Biohimiâ

Gastric cancer (GC) is one of the most common malignant tumors worldwide and ranks fifth in the structure of cancer mortality. MicroRNAs are involved in the pathogenesis and progression of GC as epigenetic factors, and are considered as potential noninvasive markers. We selected microRNAs involved in the regulation of epigenetic mechanisms in GC (miR-1301-3p, miR-106a-5p, miR-129-5p, miR-3613-3p, miR-647) and analyzed their expression in plasma of GC patients. To assess their diagnostic and prognostic potential, we estimated correlations of differential expression with the clinical and pathological characteristics of GC tumors. The study included 65 plasma samples from GC patients and 48 plasma samples obtained from individuals without tumor lesions, which were used as a control group. The expression was analyzed by using the reverse transcription polymerase chain reaction (RT-PCR) method. When comparing the expression levels of selected microRNAs in the plasma of GC patients and the control group, significant differences were found for miR-1301-3p (p = 0.04), miR-106a-5p (p = 0.029), miR-129-5p (p < 0.0001), miR-647 (p < 0.0001). MiR-129-5p expression was significantly associated with the prevalence of a primary tumor (p = 0.002), with the development of metastases to regional lymph nodes (p = 0.003) and distant metastases (p = 0.003), as well as a late clinical stage (p = 0.003). There was a significant correlation between miR-3613-3p expression and the clinical stage of GC (p = 0.049). ROC analysis revealed that combining miR-106a-5p, miR-129-5p, miR-1301-3p and miR-647 improves the diagnostic and prognostic properties of a potential panel of markers.

Telomere biology research remains a hot topic. Analysis of several thousand clinical samples from healthy individuals performed in recent years has shown that the telomere length (TL) of peripheral blood leukocytes correlates with the TL of human internal organ cells and reflects their condition, decreases under the influence of damaging factors and can serve as an indicator of health status. A decrease in TL leads to proliferation arrest and is considered as an indicator of replicative aging of proliferating cells, and a decrease in TL of peripheral blood leukocytes is considered as an indicator of aging. Novel fundamental research data enabled to formulate the concepts about the role of the enzyme CST–Polymerase alpha/primase in the complementary C-strand synthesis after completion of the 3′G overhang synthesis by telomerase during telomere replication. The discovery of telomeric TERRA RNA and its role in the regulation of telomerase activity (TA) and alternative lengthening of telomeres, as well as the possibility of TERRA translation, provided evidence of the complex epigenetic regulation of TL maintenance. The published data analysis allows concluding that telomeres are dynamic structures and that TL undergoes significant changes under the influence of damaging factors and is determined not only by the chronological age, but by the total effect of exposure to all exogenous and endogenous damaging factors during the life. An inheritable decrease in TL due to mutations in the genes of proteins that determine the telomere structure and participate in the telomere replication, primarily the proteins of the shelterin complex, the CST complex and telomerase, has been found in a number of hereditary diseases – telomeropathies. Analysis of TL and TA is of great importance for the diagnosis of telomeropathies and may be useful in cancer diagnostics, and analysis of TL – for monitoring the health status, including exposure to ionizing radiation and space flight factors, together with the prediction of individual sensitivity to the action of damaging factors of different nature. The modern advanced genetic technologies for the analysis of TL and TA are available for use in clinical and epidemiological studies, are actively used in the telomeropathies diagnostics and astronauts’ health monitoring, and continue to improve.

Tuberculosis is a leading bacterial infection in terms of lethality. The development of new tuberculosis vaccines may reduce the number of new cases and deaths from it. One of the most promising areas of vaccination is represented by mRNA vaccines, which have already proven their high effectiveness against COVID-19 and other viral infections. In our study, we developed four new multiepitope mRNA antituberculosis vaccines by modern immunoinformatic methods; the vaccines differ from each other in sequences of encoded adjuvants and codon composition. Their immunogenicity and protectivity were tested in experiments on mice. Most of the developed mRNA vaccines have led to the formation of both cellular and humoral immunity. Meanwhile, the adaptive response was stronger in the case of vaccines with adjuvant RpfE. Nonetheless, the best protective response was elicited by the mRNA-mEp21-FL-IDT vaccine (with adjuvant FL), which decreased mycobacterial load in the lungs after infection of mice with M. tuberculosis and increased animal survival. Altogether, our results indicate that the mRNA-mEp21-FL-IDT vaccine, developed by mRNA immunoinformatic methods, provides effective protection comparable to that seen after BCG vaccination.

The worldwide number of deaths from complications caused by severe influenza and COVID-19 is about 1 million cases annually. The development of effective antiviral therapy strategies for the disease treatment is one of the most important tasks. The use of the CRISPR/Cas13 system, which specifically degrades viral RNA and significantly reduces the titer of the virus, can be a solution of this problem. Despite the recent discovery, Cas13 nucleases have already shown their high efficiency in suppressing viral transcripts in cell cultures. Recent advances in mRNA technology and improvements in non-viral delivery systems have made it possible to effectively use CRISPR/Cas13 in animal models as well. In this review, we have analyzed the experimental in vitro and in vivo studies on the use of CRISPR/Cas13 systems as antiviral agent in cell cultures and animals and discussed the main directions for improving the CRISPR/Cas13 system. These data allow us to understand the prospects and limitations of further use of CRISPR/Cas13 in the treatment of viral diseases.

This paper reports design of a thermostable temperature-activated mini-intein based on the full-length intein DnaE1 from Thermus thermophilus HB27 (TthDnaE1). We performed rational design of three mini-inteins TthDnaE1 Δ272, Δ280 and Δ287 through deletion mutations in the full-length intein sequence. Two mini-inteins (Δ272 and Δ280) were capable of efficient protein splicing at temperatures above 50°C. The most active mini-intein with the Δ280 deletion was chosen as the basis for further design of a self-cleaving carrier of affinity tags through single-point mutagenesis. We performed the C1A, D405G and C1A/D405G mutations, which were proposed to eliminate the intein’s capability of N-terminal extein cleavage and extein ligation. As a result, the mini-intein Δ280 with double mutation C1A/D405G displayed the highest efficiency of C-terminal extein cleavage at its temperature optimum around 60°C. Thus, we constructed thermostable temperature-activated mini-inteins capable of efficient protein splicing or cleavage of C-terminal extein. The engineered TthDnaE1 Δ280 C1A/D405G mini-intein can serve as basis for the development of new expression system for intein-mediated production of pharmaceutical recombinant proteins and peptides.