Development and Application of Real-Time PCR-Based Screening for Identification of Omicron SARS-CoV-2 Variant Sublineages.

The Omicron strain is currently the main dominant variant of SARS-CoV-2, with a large number of sublineages. In this article, we present our experience in tracing it in Russia using molecular diagnostic methods. For this purpose, different approaches were used; for example, we developed multiprimer panels for RT-PCR and Sanger and NGS sequencing methods. For the centralized collection and analysis of samples, the VGARus database was developed, which currently includes more than 300,000 viral sequences.

Pyrene-Modified DNA Aptamers with High Affinity to Wild-Type EGFR and EGFRvIII.

Nucleic acid aptamers have been proven to be a useful tool in many applications. Particularly, aptamers to epidermal growth factor receptor (EGFR) have been successfully used for the recognition of EGFR-expressing cells, the inhibition of EGFR-dependent pathways, and targeted drug delivery into EGFR-positive cells. Several aptamers are able to discriminate wild-type EGFR from its mutant form, EGFRvIII. Aptamers to EGFR have hairpin-like secondary structures with several possible folding variations. Here, an aptamer, previously selected to EGFRvIII, was chosen as a lead compound for extensive post-SELEX maturation. The aptamer was 1.5-fold truncated, the ends of the hairpin stem were appended with GC-pairs to increase thermal stability, and single pyrene modification was introduced into the aptamer to increase affinity to the target protein. Pyrene modification was selected from extensive computer docking studies of a library of thousands of chemicals to EGFR near the EGF-binding interface. The resulting aptamers bound extracellular domains of both variants of EGFR: EGFRwt and EGFRvIII with subnanomolar apparent dissociation constants. Compared with the initial aptamer, affinity to EGFRwt was increased up to 7.5-fold, whereas affinity to EGFRvIII was increased up to 4-fold.

The Evaluation of Pharmacodynamics and Pharmacokinetics of Anti-thrombin DNA Aptamer RA-36.

Anticoagulants are a vital class of drugs, which are applied for short-term surgical procedures, and for long-term treatments for thrombosis prevention in high risk groups. Several anticoagulant drugs are commercially available, but all have intrinsic disadvantages, e.g., bleeding risks, as well as specific ones, e.g., immune response to peptide/protein drugs. Therefore, the search for novel, efficient and safe anticoagulants is essential. Nucleic acid aptamers are an emerging class of contemporary pharmaceuticals which are fully biocompatible and biodegradable; they have low toxicity, and are as efficient as many protein-based drugs. The anti-thrombin DNA aptamer RA-36 has been created using a combination of rational design and molecular dynamics, showing several extra-features over existing aptamers. Aptamer RA-36 has a bimodular structure; the first G-quadruplex binds and inhibits thrombin, whereas the second G-quadruplex varies the properties of the first. This bimodular structure provides a favorable dose-effect dependence allowing the risk of bleeding to be potentially decreased. Here, the results of efficiency trials of the aptamer are presented. The aptamer RA-36 has a distinctive species specificity; therefore, the careful selection of experimental animals was required. The anticoagulant activity was characterized in rats and monkeys in vivo. Antithrombotic activity was evaluated in the live murine model of the induced thrombosis. Pharmacokinetics was estimated by tracking radionuclide labeled aptamer in rats. The aptamer was thoroughly characterized using bivalirudin as a reference drug. Despite the different profiles of anticoagulant activity, these two compounds could refer to each other, and the corresponding doses could be estimated. Bivalirudin turned out to have 10-fold higher anticoagulant and antithrombotic activity. The difference in activity is easy to explain due to the pharmacokinetic profiles of the substances: the aptamer RA-36 has 20-fold faster elimination from blood with a half-life of 1 min. The entire dataset revealed that the non-modified DNA aptamer could be an alternative to the currently used bivalent peptide inhibitor; the dosage profile could be improved by manipulating aptamer pharmacokinetics. The study has revealed aptamer RA-36 to be one of the most promising candidates for further development as a new generation of anticoagulants.

No-reflow phenomenon and endothelial glycocalyx of microcirculation.

The progress in reperfusion therapy dictated the necessity for developing new tools and procedures for adjacent/additional therapy of acute cardiovascular disorders. The adjacent therapy is targeted on the damage of the microcirculation, leading to the unfavorable prognosis for the patients. The no-reflow phenomenon holds special place in the multifactorial etiology of the microcirculation disorders, offering a new challenge in treating the patients associated with ST-segment elevation on ECG at myocardial infarction. One of the numerous causes of no-reflow, the influence of the endothelial glycocalyx of the microcirculation, is analyzed. The results obtained in the studies of the endothelial glycocalyx ultrastructure are generalized, the effect that the fragments of the glycocalyx glycosaminoglycans have on the function of the vascular wall is demonstrated. The trends in searching for correlations between the thickness of the capillary glycocalyx and the cardiovascular disease risk are noted.