Transcriptomic analysis of HEK293A cells with a CRISPR/Cas9-mediated TDP1 knockout.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a human DNA repair protein. It is a member of the phospholipase D family based on structural similarity. TDP1 is a key enzyme of the repair of stalled topoisomerase 1 (TOP1)-DNA complexes. Previously, with the CRISPR/Cas9 method, we obtained HEK293A cells with a homozygous knockout of the TDP1 gene and used the TDP1 knockout cells as a cellular model for studying mechanisms of action of an anticancer therapy. In the present work, we hypothesized that the TDP1 knockout would alter the expression of DNA repair-related genes. By transcriptomic analysis, we investigated for the first time the effect of the TDP1 gene knockout on genes' expression changes in the human HEK293A cell line. We obtained original data implying a role of TDP1 in other processes besides the repair of the DNA-TOP1 complex. Differentially expressed gene analysis revealed that TDP1 may participate in cell adhesion and communication, spermatogenesis, mitochondrial function, neurodegeneration, a cytokine response, and the MAPK signaling pathway.

Genetic Diversity of the Human Adenovirus C Isolated from Hospitalized Children in Russia (2019-2022).

The human adenovirus (HAdV) is a common pathogen in children that can cause acute respiratory virus infection (ARVI). However, the molecular epidemiological and clinical information relating to HAdV among hospitalized children with ARVI is rarely reported in Russia. A 4-year longitudinal (2019-2022) study among hospitalized children (0-17 years old) with ARVI in Novosibirsk, Russia, was conducted to evaluate the epidemiological and molecular characteristics of HAdV. Statistically significant differences in the detection rates of epidemiological and virological data of all positive viral detections of HAdV were analyzed using a two-tailed Chi-square test. The incidence of HAdV and other respiratory viruses such as human influenza A and B viruses, respiratory syncytial virus, coronavirus, parainfluenza virus, metapneumovirus, rhinovirus, bocavirus, and SARS-CoV-2 was investigated among 3190 hospitalized children using real-time polymerase chain reaction. At least one of these respiratory viruses was detected in 74.4% of hospitalized cases, among which HAdV accounted for 4%. A total of 1.3% co-infections with HAdV were also registered. We obtained full-genome sequences of 12 HAdVs, which were isolated in cell cultures. Genetic analysis revealed the circulation of adenovirus of genotypes C1, C2, C5, C89, and 108 among hospitalized children in the period from 2019-2022.

Deficiency of the ribosomal protein uS10 (RPS20) reorganizes human cells translatome according to the abundance, CDS length and GC content of mRNAs.

Ribosomal protein uS10, a product of the RPS20 gene, is an essential constituent of the small (40S) subunit of the human ribosome. Disruptive mutations in its gene are associated with a predisposition to hereditary colorectal carcinoma. Here, using HEK293T cells, we show that a deficiency of this protein leads to a decrease in the level of ribosomes (ribosomal shortage). RNA sequencing of the total and polysome-associated mRNA samples reveals hundreds of genes differentially expressed in the transcriptome (t)DEGs and translatome (p)DEGs under conditions of uS10 deficiency. We demonstrate that the (t)DEG and (p)DEG sets partially overlap, determine genes with altered translational efficiency (TE) and identify cellular processes affected by uS10 deficiency-induced ribosomal shortage. We reveal that translated mRNAs of upregulated (p)DEGs and genes with altered TE in uS10-deficient cells are generally more abundant and that their GC contents are significantly lower than those of the respective downregulated sets. We also observed that upregulated (p)DEGs have longer coding sequences. Based on our findings, we propose a combinatorial model describing the process of reorganization of mRNA translation under conditions of ribosomal shortage. Our results reveal rules according to which ribosomal shortage reorganizes the transcriptome and translatome repertoires of actively proliferating cells.

