Vol 59, No 3 (2025)

In the current world, antibiotic resistance is one of the most serious threats to both human health and food security. Finding new ways to prevent and overcome the formation of pathogen resistance to antibiotics is an extremely important and urgent task of modern medical science. All bacteria, except mycoplasmas, have a cell wall in which various enzymes, receptors, transporters, channels and antigens are localized. This review is devoted to describing the structure of the major elements of bacterial cell walls and enzymes involved in their biosynthesis and used as molecular targets for screening and selection of new effective antibiotics. Special attention is paid to methods for studying the functional activity and inhibition of these targets. In addition, the review describes the functional characteristics of pore-forming proteins from the outer membrane of Gram-negative bacteria and the molecular mechanisms of antibiotic penetration through porin channels. Analysis of the structure and functional features of targets of different classes of antibiotics is the basis for developing new strategies to overcome bacterial resistance.

The etiology of transsexualism remains unknown to this day, as this disease is multifactorial and is caused by a complex of factors influencing the sexual differentiation of brain tissue during fetal development. It has been shown that sexual differentiation of the brain occurs at a much later stage of development than sexual differentiation of the genitals; these two processes are influenced independently of each other. Various sexual characteristics, such as gender identity (identification of oneself as a male or female role), sexual orientation (heterosexuality, homosexuality, or bisexuality), as well as the risks of developing neuropsychiatric disorders, are programmed in the brain at an early stage of development. It has been established that the structure of some areas of the brain in individuals with transsexualism differs from the structure of the corresponding areas of the brain in cisgender men and women and is close to (although not identical to) the structure of these areas in people of the opposite anatomical and genetic sex. It is shown that various effects of physiologically active substances on the developing brain result in irreversible or partially reversible modification of its neurochemical systems. Family studies have confirmed the role of genetic factors in gender identity disorders. The review provides a detailed analysis of known loci of candidate genes presumably associated with transsexualism. It is shown that for most candidate genes both positive and negative correlations with transsexualism were revealed, while for other markers no positive associations were found. The inconsistency of the research results may be due to several factors, such as the “blurred” samples of transsexuals, the choice of neutral markers that do not have functionally significant polymorphisms affecting the expression and functionality of these genes, etc. The review presents current data on the problem of transsexualism; possible prospects for further scientific research of this phenomenon at the genetic level are also outlined for the purpose of practical application in the process of diagnosis verification.

Despite active research, so far the detailed mechanisms of TBEV pathogenesis have not been fully disclosed. Recently, extracellular vesicles, especially small-sized vesicles, which have been shown to play an important role in the pathogenesis of many viruses, have attracted the attention of scientists. In this study, we investigated the effect of nonstructural protein 1 (NS1) expression of tick-borne encephalitis virus on the release of extracellular vesicles by cells, and assessed the possibility of these vesicles affecting other cells. NS1 expression by TBEV was found to increase the release of extracellular vesicles by HEK293T cells; however, no changes in the size profile of released vesicles were detected. In addition, NS1 is detected in both large and small vesicle size fractions. It was found that NS1 TBEV is not present inside the vesicles, but is associated with their outer surface. Small-sized vesicles derived from the culture medium of NS1-expressing HEK293T cells are able to induce an increase in mRNA content and interleukin-1β (IL-1β) secretion in human neuroblastoma SHSY5Y cells. The results indicate the involvement of NS1 protein and vesicles in the development of neuroinflammation and are important for understanding the mechanisms of tick–borne encephalitis virus pathogenesis.

Infection with tick-borne encephalitis virus (TBEV) can lead to severe neurological complications, largely associated with the activation of innate immunity and inflammatory reactions in the tissues of the nervous system. In this regard, the study of factors, including viral factors, influencing these processes is underway. We analyzed the possible role of non-structural protein 1 (NS1) of TBEV in the activation of innate immune response reactions in cells of the nervous system. SH-SY5Y neuroblastoma and DBTRG-05MG glioblastoma cells were transfected with a plasmid encoding NS1 or treated with extracellular vesicles of NS1-expressing HEK293T cells and then stimulated with polyinosinic-polycytidylic acid [poly(I:C)] to activate the innate immune response. It was found that poly(I:C) stimulation of NS1-expressing SH-SY5Y cells resulted in lower mRNA levels of the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), as well as the innate immune response cytokine interferon-β (IFN-β) and interferon-stimulated gene 15 product (ISG15), compared to stimulated cells without NS1 expression. In addition, transcription of the sensor gene MDA5, which is responsible for activating gene transcription of these cytokines, was reduced in these cells. In NS1-expressing DBTRG-05MG stimulated cells, only IL-1β mRNA content was reduced. Treatment of SH-SY5Y cells with extracellular vesicles from NS1-expressing cells followed by poly(I:C) stimulation resulted in increased mRNA levels of IL-6, TNF-α, and IFN-β, compared with stimulated cells treated with vesicles from non-NS1-expressing cells. No differences were detected in DBTRG-05MG cells with similar treatment. Based on these data, we can assume that TBEV NS1 plays a dual role in the formation of neuroinflammation during the infection, and consider this protein as a potential therapeutic target.

