Molekulyarnaya Biologiya最新文献

The rapidly evolving CRISPR/Cas-based genome editing technologies, which have dominated nearly all areas of molecular biology over the past decade, still face several unresolved challenges. One of the major limitations of current genome editing tools is the low efficiency of targeted long-sequence insertions. This issue is particularly critical in plant systems, where genome editing efficiency is hindered by specific cellular characteristics. Site-specific recombinases (SSRs), which have long been employed in genetic engineering to mediate various genomic rearrangements-including deletions, duplications, insertions, and inversions-are limited in their application by the requirement for preexisting recombination recognition sites in the genome. However, CRISPR/Cas and recombinase tools complement each other, and their combined use offers a powerful strategy to overcome key limitations of genome editing. The discovery of CRISPR-associated transposons such as CAST and OMEGA, which naturally utilize their own recombinases, marks a significant advance in genome engineering, providing an elegant example of the natural convergence between CRISPR and recombinase technologies.

We present GeneLens, a Python package for comprehensive analysis of differentially expressed genes and biomarker discovery. The package consists of two core modules, FSelector for biomarker identification by utilizing Monte Carlo simulations of L1-regularized models and NetAnalyzer for functional prediction of selected gene sets based on the topology of their protein-protein interaction networks. FSelector includes: (1) automated gene selection through iterative bootstrap sampling, (2) calculation of gene significance weights by taking account of ROC-AUC models and their number in simulations, and (3) adaptive thresholding for feature space reduction. NetAnalyzer performs a pathway enrichment analysis while integrating the significance weights from FSelector. Implemented as a PIP module, GeneLens provides standardized algorithms for applying machine learning and network analysis methods in differential gene expression studies, along with automated model hyperparameter tuning and visualization tools.

Traditional methods of treatment and prevention of cardiovascular disease (CVD) are not always effective, especially in severe myocardial injury. One of the promising areas for the treatment of cardiac pathologies is cell transplantation using tissue-engineered constructs from allogeneic stem cells, such as cell sheets. The success of cell therapy depends on the severity of local inflammatory reactions, angiogenesis activity, and the resistance of transplant cells to hypoxia and apoptosis, as well as on their production of the extracellular matrix. Single nucleotide polymorphisms (SNPs) in genes involved in the processes associated with CVD can serve as markers of genetic dysfunction of these genes in the cardiovascular system and be used to predict the efficacy of therapy for heart disease based on tissue-engineered constructs. This review systematizes the information, allowing us to form a panel of such SNPs and analyze it. We identified seven genes at the intersection of pathways that are key to the survival of cellular constructs, VEGFA, TGFB1, FN1, IL6R, ITIH4, NRP1, and CDH13, and selected SNPs rs998584, rs8108632, rs1250259, rs6689306, rs77347777, rs75082222, and rs6565060, which are located in the regions of these genes and associated with CVD according to the Genome-Wide Association Studies (GWAS). These polymorphisms may constitute a minimal panel to search for an association with the efficacy of cell therapy in heart disease.

-Escherichia coli purine nucleoside phosphorylase (PNP) is used as a model enzyme to investigate metabolism and stability of biologically active nucleosides in the cell. The substrate specificity of E. coli PNP was studied in the reactions of phosphorolytic cleavage of the glycoside bond in adenosine derivatives containing a cyclic terpene moiety as precursors of biologically active purine derivatives. A number of N^(6)-ter- pene-substituted adenosine derivatives were obtained, differing in hydrocarbon substituent structure. Kinetic parameters of the phosphorolysis reaction were measured, and adenosine derivatives with a bicyclic hydrocarbon moiety were found to bind to the enzyme more efficiently than monocyclic derivatives. The results make it possible to produce new purine derivatives containing a lipophilic terpene moiety in mild reaction conditions.

The DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) removes various adducts from the 3'-end of DNA, including those induced by anticancer chemotherapeutics and is therefore considered an important therapeutic target. Previously, we investigated TDP1 inhibitors as sensitizers for the anticancer drug topotecan. Now, we have demonstrated that usnic acid derivatives containing a thiazole ring with an amide linker exhibited inhibitory activity against TDP1 at micromolar and submicromolar concentrations. Moreover, the lead compound OL11-119, (R)-N-(4-(8-acetyl-1,3,7-trihydroxy-2,9a-dimethyl-9-oxo-9,9a- dihydrodibenzo[b,d]furan-4-yl)thiazol-2-yl)-4-bromobenzamide, was found to enhance topotecan-induced tumor cell death at a nontoxic concentration. Molecular docking of OL11-119 and its analog with hydrazone linker, OL9-119, a previously identified potent TDP1 inhibitor, was performed. The binding energy of OL9- 119 to the enzyme active site was shown to be lower than that of OL11-119, which correlated with the higher inhibitory activity of OL9-119 against TDP1.

The Nε-acetylation of lysine residues is one of the most common processes of post-translational protein modification. As a result of the reaction between the ε-amino group of the side chain of Lys and the activated acetyl, an amide bond is formed, which leads to a change in the charge of the protein in the region of the modification. The growing interest in such sites is due to the influence of Nε-acetylation of Lys residues on the regulation of cellular activity, the disruption of which can lead to pathological conditions. Furthermore, the prediction of the Nε-acetylation sites of Lys residues serves as a tool for planning an experiment design in modern proteomics, since the presence of a forecast simplifies the choice of proteolysis strategy, the interpretation of controversial mass spectra, and the selection of proteotypic peptides. Here, we propose a new approach for predicting the Nε-acetylation sites of Lys residues in human proteins using machine learning techniques. A feature of this approach is the use of structural formulas of peptides containing a potential Nε-acetylation site and their description in the form of Multilevel Neighborhoods of Atoms (MNA) descriptors. Such descriptors are recursively generated for each atom of the molecule. A level zero descriptor represents the atom itself, the first level descriptor includes the atom and all atoms one bond away from it, and so on. Classification models for predicting Nε-acetylation sites of Lys residues were built using the previously developed MultiPASS program based on the analysis of more than 23000 sites from the PhosphoSitePlus database. The best model was obtained with a peptide length of 35 amino acid residues and using level 9 MNA descriptors. In fivefold cross-validation, the sensitivity, specificity, and ROC-AUC of the developed model were 0.71, 0.74, and 0.82, respectively. The model identified 1136 previously unknown potential sites in 418 proteins of the human reference proteome at a classification threshold defined as the difference in the probabilities of site assignment to positive (Pa) and negative (Pi) classes, (Pa - Pi) ≥ 0.7. The obtained data can serve as a basis for further proteomic studies aimed at identifying and functionally annotating Nε-acetylation sites of Lys in human proteins.

Antimicrobial peptides (AMPs) and, in particular, proline-arginine-rich cationic AMPs (PrAMPs) have recently attracted attention as potential candidates for developing new-generation antibacterial drugs. This interest stems from the fact that PrAMPs can target antibiotic-resistant bacteria and utilize a unique mechanism, which involves an interaction with bacterial ribosomes and inhibition of protein synthesis. Additionally, PrAMPs have a broad spectrum of activity against Gram-negative bacteria, show low rates of bacterial resistance, and are relatively easy to modify structurally. Several factors limit PrAMPs application, including their susceptibility to proteolytic degradation in biological media and their insufficiently broad spectrum of antibacterial activity against Gram-positive bacteria. Bacteria may develop PrAMP resistance, and toxic effects may result from the interactions of PrAMPs with certain components of eukaryotic cells. To overcome these challenges, it is possible to modify the PrAMP structure or create conjugates of PrAMPs with other molecules. The review summarizes the recent literature on PrAMP analogs and conjugates and considers the methods of modifying PrAMPs. New properties of compounds derived from PrAMPs highlight their potential for creating effective antibacterial agents.

