Molekulârnaâ biologiâ

Successful application of the CRISPR/Cas genome editing system to various crops largely depends on the correct choice of target genes that may be purposefully changed to improve yield, quality, and resistance to biotic and abiotic stressors. The objective of this work was systematizing and cataloguing the information on the confirmed target genes for crop improvement. The latest systematic review was presented on peer-reviewed scientific papers (indexed in the Scopus database) published before August 17, 2019. The present study covers the period from August 18, 2019 to March 15, 2022. The search according to the given algorithm revealed 2090 publications, and their analysis showed that only 685 original papers contained the results of gene editing for 28 crops (the search included 56 crops). A significant part of these publications described the application of genome editing to target genes previously identified in similar works or the studies were associated with reverse genetics, while only 136 publications contained data on editing new target genes whose modification was aimed at improving plant traits important for breeding. The total number of target genes in cultivated plants that were edited to improve properties of breeding value over the entire period of the CRISPR/Cas system application was 287. A detailed analysis of the editing of new target genes is presented in this review. The studies were most often aimed at increasing plant productivity and disease resistance as well as improving the properties of plant materials. Observations are made whether it was possible to obtain stable transformants at the time of publication and whether the editing technique was applied to non-model cultivars. For a number of crops, however, the range of modified cultivars was significantly expanded, specifically for wheat, rice, soybean, tomato, potato, rapeseed, grapevine, and maize. In a vast majority of cases, agrobacterium-mediated transformation was used to deliver the editing construct; less often it was bioballistics, protoplast transfection or haploinducers. The desired change in traits was most often achieved by gene knockout. In some cases, knockdown and nucleotide substitutions were applied. The base-editing and prime-editing approaches have increasingly been used to make nucleotide substitutions in crop genes. The emergence of a convenient CRISPR/Cas editing system helped to significantly intensify the development of molecular genetics specific to many crop species.

The key role of histone deacetylases (HDACs) in the regulation of the cellular response to infection with the hepatitis C virus (HCV) was first demonstrated in 2008. Studying the metabolism of iron in the liver tissues of patients with chronic hepatitis C, the authors found that the expression of the hepcidin gene (HAMP), a hormone regulator of iron export, is markedly reduced in hepatocytes under conditions of oxidative stress caused by viral infection. HDACs were involved in the regulation of hepcidin expression through the control of acetylation level of histones and transcription factors, primarily STAT3, associated with the HAMP promoter. The purpose of this review is to summarize current data on the functioning of the HCV-HDAC3-STAT3-HAMP regulatory circuit as an example of a well-characterized interaction between the virus and the epigenetic apparatus of the host cell.

Invertebrate iridescent virus 6 (IIV6) is a member of the genus Iridovirus and belongs to the Iridoviridae family. The entirely sequenced dsDNA genome, composed of 212 482 bp, encodes 215 putative open reading frames (ORFs). ORF458R encodes a putative myristoylated membrane protein. RT-PCR analysis of ORF458R expression in the presence of DNA replication and protein synthesis inhibitors showed that this gene is transcribed in the late phase of the virus infection. Time course analysis showed that transcription of ORF458R initiates between 12 and 24 h p.i. and starts to decrease after this point. Transcription of ORF458R initiated 53 nucleotides upstream of the translation start site and ended 40 nucleotides after the stop codon. Dual luciferase reporter gene assay showed that sequences between ‒61^st and +18^th nucleotides are essential for promoter activity. Interestingly, a remarkable decrease in promoter activity, in the presence of sequences between ‒299^th and ‒143^rd nucleotides, suggested a repressor activity between these regions. Our results showed that ORF458R is transcriptionally active, and separately located sequences at its upstream region with promoter and repressor activities regulating its expression. This information on the transcriptional analysis of ORF458R will contribute to our understanding of the molecular mechanisms of IIV6 replication.

