Molekulyarnaya Biologiya最新文献

Leucine-rich repeat kinase 2 (LRRK2) belongs to the subfamily of tyrosine kinase-like kinases, the main function of which is to catalyze the transfer of γ-phosphate from ATP to the substrate in the cell due to the kinase domains. The exact functions of LRRK2 in the cell remain unknown. It has been shown that mutations in the LRRK2 gene, which are the cause of the development of the most common autosomal dominant form of neurodegenerative disease, Parkinson's disease (PD), mainly lead to a pathological increase in kinase activity. This review describes the structure of LRRK2 and the functional activity of LRRK2 kinase in the form of a monomer, dimer, and even a tetramer and describes the effect of mutations in the LRRK2 gene on the structure and kinase activity of the LRRK2 enzyme. Understanding the structure and functions of LRRK2 opens up new prospects for using it as a target for PD therapy.

In the modern-day 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 in modern medical science. All bacteria, except myco-plasmas, have cell walls in which various enzymes, receptors, transporters, channels, and antigens are located. This review is devoted to describing the structure of the major elements of bacterial cell walls and the enzymes involved in their biosynthesis, and is used as molecular targets for screening and selection of new effective antibiotics. Special attention has been paid to methods for studying the functional activity and inhibition of these targets. In addition, this review describes the functional characteristics of pore-forming proteins in 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 the targets of different classes of antibiotics is the basis for developing new strategies to overcome bacterial resistance.

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 nonstructural 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 of the cytokine interferon-1β(IFN-β) and the 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 the 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 we can consider this protein as a potential therapeutic target.

The study of the p53 protein and its interactions with other proteins is key to understanding the mechanisms by which p53 affects tumorigenesis. Mutations in the TP53 gene, which occur in approximately 50% of human cancers, often disrupt its function, highlighting its key role in tumorigenesis. Although structurally challenging due to the presence of unstructured regions, p53 has a well-documented role in DNA damage signaling and cancer progression. In this study, the interaction between p53 and 14-3-3γ monomers was studied using in silico methods. Using tertiary structure modeling, molecular dynamics, molecular docking, and virtual ligand screening, we identified small molecule compounds that can modulate the interaction of p53 with 14-3-3γ. Key findings of the study include identification of a ligand binding pocket in the p53-14-3-3γ interaction interface, generation of full-length models of 14-3-3γ and p53 using in silico methods, and selection of potential protein-protein modulators with high affinity for the proteins under study.

The modified vaccinia virus Ankara (MVA), characterized by high immunogenicity and proven safety, is considered a promising vector for vaccine development. An undeniable advantage of the MVA vector is its high capacity and the ability to incorporate several transgenes into different loci of the viral genome, enabling the creation of multivalent vaccines that encode multiple antigens simultaneously. This study examined the expression of a transgene encoding influenza virus protein epitopes after its integration into five loci of the MVA genome. The results demonstrated that the level of transgene expression is independent of the integration locus. Additionally, the dynamics of the expression of a reporter gene encoding the Enhanced Green Fluorescent Protein (EGFP) were determined under the control of the vaccinia virus promoters p11, p13.5, pLEO160, p7.5, and mH5 upon insertion of the expression cassette into the F13L gene locus of MVA. The highest expression level, though with a delayed onset of protein synthesis, was achieved with the late promoter p11. Using the p13.5 promoter resulted in earlier synthesis of EGFP in cells and higher gene expression level compared to the promoters pLEO160, p7.5, and mH5, which provided similar levels and dynamics of the reporter gene expression. These findings may be useful for developing multi-antigenic MVA-vectored vaccines.

In addition to participating in dosage compensation, the MLE helicase in D. melanogaster performs many functions in the regulation of gene expression, as does its human ortholog DHX9. Many of these functions are evolutionarily conserved and poorly explored. MLE has previously been shown to be involved in the regulation of inducible transcription of the ftz-f1 gene encoding the evolutionarily conserved nuclear receptor NR5A3. The ftz-f1 gene also encodes a constitutive transcript synthesized from an alternative promoter. The present work is devoted to the investigation of the role of MLE in the regulation of constitutive transcription of the ftz-f1 gene. This work shows that in S2 cell culture, MLE binds to the constitutive promoter and controls both inducible and constitutive transcription of the ftz-f1 gene. A novel MLE-binding cis- regulatory element of the ftz-f1 gene, enhancer 663, was identified. Using chromosome conformation capture technique the interaction of enhancer 663 with constitutive and inducible promoters of ftz-f1 gene in S2 cell culture was demonstrated. Examination of enhancer 663 histone H3 acetylation showed that it is involved in the activity of both promoters. Knockdown of MLE in S2 cell culture causes an increase in constitutive transcription. The effect of MLE on transcription beyond dosage compensation in vivo at the adult stage was shown for the first time. It was shown that at the adult stage MLE binds to both inducible and constitutive promoters and to enhancer 663. Mutation in the mle gene leads to increased expression of both transcripts of the ftz-f1 gene in females. The data obtained are important for understanding and further study of the evolutionarily conserved functions of MLE and its human ortholog DHX9.

