Biosensing platforms for DNA diagnostics based on CRISPR/Cas nucleases: towards the detection of nucleic acids at the level of single molecules in non-laboratory settings.

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.

Systems Biology for Drug Target Discovery in Acute Myeloid Leukemia.

Combining new therapeutics with all--retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells.

Combining recombinase polymerase amplification with tyrosine modified 2'-deoxyuridine-5'-triphosphate for direct voltammetric detection of double-stranded DNA: Application to potato pathogen Dickeya solani.

The approach based on a combination of isothermal recombinase polymerase amplification (RPA), 2'-deoxyuridine-5'-triphosphate modified with tyrosine aromatic group (dUTP-Y1), and direct voltammetric detection of RPA product carrying electroactive labels was successfully applied to the potato pathogen Dickeya solani. The artificial nucleotide dUTP-Y1 demonstrated a good compatibility with RPA, enabling by targeting a section of D. solani genome with a unique sequence to produce the full-size modified products at high levels of substitution of dTTP by dUTP-Y1 (up to 80-90 %) in the reaction mixture.

Polymerase incorporation of 4-nitrophenyl modified 2'-deoxyuridine-5'-triphosphates into double-stranded DNA for direct electrochemical detection.

Three novel 2'-deoxyuridine-5'-triphosphates modified with 4-nitrophenyl groups via various linkers (dUTP-N1, dUTP-N2, and dUTP-N3) were tested as bearers of reducible electroactive labels as well as substrates suitable for enzymes used in polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) with a potential application to direct electrochemical detection of double-stranded deoxyribonucleic acid (dsDNA). In cyclic and square wave voltammograms on carbon screen printed electrodes, the labeled dUTP have demonstrated distinct reduction peaks at potentials of -0.7 V to -0.

System analysis of surface CD markers during the process of granulocytic differentiation.

Plasma membrane proteins with extracellular-exposed domains are responsible for transduction of extracellular signals into intracellular responses, and their accessibility to therapeutic molecules makes them attractive targets for drug development. In this work, using omics technologies and immunochemical methods, we have studied changes in the content of markers of clusters of differentiation (CD markers) of neutrophils (CD33, CD97, CD54, CD38, CD18, CD11b, CD44, and CD71) at the level of transcripts and proteins in NB4, HL-60 and K562 cell lines, induced by the treatment with all-trans-retinoic acid (ATRA). Transcriptomic analysis revealed the induction of CD38, CD54, CD11b, and CD18 markers as early as 3 h after the addition of the inducer in the ATRA-responsive cell lines HL-60 and NB4.

Secondary metabolites of plants and their possible role in the "age of superbugs".

Bacterial infections are a serious cause of high morbidity and mortality worldwide. Over the past decades, the drug resistance of bacterial pathogens has been steadily increasing, while the rate of development of new effective antibacterial drugs remains consistently low. The plant kingdom is sometimes called a bottomless well for the search for new antimicrobial therapies.

Proteomic Approach to Investigating Expression, Localization, and Functions of the SOWAHD Gene Protein Product during Granulocytic Differentiation.

Cataloging human proteins and evaluation of their expression, cellular localization, functions, and potential medical significance are important tasks for the global proteomic community. At present, localization and functions of protein products for almost half of protein-coding genes remain unknown or poorly understood. Investigation of organelle proteomes is a promising approach to uncovering localization and functions of human proteins.

Principal Component Analysis of Alternative Splicing Profiles Revealed by Long-Read ONT Sequencing in Human Liver Tissue and Hepatocyte-Derived HepG2 and Huh7 Cell Lines.

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.

Polymerase incorporation of fluorescein or rhodamine modified 2'-deoxyuridine-5'-triphosphates into double-stranded DNA for direct electrochemical detection.

The 2'-deoxyuridine-5'-triphosphates modified with fluorescein (dUTP-Fl) or rhodamine (dUTP-Rh) were tested as bearers of electroactive labels and as proper substrates for polymerases used in polymerase chain reaction (PCR) and isothermal recombinase polymerase amplification (RPA) with the aim of electrochemical detection of double-stranded DNA (dsDNA) amplification products. For this purpose, electrochemical behavior of free fluorescein and rhodamine as well as the modified nucleotides, dUTP-Fl and dUTP-Rh, was studied by cyclic (CV) and square wave (SWV) voltammetry on carbon screen printed electrodes. Both free fluorescein and dUTP-Fl underwent a two-step oxidation at the peak potentials (E) of 0.