miRNAs are vital molecules of gene expression. They are involved in the pathogenesis of various common diseases, including atherosclerosis, its risk factors and complications. A detailed characterization of the spectrum of functionally significant polymorphisms of miRNA genes of patients with advanced carotid atherosclerosis is an actual research task. We analyzed miRNA expression and exome sequencing data of carotid atherosclerotic plaques of the same male patients (n = 8, 66–71 years of age, 67‒90% degree of carotid artery stenosis). For further study and analysis of the association between rs2910164 polymorphism of the MIR146A gene and advanced carotid atherosclerosis, we recruited 112 patients and 72 relatively healthy Slavic residents of Western Siberia. 321 and 97 single nucleotide variants (SNVs) were detected in the nucleotide sequences of pre- and mature miRNAs in carotid atherosclerotic plaques. These variants were located in 206 and 76 miRNA genes, respectively. Integration the data of exome sequencing and miRNA expression revealed 24 SNVs of 18 miRNA genes which were processed to mature form in carotid atherosclerotic plaques. SNVs with the greatest potential functional significance for miRNA expression predicted in silico were rs2910164:C>G (MIR146A), rs2682818:A>C (MIR618), rs3746444:A>G (MIR499A), rs776722712:C>T (MIR186), rs199822597:G>A (MIR363). The expression of miR-618 was lower in carotid atherosclerotic plaques of patients with the AC rs2682818 genotype of the MIR618 gene compared with the CC genotype (log₂FC = 4.8; p = 0.012). We also found the association of rs2910164:C (MIR146A) with the risk of advanced carotid atherosclerosis (OR = 2.35; 95% CI: 1.43–3.85; p = 0.001). Integrative analysis of polymorphism in miRNA genes and miRNA expression is informative for identifying functionally significant polymorphisms in miRNA genes. The rs2682818:A>C (MIR618) is a candidate for regulating miRNA expression in carotid atherosclerotic plaques. The rs2910164:C (MIR146A) is associated with the risk of advanced carotid atherosclerosis.

The Gagr gene is a domesticated gag retroelement gene in Drosophila melanogaster, whose function is associated with a stress response. The protein products of the Gagr gene and its homologues in different Drosophila species have a highly conserved structure; however, they demonstrate a certain variability in the promoter region of the gene, apparently associated with the gradual acquisition of a new function and involvement in new signaling pathways. In this work we studied the effect of oxidative stress caused by ammonium persulfate on the survival of various species of the genus Drosophila (D. melanogaster, D. mauritiana, D. simulans, D. yakuba, D. teissieri, D. pseudoobscura), analyzed the correlation between the structure of promoter regions and stress-induced changes in the expression of the Gagr gene and its homologues in different Drosophila species and comparison of stress-induced changes in the expression of oxidative stress markers: Jak-STAT signaling pathway activator gene upd3, Jak-STAT pathway effector vir-1, and signaling pathway target IMD Rel. It was found that in D. simulans and D. mauritiana sensitivity to ammonium persulfate is significantly increased, which correlates with a reduced level of transcription of vir-1 gene orthologues. The latter is due to a decrease in the number of binding sites for the transcription factor STAT92E, a component of the Jak-STAT signaling pathway, in the vir-1 promoter region. Consistent changes in the expression of the Gagr, upd3, vir-1 genes are observed in all species of the melanogaster subgroup, except D. pseudoobscura, which indicates an increase in the role of Gagr in the regulation of stress response pathways during the phylogenesis of the genus Drosophila.

Angiopoietin-like protein 4 (ANGPTL4) is considered to be one of the important circulating mediators linking intestinal microorganisms and host lipid metabolism. The objective of this study was to assess the effects of peroxisome proliferator-activated receptor γ (PPARγ) on modulating ANGPTL4 synthesis in Caco-2 cells exposed to Clostridium butyricum. The viability of Caco-2 cells and the expression of PPARγ and ANGPTL4 in Caco-2 cells were detected after the Caco-2 cells were co-cultured with C. butyricum at the concentration of 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ CFU/mL. The results showed that cell viability was enhanced by C. butyricum. Besides, PPARγ and ANGPTL4 expression and secretion in Caco-2 cells was significantly increased by 1 × 10⁷ and 1 × 10⁸ CFU/mL of C. butyricum. Furthermore, the effects of PPARγ on modulating ANGPTL4 synthesis in Caco-2 cells regulated by 1 × 10⁸ CFU/mL of C. butyricum was also be expounded in PPARγ activation/inhibition model based on Caco-2 cells and via ChIP technique. It was found that C. butyricum promoted the binding of PPARγ to the PPAR binding site (chr19: 8362157-8362357, located upstream of the transcriptional start site of angptl4) of the angptl4 gene in Caco-2 cells. However, the PPARγ was not the only way for C. butyricum to stimulate ANGPTL4 production. Taken together, PPARγ played a role in the regulation of ANGPTL4 synthesis by C. butyricum in Caco-2 cells.

