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In recent years modified bacteriophage lysins have widely been investigated for the purposes of development of antibacterial therapy. Thus, effective and precise methods for the quantitative analysis of these enzymes are in high demand. The enzyme-linked immunosorbent assay (ELISA) method has been developed for the detection of recombinant modified endolysin LysAm24-SMAP in biological samples. The optimal parameters for protein detection were determined, in particular, the influence of salt and the composition of the buffer system for preparation of the samples was studied. The applicability of the immunodetection system of the genetically engineered endolysin LysAm24-SMAP in various biological samples with enzyme concentrations from 0.4 ng/mL was demonstrated. In addition, the influence of matrix effects in samples of animal organs and tissue homogenates and producer strain lysates and their individual components during the analysis was assessed and it was shown that 0.65 M NaCl addition in the ELISA buffer is crucial for achieving correct results and reduces nonspecific interactions in the case of LysAm24-SMAP. The effectiveness of the developed system in the immunochemical control of the bacteriolytic enzyme was confirmed. [ABSTRACT FROM AUTHOR]

The bactericidal activity of recombinant endolysins LysECD7, LysAm24, LysAp22, LysSi3 and LysSt11 was assayed in multidrug resistant strains (n=120) of Salmonella enterica, E. coli, Acinetobacter baumannii, Enterobacter spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, and Campylobacter jejuni. The assay showed that the recombinant endolysins had a wide spectrum of bactericidal activity compared to endolysins of their progenitor phages. Among examined endolysins, we selected the active pharmaceutical substances with broad spectrum of bactericidal activity. Most strains were sensitive to LysECD7 (70.7%), LysAm24 (65%), and LysAp22 (58.6%), which seems to be promising causative agents for the development of finished dosage form. [ABSTRACT FROM AUTHOR]

Activity of the telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase) can be regulated by alternative splicing of its mRNA. At the present time, the exact mechanism of hTERT splicing is not fully understood. Apoptotic endonuclease EndoG is known to participate this process. EndoG expression is induced by DNA damages. The aim of this work was to investigate the ability of DNA-damaging agents with different mechanism of action to induce EndoG expression and to inhibit telomerase activity due to the activation of hTERT alternative splicing in normal activated human CD4+ and CD8+ Т lymphocytes. All investigated DNA-damaging agents were able to induce EndoG expression. Cisplatin, a therapeutic compound, producing DNA cross-links induced the highest level of DNA damages and EndoG expression. Incubation of CD4+ and CD8+ Т cells with cisplatin caused the changes in proportion of hTERT splice variants and inhibition of telomerase activity. [ABSTRACT FROM AUTHOR]

The avidity index (AI) of IgG to the RBD of SARS-CoV-2 was determined for 71 patients with a mild (outpatient) course of COVID-19, including 39 primarily and 36 secondarily reinfected, and 92 patients with a severe (hospital) course of COVID-19, including 82 primarily and 10 secondarily infected. The AI was shown to correlate with the severity of repeated disease. In the group of outpatients with a mild course, the reinfected patients had significantly higher median AIs than those with primary infections (82.3% vs. 37.1%, p < 0.0001). At the same time, in patients with a severe course of COVID-19, reinfected patients still had low-avidity antibodies (median AI of 28.4% vs. 25% in the primarily infected, difference not significant, p > 0.05). This suggests that the presence of low-avidity IgG to RBD during reinfection is a negative prognostic factor, in which a patient’s risk of developing COVID-19 in a severe form is significantly increased. Thus, patients with IgG of low avidity (AI ≤ 40%) had an 89 ± 20.5% chance of a severe course of recurrent COVID-19, whereas the detection of high-avidity antibodies (AI ≥ 50%) gave a probability of 94 ± 7.9% for a mild course of recurrent disease (p < 0.05).

Background: First vaccines for prevention of Coronavirus disease 2019 (COVID-19) are becoming available but there is a huge and unmet need for specific forms of treatment. In this study we aimed to evaluate the anti-SARS-CoV-2 effect of siRNA both in vitro and in vivo., Methods: To identify the most effective molecule out of a panel of 15 in silico designed siRNAs, an in vitro screening system based on vectors expressing SARS-CoV-2 genes fused with the firefly luciferase reporter gene and SARS-CoV-2-infected cells was used. The most potent siRNA, siR-7, was modified by Locked nucleic acids (LNAs) to obtain siR-7-EM with increased stability and was formulated with the peptide dendrimer KK-46 for enhancing cellular uptake to allow topical application by inhalation of the final formulation - siR-7-EM/KK-46. Using the Syrian Hamster model for SARS-CoV-2 infection the antiviral capacity of siR-7-EM/KK-46 complex was evaluated., Results: We identified the siRNA, siR-7, targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) as the most efficient siRNA inhibiting viral replication in vitro. Moreover, we showed that LNA-modification and complexation with the designed peptide dendrimer enhanced the antiviral capacity of siR-7 in vitro. We demonstrated significant reduction of virus titer and lung inflammation in animals exposed to inhalation of siR-7-EM/KK-46 in vivo., Conclusions: Thus, we developed a therapeutic strategy for COVID-19 based on inhalation of a modified siRNA-peptide dendrimer formulation. The developed medication is intended for inhalation treatment of COVID-19 patients., (© 2021 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

In the present article, we report data on the whole-genome sequencing of wood-rotting (white-rot) fungus Steccherinum ochraceum LE-BIN 3174. The S. ochraceum LE-BIN 3174 genome consists of 770 scaffolds (N50 = 62,812 bp) with the total length of assembly ∼35 Mb. The structural annotation of the genome resulted in the prediction of 12,441 gene models, among which 181 were models of tRNA-coding genes, and 12,260 - protein-coding genes. The protein-coding genes were annotated with different databases (Pfam, InterPro, eggNOG, dbCAN, and MEROPS). The whole genome sequence and functional annotation provide an important information for the deep investigation of biochemical processes that take place during the late stages of wood decomposition by Basidiomycetes. The Whole Genome project of S. ochraceum LE-BIN 3174 had been deposited at DDBJ/ENA/GenBank under the accession RWJN00000000. The version described in this work is version RWJN00000000.1. For further interpretation of the data provided in this article, please refer to the research article "Fungal Adaptation to the Advanced Stages of Wood Decomposition: Insights from the Steccherinum ochraceum " [1]., (© 2020 The Author(s).)