Doxorubicin (DOX) is a widely used cytotoxic drug known for its high antitumor activity; however, its use is associated with side effects. The development of DOX delivery systems that can minimize systemic toxicity and enhance therapeutic efficacy is an urgent task in modern oncology. We studied the process of loading nanoparticles (NPs) with DOX under conditions that promote DOX precipitation to achieve maximum sorption efficiency. For this purpose, polymer-stabilized magnetic NPs were synthesized, and the efficiency of loading and sedimentation was examined based on the buffer type, DOX concentration, and incubation time with the drug. Our findings indicated that in solutions with the most pronounced DOX sedimentation (phosphate and borate buffers), loading was most effective. In a phosphate buffer with an initial DOX concentration of 667 μg/mL, the loading was 886 mg DOX/g NP. The sorption of DOX on NPs under these conditions reached 85% within the first hour and increased to 90% within 3 hours. The release of DOX from NPs was 25% at pH 7.4 and 96% at pH 5.4. Analysis of the survival of EMT-HER2 breast cancer cells demonstrated that the cytotoxicity of NPs loaded with DOX under sedimentation conditions was eight times higher than that of NPs loaded at a concentration of 20 μg/mL, where DOX did not form a sediment. These results suggest that NPs loaded with DOX under sedimentation conditions can be considered an effective delivery system that not only maintains the cytotoxic properties of DOX but also significantly enhances the content and release of the delivered drug.

Advances in CRISPR/Cas-mediated genome editing have opened up treatment alternatives for many human diseases, including HIV infection. Knockout of the CCR5 gene as a potential way to treat HIV infection has long been studied. Here we analyzed guide RNAs for SpCas9 and AsCas12a nucleases targeting CCR5 gene which had been previously studied and selected the most effective among them. We also designed novel guide RNAs for the same nucleases using bioinformatics resources. We compared the efficiency of target site cleavage for all selected gRNAs using three nucleases: wt SpCas9, SpCas9-HF1-plus, and AsCas12a, as well as their off- target activities. We demonstrated that among the tested guide RNAs two for SpCas9- HF1-plus and three for AsCas12a exhibited high cleavage activity, cutting CCR5 gene in 60-72% of cells, and had off-target activities below the limit of detection. Thus, these guide RNAs may be candidates for future development of gene therapies against HIV infection.

MicroRNAs in tissues and biological fluids represent a promising class of biomarkers for the molecular diagnostics and therapy of numerous diseases, including oncological diseases. Biomarkers based on easily accessible biological fluids, primarily blood-based biomarkers, are of particular value for diagnostic and prognostic purposes. To explore the potential of microRNAs as prognostic cancer markers and targets for molecular therapy, global microRNA profiling is required, which is provided by next-generation sequencing (NGS). NGS offers high sensitivity, single nucleotide resolution, and the possibility of profiling a large number of samples in parallel. Despite the promising potential of microRNAs as biomarkers and the growing number of works in this area, the literature does not address in sufficient detail the problems associated with sample preparation methods, the specifics of library preparation for microRNA sequencing, and the difficulties of quantitative analysis. Protocols for creating libraries for microRNA sequencing present specific challenges and require selecting conditions for each type of biological sample. Here, we present in detail the preparation of libraries for microRNA sequencing from pituitary adenoma tumor tissue and blood plasma of patients with acromegaly on the Illumina platform. We discuss the difficulties and limitations of the methods and evaluate the effectiveness of sequencing plasma and brain samples. This work can serve as a guide for researchers studying the mechanisms of microRNA regulation in endocrine diseases of the pituitary gland and will also allow for the adaptation of technical procedures for various biological samples in relation to other pathologies.

Thrombogenic risk factors (blood coagulation disorders and thrombophilia) are the cause of cardiovascular diseases, among which genetic factors are worth highlighting (genetic polymorphism of the blood coagulation system, angiogenesis factors, and components of the lipid metabolism system). Early identification of clinically significant polymorphisms in genes that cause predisposition to thrombogenic diseases allows for preventive measures and timely diagnosis even before the onset of the clinical picture of the disease, and for patients with an already confirmed diagnosis, genetic diagnostics makes it possible to check the hereditary nature of the disease, select treatment tactics, and predict the risk of developing of adverse drug reactions. This article describes the process of developing the "SNP2-TMG" kit, designed to identify ten genetic markers of susceptibility to thrombogenic diseases (rs1801131, rs6025, rs11549465, rs429358, rs7412, rs1799963, rs6050, rs1799762, rs2010963, and rs1801133), by the minisequencing technique (SNaPshot technology). This kit has passed clinical trials and is approved for medical use.