Bacillus subtilis and Bacillus amyloliquefaciens Mix Suppresses Rhizoctonia Disease and Improves Rhizosphere Microbiome, Growth and Yield of Potato (Solanum tuberosum L.).

Black scurf and stem canker caused by Rhizoctonia solani is a significant disease problem of potatoes. Currently, chemical methods are the primary means of controlling this pathogen. This study sought to explore an alternative approach by harnessing the biocontrol potential of a bacterial mix of Bacillus subtilis and Bacillus amyloliquefaciens against black scurf, and to determine their effect on rhizosphere microorganisms of soil microbiota. This study showed that these bacteria demonstrate antagonistic activity against Rhizoctonia solani. Reduced damage to potato plants during the growing season in Siberia was observed. The index of disease development decreased from 40.9% to 12.0%. The treatment of tubers with this mix of bacteria also led to a change in the composition of the rhizosphere microbiota (according to CFU, 16S and ITS sequencing). This effect was accompanied by a positive change in plant physiological parameters (spectrophotometric analysis). The concentration of chlorophyll in potatoes with the bacterial mix treatment increased by 1.3 fold (p ≤ 0.001), and of carotenoids by 1.2 fold (p ≤ 0.01) compared with the control. After bacterial mix treatment, the length of the aerial parts of plants was 1.3 fold higher (p ≤ 0.001), and the number of stems 1.4 fold higher (p ≤ 0.05). The yield of potatoes was increased by 8.2 t/ha, while the large tuber fraction was increased by 16% (p ≤ 0.05). The bacteria mix of Bacillus subtilis and Bacillus amyloliquefaciens suppressed the plant pathogenic fungus Rhizoctonia solani, and simultaneously enhanced the physiological parameters of potato plants. This treatment can be used to enhance the yield/quality of potato tubers under field conditions.

A Diet with Amikacin Changes the Bacteriobiome and the Physiological State of Galleria mellonella and Causes Its Resistance to Bacillus thuringiensis.

Environmental pollution with antibiotics can cause antibiotic resistance in microorganisms, including the intestinal microbiota of various insects. The effects of low-dose aminoglycoside antibiotic (amikacin) on the resident gut microbiota of Galleria mellonella, its digestion, its physiological parameters, and the resistance of this species to bacteria Bacillus thuringiensis were investigated. Here, 16S rDNA analysis revealed that the number of non-dominant Enterococcus mundtii bacteria in the eighteenth generation of the wax moth treated with amikacin was increased 73 fold compared to E. faecalis, the dominant bacteria in the native line of the wax moth. These changes were accompanied by increased activity of acidic protease and glutathione-S-transferase in the midgut tissues of larvae. Ultra-thin section electron microscopy detected no changes in the structure of the midgut tissues. In addition, reduced pupa weight and resistance of larvae to B. thuringiensis were observed in the eighteenth generation of the wax moth reared on a diet with amikacin. We suggest that long-term cultivation of wax moth larvae on an artificial diet with an antibiotic leads to its adaptation due to changes in both the gut microbiota community and the physiological state of the insect organism.

Links between Soil Bacteriobiomes and Fungistasis toward Fungi Infecting the Colorado Potato Beetle.

Entomopathogenic fungi can be inhibited by different soil microorganisms, but the effect of a soil microbiota on fungal growth, survival, and infectivity toward insects is insufficiently understood. We investigated the level of fungistasis toward Metarhizium robertsii and Beauveria bassiana in soils of conventional potato fields and kitchen potato gardens. Agar diffusion methods, 16S rDNA metabarcoding, bacterial DNA quantification, and assays of Leptinotarsa decemlineata survival in soils inoculated with fungal conidia were used. Soils of kitchen gardens showed stronger fungistasis toward M. robertsii and B. bassiana and at the same time the highest density of the fungi compared to soils of conventional fields. The fungistasis level depended on the quantity of bacterial DNA and relative abundance of Bacillus, Streptomyces, and some Proteobacteria, whose abundance levels were the highest in kitchen garden soils. Cultivable isolates of bacilli exhibited antagonism to both fungi in vitro. Assays involving inoculation of nonsterile soils with B. bassiana conidia showed trends toward elevated mortality of L. decemlineata in highly fungistatic soils compared to low-fungistasis ones. Introduction of antagonistic bacilli into sterile soil did not significantly change infectivity of B. bassiana toward the insect. The results support the idea that entomopathogenic fungi can infect insects within a hypogean habitat despite high abundance and diversity of soil antagonistic bacteria.

