Molekulyarnaya Biologiya最新文献

Efficient analysis of large amounts of transcriptome data requires fast and easy access to raw gene expression data. In our work, we localized the available expression data of more than 50 000 genes in 54 tissues from approximately 1000 individuals from the GTEx system and created an easy-to-use interface for accessing and selecting these data. Using the capabilities of the localized system, we selected seven genes with highly stable expression from the housekeeping genes, investigated the changes in the number and activity of mast cells in the tibial artery with aging, and studied changes in the components of the intestinal barrier and the state of mucosal immunity in old age in connection with the increased incidence of ulcerative colitis after 60 years. These examples demonstrate the applicability of the localized GTEx database in various biomedical projects and applications.

The pogo superfamily is one of the most common groups of DNA transposons. Ten pogo transposons are known to occur in the genome of the Pacific oyster Crassostrea (Magallana) gigas (Thunberg, 1793). The oyster pogo elements were shown to belong predominantly to three families: pogoR, Passer, and Fot. A genome analysis in five oysters detected nine pogo elements in each oyster, while one element (Mariner-38_CGi) was found in only two genome assemblies. The genome assemblies differed in the copy number and lengths of the pogo transposons and the presence and lengths of inverted repeats. Data on the evolutionary dynamics of the pogo transposons and their low copy number in individual genomes suggested their low activity throughout the life cycle. A transcriptional activity analysis showed that the Mariner-30_CGi and Mariner-34_CGi elements were expressed permanently or in a condition-dependent manner in more than half of the cases. Transcriptional activities of five elements were found to depend on the developmental stage. It was assumed that the elements could have been domesticated by the oyster genome. The study expands the understanding of evolution of the pogo transposons and their role in animal genome diversification.

Enveloped virus entry into the host cell mediated by the viral fusion glycoproteins represents an earliest step in viral infection, the inhibition of which offers a number of advantages over the antivirals with other mechanisms of action. Viral glycoproteins are classified into three classes with rather different structures, but, despite that, they share some functional features, such as the separation of receptor recognition/binding function and membrane fusion function into two different subunits or domains. All of them are transmembrane proteins anchored in the virion's membrane, and possessing a hydrophobic structure (fusion peptide or fusion loop), which is inserted in target cell membrane early in fusion. Here, we describe the membrane fusion machinery of all 3 classes of viral glycoproteins and indicate their domains and structures, which can serve as the targets for entry inhibitors with different mechanisms of action. The examples of large and small molecule entry inhbitiors belonging to the groups of affinity blockers, inhibitors of glycoprotein-receptor binding, fusion inhibitors, anchor inhibitors and compounds blocking the function of membrane-proximal external region (MPER) of viral glycoproteins are provided. Finally, the perspectives of developing broadly acting entry inhibitors are discussed.

Reactive oxygen and nitrogen species accumulate in cells during oxidative stress and cause oxidative damage to various cell components, including DNA, proteins, and lipids, thus leading to a number of severe diseases, such as atherosclerosis. Protein C3 is a central component of the complement cascade and a key player in the immune system. Proinflammatory activity of C3 can also contribute to the development of metabolic syndrome. Although hepatocytes are the main source of C3 circulating in the blood, the regulation of C3 gene expression in hepatocytes under oxidative stress remains unexplored. Suppression of C3 gene transcription and C3 protein secretion were observed during hydrogen peroxide-induced oxidative stress in HepG2 human hepatoma cells. The transcription factor FOXO1 promoted C3 expression, and C3 repression by oxidative stress was mediated through the regulation of FOXO1/HNF4α complex binding to the C3 promoter. A novel cluster of FOXO1 binding sites was identified in the distal region of the C3 promoter and found to be essential for the regulation of C3 expression by the FOXO1/HNF4α complex. Activation of the main MAP kinase cascades (ERK1/2, p38, and JNK), AMP kinase, and the transcription factor NF-κB were necessary for C3 suppression in oxidative stress. Thus, the molecular mechanisms and transcription factors that mediate suppression of C3 production in HepG2 cells during oxidative stress were identified.

The development of resistance to trastuzumab in HER2-positive breast cancer is a serious clinical problem that limits the effectiveness of targeted therapy. In a significant proportion of patients, the mechanisms in the development of resistance remain poorly understood. The BT-474 cell line was selected as an optimal model for study because it represents a HER2-positive luminal B subtype breast cancer cell line. To identify the molecular mechanisms of resistance, a comprehensive transcriptomic analysis based on RNA-seq data comparison of three independent datasets including both sensitive and trastuzumab-resistant variants was applied. The methodological approach included multistep bioinformatics analysis followed by identification of regulatory interactions. The study identified genes with increased expression (FUCA2, HSPE1, SHLD1, NMD3) and genes with decreased expression (GPC5, FSTL1, ATG16L2, POLD2) in resistant cells. Key transcription factors (E2F1, MYC, YBX1, HEY1, NFIC, TFAP2A, AP-1/JUN, NCOA1) regulating the expression of the detected genes during the development of resistance were identified. The changes identified indicate a complex reprogramming of transcriptional activity affecting cell cycle processes, DNA repair, metabolism, and the epithelial-mesenchymal transition. The findings expand our understanding of the molecular mechanisms of trastuzumab resistance and open prospects for the development of novel therapeutic strategies to overcome drug resistance in HER2-positive breast cancer.

