Biochemistry (Moscow)最新文献

Quantitative analysis of gene transcription is widely used across various fields of biology and, in particular, in medicine, it serves as a tool for diagnostics and transcriptomic profiling of diseases. In recent years, transcriptome analysis methods based on large-scale next-generation sequencing have become widely adopted. Transcriptomic studies enable the identification of cellular processes that are active at specific time points, the investigation of transcriptome dynamics in different tissues or physiological states (such as during ontogenesis or adaptive responses) and the detection of differentially expressed genes in pathological conditions. A pronounced change in the transcription level of one or more genes under pathological conditions may be sufficient for diagnosis, serving as a transcriptional biomarker of disease. However, in some cases, altered transcription levels may indicate the presence of mutations, including those leading to disruption of splicing, activation of mobile elements, or pseudogenes. This review discusses cases in which transcriptional changes can provide insights into the genetic causes of disease, as well as the challenges that must be considered when using transcription as a diagnostic marker. In the future, specialized targeted panels based on transcriptome analysis are expected to be used not only as diagnostic and prognostic tools, but also as predictors of structural genomic abnormalities, thereby contributing to the development of novel strategies for effective disease treatment.

Tuberculosis is a leading cause of death from a bacterial infection agent. The development of new tuberculosis vaccines can reduce the number of new cases and tuberculosis-related deaths. One of the most promising areas in vaccination is development of mRNA vaccines, which have already proven their high effectiveness against COVID-19 and other viral infections. Using modern immunoinformatic methods, we developed four new antituberculosis multiepitope mRNA vaccines differing in the encoded adjuvants and codon composition and tested their immunogenicity and protectivity in mice. Most of the developed mRNA vaccines induced the formation of both cellular and humoral immunity. The adaptive response was stronger for the vaccines with the RpfE adjuvant; however, the best protective response was elicited by the mRNA-mEp21-FL-IDT vaccine with the FL adjuvant. This vaccine reduced the mycobacterial load in the lungs of mice infected with Mycobacterium tuberculosis and increased their survival rate. Altogether, our results indicate that the mRNA-mEp21-FL-IDT vaccine ensures effective protection against tuberculosis comparable to that provided by the BCG vaccine.

Telomere biology still remains a topic of interest in life sciences. Analysis of several thousand clinical samples from healthy individuals performed in recent years has shown that the telomere length (TL) in peripheral blood leukocytes correlates with the TL in cells of internal organ and reflects their condition. TL decreases under the influence of damaging factors and can serve as an indicator of health status. The telomere shortening leads to the cell proliferation arrest and is considered as a marker of replicative aging of proliferating cells. A decrease in the TL in peripheral blood leukocytes is viewed as an indicator of organism aging. Recent studies have allowed to formulate the concept on the role of the CST–polymerase α/primase in the C-strand fill in after completion of 3′G overhang synthesis by telomerase during telomere replication. The discovery of the telomeric RNA (TERRA) and its role in the regulation of telomerase activity (TA) and alternative lengthening of telomeres, as well as the possibility of TERRA translation, has provided evidence of the complex epigenetic regulation of the TL maintenance. Analysis of the published data indicates that telomeres are dynamic structures, whose length undergoes significant changes under the influence of damaging factors. TL is determined not only by the chronological age, but also by the exposure to the exogenous and endogenous deleterious factors during the lifetime. A decrease in the TL due to inherited mutations in the genes coding for proteins involved in the telomere structure formation and telomere replication (primarily, proteins of the shelterin and CST complexes and telomerase) has been found in a number of hereditary diseases – telomeropathies. The assessment of TL and TA is of great importance for the diagnostics of telomeropathies and can be useful in the diagnostics of cancer. Analysis of TL can be used for monitoring the health status (e.g., in the case of exposure to ionizing radiation and space flight factors), as well as predicting individual’s sensitivity to the action of various damaging agents. The application of modern advancement in genetic technologies in the analysis of TL and TA makes it available for the use in clinical and epidemiological studies, diagnostics of telomeropathies, and monitoring of astronauts’ health.

The worldwide number of deaths from complications caused by severe influenza and COVID-19 is about 1 million cases annually. Development of the effective antiviral therapy strategies for the disease treatment is one of the most important tasks. Use of the CRISPR/Cas13 system, which specifically degrades viral RNA and significantly reduces titer of the virus, could be a solution of this problem. Despite the fact that Cas13 nucleases have been discovered only recently, they already have shown high efficiency in suppressing viral transcripts in cell cultures. The 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 analyzed experimental in vitro and in vivo studies on the use of CRISPR/Cas13 systems as an antiviral agent in cell cultures and animal models and discussed main directions for improving the CRISPR/Cas13 system. These data allow us to understand prospects and limitations of the further use of CRISPR/Cas13 in the treatment of viral diseases.

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 clinical and pathological characteristics of GC tumors. The study included 65 plasma samples from the GC patients and 48 plasma samples obtained from the individuals without tumor lesions, which were used as a control group. The expression was analyzed by using real-time 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 = 0.040), 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 with the late clinical stage (p = 0.003). There was a significant correlation between the 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 diagnostic and prognostic properties of the potential panel of markers.

