Molekulyarnaya Biologiya最新文献

Despite increasing data on the properties of the origins of replication, the molecular mechanisms underlying the origin recognition complex (ORC) positioning in the genome are still poorly understood. It has been suggested that the key factors determining the positioning of ORC in the genome are DNA-binding proteins that form various DNA regulatory elements, including insulators, promoters, and enhancers, thereby linking the replication program to different levels of transcriptional regulation. Previously, we demonstrated that the Su(Hw) protein is the first example of such a protein. Subsequent studies identified a number of other DNA-binding proteins, including CG10543, which may be responsible for the formation of the corresponding regulatory elements and the recruitment of transcriptional and replication complexes to their binding sites. It has been shown that the Drosophila CG10543 protein interacts with the deubiquitinating (DUB) module of the SAGA complex. The binding sites of the CG10543 protein are predominantly located in the promoter regions of active genes and colocalize with the SAGA and dSWI/SNF chromatin modification and remodeling complexes, as well as with the ORC replication complex. To investigate the role of the CG10543 protein in transcriptional regulation, an RNA-Seq experiment was conducted in Drosophila S2 cells under normal conditions and upon RNA interference with the CG10543 protein. It was shown that the CG10543 protein affects the transcription of 469 genes, with a significant portion of these genes (23%) being ecdysone-dependent genes. Ecdysone is the main steroid hormone in Drosophila, is responsible for Drosophila metamorphosis, and has a significant effect on the expression of many genes during development. We demonstrated that CG10543 sites colocalize with the CBP protein and the histone mark H3K27Ac, which are characteristic of active regulatory elements. The CG10543 protein also colocalizes with the CP190 protein, suggesting a potential mechanism of transcriptional regulation through the formation of long-range interactions between regulatory elements.

Era GTPase of Staphylococcus aureus is a ribosome maturation factor. Era GTPase is involved in the late stages of small (30S) ribosome subunit assembly and regulates formation of the entire 70S ribosome. Study of the structure of the Era and 30S subunit complex from S. aureus will help in studying the processes of ribosome maturation and the mechanisms of protein synthesis regulation in this pathogenic microorganism. This paper presents protocols for obtaining Era GTPase and immature 30S ribosome subunits from S. aureus, as well as a protocol for assembling their complex for further structural studies.

Vectors based on adeno-associated viruses (AAV) have proven to be a convenient tool for genomic editing. However, for transgenesis of laboratory animals or gene therapy for human diseases, a high AAV titer is required. We optimized the conditions for producing AAV serotype 9 (AAV9) to increase the yield of the virus. A common approach is based on transfection of producer cells with three plasmids. We performed transfection with two plasmids encoding all the necessary components for AAV9 production. This allowed us to increase the final title fourfold. Optimization of the molar ratio of the plasmid containing the gene of interest to the plasmid encoding the proteins of the replicative complex, capsid, and auxiliary factors led to a twofold increase in the titer. Optimization of the composition of the cultivation medium for AAV9 production allowed us to increase significantly the yield of the virus. When the DMEM-F12 production medium with a less nutritious DMEM was supplemented with fetal bovine serum, the yield of AAV9 increased by approximately three orders of magnitude. Thus, optimization of the number and ratio of plasmids, as well as the composition of the cell culture medium, made it possible to increase significantly the production of AAV9 and achieve a final titer of 2.5 x 10^(12) vector genomes in 1 mL of the production medium of the adhesive HEK293T cell line.

Seasonal influenza is an acute respiratory illness caused by the influenza A and B viruses that circulate worldwide. Due to high variability, new strains of the virus emerge every year. Therefore, vaccine formulation has to be revised every year. The advantages of mRNA vaccines are that they can be produced quickly and without preliminary adaptation of the vaccine strain to chicken embryos. Here, the results of developing and studying the mRNA-C3-H1 vaccine encoding the hemagglutinin (HA) of the influenza A(H1N1)pdm09 virus are presented. The design and production of a DNA-template for the synthesis of mature HA mRNA in one step were described. The obtained mRNA was purified from double-stranded RNA impurities using a method based on the use of cellulose powder. The efficacy of the vaccine was assessed on BALB/c mice. The mice were immunized with a "naked" mRNA vaccine using a needle-free jet injector. According to the ELISA results, the average antibody titer in the serum of immunized animals was 4.6 x 10^(5). Sera of immunized animals neutralized the mouse-adapted influenza A/California/04/09 (H1N1) MA8 virus with an average titer of 6 x 10^(2). As shown by the ELISpot method, the developed mRNA vaccine induced a T-cell immune response in mice. After stimulation of splenocytes with specific peptides, the average number of T-lymphocytes secreting IFN-γ was 236 per 10^(6) cells. Immunization with the mRNA vaccine was shown to protect mice from infection with a lethal dose of the influenza A/California/04/09 (H1N1) MA8 virus. Thus, the developed experimental mRNA-C3-H1 vaccine is immunogenic and prevents morbidity and mortality in mice after infection with a homologous strain of the influenza virus.