Methods for correcting errors in synthetic oligonucleotides and genetic constructs derived from them are described. Methods for isolating oligonucleotides with a perfect structure from oligonucleotide pools are discussed. Methods for correcting errors in the DNA structure using mismatch-specific endonucleases and proteins of the bacterial DNA repair system are considered. Examples of practical application of the methods developed for adjusting the structure of synthesized genetic constructs are given.

Stimulus-sensitive smart materials are activated by input signals and remain inactive until they arrive. Such materials are of great interest for the analysis of biochemical data in diagnostics and therapy. To develop nanomaterial-based smart theranostic agents, it is necessary to know the affinity of interaction and the kinetics of binding of agents to the biochip surface. However, the assessment of kinetic parameters of nanoparticle-substrate and nanoparticle-nanoparticle interactions remains a challenging task. Here, a label-free interferometry biosensor for analyzing the kinetics of binding of smart nanomaterials to the biochip surface has been developed. Using the developed biosensor, we optimized the work of molecular beacons on nanoparticles. For these smart materials, a sevenfold increase in the adsorption rate was demonstrated when the molecular beacons were switched from the "off" state (without ligand) to the "on" state (in the presence of DNA analyte). This change in adsorption rate was used to develop a kinetic biosensor that detected input DNA with a threshold of 50 ± 10 pM and a linear dynamic range of three orders of magnitude. The designed nanoparticle beacons open up new possibilities for the creation of improved theranostic nanorobots, due to their high sensitivity to the analytes and efficient work at physiological ionic strength. The latter distinguishes them favorably from previously developed nanobeacons, which were effective only in solutions with a high salt content. In the future, the biosensor can be used as a next-generation diagnostic tool.

In bacteria, glycine and proline codons, located upstream of stop codons, suppress translation termination. However, the effects of those codons in eukaryotes have not been systematically investigated. In this study, we demonstrate that preceding stop codon CCC codon of proline suppresses translation termination in eukaryotes during the synthesis of long protein. Conversely, during the synthesis of short peptide, a upstream proline codon stimulates the formation of termination complexes. Furthermore, we investigated the role of poly(A)-binding protein (PABP), a key regulator of eukaryotic translation termination associated with the poly(A) tail of mRNA, in modulation of translation termination by the 5' context of stop codons. Our findings reveal that during the synthesis of short peptides PABP reduces dependence of translation termination on the 5' stop codon contexts and promotes translation termination independently of the 5' stop codon context during the synthesis of long proteins.

The unconventional halotolerant yeast Debaryomyces hansenii is of great importance in biotechnology and the food industry, and in basic research it serves as a model for studying the molecular mechanisms of resistance to increased salinity and osmotic stress. We have previously established an efficient method for editing the D. hansenii genome using the CRISPR/Cas9 system. In turn, this has stimulated further investigation of the structure and physiological role of DNA double-strand break repair pathways in D. hansenii. The aim of the present work was to evaluate the involvement of key components of the DNA double-stranded break repair system by the non-homologous end joining (NHEJ) mechanism in the resistance of D. hansenii to DNA-damaging compounds and compounds that induce oxidative, high salinity, and osmotic stress. Using the CRISPR/Cas9 system, mutant strains with knockout of the DEHA2F10208g (DhKU70), DEHA2B01584g (DhKU80) , and DEHA2G04224g (DhLIG4) genes encoding key components of NHEJ were obtained. It was found that mutant strains, unlike the wild-type strain, are sensitive to chemical compounds that damage DNA, as well as to compounds that cause oxidative stress. Osmotic and high salinity stresses and vanillin do not cause significant changes in the rate of colony formation of mutant strains. Unexpectedly, mutant strains exhibit increased resistance to caffeine compared to the wild-type strain. The data indicate that the NHEJ systems of D. hansenii play a significant role in the response to DNA-damaging and oxidative types of stress. The importance of the NHEJ system in the processes of maintaining yeast cell homeostasis should be taken into account when creating strains producing valuable substances.

Proper assembly and maturation of ribosomal subunits are critical processes that ensure functional activity, translation efficiency, and fidelity of the ribosome. The GTP-binding protein YsxC is found in many bacteria and is one of the protein factors involved in maturation of the large ribosomal subunit. Immature ribosomal intermediates, designated 45S subunits, are known to accumulate within the cell in the absence of YsxC. The 45S subunits cannot associate with the small ribosomal subunit and thus fail to form ribosomes capable of their necessary functions. A deletion of the ysxC gene is lethal in Staphylococcus aureus. The mechanism of YsxC interactions with the S. aureus ribosome remains to be elucidated. A protocol was devised to isolate, purify, and assembly the YsxC protein complex with the 50S subunit of the S. aureus ribosome. A sample obtained according to the protocol proved suitable for data collection by transmission cryo-electron microscopy.