Breast cancer remains one of the leading causes of cancer mortality among women, and the study of epigenetic mechanisms is an important task of molecular oncology in breast cancer. In this study, we analyzed the expression levels of eight microRNAs (miR-125b-5p, -127-5p, -129-5p, -132-3p, -148a-3p, -193a- 5p, -24-2-5p, and -34b-3p) and methylation of promoter regions of seven microRNA genes in a representative set of 40 and 70 paired samples of tumor and normal breast tissue, respectively, and showed hypermethylation of promoter regions of seven genes and a statistically significant decrease in the expression levels of eight microRNAs in tumors. For three genes (MIR125B-1, MIR129-2, MIR148A), inverse relationships between methylation and expression (rs < -0.5) were revealed, indicating their possible epigenetic regulation. Statistically significant positive correlations of expression levels were revealed for seven pairwise combinations of miRNAs, suggesting their coordinated functioning. Indeed, for the pairs miR-127-5p/miR-125b-5p, miR-148a-3p/miR-125b-5p, miR-148a-3p/miR-132-3p, and miR-34b-3p/miR-193a-5p, common mRNA targets and involvement in biological processes, including pathways associated with epigenetic regulation, proliferation, and metastasis, were revealed. The miRNA-mRNA regulatory network constructed involving DNMTs and EZH2 highlights their potential role in breast cancer progression and demonstrates diagnostic and prognostic significance.

In 1976, the antifungal hydroxamic antibiotic trichostatin A (TSA) was isolated from the metabolites of the bacterium Streptomyces hygroscopicus. It took 14 years to find that TSA has an effect on the proliferation and differentiation of mammalian cells by inhibiting histone deacetylase (HDAC) activity. By 2015, a single database containing the structures of about 1050 synthetic and 400 natural HDAC inhibitors had been created. Currently, five inhibitors are approved for use as anticancer agents, with dozens more compounds undergoing clinical trials. However, the implementation of new agents is severely hampered by their multidirectional action and the severity of side effects. New strategies are needed to overcome these problems, including the development of inhibitors targeting a specific class of HDACs. In addition to the most important characteristics of histone deacetylases and their natural inhibitors, this review considers current approaches to the design of selective HDAC inhibitors and the methods used to test them.

Pancreatic Ductal AdenoCarcinoma (PDAC) is one of the most therapy-resistant tumors. Cultured cells originating from different stages of PDAC development are characterized by different levels of expression of a number of transcription factors. In particular, poorly differentiated high-grade PDAC cells are characterized by increased expression of ZEB1 gene encoding multifunctional transcription factor ZEB1, one of the main regulators of epithelial-mesenchymal transition. By the method of Circular Chromosome Conformation Capture (4C-seq) we studied the spatial organization of chromatin of regulatory region of ZEB1 gene in cultures of highly differentiated PDAC cells (Capan2) with low level of ZEB1 expression and poorly differentiated PDAC MIA PaCa2 cells with a high level of expression of this gene, and compared it with the chromatin organization of KLF5 gene. The number and distribution of contacts of the ZEB1 regulatory region with other chromatin regions are similar in these cell types and differ significantly from the pattern of distribution of contacts characteristic for KLF5 gene studied earlier. In Capan2 cells, the contacts of the regulatory region of the ZEB1 gene are tend to locate in regions with an increased level of H3K27ac modification, whereas in MIA PaCa2 cells these contacts are predominantly located in regions with a decreased level of H3K27ac. Consequently, the probability of contact of distant chromatin regions is primarily determined not by the degree of chromatin openness/activity of this region. To explain the data obtained, we assumed that the main regulator of the ZEB1 gene transcription level in the studied cells is a transcriptional repressor, whereas for the KLF5 gene main regulator is a transcriptional activator. According to a number of properties, one of the possible candidates for the role of this repressor may be the product of the ZNF438 gene. In addition, we have characterized a number of regions in contact with the ZEB1 promoter that are specific for MIA PaCa2 cells and contain potential regulators of this gene activity.