Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method’s application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: (1) the DNA extraction method greatly influences amplification success; (2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and (3) the “non-amplifiable” samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.

Novel variously substituted thiohydantoin-based dispiro-indolinones were prepared using a regio- and diastereoselective synthetic route from 5-arylidene-2-thiohydantoins, isatines, and sarcosine. The obtained molecules were subsequently evaluated in vitro against the cancer cell lines LNCaP, PC3, HCT^wt, and HCT^(−/−). Several compounds demonstrated a relatively high cytotoxic activity vs. LNCaP cells (IC₅₀ = 1.2–3.5 µM) and a reasonable selectivity index (SI = 3–10). Confocal microscopy revealed that the conjugate of propargyl-substituted dispiro-indolinone with the fluorescent dye Sulfo-Cy5-azide was mainly localized in the cytoplasm of HEK293 cells. P388-inoculated mice and HCT116-xenograft BALB/c nude mice were used to evaluate the anticancer activity of compound 29 in vivo. Particularly, the TGRI value for the P388 model was 93% at the final control timepoint. No mortality was registered among the population up to day 31 of the study. In the HCT116 xenograft model, the compound (170 mg/kg, i.p., o.d., 10 days) provided a T/C ratio close to 60% on day 8 after the treatment was completed. The therapeutic index—estimated as LD₅₀/ED₅₀—for compound 29 in mice was ≥2.5. Molecular docking studies were carried out to predict the possible binding modes of the examined molecules towards MDM2 as the feasible biological target. However, such a mechanism was not confirmed by Western blot data and, apparently, the synthesized compounds have a different mechanism of cytotoxic action.

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using16SrRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.

Recombination is one of the mechanisms of SARS-CoV-2 evolution along with the occurrence of point mutations, insertions, and deletions. Recently, recombinant variants of SARS-CoV-2 have been registered in different countries, and some of them have become circulating forms. In this work, we performed screening of SARS-CoV-2 genomic sequences to identify recombination events and co-infections with various strains of the SARS-CoV-2 virus detected in Russia from February 2020 to March 2022. The study included 9336 genomes of the COVID-19 pathogen obtained as a result of high-throughput sequencing on the Illumina platform. For data analysis, we used an algorithm developed by our group that can identify viral recombination variants and cases of co-infections by estimating the frequencies of characteristic substitutions in raw read alignment files and VCF files. The detected cases of recombination were confirmed by alternative sequencing methods, principal component analysis, and phylogenetic analysis. The suggested approach allowed for the identification of recombinant variants of strains BA.1 and BA.2, among which a new recombinant variant was identified, as well as a previously discovered one. The results obtained are the first evidence of the spread of recombinant variants of SARS-CoV-2 in Russia. In addition to cases of recombination we identified cases of coinfection: eight of them contained the genome of the Omicron line as one of the variants, six of them the genome of the Delta line, and two with the genome of the Alpha line.

Next generation sequencing (NGS) technologies have greatly enhanced our ability to identify new viral pathogens in various types of biological samples. This approach has led to the discovery of new viruses and has revealed hidden associations of viromes with many diseases. However, unlike the 16S rRNA, which allows for bacterial detection by metabarcoding, the diversity and variability of viral genomes render the creation of universal oligonucleotides for targeting all known and novel viruses impossible. While whole­genome sequencing solves this problem, its efficiency is inadequate due to the high cost per sample and relatively low sensitivity. Furthermore, the existing approaches to designing oligonucleotides for targeted PCR enrichment are usually incomprehensive, being oriented at detecting a particular viral species or a genus based on the presumption of its presence in the sample. In this study, we developed a computational pipeline for designing genus-specific oligonucleotides that would simultaneously cover a multitude of known viruses from different taxonomic groups. This new tool was used to design an oligonucleotide panel for targeted enrichment of viral nucleic acids in different types of samples, and its applicability for the detection of multiple viral genera at once was demonstrated. Next, we created a custom protocol for NGS library preparation adapted to the new primer panel, which was tested together on a number of samples and proved highly efficient in pathogen detection and identification. Since a reliable algorithm for bioinformatic analysis is crucial for rapid classification of the sequences, in this work, we developed an NGS­based data analysis module and demonstrated its functionality both for detecting novel viruses and analyzing virome diversity. This work was supported by an RSF (Russian Science Foundation) grant (No. 17-74-20096).