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New personnel management systems are being built in the changing socio-economic conditions. Scientific and methodological approaches that make it possible to perform in-depth research on the labor opportunities of employees are developed. The article deals with the cultural aspects of the formation of competitive advantages of the employee in the conditions of the intersection of cultures. Representation of labor potential as a socio-economic category that reflects the ability of personnel realize the goals of the enterprise is given. The provision according to which competitive advantages are a means of improving the quality of relations in the labor sphere on the basis of labor potential, considering the cultural aspects of the competitive advantages of employees is substantiated. In what way it is possible to identify the content of the components of the labor potential of workers using the tools of the competence-based approach it is shown.

Herein, we report the synthesis, structure-activity relationship study, and biological evaluation of neurosteroid inhibitors of N-methyl-D-aspartate receptors (NMDARs) receptors that employ an amide structural motif, relative to pregnanolone glutamate (PAG) – a compound with neuroprotective properties. All compounds were found to be more potent NMDAR inhibitors (IC50 values varying from 1.4 to 21.7 μM) than PAG (IC50 = 51.7 μM). Selected compound 6 was evaluated for its NMDAR subtype selectivity and its ability to inhibit AMPAR/GABAR responses. Compound 6 inhibits the NMDARs (8.3 receptors (8.3 ± 2.1 μM) more strongly than it does at the GABAR and AMPARs (17.0 receptors (17.0 ± 0.2 μM and 276.4 ± 178.7 μM, respectively). In addition, compound 6 (10 μM) decreases the frequency of action potentials recorded in cultured hippocampal neurons. Next, compounds 3, 5–7, 9, and 10 were not associated with mitotoxicity, hepatotoxicity nor ROS induction. Lastly, we were able to show that all compounds have improved rat and human plasma stability over PAG.

The use of CRISPR/Cas nucleases for the development of DNA diagnostic systems in out-of-laboratory conditions (point-of-need testing, PONT) has demonstrated rapid growth in the last few years, starting with the appearance in 2017-2018 of the first diagnostic platforms known as DETECTR and SHERLOCK. The platforms are based on a combination of methods of nucleic acid isothermal amplification with selective CRISPR/Cas detection of target amplicons. This significantly improves the sensitivity and specificity of PONT, making them comparable with or even superior to the sensitivity and specificity of polymerase chain reaction, considered as the "gold standard" of DNA diagnostics. The review considers modern approaches to the coupling of CRISPR/Cas detection using Cas9, Cas12a, Cas12b, Cas13a, Cas14, and Cas3 nucleases to various methods of nucleic acid isothermal amplification, with an emphasis on works in which sensitivity at the level of single molecules (attomolar and subattomolar concentrations of the target) is achieved. The properties of CRISPR/Cas nucleases used for targeted DNA diagnostics and the features of methods of nucleic acid isothermal amplification are briefly considered in the context of the development of diagnostic biosensing platforms. Special attention is paid to the most promising directions for the development of DNA diagnostics using CRISPR/Cas nuclease.

Genitourinary cancer (GUC) represents more than one fifth of all human cancers. This makes the development of approaches to its early diagnosis an important task of modern biomedicine. Circulating microRNAs, short (17-25 nucleotides) non-coding RNA molecules found in human biological fluids and performing a regulatory role in the cell, are considered as promising diagnostic and prognostic biomarkers of cancers, including GUC. In this review we have considered the current state of research aimed at assessing microRNAs as biomarkers of such human GUC types as malignant tumors of the bladder, kidney, prostate, testicles, ovaries, and cervix. A special attention has been paid to studies devoted to the identification of microRNAs in urine as a surrogate "liquid biopsy" that may provide the simplest and cheapest approach to mass non-invasive screening of human GUC. The use of microRNA panels instead of single types of microRNA generally leads to higher sensitivity and specificity of the developed diagnostic tests. However, to date, work on the microRNAs assessment as biomarkers of human GUC is still of a research nature, and the further introduction of diagnostic tests based on microRNAs into practice requires successful clinical trials.