Microarrays with immobilized protein probes are used to analyze protein samples. Selection of materials for biochip fabrication, functionalization of the carrier surface, construction of ordered cell matrices, immobilization of protein molecular probes in cells, and improvement of the sensitivity of protein sample analysis are key tasks of biochip technology. The following methodological approaches were used in this work. To preserve affinity of protein probes, mild conditions were used to immobilize the probes in biochip cells. To achieve a high concentration and spatial accessibility of the probes, three-dimensional cells to immobilize the probes were made of dynamically mobile polymer brushes, in which only one end of a polymer molecule is attached to the substrate. A cell array was formed on a substrate surface by photoinduced radical polymerization of monomers with reactive chemical groups (photolithography), according to a photomask template. A comparative analysis was performed for polymer brush structures prepared on a polybutylene terephthalate substrate by photoinduced radical polymerization. The structures consisted of units formed by one or more monomers. The efficiency of protein probe immobilization in cells was studied as dependent on the method used to activate the reactive groups in polymer chains. The composition of acrylate monomers used to form biochip cells was tested for the effect on the specific binding of response proteins to protein probes immobilized in the cells. A new method of biochip fabrication was developed. Substrates made of nonphotoactive polybutylene terephthalate were coated with a thin layer of the photoactive polymer polyvinyl acetate. Cells obtained by monomer photopolymerization on the modified substrate showed no degradation or detachment from the surface in aqueous solutions. Substrates coated with polyvinyl acetate did not adsorb proteins. Streptavidin and human immunoglobulins were used as model protein probes, and biotinylated goat immunoglobulins and goat antibodies against human immunoglobulins served as response proteins. Polymers with irregular structures were found to promote a higher concentration of protein probes and their uniform distribution within the cells, thus ensuring a higher efficiency of specific binding with response proteins. Biochips with cells made of brush polymers on black polybutylene terephthalate substrates were found to be promising for further improvement, aiming to employ them in immunofluorescence analyses of target proteins and to develop the lab-on-a-chip microanalysis technologies.

During the COVID-19 pandemic, it became clear that, to ensure global health security, it is essential to have a developed platform that can be used to develop a safe, low-cost, effective vaccine quickly. DNA vaccines have several advantages over other platforms, including rapid development and ease of production. They are more stable than mRNA vaccines. Unlike viral vector-based vaccines, DNA vaccines do not induce antivector immunity. One of the disadvantages of DNA vaccines is their relatively low immunogenicity. This problem can be solved using jet injection. Here, we evaluated and confirmed the efficiency of an inexpensive, simple, and safe method for delivering the naked DNA vaccine pVAXrbd encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein using a spring-loaded jet injector. Based on the results of histological analysis, optimal conditions were determined that ensure low tissue trauma in laboratory animals upon administration of pVAXrbd. An optimized immunization protocol for BALB/c mice was used to compare the immunogenicity of pVAXrbd with two different administration schemes: using a jet injector under the skin and into the adjacent muscle layers or intramuscularly using a syringe with a needle. Mice immunized with "naked" pVAX-RBD were shown to produce high levels of specific virus-neutralizing antibodies. The vaccine also induced a strong RBD-specific T-cell response. As shown by quantitative PCR analysis of viral RNA, vaccinated mice infected with the Gamma variant of SARS-CoV-2 developed a protective immune response; moreover, it was more pronounced in animals to which the DNA-vaccine was administered using a jet injector compared to those immunized intramuscularly. Thus, the introduction of a DNA-vaccine using jet injection effectively activates both types of immune response and leads to a decrease in the viral load. Jet injection is a promising method for delivering DNA vaccines, characterized by low cost, simplicity, technological administration, and minimal pain for the patient.

