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The aim of the research was the experimental investigation of test-system on the panels of blood serа from healthy, leukemic, and experimentally infected with various Mycobacterium species cows. The results of serological testing of biological samples from cattle infected with different strains of Mycobacterium are presented. The analysis of informative indicators (sensitivity and specificity) of test system for the diagnosis of tuberculosis based on the determination of the immunoglobulins G to bovine tuberculosis pathogen Mycobacterium bovis, was done using chimeric recombinant protein MPT83(115-220)-MPT63 as an antigen. Above-mentioned recombinant protein is based on antigens of M. bovis/M. tuberculosis and achieves high sensitivity and specificity of the test system because it reduces the likelihood of false positive results caused by infection with atypical mycobacteria. The developed test system “IB-Chem Anti-Mycobacterium bovis” was tested at the State Scientific Control Institute of Biotechnology and Strains of Microorganisms and is recommended for use. [ABSTRACT FROM AUTHOR]

The continuous evolution of SARS-CoV-2 has led to the emergence of several variants of concern (VOCs) that significantly affect global health. This study aims to investigate how these VOCs affect host cells at proteome level to better understand the mechanisms of disease. To achieve this, we first analyzed the (phospho)proteome changes of host cells infected with Alpha, Beta, Delta, and Omicron BA.1 and BA.5 variants over time frames extending from 1 to 36 h post infection. Our results revealed distinct temporal patterns of protein expression across the VOCs, with notable differences in the (phospho)proteome dynamics that suggest variant-specific adaptations. Specifically, we observed enhanced expression and activation of key components within crucial cellular pathways such as the RHO GTPase cycle, RNA splicing, and endoplasmic reticulum-associated degradation (ERAD)-related processes. We further utilized proximity biotinylation mass spectrometry (BioID-MS) to investigate how specific mutation of these VOCs influence viral-host protein interactions. Our comprehensive interactomics dataset uncovers distinct interaction profiles for each variant, illustrating how specific mutations can change viral protein functionality. Overall, our extensive analysis provides a detailed proteomic profile of host cells for each variant, offering valuable insights into how specific mutations may influence viral protein functionality and impact therapeutic target identification. These insights are crucial for the potential use and design of new antiviral substances, aiming to enhance the efficacy of treatments against evolving SARS-CoV-2 variants., Competing Interests: Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s).)

Treatment options for COVID-19, caused by SARS-CoV-2, remain limited. Understanding viral pathogenesis at the molecular level is critical to develop effective therapy. Some recent studies have explored SARS-CoV-2-host interactomes and provided great resources for understanding viral replication. However, host proteins that functionally associate with SARS-CoV-2 are localized in the corresponding subnetwork within the comprehensive human interactome. Therefore, constructing a downstream network including all potential viral receptors, host cell proteases, and cofactors is necessary and should be used as an additional criterion for the validation of critical host machineries used for viral processing. This study applied both affinity purification mass spectrometry (AP-MS) and the complementary proximity-based labeling MS method (BioID-MS) on 29 viral ORFs and 18 host proteins with potential roles in viral replication to map the interactions relevant to viral processing. The analysis yields a list of 693 hub proteins sharing interactions with both viral baits and host baits and revealed their biological significance for SARS-CoV-2. Those hub proteins then served as a rational resource for drug repurposing via a virtual screening approach. The overall process resulted in the suggested repurposing of 59 compounds for 15 protein targets. Furthermore, antiviral effects of some candidate drugs were observed in vitro validation using image-based drug screen with infectious SARS-CoV-2. In addition, our results suggest that the antiviral activity of methotrexate could be associated with its inhibitory effect on specific protein-protein interactions., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

P element is a DNA transposon, known to spread in genome using transposase activity. Its activity is tissue-specific and normally observed at high temperatures within 24 degrees C to 29 degrees C. Here, we present a predicted RNA secondary structure domain of P element pre-mRNA which could potentially regulate the temperature sensitivity of the P element activity. In canonical P elements, the structure is a small hairpin with double-helical part interrupted by a symmetric loop and a mismatch. In M type P elements, the A.A mismatch is substituted by an A-U base pair, stabilizing the structure. The hairpin structure covers the region involving the IVS-3 5' splice site and both pseudo-splice sites F1 and F2. While the IVS-3 and F1 binding sites of U1 snRNA are located in the double-stranded part of the structure, the F2 site is exposed in the hairpin loop. The formation of this structure may interfere with landing of U1 snRNA on IVS-3 site, while F2 is positioned for the interaction. Alignment of P element sequences supports the proposed existence of the hairpin, showing high similarity for this region. The hairpin structure, stable at low temperatures, may prevent correct IVS-3 splicing. Conversely, temperature-induced destabilization of the hairpin structure may result in the splicing at the proper IVS-3 splice site. Taking into account the increasing amount of data demonstrating the important influence of RNA folding on phenotypes determined by alternative splicing a model for possible regulation of the activity of mobile elements by pre-mRNA secondary structure seems intriguing.

DNA methylation has been discovered in Drosophila only recently. Current evidence indicates that de novo methylation patterns in drosophila are maintained in a different way compared to vertebrates and plants. As the genomic role and determinants of DNA methylation are poorly understood in invertebrates, its link with several factors has been suggested. In this study, we tested for the putative link between DNA methylation patterns in Drosophila melanogaster and radiation or the activity of P transposon. Neither of the links was apparent from the results, however, we obtained some hints on a possible link between DNA methylation pattern and genomic heterogeneity of fly lineages.

Antibody responses to purified protein derivate PPD of tuberculin and to antigens MPB63 and MPB83 of Mycobacterium bovis were determined in bovine herd (94 adult animals). Statistical approach based on approximation by multiple Gaussians with Levenberg-Marquardt algorithm for analysis of antibody level distribution against antigens examined was provided. Our results confirm that indirect ELISA with recombinant MPB83 and MPB63 as well as conventional PPD could be used for test-systems development for detection of cow tuberculosis infection at the herd level.

The aim of this work was to obtain the panel of recombinant single-chain Fv-antibodies against diphtheria toxin B subunit, the main diagnostic and pathogenic antigen of Corynebacterium diphtheriae. For this purpose we have constructed the immune library of murine immunoglobulin genes. A number of scFv specific to diphtheria toxin B subunit were acquired from the obtained library after one round of selection by phage-display. ScFv encoding DNA-fragments of eight clones were subcloned into plasmid pET-22b(+). It was shown that selected scFv were highly specific to diphtheria toxin B subunit, with affinity constant for different clones ranged from 10(7) to 10(9) M(-1).

Diphtheria toxin's B subunit provides toxin interaction with its receptor on the cell surface and translocation of toxin's A subunit from endosome to cytozole of sensitive cells. Functional analogues of B subunit with fluorescent label are considered as perspective tools for studying the above mentioned processes. The aim of the work was to obtain fluorescent B subunit analogues and to detect the specificity of their interaction with Vero line cells. B subunit fluorescent analogues were obtained in two different ways. The first one was B subunit chemical conjugation with fluorescein isothiocyanate and the second one was genetic fusion of recombinant B subunit chain with enhanced green fluorescent protein chain. Specific interaction of B subunit fluorescent derivatives with Vero cells was studied by flow cytometry and confocal microscopy. Using competitive analysis it was shown that B subunit fluorescent analogues possessed different affinity for cells. The affinity of EGFP-SbB was higher than FITC-SbB. Our results indicate the possibility to use the fluorescent derivatives of B subunit as tools for identification of diphtheria toxin's receptor (HB-EGF) expression on the cell surface as well as for studying the interaction and penetration of diphtheria toxin to the cell.