The long-read RNA sequencing developed by Oxford Nanopore Technologies provides a direct quantification of transcript isoforms, thereby making it possible to present alternative splicing (AS) profiles as arrays of single splice variants with different abundances. Additionally, AS profiles can be presented as arrays of genes characterized by the degree of alternative splicing (the DAS-the number of detected splice variants per gene). Here, we successfully utilized the DAS to reveal biological pathways influenced by the alterations in AS in human liver tissue and the hepatocyte-derived malignant cell lines HepG2 and Huh7, thus employing the mathematical algorithm of gene set enrichment analysis. Furthermore, analysis of the AS profiles as abundances of single splice variants by using the graded tissue specificity index τ provided the selection of the groups of genes expressing particular splice variants specifically in liver tissue, HepG2 cells, and Huh7 cells. The majority of these splice variants were translated into proteins products and appeal to be in focus regarding further insights into the mechanisms underlying cell malignization. The used metrics are intrinsically suitable for transcriptome-wide AS profiling using long-read sequencing.

The long-read RNA sequencing developed by Oxford Nanopore Technology provides a direct quantification of transcript isoforms. That makes the number of transcript isoforms per gene an intrinsically suitable metric for alternative splicing (AS) profiling in the application to this particular type of RNA sequencing. By using this simple metric and recruiting principal component analysis (PCA) as a tool to visualize the high-dimensional transcriptomic data, we were able to group biospecimens of normal human liver tissue and hepatocyte-derived malignant HepG2 and Huh7 cells into clear clusters in a 2D space. For the transcriptome-wide analysis, the clustering was observed regardless whether all genes were included in analysis or only those expressed in all biospecimens tested. However, in the application to a particular set of genes known as pharmacogenes, which are involved in drug metabolism, the clustering worsened dramatically in the latter case. Based on PCA data, the subsets of genes most contributing to biospecimens' grouping into clusters were selected and subjected to gene ontology analysis that allowed us to determine the top 20 biological processes among which translation and processes related to its regulation dominate. The suggested metrics can be a useful addition to the existing metrics for describing AS profiles, especially in application to transcriptome studies with long-read sequencing.

The analysis of cytochrome P450 transcripts was carried out by the nanopore sequencing in liver tissue samples of three donors and HepG2 line cells. It has been demonstrated that direct mRNA sequencing with a MinION nanopore sequencer (Oxford Nanopore Technologies) allows one to obtained quantitative profiles for transcripts (and their splice variants) of cytochrome P450 superfamily genes encoding isoforms involved in metabolism of the large (~80%) part of drugs. The splice variant profiles substantially differ for donors. The cytochrome P450 gene expression at the transcript level is significantly weaker in cells of the HepG2 line compared with that in the normal liver tissue. This limits the capability of the direct mRNA nanopore sequencing for studying alternative splicing of cytochrome P450 transcripts in HepG2 cells. Both quantitative and qualitative profiles of the cytochrome P450 gene expression at the transcript level are notably differ in human liver tissue and HepG2 cells.

Using human chromosome 18 (Ch18) genes as an example, a PCR analysis of the interindividual variability of gene expression in liver tissue was performed. Although the quantitative profiles of the Ch18 transcriptome, expressed in the number of cDNA copies per single cell, showed a high degree of correlation between donors (Pearson correlation coefficients ranged from 0.963 to 0.966), the expression of the significant number of genes (from 13% to 19%, depending on the method of experimental data normalization) varied by more than 4-fold when comparing donors pairwise. At the same time, the proportion of differentially expressed genes increased with a decrease in the level of their expression. It is shown that the higher quantitative variability of low-abundance transcripts is mainly not technical, but biological. Bioinformatic analysis of the interindividual variability of the differential expression of chromosome 18 genes in human liver tissue did not reveal any statistically significant groups of genes related to certain biological processes that indicated a rather transient nature of the interindividual variability of their expression, probably reflecting the response of cells of an individual to specific external stimuli.

