tissue markers of tumor stroma is promising in prognosticating the course of GC. The search for combinations of markers seems to be highly promising, with their comprehensive analysis capable of helping personalize advanced antitumor therapy.

Brain-derived neurotrophic factor (BDNF) is known to be involved in the pathogenesis of Alzheimer’s disease (AD). However, the pharmacological use of full-length neurotrophin is limited, because of its macromolecular protein nature. A dimeric dipeptide mimetic of the BDNF loop 1, bis-(N-monosuccinyl-L-methionyl-L-serine) heptamethylene diamide (GSB-214), was designed at the Zakusov Research Institute of Pharmacology. GSB-214 activates TrkB, PI3K/AKT, and PLC-γ1 in vitro. GSB-214 exhibited a neuroprotective activity during middle cerebral artery occlusion in rats when administered intraperitoneally (i.p.) at a dose of 0.1 mg/kg and improved memory in the novel object recognition test (0.1 and 1.0 mg/kg, i.p.). In the present study, we investigated the effects of GSB-214 on memory in the scopolamine- and steptozotocin-induced AD models, with reference to activation of TrkB receptors. AD was modeled in rats using a chronic i.p. scopolamine injection or a single streptozotocin injection into the cerebral ventricles. GSB-214 was administered within 10 days after the exposure to scopolamine at doses of 0.05, 0.1, and 1 mg/kg (i.p.) or within 14 days after the exposure to streptozotocin at a dose of 0.1 mg/kg (i.p.). The effect of the dipeptide was evaluated in the novel object recognition test; K252A, a selective inhibitor of tyrosine kinase receptors, was used to reveal a dependence between the mnemotropic action and Trk receptors. GSB-214 at doses of 0.05 and 0.1 mg/kg statistically significantly prevented scopolamine-induced long-term memory impairment, while not affecting short-term memory. In the streptozotocin-induced model, GSB-214 completely eliminated the impairment of short-term memory. No mnemotropic effect of GSB-214 was registered when Trk receptors were inhibited by K252A.

The spread of the monkeypox virus infection among humans in many countries outside of Africa, which started in 2022, is now drawing the attention of the medical and scientific communities to the fact that immunization against this infection is sorely needed. According to current guidelines, immunization of people with the first-generation smallpox vaccine based on the vaccinia virus (VACV) LIVP strain, which is licensed in Russia, should be performed via transepidermal inoculation (skin scarification, s.s.). However, the long past experience of using this vaccination technique suggests that it does not ensure virus inoculation into patients’ skin with enough reliability. The procedure of intradermal (i.d.) injection of a vaccine can be an alternative to s.s. inoculation. The effectiveness of i.d. vaccination can depend on the virus injection site on the body. Therefore, the aim of this study was to compare the development of the humoral and cellular immune responses in BALB/c mice immunized with the LIVP VACV strain, which was administered either by s.s. inoculation or i.d. injection into the same tail region of the animal. A virus dose of 10⁵ pfu was used in both cases. ELISA of serum samples revealed no significant difference in the dynamics and level of production of VACV-specific IgM and IgG after i.d. or s.s. vaccination. A ELISpot analysis of splenocytes from the vaccinated mice showed that i.d. administration of VACV LIVP to mice induces a significantly greater T-cell immune response compared to s.s. inoculation. In order to assess the protective potency, on day 45 post immunization, mice were intranasally infected with lethal doses of either the cowpox virus (CPXV) or the ectromelia virus (ECTV), which is evolutionarily distant from the VACV and CPXV. Both vaccination techniques ensured complete protection of mice against infection with the CPXV. However, when mice were infected with a highly virulent strain of ECTV, 50% survived in the i.d. immunized group, whereas only 17% survived in the s.s. immunized group. It appears, therefore, that i.d. injection of the VACV can elicit a more potent protective immunity against orthopoxviruses compared to the conventional s.s. technique.