Mutation at the Site of Hydroxylation in the Ribosomal Protein uL15 (RPL27a) Causes Specific Changes in the Repertoire of mRNAs Translated in Mammalian Cells.

Ribosomal protein uL15 (RPL27a) carries a specific modification, hydroxylation, at the His39 residue, which neighbors the CCA terminus of the E-site-bound tRNA at the mammalian ribosome. Under hypoxia, the level of hydroxylation of this protein decreases. We transiently transfected HEK293T cells with constructs expressing wild-type uL15 or mutated uL15 (His39Ala) incapable of hydroxylation, and demonstrated that ribosomes containing both proteins are competent in translation. By applying RNA-seq to the total cellular and polysome-associated mRNAs, we identified differentially expressed genes (DEGs) in cells containing exogenous uL15 or its mutant form. Analyzing mRNA features of up- and down-regulated DEGs, we found an increase in the level of more abundant mRNAs and shorter CDSs in cells with uL15 mutant for both translated and total cellular mRNAs. The level of longer and rarer mRNAs, on the contrary, decreased. Our data show how ribosome heterogeneity can change the composition of the translatome and transcriptome, depending on the properties of the translated mRNAs.

Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.

Soil Microbiome in Conditions of Oil Pollution of Subarctic Ecosystems.

The present study aimed to investigate the recovery of soil quality and the bacterial and fungal communities following various recultivation methods in areas contaminated with oil. Oil spills are known to have severe impacts on ecosystems; thus, the restoration of contaminated soils has become a significant challenge nowadays. The study was conducted in the forest-tundra zone of the European North-East, where 39 soil samples from five oil-contaminated sites and reference sites were subjected to metagenomic analyses. The contaminated sites were treated with different biopreparations, and the recovery of soil quality and microbial communities were analyzed. The analysis of bacteria and fungi communities was carried out using 16S rDNA and ITS metabarcoding. It was found that 68% of bacterial OTUs and 64% of fungal OTUs were unique to the reference plot and not registered in any of the recultivated plots. However, the species diversity of recultivated sites was similar, with 50-80% of bacterial OTUs and 44-60% of fungal OTUs being common to all sites. New data obtained through soil metabarcoding confirm our earlier conclusions about the effectiveness of using biopreparations with indigenous oil-oxidizing micro-organisms also with mineral fertilizers, and herbaceous plant seeds for soil remediation. It is possible that the characteristics of microbial communities will be informative in the bioindication of soils reclaimed after oil pollution.

Flow-Seq Evaluation of Translation Driven by a Set of Natural Escherichia coli 5'-UTR of Variable Length.

Flow-seq is a method that combines fluorescently activated cell sorting and next-generation sequencing to deduce a large amount of data about translation efficiency from a single experiment. Here, we constructed a library of fluorescent protein-based reporters preceded by a set of 648 natural 5'-untranslated regions (5'-UTRs) of Escherichia coli genes. Usually, Flow-seq libraries are constructed using uniform-length sequence elements, in contrast to natural situations, where functional elements are of heterogenous lengths. Here, we demonstrated that a 5'-UTR library of variable length could be created and analyzed with Flow-seq. In line with previous Flow-seq experiments with randomized 5'-UTRs, we observed the influence of an RNA secondary structure and Shine-Dalgarno sequences on translation efficiency; however, the variability of these parameters for natural 5'-UTRs in our library was smaller in comparison with randomized libraries. In line with this, we only observed a 30-fold difference in translation efficiency between the best and worst bins sorted with this factor. The results correlated with those obtained with ribosome profiling.

