Vol 53, No 3 (2019)

A serious problem in the treatment of HIV infection is the emergence of drug-resistant forms of the virus. One promising approach to solving this problem is the development of inhibitors of the interaction between viral proteins with cellular co-factors. However, the development of this approach is hampered due to the lack of knowledge about the involvement of cellular proteins in the pathogenesis of HIV infection. In particular, it is known that the integration of viral DNA into the host genome generates numerous lesions in the cellular DNA, the repair of which is absolutely necessary for successful replication of the virus. However, it is still unknown which cellular proteins are involved in repairing this damage. In this review, we summarize what is known to date about the role of cellular repair systems in the replication of HIV-1 in general, and in the repair of damage that occurs during the integration of viral DNA into a cell’s genome, in particular.

The prion properties of alpha-synuclein, a key aggregating protein involved in the pathogenesis of so-called synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy, and its various conformers are discussed. It is shown that alpha-synuclein may be transferred between cells by prion-like propagation. Similarly to other prions, alpha-synuclein aggregation develops from the initial lag-phase (nucleation) to the subsequent growth phase (elongation), and to the stationary phase where the aggregates and monomers exist in equilibrium. Similarly to prions, alpha-synuclein undergoes conformational changes from an alpha-helix to its beta-folded structure. However, there is currently no evidence that alpha-synuclein-dependent PD can be transmitted from person-to-person. This review describes the prion properties of alpha-synuclein, possible ways of its intercellular propagation, and novel approaches to PD diagnostics.

The causative agent of tick-borne encephalitis (a neurotropic RNA virus from the Flavivirus genus) can cause both severe paralytic forms of the disease (meningoencephalitis, etc.) and milder nonparalytic forms (fever and meningitis). The organism response to viral infection (and, as a consequence, the nature and outcome of the disease) significantly depends on individual peculiarities of the human organism protective systems predetermined by genome structure. Human genetic predisposition to tick-borne encephalitis has been poorly studied. In the present work, the results of the search for new genes that predetermine the peculiarities and outcome of tick-borne encephalitis in humans are presented. The aim of the work was to verify the association between three previously detected (using the exome sequencing on a limited sample of tick-borne encephalitis patients with severe forms) SNPs: intronic rs3109675 (C/T) in the COL5A1 gene, intronic rs41554313 (A/G) in the POLRMT gene, and intergenic rs10006630 (C/A), and the predisposition to tick-borne encephalitis in a Russian population (using an extended sample of patients with different forms of tick-borne encephalitis). The association of the rs10006630 SNP located in chromosome 4 between the FABP2 and LINC01061 genes with a predisposition to tick-borne encephalitis was confirmed. This SNP can be considered as a new genetic marker of a human predisposition to severe forms of tick-borne encephalitis. The possible regulatory role of this SNP in the functioning of neighboring genes and a mechanism of its effect on the development of predisposition to severe forms of tick-borne encephalitis require further study.

The discovery of novel significant molecular and genetic markers is important for the diagnostics, prognosis, and therapy selection in hematological malignancies. Distinct cytogenetic aberrations leading to the formation of fusion genes are found in more than 40% of pediactric cases of acute myeloid leukemia (AML); however, the tumor cells in approximately 20% of these patients display cytogenetically normal karyotype (NK-AML). Here we present the analysis of the mutational profiles of leukemic cells collected from pediatric AML cases without known clinically significant chromosomal aberrations aimed at identifying AML specific markers. In 34 pediatric cases of different AML types, the coding regions of 26 genes involved in the AML pathogenesis were analyzed by massive parallel sequencing. Sequencing revealed the somatic mutations in genes that are involved in various intracellular signaling pathways, including the CEBPA, ETV, IDH1, JAK2, and NRAS genes. In addition, rare genetic variants were found in CUX1, FLT3, TET2, PTPN11, and NUP98 genes. This data may contribute to the understanding of the mechanisms of malignant cell transformation in the case of leukemogenesis.

miRNA genes play an important role in cancer pathogenesis, while they may be suppressed by hypermethylation. Here, we assess the diagnostic potential of a group of hypermethylated miRNA genes (MIR-124-1, MIR-124-3, MIR-125B-1, MIR-127, MIR-132, MIR-193a, and MIR-34b/c) in a representative set of 70 breast cancer samples and 17 breast tissue samples from deceased donors with no malignancies. For these seven genes, the methylation status is determined using the methylation-specific PCR. Methylation reached 26–76% in tumor specimens, 1‒27% in paired considered normal breast tissues, and 0–18% in breast tissue from deceased donors. By quantitative RT-PCR, reduced expression levels of the investigated miRNAs are detected, with a negative correlation of expression levels with gene hypermethylation. Combinations of three or four hypermethylation biomarkers, namely, MIR-124-1, MIR-125B-1, MIR-127, and MIR-34b/c are found suitable for breast cancer diagnostics; with sensitivity (76‒93%), specificity (88‒100%), and AUC (0.88‒0.94). Notably, the MIR-127 gene was hypermethylated only in the tumor samples of patients with metastases, and, therefore, should be tested as a marker of breast cancer dissemination. These findings may lead to improvement in the management of breast cancer.

