Molekulyarnaya Biologiya最新文献

DNA methylation is one of the most important mechanisms closely involved in the epigenetic regulation of gene expression. However, the relationship between DNA methylation and expression is not completely understood. There are reported examples of changes in DNA methylation being the cause of gene expression, and vice versa - examples of changes in gene expression to entail changes in methylation. Earlier, we introduced the concept of CpG traffic lights - individual CpG sites methylation levels of which significantly correlate with the expression of a gene nearby - and showed their important role in the regulation of enhancers. In this work, we show that the levels of CpG traffic lights methylation are heterogeneous in the cell population, and suggest that this feature is caused by their dynamic demethylation. The 5hmC enrichment and TET2 localization sites in CpG traffic lights confirm our hypothesis. In order to find out whether methylation of CpG sites is the cause or the consequence of the gene expression, we applied the assessment of the causation direction method developed by Jonas Peters and co-authors. We determined that in CpG sites where changes in methylation are predicted to be the cause of gene expression changes (M→E CpG sites), methylation levels are more stable in different cells and active demethylation occurs less often than in CpG sites where presumably changes in gene expression cause changes in methylation (E→M CpG sites). We also show a greater share of M→E CpG sites in the promoter regions than in the bodies of the genes where E→M CpG sites are more common. Based on these observations, we believe that methylation of M→E CpG sites is stable and functions as an "on/off" switch. On the contrary methylation of E→M sites is dynamic, variable between cells in a cell population, and is caused primarily by active demethylation.

Bacillus cereus sensu lato comprises genetically, morphologically, and physiologically similar gram-positive spore-forming bacterial species with high pathogenic potential, such as B. anthracis, B. cereus, and B. thuringiensis. Toxin-producing strains of B. cereus s.I. pose a major threat to human health. The high degree of similarity between these species makes it very difficult to identify them and to take adequate measures to treat the diseases they cause. Previously, we characterized the clinical isolate CCGC 19/16 belonging to B. cereus s.l. that exhibited features of both B. cereus and B. cytotoxicus. In the present work, CCGC 19/16 was identified as B. cytotoxicus using multilocus sequence typing (MLST) and mass spectrometric analysis. It was also shown that, unlike other representatives of the B. cytotoxicus species, strain CCGC 19/16 is not thermotolerant. Unlike B. cereus, strain CCGC 19/16 is sensitive to most antibiotics and shows increased motility. Like B. cereus strain CCGC 19/16 forms β-hemolysis zones in blood agar. In addition, it has been shown that prolonged storage of samples prior to analysis can lead to misidentification of the isolate. Our results indicate that "rapid methods" of analysis using single genes have insufficient resolving power in the identification of B. cereus s.l. species. The combination of MLST analysis with MALDI-TOF MS provides sufficient resolution.

Cystathionine-γ-lyase (CSE) is a key enzyme for H2S generation in the pathogenic bacteria Staphylococcus aureus, Pseudomonas aeruginosa, etc. Suppression of CSE activity significantly increases the antibiotic susceptibility of bacteria. In this work a method to synthesize a novel indole-based CSE inhibitor, 3-ammo-5-[(6-bromo-1H-indol-1-yl)methyl]thiophene, named MNS1, has been developed. The synthesis of MNS1 is based on the modification of substituted thiophene as a main structural fragment, which is involved in alkylation of 6-bromoindole at final steps. The dissociation constant of the MNS1 complex with S. aureus CSE (SaCSE) is 0.5 μM, one order of magnitude lower than with human CSE (hCSE). MNS1 was shown to efficiently enhance the antibacterial effect of gentamicin against Bacillus subtilis, suggesting its possible use as an antibiotic potentiator to inhibit the growth of CSE-expressing bacterial cells.

Adequate empiric therapy is difficult to choose without monitoring the local distribution of antibiotic-resistant bacteria in each hospital. The frequency of β-lactamase-producing Enterobacterales was compared in patients of therapeutic and surgical units. Antibiotic susceptibility was evaluated by the disk diffusion test. Production of extended-spectrum β-lactamases (ESBLs) was detected by the double-disk test, and carbapenemases were determined by a modified carbapenem inactivation method. Carbapenemase genes and their expression were quantified by real-time PCR and immunochromatography. More than one-third of Enterobacterales isolates produced ESBLs in both the therapeutic (35.51%) and surgical (39.85%) units. The proportions of carbapenemase producers was comparable in the two groups, amounting to 8.41 and 9.77%, respectively. Metallo-β-lactamases predominated in the surgical units; and serine β-lactamases, in the therapeutic units. β-Lactamase producers were less frequent among community-acquired isolates than among nosocomial ones in both therapeutic (31.48 and 56.6%) and surgical (45.45 and 51%) units, but the differences were nonsignificant. Although the proportion of β-lactamase producers in the surgical and therapeutic units was not found to increase over three years of the study, local monitoring should certainly be continued in order to develop a local strategy for adequate use of antibacterial drugs.