Transcriptional cyclin-dependent kinases 8 and 19 (CDK8 and CDK19) are enzymatic components of the Mediator complex. CDK19 is presumably a minor paralog of CDK8 and is thought to compensate for lack of CDK8 in certain cases, but evidence for unique functions of the two kinases remains sporadic. Experiments with tumor cell lines have yielded the vast majority of information on the role and mechanisms of action of the kinases, and the results might be irrelevant because of the changes accumulated by tumor cells. Mice with an inducible Cdk8 knockout and a constitutive Cdk19 knockout (Cdk8^( fl/fl)/Cdk19^(-/-)/ Rosa26/Cre/ER^(T2)) were therefore engineered for the first time to study the joint role of CDK8 and CDK19 in the while organism and primary cell cultures. The mice were used to obtain Cdk19^(-/-) mouse embryonic fibroblasts (MEFs) in which a Cdk8 knockout could be induced with 4-hydroxytamoxifen (4-OHT). Unlike most tumor cells, MEFs were sensitive to CDK8/19 inhibition and knockout: the inhibition led to significant cell death within 5 days, while the knockout decreased the cell proliferation rate. RNA sequencing revealed alterations in the Wnt signaling pathway, cytokine response, and osteoclast differentiation in Cdk8/Cdk19 knockout MEFs. Expression of genes associated with steroidogenesis was lower, consistent with earlier data. Changes associated with the cytoskeleton, adipogenic differentiation, osteogenic differentiation, cell adhesion, extracellular matrix formation, and mitochondrial biogenesis were described for the first time. Studies of the stress responses in MEFs showed that responses to DNA damage from X-ray irradiation and to serum stimulation after starvation were also mediated by CDK8/19 and were significantly reduced in Cdk8/Cdk19 knockout cells.

The discovery of a class of long noncoding RNAs (lncRNAs), including lncRNAs of the small nucleolar RNA (snoRNA) host gene family, SNHG, has led to growing interest in the study of both snoRNAs themselves and the genes encoding them. Currently, of the 232 known snoRNA genes, only 32 have been confirmed to have lncRNAs. At the same time, a positive correlation has been shown between the expression of lncRNAs and snoRNAs encoded by a common host gene of the SNHG family. Thus, lncRNA of the SNHG1 gene correlates with snoRNAs SNORD22 and SNORD25-31, and lncRNA of the SNHG16 gene, with snoRNAs SNORD1A, SNORD1B, and SNORD1C. There is evidence that SNHG lncRNAs can participate in oncogenesis both through regulatory functions inherent to lncRNAs and by influencing ribosome biogenesis. At the same time, information has accumulated on the "extraribosomal" functions of snoRNAs. In addition to a brief excursion into the biological functions of snoRNAs and SNHG lncRNAs, we present a comprehensive review of data on the role of these two types of noncoding RNAs in the pathogenesis of ovarian cancer, the most insidious cancer of the female reproductive system. The influence of these regulatory RNAs on the main processes of ovarian oncogenesis, such as apoptosis, epithelial-mesenchymal transition, cell cycle control, and DNA methylation mechanisms in this type of cancer is considered. The prospects for clinical application of regulatory RNAs due to their influence on the level of drug resistance are also discussed.

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 the expression of nonstructural protein 1 (NS1) 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 the mRNA content and interleukin-1β(IL-1β) secretion in human neuroblastoma SHSY5Y cells. The results indicate the involvement of the NS1 protein and vesicles in the development of neuroinflammation and are important for understanding the mechanisms of pathogenesis of tick-borne encephalitis virus.

The etiology of transsexualism (TS) remains unknown today because the disease is multifactorial and is caused by a set of factors, including those affecting sexual differentiation of brain tissue during fetal development. Sexual differentiation of the brain has been shown to occur at a much later developmental stage than sexual differentiation of the genitals, and the two processes are regulated independently of each other. Various sexual characteristics, such as gender identity (self-identification of oneself as male or female) and sexual orientation (heterosexuality, homosexuality, or bisexuality), as well as risks of developing neuropsychiatric disorders, are programmed in the brain at an early developmental stage. The structure of certain brain areas in transsexual individuals has been found to differ from that in cisgender men and women and is close, although not identical, to that in humans of the opposite anatomical and genetic sex. Various effects of physiologically active substances on the developing brain have been shown to result in irreversible or partly 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 the known loci of candidate genes presumably associated with TS. Both positive and negative correlations with TS have been revealed for most candidate genes, while only negative correlations are known for other markers. The inconsistency of the research results may be due to several factors, including "blurred" samples of transsexuals, the choice of neutral markers lacking the functionally significant polymorphisms that affect their expression and functionality, etc. The review considers the current data on the problem of TS and outlines the possible prospects for further research of the phenomenon at the genetic level with the aim of using the results to verify the diagnosis.