European seabass (Dicentrarchus labrax, Linnaeus, 1758) (L) and gilthead seabream (Sparus aurata, Linnaeus, 1758) (C) muscles were hydrolysated by Alcalase (L alc , C alc ) and Chymotrypsin (L ch , C ch ) then hydrolysates were examined and their peptide profiles obtained. A total of 765, 794, 132 and 232 peptides were identified in C alc , L alc , C ch and L ch , respectively. Although, L ch and C ch were expected to have more antioxidant capacity because of their peptide profiles, Alcalase hydrolysates observed in vitro, were slightly higher (TEAC assay for C alc : 848.11 ± 60.78 μmol TE/g protein). Maximum inhibition of oxidative stress was determined for L alc (12.8% ± 4.5%) in MDCK1 cell lines. Highest proliferative capacity observed for C alc (147.0% ± 3.1%) at MTT assay in MDCK1 cell culture. L ch showed the highest chemopreventive effect with a 40-60% decrease for human colon adenocarcinoma cell line HT-29. This research points out the importance of aquatic sources as raw materials for peptide researches., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Activity of telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase) can be regulated by alternative splicing of its mRNA. At present time exact mechanism of hTERT splicing is not fully understood. Apoptotic endonuclease EndoG is known to participate this process. EndoG expression is induced by DNA damages. The aim of this work was to investigate the ability of DNA-damaging agents with different mechanism of action to induce EndoG expression and inhibit telomerase activity due to the activation of hTERT alternative splicing in normal activated human CD4+ and CD8+ T-lymphocytes. All investigated DNA-damaging agents were able to induce EndoG expression. Cisplatin, a therapeutic compound, producing DNA cross-links induced the highest level of DNA damages and EndoG expression. Incubation of CD4+ and CD8+ T-cells with cisplatin caused the changes in proportion of hTERT splice variants and inhibition of telomerase activity.

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.

Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of b-deletion splice variant was determined during EndoG over-expression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei and after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.

The consistent application of homogenization and enzymatic treatment is required to obtain protoplasts from the basidiomycete fungus Trametes hirsuta. The maximum yield of protoplasts (∼2.5 × 107/mL) was achieved when mycelium in the exponential growth phase (60 h) was used. The maximum stability was observed in MES+ buffer during 4 h of incubation; in this case the titer reduction was 5–7%. Studies of the effect of antioxidants with different antioxidant capacities expressed in mmol equivalents of Trolox (ascorbate, 0.99; α-tocopherol, 1.0; β-carotene, 2.14; quercetin, 3.98) indicated that the yield of protoplasts was increased in the presence of β-carotene and quercetin by 18–24%. The studied antioxidants did not affect the protoplasts stability. The degree of regeneration of protoplasts correlated with the antioxidant capacity of the studied antioxidants and was maximal (0.4%) in the presence of β-carotene and quercetin; it was 0.1% in the presence of MES+. The rate of protoplast growth was two times higher in the presence of β-carotene and quercetin.

Telomerase activity is known to be regulated by alternative splicing of its catalytic subunit hTERT (human Telomerase Reverse Transcriptase) mRNA. Induction of non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. Strong correlation was found between expression of EndoG and hTERT splice-variants in 12 colon cancer cell lines. Overexpression of EndoG in СаСо-2 cells downregulated the expression of active full-length hTERT variant and upregulated non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, dramatically shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. These data indicated the participation of EndoG in alternative splicing of mRNA of telomerase catalytic subunit, regulation of telomerase activity and cell fate.

There were investigated morphofunctional indices of liver and kidney in male outbred rats in the dynamics of the 6-months consumption of water after its noncontact activation. There were studied 4 experimental groups of animals consumed waters named as "Anolyte" and in dependence on the activation time, 3 types of catholyte water ("Catholyte--5", "Catholyte--25", "Catholyte--40"). Moscow tap water settled for a week served as control. "Anolyte" water was found to increase in the kidney the number of hypertrophied gromeruli only in 6 months, while the consumption of "Catholyte--25" water and especially, "Catholyte--40" in 1 and 6 months caused the damage of liver and kidney, and for the index of alteration of renal glomeruli after 6 months of water consumption there was revealed the dependence on the activation time of "Catalytes".

A comparative analysis of transcripts from the basidiomycota T. hirsuta grown with and without an inducer of the laccase biosynthesis was carried out. Methods of subtraction hybridization and massive parallel sequencing were used for this purpose. Unique transcripts encoded by genes that have a relatively high level of expression and belong to different gene ontology categories were identified. Also, a large number of transcripts were found to encode for predicted proteins, as well as noncoding transcripts. The latter may represent regulatory RNA molecules. Transcripts that increase their abundance when the laccase synthesis is induced are selected as gene-candidates involved in the laccase biosynthetic pathway.