Molekulârnaâ biologiâ

Breast cancer (BC) is a multifactorial disease that is characterized by various genetic and epigenetic changes that occur due to the effect various factors including that of environmental etiological agents. The obtained scientific data speak volumes for epigenetic dysregulation in the BC pathogenesis. Out of all epigenetic markers, various microRNA regulating a wide spectrum of biological processes in a cell could be viewed as one of the potential risk predictors. Understanding the functional role of these molecules will provide valuable information about complex molecular mechanisms underlying the appearance and development of BC. The given review summarizes currently existing publicly available data on aberrant expression of miR-125b, miR-181a, miR-16 in case of various cancer localizations; analyzes their role in BC pathogenesis; presents the annotation of the target-genes, evaluates the repression potential of microRNA and their diagnostic significance in case of BC and an analysis of changes in miRNA expression during radiation exposure was conducted. Interest in examining specific miRNAs is due to the results of long-term monitoring of the health of people living in radioactively contaminated areas of the Southern Urals, as well as data on the expression profiles of miR-125b, miR-181a and miR-16 in the remote period in irradiated people exposure in the long term in exposed people.

Thrombogenic risk factors (blood coagulation disorders and thrombophilia) are the cause of cardiovascular diseases, among which genetic factors are worth highlighting (genetic polymorphism of the blood coagulation system, angiogenesis factors, components of the lipid metabolism system). Early identification of clinically significant polymorphisms in genes that cause predisposition to thrombogenic diseases allows for preventive measures and timely diagnosis even before the onset of the clinical picture of the disease, and for patients with an already confirmed diagnosis genetic diagnostics makes it possible to check the hereditary nature of the disease, select treatment tactics, predict the risk of developing of adverse drug reactions. This article describes the process of developing the “SNP2-TMG” kit, designed to identify ten genetic markers of susceptibility to thrombogenic diseases (rs1801131, rs6025, rs11549465, rs429358, rs7412, rs1799963, rs6050, rs1799762, rs2010963, rs1801133), by minisequencing technique (SNaPshot technology). This kit has passed clinical trials and is approved for medical use.

Advances in CRISPR/Cas-mediated genome editing have opened up treatment alternatives for many human diseases, including HIV infection. Knockout of the CCR5 gene as a potential way to treat HIV infection has long been studied. Here we analyzed guide RNAs for SpCas9 and AsCas12a nucleases targeting CCR5 gene which had been previously studied and selected the most effective among them. We also designed novel guide RNAs for the same nucleases using bioinformatics resources. We compared the efficiency of target site cleavage for all selected gRNAs using three nucleases: wt SpCas9, SpCas9-HF1-plus, and AsCas12a, as well as their off- target activities. We demonstrated that among the tested guide RNAs two for SpCas9-HF1-plus and three for AsCas12a exhibited high cleavage activity, cutting CCR5 gene in 60–72% of cells, and had off-target activities below the limit of detection. Thus, these guide RNAs may be candidates for future development of gene therapies against HIV infection.

One of the promising new approaches to HIV infection treatment is the CRISPR/Cas-mediated knockout of the CCR5 receptor gene followed by the integration of an anti-HIV gene into the break site. Numerous studies have focused on the knockout of the CCR5 gene; however, the efficiency of subsequent targeted integration of long fragments remains poorly studied. To evaluate the efficiency of this approach, we used HT1080 cells and investigated the integration of a cassette expressing the EGFP gene into the CCR5 locus using two different nucleases (SpCas9 and AsCpf1) and various donor DNA constructs delivered by recombinant adeno-associated viral vectors (rAAV). For each nuclease, we designed five variants of donor DNA differing in the length (ranging from 150 to 1000 bp) or structure of the homology arms. The efficiency of transgene integration with 150 bp homology arms was the lowest for both nucleases and significantly differed from constructs with longer homology arms. Furthermore, it was shown that the presence of nuclease cleavage sites in the donor DNA flanking the cassette with homology arms did not affect the efficiency of transgene integration during AAV delivery. We demonstrated that the AsCpf1 nuclease provided higher efficiency of EGFP transgene integration than SpCas9, despite lower efficiency of CCR5 knockout. The maximum percentage of cells with integrated transgene was achieved using the AsCpf1 nuclease and an expression cassette with 600 bp homology arms, reaching 59 ± 6%.