Pectobacterium versatile Bacteriophage Possum: A Complex Polysaccharide-Deacetylating Tail Fiber as a Tool for Host Recognition in Pectobacterial Schitoviridae.

Novel, closely related phages Possum and Horatius infect Pectobacterium versatile, a phytopathogen causing soft rot in potatoes and other essential plants. Their properties and genomic composition define them as N4-like bacteriophages of the genus Cbunavirus, a part of a recently formed family Schitoviridae. It is proposed that the adsorption apparatus of these phages consists of tail fibers connected to the virion through an adapter protein. Tail fibers possess an enzymatic domain. Phage Possum uses it to deacetylate O-polysaccharide on the surface of the host strain to provide viral attachment. Such an infection mechanism is supposed to be common for all Cbunavirus phages and this feature should be considered when designing cocktails for phage control of soft rot.

Deconvolution of B cell receptor repertoire in multiple sclerosis patients revealed a delay in tBreg maturation.

Background: B lymphocytes play a pivotal regulatory role in the development of the immune response. It was previously shown that deficiency in B regulatory cells (Bregs) or a decrease in their anti-inflammatory activity can lead to immunological dysfunctions. However, the exact mechanisms of Bregs development and functioning are only partially resolved. For instance, only a little is known about the structure of their B cell receptor (BCR) repertoires in autoimmune disorders, including multiple sclerosis (MS), a severe neuroinflammatory disease with a yet unknown etiology. Here, we elucidate specific properties of B regulatory cells in MS. Methods: We performed a prospective study of the transitional Breg (tBreg) subpopulations with the CD19+CD24highCD38high phenotype from MS patients and healthy donors by (i) measuring their content during two diverging courses of relapsing-remitting MS: benign multiple sclerosis (BMS) and highly active multiple sclerosis (HAMS); (ii) analyzing BCR repertoires of circulating B cells by high-throughput sequencing; and (iii) measuring the percentage of CD27+ cells in tBregs. Results: The tBregs from HAMS patients carry the heavy chain with a lower amount of hypermutations than tBregs from healthy donors. The percentage of transitional CD24highCD38high B cells is elevated, whereas the frequency of differentiated CD27+ cells in this transitional B cell subset was decreased in the MS patients as compared with healthy donors. Conclusions: Impaired maturation of regulatory B cells is associated with MS progression.

Reorganization of the Landscape of Translated mRNAs in NSUN2-Deficient Cells and Specific Features of NSUN2 Target mRNAs.

The RNA cytosine C5 methyltransferase NSUN2 has a variety of RNA substrates and plays an important role in mRNA metabolism. NSUN2 binds to specific sequences enriched in exosomal mRNAs, suggesting its possible involvement in the sorting of mRNAs into exosomes. We applied the photoactivatable.4-thiouridine-enhanced cross-linking and immunoprecipitation assay involving high-throughput RNA sequencing (RNA-seq) to HEK293T cells to determine NSUN2 mRNA targets. NSUN2 cross-linking sites were found in more than one hundred relatively abundant mRNAs with a high GC content and a pronounced secondary structure. Then, utilizing RNA-seq for the total and polysome-associated mRNA from HEK293T cells with and without the knockdown of NSUN2, we identified differentially expressed genes, as well as genes with altered translational efficiency (GATEs). It turned out that the up-regulated GATE mRNAs were much shorter on average than the down-regulated ones, and their GC content was higher; moreover, they contained motifs with C residues located in GC-rich environments. Our findings reveal the specific features of mRNAs that make them potential targets for NSUN2 and expand our understanding of the role of NSUN2 in controlling translation and, possibly, in mRNA sorting into exosomes implemented through the methylation of cytosine residues.

