Molekulârnaâ biologiâ

The COVID-19 pandemic has triggered the development of new directions in vaccine development, among which DNA- and mRNA-based technologies are particularly noteworthy. The platform based on DNA vaccines is developing particularly intensively due to their high stability at ambient temperature and the ability to activate both humoral and cellular immunity. The full cycle of DNA vaccine creation, which includes the construction of plasmid DNA, obtaining a producer strain, fermentation and purification, takes 2‒4 weeks. In addition, the production technology of such vaccines does not require working with dangerous pathogens, which significantly simplifies the process of their production and reduces the overall cost. Over more than 30 years of rapid development, DNA vaccine technology continues to undergo changes. Currently, there is a licensed DNA vaccine for the prevention of COVID-19, and many candidate prophylactic vaccines against viral and bacterial diseases are in clinical trials. The review covers not only the principles of constructing plasmid DNA vaccines, but also new technologies for obtaining DNA constructs, such as minicircular DNA, MIDGE DNA and Doggybone^TM DNA. New types of DNA vaccines are interesting because they consist only of the most essential elements for activating the immune response. Such constructs completely lack the sequences necessary for the production of plasmid DNA in bacterial cells — for example, the antibiotic resistance gene. One of the key problems in the development of a DNA vaccine is the method of its delivery to target cells. Currently, various delivery methods are used, both chemical and physical, which are rapidly developing and have already proven themselves to be reliable and effective. The characteristics of some of the most promising methods are also presented in the review.

Interactions of intercellular adhesion molecules of the selectin family with glycoconjugates of cell membranes mediate the initial stage of the adhesion cascade, which recruits leukocytes, circulating in the bloodstream, to the sites of infection or damage. The formation of heterotypic cell aggregates between individual cells of hematopoietic and non-hematopoietic origin may be involved in processes, leading to inflammation, thrombosis and metastasis. A key protein, plays an important role in the binding of selectins, which serves as a ligand for all three selectins, dimeric glycoprotein, P-selectin glycoprotein ligand — PSGL-1. PSGL-1 combines signals activating various biochemical pathways during binding and rolling of leukocytes. The integration of these signals leads to activation of leukocytes, integrin-mediated arrest, restructuring of the cytoskeleton of interacting cells, polarization and subsequent diapedesis of leukocytes into surrounding tissues. The multilevel effect of PSGL-1 on cellular traffic in the physiological and inflammatory states is largely determined by posttranslational modifications, among which an important place is given to specific O- and N-glycosylation and sulfation. In this review, we discuss modifications of PSGL-1 associated with the initiation of biochemical pathways, as well as its interactions, which make it possible to classify this molecule as signaling, paying special attention to the mechanisms leading to pathology, including cardiovascular.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders characterized by progressive motor impairment due to the death of dopaminergic neurons in the substantia nigra of the brain. PD affects more than 1% of the population over 60 years of age worldwide. Despite significant progress in understanding the pathogenesis of PD, including genetic and biochemical aspects, current therapies are limited to symptomatic treatment. Recent evidence suggests that impaired autophagy leads to the accumulation of abnormal proteins, particularly α-synuclein, aggregated forms of which are neurotoxic to dopaminergic neurons in the substantia nigra. Notably, PD is predominantly sporadic. However, monogenic forms of the disease have also been described. Among the most common PD forms with known etiology are PD associated with mutations in the GBA1 gene and PD associated with mutations in the LRRK2 gene. Leucine-rich repeat kinase 2 (LRRK2), encoded by the LRRK2 gene, and the lysosomal enzyme glucocerebrosidase (GCase), encoded by the GBA1 gene, are involved in the same endolysosomal pathway. The LRRK2 and GCase dysfunction reported in PD, especially in the case of mutations in the genes encoding them, can lead to impairment of the endolysosomal pathway, lysosomal function, and possibly autophagy. This review highlights the molecular mechanisms of autophagy and prospects for targeted therapy of PD based on the induction of autophagy by influencing key players in this process.