Countering the spread of new respiratory infections and reducing the damage they cause to society requires efficient strategies for rapid development of targeted therapeutics, such as monoclonal antibodies. Nanobodies, defined as variable fragments of heavy-chain camelid antibodies, have a set of characteristics that make them particularly convenient for this purpose. The speed at which the SARS-CoV-2 pandemic had spread has confirmed that a key factor in the development of therapeutics is obtaining highly effective blocking agents as soon as possible, as well as the diversity of epitopes to which these agents bind. We have optimized the process of selection of blocking nanobodies from the genetic material of camelids and obtained a panel of nanobody structures with affinity to spike protein in the lower nanomolar and picomolar ranges and high binding specificity. The subset of nanobodies that demonstrate the ability to block the interaction between the spike protein and the cellular ACE2 receptor was selected in experiments in vitro and in vivo. It has been established that the epitopes bound by the nanobodies are located in the RBD domain of the spike protein and have little overlap. The diversity of binding regions may allow the mixture of nanobodies to retain potential therapeutic efficacy towards new variants of the spike protein, and the structural features of nanobodies, in particular, their compact size and high stability, indicate the possibility of their utilization in the form of aerosols.

In this work, we synthesized and characterized the properties of a series of new fluorescent narrow-groove ligands DB₃(n). DB₃(n) compounds based on dimeric trisbenzimidazoles have the ability to bind to the AT regions of DNA. The synthesis of DB₃(n), trisbenzimidazole fragments of which are linked by oligomethylene linkers of different lengths (n = 1, 5, 9), is based on the condensation of monomeric trisbenzimidazole MB₃ with α,ω-alkyldicarboxylic acids. DB₃(n) proved to be effective inhibitors of the catalytic activity of HIV-1 integrase at submicromolar concentrations (0.20–0.30 µM). DB₃(n) was found to inhibit the catalytic activity of DNA topoisomerase I at low micromolar concentrations.

Transcatheter arterial chemoembolization is one of the interventional treatments for hepatocellular carcinoma (HCC). This treatment is generally used for patients with intermediate to advanced hepatocellular carcinoma, and identifying the role of HCC-related genes can help improve the efficiency of transcatheter arterial chemoembolization. To investigate the role of HCC-related genes and to provide valid evidence for transcatheter arterial chemoembolization treatment, we performed a comprehensive bioinformatics analysis. Through text mining (“hepatocellular carcinoma”) and microarray data analysis (GSE104580), we obtained a standard gene set, which was followed by gene ontology and Kyoto Gene and Genome Encyclopedia analysis. The significant 8 genes clustered in protein-protein interactions network were chosen to be used in the follow-up analysis. Through survival analysis low expression of the key genes were found to be strongly associated with survival in HCC patients in this study. The correlation between the expression of the key genes and tumor immune infiltration was assessed by Pearson correlation analysis. As a result, 15 drugs targeting seven of the eight genes have been identified, and therefore can be considered as potential components for transcatheter arterial chemoembolization treatment of HCC.

A diagnostic system based on recombinase polymerase amplification (RPA) has been developed to identify six bacterial pathogens of human pneumonia. Species-specific primers have been designed and optimized to conduct a multiplex reaction in one common volume. Labeled primers were used for reliable discrimination of amplification products close in size. Identification of the pathogen was carried out by visual analysis of the electrophoregram. The analytical sensitivity of the developed multiplex RPA was 10²‒10³ copies of DNA. The specificity of the system was determined by the absence of cross-amplification of the studied DNA samples of pneumonia pathogens for each pair of primers, as well as for the DNA of Mycobacterium tuberculosis H37_rν, and amounted to 100%. The analysis execution time is less than an 1 h, including electrophoretic reaction control. The test system can be used in specialized clinical laboratories for rapid analysis of samples from patients with suspected pneumonia.