Changes in the Transcriptome Caused by Mutations in the Ribosomal Protein uS10 Associated with a Predisposition to Colorectal Cancer.

A number of mutations in the RPS20 gene encoding the ribosomal protein uS10 have been found to be associated with a predisposition to hereditary non-polyposis colorectal carcinoma (CRC). We transfected HEK293T cells with constructs carrying the uS10 minigene with mutations identical to those mentioned above and examined the effects of the produced proteins on the cellular transcriptome. We showed that uS10 with mutations p.V50SfsX23 or p.L61EfsX11 cannot be incorporated into 40S ribosomal subunits, while the protein with the missense mutation p.V54L functionally replaces the respective endogenous protein in the 40S subunit assembly and the translation process. The comparison of RNA-seq data obtained from cells producing aberrant forms of uS10 with data for those producing the wild-type protein revealed overlapping sets of upregulated and downregulated differently expressed genes (DEGs) related to several pathways. Among the limited number of upregulated DEGs, there were genes directly associated with the progression of CRC, e.g., PPM1D and PIGN. Our findings indicate that the accumulation of the mutant forms of uS10 triggers a cascade of cellular events, similar to that which is triggered when the cell responds to a large number of erroneous proteins, suggesting that this may increase the risk of cancer.

Complete Genome of Sinorhizobium meliloti AK76, a Symbiont of Wild Diploid Medicago lupulina from the Mugodgary Mountain Region.

Sinorhizobium meliloti is a symbiotic bacterial species forming nitrogen-fixing nodules on roots of annual and perennial Medicago spp. We report the full genome sequence of S. meliloti strain AK76, an effective symbiont of the wild diploid plant Medicago lupulina grown in the Mugodgary Mountain region, Kazakhstan.

Deficiency of the Ribosomal Protein uL5 Leads to Significant Rearrangements of the Transcriptional and Translational Landscapes in Mammalian Cells.

Protein uL5 (formerly called L11) is an integral component of the large (60S) subunit of the human ribosome, and its deficiency in cells leads to the impaired biogenesis of 60S subunits. Using RNA interference, we reduced the level of uL5 in HEK293T cells by three times, which caused an almost proportional decrease in the content of the fraction corresponding to 80S ribosomes, without a noticeable diminution in the level of polysomes. By RNA sequencing of uL5-deficient and control cell samples, which were those of total mRNA and mRNA from the polysome fraction, we identified hundreds of differentially expressed genes (DEGs) at the transcriptome and translatome levels and revealed dozens of genes with altered translational efficiency (GATEs). Transcriptionally up-regulated DEGs were mainly associated with rRNA processing, pre-mRNA splicing, translation and DNA repair, while down-regulated DEGs were genes of membrane proteins; the type of regulation depended on the GC content in the 3' untranslated regions of DEG mRNAs. The belonging of GATEs to up-regulated and down-regulated ones was determined by the coding sequence length of their mRNAs. Our findings suggest that the effects observed in uL5-deficient cells result from an insufficiency of translationally active ribosomes caused by a deficiency of 60S subunits.

Gene Analysis, Cloning, and Heterologous Expression of Protease from a Micromycete Aspergillus ochraceus Capable of Activating Protein C of Blood Plasma.

Micromycetes are known to secrete numerous enzymes of biotechnological and medical potential. Fibrinolytic protease-activator of protein C (PAPC) of blood plasma from micromycete Aspergillus ochraceus VKM-F4104D was obtained in recombinant form utilising the bacterial expression system. This enzyme, which belongs to the proteinase-K-like proteases, is similar to the proteases encoded in the genomes of Aspergillus fumigatus ATCC MYA-4609, A. oryzae ATCC 42149 and A. flavus 28. Mature PAPC-4104 is 282 amino acids long, preceded by the 101-amino acid propeptide necessary for proper folding and maturation. The recombinant protease was identical to the native enzyme from micromycete in terms of its biological properties, including an ability to hydrolyse substrates of activated protein C (pGlu-Pro-Arg-pNA) and factor Xa (Z-D-Arg-Gly-Arg-pNA) in conjugant reactions with human blood plasma. Therefore, recombinant PAPC-4104 can potentially be used in medicine, veterinary science, diagnostics, and other applications.