Disordered proteins are of great interest due to their structural features, as they do not have well-defined three-dimensional structures. These proteins, often called intrinsically disordered proteins or regions, play critical roles in various cellular processes and are associated with the development of a number of diseases. Our in silico research focused on the investigation of protein complexes that include both the ordered protein, such as 14-3-3γ, and proteins with intrinsically disordered regions, such as nucleocapsid (N) of SARS-CoV-2 and p53. Our findings demonstrate that complexes modeled by AlphaFold2 and refined using discrete molecular dynamics simulations acquire assembled structures in disordered regions. After refinement, the modeled complexes exhibit a degree of structural assembly that addresses a key challenge in studying disordered proteins – their propensity to evade stable conformations.

It is generally accepted that the existing set of proteinogenic amino acids encoded by the standard genetic code was formed step by step in the course of evolution. Most studies name Ala, Asp, Glu, Gly, Ile, Leu, Pro, Ser, Thr and Val as early amino acids, presumably of extraterrestrial origin. However, other studies have chosen a consensus list of early amino acids in which Ile is replaced by Arg. We compared the differences between early and late amino acids for the lists with Ile and with Arg based on their physicochemical properties (AAindex database). The point-biserial correlation coefficient rpb, Student’s t-test and its reliability, p-value, were calculated between the binary lists with Ile and Arg and each AA index. Since a total of 2×553 p-values were obtained, the problem of multiple comparisons was solved using the Bonferroni correction and the Benjamini-Hochberg method. Next, we used the 2B-PLS method, which is applied to two different sets of variables related to the same objects, to find information common to both sets. The first set was the binary lists of Trifonov (Arg) and Wong (Ile), and the second set was 553 AA indexes. The maximum correlation with both the list with Ile and with Arg (1.0 and 0.8, respectively) was demonstrated by the binary AA index CHAM830108, which characterizes the ability of an amino acid to be a charge donor: late amino acids are capable of being donors, while early ones are not. Apparently, this is due to the differences in the conditions under which the standard set of amino acids evolved: prebiotic and biotic. The results of the 2B-PLS analysis also show that in the list of 10 evolutionarily early amino acids, Ile looks preferable to Arg. The allocation of the last 6 amino acids (Cys, His, Met, Phe, Trp, Tyr) obtained on the basis of the reduction of the HOMO-LUMO gap in a separate, third stage of the evolution of the set of standard amino acids is confirmed. A compact arrangement on the 2B-PLS plane of the physicochemical properties of three groups of amino acids, in which adenine, thymine and cytosine are located in the second position of the codons, respectively, as well as the maximum dispersion of amino acids with guanine in the second position of the codons, is revealed.

MicroRNAs in tissues and biological fluids represent a promising class of biomarkers for the molecular diagnostics and therapy of numerous diseases, including oncological diseases. Biomarkers based on easily accessible biological fluids, primarily blood-based biomarkers, are of particular value for diagnostic and prognostic purposes. To explore the potential of microRNAs as prognostic cancer markers and targets for molecular therapy, global microRNA profiling is required, which is provided by next-generation sequencing (NGS). NGS offers high sensitivity, single nucleotide resolution, and the possibility of profiling a large number of samples in parallel. Despite the promising potential of microRNAs as biomarkers and the growing number of works in this area, the literature does not sufficiently address in detail the problems associated with sample preparation methods, the specifics of library preparation for microRNA sequencing, and the difficulties of quantitative analysis. Protocols for creating libraries for microRNA sequencing present specific challenges and require selecting conditions for each type of biological sample. Here, we present in detail the preparation of libraries for microRNA sequencing from pituitary adenoma tumor tissue and blood plasma of patients with acromegaly on the Illumina platform. We discuss the difficulties and limitations of the methods and evaluate the effectiveness of sequencing plasma and brain samples. The work can serve as a guide for researchers studying the mechanisms of microRNA regulation in endocrine diseases of the pituitary gland and will also allow for the adaptation of technical procedures for various biological samples in relation to other pathologies.