The cutoff level applied in sequencing analysis varies according to the sequencing technology, sample type, and study purpose, which can largely affect the coverage and reliability of the data obtained. In this study, we aimed to determine the optimal combination of parameters for reliable RNA transcriptome data analysis. Toward this end, we compared the results obtained from different transcriptome analysis platforms (quantitative polymerase chain reaction, Illumina RNASeq, and Oxford Nanopore Technologies MinION) for the transcriptome encoded by human chromosome 18 (Chr 18) using the same sample types (HepG2 cells and liver tissue). A total of 275 protein-coding genes encoded by Chr 18 was taken as the gene set for evaluation. The combination of Illumina RNASeq and MinION nanopore technologies enabled the detection of at least one transcript for each protein-coding gene encoded by Chr 18. This combination also reduced the probability of false-positive detection of low-copy transcripts due to the simultaneous confirmation of the presence of a transcript by the two fundamentally different technologies: short reads essential for reliable detection (Illumina RNASeq) and long-read sequencing data (MinION). The combination of these technologies achieved complete coverage of all 275 protein-coding genes on Chr 18, identifying transcripts with non-zero expression levels. This approach can improve distinguishing the biological and technical reasons for the absence of mRNA detection for a given gene in transcriptomics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ilgisonis, Vavilov, Ponomarenko, Lisitsa, Poverennaya, Zgoda, Radko and Archakov.)

The chromosome-centric dataset was created by applying several technologies of transcriptome profiling. The described dataset is available at NCBI repository (BioProject ID PRJNA635536). The dataset referred to the same type of tissue, cell lines, transcriptome sequencing technologies, and was accomplished in a period of 8 years (the first data were obtained in 2013 while the last ones - in 2020). The high-throughput sequencing technologies were employed along with the quantitative PCR (qPCR) approach, for data generation using the gene expression level assessment. qPCR was performed for a limited group of genes, encoded on human chromosome 18, for the Russian part of the Chromosome-Centric Human Proteome Project. The data of high-throughput sequencing are provided as Excel spreadsheets, where the data on FPKM and TMP values were evaluated for the whole transcriptome with both Illumina HiSeq and Oxford Nanopore Technologies MinION sequencing., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article., (© 2021 The Author(s). Published by Elsevier Inc.)

Selected reaction monitoring (SRM) is a mass spectrometric technique characterized by the exceptionally high selectivity and sensitivity of protein detection. However, even with this technique, the quantitative detection of low- and ultralow-abundance proteins in blood plasma, which is of great importance for the search and verification of novel protein disease markers, is a challenging task due to the immense dynamic range of protein abundance levels. One approach used to overcome this problem is the immunoaffinity enrichment of target proteins for SRM analysis, employing monoclonal antibodies. Aptamers appear as a promising alternative to antibodies for affinity enrichment. Here, using recombinant protein SMAD4 as a model target added at known concentrations to human blood plasma and SRM as a detection method, we investigated a relationship between the initial amount of the target protein and its amount in the fraction enriched with SMAD4 by an anti-SMAD4 DNA-aptamer immobilized on magnetic beads. It was found that the aptamer-based enrichment provided a 30-fold increase in the sensitivity of SRM detection of SMAD4. These results indicate that the aptamer-based affinity enrichment of target proteins can be successfully employed to improve quantitative detection of low-abundance proteins by SRM in undepleted human blood plasma.

Using random (combinatorial) DNA-libraries with various degrees of diversity, it was shown that their amplification by polymerase chain reaction in real time resulted in appearance of a maximum on amplification curves. The relative decrease of fluorescence after passing the maximum was directly proportional to the logarithm of the number of oligonucleotide sequence variants in the random DNA-library provided that this number was within in the interval from 1 to 104 and remained practically unaltered when the number of variants was in the interval from 105 to 108. The obtained dependence was used in the course of SELEX to evaluate changes in the diversity of random DNA-libraries from round to round in selection of DNA-aptamers to the recombinant SMAD4 protein. As a result, oligonucleotides containing sequences able to form a site of SMAD4-DNA interactions known as SBE (SMAD-binding element) have been selected thus indicating that the SMAD4-SBE interaction dominates the aptamer selection.

Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Far-upstream element (FUSE) binding protein 1 (FUBP1) was originally identified as a regulator of the oncogene c-Myc via binding to the FUSE within the c-Myc promoter and activating the expression of the gene. Recent studies have identified FUBP1 as a regulator of transcription, translation, and splicing via its DNA and RNA binding activities. Here we report the identification of FUBP1 as a novel binding partner of E1A. FUBP1 binds directly to E1A via the N terminus (residues 1 to 82) and conserved region 3 (residues 139 to 204) of adenovirus 5 E1A. The depletion of FUBP1 via short interfering RNAs (siRNA) reduces virus growth and drives the upregulation of the cellular stress response by activating the expression of p53-regulated genes. During infection, FUBP1 is relocalized within the nucleus, and it is recruited to viral promoters together with E1A while at the same time being lost from the FUSE upstream of the c-Myc promoter. The depletion of FUBP1 affects viral and cellular gene expression. Importantly, in FUBP1-depleted cells, p53-responsive genes are upregulated, p53 occupancy on target promoters is enhanced, and histone H3 lysine 9 is hyperacetylated. This is likely due to the loss of the FUBP1-mediated suppression of p53 DNA binding. We also observed that E1A stabilizes the FUBP1-p53 complex, preventing p53 promoter binding. Together, our results identify, for the first time, FUBP1 as a novel E1A binding protein that participates in aspects of viral replication and is involved in the E1A-mediated suppression of p53 function. IMPORTANCE Viral infection triggers innate cellular defense mechanisms that have evolved to block virus replication. To overcome this, viruses have counterevolved mechanisms that ensure that cellular defenses are either disarmed or not activated to guarantee successful replication. One of the key regulators of cellular stress is the tumor suppressor p53 that responds to a variety of cellular stress stimuli and safeguards the integrity of the genome. During infection, many viruses target the p53 pathway in order to deactivate it. Here we report that human adenovirus 5 coopts the cellular protein FUBP1 to prevent the activation of the p53 stress response pathway that would block viral replication. This finding adds to our understanding of p53 deactivation by adenovirus and highlights its importance in infection and innate immunity., (Copyright © 2018 American Society for Microbiology.)

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of target protein with aptamers immobilized on a solid phase was studied. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as model target protein. It has been demonstrated that the affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads results in an approximately 10-fold increase of the concentration of target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM is possible without an interference from other peptides present in a tryptic digest.

Background: Timely identification of respiratory virus infection is essential to mitigate inappropriate antibiotic use and to implement appropriate treatment and/or infection control procedures. As such, multiplexed PCR assays have become standard in many virology laboratories., Objectives: To compare the Seeplex RV15 (test of record) with two newer generation multiplex assays, the Anyplex II RV16 and the xTAG respiratory virus panels., Study Design: Two hundred and three retrospective and 36 prospective respiratory samples were tested by all three assays. Samples were deemed to be positive if they tested positive for a virus by at least two of the three respective assays. Negative samples also had to test negative by at least two of the three assays. Inconclusive samples were those that showed band signal intensity between 0 and 100 on the RV15, but had not been previously tested on the RV16 or xTAG., Results and Conclusions: Overall sensitivity and specificity of all three assays were similar (∼85% and 100%, respectively). Given each assay can identify multiple different viruses, the targets reported by one assay did not always agree with each target from another assay. Partial discordant rates were 47% and 21% for positive and negative samples, respectively. These higher than expected partial discordant rates may be due to primer or chemistry differences amongst the three multiplex assays., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology.

Suppression of interferon signaling is of paramount importance to a virus. Interferon signaling significantly reduces or halts the ability of a virus to replicate; therefore, viruses have evolved sophisticated mechanisms that suppress activation of the interferon pathway or responsiveness of the infected cell to interferon. Adenovirus has multiple modes of inhibiting the cellular response to interferon. Here, we report that E1A, previously shown to regulate interferon signaling in multiple ways, inhibits interferon-stimulated gene expression by modulating RuvBL1 function. RuvBL1 was previously shown to affect type I interferon signaling. E1A binds to RuvBL1 and is recruited to RuvBL1-regulated promoters in an interferon-dependent manner, preventing their activation. Depletion of RuvBL1 impairs adenovirus growth but does not appear to significantly affect viral protein expression. Although RuvBL1 has been shown to play a role in cell growth, its depletion had no effect on the ability of the virus to replicate its genome or to drive cells into S phase. E1A was found to bind to RuvBL1 via the C terminus of E1A, and this interaction was important for suppression of interferon-stimulated gene transcriptional activation and recruitment of E1A to interferon-regulated promoters. Here, we report the identification of RuvBL1 as a new target for adenovirus in its quest to suppress the interferon response. IMPORTANCE For most viruses, suppression of the interferon signaling pathway is crucial to ensure a successful replicative cycle. Human adenovirus has evolved several different mechanisms that prevent activation of interferon or the ability of the cell to respond to interferon. The viral immediate-early gene E1A was previously shown to affect interferon signaling in several different ways. Here, we report a novel mechanism reliant on RuvBL1 that E1A uses to prevent activation of interferon-stimulated gene expression following infection or interferon treatment. This adds to the growing knowledge of how viruses are able to inhibit interferon and identifies a novel target used by adenovirus for modulation of the cellular interferon pathway., (Copyright © 2017 American Society for Microbiology.)