Transcriptional cyclin-dependent kinases CDK8 and CDK19 are enzymatic components of the Mediator complex. CDK19 is supposed to be a minor paralog of CDK8, but, probably, in some situations it can compensate for the absence of CDK8, while evidence for unique functions of both kinases remains sporadic. The vast majority of information on the role and mechanisms of action of these proteins was obtained in experiments using tumor cell lines, which may give irrelevant results due to the changes accumulated by tumor cells. In this regard, we first obtained mice (Cdk8fl/fl/Cdk19–/–/Rosa26/Cre/ERT2) with inducible Cdk8 knockout on the background of constitutive Cdk19 knockout to study their joint role in the whole organism and in primary cells. Using these mice, we obtained Cdk19–/– embryonic fibroblasts, with Cdk8 knockout inducible by 4-hydroxytamoxifen. We found that, unlike most tumor cells, embryonic fibroblasts are sensitive to CDK8/19 inhibition and knockout: inhibition resulted in a significant cell death already at day 5, while knockout resulted in a decrease in their proliferation rate. RNA sequencing revealed, consistent with previously published studies, alterations in the Wnt signaling pathway, cytokine response, and osteoclast differentiation. Consistent with our previously published results, expression of genes associated with steroidogenesis was reduced. Changes associated with the cytoskeleton, adipogenic differentiation, osteogenic differentiation, cell adhesion, extracellular matrix formation, and mitochondrial biogenesis were previously undescribed. When studying the stress response of embryonic fibroblasts, we found that the response to DNA damage from X-ray irradiation and to serum stimulation after starvation was also mediated by CDK8/19 and was significantly reduced in knockout cells.

Microarrays with immobilised protein probes are used in the analysis of protein samples. Selection of materials for biochip fabrication, functionalisation of the carrier surface, obtaining ordered cell matrices, immobilisation of protein molecular probes in cells, achieving high sensitivity of protein sample analysis are the key tasks of biochip technology. The following methodological approaches were used in this work. To preserve affinity properties, protein probes were immobilised in biochip cells under ‘soft’ conditions, after cell preparation. In order to achieve high concentration and prostanse accessibility, probes were immobilised in three-dimensional cells obtained from dynamically mobile brush polymers fixed on the substrate only at one end. The cell matrix was obtained on the substrate surface by photoinduced radical polymerisation of monomers with reactive chemical groups, photolithographically, according to the photomask template. We carried out a comparative analysis of polymer brush structures prepared on a polybutylene terephthalate substrate by photoinduced radical polymerisation. These structures consisted of links of one or more monomers. The influence of the method of activation of reactive groups on the polymer chains on the efficiency of immobilisation of molecular protein probes in the cells was investigated. The influence of the composition of acrylate monomers, from which the cells were obtained, on the specific binding of response proteins to protein probes immobilised in biochip cells was studied. A new method of biochip fabrication was developed. Substrates made of photoactive polybutylene terephthalate were coated with a thin layer of photoactive polymer polyvinyl acetate. The cells, which were obtained by photopolymerisation of monomers on the modified substrate, did not degrade or peel from the surface in aqueous solutions. The substrates coated with polyvinyl acetate do not adsorb proteins. Streptavidin and human immunoglobulins were used as models of protein probes, and biotinylated goat immunoglobulins and goat antibodies against human immunoglobulins were used as response proteins. The study found that polymers with irregular structure promoted higher concentration of protein probes and their uniform distribution within the cells, which positively influenced the efficiency of specific binding to response proteins. Biochips with cells of their brush polymers on black polybutylene terephthalate substrate appear promising for further improvement for use in immunofluorescence analysis of protein targets for the development of ‘lab-on-a-chip’ microanalysis technologies.