Can Potato Plants Be Colonized with the Fungi Metarhizium and Beauveria under Their Natural Load in Agrosystems?

Beauveria and Metarhizium fungi are facultative plant endophytes that provide plant growth-stimulating, immunomodulatory, and other beneficial effects. However, little is known about the level of plant colonization by these fungi under natural conditions. We assessed the endophytic colonization of potatoes (Solanum tuberosum) with entomopathogenic fungi at their natural load in soils (102-104 colony-forming units per g). Microbiological analyses of soils and plant organs, as well as a metagenomic analysis of potato roots and leaves, were conducted in three locations in Western Siberia, consisting of conventional agrosystems and kitchen gardens. The fungi were isolated at a relatively high frequency from unsterilized roots (up to 53% of Metarhizium-positive plants). However, the fungi were sparsely isolated from the internal tissues of roots, stems, and leaves (3%). Among the genus Metarhizium, two species, M. robertsii and M. brunneum, were detected in plants as well as in soils, and the first species was predominant. A metagenomic analysis of internal potato tissues showed a low relative abundance of Beauveria and Metarhizium (<0.3%), and the communities were represented primarily by phytopathogens. We suggest that colonization of the internal tissues of potatoes occurs sporadically under a natural load of entomopathogenic fungi in soils. The lack of stable colonization of potato plants with Beauveria and Metarhizium may be due to competition with phytopathogens.

Knockdown of the Ribosomal Protein eL38 in HEK293 Cells Changes the Translational Efficiency of Specific Genes.

The protein eL38 is one of the smallest proteins of the mammalian ribosome, which is a component of its large (60S) subunit. The haploinsufficiency of eL38 in mice leads to the Tail-short mutant phenotype characterized by defects in the development of the axial skeleton caused by the poor translation of mRNA subsets of Hox genes. Using the ribosome profiling assay applied to HEK293 cells knocked down of eL38, we examined the effects of the lack of eL38 in 60S subunits on gene expression at the level of translation. A four-fold decrease in the cell content of eL38 was shown to result in significant changes in the translational efficiencies of 150 genes. Among the genes, whose expression at the level of translation was enhanced, there were mainly those associated with basic metabolic processes; namely, translation, protein folding, chromosome organization, splicing, and others. The set of genes with reduced translation efficiencies contained those that are mostly involved in the processes related to the regulation of transcription, including the activation of Hox genes. Thus, we demonstrated that eL38 insufficiency significantly affects the expression of certain genes at the translational level. Our findings facilitate understanding the possible causes of some anomalies in eL38-deficient animals.

Extended Evaluation of Viral Diversity in Lake Baikal through Metagenomics.

Lake Baikal is a unique oligotrophic freshwater lake with unusually cold conditions and amazing biological diversity. Studies of the lake's viral communities have begun recently, and their full diversity is not elucidated yet. Here, we performed DNA viral metagenomic analysis on integral samples from four different deep-water and shallow stations of the southern and central basins of the lake. There was a strict distinction of viral communities in areas with different environmental conditions. Comparative analysis with other freshwater lakes revealed the highest similarity of Baikal viromes with those of the Asian lakes Soyang and Biwa. Analysis of new data, together with previously published data allowed us to get a deeper insight into the diversity and functional potential of Baikal viruses; however, the true diversity of Baikal viruses in the lake ecosystem remains still unknown. The new metaviromic data will be useful for future studies of viral composition, distribution, and the dynamics associated with global climatic and anthropogenic impacts on this ecosystem.