Using reverse transcription in conjunction with the quantitative real-time PCR or digital droplet PCR, the transcriptome profiling of human chromosome 18 has been carried out in liver hepatocytes and hepatoblastoma cells (HepG2 cell line) in terms of the absolute number of each transcript per cell. The transcript abundance varies within the range of 0.006 to 9635 and 0.011 to 4819 copies per cell for HepG2 cell line and hepatocytes, respectively. The expression profiles for genes of chromosome 18 in hepatocytes and HepG2 cells were found to significantly correlate: the Spearman's correlation coefficient was equal to 0.81. The distribution of frequency of transcripts over their abundance was bimodal for HepG2 cells and unimodal for liver hepatocytes. Bioinformatic analysis of the differential gene expression has revealed that genes of chromosome 18, overexpressed in HepG2 cells compared to hepatocytes, are associated with cell division and cell adhesion processes. It is assumed that the enhanced expression of those genes in HepG2 cells is related to the proliferation activity of cultured cells. The differences in transcriptome profiles have to be taken into account when modelling liver hepatocytes with cultured HepG2 cells.

MicroRNA is a special type of regulatory molecules governing gene expression. Circulating microRNAs found in blood and other biological fluids are considered today as potential biomarkers of human pathology. Presently, quantitative alterations of particular microRNAs are revealed for a large number of oncological diseases and other disorders. The recently emerged method of digital droplet PCR (ddPCR) possesses a number of advantages making this method the most suitable for verification and validation of perspective microRNA markers of human pathologies. Among these advantages are the high accuracy and reproducibility of microRNA quantification as well as the capability to directly measure the absolute number of microRNA copies with the large dynamic range and a high throughput. The paper reviews microRNA biogenesis, the origin of circulating microRNAs, and methods used for their quantification. The special technical features of ddPCR, which make it an attractive method both for studying microRNAs as biomarkers of human pathologies and for basic research devoted to aspects of gene regulation by microRNA molecules, are also discussed.

Unlabelled: To successfully replicate in an infected host cell, a virus must overcome sophisticated host defense mechanisms. Viruses, therefore, have evolved a multitude of devices designed to circumvent cellular defenses that would lead to abortive infection. Previous studies have identified Nek9, a cellular kinase, as a binding partner of adenovirus E1A, but the biology behind this association remains a mystery. Here we show that Nek9 is a transcriptional repressor that functions together with E1A to silence the expression of p53-inducible GADD45A gene in the infected cell. Depletion of Nek9 in infected cells reduces virus growth but unexpectedly enhances viral gene expression from the E2 transcription unit, whereas the opposite occurs when Nek9 is overexpressed. Nek9 localizes with viral replication centers, and its depletion reduces viral genome replication, while overexpression enhances viral genome numbers in infected cells. Additionally, Nek9 was found to colocalize with the viral E4 orf3 protein, a repressor of cellular stress response. Significantly, Nek9 was also shown to associate with viral and cellular promoters and appears to function as a transcriptional repressor, representing the first instance of Nek9 playing a role in gene regulation. Overall, these results highlight the complexity of virus-host interactions and identify a new role for the cellular protein Nek9 during infection, suggesting a role for Nek9 in regulating p53 target gene expression., Importance: In the arms race that exists between a pathogen and its host, each has continually evolved mechanisms to either promote or prevent infection. In order to successfully replicate and spread, a virus must overcome every mechanism that a cell can assemble to block infection. On the other hand, to counter viral spread, cells must have multiple mechanisms to stifle viral replication. In the present study, we add to our understanding of how the human adenovirus is able to circumvent cellular roadblocks to replication. We show that the virus uses a cellular protein, Nek9, in order to block activation of p53-regulated gene GADD45A, which is an important player in stress response and p53-mediated cell cycle arrest. Importantly, our study also identifies Nek9 as a transcriptional repressor., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)