N⁶-methyladenosine (m⁶A) has been identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, and plays important biological roles in the regulation of cellular metabolic processes. However, its role in myogenic differentiation is unclear. Here, we altered the m⁶A RNA methylation level by overexpression of METTL3, and explored the effect of m⁶A RNA methylation on myogenic differentiation of murine myoblasts in vitro. The m⁶A RNA methylation level is regulated by exogenous methylation inhibitor cycloleucine (Cyc) and methyl donor betaine (Bet). Therefore, chemical reagents of Cyc and Bet were used to test the regulatory effect of m⁶A RNA methylation on myogenic differentiation. Results showed that METTL3 and Bet positively regulated the m⁶A RNA methylation levels, and Cyc negatively regulated m⁶A RNA methylation levels. In addition, m⁶A methylation positively regulated myogenic differentiation in murine myoblasts. These findings provide insight in the mechanisms underlying the effect of m⁶A RNA methylation on myogenesis.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for hematopoietic malignancies. The graft-derived donor lymphocytes are capable of eliminating the residual recipient malignant cells in the course of allogeneic immune response, thus decreasing the chances of a relapse of the disease. Foreign peptides of the recipient presented by the MHC molecules are able to elicit the immune response immunologically. These polymorphic peptides are known as minor histocompatibility antigens (MiHAs). MiHAs occur due to the nonsynonymous single nucleotide polymorphisms in human genome. Transfusion of T cells specific to MiHAs presented predominantly in the cells of hematopoietic origin will allow the targeted elimination of residual malignant clones avoiding undesirable damage to healthy tissues. To induce the immune response, the donor must be homozygous by the MiHA allele and the recipient must either be homozygous or heterozygous by the alternative MiHA allele. The therapeutic mismatch occurs in 25% of cases under the optimal frequency of allelic variants. Minor antigen ACC-1Y originates from polymorphism in the BCL-2A1 gene; its immunogenic mismatch occurrence approaches the theoretical maximum. In addition, BCL2A1 is overexpressed in cells of various lymphomas. ACC-1Y is presented on allele HLA-A*24:02, which is relatively frequent in the Russian population. Combination of these factors makes the minor antigen ACC-1Y a promising target for immunotherapy. Transfusion of donor CD8⁺ lymphocytes modified with transgenic MiHA-specific TCR is one of the promising methods of posttransplant leukemia therapy and relapse prophylaxis. We obtained a sequence of high-affinity ACC-1Y-specific TCR after the antigen-specific expansion of T cells derived from a healthy ACC-1Y^–/– donor. We cloned this sequence into the lentiviral vector and obtained the assembled viral particles. Further, we transduced the CD8⁺ lymphocyte culture and demonstrated its antigen-specific cytotoxic activity. It is suggested that CD8⁺ lymphocytes modified by the described method could be potentially transferred to recipients as a therapy against relapse after allo-HSCT.

It is known that long (200–300 nucleotides and longer) non-protein-coding RNAs (ncRNAs) tissue-specifically expressed from the regulatory regions of developmental genes can regulate the transcription of the mRNA of these genes. In this study, an attempt is made to identify differentially expressed ncRNAs in the extended promoter region of the fork head (fkh) gene of the fruit fly Drosophila melanogaster. We investigated four preparations of the total RNA: from embryos, from adult flies (separately from females and males), and from the S2 cell line of cultured Drosophila cells. In the total RNA preparations from embryos and adult flies, the levels of fkh expression differed substantially, whereas in S2 cells its expression is not detected at all (shown in this work). We perform classical Northern blot analysis of gel-separated RNAs hybridized to a series of radioactively labeled DNA fragments corresponding to the adjacent and partially overlapping regions of the promoter region of the fkh gene. Several previously unknown differentially expressed ncRNAs are detected, including those in the regions overlapping with the previously detected regulatory elements (TRE1 and salivary gland enhancer sgE) and the transcription start site of the fkh gene. The collected data complement and clarify the results of the previously conducted RNA-seq experiments, in particular, in terms of the length of the detected RNAs. These results may serve as a foundation for further studies of the mechanisms of tissue-specific regulation of the fkh gene expression.

Homeodomain transcription factors play a significant role in adipocyte differentiation. The role of Pbx1 and Prep1, proteins of the TALE family (the three amino acid loop extension), was previously established in adipocyte differentiation of mesenchymal stromal cells and 3T3-L1 cell line. In this study, with the use of RNA interference technology we show that another transcription factor from the same family, Meis1, which is a core protein of mature cardiomyocytes, represses adipogenesis to a greater degree than its paralog Meis2. A number of Meis target genes, markers of adipocytes, are identified. This may indicate the transcriptional mechanism of the effect of Meis1 on the adipocyte differentiation of mouse preadipocytes.

To develop a quantum-dot-based multiplexed imaging system for the simultaneous monitoring of Hsf-1/Hsp70 after heat shock, and to evaluate the effects of combined thermotherapy and leucine deprivation therapy on Hsf-1 inactivation. SCC-25 cells were leucine starved for 0, 1, 2, 3 or 4 days following which the cells underwent heat shock at 42°C for 30 min. At 6 h after heat shock, Hsf-1 activation and translocation to the nucleus was observed in cells that were leucine starved for 0, 1 and 2 days, and the synthesis of Hsp70 and Hsf-1 reached their maximum values and had a tendency to gather in the nucleus. However, in cells that were leucine starved for 3 and 4 days, Hsf-1 activity and Hsp70 synthesis level was dramatically decreased. Dietary restriction of leucine for at least three days could result in the inactivation of Hsf-1, leading to a reduction in Hsp70 synthesis. The combination of thermotherapy and short-term leucine deprivation therapy may become effective approach for the treatment of oral tumors.

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP–template–polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature–time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).

Proteins with homo-repeats of more than 4 amino acid residues in length were examined to understand whether some splicing sites in pre-mRNA may be attributed to homo-repeats in human proteins. The human proteome was found to contain a total of 404 proteins with homo-repeats that account for at least one splicing site in pre-mRNA. Pre-mRNA splicing sites were more often found in the C-terminal part (67%) than in the middle or N-terminal part of a homo-repeat. Ten homo-repeats were identified to have two splicing sites per repeat. The repeats were lysine homo-repeats in all but one case.