Bladder cancer (BCa) remains a significant clinical challenge because of high recurrence rates and variable response to immunotherapy and chemotherapy. Recent studies have highlighted the role of N6-methyladenosine (m⁶A) modification in RNA in the regulation of various cellular processes, including tumor progression and drug resistance. The review examines the impact of m⁶A methylation on BCa pathogenesis, with a particular special focus on the role of m⁶A pathway factors and m⁶A-modified RNAs in tumorigenesis, proliferation, invasion, and migration of cancer cells. The mechanisms of m⁶A-mediated chemotherapy resistance in BCa cells are discussed, including single nucleotide polymorphisms in m⁶A-associated patterns. Significant advances in the high-throughput analysis of m⁶A methylation have enabled development of novel m⁶A-based biomarkers for the risk assessment, early diagnostics, and prediction of relapse and treatment response in BCa. The review outlines the prospects of the m⁶A-based molecular diagnostics in BCa.

Prime editing is a highly promising strategy for treating hereditary disorders due to its superior efficiency and safety profile compared to the conventional CRISPR-Cas9 systems. This study is dedicated to development of a causal therapy for cystic fibrosis by targeting the F508del variant of the CFTR gene using prime editing, as this specific deletion accounts for a substantial proportion of cystic fibrosis cases. While prime editing has shown remarkable precision in introducing targeted genetic modifications, its application in AT-rich genomic regions, such as the one containing the F508del variant, remains challenging. To overcome this limitation, we systematically evaluated 24 pegRNAs designed for two distinct prime editing systems, PEmax and PE2-NG. Efficiency of the F508del variant correction reached 2.81% (without normalization for transfection efficiency) in the airway basal cells from the patients with homozygous F508del mutation. However, the average transfection efficiency was only 11.9%, emphasizing critical need for the advancements in delivery methodologies. These findings highlight potential of prime editing as an approach for treating cystic fibrosis, while also underscoring necessity for further optimization of both editing constructs and delivery vectors to achieve clinically relevant correction levels.

Established genome editing technologies, such as CRISPR-Cas and RNA interference (RNAi), have significantly advanced research studies in nearly all fields of life sciences, including biotechnology and medicine, and have become increasingly in demand in plant biology. In the review, we present the main principles of the CRISPR-Cas and RNAi technologies and their application in model plants and crops for the control of viral diseases. The review explores the antiviral effects they provide, including direct suppression of genomes of DNA- and RNA-containing viruses and inhibition of activity of host genes that increase plant susceptibility to viruses. We also provide a detailed comparison of the effectiveness of CRISPR-Cas and RNAi methods in plant protection, as well as discuss their advantages and disadvantages, factors limiting their application in practice, and possible approaches to overcome such limitations.

Emergence of resistance in acute monocytic leukemia cells (AMoL, AML-M5) to the action of antitumor agents is one of the main reasons for the extremely low survival and curability of the patients diagnosed with AMoL. It is well known that the AML cells have an “inflammatory” phenotype and form a unique pro-inflammatory microenvironment. Previously, we identified increase in the resistance of the THP-1 human AML-M5 cells to the action of DNA topoisomerase I and II inhibitors (topotecan, etoposide, doxorubicin) in the in vitro model simulating conditions of pro-inflammatory microenvironment – a three-dimensional long-term high-density cell culture. In this research, we investigated the mechanisms of this phenomenon using fluorescence microscopy and spectrophotometry, DNA comet assay, Western blot analysis, differential gene expression analysis, and flow cytometry. The results showed that the increase in resistance to the action of DNA topoisomerase inhibitors, in particular etoposide, in the THP-1 AML-M5 cells in a hypercellular proinflammatory microenvironment is realized through reduced accumulation of the single- and double-strand DNA breaks and, accordingly, reduced response to DNA damage. It may also be due to the pronounced activation of the signaling pathways of interferon types 1 and 2, NF-κB/STAT-dependent signaling pathways, occurring against the background of a significant suppression of the activity of transcription factors of the Myc and E2F families. The results of this work provide new ideas about the role of pro-inflammatory activation in the increased resistance of AML cells to the death induced by the action of DNA topoisomerase inhibitors.

Cryopreservation of human B cells is widely employed in clinical and research applications. However, it is still commonly believed that only freshly isolated B cells should be analyzed to accurately evaluate, both quantitatively and qualitatively, the immunoglobulin (Ig) repertoire. In this study, we use next-generation sequencing to investigate how the Ig repertoire reshapes after cryopreservation of B cells, with special focus on the clonotype representation after freeze-thawing, either with and without subsequent restimulation. Our findings indicated that the Ig repertoire was preserved after cell freezing, which might encourage scientists to use cryopreserved, patient-derived B cells in the studies of Ig repertoire, as well as demonstrated potential clinical and experimental applications of monitoring the Ig repertoire of cryopreserved B cells.