Implication of integrin α5β1 in senescence of SK-Mel-147 human melanoma cells.

Downregulation of α5β1 integrin in the SK-Mel-147 human melanoma culture model sharply inhibits the phenotypic manifestations of tumor progression: cell proliferation and clonal activity. This was accompanied by a 2-3-fold increase in the content of SA-β-Gal positive cells thus indicating an increase in the cellular senescence phenotype. These changes were accompanied by a significant increase in the activity of p53 and p21 tumor suppressors and components of the PI3K/Akt/mTOR/p70 signaling pathway.

Bacterial Therapy of Cancer: A Way to the Dustbin of History or to the Medicine of the Future?

Bacteria are the constant companions of the human body throughout its life and even after its death. The history of a human disease such as cancer and the history of microorganisms, particularly bacteria, are believed to closely intertwined. This review was conceived to highlight the attempts of scientists from ancient times to the present day to discover the relationship between bacteria and the emergence or development of tumors in the human body.

Exploiting Multi-Omics Profiling and Systems Biology to Investigate Functions of TOMM34.

Although modern biology is now in the post-genomic era with vastly increased access to high-quality data, the set of human genes with a known function remains far from complete. This is especially true for hundreds of mitochondria-associated genes, which are under-characterized and lack clear functional annotation. However, with the advent of multi-omics profiling methods coupled with systems biology algorithms, the cellular role of many such genes can be elucidated.

Nuclear Proteomics of Induced Leukemia Cell Differentiation.

Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell maturation. The nuclear proteome represents a rich source of regulatory molecules, including transcription factors (TFs). It is important to have an understanding of molecular perturbations at the early stages of the differentiation processes.

TRIM28 Is a Novel Regulator of CD133 Expression Associated with Cancer Stem Cell Phenotype.

CD133 is an extensively studied marker of the most malignant tumor cell population, designated as cancer stem cells (CSCs). However, the function of this glycoprotein and its involvement in cell regulatory cascades are still poorly understood. Here we show a positive correlation between the level of CD133 plasma membrane expression and the proliferative activity of cells of the Caco-2, HT-29, and HUH7 cancer cell lines.

[Splice variants of mRNA of cytochrome P450 genes: analysis by the nanopore sequencing method in human liver tissue and HepG2 cell line].

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.

[PCR analysis of the expression of chromosome 18 genes in human liver tissue: interindividual variability].

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.

Omics Technologies to Decipher Regulatory Networks in Granulocytic Cell Differentiation.

Induced granulocytic differentiation of human leukemic cells under all--retinoid acid (ATRA) treatment underlies differentiation therapy of acute myeloid leukemia. Knowing the regulation of this process it is possible to identify potential targets for antileukemic drugs and develop novel approaches to differentiation therapy. In this study, we have performed transcriptomic and proteomic profiling to reveal up- and down-regulated transcripts and proteins during time-course experiments.

Human Chr18 transcriptome dataset combined from the Illumina HiSeq, ONT MinION, and qPCR data.

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).

Contact-Dependent Growth Inhibition in Bacteria: Do Not Get Too Close!

Over millions of years of evolution, bacteria have developed complex strategies for intra-and interspecies interactions and competition for ecological niches and resources. Contact-dependent growth inhibition systems (CDI) are designed to realize a direct physical contact of one bacterial cell with other cells in proximity via receptor-mediated toxin delivery. These systems are found in many microorganisms including clinically important human pathogens.

A Neuroprotective Dose of Isatin Causes Multilevel Changes Involving the Brain Proteome: Prospects for Further Research.

Isatin (indole-2,3-dione) is an endogenous regulator, exhibiting a wide range of biological and pharmacological activities. At doses of 100 mg/kg and above, isatin is neuroprotective in different experimental models of neurodegeneration. Good evidence exists that its effects are realized via interaction with numerous isatin-binding proteins identified in the brain and peripheral tissues studied.

Evaluation of Aptamers as Affinity Reagents for an Enhancement of SRM-Based Detection of Low-Abundance Proteins in Blood Plasma.

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.

[Evaluation of the diversity of random DNA-libraries by the shape of amplification curves for estimation of the efficiency of aptamer selection].

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.