One of the current trends in oncogynecology is the search for effective biomarkers of ovarian cancer. Metabolic profiling by ultra-high performance liquid chromatography and mass spectrometry allows obtaining information about the totality of all low molecular weight metabolites of patient's biological fluids sample. These metabolites can become potential disease markers, while their combination with microRNA level data significantly increases the diagnostic value. Therefore, the aim of the study was to analyze the metabolomic profile and microRNA transcripts level in urine of serous ovarian adenocarcinoma patients to identify potential non-invasive diagnostic markers of the disease. The study included 60 patients diagnosed with serous ovarian adenocarcinoma and 20 individuals without cancer history. Chromatographic separation of metabolites was performed on a Vanquish Flex UHPLC System chromatograph coupled to an Orbitrap Exploris 480 mass spectrometer. The search for gene regulators of metabolites and microRNA regulators of genes was carried out using the Random forest machine learning method. The microRNA transcripts level in urine was determined by real-time PCR. LASSO-penalized logistic regression was used to build predictive models. In patients with ovarian cancer, 26 compounds had an abnormal concentration compared to the control group (kynurenine, phenylalanyl-valine, lysophosphatidylcholines 18:3, 18:2, 20:4 and 14:0, alanyl-leucine, L-phenylalanine, phosphatidylinositol (34:1), 5-methoxytryptophan, 2-hydroxymyristic acid, 3-oxocholic acid, indoleacrylic acid, lysophosphatidylserine (20:4), L-beta-aspartyl-L-phenylalanine, myristic acid, decanoylcarnitine, aspartyl-glycine, malonylcarnitine, 3-hydroxybutyrylcarnitine, 3-methylxanthine, 2,6 dimethylheptanoylcarnitine, 3-oxododecanoic acid, N-acetylproline, L-octanoylcarnitine and capryloylglycine). Using the Random forest method, metabolite-gene regulator (47 genes) and metabolite-microRNA regulator (613 unique microRNA) relationships were established. The identified 85 microRNAs were validated by real-time PCR. Changes in the levels of miR-382-5p, miR-593-3p, miR-29a-5p, miR-2110, miR-30c-5p, miR-181a-5p, let-7b-5p, miR-27a-3p transcripts were detected. miR-370-3p, miR-6529-5p, miR-653-5p, miR-4742-5p, miR-2467-3p, miR-1909-5p, miR-6743-5p, miR-875-3p, miR- 19a-3p, miR-208a-5p, miR-330-5p, miR-1207-5p, miR-4668-3p, miR-3193, miR-23a-3p, miR-12132, miR-765, miR-181b- 5p, miR-4529-3p, miR-33b-5p, miR-17-5p, miR-6866-3p, miR-4753-5p, miR-103a-3p, miR-423-5p, miR-491-5p, miR-196b-5p, miR-6843-3p, miR-423-5p and miR-3184-5p in the urine of patients with ovarian cancer relative to the control group was found. Thus, in ovarian serous adenocarcinoma patients a significant metabolomic imbalance of urine was found associated with changes in the levels of microRNAs that regulate the signaling pathways of these metabolites. At the same time, 26 compounds with abnormal concentration and levels of microRNA transcripts miR-33b-5p, miR-423-5p, miR-6843-3p, miR-4668-3p, miR-30c-5p, miR-6743-5p, miR-4742-5p, miR-1207-5p and miR-17-5p in urine can serve as non-invasive diagnostic markers for ovarian cancer.

The kinetics of amplification and the features of individual and simultaneous incorporation of modified deoxynucleoside triphosphates in DNA during rolling circle amplification (RCA) have been studied. The study was carried out for six pairs of Sy5-labeled triphosphates of deoxyuridine (dU) and deoxycytidine (dC) previously synthesized with similar fluorescent substituents inside the pair. The effect of the linker length between the fluorophore and the pyrimidine base on the incorporation density was determined: nucleotides with a linker length of six carbon atoms are embedded in a growing DNA chain better than with three carbon atoms. It was found that the combined introduction of triphosphates into the reaction in an equivalent total concentration does not enhance the inhibitory effect, which gives grounds for a more detailed study of the simultaneous use of labeled dU and dC.

Thiocyanate dehydrogenase is enzyme catalyzing transformation of a thiocyanate ion into a cyanate ion with outcome of two electrons, two protons and a neutral atom of sulphur. Earlier structures of thiocyanate dehydrogenase from Thioalkalivibrio paradoxus were solved. Despite not perfect quality of the structures (twinning and pronounced anisotropy of the crystals, incomplete occupancy of the copper ions, absence of data for complexes with analogues of the substrate), there was suggested a mechanism of the enzyme functioning based on those structures. Recently at atomic resolution there have been solved structures of a gene-modified copy of relative enzyme from Pelomicrobium methylotrophicum for free protein and its complex with thiourea. In the new structures copper ions of the active site possess complete occupancy. In these structures it is possible to reliably identify two conformations of the protein molecule with opened and closed active sites. The new structural high resolution data also allowed us to determine the presence of the superposition of different states of the copper ions for each of the two conformations. In each state the copper ions have different oxidation degrees, different corresponding ligands and partial occupancies. The ion charges were determined according to the ions coordination. In the protein molecule with the closed active site the complexes with inhibitor (thiourea ion) and molecular oxygen are observed. The complex with thiourea allows us to model binding of thiocyanate ion to the enzyme molecule. Taking into account the changes of the structures in the opened and closed conformations, a mechanism of the attacking oxygen ligand activation is suggested. A new scheme of the enzymatic reaction is discussed.

High-throughput transcriptomic research methods provide the assessment of a vast number of factors, valuable for researchers. At the same time the “curse of dimensionality” issues arise, which lead to increasing requirements on data processing and analysis methods. In this study, we propose a new algorithm that combines Monte Carlo methods and machine learning. This algorithm will enable feature space reduction by highlighting genes most likely associated with the investigated diseases. Our approach allows not only to generate a set of “interesting” genes but also to assign weight to each gene, indicating its “importance”. This measure can be used in subsequent statistical analysis, visualization, and interpretation of results. Algorithm performance was demonstrated on open transcriptomic data of patients with HCM (GSE36961 and GSE1145). The analysis revealed genes MYH6, FCN3, RASD1, and SERPINA3, which is in good agreement with the available literature.

Immune response gene expression analysis is an important task in studies of interactions between host and an infectious agent. Many approaches to this task have been developed, but despite significant progress, the problem of selecting a single standard for data normalization remain unsolved. In the present work, HPRT1, SDHA, GAPDH and TBP were selected as candidates for reference genes with stable expression and a system for their analysis based on multiplex real-time RT-PCR was developed. The results of calculations using geNorm and BestKeeper algorithms allowed to create a stable index based on two genes — HPRT1 and SDHA. It was used for normalization of expression level of target genes of Toll-like receptors: TLR1, TLR2, TLR4, TLR6 and TLR8. The obtained expression values of Toll-like receptor genes in the sample of conditionally healthy individuals were characterized by high stability and positive mutual correlation (except TLR6), which may indicate common mechanisms of expression regulation and also confirms the possibility of using the developed multiplex system to analyze the expression of immune response genes.