Resistance to antimicrobial drugs is an urgent problem not only in public health, but also in animal husbandry. The widespread use of antimicrobials in feed additives is one of the main reasons for the rapid spread of antibiotic resistance in the microbiota of the gastrointestinal tract of farm animals. To characterize antibiotic resistance genes (resistome), we performed metagenomic analysis of the feces of 24 cattle from different regions of Russia, including cows of different breeds and yaks. Animals differed in the type of housing: year-round on pastures or in barns of conventional farms, with consumption of feed additives. Although genes of resistance to aminoglycosides, β-lactams, glycopeptides, MLS antibiotics (macrolides, lincosamides, and streptogramins), phenicols, and tetracyclines were detected in samples from both groups of animals, the content of the resistome in the fecal microbiome of stall-bred cattle was about ten times higher than in animals kept on pastures. The resistome of stall cattle was dominated by β-lactamases and tetracycline resistance genes, the content of which in the microbiome was 24 and 60 times higher, respectively, than in animals kept on pastures. Apparently, the spread of resistance to β-lactams and tetracyclines in stall cattle reflects the active use of these antibiotics in livestock production. Metagenomic analysis of livestock feces can be used to quantify antibiotic resistance genes for the purpose of monitoring antimicrobial drugs used in animal husbandry.

Antibiotic and, more broadly, antimicrobial resistance is a naturally occurring biological phenomenon and a major public health problem. Mass emergence of drug-resistant bacterial strains was first observed in the mid-20th century. Since then, cases of resistance have been reported worldwide, and multidrug resistance has been increasingly reported over the past two decades. Overuse of antibacterial agents and their release into the environment contribute to the development of bacterial resistance. Unfortunately, a search and design of new effective antibiotics are declining, while it is necessary to intensify such studies and to search for alternative methods to treat infectious diseases.

Issues related to the spread of antibiotic resistance genes in environmental microbial communities are considered. "Hotspots" of adaptive evolution, accumulation, and spread of antibiotic-resistant bacteria and genetic material of antibiotic resistance are highlighted. Such "hotspots" include anthropogenic ecosystems, such as municipal wastewater treatment plants, municipal solid waste landfills, livestock enterprises, and agrocenoses. The influence of various types of pollutants and biotic factors on enhancement of mutagenesis and horizontal transfer of antibiotic resistance genes is considered. The role of mobile genetic elements in mobilization and accelerated spread of resistance determinants is shown. Special attention is paid to the role of oxidative stress and stress regulons, which are activated for realization and control of molecular genetic mechanisms of adaptive evolution of bacteria and the horizontal distribution of genetic material in bacterial populations. Oxidative stress is identified as one of the main activators of genome destabilization and adaptive evolution of bacteria.

Mouse genome modification requires costly equipment and highly skilled personnel to manipulate zygotes. A number of zygote electroporation techniques were reported to be highly efficient in gene delivery. One of these methods called i-GONAD (improved Genome-editing via Oviductal Nucleic Acids Delivery) describes electroporation-based gene transfer to zygotes in utero. Here we adopted this technology to develop an easy-to-use and cost-effective pipeline enabling mouse genome-editing from scratch with minimal requirements to operator skills and animal use. We chose the CRISPR/Cas9 system as a genome editing tool and i-GONAD as a gene delivery method to produce Il10 knockout in C57BL/6 mice. Three animals out of 13 delivered pups (23%) were genetically compromised at Il10 locus suggesting the feasibility of the approach. This protocol provides detailed description of the used technical settings paired with troubleshooting tips and could be of interest to those who aim at establishing in-house mouse transgenesis pipeline at minimal equipment cost from scratch.

Increased resistance to polycyclic glycopeptide antibiotics has become a serious problem for chemotherapy of infections caused by resistant Gram-positive bacteria. Chemical modification of known natural antibiotics is the main direction in the creation of the next generation of anti-infective drugs. Over the past two decades, a series of hydrophobic glycopeptide analogues active against resistant strains of Gram-positive bacteria have been developed, three of which - oritavancin, telavancin, and dalbavancin - were approved by the US Food and Drug Administration (FDA) in 2013-2014 for the treatment of infections caused by sensitive and resistant strains of staphylococci and enterococci. It has been established that hydrophobic derivatives of glycopeptides can act on resistant strains of bacteria by a mechanism that does not allow binding to the modified target of resistant bacteria. Understanding the mechanism of action of natural and modified glycopeptides is considered as the basis for the rational design of compounds with valuable properties to achieve fundamental results. The possibility of using semi-synthetic glycopeptide analogues in the fight against viral infections caused by enveloped viruses is also considered. This review outlines the main ways of chemical design in creating a new generation of glycopeptide antibiotics that overcome resistance to Gram-positive pathogens and the mechanisms of their action.

Acute and subchronic toxicity of the pharmacological pair based on encapsulated Citrobacter freundii C115H methionine γ-lyase enzyme/prodrug (methiin) was studied in female ICR mice. The drug showed a weak/moderate dose-dependent hepatotoxic effect. Most of the changes in liver morphology identified were insignificant or mild deviations from the norm. Long-term use of a single therapeutic dose per mouse of 1.5 U C. freundii C115H methionine γ-lyase @ (PEG-P(Asp)70/PLL70)-PIC-some/2 mg methiin led to a slight decrease in the weight of animals without obvious signs of intoxication. A quarter of the animals in this group had no deviations from the norm in liver morphology. No nephrotoxic effect was found in any study group.